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Sample records for alk inhibitor crizotinib

  1. Crizotinib-resistant NPM-ALK mutants confer differential sensitivity to unrelated Alk inhibitors.

    PubMed

    Ceccon, Monica; Mologni, Luca; Bisson, William; Scapozza, Leonardo; Gambacorti-Passerini, Carlo

    2013-02-01

    The dual ALK/MET inhibitor crizotinib was recently approved for the treatment of metastatic and late-stage ALK+ NSCLC, and is currently in clinical trial for other ALK-related diseases. As predicted after other tyrosine kinase inhibitors' clinical experience, the first mutations that confer resistance to crizotinib have been described in patients with non-small cell lung cancer (NSCLC) and in one patient inflammatory myofibroblastic tumor (IMT). Here, we focused our attention on the anaplastic large cell lymphoma (ALCL), where the oncogenic fusion protein NPM-ALK, responsible for 70% to 80% of cases, represents an ideal crizotinib target. We selected and characterized 2 human NPM-ALK+ ALCL cell lines, KARPAS-299 and SUP-M2, able to survive and proliferate at different crizotinib concentrations. Sequencing of ALK kinase domain revealed that a single mutation became predominant at high crizotinib doses in each cell line, namely L1196Q and I1171N in Karpas-299 and SUP-M2 cells, respectively. These mutations also conferred resistance to crizotinib in Ba/F3 cells expressing human NPM-ALK. The resistant cell populations, as well as mutated Ba/F3 cells, were characterized for sensitivity to two additional ALK inhibitors: the dual ALK/EGFR inhibitor AP26113 and NVP-TAE684. While L1196Q-positive cell lines were sensitive to both inhibitors, cells carrying I1171N substitution showed cross-resistance to all ALK inhibitors tested. This study provides potentially relevant information for the management of patients with ALCL that may relapse after crizotinib treatment.

  2. Activity of second-generation ALK inhibitors against crizotinib-resistant mutants in an NPM-ALK model compared to EML4-ALK.

    PubMed

    Fontana, Diletta; Ceccon, Monica; Gambacorti-Passerini, Carlo; Mologni, Luca

    2015-07-01

    Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor involved in both solid and hematological tumors. About 80% of ALK-positive anaplastic large-cell lymphoma (ALCL) cases are characterized by the t(2;5)(p23;q35) translocation, encoding for the aberrant fusion protein nucleophosmin (NPM)-ALK, whereas 5% of non-small-cell lung cancer (NSCLC) patients carry the inv(2)(p21;p23) rearrangement, encoding for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion. The ALK/c-MET/ROS inhibitor crizotinib successfully improved the treatment of ALK-driven diseases. However, several cases of resistance appeared in NSCLC patients, and ALK amino acid substitutions were identified as a leading cause of resistance to crizotinib. Second-generation ALK inhibitors have been developed in order to overcome crizotinib resistance. In this work, we profiled in vitro the activity of crizotinib, AP26113, ASP3026, alectinib, and ceritinib against six mutated forms of ALK associated with clinical resistance to crizotinib (C1156Y, L1196M, L1152R, G1202R, G1269A, and S1206Y) and provide a classification of mutants according to their level of sensitivity/resistance to the drugs. Since the biological activity of ALK mutations extends beyond the specific type of fusion, both NPM-ALK- and EML4-ALK-positive cellular models were used. Our data revealed that most mutants may be targeted by using different inhibitors. One relevant exception is represented by the G1202R substitution, which was highly resistant to all drugs (>10-fold increased IC50 compared to wild type) and may represent the most challenging mutation to overcome. These results provide a prediction of cross-resistance of known crizotinib-resistant mutations against all second-generation tyrosine kinase inhibitors (TKIs) clinically available, and therefore could be a useful tool to help clinicians in the management of crizotinib-resistance cases.

  3. Novel ALK inhibitor AZD3463 inhibits neuroblastoma growth by overcoming crizotinib resistance and inducing apoptosis

    PubMed Central

    Wang, Yongfeng; Wang, Long; Guan, Shan; Cao, Wenming; Wang, Hao; Chen, Zhenghu; Zhao, Yanling; Yu, Yang; Zhang, Huiyuan; Pang, Jonathan C.; Huang, Sophia L.; Akiyama, Yo; Yang, Yifan; Sun, Wenjing; Xu, Xin; Shi, Yan; Zhang, Hong; Kim, Eugene S.; Muscal, Jodi A.; Lu, Fengmin; Yang, Jianhua

    2016-01-01

    ALK receptor tyrosine kinase has been shown to be a therapeutic target in neuroblastoma. Germline ALK activating mutations are responsible for the majority of hereditary neuroblastoma and somatic ALK activating mutations are also frequently observed in sporadic cases of advanced NB. Crizotinib, a first-line therapy in the treatment of advanced non-small cell lung cancer (NSCLC) harboring ALK rearrangements, demonstrates striking efficacy against ALK-rearranged NB. However, crizotinib fails to effectively inhibit the activity of ALK when activating mutations are present within its kinase domain, as with the F1174L mutation. Here we show that a new ALK inhibitor AZD3463 effectively suppressed the proliferation of NB cell lines with wild type ALK (WT) as well as ALK activating mutations (F1174L and D1091N) by blocking the ALK-mediated PI3K/AKT/mTOR pathway and ultimately induced apoptosis and autophagy. In addition, AZD3463 enhanced the cytotoxic effects of doxorubicin on NB cells. AZD3463 also exhibited significant therapeutic efficacy on the growth of the NB tumors with WT and F1174L activating mutation ALK in orthotopic xenograft mouse models. These results indicate that AZD3463 is a promising therapeutic agent in the treatment of NB. PMID:26786851

  4. Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK-mutated neuroblastoma.

    PubMed

    Moore, Nathan F; Azarova, Anna M; Bhatnagar, Namrata; Ross, Kenneth N; Drake, Lauren E; Frumm, Stacey; Liu, Qinsong S; Christie, Amanda L; Sanda, Takaomi; Chesler, Louis; Kung, Andrew L; Gray, Nathanael S; Stegmaier, Kimberly; George, Rani E

    2014-09-30

    Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALK(F1174L) mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALK(F1174L)/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALK(F1174L)/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.

  5. The use of cellular thermal shift assay (CETSA) to study Crizotinib resistance in ALK-expressing human cancers.

    PubMed

    Alshareef, Abdulraheem; Zhang, Hai-Feng; Huang, Yung-Hsing; Wu, Chengsheng; Zhang, Jing Dong; Wang, Peng; El-Sehemy, Ahmed; Fares, Mohamed; Lai, Raymond

    2016-01-01

    Various forms of oncogenic ALK proteins have been identified in various types of human cancers. While Crizotinib, an ALK inhibitor, has been found to be therapeutically useful against a subset of ALK(+) tumours, clinical resistance to this drug has been well recognized and the mechanism of this phenomenon is incompletely understood. Using the cellular thermal shift assay (CETSA), we measured the Crizotinib-ALK binding in a panel of ALK(+) cell lines, and correlated the findings with the ALK structure and its interactions with specific binding proteins. The Crizotinib IC50 significantly correlated with Crizotinib-ALK binding. The suboptimal Crizotinib-ALK binding in Crizotinib-resistant cells is not due to the cell-specific environment, since transfection of NPM-ALK into these cells revealed substantial Crizotinib-NPM-ALK binding. Interestingly, we found that the resistant cells expressed higher protein level of β-catenin and siRNA knockdown restored Crizotinib-ALK binding (correlated with a significant lowering of IC50). Computational analysis of the crystal structures suggests that β-catenin exerts steric hindrance to the Crizotinib-ALK binding. In conclusion, the Crizotinib-ALK binding measurable by CETSA is useful in predicting Crizotinib sensitivity, and Crizotinib-ALK binding is in turn dictated by the structure of ALK and some of its binding partners. PMID:27641368

  6. Increased oral availability and brain accumulation of the ALK inhibitor crizotinib by coadministration of the P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) inhibitor elacridar.

    PubMed

    Tang, Seng Chuan; Nguyen, Luan N; Sparidans, Rolf W; Wagenaar, Els; Beijnen, Jos H; Schinkel, Alfred H

    2014-03-15

    Crizotinib is an oral tyrosine kinase inhibitor approved for treating patients with non-small cell lung cancer (NSCLC) containing an anaplastic lymphoma kinase (ALK) rearrangement. We used knockout mice to study the roles of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) in plasma pharmacokinetics and brain accumulation of oral crizotinib, and the feasibility of improving crizotinib kinetics using coadministration of the dual ABCB1/ABCG2 inhibitor elacridar. In vitro, crizotinib was a good transport substrate of human ABCB1, but not of human ABCG2 or murine Abcg2. With low-dose oral crizotinib (5 mg/kg), Abcb1a/1b(-/-) and Abcb1a/1b;Abcg2(-/-) mice had an approximately twofold higher plasma AUC than wild-type mice, and a markedly (~40-fold) higher brain accumulation at 24 hr. Also at 4 hr, crizotinib brain concentrations were ∼25-fold, and brain-to-plasma ratios ~14-fold higher in Abcb1a/1b(-/-) and Abcb1a/1b;Abcg2(-/-) mice than in wild-type mice. High-dose oral crizotinib (50 mg/kg) resulted in comparable plasma pharmacokinetics between wild-type and Abcb1a/1b(-/-) mice, suggesting saturation of intestinal Abcb1. Nonetheless, brain accumulation at 24 hr was still ~70-fold higher in Abcb1a/1b(-/-) than in wild-type mice. Importantly, oral elacridar coadministration increased the plasma and brain concentrations and brain-to-plasma ratios of crizotinib in wild-type mice, equaling the levels in Abcb1a/1b;Abcg2(-/-) mice. Our results indicate that crizotinib oral availability and brain accumulation were primarily restricted by Abcb1 at a non-saturating dose, and that coadministration of elacridar with crizotinib could substantially increase crizotinib oral availability and delivery to the brain. This principle might be used to enhance therapeutic efficacy of crizotinib against brain metastases in NSCLC patients.

  7. Multiple Endocrine Disruption by the MET/ALK Inhibitor Crizotinib in Patients With Non–Small Cell Lung Cancer

    PubMed Central

    Sargis, Robert M.; Salgia, Ravi

    2014-01-01

    Objectives Non–small cell lung cancer (NSCLC) is a heterogenous group of disorders that can be subclassified based upon molecular characterization. Anaplastic lymphoma kinase translocation and MET aberrations occur in a subset of NSCLC. Anaplastic lymphoma kinase/ MET have been shown to be inhibited by the small molecule tyrosine kinase inhibitor crizotinib. Recently, crizotinib was shown to decrease testosterone in males. Herein, we describe the effects of crizotinib on multiple hormonal axes. Materials and Methods Seven consecutive patients with NSCLC who were receiving crizotinib as part of their standard care were evaluated for hormonal disruptions. Results Primary hypogonadism was detected in 4/5 of males, whereas mildly elevated prolactin was observed in 4/7 patients. Hypocalcemia was observed in 3/7 patients. Interestingly, 5/7 patients had elevated levels of insulin-like growth factor-1 (IGF-1) levels, and the remaining 2 individuals had levels that were near the upper limits of the normal range. Conclusions Because of cellular cross-talk between MET and IGF-1 signaling, elevated IGF-1 levels induced by crizotinib treatment may have implications for long-term drug efficacy. Furthermore, this finding suggests a potential avenue of therapeutic synergy, namely coordinate inhibition of the MET and IGF-1 signaling pathways. Finally, as crizotinib has been recently approved, it is prudent to check hormone and calcium biomarkers and correct noted deficiencies for improved outcomes and quality of life. PMID:23934135

  8. A molecular dynamics investigation on the crizotinib resistance mechanism of C1156Y mutation in ALK

    SciTech Connect

    Sun, Hui-Yong; Ji, Feng-Qin

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer The study revealed the detailed resistance mechanism of the non-active mutation C1156Y in ALK. Black-Right-Pointing-Pointer C1156Y leads to crizotinib displacement and conformational changes in the binding cavity. Black-Right-Pointing-Pointer The conformations cause a decline in the vdW and electrostatic energy between crizotinib and ALK. -- Abstract: Crizotinib is an anaplastic lymphoma kinase (ALK) inhibitor that has recently been approved in the US for the treatment of non-small cell lung carcinoma (NSCLC). Despite its outstanding safety and efficacy, several resistant mutations against crizotinib have been detected in the treatment of NSCLC. However, in contrast to the widely accepted mechanism of steric hindrance by mutations at the active site, the mechanism by which the C1156Y non-active site mutation confers resistance against crizotinib remains unclear. In the present study, the resistance mechanism of C1156Y in ALK was investigated using molecular dynamics simulations. The results suggest that despite the non-active site mutation, C1156Y causes the dislocation of crizotinib as well as the indirect conformational changes in the binding cavity, which results in a marked decrease in the van der Waals and electrostatic interactions between crizotinib and ALK. The obtained results provide a detailed explanation of the resistance caused by C1156Y and may give a vital clue for the design of drugs to combat crizotinib resistance.

  9. A novel acquired ALK F1245C mutation confers resistance to crizotinib in ALK-positive NSCLC but is sensitive to ceritinib.

    PubMed

    Kodityal, Sandeep; Elvin, Julia A; Squillace, Rachel; Agarwal, Nikita; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J; Ou, Sai-Hong Ignatius

    2016-02-01

    The emergence of acquired anaplastic lymphoma kinase (ALK) resistant mutations is a common molecular mechanism underpinning disease progression during crizotinib treatment of ALK-positive (ALK+) non-small cell lung cancer (NSCLC) patients. Identifying acquired resistance mutations in ALK is paramount for tailoring future therapy with second generation ALK inhibitors and beyond. Comprehensive genomic profiling using hybrid-capture next generation sequencing has been successful in identifying acquired ALK resistance mutations. Here we described the emergence of an ALK F1245C mutation in an advanced ALK+ NSCLC patient (EML4-ALK variant 3a/b) who developed slow disease progression after a durable response to crizotinib. The patient was eventually switched to ceritinib with on-going clinical response. This is the first patient report that ALK F1245C is an acquired resistance mutation to crizotinib that can be overcome by ceritinib. PMID:26775591

  10. The use of cellular thermal shift assay (CETSA) to study Crizotinib resistance in ALK-expressing human cancers

    PubMed Central

    Alshareef, Abdulraheem; Zhang, Hai-Feng; Huang, Yung-Hsing; Wu, Chengsheng; Zhang, Jing Dong; Wang, Peng; El-Sehemy, Ahmed; Fares, Mohamed; Lai, Raymond

    2016-01-01

    Various forms of oncogenic ALK proteins have been identified in various types of human cancers. While Crizotinib, an ALK inhibitor, has been found to be therapeutically useful against a subset of ALK+ tumours, clinical resistance to this drug has been well recognized and the mechanism of this phenomenon is incompletely understood. Using the cellular thermal shift assay (CETSA), we measured the Crizotinib—ALK binding in a panel of ALK+ cell lines, and correlated the findings with the ALK structure and its interactions with specific binding proteins. The Crizotinib IC50 significantly correlated with Crizotinib—ALK binding. The suboptimal Crizotinib—ALK binding in Crizotinib-resistant cells is not due to the cell-specific environment, since transfection of NPM-ALK into these cells revealed substantial Crizotinib—NPM-ALK binding. Interestingly, we found that the resistant cells expressed higher protein level of β-catenin and siRNA knockdown restored Crizotinib—ALK binding (correlated with a significant lowering of IC50). Computational analysis of the crystal structures suggests that β-catenin exerts steric hindrance to the Crizotinib—ALK binding. In conclusion, the Crizotinib—ALK binding measurable by CETSA is useful in predicting Crizotinib sensitivity, and Crizotinib—ALK binding is in turn dictated by the structure of ALK and some of its binding partners. PMID:27641368

  11. Three Years Sustained Complete Remission Achieved in a Primary Refractory ALK-Positive Anaplastic T Large Cell Lymphoma Treated with Crizotinib

    PubMed Central

    Mahuad, Carolina Valeria; Repáraz, María de los Ángeles Vicente; Zerga, Marta E.; Aizpurua, María Florencia; Casali, Claudia; Garate, Gonzalo

    2016-01-01

    The prognosis of the primary refractory anaplastic lymphoma kinase (ALK+) anaplastic T large cell lymphoma is ominous. The identification of molecular targets with potential to drive oncogenesis remains a cornerstone for the designing of new selective cancer therapies. Crizotinib is a selective ATP-competitive inhibitor for ALK, approved for its use in lung cancer with rearrangements on ALK gene. The reported cases describe the use of crizotinib as a bridging strategy prior to allotransplantation; there are no reported prolonged survivals under monotherapy with Crizotinib. We report a case of a primary refractory ALK+ anaplastic large-cell lymphoma that sustains complete response after 3 years of crizotinib monotherapy. PMID:27441079

  12. Three Years Sustained Complete Remission Achieved in a Primary Refractory ALK-Positive Anaplastic T Large Cell Lymphoma Treated with Crizotinib.

    PubMed

    Mahuad, Carolina Valeria; Repáraz, María de Los Ángeles Vicente; Zerga, Marta E; Aizpurua, María Florencia; Casali, Claudia; Garate, Gonzalo

    2016-06-28

    The prognosis of the primary refractory anaplastic lymphoma kinase (ALK+) anaplastic T large cell lymphoma is ominous. The identification of molecular targets with potential to drive oncogenesis remains a cornerstone for the designing of new selective cancer therapies. Crizotinib is a selective ATP-competitive inhibitor for ALK, approved for its use in lung cancer with rearrangements on ALK gene. The reported cases describe the use of crizotinib as a bridging strategy prior to allotransplantation; there are no reported prolonged survivals under monotherapy with Crizotinib. We report a case of a primary refractory ALK+ anaplastic large-cell lymphoma that sustains complete response after 3 years of crizotinib monotherapy. PMID:27441079

  13. Reversal of microRNA-150 silencing disadvantages crizotinib-resistant NPM-ALK(+) cell growth.

    PubMed

    Hoareau-Aveilla, Coralie; Valentin, Thibaud; Daugrois, Camille; Quelen, Cathy; Mitou, Géraldine; Quentin, Samuel; Jia, Jinsong; Spicuglia, Salvatore; Ferrier, Pierre; Ceccon, Monica; Giuriato, Sylvie; Gambacorti-Passerini, Carlo; Brousset, Pierre; Lamant, Laurence; Meggetto, Fabienne

    2015-09-01

    The regulatory microRNA miR-150 is involved in the development of hemopathies and is downregulated in T-lymphomas, such as anaplastic large-cell lymphoma (ALCL) tumors. ALCL is defined by the presence or absence of translocations that activate the anaplastic lymphoma kinase (ALK), with nucleophosmin-ALK (NPM-ALK) fusions being the most common. Here, we compared samples of primary NPM-ALK(+) and NPM-ALK(-) ALCL to investigate the role of miR-150 downstream of NPM-ALK. Methylation of the MIR150 gene was substantially elevated in NPM-ALK(+) biopsies and correlated with reduced miR-150 expression. In NPM-ALK(+) cell lines, DNA hypermethylation-mediated miR-150 repression required ALK-dependent pathways, as ALK inhibition restored miR-150 expression. Moreover, epigenetic silencing of miR-150 was due to the activation of STAT3, a major downstream substrate of NPM-ALK, in cooperation with DNA methyltransferase 1 (DNMT1). Accordingly, miR-150 repression was turned off following treatment with the DNMT inhibitor, decitabine. In murine NPM-ALK(+) xenograft models, miR-150 upregulation induced antineoplastic activity. Treatment of crizotinib-resistant NPM-ALK(+) KARPAS-299-CR06 cells with decitabine or ectopic miR-150 expression reduced viability and growth. Altogether, our results suggest that hypomethylating drugs, alone or in combination with other agents, may benefit ALK(+) patients harboring tumors resistant to crizotinib and other anti-ALK tyrosine kinase inhibitors (TKIs). Moreover, these results support further work on miR-150 in these and other ALK(+) malignancies.

  14. Reversal of microRNA-150 silencing disadvantages crizotinib-resistant NPM-ALK(+) cell growth

    PubMed Central

    Hoareau-Aveilla, Coralie; Valentin, Thibaud; Daugrois, Camille; Quelen, Cathy; Mitou, Géraldine; Quentin, Samuel; Jia, Jinsong; Spicuglia, Salvatore; Ferrier, Pierre; Ceccon, Monica; Giuriato, Sylvie; Gambacorti-Passerini, Carlo; Brousset, Pierre; Lamant, Laurence; Meggetto, Fabienne

    2015-01-01

    The regulatory microRNA miR-150 is involved in the development of hemopathies and is downregulated in T-lymphomas, such as anaplastic large-cell lymphoma (ALCL) tumors. ALCL is defined by the presence or absence of translocations that activate the anaplastic lymphoma kinase (ALK), with nucleophosmin-ALK (NPM-ALK) fusions being the most common. Here, we compared samples of primary NPM-ALK(+) and NPM-ALK(–) ALCL to investigate the role of miR-150 downstream of NPM-ALK. Methylation of the MIR150 gene was substantially elevated in NPM-ALK(+) biopsies and correlated with reduced miR-150 expression. In NPM-ALK(+) cell lines, DNA hypermethylation–mediated miR-150 repression required ALK-dependent pathways, as ALK inhibition restored miR-150 expression. Moreover, epigenetic silencing of miR-150 was due to the activation of STAT3, a major downstream substrate of NPM-ALK, in cooperation with DNA methyltransferase 1 (DNMT1). Accordingly, miR-150 repression was turned off following treatment with the DNMT inhibitor, decitabine. In murine NPM-ALK(+) xenograft models, miR-150 upregulation induced antineoplastic activity. Treatment of crizotinib-resistant NPM-ALK(+) KARPAS-299-CR06 cells with decitabine or ectopic miR-150 expression reduced viability and growth. Altogether, our results suggest that hypomethylating drugs, alone or in combination with other agents, may benefit ALK(+) patients harboring tumors resistant to crizotinib and other anti-ALK tyrosine kinase inhibitors (TKIs). Moreover, these results support further work on miR-150 in these and other ALK(+) malignancies. PMID:26258416

  15. Targeting autophagy enhances the anti-tumoral action of crizotinib in ALK-positive anaplastic large cell lymphoma

    PubMed Central

    Desquesnes, Aurore; Le Gonidec, Sophie; AlSaati, Talal; Beau, Isabelle; Lamant, Laurence; Meggetto, Fabienne; Espinos, Estelle; Codogno, Patrice; Brousset, Pierre; Giuriato, Sylvie

    2015-01-01

    Anaplastic Lymphoma Kinase-positive Anaplastic Large Cell Lymphomas (ALK+ ALCL) occur predominantly in children and young adults. Their treatment, based on aggressive chemotherapy, is not optimal since ALCL patients can still expect a 30% 2-year relapse rate. Tumor relapses are very aggressive and their underlying mechanisms are unknown. Crizotinib is the most advanced ALK tyrosine kinase inhibitor and is already used in clinics to treat ALK-associated cancers. However, crizotinib escape mechanisms have emerged, thus preventing its use in frontline ALCL therapy. The process of autophagy has been proposed as the next target for elimination of the resistance to tyrosine kinase inhibitors. In this study, we investigated whether autophagy is activated in ALCL cells submitted to ALK inactivation (using crizotinib or ALK-targeting siRNA). Classical autophagy read-outs such as autophagosome visualization/quantification by electron microscopy and LC3-B marker turn-over assays were used to demonstrate autophagy induction and flux activation upon ALK inactivation. This was demonstrated to have a cytoprotective role on cell viability and clonogenic assays following combined ALK and autophagy inhibition. Altogether, our results suggest that co-treatment with crizotinib and chloroquine (two drugs already used in clinics) could be beneficial for ALK-positive ALCL patients. PMID:26338968

  16. The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

    PubMed

    Guan, J; Tucker, E R; Wan, H; Chand, D; Danielson, L S; Ruuth, K; El Wakil, A; Witek, B; Jamin, Y; Umapathy, G; Robinson, S P; Johnson, T W; Smeal, T; Martinsson, T; Chesler, L; Palmer, R H; Hallberg, B

    2016-09-01

    The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALK(F1174L)/MYCN Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients. PMID:27483357

  17. Crizotinib for the Treatment of ALK-Rearranged Non-Small Cell Lung Cancer: A Success Story to Usher in the Second Decade of Molecular Targeted Therapy in Oncology

    PubMed Central

    Bartlett, Cynthia Huang; Mino-Kenudson, Mari; Cui, Jean; Iafrate, A. John

    2012-01-01

    Crizotinib, an ALK/MET/ROS1 inhibitor, was approved by the U.S. Food and Drug Administration for the treatment of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) in August 2011, merely 4 years after the first publication of ALK-rearranged NSCLC. The crizotinib approval was accompanied by the simultaneous approval of an ALK companion diagnostic fluorescent in situ hybridization assay for the detection of ALK-rearranged NSCLC. Crizotinib continued to be developed as an ALK and MET inhibitor in other tumor types driven by alteration in ALK and MET. Crizotinib has recently been shown to be an effective ROS1 inhibitor in ROS1-rearranged NSCLC, with potential future clinical applications in ROS1-rearranged tumors. Here we summarize the heterogeneity within the ALK- and ROS1-rearranged molecular subtypes of NSCLC. We review the past and future clinical development of crizotinib for ALK-rearranged NSCLC and the diagnostic assays to detect ALK-rearranged NSCLC. We highlight how the success of crizotinib has changed the paradigm of future drug development for targeted therapies by targeting a molecular-defined subtype of NSCLC despite its rarity and affected the practice of personalized medicine in oncology, emphasizing close collaboration between clinical oncologists, pathologists, and translational scientists. PMID:22989574

  18. The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN

    PubMed Central

    Guan, J.; Tucker, E. R.; Wan, H.; Chand, D.; Danielson, L. S.; Ruuth, K.; El Wakil, A.; Witek, B.; Jamin, Y.; Umapathy, G.; Robinson, S. P.; Johnson, T. W.; Smeal, T.; Martinsson, T.; Chesler, L.; Palmer, R. H.

    2016-01-01

    ABSTRACT The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo. In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALKF1174L/MYCN. Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients. PMID:27483357

  19. ALK and crizotinib: after the honeymoon…what else? Resistance mechanisms and new therapies to overcome it.

    PubMed

    Rolfo, Christian; Passiglia, Francesco; Castiglia, Marta; Raez, Luis E; Germonpre, Paul; Gil-Bazo, Ignacio; Zwaenepoel, Karen; De Wilde, Annemieke; Bronte, Giuseppe; Russo, Antonio; Van Meerbeeck, Jan P; Van Schil, Paul; Pauwels, Patrick

    2014-08-01

    The last few decades have witnessed a silent revolution in the war against NSCLC, thanks to the discovery of "oncogenic drivers" and the subsequent development of targeted therapies. The discovery of the EML4-ALK fusion gene in a subgroup of patients with NSCLC and the subsequent clinical development of crizotinib has been an amazing success story in lung cancer translational-research, and its accelerated approval [only 4 years from the discovery of ALK rearrangement in NSCLC to the approval by the Food and Drug Administration (FDA)] marked the beginning of the new decade of targeted therapy. However, common to all targeted therapies, despite an initial benefit, patients inevitably experience tumor progression, due to the development of resistance. Several molecular mechanisms are responsible for acquired resistance, such as secondary mutations of ALK kinase domain or amplification of ALK fusion gene, or the activation of other oncogenic drivers, which may cause resistance independently of ALK genetic alterations. Pre-clinical data and early clinical trials showed the promising efficacy of a new class of ALK-inhibitors in overcoming acquired resistance. The inhibition of the molecular chaperone, HSP90, represents another promising strategy to overcome crizotinib resistance in ALK-rearranged NSCLC. Several molecules are currently under investigation in order to establish their specific role in the treatment of ALK-rearranged NSCLC.

  20. Crizotinib (PF-2341066) induces apoptosis due to downregulation of pSTAT3 and BCL-2 family proteins in NPM-ALK(+) anaplastic large cell lymphoma.

    PubMed

    Hamedani, Farid Saei; Cinar, Munevver; Mo, Zhicheng; Cervania, Melissa A; Amin, Hesham M; Alkan, Serhan

    2014-04-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is an aberrant fusion gene product with tyrosine kinase activity and is expressed in substantial subset of anaplastic large cell lymphomas (ALCL). It has been shown that NPM-ALK binds to and activates signal transducer and activator of transcription 3 (STAT3). Although NPM-ALK(+) ALCL overall shows a better prognosis, there is a sub-group of patients who relapses and is resistant to conventional chemotherapeutic regimens. NPM-ALK is a potential target for small molecule kinase inhibitors. Crizotinib (PF-2341066) is a small, orally bioavailable molecule that inhibits growth of tumors with ALK activity as shown in a subgroup of non-small lung cancer patients with EML4-ALK expression. In this study, we have investigated the in vitro effects of Crizotinib in ALCL cell line with NPM-ALK fusion. Crizotinib induced marked downregulation of STAT3 phosphorylation, which was associated with significant apoptotic cell death. Apoptosis induction was attributed to caspase-3 cleavage and marked downregulation of the Bcl-2 family of proteins including MCL-1. These findings implicate that Crizotinib has excellent potential to treat patients with NPM-ALK(+) ALCL through induction of apoptotic cell death and downregulation of major oncogenic proteins in this aggressive lymphoma.

  1. Metachronous primary uterine cancer surgically resected during Crizotinib treatment in a ALK-rearranged advanced lung adenocarcinoma

    PubMed Central

    Misino, Andrea; Scattone, Anna; Caldarola, Lucia; Petroni, Stella; Logroscino, Antonio; Montagna, Elisabetta Sara; Serio, Gabriella; Simone, Giovanni; Galetta, Domenico

    2016-01-01

    Rearrangements of the anaplastic lymphoma kinase (ALK) gene are present in 3% to 7% of non-small-cell lung cancers (NSCLCs). Patients harboring ALK rearrangements show very favourable outcomes if treated with targeted agents, among which crizotinib is the first and best studied. Crizotinib, an oral small-molecule tyrosine kinase inhibitor of ALK, MET, and ROS1 kinases, is a very active and well tolerated drug. Nevertheless, the optimal therapy management with this new drug is still partially unknown, especially with regard to the safety of combined treatments. Recently, the integration of locoregional treatments has been proposed as a feasible multimodality strategy in selected patients with good clinical conditions and slow-growing or oligoprogressive disease. In this report, a case of advanced lung adenocarcinoma, progressed after first line chemotherapy and re-biopsied detecting ALK rearrangement, is described. During crizotinib treatment the primary lung tumor showed an excellent regression; meanwhile a major surgery for a metachronous uterine cancer was safely and successfully carried out. PMID:26958511

  2. A patient previously treated with ALK inhibitors for central nervous system lesions from ALK rearranged lung cancer: a case report

    PubMed Central

    Kashima, Jumpei; Okuma, Yusuke; Hishima, Tsunekazu

    2016-01-01

    Background Patients with anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC) are now preferentially treated with tyrosine kinase inhibitors (TKIs). However, patients treated with ALK inhibitors end up with acquired resistance. Case presentation We present a patient with recurrent ALK-rearranged NSCLC that developed multiple brain metastases and meningitis carcinomatosa after sequential treatment with several lines of cytotoxic chemotherapy, crizotinib, and alectinib. After the patient underwent retreatment with crizotinib as salvage therapy because of poor performance status, the intracranial metastatic foci and meningeal thickening were shrank within 1 week. Conclusion Our experience with this case suggests that alectinib may restore sensitivity to crizotinib or amplified pathway such as MET which bestowed alectinib resistance was inhibited with crizotinib. PMID:27785052

  3. Evidence Suggesting That Discontinuous Dosing of ALK Kinase Inhibitors May Prolong Control of ALK+ Tumors.

    PubMed

    Amin, Amit Dipak; Rajan, Soumya S; Liang, Winnie S; Pongtornpipat, Praechompoo; Groysman, Matthew J; Tapia, Edgar O; Peters, Tara L; Cuyugan, Lori; Adkins, Jonathan; Rimsza, Lisa M; Lussier, Yves A; Puvvada, Soham D; Schatz, Jonathan H

    2015-07-15

    The anaplastic lymphoma kinase (ALK) is chromosomally rearranged in a subset of certain cancers, including 2% to 7% of non-small cell lung cancers (NSCLC) and ∼70% of anaplastic large cell lymphomas (ALCL). The ALK kinase inhibitors crizotinib and ceritinib are approved for relapsed ALK(+) NSCLC, but acquired resistance to these drugs limits median progression-free survival on average to ∼10 months. Kinase domain mutations are detectable in 25% to 37% of resistant NSCLC samples, with activation of bypass signaling pathways detected frequently with or without concurrent ALK mutations. Here we report that, in contrast to NSCLC cells, drug-resistant ALCL cells show no evidence of bypassing ALK by activating alternate signaling pathways. Instead, drug resistance selected in this setting reflects upregulation of ALK itself. Notably, in the absence of crizotinib or ceritinib, we found that increased ALK signaling rapidly arrested or killed cells, allowing a prolonged control of drug-resistant tumors in vivo with the administration of discontinuous rather than continuous regimens of drug dosing. Furthermore, even when drug resistance mutations were detected in the kinase domain, overexpression of the mutant ALK was toxic to tumor cells. We confirmed these findings derived from human ALCL cells in murine pro-B cells that were transformed to cytokine independence by ectopic expression of an activated NPM-ALK fusion oncoprotein. In summary, our results show how ALK activation functions as a double-edged sword for tumor cell viability, with potential therapeutic implications. PMID:26018086

  4. Evidence Suggesting that Discontinuous Dosing of ALK Kinase Inhibitors May Prolong Control of ALK+ Tumors

    PubMed Central

    Amin, Amit Dipak; Rajan, Soumya S.; Liang, Winnie S.; Pongtornpipat, Praechompoo; Groysman, Matthew J.; Tapia, Edgar O.; Peters, Tara L.; Cuyugan, Lori; Adkins, Jonathan; Rimsza, Lisa M.; Lussier, Yves A.; Puvvada, Soham D.; Schatz, Jonathan H.

    2015-01-01

    The anaplastic lymphoma kinase ALK is chromosomally rearranged in a subset of certain cancers, including 2–7% non-small cell lung cancers (NSCLC) and ~70% of anaplastic large cell lymphomas (ALCL). The ALK kinase inhibitors crizotinib and ceritinib are approved for relapsed ALK+ NSCLC, but acquired resistance to these drugs limits median progression-free survival on average to ~10 months. Kinase domain mutations are detectable in 25–37% of resistant NSCLC samples, with activation of bypass signaling pathways detected frequently with or without concurrent ALK mutations. Here we report that, in contrast to NSCLC cells, drug resistant ALCL cells show no evidence of bypassing ALK by activating alternate signaling pathways. Instead, drug resistance selected in this setting reflects upregulation of ALK itself. Notably, in the absence of crizotinib or ceritinib, we found that increased ALK signaling rapidly arrested or killed cells, allowing a prolonged control of drug-resistant tumors in vivo with the administration of discontinuous rather than continuous regimens of drug dosing. Furthermore, even when drug resistance mutations were detected in the kinase domain, overexpression of the mutant ALK was toxic to tumor cells. We confirmed these findings derived from human ALCL cells in murine pro-B cells that were transformed to cytokine independence by ectopic expression of an activated NPM-ALK fusion oncoprotein. In summary, our results show how ALK activation functions as a double-edged sword for tumor cell viability, with potential therapeutic implications. PMID:26018086

  5. Fatal Haemoptysis Associated with Dramatic Response to Crizotinib in an ALK-Rearranged Lung Adenocarcinoma

    PubMed Central

    Mussat, Elodie; Giraud, Violaine; Julie, Catherine; Chinet, Thierry

    2016-01-01

    The presence of an ALK (Anaplastic Lymphoma Kinase) rearrangement is a rare molecular feature in Non-Small Cell Lung Carcinoma (NSCLC), and concerns mainly non- or light smokers, young patients, with adenocarcinoma histological type. These tumours are particularly sensitive to Alk-targeted therapies, as crizotinib. Crizotinib is usually well-tolerated. We report a case of fatal haemoptysis associated with dramatic response to crizotinib in a patient with an ALK-rearranged lung adenocarcinoma. The patient presented a mediastinal invasion with tracheal involvement and compression of the right pulmonary artery. The initial evolution under crizotinib was good with tumour response. At 6 weeks of crizotinib the patient presented a massive haemoptysis with a tracheobronchial fistula and pneumomediastinum. She died of acute respiratory failure. Our case is the first to report a fatal effect of crizotinib associated with tumour necrosis and good tumour response on a massive mediastinal infiltration. Precautions are recommended with the use of crizotinib in proximal lung tumours with vascular invasion. PMID:27134984

  6. Tackling ALK in non-small cell lung cancer: the role of novel inhibitors

    PubMed Central

    Facchinetti, Francesco; Di Maio, Massimo; Graziano, Paolo; Bria, Emilio; Rossi, Giulio; Novello, Silvia

    2016-01-01

    Crizotinib is an oral inhibitor of anaplastic lymphoma kinase (ALK) with remarkable clinical activity in patients suffering from ALK-rearranged non-small cell lung cancer (NSCLC), accounting to its superiority compared to chemotherapy. Unfortunately, virtually all ALK-rearranged tumors acquire resistance to crizotinib, frequently within one year since the treatment initiation. To date, therapeutic strategies to overcome crizotinib resistance have focused on the use of more potent and structurally different compounds. Second-generation ALK inhibitors such as ceritinib (LDK378), alectinib (CH5424802/RO5424802) and brigatinib (AP26113) have shown relevant clinical activity, consequently fostering their rapid clinical development and their approval by health agencies. The third-generation inhibitor lorlatinib (PF-06463922), selectively active against ALK and ROS1, harbors impressive biological potency; its efficacy in reversing resistance to crizotinib and to other ALK inhibitors is being proven by early clinical trials. The NTRK1-3 and ROS1 inhibitor entrectinib (RXDX-101) has been reported to act against NSCLC harboring ALK fusion proteins too. Despite the quick development of these novel agents, several issues remain to be discussed in the treatment of patients suffering from ALK-rearranged NSCLC. This position paper will discuss the development, the current evidence and approvals, as long as the future perspectives of new ALK inhibitors beyond crizotinib. Clinical behaviors of ALK-rearranged NSCLC vary significantly among patients and differential molecular events responsible of crizotinib resistance account for the most important quote of this heterogeneity. The precious availability of a wide range of active anti-ALK compounds should be approached in a critical and careful perspective, in order to develop treatment strategies tailored on the disease evolution of every single patient. PMID:27413712

  7. Overcoming the resistance to crizotinib in patients with non-small cell lung cancer harboring EML4/ALK translocation.

    PubMed

    Perez, Cesar A; Velez, Michel; Raez, Luis E; Santos, Edgardo S

    2014-05-01

    The large knowledge learned in molecular biology specifically in the oncology field during the last ten years has resulted in fruitful results for the treatment of non-small cell lung cancer. The first pathway to be effectively targeted in lung cancer was the epidermal growth factor receptor. The acceptance of epidermal growth factor receptor mutation as a strong predictive biomarker in non-small cell lung carcinoma has encouraged the search for more targets. In 2011, regulatory entities granted conditional approval to an anaplastic lymphoma kinase inhibitor (crizotinib) based on an impressive overall response rate in previously treated non-small cell lung cancer patients whose tumors harbored EML4/ALK translocations. The landmark approval of crizotinib based on early promising clinical data highlights the remarkable success of molecular medicine in lung cancer therapeutics. The cumulative data developed after that approval has confirmed the appropriateness of this decision as recently reported phase III has now demonstrated. Unfortunately, resistance to this agent invariably develops and we now face the challenge of understanding several resistance pathways and overcoming them with new and more potent compounds. New agents in clinical development such as alectinib, LDK378, AP26113, and AUY922 have not only demonstrated promising activity in crizotinib resistant patients, but also crossing new pharmacokinetic boundaries in ALK inhibition as potent CNS penetration.

  8. Overcoming the resistance to crizotinib in patients with non-small cell lung cancer harboring EML4/ALK translocation.

    PubMed

    Perez, Cesar A; Velez, Michel; Raez, Luis E; Santos, Edgardo S

    2014-05-01

    The large knowledge learned in molecular biology specifically in the oncology field during the last ten years has resulted in fruitful results for the treatment of non-small cell lung cancer. The first pathway to be effectively targeted in lung cancer was the epidermal growth factor receptor. The acceptance of epidermal growth factor receptor mutation as a strong predictive biomarker in non-small cell lung carcinoma has encouraged the search for more targets. In 2011, regulatory entities granted conditional approval to an anaplastic lymphoma kinase inhibitor (crizotinib) based on an impressive overall response rate in previously treated non-small cell lung cancer patients whose tumors harbored EML4/ALK translocations. The landmark approval of crizotinib based on early promising clinical data highlights the remarkable success of molecular medicine in lung cancer therapeutics. The cumulative data developed after that approval has confirmed the appropriateness of this decision as recently reported phase III has now demonstrated. Unfortunately, resistance to this agent invariably develops and we now face the challenge of understanding several resistance pathways and overcoming them with new and more potent compounds. New agents in clinical development such as alectinib, LDK378, AP26113, and AUY922 have not only demonstrated promising activity in crizotinib resistant patients, but also crossing new pharmacokinetic boundaries in ALK inhibition as potent CNS penetration. PMID:24598368

  9. Development of potent ALK inhibitor and its molecular inhibitory mechanism against NSCLC harboring EML4-ALK proteins

    SciTech Connect

    Kang, Chung Hyo; Yun, Jeong In; Lee, Kwangho; Lee, Chong Ock; Lee, Heung Kyoung; Yun, Chang-Soo; Hwang, Jong Yeon; Cho, Sung Yun; Jung, Heejung; Kim, Pilho; Ha, Jae Du; Jeon, Jeong Hee; Choi, Sang Un; Jeong, Hye Gwang; Kim, Hyoung Rae; Park, Chi Hoon

    2015-08-28

    Here, we show the newly synthesized and potent ALK inhibitor having similar scaffold to KRCA-0008, which was reported previously, and its molecular mechanism against cancer cells harboring EML4-ALK fusion protein. Through ALK wild type enzyme assay, we selected two compounds, KRCA-0080 and KRCA-0087, which have trifluoromethyl instead of chloride in R2 position. We characterized these newly synthesized compounds by in vitro and in vivo assays. Enzyme assay shows that KRCA-0080 is more potent against various ALK mutants, including L1196M, G1202R, T1151-L1152insT, and C1156Y, which are seen in crizotinib-resistant patients, than KRCA-0008 is. Cell based assays demonstrate our compounds downregulate the cellular signaling, such as Akt and Erk, by suppressing ALK activity to inhibit the proliferation of the cells harboring EML4-ALK. Interestingly, our compounds induced strong G1/S arrest in H3122 cells leading to the apoptosis, which is proved by PARP-1 cleavage. In vivo H3122 xenograft assay, we found that KRCA-0080 shows significant reduction in tumor size compared to crizotinib and KRCA-0008 by 15–20%. Conclusively, we report a potent ALK inhibitor which shows significant in vivo efficacy as well as excellent inhibitory activity against various ALK mutants. - Highlights: • We synthesized KRCA-0008 derivatives having trifluoromethyl instead of chloride. • KRCA-0080 shows superior activity against several ALK mutants to KRCA-0008. • Cellular assays show our ALK inhibitors suppress only EML4-ALK positive cells. • Our ALK inhibitors induce G1/S arrest to lead apoptosis in H3122 cells. • KRCA-0080 has superior in vivo efficacy to crizotinib and KRCA-0008 by 15–20%.

  10. Structure Based Drug Design of Crizotinib (PF-02341066), a Potent and Selective Dual Inhibitor of Mesenchymal-Epithelial Transition Factor (c-MET) Kinase and Anaplastic Lymphoma Kinase (ALK)

    SciTech Connect

    Cui, J Jean; Tran-Dube,; #769; Michelle,; Shen, Hong; Nambu, Mitchell; Kung, Pei-Pei; Pairish, Mason; Jia, Lei; Meng, Jerry; Funk, Lee; Botrous, Iriny; McTigue, Michele; Grodsky, Neil; Ryan, Kevin; Padrique, Ellen; Alton, Gordon; Timofeevski, Sergei; Yamazaki, Shinji; Li, Qiuhua; Zou, Helen; Christensen, James; Mroczkowski, Barbara; Bender, Steve; Kania, Robert S; Edwards, Martin P

    2011-08-03

    Because of the critical roles of aberrant signaling in cancer, both c-MET and ALK receptor tyrosine kinases are attractive oncology targets for therapeutic intervention. The cocrystal structure of 3 (PHA-665752), bound to c-MET kinase domain, revealed a novel ATP site environment, which served as the target to guide parallel, multiattribute drug design. A novel 2-amino-5-aryl-3-benzyloxypyridine series was created to more effectively make the key interactions achieved with 3. In the novel series, the 2-aminopyridine core allowed a 3-benzyloxy group to reach into the same pocket as the 2,6-dichlorophenyl group of 3 via a more direct vector and thus with a better ligand efficiency (LE). Further optimization of the lead series generated the clinical candidate crizotinib (PF-02341066), which demonstrated potent in vitro and in vivo c-MET kinase and ALK inhibition, effective tumor growth inhibition, and good pharmaceutical properties.

  11. Crizotinib resistance: implications for therapeutic strategies.

    PubMed

    Dagogo-Jack, I; Shaw, A T

    2016-09-01

    In 2007, a chromosomal rearrangement resulting in a gene fusion leading to expression of a constitutively active anaplastic lymphoma kinase (ALK) fusion protein was identified as an oncogenic driver in non-small-cell lung cancer (NSCLC). ALK rearrangements are detected in 3%-7% of patients with NSCLC and are particularly enriched in younger patients with adenocarcinoma and a never or light smoking history. Fortuitously, crizotinib, a small molecule tyrosine kinase inhibitor initially developed to target cMET, was able to be repurposed for ALK-rearranged (ALK+) NSCLC. Despite dramatic and durable initial responses to crizotinib; however, the vast majority of patients will develop resistance within a few years. Diverse molecular mechanisms underlie resistance to crizotinib. This review will describe the clinical activity of crizotinib, review identified mechanisms of crizotinib resistance, and end with a survey of emerging therapeutic strategies aimed at overcoming crizotinib resistance. PMID:27573756

  12. Bilateral breast adenocarcinomas with EML4–ALK fusion in a patient with multiple metastases successfully treated with crizotinib: is lung the primary site?

    PubMed Central

    Liu, Chao; Ding, Lijuan; Sun, Bing; Wu, Shikai

    2016-01-01

    Breast metastases from non-mammary cancers are rare, especially when they appear synchronously. Clinically, it is vitally important to accurately diagnose these patients, as this will directly influence their treatment and survival. We present a very rare and complex case of bilateral breast adenocarcinomas with an EML4–ALK fusion, which was diagnosed as bilateral breast metastases of non-small-cell lung cancer by immunohistochemistry and comprehensive genomic investigation. The patient was successfully treated with an ALK inhibitor (crizotinib); symptoms improved quickly after initiation of crizotinib therapy, and a partial response was observed after 3 months. The experience of diagnosis and treatment of this case indicates the importance and necessity of genomic investigations in such patients, and suggests that we need to consider the rare possibility of this kind of metastasis in order to provide optimal treatment. PMID:27366096

  13. ALK kinase domain mutations in primary anaplastic large cell lymphoma: consequences on NPM-ALK activity and sensitivity to tyrosine kinase inhibitors.

    PubMed

    Lovisa, Federica; Cozza, Giorgio; Cristiani, Andrea; Cuzzolin, Alberto; Albiero, Alessandro; Mussolin, Lara; Pillon, Marta; Moro, Stefano; Basso, Giuseppe; Rosolen, Angelo; Bonvini, Paolo

    2015-01-01

    ALK inhibitor crizotinib has shown potent antitumor activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL) and the opportunity to include ALK inhibitors in first-line therapies is oncoming. However, recent studies suggest that crizotinib-resistance mutations may emerge in ALCL patients. In the present study, we analyzed ALK kinase domain mutational status of 36 paediatric ALCL patients at diagnosis to identify point mutations and gene aberrations that could impact on NPM-ALK gene expression, activity and sensitivity to small-molecule inhibitors. Amplicon ultra-deep sequencing of ALK kinase domain detected 2 single point mutations, R335Q and R291Q, in 2 cases, 2 common deletions of exon 23 and 25 in all the patients, and 7 splicing-related INDELs in a variable number of them. The functional impact of missense mutations and INDELs was evaluated. Point mutations were shown to affect protein kinase activity, signalling output and drug sensitivity. INDELs, instead, generated kinase-dead variants with dominant negative effect on NPM-ALK kinase, in virtue of their capacity of forming non-functional heterocomplexes. Consistently, when co-expressed, INDELs increased crizotinib inhibitory activity on NPM-ALK signal processing, as demonstrated by the significant reduction of STAT3 phosphorylation. Functional changes in ALK kinase activity induced by both point mutations and structural rearrangements were resolved by molecular modelling and dynamic simulation analysis, providing novel insights into ALK kinase domain folding and regulation. Therefore, these data suggest that NPM-ALK pre-therapeutic mutations may be found at low frequency in ALCL patients. These mutations occur randomly within the ALK kinase domain and affect protein activity, while preserving responsiveness to crizotinib.

  14. NPM/ALK mutants resistant to ASP3026 display variable sensitivity to alternative ALK inhibitors but succumb to the novel compound PF-06463922.

    PubMed

    Mologni, Luca; Ceccon, Monica; Pirola, Alessandra; Chiriano, Gianpaolo; Piazza, Rocco; Scapozza, Leonardo; Gambacorti-Passerini, Carlo

    2015-03-20

    ALK is involved in the onset of several tumors. Crizotinib (XalkoriTM), a potent ALK inhibitor, represents the current front-line treatment for ALK+ NSCLC and shows great clinical efficacy. However, resistant disease often develops after initial response. ASP3026 is a novel second-generation ALK inhibitor with activity on crizotinib-resistant ALK-L1196M gatekeeper mutant. As resistance is likely to be a relevant hurdle for any drug, we sought to determine the resistance profile of ASP3026 in the context of NPM/ALK+ ALCL. We selected six ASP3026-resistant cell lines by culturing human ALCL cells in the presence of increasing concentrations of drug. The established resistant cell lines carry several point mutations in the ALK kinase domain (G1128S, C1156F, I1171N/T, F1174I, N1178H, E1210K and C1156F/D1203N were the most frequent) that are shown to confer resistance to ASP3026 in the Ba/F3 cell model. All mutants were profiled for cross-resistance against a panel of clinically relevant inhibitors including ceritinib, alectinib, crizotinib, AP26113 and PF-06463922. Finally, a genetically heterogeneous ASP3026-resistant cell line was exposed to second-line treatment simulations with all inhibitors. The population evolved according to relative sensitivity of its mutant subclones to the various drugs. Compound PF-06463922 did not allow the outgrowth of any resistant clone, at non-toxic doses.

  15. Chromoplectic TPM3–ALK rearrangement in a patient with inflammatory myofibroblastic tumor who responded to ceritinib after progression on crizotinib

    PubMed Central

    Mansfield, A. S.; Murphy, S. J.; Harris, F. R.; Robinson, S. I.; Marks, R. S.; Johnson, S. H.; Smadbeck, J. B.; Halling, G. C.; Yi, E. S.; Wigle, D.; Vasmatzis, G.; Jen, J.

    2016-01-01

    Background Inflammatory myofibroblastic tumors (IMTs) are rare sarcomas that can occur at any age. Surgical resection is the primary treatment for patients with localized disease; however, these tumors frequently recur. Less commonly, patients with IMTs develop or present with metastatic disease. There is no standard of care for these patients and traditional cytotoxic therapy is largely ineffective. Most IMTs are associated with oncogenic ALK, ROS1 or PDGFRβ fusions and may benefit from targeted therapy. Patient and methods We sought to understand the genomic abnormalities of a patient who presented for management of metastatic IMT after progression of disease on crizotinib and a significant and durable partial response to the more potent ALK inhibitor ceritinib. Results The residual IMT was resected based on the recommendations of a multidisciplinary tumor sarcoma tumor board and analyzed by whole-genome mate pair sequencing. Analysis of the residual, resected tumor identified a chromoplectic TPM3–ALK rearrangement that involved many other known oncogenes and was confirmed by rtPCR. Conclusions In our analysis of the treatment-resistant, residual IMT, we identified a complex pattern of genetic rearrangements consistent with chromoplexy. Although it is difficult to know for certain if these chromoplectic rearrangements preceded treatment, their presence suggests that chromoplexy has a role in the oncogenesis of IMTs. Furthermore, this patient's remarkable response suggests that ceritinib should be considered as an option after progression on crizotinib for patients with metastatic or unresectable IMT and ALK mutations. PMID:27742657

  16. Crizotinib resistance in acute myeloid leukemia with inv(2)(p23q13)/RAN binding protein 2 (RANBP2) anaplastic lymphoma kinase (ALK) fusion and monosomy 7.

    PubMed

    Takeoka, Kayo; Okumura, Atsuko; Maesako, Yoshitomo; Akasaka, Takashi; Ohno, Hitoshi

    2015-03-01

    This is the first report on the development of a p.G1269A mutation within the kinase domain (KD) of ALK after crizotinib treatment in RANBP2-ALK acute myeloid leukemia (AML). An elderly woman with AML with an inv(2)(p23q13)/RANBP2-ALK and monosomy 7 was treated with crizotinib. After a short-term hematological response and the restoration of normal hematopoiesis, she experienced a relapse of AML. Fluorescence in situ hybridization using the ALK break-apart probe confirmed the inv(2)(p23q13), while G-banded karyotyping revealed the deletion of a segment of the short arm of chromosome 1 [del(1)(p13p22)] after crizotinib therapy. The ALK gene carried a heterozygous mutation at the nucleotide position g.716751G>C within exon 25, causing the p.G1269A amino acid substitution within the ALK-KD. Reverse transcriptase PCR revealed that the mutated ALK allele was selectively transcribed and the mutation occurred in the ALK allele rearranged with RANBP2. As both the del(1)(p13p22) at the cytogenetic level and p.G1269A at the nucleotide level newly appeared after crizotinib treatment, it is likely that they were secondarily acquired alterations involved in crizotinib resistance. Although secondary genetic abnormalities in ALK are most frequently described in non-small cell lung cancers harboring an ALK alteration, this report suggests that an ALK-KD mutation can occur independently of the tumor cell type or fusion partner after crizotinib treatment.

  17. Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK-positive neuroblastoma cells, Drosophila and mice

    PubMed Central

    Pfeifer, Kathrin; Rivera, Victor M.; Guan, Jikui; Palmer, Ruth H.; Hallberg, Bengt

    2016-01-01

    Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor which has been implicated in numerous solid and hematologic cancers. ALK mutations are reported in about 5-7% of neuroblastoma cases but the ALK-positive percentage increases significantly in the relapsed patient population. Crizotinib, the first clinically approved ALK inhibitor for the treatment of ALK-positive lung cancer has had less dramatic responses in neuroblastoma. Here we investigate the efficacy of a second-generation ALK inhibitor, brigatinib, in a neuroblastoma setting. Employing neuroblastoma cell lines, mouse xenograft and Drosophila melanogaster model systems expressing different constitutively active ALK variants, we show clear and efficient inhibition of ALK activity by brigatinib. Similar abrogation of ALK activity was observed in vitro employing a set of different constitutively active ALK variants in biochemical assays. These results suggest that brigatinib is an effective inhibitor of ALK kinase activity in ALK addicted neuroblastoma that should be considered as a potential future therapeutic option for ALK-positive neuroblastoma patients alone or in combination with other treatments. PMID:27049722

  18. Overcoming resistance to first/second generation epidermal growth factor receptor tyrosine kinase inhibitors and ALK inhibitors in oncogene-addicted advanced non-small cell lung cancer

    PubMed Central

    Romanidou, Ourania; Landi, Lorenza; Cappuzzo, Federico; Califano, Raffaele

    2016-01-01

    Epidermal growth factor receptor (EGFR) activating mutations and anaplastic lymphoma kinase (ALK) gene rearrangement in advanced non-small cell lung cancer (NSCLC) represent the two oncogenic events with an impact on current clinical practice. EGFR tyrosine kinase inhibitors (TKIs) and crizotinib are the standard of care for the treatment of EGFR mutant and ALK gene rearranged advanced NSCLC patients. Unfortunately, despite initial clinical benefit, acquired resistance to EGFR-TKIs or crizotinib usually develops after an average of 10–12 months of treatment. The aim of this review is to describe the mechanisms of resistance to first/second generation EGFR-TKIs and crizotinib. In particular, we focus on strategies to overcome resistance due to secondary EGFR T790M mutation and mutations of the ALK domain. PMID:27239236

  19. Elucidation of Resistance Mechanisms to Second-Generation ALK Inhibitors Alectinib and Ceritinib in Non-Small Cell Lung Cancer Cells.

    PubMed

    Dong, Xuyuan; Fernandez-Salas, Ester; Li, Enxiao; Wang, Shaomeng

    2016-03-01

    Crizotinib is the first anaplastic lymphoma kinase (ALK) inhibitor to have been approved for the treatment of non-small cell lung cancer (NSCLC) harboring an ALK fusion gene, but it has been found that, in the clinic, patients develop resistance to it. Alectinib and ceritinib are second-generation ALK inhibitors which show remarkable clinical responses in both crizotinib-naive and crizotinib-resistant NSCLC patients harboring an ALK fusion gene. Despite their impressive activity, clinical resistance to alectinib and ceritinib has also emerged. In the current study, we elucidated the resistance mechanisms to these second-generation ALK inhibitors in the H3122 NSCLC cell line harboring the EML4-ALK variant 1 fusion in vitro. Prolonged treatment of the parental H3122 cells with alectinib and ceritinib led to two cell lines which are 10 times less sensitive to alectinib and ceritinib than the parental H3122 cell line. Although mutations of ALK in its kinase domain are a common resistance mechanism for crizotinib, we did not detect any ALK mutation in these resistant cell lines. Rather, overexpression of phospho-ALK and alternative receptor tyrosine kinases such as phospho-EGFR, phospho-HER3, and phospho-IGFR-1R was observed in both resistant cell lines. Additionally, NRG1, a ligand for HER3, is upregulated and responsible for resistance by activating the EGFR family pathways through the NRG1-HER3-EGFR axis. Combination treatment with EGFR inhibitors, in particular afatinib, was shown to be effective at overcoming resistance. Our study provides new mechanistic insights into adaptive resistance to second-generation ALK inhibitors and suggests a potential clinical strategy to combat resistance to these second-generation ALK inhibitors in NSCLC.

  20. Elucidation of Resistance Mechanisms to Second-Generation ALK Inhibitors Alectinib and Ceritinib in Non–Small Cell Lung Cancer Cells

    PubMed Central

    Dong, Xuyuan; Fernandez-Salas, Ester; Li, Enxiao; Wang, Shaomeng

    2016-01-01

    Crizotinib is the first anaplastic lymphoma kinase (ALK) inhibitor to have been approved for the treatment of non–small cell lung cancer (NSCLC) harboring an ALK fusion gene, but it has been found that, in the clinic, patients develop resistance to it. Alectinib and ceritinib are second-generation ALK inhibitors which show remarkable clinical responses in both crizotinib-naive and crizotinib-resistant NSCLC patients harboring an ALK fusion gene. Despite their impressive activity, clinical resistance to alectinib and ceritinib has also emerged. In the current study, we elucidated the resistance mechanisms to these second-generation ALK inhibitors in the H3122 NSCLC cell line harboring the EML4-ALK variant 1 fusion in vitro. Prolonged treatment of the parental H3122 cells with alectinib and ceritinib led to two cell lines which are 10 times less sensitive to alectinib and ceritinib than the parental H3122 cell line. Although mutations of ALK in its kinase domain are a common resistance mechanism for crizotinib, we did not detect any ALK mutation in these resistant cell lines. Rather, overexpression of phospho-ALK and alternative receptor tyrosine kinases such as phospho-EGFR, phospho-HER3, and phospho-IGFR-1R was observed in both resistant cell lines. Additionally, NRG1, a ligand for HER3, is upregulated and responsible for resistance by activating the EGFR family pathways through the NRG1-HER3-EGFR axis. Combination treatment with EGFR inhibitors, in particular afatinib, was shown to be effective at overcoming resistance. Our study provides new mechanistic insights into adaptive resistance to second-generation ALK inhibitors and suggests a potential clinical strategy to combat resistance to these second-generation ALK inhibitors in NSCLC. PMID:26992917

  1. Rearranged EML4-ALK fusion transcripts sequester in circulating blood platelets and enable blood-based crizotinib response monitoring in non-small-cell lung cancer

    PubMed Central

    Nilsson, R. Jonas A.; Karachaliou, Niki; Berenguer, Jordi; Gimenez-Capitan, Ana; Schellen, Pepijn; Teixido, Cristina; Tannous, Jihane; Kuiper, Justine L.; Drees, Esther; Grabowska, Magda; van Keulen, Marte; Heideman, Danielle A.M.; Thunnissen, Erik; Dingemans, Anne-Marie C.; Viteri, Santiago; Tannous, Bakhos A.; Drozdowskyj, Ana; Rosell, Rafael; Smit, Egbert F.; Wurdinger, Thomas

    2016-01-01

    Purpose: Non-small-cell lung cancers harboring EML4-ALK rearrangements are sensitive to crizotinib. However, despite initial response, most patients will eventually relapse, and monitoring EML4-ALK rearrangements over the course of treatment may help identify these patients. However, challenges associated with serial tumor biopsies have highlighted the need for blood-based assays for the monitoring of biomarkers. Platelets can sequester RNA released by tumor cells and are thus an attractive source for the non-invasive assessment of biomarkers. Methods: EML4-ALK rearrangements were analyzed by RT-PCR in platelets and plasma isolated from blood obtained from 77 patients with non-small-cell lung cancer, 38 of whom had EML4-ALK-rearranged tumors. In a subset of 29 patients with EML4-ALK-rearranged tumors who were treated with crizotinib, EML4-ALK rearrangements in platelets were correlated with progression-free and overall survival. Results: RT-PCR demonstrated 65% sensitivity and 100% specificity for the detection of EML4-ALK rearrangements in platelets. In the subset of 29 patients treated with crizotinib, progression-free survival was 3.7 months for patients with EML4-ALK+ platelets and 16 months for those with EML4-ALK− platelets (hazard ratio, 3.5; P = 0.02). Monitoring of EML4-ALK rearrangements in the platelets of one patient over a period of 30 months revealed crizotinib resistance two months prior to radiographic disease progression. Conclusions: Platelets are a valuable source for the non-invasive detection of EML4-ALK rearrangements and may prove useful for predicting and monitoring outcome to crizotinib, thereby improving clinical decisions based on radiographic imaging alone. PMID:26544515

  2. Successful Management of Crizotinib-Induced Neutropenia in a Patient with Anaplastic Lymphoma Kinase-Positive Non-Small Cell Lung Cancer: A Case Report

    PubMed Central

    Osugi, Jun; Owada, Yuki; Yamaura, Takumi; Muto, Satoshi; Okabe, Naoyuki; Matsumura, Yuki; Higuchi, Mitsunori; Suzuki, Hiroyuki; Gotoh, Mitsukazu

    2016-01-01

    Crizotinib, the first clinically available inhibitor of anaplastic lymphoma kinase (ALK) gene rearrangement, is generally well tolerated. In contrast, neutropenia induced by crizotinib is a commonly reported grade 3 or 4 adverse event. In such cases, interruption and dose reduction of crizotinib might be necessary for some patients with severe neutropenia. However, information concerning clinical experience and management of severe neutropenia is currently limited. In this report, the successful management of crizotinib-induced neutropenia by dose reduction of crizotinib in a patient with ALK-positive non-small cell lung cancer is described. PMID:26933419

  3. The ALK gene, an attractive target for inhibitor development.

    PubMed

    Tartari, Carmen J; Scapozza, Leonardo; Gambacorti-Passerini, Carlo

    2011-01-01

    Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase that belongs to the Insulin receptor subfamily involved as full length receptor in neural development. Even if the expression of ALK protein is down-regulated in the adults, the ALK full length is expressed in different types of tumors. Moreover, chromosomal rearrangements, involving the alk gene, can occur leading the formation of different ALK fusion proteins characterized by the kinase domain of ALK fused to several partners that determine cellular localization. Structural investigation and characterization of the ALK kinase domain in absence of its crystal structure constituted the basis of development of ALK small molecule inhibitors. Here, we described normal function of the ALK receptor and its role in tumors; formation of the constitutively activated ALK fusion proteins and we reported an update of developed small molecule inhibitors of the ALK kinase activity. PMID:21513493

  4. New treatment options for ALK+ advanced non-small-cell lung cancer: critical appraisal of ceritinib

    PubMed Central

    Rothschild, Sacha I

    2016-01-01

    Rearrangements in ALK gene and EML4 gene were first described in 2007. This genomic aberration is found in about 2%–8% of non-small-cell lung cancer (NSCLC) patients. Crizotinib was the first ALK tyrosine kinase inhibitor licensed for the treatment of metastatic ALK-positive NSCLC based on a randomized Phase III trial. Despite the initial treatment response of crizotinib, disease progression inevitably develops after approximately 10 months of therapy. Different resistance mechanisms have recently been described. One relevant mechanism of resistance is the development of mutations in ALK. Novel ALK tyrosine kinase inhibitors have been developed to overcome these mutations. Ceritinib is an oral second-generation ALK inhibitor showing clinical activity not only in crizotinib-resistant ALK-positive NSCLC but also in treatment-naïve ALK-positive disease. In this paper, preclinical and clinical data of ceritinib are reviewed, and its role in the clinical setting is put into perspective. PMID:27217763

  5. Anaplastic lymphoma kinase-positive lung adenocarcinoma patient with development of sick sinus syndrome while on targeted treatment with crizotinib.

    PubMed

    Jiang, Hao; Li, Mei-Mei; Jin, Shu-Xian

    2015-03-01

    The anaplastic lymphoma kinase (ALK) positive non-small cell lung cancer (NSCLC) patients are younger and have never smoked, while pathologically are predominately adenocarcinomas. Crizotinib as an ALK inhibitor has been used in treating ALK positive NSCLC patients for several years and some adverse effects should be paid attention to. We now describe a case of ALK positive NSCLC patient with development of sick sinus syndrome (SSS) while on targeted treatment with crizotinib. A 46-year-old non-smoking woman with right hilar mass and underwent transesophageal endoscopic ultrasonography lymph node biopsy showed low differentiation adenocarcinoma, immunohistochemistry (IHC) of tumor samples revealed the ALK overexpression. The severe sinus bradycardia and RR interval prolongation were detected 3 months after crizotinib treatment, she underwent pacemaker implantation. Although the severe sinus bradycardia and RR interval prolongation were unusual adverse effects, physicians should be aware of these side effects when using crizotinib.

  6. [Crizotinib - molecular therapy for lung cancer].

    PubMed

    Gröschel, A; Warth, A; Reinmuth, N

    2013-04-01

    The anaplastic lymphoma kinase (ALK) can act as a key oncogenic driver after activation by means of processes such as gene rearrangement. In approximately 5 % of patients with advanced non-small cell lung cancer (NSCLC), an oncogenic fusion gene of echinoderm microtubule-associated protein-like 4 (EML4) and ALK has been detected using fluorescence in situ hybridisation (FISH). Moreover, various methods including immunohistochemistry and PCR-based assays can be used for analysing ALK expression. Clinical data have been generated for crizotinib, a small molecule inhibitor of the ALK receptor tyrosine kinase, demonstrating a substantial improvement of objective response rate and prolonged progression-free survival (PFS) compared to standard chemotherapy in pretreated NSCLC patients harbouring EML4-ALK fusion genes. In the current review, recent data on the detection and inhibition of ALK in advanced NSCLC are summarised. PMID:23576200

  7. ALK-rearrangements and testing methods in non-small cell lung cancer: a review

    PubMed Central

    Shackelford, Rodney E.; Vora, Moiz; Mayhall, Kim; Cotelingam, James

    2014-01-01

    The anaplastic lymphoma tyrosine kinase (ALK) gene was first described as a driver mutation in anaplastic non-Hodgkin's lymphoma. Dysregulated ALK expression is now an identified driver mutation in nearly twenty different human malignancies, including 4-9% of non-small cell lung cancers (NSCLC). The tyrosine kinase inhibitor crizotinib is more effective than standard chemotherapeutic agents in treating ALK positive NSCLC, making molecular diagnostic testing for dysregulated ALK expression a necessary step in identifying optimal treatment modalities. Here we review ALKmediated signal transduction pathways and compare the molecular protocols used to identify dysregulated ALK expression in NSCLC. We also discuss the use of crizotinib and second generation ALK tyrosine kinase inhibitors in the treatment of ALK positive NSCLC, and the known mechanisms of crizotinib resistance in NSCLC. PMID:24955213

  8. Insight into drug resistance mechanisms and discovery of potential inhibitors against wild-type and L1196M mutant ALK from FDA-approved drugs.

    PubMed

    Li, Jianzong; Liu, Wei; Luo, Hao; Bao, Jinku

    2016-09-01

    Anaplastic lymphoma kinase (ALK) plays a crucial role in multiple malignant cancers. It is known as a well-established target for the treatment of ALK-dependent cancers. Even though substantial efforts have been made to develop ALK inhibitors, only crizotinib, ceritinib, and alectinib had been approved by the U.S. Food and Drug Administration for patients with ALK-positive non-small cell lung cancer (NSCLC). The secondary mutations with drug-resistance bring up difficulties to develop effective drugs for ALK-positive cancers. To give a comprehensive understanding of molecular mechanism underlying inhibitor response to ALK tyrosine kinase mutations, we established an accurate assessment for the extensive profile of drug against ALK mutations by means of computational approaches. The molecular mechanics-generalized Born surface area (MM-GBSA) method based on molecular dynamics (MD) simulation was carried out to calculate relative binding free energies for receptor-drug systems. In addition, the structure-based virtual screening was utilized to screen effective inhibitors targeting wild-type ALK and the gatekeeper mutation L1196M from 3180 approved drugs. Finally, the mechanism of drug resistance was discussed, several novel potential wild-type and L1196M mutant ALK inhibitors were successfully identified. PMID:27585676

  9. An Unusual Case of Systemic Inflammatory Myofibroblastic Tumor with Successful Treatment with ALK-Inhibitor.

    PubMed

    Jacob, Sanjivini V; Reith, John D; Kojima, Angerika Y; Williams, William D; Liu, Chen; Vila Duckworth, Lizette

    2014-01-01

    Systemic inflammatory myofibroblastic tumor is an exceedingly rare entity. A 45-year-old Hispanic female presented with a 6-month history of left-sided thigh pain, low back pain, and generalized weakness. PET/CT scan revealed abnormal activity in the liver, adrenal gland, and pancreas. MRI of the abdomen demonstrated two 6-7 cm masses in the liver. MRI of the lumbar spine demonstrated lesions in the L2 to L4 spinous processes, paraspinal muscles, and subcutaneous tissues, as well as an 8 mm enhancing intradural lesion at T11, all thought to be metastatic disease. A biopsy of the liver showed portal tract expansion by a spindle cell proliferation rich in inflammation. Tumor cells showed immunoreactivity for smooth muscle actin and anaplastic lymphoma kinase 1 (ALK1). Tissue from the L5 vertebra showed a process histologically identical to that seen in the liver. FISH analysis of these lesions demonstrated an ALK (2p23) gene rearrangement. The patient was successfully treated with an ALK-inhibitor, Crizotinib, and is now in complete remission. We present the first reported case, to our knowledge, of inflammatory myofibroblastic tumor with systemic manifestations and ALK translocation. This case is a prime example of how personalized medicine has vastly improved patient care through the use of molecular-targeted therapy.

  10. An Unusual Case of Systemic Inflammatory Myofibroblastic Tumor with Successful Treatment with ALK-Inhibitor

    PubMed Central

    Jacob, Sanjivini V.; Reith, John D.; Kojima, Angerika Y.; Williams, William D.; Liu, Chen; Vila Duckworth, Lizette

    2014-01-01

    Systemic inflammatory myofibroblastic tumor is an exceedingly rare entity. A 45-year-old Hispanic female presented with a 6-month history of left-sided thigh pain, low back pain, and generalized weakness. PET/CT scan revealed abnormal activity in the liver, adrenal gland, and pancreas. MRI of the abdomen demonstrated two 6-7 cm masses in the liver. MRI of the lumbar spine demonstrated lesions in the L2 to L4 spinous processes, paraspinal muscles, and subcutaneous tissues, as well as an 8 mm enhancing intradural lesion at T11, all thought to be metastatic disease. A biopsy of the liver showed portal tract expansion by a spindle cell proliferation rich in inflammation. Tumor cells showed immunoreactivity for smooth muscle actin and anaplastic lymphoma kinase 1 (ALK1). Tissue from the L5 vertebra showed a process histologically identical to that seen in the liver. FISH analysis of these lesions demonstrated an ALK (2p23) gene rearrangement. The patient was successfully treated with an ALK-inhibitor, Crizotinib, and is now in complete remission. We present the first reported case, to our knowledge, of inflammatory myofibroblastic tumor with systemic manifestations and ALK translocation. This case is a prime example of how personalized medicine has vastly improved patient care through the use of molecular-targeted therapy. PMID:25045570

  11. ALK F1174V mutation confers sensitivity while ALK I1171 mutation confers resistance to alectinib. The importance of serial biopsy post progression.

    PubMed

    Ou, Sai-Hong; Milliken, Jeffrey C; Azada, Michele C; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J

    2016-01-01

    Many acquired resistant mutations to the anaplastic lymphoma kinase (ALK) gene have been identified during treatment of ALK-rearranged non-small cell lung cancer (NSCLC) patients with crizotinib, ceritinib, and alectinib. These various acquired resistant ALK mutations confer differential sensitivities to various ALK inhibitors and may provide guidance on how to sequence the use of many of the second generation ALK inhibitors. We described a patient who developed an acquired ALK F1174V resistant mutation on progression from crizotinib that responded to alectinib for 18 months but then developed an acquired ALK I1171S mutation to alectinib. Both tumor samples had essentially the same genomic profile by comprehensive genomic profiling otherwise. This is the first patient report that demonstrates ALK F1174V mutation is sensitive to alectinib and further confirms missense acquired ALK I1171 mutation is resistant to alectinib. Sequential tumor re-biopsy for comprehensive genomic profiling (CGP) is important to appreciate the selective pressure during treatment with various ALK inhibitors underpinning the evolution of the disease course of ALK+NSCLC patients while on treatment with the various ALK inhibitors. This approach will likely help inform the optimal sequencing strategy as more ALK inhibitors become available. This case report also validates the importance of developing structurally distinct ALK inhibitors for clinical use to overcome non-cross resistant ALK mutations. PMID:26464158

  12. ALK F1174V mutation confers sensitivity while ALK I1171 mutation confers resistance to alectinib. The importance of serial biopsy post progression.

    PubMed

    Ou, Sai-Hong; Milliken, Jeffrey C; Azada, Michele C; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J

    2016-01-01

    Many acquired resistant mutations to the anaplastic lymphoma kinase (ALK) gene have been identified during treatment of ALK-rearranged non-small cell lung cancer (NSCLC) patients with crizotinib, ceritinib, and alectinib. These various acquired resistant ALK mutations confer differential sensitivities to various ALK inhibitors and may provide guidance on how to sequence the use of many of the second generation ALK inhibitors. We described a patient who developed an acquired ALK F1174V resistant mutation on progression from crizotinib that responded to alectinib for 18 months but then developed an acquired ALK I1171S mutation to alectinib. Both tumor samples had essentially the same genomic profile by comprehensive genomic profiling otherwise. This is the first patient report that demonstrates ALK F1174V mutation is sensitive to alectinib and further confirms missense acquired ALK I1171 mutation is resistant to alectinib. Sequential tumor re-biopsy for comprehensive genomic profiling (CGP) is important to appreciate the selective pressure during treatment with various ALK inhibitors underpinning the evolution of the disease course of ALK+NSCLC patients while on treatment with the various ALK inhibitors. This approach will likely help inform the optimal sequencing strategy as more ALK inhibitors become available. This case report also validates the importance of developing structurally distinct ALK inhibitors for clinical use to overcome non-cross resistant ALK mutations.

  13. Personalized treatment in advanced ALK-positive non-small cell lung cancer: from bench to clinical practice

    PubMed Central

    Passaro, Antonio; Lazzari, Chiara; Karachaliou, Niki; Spitaleri, Gianluca; Pochesci, Alessia; Catania, Chiara; Rosell, Rafael; de Marinis, Filippo

    2016-01-01

    The discovery of anaplastic lymphoma kinase (ALK) gene rearrangements and the development of tyrosine kinase inhibitors (TKI) that target them have achieved unprecedented success in the management of patients with ALK-positive non-small cell lung cancer (NSCLC). Despite the high efficacy of crizotinib, the first oral ALK TKI approved for the treatment of ALK-positive NSCLC, almost all patients inevitably develop acquired resistance, showing disease progression in the brain or in other parenchymal sites. Second- or third-generation ALK TKIs have shown to be active in crizotinib-pretreated or crizotinib-naïve ALK-positive patients, even in those with brain metastases. In this review, the current knowledge regarding ALK-positive NSCLC, focusing on the biology of the disease and the available therapeutic options are discussed. PMID:27799783

  14. Rationale for co-targeting IGF-1R and ALK in ALK fusion positive lung cancer

    PubMed Central

    Lovly, Christine M.; McDonald, Nerina T.; Chen, Heidi; Ortiz-Cuaran, Sandra; Heukamp, Lukas C.; Yan, Yingjun; Florin, Alexandra; Ozretić, Luka; Lim, Diana; Wang, Lu; Chen, Zhao; Chen, Xi; Lu, Pengcheng; Paik, Paul K.; Shen, Ronglai; Jin, Hailing; Buettner, Reinhard; Ansén, Sascha; Perner, Sven; Brockmann, Michael; Bos, Marc; Wolf, Jürgen; Gardizi, Masyar; Wright, Gavin M.; Solomon, Benjamin; Russell, Prudence A.; Rogers, Toni-Maree; Suehara, Yoshiyuki; Red-Brewer, Monica; Tieu, Rudy; de Stanchina, Elisa; Wang, Qingguo; Zhao, Zhongming; Johnson, David H.; Horn, Leora; Wong, Kwok-Kin; Thomas, Roman K.; Ladanyi, Marc; Pao, William

    2014-01-01

    The ALK tyrosine kinase inhibitor (TKI), crizotinib, shows significant activity in patients whose lung cancers harbor ALK fusions but its efficacy is limited by variable primary responses and acquired resistance. In work arising from the intriguing clinical observation of a patient with ALK fusion+ lung cancer who had an ‘exceptional response’ to an IGF-1R antibody, we define a therapeutic synergism between ALK and IGF-1R inhibitors. Similar to IGF-1R, ALK fusion proteins bind to the adaptor, IRS-1, and IRS-1 knockdown enhances the anti-tumor effects of ALK inhibitors. In models of ALK TKI resistance, the IGF-1R pathway is activated, and combined ALK/IGF-1R inhibition improves therapeutic efficacy. Consistent with this finding, IGF-1R/IRS-1 levels are increased in biopsy samples from patients progressing on crizotinib therapy. Collectively, these data support a role for the IGF-1R/IRS-1 pathway in both ALK TKI-sensitive and TKI-resistant states and provide biological rationale for further clinical development of dual ALK/IGF-1R inhibitors. PMID:25173427

  15. Targeting stemness is an effective strategy to control EML4-ALK+ non-small cell lung cancer cells.

    PubMed

    Oh, Se Jin; Noh, Kyung Hee; Lee, Young-Ho; Hong, Soon-Oh; Song, Kwon-Ho; Lee, Hyo-Jung; Kim, Soyeon; Kim, Tae Min; Jeon, Ju-Hong; Seo, Jae Hong; Kim, Dong-Wan; Kim, Tae Woo

    2015-11-24

    The fusion between anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) is a causative factor in a unique subset of patients with non-small cell lung carcinoma (NSCLC). Although the inhibitor crizotinib, as it blocks the kinase activity of the resulting EML4-ALK fusion protein, displays remarkable initial responses, a fraction of NSCLC cases eventually become resistant to crizotinib by acquiring mutations in the ALK domain or activating bypass pathways via EGFR, KIT, or KRAS. Cancer stem cell (CSC) theory provides a plausible explanation for acquisition of tumorigenesis and resistance. However, the question as to whether EML4-ALK-driven tumorigenesis is linked with the stem-like property and whether the stemness is an effective target in controlling EML4-ALK+ NSCLC including crizotinib-resistant NSCLC cells has not been addressed. Here, we report that stem-like properties stem from ALK activity in EML4-ALK+ NSCLC cells. Notably, treatment with rapamycin, a CSC targeting agent, attenuates stem-like phenotypes of the EML4-ALK+ cells, which increased capability of tumor formation and higher expression of stemness-associated molecules such as ALDH, NANOG, and OCT4. Importantly, combinational treatment with rapamycin and crizotinib leads to synergistic anti-tumor effects on EML4-ALK+ NSCLC cells as well as on those resistant to crizotinib. Thus, we provide a proof of principle that targeting stemness would be a novel strategy to control intractable EML4-ALK+ NSCLC.

  16. Crizotinib: from discovery to accelerated development to front-line treatment.

    PubMed

    Blackhall, F; Cappuzzo, F

    2016-09-01

    Non-small-cell lung cancer (NSCLC) is associated with a poor prognosis and low survival rates, providing a strong rationale for the development of new treatment options. The discovery of ALK gene rearrangements in a subset of NSCLC specimens and the identification and development of the first-in-class ALK inhibitor crizotinib provided a personalised treatment option for patients with advanced ALK-positive NSCLC. Crizotinib demonstrated rapid and durable responses in advanced ALK-positive NSCLC patients in phase I and II studies, leading to accelerated FDA approval. Subsequent evaluation in phase III studies showed that crizotinib improved progression-free survival compared with platinum-based doublet chemotherapy in previously untreated patients and compared with pemetrexed or docetaxel in previously treated patients. Crizotinib was shown to have an acceptable safety profile and also to improve quality of life and symptom scores. Overall, crizotinib has been shown to provide a valuable first- and second-line treatment option and is now the first-line standard of care for patients with advanced ALK-positive NSCLC. PMID:27573754

  17. ALK Positive Lung Cancer: Clinical Profile, Practice and Outcomes in a Developing Country

    PubMed Central

    Chougule, Anuradha; Kane, Subhadha; Kumar, Rajiv; Mahajan, Abhishek; Janu, Amit

    2016-01-01

    Objectives To evaluate the performance and treatment profile of advanced EML4—ALK positive Non-small cell lung cancer (NSCLC) patients in a developing country with potentially restricted access to Crizotinib. Materials and Methods A retrospective analysis of advanced ALK positive NSCLC patients who were treated from June 2012 to September 2015 was conducted. The primary goal was to evaluate outcomes of advanced ALK positive NSCLC in our practice and examine the logistic constraints in procuring Crizotinib. Results 94 patients were available for analysis. 21 (22.3%) patients were started on Crizotinib upfront, 60 (63.8%) on chemotherapy, 10 (10.6%) on Tyrosine kinase inhibitors (in view of poor PS) and 3 (3.2%) patients were offered best supportive care. Reasons for not starting Crizotinib upfront included symptomatic patients needing early initiation of therapy (23.3%), ALK not tested upfront (23.3%) and financial constraints (21.9%). 69 patients (73.4%) received Crizotinib at some stage during treatment. Dose interruptions (> 1 week) with Crizotinib were seen in 20 patients (29%), with drug toxicity being the commonest reason (85%). Median Progression free survival (PFS) on first line therapy for the entire cohort was 10 months, with a significant difference between patients receiving Crizotinib and those who did not ever receive Crizotinib (10 months vs. 2 months, p = 0.028). Median Overall Survival (OS) was not reached for the entire cohort, with 1 year survival being 81.2%. Patients with an ECOG Performance Status (PS) of >2 had a significantly reduced PFS compared to patients with PS < = 2 (1.5 months vs. 11 months, p< 0.001). 47 patients with financial constraints (68.1%) received Crizotinib completely free via various extramural support schemes. Conclusion A majority of our ALK positive NSCLC patients were exposed to Crizotinib through the help of various support mechanisms and these patients had similar outcomes to that reported from previously published

  18. CRKL mediates EML4-ALK signaling and is a potential therapeutic target for ALK-rearranged lung adenocarcinoma

    PubMed Central

    Voeller, Donna; Gower, Arjan; Kim, In-Kyu; Zhang, Yu-Wen; Giaccone, Giuseppe

    2016-01-01

    Anaplastic lymphoma kinase (ALK) gene rearrangements are oncogenic drivers in a small subset of patients with non-small-cell lung cancer (NSCLC). The ALK inhibitors are highly effective in NSCLC patients harboring ALK rearrangements; however, most patients acquire resistance to the therapy following an initial response. Mechanisms of acquired resistance are complex. We used LC-MS/MS-based phosphotyrosine-peptide profiling in the EML4-ALK rearranged H3122 and H2228 cells treated with ALK inhibitors, to identify downstream effectors of ALK. We then used Western blot, siRNA experiments, cell proliferation, viability and migration assays to validate our findings. We identified CRKL as a novel downstream effector of ALK signaling. We demonstrated that CRKL tyrosine phosphorylation was repressed by pharmacological inhibition or small interfering RNA (siRNA) knockdown of ALK in the ALK-rearranged cells. More importantly, CRKL knockdown attenuated their cell proliferation, viability, and migration, but it had no effect on ALK phosphorylation and expression in these cells. Furthermore, CRKL tyrosine phosphorylation was inhibited by dasatinib (an inhibitor of ABL and SRC kinases), which in combination with the ALK inhibitor crizotinib displayed a synergistic inhibitory effect in vitro. In conclusion, our study suggests that CRKL is a key downstream effector of ALK, and combined inhibition of ALK and CRKL may represent an effective strategy for treating ALK-rearranged NSCLC patients. PMID:27078848

  19. Crizotinib-Induced Abnormal Signal Processing in the Retina.

    PubMed

    Ishii, Toshiyuki; Iwasawa, Shunichiro; Kurimoto, Ryota; Maeda, Akemi; Takiguchi, Yuichi; Kaneda, Makoto

    2015-01-01

    Molecular target therapy for cancer is characterized by unique adverse effects that are not usually observed with cytotoxic chemotherapy. For example, the anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor crizotinib causes characteristic visual disturbances, whereas such effects are rare when another ALK-tyrosine kinase inhibitor, alectinib, is used. To elucidate the mechanism responsible for these visual disturbances, the responses to light exhibited by retinal ganglion cells treated with these agents were evaluated using a C57BL6 mouse ex vivo model. Both crizotinib and alectinib changed the firing rate of ON and OFF type retinal ganglion cells. However, the ratio of alectinib-affected cells (15.7%) was significantly lower than that of crizotinib-affected cells (38.6%). Furthermore, these drugs changed the response properties to light stimuli of retinal ganglion cells in some of the affected cells, i.e., OFF cells responded to both ON and OFF stimuli, etc. Finally, the expressions of ALK (a target receptor of both crizotinib and alectinib) and of MET and ROS1 (additional target receptors of crizotinib) were observed at the mRNA level in the retina. Our findings suggest that these drugs might target retinal ganglion cells and that the potency of the drug actions on the light responses of retinal ganglion cells might be responsible for the difference in the frequencies of visual disturbances observed between patients treated with crizotinib and those treated with alectinib. The present experimental system might be useful for screening new molecular target agents prior to their use in clinical trials.

  20. Crizotinib-Induced Abnormal Signal Processing in the Retina

    PubMed Central

    Ishii, Toshiyuki; Iwasawa, Shunichiro; Kurimoto, Ryota; Maeda, Akemi; Takiguchi, Yuichi; Kaneda, Makoto

    2015-01-01

    Molecular target therapy for cancer is characterized by unique adverse effects that are not usually observed with cytotoxic chemotherapy. For example, the anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor crizotinib causes characteristic visual disturbances, whereas such effects are rare when another ALK-tyrosine kinase inhibitor, alectinib, is used. To elucidate the mechanism responsible for these visual disturbances, the responses to light exhibited by retinal ganglion cells treated with these agents were evaluated using a C57BL6 mouse ex vivo model. Both crizotinib and alectinib changed the firing rate of ON and OFF type retinal ganglion cells. However, the ratio of alectinib-affected cells (15.7%) was significantly lower than that of crizotinib-affected cells (38.6%). Furthermore, these drugs changed the response properties to light stimuli of retinal ganglion cells in some of the affected cells, i.e., OFF cells responded to both ON and OFF stimuli, etc. Finally, the expressions of ALK (a target receptor of both crizotinib and alectinib) and of MET and ROS1 (additional target receptors of crizotinib) were observed at the mRNA level in the retina. Our findings suggest that these drugs might target retinal ganglion cells and that the potency of the drug actions on the light responses of retinal ganglion cells might be responsible for the difference in the frequencies of visual disturbances observed between patients treated with crizotinib and those treated with alectinib. The present experimental system might be useful for screening new molecular target agents prior to their use in clinical trials. PMID:26271036

  1. Insights into brain metastasis in patients with ALK+ lung cancer: is the brain truly a sanctuary?

    PubMed

    Toyokawa, Gouji; Seto, Takashi; Takenoyama, Mitsuhiro; Ichinose, Yukito

    2015-12-01

    Anaplastic lymphoma kinase (ALK) has been identified to exert a potent transforming activity through its rearrangement in non-small cell lung cancer (NSCLC), and patients (pts) with ALK rearrangement can be treated more successfully with ALK inhibitors, such as crizotinib, alectinib, and ceritinib, than with chemotherapy. Despite the excellent efficacy of ALK inhibitors, resistance to these drugs is inevitably encountered in most ALK-rearranged pts. Cases of resistance are subtyped into three groups, i.e., systemic, oligo, and central nervous system (CNS) types, with the CNS being used to be considered a sanctuary. With regard to the management of CNS lesions in pts with ALK+ NSCLC, a growing body of evidence has gradually demonstrated the intracranial (IC) efficacy of ALK inhibitor (ALKi) in ALK+ NSCLC pts with brain metastases (BMs). Although the efficacy of crizotinib for the CNS lesions remains controversial, a recent retrospective investigation of ALK+ pts with BM enrolled in PROFILE 1005 and PROFILE 1007 demonstrated that crizotinib is associated with a high disease control rate for BM. However, BM comprises the most common site of progressive disease in pts with or without baseline BMs, which is a serious problem for crizotinib. Furthermore, alectinib can be used to achieve strong and long-lasting inhibitory effects on BM. In addition to alectinib, the IC efficacy of other next-generation ALK inhibitors, such as ceritinib, AP26113 and PF-06463922, has been demonstrated. In this article, we review the latest evidence regarding the BM and IC efficacy of ALK inhibitors in pts with ALK+ NSCLC.

  2. The role of the ALK receptor in cancer biology.

    PubMed

    Hallberg, B; Palmer, R H

    2016-09-01

    A vast array of oncogenic variants has been identified for anaplastic lymphoma kinase (ALK). Therefore, there is a need to better understand the role of ALK in cancer biology in order to optimise treatment strategies. This review summarises the latest research on the receptor tyrosine kinase ALK, and how this information can guide the management of patients with cancer that is ALK-positive. A variety of ALK gene alterations have been described across a range of tumour types, including point mutations, deletions and rearrangements. A wide variety of ALK fusions, in which the kinase domain of ALK and the amino-terminal portion of various protein partners are fused, occur in cancer, with echinoderm microtubule-associated protein-like 4 (EML4)-ALK being the most prevalent in non-small-cell lung cancer (NSCLC). Different ALK fusion proteins can mediate different signalling outputs, depending on properties such as subcellular localisation and protein stability. The ALK fusions found in tumours lack spatial and temporal regulation, which can also affect dimerisation and substrate specificity. Two ALK tyrosine kinase inhibitors (TKIs), crizotinib and ceritinib, are currently approved in Europe for use in ALK-positive NSCLC and several others are in development. These ALK TKIs bind slightly differently within the ATP-binding pocket of the ALK kinase domain and are associated with the emergence of different resistance mutation patterns during therapy. This emphasises the need to tailor the sequence of ALK TKIs according to the ALK signature of each patient. Research into the oncogenic functions of ALK, and fast paced development of ALK inhibitors, has substantially improved outcomes for patients with ALK-positive NSCLC. Limited data are available surrounding the physiological ligand-stimulated activation of ALK signalling and further research is needed. Understanding the role of ALK in tumour biology is key to further optimising therapeutic strategies for ALK

  3. Treating patients with ALK-positive non-small cell lung cancer: latest evidence and management strategy

    PubMed Central

    Liao, Bin-Chi; Shih, Jin-Yuan; Yang, James Chih-Hsin

    2015-01-01

    Rearrangements in anaplastic lymphoma kinase (ALK) gene and echinoderm microtubule-associated protein-like 4 (EML4) gene were first described in a small portion of patients with non-small cell lung cancer (NSCLC) in 2007. Fluorescence in situ hybridization is used as the diagnostic test for detecting an EML4–ALK rearrangement. Crizotinib, an ALK inhibitor, is effective in treating advanced ALK-positive NSCLC, and the US Food and Drug Administration approved it for treating ALK-positive NSCLC in 2011. Several mechanisms of acquired resistance to crizotinib have recently been reported. Second-generation ALK inhibitors were designed to overcome these resistance mechanisms. Two of them, ceritinib and alectinib, were approved in 2014 for advanced ALK-positive NSCLC in the US and Japan, respectively. Heat shock protein 90 (Hsp90) inhibitors also showed activity against ALK-positive NSCLC. Here we review the recent development of crizotinib, ceritinib, alectinib and other second-generation ALK inhibitors as well as Hsp90 inhibitors. We also discuss management strategies for advanced ALK-positive NSCLC. PMID:26327925

  4. PF-06463922, an ALK/ROS1 inhibitor, overcomes resistance to 1st and 2nd generation ALK inhibitors in pre-clinical models

    PubMed Central

    Zou, Helen Y.; Friboulet, Luc; Kodack, David P.; Engstrom, Lars D.; Li, Qiuhua; West, Melissa; Tang, Ruth W.; Wang, Hui; Tsaparikos, Konstantinos; Wang, Jinwei; Timofeevski, Sergei; Katayama, Ryohei; Dinh, Dac M.; Lam, Hieu; Lam, Justine L.; Yamazaki, Shinji; Hu, Wenyue; Patel, Bhushankumar; Bezwada, Divya; Frias, Rosa L.; Lifshits, Eugene; Mahmood, Sidra; Gainor, Justin F.; Affolter, Timothy; Lappin, Patrick B.; Gukasyan, Hovhannes; Lee, Nathan; Deng, Shibing; Jain, Rakesh K; Johnson, Ted W.; Shaw, Alice T.; Fantin, Valeria R.; Smeal, Tod

    2015-01-01

    SUMMARY We report the preclinical evaluation of PF-06463922, a potent and brain penetrant ALK/ROS1 inhibitor. Compared to other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors due to secondary ALK kinase domain mutations and/or due to the failed control of brain metastases. PMID:26144315

  5. Inhibition of Axl improves the targeted therapy against ALK-mutated neuroblastoma

    SciTech Connect

    Xu, Fei; Li, Hongling; Sun, Yong

    2014-11-28

    Highlights: • First reported Axl is co-expressed with ALK in neuroblastoma tissues and cell lines. • Axl activation promotes cell growth and impairs the efficiency of ALK inhibitor. • Further found silence of Axl leads to increased sensitivity to ALK inhibitors. • Axl inhibitor promotes the efficiency of targeted therapy in vitro and in vivo. • Axl activation should be considered in the clinical application of ALK inhibitors. - Abstract: Neuroblastoma (NB) patients harboring mutated ALK can be expected to potentially benefit from targeted therapy based on ALK tyrosine kinase inhibitor (TKI), such as crizotinib and ceritinib. However, the effect of the treatment varies with different individuals, although with the same genic changes. Axl receptor tyrosine kinase is expressed in a variety of human cancers, but little data are reported in NB, particularly in which carrying mutated ALK. In this study, we focus on the roles of Axl in ALK-mutated NB for investigating rational therapeutic strategy. We found that Axl is expressed in ALK-positive NB tissues and cell lines, and could be effectively activated by its ligand GAS6. Ligand-dependent Axl activation obviously rescued crizotinib-mediated suppression of cell proliferation in ALK-mutated NB cells. Genetic inhibition of Axl with specific small interfering RNA markedly increased the sensitivity of cells to ALK-TKIs. Furthermore, a small-molecule inhibitor of Axl significantly enhanced ALK-targeted therapy, as an increased frequency of apoptosis was observed in NB cells co-expressing ALK and Axl. Taken together, our results demonstrated that activation of Axl could lead to insensitivity to ALK inhibitors, and dual inhibition of ALK and Axl might be a potential therapeutic strategy against ALK-mutated NB.

  6. The ALK inhibitor ASP3026 eradicates NPM-ALK⁺ T-cell anaplastic large-cell lymphoma in vitro and in a systemic xenograft lymphoma model.

    PubMed

    George, Suraj Konnath; Vishwamitra, Deeksha; Manshouri, Roxsan; Shi, Ping; Amin, Hesham M

    2014-07-30

    NPM-ALK⁺ T-cell anaplastic large-cell lymphoma (ALCL) is an aggressive type of cancer. Standard treatment of NPM-ALK⁺ ALCL is CHOP polychemotherapy. Although patients initially respond favorably to CHOP, resistance, relapse, and death frequently occur. Recently, selective targeting of ALK has emerged as an alternative therapeutic strategy. ASP3026 is a second-generation ALK inhibitor that can overcome crizotinib resistance in non-small cell lung cancer, and is currently being evaluated in clinical trials of patients with ALK⁺ solid tumors. However, NPM-ALK⁺ ALCL patients are not included in these trials. We studied the effects of ASP3026 on NPM-ALK⁺ ALCL cell lines in vitro and on systemic lymphoma growth in vivo. ASP3026 decreased the viability, proliferation, and colony formation, as well as induced apoptotic cell death of NPM-ALK⁺ ALCL cells. In addition, ASP3026 significantly reduced the proliferation of 293T cells transfected with NPM-ALK mutants that are resistant to crizotinib and downregulated tyrosine phosphorylation of these mutants. Moreover, ASP3026 abrogated systemic NPM-ALK⁺ ALCL growth in mice. Importantly, the survival of ASP3026-treated mice was superior to that of control and CHOP-treated mice. Our data suggest that ASP3026 is an effective treatment for NPM-ALK⁺ ALCL, and support the enrollment of patients with this lymphoma in the ongoing clinical trials.

  7. Total Body Metabolic Tumor Response in ALK Positive Non-Small Cell Lung Cancer Patients Treated with ALK Inhibition

    PubMed Central

    Koole, Michel J. B.; Bongaerts, Alphons H. H.; Pruim, Jan; Groen, Harry J. M.

    2016-01-01

    Background In ALK-positive advanced NSCLC, crizotinib has a high response rate and effectively increases quality of life and survival. CT measurement of the tumor may insufficiently reflect the actual tumor load changes during targeted therapy with crizotinib. We explored whether 18F-FDG PET measured metabolic changes are different from CT based changes and studied the impact of these changes on disease progression. Methods 18F-FDG PET/CT was performed prior to and after 6 weeks of crizotinib treatment. Tumor response on CT was classified with RECIST 1.1, while 18F-FDG PET response was assessed according to the 1999 EORTC recommendations and PERCIST criteria. Agreement was assessed using McNemars test. During follow-up, patients received additional PET/CT during crizotinib treatment and second generation ALK inhibition. We assessed whether PET was able to detect progression earlier then CT. Results In this exploratory study 15 patients were analyzed who were treated with crizotinib. There was a good agreement in the applicability of CT and 18F-FDG PET/CT using the EORTC recommendations. During first line crizotinib and subsequent second line ALK inhibitors, PET was able to detect progression earlier then CT in 10/22 (45%) events of progression and in the others disease progression was detected simultaneously. Conclusion In advanced ALK positive NSCLC PET was able to detect progressive disease earlier than with CT in nearly half of the assessments while both imaging tests performed similar in the others. PMID:27137772

  8. Targeting brain metastases in ALK-rearranged non-small-cell lung cancer.

    PubMed

    Zhang, Isabella; Zaorsky, Nicholas G; Palmer, Joshua D; Mehra, Ranee; Lu, Bo

    2015-10-01

    The incidence of brain metastases has increased as a result of improved systemic control and advances in imaging. However, development of novel therapeutics with CNS activity has not advanced at the same rate. Research on molecular markers has revealed many potential targets for antineoplastic agents, and a particularly important aberration is translocation in the ALK gene, identified in non-small-cell lung cancer (NSCLC). ALK inhibitors have shown systemic efficacy against ALK-rearranged NSCLC in many clinical trials, but the effectiveness of crizotinib in CNS disease is limited by poor blood-brain barrier penetration and acquired drug resistance. In this Review, we discuss potential pathways to target ALK-rearranged brain metastases, including next generation ALK inhibitors with greater CNS penetration and mechanisms to overcome resistance. Other important mechanisms to control CNS disease include targeting pathways downstream of ALK phosphorylation, increasing the permeability of the blood-brain barrier, modifying the tumour microenvironment, and adding concurrent radiotherapy.

  9. In vivo imaging models of bone and brain metastases and pleural carcinomatosis with a novel human EML4-ALK lung cancer cell line.

    PubMed

    Nanjo, Shigeki; Nakagawa, Takayuki; Takeuchi, Shinji; Kita, Kenji; Fukuda, Koji; Nakada, Mitsutoshi; Uehara, Hisanori; Nishihara, Hiroshi; Hara, Eiji; Uramoto, Hidetaka; Tanaka, Fumihiro; Yano, Seiji

    2015-03-01

    EML4-ALK lung cancer accounts for approximately 3-7% of non-small-cell lung cancer cases. To investigate the molecular mechanism underlying tumor progression and targeted drug sensitivity/resistance in EML4-ALK lung cancer, clinically relevant animal models are indispensable. In this study, we found that the lung adenocarcinoma cell line A925L expresses an EML4-ALK gene fusion (variant 5a, E2:A20) and is sensitive to the ALK inhibitors crizotinib and alectinib. We further established highly tumorigenic A925LPE3 cells, which also have the EML4-ALK gene fusion (variant 5a) and are sensitive to ALK inhibitors. By using A925LPE3 cells with luciferase gene transfection, we established in vivo imaging models for pleural carcinomatosis, bone metastasis, and brain metastasis, all of which are significant clinical concerns of advanced EML4-ALK lung cancer. Interestingly, crizotinib caused tumors to shrink in the pleural carcinomatosis model, but not in bone and brain metastasis models, whereas alectinib showed remarkable efficacy in all three models, indicative of the clinical efficacy of these ALK inhibitors. Our in vivo imaging models of multiple organ sites may provide useful resources to analyze further the pathogenesis of EML4-ALK lung cancer and its response and resistance to ALK inhibitors in various organ microenvironments.

  10. ALK(R1275Q) perturbs extracellular matrix, enhances cell invasion and leads to the development of neuroblastoma in cooperation with MYCN.

    PubMed

    Ueda, T; Nakata, Y; Yamasaki, N; Oda, H; Sentani, K; Kanai, A; Onishi, N; Ikeda, K; Sera, Y; Honda, Z-I; Tanaka, K; Sata, M; Ogawa, S; Yasui, W; Saya, H; Takita, J; Honda, H

    2016-08-25

    Overexpression of MYCN is a hallmark of neuroblastoma (NB). ALK(R1275Q), an activating mutation of ALK (anaplastic lymphoma kinase), has been found in sporadic and familial NB patients. In this report, we demonstrated that ALK(R1275Q) knock-in, MYCN transgenic compound mice developed NB with complete penetrance. Transcriptome analysis revealed that ALK(R1275Q) globally downregulated the expression of extracellular matrix (ECM)- and basement membrane (BM)-associated genes in both primary neuronal cells and NB tumors. Accordingly, ALK(R1275Q)/MYCN tumors exhibited reduced expression of ECM/BM-related proteins as compared with MYCN tumors. In addition, on MYCN transduction, ALK(R1275Q)-expressing neuronal cells exhibited increased migratory and invasive activities. Consistently, enhanced invasion and metastasis were demonstrated in ALK(R1275Q)/MYCN mice. These results collectively indicate that ALK(R1275Q) confers a malignant potential on neuronal cells that overexpress MYCN by impairing normal ECM/BM integrity and enhancing tumor growth and dissemination. Moreover, we found that crizotinib, an ALK inhibitor, almost completely inhibited the growth of ALK(R1275Q)/MYCN tumors in an allograft model. Our findings provided insights into the cooperative mechanism of the mutated ALK and overexpressed MYCN in the pathogenesis of NB and demonstrated the effectiveness of crizotinib on ALK(R1275Q)-positive tumors. PMID:26829053

  11. Canadian consensus: inhibition of ALK-positive tumours in advanced non-small-cell lung cancer

    PubMed Central

    Melosky, B.; Agulnik, J.; Albadine, R.; Banerji, S.; Bebb, D.G.; Bethune, D.; Blais, N.; Butts, C.; Cheema, P.; Cheung, P.; Cohen, V.; Deschenes, J.; Ionescu, D.N.; Juergens, R.; Kamel-Reid, S.; Laurie, S.A.; Liu, G.; Morzycki, W.; Tsao, M.S.; Xu, Z.; Hirsh, V.

    2016-01-01

    Anaplastic lymphoma kinase (alk) is an oncogenic driver in non-small-cell lung cancer (nsclc). Chromosomal rearrangements involving the ALK gene occur in up to 4% of nonsquamous nsclc patients and lead to constitutive activation of the alk signalling pathway. ALK-positive nsclc is found in relatively young patients, with a median age of 50 years. Patients frequently have brain metastasis. Targeted inhibition of the alk pathway prolongs progression-free survival in patients with ALK-positive advanced nsclc. The results of several recent clinical trials confirm the efficacy and safety benefit of crizotinib and ceritinib in this population. Canadian oncologists support the following consensus statement: All patients with advanced nonsquamous nsclc (excluding pure neuroendocrine carcinoma) should be tested for the presence of an ALK rearrangement. If an ALK rearrangement is present, treatment with a targeted alk inhibitor in the first-line setting is recommended. As patients become resistant to first-generation alk inhibitors, other treatments, including second-generation alk inhibitors can be considered. PMID:27330348

  12. Synergism between ivermectin and the tyrosine kinase/P-glycoprotein inhibitor crizotinib against Haemonchus contortus larvae in vitro.

    PubMed

    Raza, Ali; Kopp, Steven R; Kotze, Andrew C

    2016-08-30

    Anthelmintic resistance is a major problem in parasitic nematodes of livestock worldwide. One means to counter resistance is to use synergists that specifically inhibit resistance mechanisms in order to restore the toxicity, and hence preserve the usefulness, of currently available anthelmintics. P-glycoproteins (P-gps) eliminate a wide variety of structurally unrelated xenobiotics from cells, and have been implicated in anthelmintic resistance. Crizotinib is a tyrosine kinase inhibitor under development as a cancer therapeutic. The compound also inhibits P-gps, and has been shown to reverse multidrug resistance in cancer cells. We were therefore interested in determining if the compound was able to increase the sensitivity of Haemonchus contortus larvae to ivermectin, as measured by in vitro larval development and migration assays with a drug-resistant and a -susceptible isolate. In migration assays, co-administration of crizotinib increased the toxicity of ivermectin to resistant larvae (up to 5.7-fold decrease in ivermectin IC50), and rendered the resistant larvae equally or more sensitive to ivermectin than the susceptible isolate. On the other hand, co-administration of crizotinib had no effect on ivermectin sensitivity in the susceptible isolate. In development assays, significant increases in the sensitivity of both the resistant (up to 1.9-fold) and susceptible (up to 1.6-fold) larvae to ivermectin were observed, although the magnitude of the observed synergism was less than seen in migration assays, and the resistant larvae retained significant levels of ivermectin resistance. By highlighting the ability of the P-gp inhibitor crizotinib to increase the sensitivity of H. contortus larvae to ivermectin, this study provides further evidence that P-gp inhibitors are potential tools for modulating the efficacy of anthelmintics. In addition, the differences in the outcomes of the two assays, with 'resistance-breaking' effects being much more marked in migration

  13. Metformin restores crizotinib sensitivity in crizotinib-resistant human lung cancer cells through inhibition of IGF1-R signaling pathway

    PubMed Central

    Zhang, Kejun; Lin, Caiyu; Han, Rui; Lu, Conghua; He, Yong

    2016-01-01

    Aim Despite the impressive efficacy of crizotinib for the treatment of ALK-positive non-small cell lung cancer, patients invariably develop therapeutic resistance. Suppression of the IGF-1R signaling pathway may abrogate this acquired mechanism of drug resistance to crizotinib. Metformin, a widely used antidiabetic agent, may reverse crizotinib resistance through inhibition of IGF-1R signaling. Results The present study revealed that metformin effectively increased the sensitivity of both crizotinib-sensitive and -resistant non-small cell lung cancer cells to crizotinib, as evidenced by decreased proliferation and invasion and enhanced apoptosis. Metformin reduced IGF-1R signaling activation in crizotinib-resistant cells. Furthermore, the addition of IGF-1 to crizotinib-sensitive H2228 cells induced crizotinib resistance, which was overcome by metformin. Experimental design The effects of metformin to reverse crizotinib resistance were examined in vitro by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), invasion assay, ki67 incorporation assay, flow cytometry analysis, Western blot analysis, and colony-forming assay. Conclusions Metformin may be used in combination with crizotinib in ALK+ NSCLC patients to overcome crizotinib resistance and prolong survival. PMID:27144340

  14. Crizotinib for Advanced Non-Small Cell Lung Cancer

    Cancer.gov

    A summary of results from an international phase III clinical trial that compared crizotinib versus chemotherapy in previously treated patients with advanced lung cancer whose tumors have an EML4-ALK fusion gene.

  15. Targeting ALK in neuroblastoma--preclinical and clinical advancements.

    PubMed

    Carpenter, Erica L; Mossé, Yael P

    2012-07-01

    Despite improvements in cancer therapies in the past 50 years, neuroblastoma remains a devastating clinical problem and a leading cause of childhood cancer deaths. Advances in treatments for children with high-risk neuroblastoma have, until recently, involved addition of cytotoxic therapy to dose-intensive regimens. In this era of targeted therapies, substantial efforts have been made to identify optimal targets for different types of cancer. The discovery of hereditary and somatic activating mutations in the oncogene ALK has now placed neuroblastoma among other cancers, such as melanoma and non-small-cell lung cancer (NSCLC), which benefit from therapies with oncogene-specific small-molecule tyrosine kinase inhibitors. Crizotinib, a small-molecule inhibitor of ALK, has transformed the landscape for the treatment of NSCLC harbouring ALK translocations and has demonstrated activity in preclinical models of ALK-driven neuroblastomas. However, inhibition of mutated ALK is complex when compared with translocated ALK and remains a therapeutic challenge. This Review discusses the biology of ALK in the development of neuroblastoma, preclinical and clinical progress with the use of ALK inhibitors and immunotherapy, challenges associated with resistance to such therapies and the steps being taken to overcome some of these hurdles.

  16. Design of novel quinazoline derivatives and related analogues as potent and selective ALK5 inhibitors

    SciTech Connect

    Gellibert, F.; Fouchet, M.-H.; Nguyen, V.-L.; Wang, R.; Krysa, G.; de Gouville, A.-C.; Huet, S.; Dodic, N.

    2009-07-23

    Starting from quinazoline 3a, we designed potent and selective ALK5 inhibitors over p38MAP kinase from a rational drug design approach based on co-crystal structures in the human ALK5 kinase domain. The quinazoline 3d exhibited also in vivo activity in an acute rat model of DMN-induced liver fibrosis when administered orally at 5 mg/kg (bid).

  17. Detection of novel and potentially actionable anaplastic lymphoma kinase (ALK) rearrangement in colorectal adenocarcinoma by immunohistochemistry screening

    PubMed Central

    Wang, Kai; Kim, Sun Young; Jang, Jiryeon; Kim, Seung Tae; Park, Joon Oh; Lim, Ho Yeong; Kang, Won Ki; Park, Young Suk; Lee, Jiyun; Lee, Woo Yong; Park, Yoon Ah; Huh, Jung Wook; Yun, Seong Hyeon; Do, In-Gu; Kim, Seok Hyung; Balasubramanian, Sohail; Stephens, Philip J.; Ross, Jeffrey S.; Li, Gang Gary; Hornby, Zachary; Ali, Siraj M.; Miller, Vincent A.; Kim, Kyoung-Mee; Ou, Sai-Hong Ignatius

    2015-01-01

    Purpose Anaplastic lymphoma kinase (ALK) rearrangement has been detected in colorectal carcinoma (CRC) using advanced molecular diagnostics tests including exon scanning, fluorescence in situ hybridization (FISH), and next generation sequencing (NGS). We investigated if immunohistochemistry (IHC) can be used to detect ALK rearrangement in gastrointestinal malignancies. Experimental designs Tissue microarrays (TMAs) from consecutive gastric carcinoma (GC) and CRC patients who underwent surgical resection at Samsung Medical Center, Seoul, Korea were screened by IHC using ALK monoclonal antibody 5A4. IHC positive cases were confirmed by FISH, nCounter assays, and NGS-based comprehensive genomic profiling (CGP). ALK IHC was further applied to CRC patients enrolled in a pathway-directed therapeutic trial. Results Four hundred thirty-two GC and 172 CRC cases were screened by IHC. No GC sample was ALK IHC positive. One CRC (0.6%) was ALK IHC positive (3+) that was confirmed by ALK FISH and a novel CAD-ALK (C35; A20) fusion variant that resulted from a paracentric inversion event inv(2)(p22–21p23) was identified by CGP. One out of 50 CRC patients enrolled in a pathway-directed therapeutic trial was ALK IHC positive (3+) confirmed by ALK FISH and found to harbor the EML4-ALK (E21, A20) fusion variant by CGP. Growth of a tumor cell line derived from this EML4-ALK CRC patient was inhibited by ALK inhibitors crizotinib and entrectinib. Conclusions ALK IHC is a viable screening strategy for identifying ALK rearrangement in CRC. ALK rearrangement is a potential actionable driver mutation in CRC based on survival inhibition of patient tumor-derived cell line by potent ALK inhibitors. PMID:26172300

  18. A Functional Landscape of Resistance to ALK Inhibition in Lung Cancer

    PubMed Central

    Wilson, Frederick H.; Johannessen, Cory M.; Piccioni, Federica; Tamayo, Pablo; Kim, Jong Wook; Van Allen, Eliezer M.; Corsello, Steven M.; Capelletti, Marzia; Calles, Antonio; Butaney, Mohit; Sharifnia, Tanaz; Gabriel, Stacey B.; Mesirov, Jill P.; Hahn, William C.; Engelman, Jeffrey A.; Meyerson, Matthew; Root, David E.; Jänne, Pasi A.; Garraway, Levi A.

    2015-01-01

    Summary We conducted a large-scale functional genetic study to characterize mechanisms of resistance to ALK inhibition in ALK-dependent lung cancer cells. We identify members of known resistance pathways and additional putative resistance drivers. Among the latter were members of the P2Y purinergic receptor family of G-protein coupled receptors (P2Y1, P2Y2, and P2Y6). P2Y receptors mediated resistance in part through a protein kinase C (PKC)-dependent mechanism. Moreover, PKC activation alone was sufficient to confer resistance to ALK inhibitors whereas combined ALK and PKC inhibition restored sensitivity. We observed enrichment of gene signatures associated with several resistance drivers (including P2Y receptors) in crizotinib-resistant ALK-rearranged lung tumors compared to treatment-naïve controls, supporting a role for identified resistance mechanisms in clinical resistance. PMID:25759024

  19. 3D-QSAR and molecular fragment replacement study on diaminopyrimidine and pyrrolotriazine ALK inhibitors

    NASA Astrophysics Data System (ADS)

    Ke, Zhipeng; Lu, Tao; Liu, Haichun; Yuan, Haoliang; Ran, Ting; Zhang, Yanmin; Yao, Sihui; Xiong, Xiao; Xu, Jinxing; Xu, Anyang; Chen, Yadong

    2014-06-01

    Over expression of anaplastic lymphoma kinase (ALK) has been found in many types of cancer, and ALK is a promising therapeutic target for the treatment of cancer. To obtain new potent inhibitors of ALK, we conducted lead optimization using 3D-QSAR modeling and molecular docking investigation of 2,4-diaminopyrimidines and 2,7-disubstituted-pyrrolo[2,1-f][1,2,4]triazine-based compounds. Three favorable 3D-QSAR models (CoMFA with q2, 0.555; r2, 0.939; CoMSIA with q2, 0.625; r2, 0.974; Topomer CoMFA with q2, 0.557; r2 0.756) have been developed to predict the biological activity of novel compounds. Topomer Search was utilized for virtual screening to obtain suitable fragments. The novel compounds generated by molecular fragment replacement (MFR) were evaluated by Topomer CoMFA prediction, Glide (docking) and further evaluated with CoMFA and CoMSIA prediction. 25 novel 2,7-disubstituted-pyrrolo[2,1-f][1,2,4]triazine derivatives as potential ALK inhibitors were finally obtained. In this paper, a combination of CoMFA, CoMSIA and Topomer CoMFA could obtain favorable 3D-QSAR models and suitable fragments for ALK inhibitors optimization. The work flow which comprised 3D-QSAR modeling, Topomer Search, MFR, molecular docking and evaluating criteria could be applied to de novo drug design and the resulted compounds initiate us to further optimize and design new potential ALK inhibitors.

  20. Identification of a novel HIP1-ALK fusion variant in Non-Small-Cell Lung Cancer (NSCLC) and discovery of ALK I1171 (I1171N/S) mutations in two ALK-rearranged NSCLC patients with resistance to Alectinib.

    PubMed

    Ou, Sai-Hong Ignatius; Klempner, Samuel J; Greenbowe, Joel R; Azada, Michele; Schrock, Alexa B; Ali, Siraj M; Ross, Jeffrey S; Stephens, Philip J; Miller, Vincent A

    2014-12-01

    Huntingtin-interacting protein 1 (HIP1) has recently been identified as a new fusion partner fused to anaplastic lymphoma kinase (ALK) in non-small-cell lung cancer (NSCLC). To date, two variants of HIP1-ALK (H21; A20) and (H28; A20) have been identified in NSCLC. However, the response of patients with NSCLC harboring HIP1-ALK to ALK inhibitors and potential resistance mechanisms to such remain unknown. Here, we report a patient with NSCLC harboring a novel HIP1-ALK fusion variant (H30; A20). This patient and another patient with EML4-ALK variant 3a/b initially responded sequentially to crizotinib and then alectinib, a next-generation ALK inhibitor, but developed acquired resistance to alectinib with the presence of a mutation in amino acid residue 1171 (I1171N and I1171S respectively) located in the hydrophobic regulatory spine (R-spine) of the ALK kinase in both the cases as identified by a comprehensive next-generation sequencing-based assay performed on biopsies of new liver metastases that developed during alectinib treatment.

  1. Novel targeted therapies for resistant ALK-rearranged non-small-cell lung cancer: ceritinib and beyond.

    PubMed

    Kanaan, Zeyad; Kloecker, Goetz H; Paintal, Ajit; Perez, Cesar A

    2015-01-01

    Lung cancer is the leading cause of cancer-related mortality in both sexes, accounting for over one quarter of cancer deaths. Non-small-cell lung cancer (NSCLC) comprises 85%-90% of lung cancer diagnoses and despite advances in multimodality therapies, 5-year survival rates remain dismal with a median survival for patients with metastatic disease of 1 year. The positive outcomes of targeted therapies against the kinase domain of epidermal growth factor receptor in NSCLC triggered consistent efforts to identify the so-called driver mutations as other potential targets. Anaplastic large-cell kinase (ALK) gene rearrangements were identified and targeted resulting in promising response rates in early studies. Unfortunately, most of the patients treated with crizotinib, the first-generation ALK inhibitor, progressed within 9 months. Ceritinib is a second-generation ALK inhibitor that has demonstrated activity in crizotinib-resistant patients, becoming a promising treatment option in this population. Furthermore, additional novel ALK inhibitors and agents targeting alternative pathways have been recruited to rechallenge this evasive disease post-crizotinib resistance.

  2. Novel targeted therapies for resistant ALK-rearranged non-small-cell lung cancer: ceritinib and beyond

    PubMed Central

    Kanaan, Zeyad; Kloecker, Goetz H; Paintal, Ajit; Perez, Cesar A

    2015-01-01

    Lung cancer is the leading cause of cancer-related mortality in both sexes, accounting for over one quarter of cancer deaths. Non-small-cell lung cancer (NSCLC) comprises 85%–90% of lung cancer diagnoses and despite advances in multimodality therapies, 5-year survival rates remain dismal with a median survival for patients with metastatic disease of 1 year. The positive outcomes of targeted therapies against the kinase domain of epidermal growth factor receptor in NSCLC triggered consistent efforts to identify the so-called driver mutations as other potential targets. Anaplastic large-cell kinase (ALK) gene rearrangements were identified and targeted resulting in promising response rates in early studies. Unfortunately, most of the patients treated with crizotinib, the first-generation ALK inhibitor, progressed within 9 months. Ceritinib is a second-generation ALK inhibitor that has demonstrated activity in crizotinib-resistant patients, becoming a promising treatment option in this population. Furthermore, additional novel ALK inhibitors and agents targeting alternative pathways have been recruited to rechallenge this evasive disease post-crizotinib resistance. PMID:25945060

  3. PD-1 Axis Inhibitors in EGFR- and ALK-Driven Lung Cancer: Lost Cause?

    PubMed

    Gettinger, Scott; Politi, Katerina

    2016-09-15

    Programmed death axis 1 (PD-1) inhibitors have ushered in a new error of cancer immunotherapeutics for advanced smoking-associated non-small cell lung cancer. Their role in treating EGFR-mutant and ALK-rearranged lung cancer has yet to be determined. Clin Cancer Res; 22(18); 4539-41. ©2016 AACRSee related article by Gainor et al., p. 4585. PMID:27470969

  4. Synergistic Effects of Crizotinib and Temozolomide in Experimental FIG-ROS1 Fusion-Positive Glioblastoma.

    PubMed

    Das, Arabinda; Cheng, Ron Ron; Hilbert, Megan L T; Dixon-Moh, Yaenette N; Decandio, Michele; Vandergrift, William Alex; Banik, Naren L; Lindhorst, Scott M; Cachia, David; Varma, Abhay K; Patel, Sunil J; Giglio, Pierre

    2015-01-01

    Glioblastoma (GB) is the most common malignant brain tumor. Drug resistance frequently develops in these tumors during chemotherapy. Therefore, predicting drug response in these patients remains a major challenge in the clinic. Thus, to improve the clinical outcome, more effective and tolerable combination treatment strategies are needed. Robust experimental evidence has shown that the main reason for failure of treatments is signal redundancy due to coactivation of several functionally linked receptor tyrosine kinases (RTKs), including anaplastic lymphoma kinase (ALK), c-Met (hepatocyte growth factor receptor), and oncogenic c-ros oncogene1 (ROS1: RTK class orphan) fusion kinase FIG (fused in GB)-ROS1. As such, these could be attractive targets for GB therapy. The study subjects consisted of 19 patients who underwent neurosurgical resection of GB tissues. Our in vitro and ex vivo models promisingly demonstrated that treatments with crizotinib (PF-02341066: dual ALK/c-Met inhibitor) and temozolomide in combination induced synergistic antitumor activity on FIG-ROS1-positive GB cells. Our results also showed that ex vivo FIG-ROS1+ slices (obtained from GB patients) when cultured were able to preserve tissue architecture, cell viability, and global gene-expression profiles for up to 14 days. Both in vitro and ex vivo studies indicated that combination blockade of FIG, p-ROS1, p-ALK, and p-Met augmented apoptosis, which mechanistically involves activation of Bim and inhibition of survivin, p-Akt, and Mcl-1 expression. However, it is important to note that we did not see any significant synergistic effect of crizotinib and temozolomide on FIG-ROS1-negative GB cells. Thus, these ex vivo culture results will have a significant impact on patient selection for clinical trials and in predicting response to crizotinib and temozolomide therapy. Further studies in different animal models of FIG-ROS1-positive GB cells are warranted to determine useful therapies for the

  5. Synergistic Effects of Crizotinib and Temozolomide in Experimental FIG-ROS1 Fusion-Positive Glioblastoma

    PubMed Central

    Das, Arabinda; Cheng, Ron Ron; Hilbert, Megan L.T.; Dixon-Moh, Yaenette N.; Decandio, Michele; Vandergrift, William Alex; Banik, Naren L.; Lindhorst, Scott M.; Cachia, David; Varma, Abhay K.; Patel, Sunil J.; Giglio, Pierre

    2015-01-01

    Glioblastoma (GB) is the most common malignant brain tumor. Drug resistance frequently develops in these tumors during chemotherapy. Therefore, predicting drug response in these patients remains a major challenge in the clinic. Thus, to improve the clinical outcome, more effective and tolerable combination treatment strategies are needed. Robust experimental evidence has shown that the main reason for failure of treatments is signal redundancy due to coactivation of several functionally linked receptor tyrosine kinases (RTKs), including anaplastic lymphoma kinase (ALK), c-Met (hepatocyte growth factor receptor), and oncogenic c-ros oncogene1 (ROS1: RTK class orphan) fusion kinase FIG (fused in GB)-ROS1. As such, these could be attractive targets for GB therapy. The study subjects consisted of 19 patients who underwent neurosurgical resection of GB tissues. Our in vitro and ex vivo models promisingly demonstrated that treatments with crizotinib (PF-02341066: dual ALK/c-Met inhibitor) and temozolomide in combination induced synergistic antitumor activity on FIG-ROS1-positive GB cells. Our results also showed that ex vivo FIG-ROS1+ slices (obtained from GB patients) when cultured were able to preserve tissue architecture, cell viability, and global gene-expression profiles for up to 14 days. Both in vitro and ex vivo studies indicated that combination blockade of FIG, p-ROS1, p-ALK, and p-Met augmented apoptosis, which mechanistically involves activation of Bim and inhibition of survivin, p-Akt, and Mcl-1 expression. However, it is important to note that we did not see any significant synergistic effect of crizotinib and temozolomide on FIG-ROS1-negative GB cells. Thus, these ex vivo culture results will have a significant impact on patient selection for clinical trials and in predicting response to crizotinib and temozolomide therapy. Further studies in different animal models of FIG-ROS1-positive GB cells are warranted to determine useful therapies for the

  6. Identification of ALK5 inhibitor via structure-based virtual screening and ADMET prediction.

    PubMed

    Lin, Aibin; Cai, Zhongqi; Hu, Guoping; Li, Qiang

    2015-01-01

    TGF-β plays a critical role in the initiation and progression of fibrosis in various organ systems such as kidney, heart, lung and liver. TGF-β and its receptors (ALK5 and TβR II) are able to control the cellular growth and promote several biological responses. To date, many pharmaceutical companies have employed virtual screening to identify potent inhibitors against ALK5. Nevertheless, none of these studies had involved the in silico ADMET evaluation and Raccoon filtering. In our experiment, all 57423 molecules were downloaded from TCM database and were filtered and converted to PDBQT formats by Raccoon software. Then 24 189 structures were run through AutoDock Vina in PyRx 0.8, 164 molecules were selected and further evaluated by ADMET Predictor 6.5, and 56 structures were selected and docked by Glide 6.2. Finally, the top 10 hits were identified as promising oral ALK5 inhibitors according to their Glide scores. The Glide scores of the best two compounds, 40686 and 33534, were -10.75 and -10.30 kcal/mol, respectively. This research provides a set of combined and detailed virtual screening protocol and is helpful for explaining the mechanism of receptor-ligand interactions.

  7. Automation of ALK gene rearrangement testing with fluorescence in situ hybridization (FISH): a feasibility study.

    PubMed

    Zwaenepoel, Karen; Merkle, Dennis; Cabillic, Florian; Berg, Erica; Belaud-Rotureau, Marc-Antoine; Grazioli, Vittorio; Herelle, Olga; Hummel, Michael; Le Calve, Michele; Lenze, Dido; Mende, Stefanie; Pauwels, Patrick; Quilichini, Benoit; Repetti, Elena

    2015-02-01

    In the past several years we have observed a significant increase in our understanding of molecular mechanisms that drive lung cancer. Specifically in the non-small cell lung cancer sub-types, ALK gene rearrangements represent a sub-group of tumors that are targetable by the tyrosine kinase inhibitor Crizotinib, resulting in significant reductions in tumor burden. Phase II and III clinical trials were performed using an ALK break-apart FISH probe kit, making FISH the gold standard for identifying ALK rearrangements in patients. FISH is often considered a labor and cost intensive molecular technique, and in this study we aimed to demonstrate feasibility for automation of ALK FISH testing, to improve laboratory workflow and ease of testing. This involved automation of the pre-treatment steps of the ALK assay using various protocols on the VP 2000 instrument, and facilitating automated scanning of the fluorescent FISH specimens for simplified enumeration on various backend scanning and analysis systems. The results indicated that ALK FISH can be automated. Significantly, both the Ikoniscope and BioView system of automated FISH scanning and analysis systems provided a robust analysis algorithm to define ALK rearrangements. In addition, the BioView system facilitated consultation of difficult cases via the internet.

  8. An integrated molecular modeling approach for in silico design of new tetracyclic derivatives as ALK inhibitors.

    PubMed

    Peddi, Saikiran Reddy; Sivan, Sree Kanth; Manga, Vijjulatha

    2016-10-01

    Anaplastic lymphoma kinase (ALK), a promising therapeutic target for treatment of human cancers, is a receptor tyrosine kinase that instigates the activation of several signal transduction pathways. In the present study, in silico methods have been employed in order to explore the structural features and functionalities of a series of tetracyclic derivatives displaying potent inhibitory activity toward ALK. Initially docking was performed using GLIDE 5.6 to probe the bioactive conformation of all the compounds and to understand the binding modes of inhibitors. The docking results revealed that ligand interaction with Met 1199 plays a crucial role in binding of inhibitors to ALK. Further to establish a robust 3D-QSAR model using CoMFA and CoMSIA methods, the whole dataset was divided into three splits. Model obtained from Split 3 showed high accuracy ([Formula: see text] of 0.700 and 0.682, [Formula: see text] of 0.971 and 0.974, [Formula: see text] of 0.673 and 0.811, respectively for CoMFA and CoMSIA). The key structural requirements for enhancing the inhibitory activity were derived from CoMFA and CoMSIA contours in combination with site map analysis. Substituting small electronegative groups at Position 8 by replacing either morpholine or piperidine rings and maintaining hydrophobic character at Position 9 in tetracyclic derivatives can enhance the inhibitory potential. Finally, we performed molecular dynamics simulations in order to investigate the stability of protein ligand interactions and MM/GBSA calculations to compare binding free energies of co-crystal ligand and newly designed molecule N1. Based on the coherence of outcome of various molecular modeling studies, a set of 11 new molecules having potential predicted inhibitory activity were designed. PMID:26758803

  9. An integrated molecular modeling approach for in silico design of new tetracyclic derivatives as ALK inhibitors.

    PubMed

    Peddi, Saikiran Reddy; Sivan, Sree Kanth; Manga, Vijjulatha

    2016-10-01

    Anaplastic lymphoma kinase (ALK), a promising therapeutic target for treatment of human cancers, is a receptor tyrosine kinase that instigates the activation of several signal transduction pathways. In the present study, in silico methods have been employed in order to explore the structural features and functionalities of a series of tetracyclic derivatives displaying potent inhibitory activity toward ALK. Initially docking was performed using GLIDE 5.6 to probe the bioactive conformation of all the compounds and to understand the binding modes of inhibitors. The docking results revealed that ligand interaction with Met 1199 plays a crucial role in binding of inhibitors to ALK. Further to establish a robust 3D-QSAR model using CoMFA and CoMSIA methods, the whole dataset was divided into three splits. Model obtained from Split 3 showed high accuracy ([Formula: see text] of 0.700 and 0.682, [Formula: see text] of 0.971 and 0.974, [Formula: see text] of 0.673 and 0.811, respectively for CoMFA and CoMSIA). The key structural requirements for enhancing the inhibitory activity were derived from CoMFA and CoMSIA contours in combination with site map analysis. Substituting small electronegative groups at Position 8 by replacing either morpholine or piperidine rings and maintaining hydrophobic character at Position 9 in tetracyclic derivatives can enhance the inhibitory potential. Finally, we performed molecular dynamics simulations in order to investigate the stability of protein ligand interactions and MM/GBSA calculations to compare binding free energies of co-crystal ligand and newly designed molecule N1. Based on the coherence of outcome of various molecular modeling studies, a set of 11 new molecules having potential predicted inhibitory activity were designed.

  10. Structure–Activity Relationship of 3,5-Diaryl-2-aminopyridine ALK2 Inhibitors Reveals Unaltered Binding Affinity for Fibrodysplasia Ossificans Progressiva Causing Mutants

    PubMed Central

    2015-01-01

    There are currently no effective therapies for fibrodysplasia ossificans progressiva (FOP), a debilitating and progressive heterotopic ossification disease caused by activating mutations of ACVR1 encoding the BMP type I receptor kinase ALK2. Recently, a subset of these same mutations of ACVR1 have been identified in diffuse intrinsic pontine glioma (DIPG) tumors. Here we describe the structure–activity relationship for a series of novel ALK2 inhibitors based on the 2-aminopyridine compound K02288. Several modifications increased potency in kinase, thermal shift, or cell-based assays of BMP signaling and transcription, as well as selectivity for ALK2 versus closely related BMP and TGF-β type I receptor kinases. Compounds in this series exhibited a wide range of in vitro cytotoxicity that was not correlated with potency or selectivity, suggesting mechanisms independent of BMP or TGF-β inhibition. The study also highlights a potent 2-methylpyridine derivative 10 (LDN-214117) with a high degree of selectivity for ALK2 and low cytotoxicity that could provide a template for preclinical development. Contrary to the notion that activating mutations of ALK2 might alter inhibitor efficacy due to potential conformational changes in the ATP-binding site, the compounds demonstrated consistent binding to a panel of mutant and wild-type ALK2 proteins. Thus, BMP inhibitors identified via activity against wild-type ALK2 signaling are likely to be of clinical relevance for the diverse ALK2 mutant proteins associated with FOP and DIPG. PMID:25101911

  11. Structure-activity relationship of 3,5-diaryl-2-aminopyridine ALK2 inhibitors reveals unaltered binding affinity for fibrodysplasia ossificans progressiva causing mutants.

    PubMed

    Mohedas, Agustin H; Wang, You; Sanvitale, Caroline E; Canning, Peter; Choi, Sungwoon; Xing, Xuechao; Bullock, Alex N; Cuny, Gregory D; Yu, Paul B

    2014-10-01

    There are currently no effective therapies for fibrodysplasia ossificans progressiva (FOP), a debilitating and progressive heterotopic ossification disease caused by activating mutations of ACVR1 encoding the BMP type I receptor kinase ALK2. Recently, a subset of these same mutations of ACVR1 have been identified in diffuse intrinsic pontine glioma (DIPG) tumors. Here we describe the structure-activity relationship for a series of novel ALK2 inhibitors based on the 2-aminopyridine compound K02288. Several modifications increased potency in kinase, thermal shift, or cell-based assays of BMP signaling and transcription, as well as selectivity for ALK2 versus closely related BMP and TGF-β type I receptor kinases. Compounds in this series exhibited a wide range of in vitro cytotoxicity that was not correlated with potency or selectivity, suggesting mechanisms independent of BMP or TGF-β inhibition. The study also highlights a potent 2-methylpyridine derivative 10 (LDN-214117) with a high degree of selectivity for ALK2 and low cytotoxicity that could provide a template for preclinical development. Contrary to the notion that activating mutations of ALK2 might alter inhibitor efficacy due to potential conformational changes in the ATP-binding site, the compounds demonstrated consistent binding to a panel of mutant and wild-type ALK2 proteins. Thus, BMP inhibitors identified via activity against wild-type ALK2 signaling are likely to be of clinical relevance for the diverse ALK2 mutant proteins associated with FOP and DIPG.

  12. Combating autophagy is a strategy to increase cytotoxic effects of novel ALK inhibitor entrectinib in neuroblastoma cells

    PubMed Central

    Aveic, Sanja; Pantile, Marcella; Seydel, Anke; Esposito, Maria Rosaria; Zanon, Carlo; Li, Gary; Tonini, Gian Paolo

    2016-01-01

    Neuroblastoma (NB) is a threatening childhood malignancy. Its prognosis is affected by several morphological, and biological characteristics, including the constitutive expression of ALK tyrosine kinase. In this study we examined the therapeutic potential of a novel ALK inhibitor, entrectinib, in obliterating NB tumor cells. Entrectinib showed the growth-inhibitory effects on NB cells with a 50% inhibitory concentration range of 0.03–5 μM. In the ALK-dependent cells, entrectinib mediated G1-arrest, which was associated with modified expression of multiple cell-cycle regulators. Down-regulation of Ki-67, and attenuated phosphorylation of ERK1/2, and STAT3, correlated with observed antiproliferative capacity of entrectinib. Initial cytostatic activity of entrectinib was followed by concentration-dependent apoptotic cell death, and Caspase-3 activation. However, we delineated a reduced sensitivity of ALK mutated NB cells to entrectinib, and demonstrated strong activation of autophagy in SH-SY5YF1174L NB cell line. Abrogation of autophagy by chloroquine increased significantly the toxicity of entrectinib, as confirmed by enhanced death rate, and PARP protein cleavage in SH-SY5YF1174L cells. In aggregate, our data show that entrectinib inhibits proliferation, and induces G1-arrest, and apoptosis in NB cells. We propose entrectinib for further consideration in treatment of NB, and recommend pharmacological inhibition of autophagy to be explored for a combined therapeutic approach in NB patients that might develop resistance to entrectinib. PMID:26735175

  13. Will the Requirement by the US FDA to Simultaneously Co-Develop Companion Diagnostics (CDx) Delay the Approval of Receptor Tyrosine Kinase Inhibitors for RTK-Rearranged (ROS1-, RET-, AXL-, PDGFR-α-, NTRK1-) Non-Small Cell Lung Cancer Globally?

    PubMed

    Ou, Sai-Hong Ignatius; Soo, Ross A; Kubo, Akihito; Kawaguchi, Tomoya; Ahn, Myung-Ju

    2014-01-01

    The discovery of anaplastic lymphoma kinase (ALK) rearrangement in non-small cell lung cancer (NSCLC) in 2007 and the approval of crizotinib for the treatment of advanced ALK-rearranged NSCLC in 2011 represents a landmark in the development of targeted oncology therapy. The approval of crizotinib was accompanied simultaneously by the approval of the Vysis (Abbott Molecular) break-apart fluorescence in situ hybridization (FISH) test as the companion diagnostic (CDx) test to detect ALK rearrangement. Pfizer, the manufacturer of crizotinib, sponsored the screening of thousands of patients and the standardization of the ALK FISH test as part of the approval process for crizotinib, a first in class ALK inhibitor. Many pharmaceutical companies are now using the Food and Drug Administration (FDA)-approved ALK FISH assay to enroll patients onto trials for their own respective ALK inhibitors. In essence they are "piggybacking" on the FDA-approved ALK FISH assay without having to pay for the development of a CDx, nor screening for ALK-rearranged NSCLC patients in the protocols because screening for ALK rearrangement is now the standard of care in NSCLC after the approval of crizotinib. Since 2007, rearrangement in more receptor tyrosine kinases (RTKs) such as ROS1, RET, AXL, PDGFR-α, and NTRK1 have been discovered in NSCLC but the incidence of each subtype of RTK-rearranged NSCLC is quite rare. Crizotinib has now demonstrated significant clinical activity in ROS1-rearranged NSCLC patients. Whether crizotinib will gain official FDA approval for use in ROS1-rearranged NSCLC, on the other hand, remains unclear as there is no test for ROS1-rearrangement currently being developed to support US FDA approval as a CDx. This may be due in part to the fact that the full cost associated with the development of a pre-market approved-approved CDx must be borne by the company seeking the first drug approval in a new indication. Given the low incidence of ROS1-rearrangement in NSCLC, and

  14. Alectinib induced CNS radiation necrosis in an ALK+NSCLC patient with a remote (7 years) history of brain radiation.

    PubMed

    Ou, Sai-Hong Ignatius; Weitz, Michael; Jalas, John R; Kelly, Daniel F; Wong, Vanessa; Azada, Michele C; Quines, Oliver; Klempner, Samuel J

    2016-06-01

    Alectinib is a second generation ALK inhibitor that has significant clinical activity in central nervous system (CNS) metastases in anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC). Pseudoprogression (PsP) due to radiation necrosis during alecitnib treatment of central nervous system (CNS) metastases from ALK-rearranged NSCLC as been reported. Hence, distinguishing radiation-related PsP from alectinib-induced radiographic changes is important to avoid erroneous early trial discontinuation and abandonment of an effective treatment. However, it remains difficult to assess casuality of radiation necrosis is related to recent direct radiation or induced by alectinib treatment or both. It is also unknown how long from previous radiation can alectinib still induce radiation necrosis. Here we reported a crizotinib-refractory ALK-positive NSCLC patient who develop radiation necrosis in one of his metastatic CNS lesions after approximately 12 months of alectinib treatment who otherwise had on-going CNS response on alectinib. His most recent radiation to his CNS metastases was 7 years prior to the start of alectinib. This case illustrates that in the setting of pror CNS radiation, given the significant clinical activity of alectinib in CNS metastases in ALK-positive NSCLC patients the risk of CNS radiation necrosis remains long after previous radiation to the CNS metastases has been completed and can occur after durable response of treatment. PMID:27133743

  15. Multi-parameter in vitro toxicity testing of crizotinib, sunitinib, erlotinib, and nilotinib in human cardiomyocytes

    SciTech Connect

    Doherty, Kimberly R.; Wappel, Robert L.; Talbert, Dominique R.; Trusk, Patricia B.; Moran, Diarmuid M.; Kramer, James W.; Brown, Arthur M.; Shell, Scott A.; Bacus, Sarah

    2013-10-01

    Tyrosine kinase inhibitors (TKi) have greatly improved the treatment and prognosis of multiple cancer types. However, unexpected cardiotoxicity has arisen in a subset of patients treated with these agents that was not wholly predicted by pre-clinical testing, which centers around animal toxicity studies and inhibition of the human Ether-à-go-go-Related Gene (hERG) channel. Therefore, we sought to determine whether a multi-parameter test panel assessing the effect of drug treatment on cellular, molecular, and electrophysiological endpoints could accurately predict cardiotoxicity. We examined how 4 FDA-approved TKi agents impacted cell viability, apoptosis, reactive oxygen species (ROS) generation, metabolic status, impedance, and ion channel function in human cardiomyocytes. The 3 drugs clinically associated with severe cardiac adverse events (crizotinib, sunitinib, nilotinib) all proved to be cardiotoxic in our in vitro tests while the relatively cardiac-safe drug erlotinib showed only minor changes in cardiac cell health. Crizotinib, an ALK/MET inhibitor, led to increased ROS production, caspase activation, cholesterol accumulation, disruption in cardiac cell beat rate, and blockage of ion channels. The multi-targeted TKi sunitinib showed decreased cardiomyocyte viability, AMPK inhibition, increased lipid accumulation, disrupted beat pattern, and hERG block. Nilotinib, a second generation Bcr-Abl inhibitor, led to increased ROS generation, caspase activation, hERG block, and an arrhythmic beat pattern. Thus, each drug showed a unique toxicity profile that may reflect the multiple mechanisms leading to cardiotoxicity. This study demonstrates that a multi-parameter approach can provide a robust characterization of drug-induced cardiomyocyte damage that can be leveraged to improve drug safety during early phase development. - Highlights: • TKi with known adverse effects show unique cardiotoxicity profiles in this panel. • Crizotinib increases ROS, apoptosis, and

  16. Targeted Disruption of ALK Reveals a Potential Role in Hypogonadotropic Hypogonadism.

    PubMed

    Witek, Barbara; El Wakil, Abeer; Nord, Christoffer; Ahlgren, Ulf; Eriksson, Maria; Vernersson-Lindahl, Emma; Helland, Åslaug; Alexeyev, Oleg A; Hallberg, Bengt; Palmer, Ruth H

    2015-01-01

    Mice lacking ALK activity have previously been reported to exhibit subtle behavioral phenotypes. In this study of ALK of loss of function mice we present data supporting a role for ALK in hypogonadotropic hypogonadism in male mice. We observed lower level of serum testosterone at P40 in ALK knock-out males, accompanied by mild disorganization of seminiferous tubules exhibiting decreased numbers of GATA4 expressing cells. These observations highlight a role for ALK in testis function and are further supported by experiments in which chemical inhibition of ALK activity with the ALK TKI crizotinib was employed. Oral administration of crizotinib resulted in a decrease of serum testosterone levels in adult wild type male mice, which reverted to normal levels after cessation of treatment. Analysis of GnRH expression in neurons of the hypothalamus revealed a significant decrease in the number of GnRH positive neurons in ALK knock-out mice at P40 when compared with control littermates. Thus, ALK appears to be involved in hypogonadotropic hypogonadism by regulating the timing of pubertal onset and testis function at the upper levels of the hypothalamic-pituitary gonadal axis.

  17. Conditional TPM3-ALK and NPM-ALK transgenic mice develop reversible ALK-positive early B-cell lymphoma/leukemia.

    PubMed

    Giuriato, Sylvie; Foisseau, Marianne; Dejean, Emilie; Felsher, Dean W; Al Saati, Talal; Demur, Cécile; Ragab, Ashraf; Kruczynski, Anna; Schiff, Claudine; Delsol, Georges; Meggetto, Fabienne

    2010-05-20

    NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) and TPM3-ALK (nonmuscular tropomyosin 3-anaplastic lymphoma kinase) are oncogenic tyrosine kinases implicated in the pathogenesis of human ALK-positive lymphoma. We report here the development of novel conditional mouse models for ALK-induced lymphomagenesis, with the use of the tetracycline regulatory system under the control of the EmuSRalpha enhancer/promoter. The expression of either oncogene resulted in the arrest of the differentiation of early B cells and lymphomagenesis. We also observed the development of skin keratoacanthoma lesions, probably because of aberrant ALK expression in keratinocytes. The inactivation of the ALK oncogene on doxycycline treatment was sufficient to induce sustained regression of both hematopoietic tumors and skin disease. Importantly, treatment with the specific ALK inhibitor (PF-2341066) also reversed the pathologic states, showing the value of these mouse models for the validation of ALK tyrosine kinase inhibitors. Thus, our results show (1) that NPM-ALK and TPM3-ALK oncogenes are sufficient for lymphoma/leukemia development and required for tumor maintenance, hence validating ALK as potentially effective therapeutic target; and (2) for the first time, in vivo, the equal tumorigenic potential of the NPM-ALK and TPM3-ALK oncogenic tyrosine kinases. Our models offer a new tool to investigate in vivo the molecular mechanisms associated with ALK-induced lymphoproliferative disorders.

  18. Alectinib's activity against CNS metastases from ALK-positive non-small cell lung cancer: a single institution case series.

    PubMed

    Metro, Giulio; Lunardi, Gianluigi; Bennati, Chiara; Chiarini, Pietro; Sperduti, Isabella; Ricciuti, Biagio; Marcomigni, Luca; Costa, Cinzia; Crinò, Lucio; Floridi, Piero; Gori, Stefania; Chiari, Rita

    2016-09-01

    In the present study we assessed the activity of the next-generation anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (-TKI) alectinib, in patients with ALK-postive, advanced non-small cell lung cancer (NSCLC) and central nervous system (CNS) metastases. NSCLCs with ALK-positive disease, as assessed by fluorescence in situ hybridization, and CNS metastases were treated with alectinib 600 mg BID. Included patients were followed prospectively in order to evaluate the efficacy of the drug, with particular emphasis on activity in the CNS. Eleven consecutive patients were enrolled. The majority of them were pretreated with crizotinib (n = 10, 90.9 %), and cranial radiotherapy (n = 8, 72.7 %). Six of the seven patients with measurable CNS disease experienced a CNS response, including three patients who were naïve for cranial radiation. Median duration of response was 8 months. For the whole population, median CNS-progression-free survival (-PFS), systemic-PFS, overall-PFS, overall survival, and 1-year survival were 8, 11, 8, 13 months, and 31.1 %, respectively. Two patients experiencing a CNS response were assessed for alectinib's concentrations in serum and cerebro-spinal fluid (CSF), and showed a CSF-to-serum ratio ranging from 0.001 to 0.003 ng/mL. Alectinib is highly active against CNS metastases from ALK-positive NSCLCs, irrespective of prior treatment(s) with ALK-TKI(s) and/or cranial radiotherapy. The low CSF-to-serum ratio of alectinib suggests that measuring the concentrations of the drug in the CSF may not be a reliable surrogate of its distribution into the CNS. PMID:27324494

  19. The analysis of ALK gene rearrangement by fluorescence in situ hybridization in non-small cell lung cancer patients

    PubMed Central

    Krawczyk, Paweł Adam; Ramlau, Rodryg Adam; Szumiło, Justyna; Kozielski, Jerzy; Kalinka-Warzocha, Ewa; Bryl, Maciej; Knopik-Dąbrowicz, Alina; Spychalski, Łukasz; Szczęsna, Aleksandra; Rydzik, Ewelina; Milanowski, Janusz

    2013-01-01

    Introduction ALK gene rearrangement is observed in a small subset (3–7%) of non-small cell lung cancer (NSCLC) patients. The efficacy of crizotinib was shown in lung cancer patients harbouring ALK rearrangement. Nowadays, the analysis of ALK gene rearrangement is added to molecular examination of predictive factors. Aim of the study The frequency of ALK gene rearrangement as well as the type of its irregularity was analysed by fluorescence in situ hybridisation (FISH) in tissue samples from NSCLC patients. Material and methods The ALK gene rearrangement was analysed in 71 samples including 53 histological and 18 cytological samples. The analysis could be performed in 56 cases (78.87%), significantly more frequently in histological than in cytological materials. The encountered problem with ALK rearrangement diagnosis resulted from the scarcity of tumour cells in cytological samples, high background fluorescence noises and fragmentation of cell nuclei. Results The normal ALK copy number without gene rearrangement was observed in 26 (36.62%) patients ALK gene polysomy without gene rearrangement was observed in 25 (35.21%) samples while in 3 (4.23%) samples ALK gene amplification was found. ALK gene rearrangement was observed in 2 (2.82%) samples from males, while in the first case the rearrangement coexisted with ALK amplification. In the second case, signet-ring tumour cells were found during histopathological examination and this patient was successfully treated with crizotinib with partial remission lasting 16 months. Conclusions FISH is a useful technique for ALK gene rearrangement analysis which allows us to specify the type of gene irregularities. ALK gene examination could be performed in histological as well as cytological (cellblocks) samples, but obtaining a reliable result in cytological samples depends on the cellularity of examined materials. PMID:24592134

  20. Liposomes ameliorate Crizotinib- and Nilotinib-induced inhibition of the cardiac IKr channel and QTc prolongation.

    PubMed

    Shopp, George M; Helson, Lawrence; Bouchard, Annie; Salvail, Dany; Majeed, Muhammad

    2014-09-01

    Crizotinib (Xalkori®) and nilotinib (Tasigna®) are tyrosine kinase inhibitors approved for the treatment of non-small cell lung cancer and chronic myeloid leukemia, respectively. Both have been shown to result in electrocardiogram rate-corrected Q-wave T-wave interval (QTc) prolongation in humans and animals. Liposomes have been shown to ameliorate drug-induced effects on the cardiac-delayed rectifier K(+) current (IKr, KV11.1), coded by the human ether-a-go-go-related gene (hERG). This study was undertaken to determine if liposomes would also decrease the effect of crizotinib and nilotinib on the IKr channel. Crizotinib and nilotinib were tested in an in vitro IKr assay using human embryonic kidney (HEK) 293 cells stably transfected with the hERG. Dose-responses were determined and the 50% inhibitory concentrations (IC50s) were calculated. When the HEK 293 cells were treated with crizotinib or nilotinib that were mixed with liposomes, there was a significant decrease in the IKr channel inhibitory effects of these two drugs. When isolated, rabbit hearts were exposed to crizotinib or nilotinib, there were significant increases in QTc prolongation. Mixing either of the drugs with liposomes ameliorated the effects of the drugs. Rabbits dosed intravenously (IV) with crizotinib or nilotinib showed QTc prolongation. When liposomes were injected prior to crizotinib or nilotinib, the liposomes decreased the effects on the QTc interval. The use of liposomal encapsulated QT-prolongation agents, or giving liposomes in combination with drugs, may decrease their cardiac liability. PMID:25202051

  1. Liposomes ameliorate Crizotinib- and Nilotinib-induced inhibition of the cardiac IKr channel and QTc prolongation.

    PubMed

    Shopp, George M; Helson, Lawrence; Bouchard, Annie; Salvail, Dany; Majeed, Muhammad

    2014-09-01

    Crizotinib (Xalkori®) and nilotinib (Tasigna®) are tyrosine kinase inhibitors approved for the treatment of non-small cell lung cancer and chronic myeloid leukemia, respectively. Both have been shown to result in electrocardiogram rate-corrected Q-wave T-wave interval (QTc) prolongation in humans and animals. Liposomes have been shown to ameliorate drug-induced effects on the cardiac-delayed rectifier K(+) current (IKr, KV11.1), coded by the human ether-a-go-go-related gene (hERG). This study was undertaken to determine if liposomes would also decrease the effect of crizotinib and nilotinib on the IKr channel. Crizotinib and nilotinib were tested in an in vitro IKr assay using human embryonic kidney (HEK) 293 cells stably transfected with the hERG. Dose-responses were determined and the 50% inhibitory concentrations (IC50s) were calculated. When the HEK 293 cells were treated with crizotinib or nilotinib that were mixed with liposomes, there was a significant decrease in the IKr channel inhibitory effects of these two drugs. When isolated, rabbit hearts were exposed to crizotinib or nilotinib, there were significant increases in QTc prolongation. Mixing either of the drugs with liposomes ameliorated the effects of the drugs. Rabbits dosed intravenously (IV) with crizotinib or nilotinib showed QTc prolongation. When liposomes were injected prior to crizotinib or nilotinib, the liposomes decreased the effects on the QTc interval. The use of liposomal encapsulated QT-prolongation agents, or giving liposomes in combination with drugs, may decrease their cardiac liability.

  2. Crizotinib

    MedlinePlus

    ... medications for human immunodeficiency virus (HIV) such as atazanavir (Reyataz), indinavir (Crixivan), nelfinavir (Viracept), ritonavir (Norvir, in ... order a lab test before you begin your treatment to see whether your cancer can be treated ...

  3. An ALK translocation positive carcinoma of the lung presenting as uremia due to bilateral renal obstruction.

    PubMed

    Rosenberg, Shilo; Katz, Ran; Pode, Dov; Gofrit, N Ofer; Pizov, Galina; Hovav, Nechushtan

    2013-01-01

    We describe an unusual presentation of metastatic lung adenocarcinoma as malignant retroperitoneal fibrosis (MRPF). The diagnostic challenge, due to the small solitary lung mass and absence of a discrete retroperitoneal mass, was overcome by diagnostic laparoscopy. Molecular analysis of tissue acquired was positive for ALK gene rearrangement. Treatment of the patient with crizotinib reversed MRPF. He was weaned off the nephrostomy tubes and is with stable renal function 11 months after diagnosis. PMID:23914266

  4. ROS1 Kinase Inhibitors for Molecular-Targeted Therapies.

    PubMed

    Al-Sanea, M M; Abdelazem, A Z; Park, B S; Yoo, K H; Sim, T; Kwon, Y J; Lee, S H

    2016-01-01

    ROS1 is a pivotal transmembrane receptor protein tyrosine kinase which regulates several cellular processes like apoptosis, survival, differentiation, proliferation, cell migration, and transformation. There is increasing evidence supporting that ROS1 plays an important role in different malignancies including glioblastoma, colorectal cancer, gastric adenocarcinoma, inflammatory myofibroblastic tumor, ovarian cancer, angiosarcoma, and non small cell lung cancer; thus, ROS1 has become a potential drug discovery target. ROS1 shares about 49% sequence homology with ALK primary structure; therefore, wide range of ALK kinase inhibitors have shown in vitro inhibitory activity against ROS1 kinase. After Crizotinib approval by FDA for the management of ALK-rearranged lung cancer, ROS1-positive tumors have been focused. Although significant advancements have been achieved in understanding ROS1 function and its signaling pathways plus recent discovery of small molecules modulating ROS1 protein, a vital need of medicinal chemistry efforts is still required to produce selective and potent ROS1 inhibitors as an important therapeutic strategy for different human malignancies. This review focuses on the current knowledge about different scaffolds targeting ROS1 rearrangements, methods to synthesis, and some biological data about the most potent compounds that have delivered various scaffold structures. PMID:26438251

  5. Benefit-Risk Summary of Crizotinib for the Treatment of Patients With ROS1 Alteration-Positive, Metastatic Non-Small Cell Lung Cancer

    PubMed Central

    Blumenthal, Gideon M.; Luo, Lola; He, Kun; Fran, Ingrid; Lemery, Steven; Pazdur, Richard

    2016-01-01

    On March 11, 2016, after an expedited 5-month review, the U.S. Food and Drug Administration expanded the crizotinib metastatic non-small cell lung cancer (mNSCLC) indication to include the treatment of patients whose tumors harbor a ROS1 rearrangement. The approval was based on a clinically meaningful, durable objective response rate (ORR) in a multicenter, single-arm clinical trial (ROS1 cohort of Trial PROFILE 1001) in patients with ROS1-positive mNSCLC. The trial enrolled 50 patients (age range: 25–77 years) whose tumors were prospectively determined to have a ROS1 gene rearrangement by break-apart fluorescence in situ hybridization (96%) or reverse transcriptase polymerase chain reaction (4%) clinical trial assays. Crizotinib demonstrated an ORR of 66% (95% confidence interval [CI]: 51%–79%) with a median duration of response of 18.3 months by independent radiology review and 72% (95% CI: 58%–84%) by investigator review. Patients received crizotinib 250 mg twice daily and had a median duration of exposure of 34.4 months. The toxicity profile in ROS1-positive patients was generally consistent with the randomized safety data in the U.S. Product Insert from two ALK-positive mNSCLC trials. The most common (≥25%) adverse reactions and laboratory test abnormalities included vision disorders, elevation of alanine transaminase and aspartate transaminase levels, nausea, hypophosphatemia, diarrhea, edema, vomiting, constipation, neutropenia, and fatigue. There were no treatment-related deaths. A favorable benefit-to-risk evaluation led to the traditional approval of crizotinib for this new supplemental indication. Implications for Practice: Given the results from the ROS1 cohort of the clinical trial PROFILE 1001, crizotinib represents a new treatment option and the first approved therapy for patients with metastatic non-small cell lung cancer whose tumors are ROS1 positive. Crizotinib demonstrated efficacy irrespective of prior treatment status. PMID:27328934

  6. A causal link from ALK to hexokinase II overexpression and hyperactive glycolysis in EML4-ALK-positive lung cancer

    PubMed Central

    Ma, Yibao; Yu, Chunrong; Mohamed, Esraa M.; Shao, Huanjie; Wang, Li; Sundaresan, Gobalakrishnan; Zweit, Jamal; Idowu, Michael; Fang, Xianjun

    2016-01-01

    A high rate of aerobic glycolysis is a hallmark of malignant transformation. Accumulating evidence suggests that diverse regulatory mechanisms mediate this cancer-associated metabolic change seen in a wide spectrum of cancer. The echinoderm microtubule associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion protein is found in approximately 3-7% of non-small cell lung carcinomas (NSCLC). Molecular evidence and therapeutic effectiveness of FDA-approved ALK inhibitors indicated that EML4-ALK is a driving factor of lung tumorigenesis. A recent clinical study showed that NSCLC harboring EML4-ALK rearrangements displayed higher glucose metabolism compared to EML4-ALK-negative NSCLC. In the current work, we presented evidence that EML4-ALK is coupled to overexpression of hexokinase II (HK2), one of the rate-limiting enzymes of the glycolytic pathway. The link from EML4-ALK to HK2 upregulation is essential for a high rate of glycolysis and proliferation of EML4-ALK-rearranged NSCLC cells. We identified hypoxia-inducible factor 1α (HIF1α) as a key transcription factor to drive HK2 gene expression in normoxia in these cells. EML4-ALK induced hypoxia-independent but glucose-dependent accumulation of HIF1α protein via both transcriptional activation of HIF1α mRNA and the PI3K-AKT pathway to enhance HIF1α protein synthesis. The EML4-ALK-mediated upregulation of HIF1α, HK2 and glycolytic metabolism was also highly active in vivo as demonstrated by FDG-PET imaging of xenografts grown from EML4-ALK-positive NSCLC cells. Our data reveal a novel EML4-ALK-HIF1α-HK2 cascade to enhance glucose metabolism in EML4-ALK-positive NSCLC. PMID:27132509

  7. Concordance of anaplastic lymphoma kinase (ALK) gene rearrangements between circulating tumor cells and tumor in non-small cell lung cancer.

    PubMed

    Tan, Chye Ling; Lim, Tse Hui; Lim, Tony Kh; Tan, Daniel Shao-Weng; Chua, Yong Wei; Ang, Mei Kim; Pang, Brendan; Lim, Chwee Teck; Takano, Angela; Lim, Alvin Soon-Tiong; Leong, Man Chun; Lim, Wan-Teck

    2016-04-26

    Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC) is routinely evaluated by fluorescent in-situ hybridization (FISH) testing on biopsy tissues. Testing can be challenging however, when suitable tissue samples are unavailable. We examined the relevance of circulating tumor cells (CTC) as a surrogate for biopsy-based FISH testing. We assessed paired tumor and CTC samples from patients with ALK rearranged lung cancer (n = 14), ALK-negative lung cancer (n = 12), and healthy controls (n = 5) to derive discriminant CTC counts, and to compare ALK rearrangement patterns. Blood samples were enriched for CTCs to be used for ALK FISH testing. ALK-positive CTCs counts were higher in ALK-positive NSCLC patients (3-15 cells/1.88 mL of blood) compared with ALK-negative NSCLC patients and healthy donors (0-2 cells/1.88 mL of blood). The latter range was validated as the 'false positive' cutoff for ALK FISH testing of CTCs. ALK FISH signal patterns observed on tumor biopsies were recapitulated in CTCs in all cases. Sequential CTC counts in an index case of lung cancer with no evaluable tumor tissue treated with crizotinib showed six, three and eleven ALK-positive CTCs per 1.88 mL blood at baseline, partial response and post-progression time points, respectively. Furthermore, ALK FISH rearrangement suggestive of gene copy number increase was observed in CTCs following progression. Recapitulation of ALK rearrangement patterns in the tumor on CTCs, suggested that CTCs might be used to complement tissue-based ALK testing in NSCLC to guide ALK-targeted therapy when suitable tissue biopsy samples are unavailable for testing. PMID:26993609

  8. Concordance of anaplastic lymphoma kinase (ALK) gene rearrangements between circulating tumor cells and tumor in non-small cell lung cancer

    PubMed Central

    Lim, Tony KH; Tan, Daniel Shao-Weng; Chua, Yong Wei; Ang, Mei Kim; Pang, Brendan; Lim, Chwee Teck; Takano, Angela; Lim, Alvin Soon-Tiong; Leong, Man Chun; Lim, Wan-Teck

    2016-01-01

    Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC) is routinely evaluated by fluorescent in-situ hybridization (FISH) testing on biopsy tissues. Testing can be challenging however, when suitable tissue samples are unavailable. We examined the relevance of circulating tumor cells (CTC) as a surrogate for biopsy-based FISH testing. We assessed paired tumor and CTC samples from patients with ALK rearranged lung cancer (n = 14), ALK-negative lung cancer (n = 12), and healthy controls (n = 5) to derive discriminant CTC counts, and to compare ALK rearrangement patterns. Blood samples were enriched for CTCs to be used for ALK FISH testing. ALK-positive CTCs counts were higher in ALK-positive NSCLC patients (3–15 cells/1.88 mL of blood) compared with ALK-negative NSCLC patients and healthy donors (0–2 cells/1.88 mL of blood). The latter range was validated as the ‘false positive’ cutoff for ALK FISH testing of CTCs. ALK FISH signal patterns observed on tumor biopsies were recapitulated in CTCs in all cases. Sequential CTC counts in an index case of lung cancer with no evaluable tumor tissue treated with crizotinib showed six, three and eleven ALK-positive CTCs per 1.88 mL blood at baseline, partial response and post-progression time points, respectively. Furthermore, ALK FISH rearrangement suggestive of gene copy number increase was observed in CTCs following progression. Recapitulation of ALK rearrangement patterns in the tumor on CTCs, suggested that CTCs might be used to complement tissue-based ALK testing in NSCLC to guide ALK-targeted therapy when suitable tissue biopsy samples are unavailable for testing. PMID:26993609

  9. Anti-ALK Antibodies in Patients with ALK-Positive Malignancies Not Expressing NPM-ALK.

    PubMed

    Damm-Welk, Christine; Siddiqi, Faraz; Fischer, Matthias; Hero, Barbara; Narayanan, Vignesh; Camidge, David Ross; Harris, Michael; Burke, Amos; Lehrnbecher, Thomas; Pulford, Karen; Oschlies, Ilske; Siebert, Reiner; Turner, Suzanne; Woessmann, Wilhelm

    2016-01-01

    Patients with Nucleophosmin (NPM)- Anaplastic Lymphoma Kinase (ALK) fusion positive Anaplastic Large Cell Lymphoma produce autoantibodies against ALK indicative of an immune response against epitopes of the chimeric fusion protein. We asked whether ALK-expression in other malignancies induces specific antibodies. Antibodies against ALK were detected in sera of one of 50 analysed ALK-expressing neuroblastoma patients, 13 of 21 ALK positive non-small cell lung carcinoma (NSCLC) patients, 13 of 22 ALK translocation-positive, but NPM-ALK-negative lymphoma patients and one of one ALK-positive rhabdomyosarcoma patient, but not in 20 healthy adults. These data suggest that boosting a pre-existent anti-ALK immune response may be more feasible for patients with ALK-positive NSCLC, lymphomas and rhabdomyosarcomas than for tumours expressing wild-type ALK.

  10. Anti-ALK Antibodies in Patients with ALK-Positive Malignancies Not Expressing NPM-ALK

    PubMed Central

    Damm-Welk, Christine; Siddiqi, Faraz; Fischer, Matthias; Hero, Barbara; Narayanan, Vignesh; Camidge, David Ross; Harris, Michael; Burke, Amos; Lehrnbecher, Thomas; Pulford, Karen; Oschlies, Ilske; Siebert, Reiner; Turner, Suzanne; Woessmann, Wilhelm

    2016-01-01

    Patients with Nucleophosmin (NPM)- Anaplastic Lymphoma Kinase (ALK) fusion positive Anaplastic Large Cell Lymphoma produce autoantibodies against ALK indicative of an immune response against epitopes of the chimeric fusion protein. We asked whether ALK-expression in other malignancies induces specific antibodies. Antibodies against ALK were detected in sera of one of 50 analysed ALK-expressing neuroblastoma patients, 13 of 21 ALK positive non-small cell lung carcinoma (NSCLC) patients, 13 of 22 ALK translocation-positive, but NPM-ALK-negative lymphoma patients and one of one ALK-positive rhabdomyosarcoma patient, but not in 20 healthy adults. These data suggest that boosting a pre-existent anti-ALK immune response may be more feasible for patients with ALK-positive NSCLC, lymphomas and rhabdomyosarcomas than for tumours expressing wild-type ALK. PMID:27471553

  11. Anti-ALK Antibodies in Patients with ALK-Positive Malignancies Not Expressing NPM-ALK.

    PubMed

    Damm-Welk, Christine; Siddiqi, Faraz; Fischer, Matthias; Hero, Barbara; Narayanan, Vignesh; Camidge, David Ross; Harris, Michael; Burke, Amos; Lehrnbecher, Thomas; Pulford, Karen; Oschlies, Ilske; Siebert, Reiner; Turner, Suzanne; Woessmann, Wilhelm

    2016-01-01

    Patients with Nucleophosmin (NPM)- Anaplastic Lymphoma Kinase (ALK) fusion positive Anaplastic Large Cell Lymphoma produce autoantibodies against ALK indicative of an immune response against epitopes of the chimeric fusion protein. We asked whether ALK-expression in other malignancies induces specific antibodies. Antibodies against ALK were detected in sera of one of 50 analysed ALK-expressing neuroblastoma patients, 13 of 21 ALK positive non-small cell lung carcinoma (NSCLC) patients, 13 of 22 ALK translocation-positive, but NPM-ALK-negative lymphoma patients and one of one ALK-positive rhabdomyosarcoma patient, but not in 20 healthy adults. These data suggest that boosting a pre-existent anti-ALK immune response may be more feasible for patients with ALK-positive NSCLC, lymphomas and rhabdomyosarcomas than for tumours expressing wild-type ALK. PMID:27471553

  12. Clinical characteristics and outcomes of patients with primary lung adenocarcinoma harboring ALK rearrangements detected by FISH, IHC, and RT-PCR.

    PubMed

    Wang, Jinghui; Cai, Yiran; Dong, Yujie; Nong, Jingying; Zhou, Lijuan; Liu, Guimei; Su, Dan; Li, Xi; Wu, Shafei; Chen, Xuejing; Qin, Na; Zeng, Xuan; Zhang, Haiqing; Zhang, Zongde; Zhang, Shucai

    2014-01-01

    EML4-ALK is a new driver gene of non-small cell lung cancer and a target of crizotinib. The objectives of this study were to determine the frequency of ALK rearrangements in a large cohort of patients with primary lung adenocarcinoma and to analyze the association of ALK rearrangements with clinicopathological characteristics and clinical outcomes. The roles of fluorescence in situ hybridization (FISH), Ventana immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR) in the detection of ALK rearrangements were evaluated. The ALK rearrangement was detected in 430 specimens from individual patients with primary lung adenocarcinoma using FISH and Ventana IHC based on tissue microarrays. The EGFR status was detected in all of the specimens through DNA sequencing. An RT-PCR was performed on 200 of the specimens and confirmed by sequencing. Of the 430 patients, 46 (10.7%) harbored ALK rearrangements. The ALK rearrangements were associated with a younger age and the EGFR wild type in comparison with ALK-negative patients. The sensitivity and specificity of the Ventana IHC were 100% and 98.2%, respectively, and the concordance rate between the FISH and the Ventana IHC was 98.4%. The sensitivity and specificity of RT-PCR were 95.5% and 87.0%, respectively, and the concordance rate between the FISH and the RT-PCR was 89.0%. The Cox analysis indicated that an early stage and EGFR-activating mutations were independently associated with a longer OS. This study demonstrated that ALK rearrangements are associated with a younger age and the EGFR wild type rather than with other clinicopathological factors. Although the FISH and Ventana IHC have better concordance, and RT-PCR is a more sensitive method and can identify different variants or partners, the IHC and RT-PCR need to be further evaluated in clinical trials to identify their roles in guiding patients' targeted therapy using crizotinib.

  13. ALK status testing in non-small cell lung carcinoma: correlation between ultrasensitive IHC and FISH.

    PubMed

    Minca, Eugen C; Portier, Bryce P; Wang, Zhen; Lanigan, Christopher; Farver, Carol F; Feng, Yan; Ma, Patrick C; Arrossi, Valeria A; Pennell, Nathan A; Tubbs, Raymond R

    2013-05-01

    ALK gene rearrangements in advanced non-small cell lung carcinomas (NSCLC) are an indication for targeted therapy with crizotinib. Fluorescence in situ hybridization (FISH) using a recently approved companion in vitro diagnostic class FISH system commonly assesses ALK status. More accessible IHC is challenged by low expression of ALK-fusion transcripts in NSCLC. We compared ultrasensitive automated IHC with FISH for detecting ALK status on 318 FFPE and 40 matched ThinPrep specimens from 296 patients with advanced NSCLC. IHC was concordant with FFPE-FISH on 229 of 231 dual-informative samples (31 positive and 198 negative) and with ThinPrep-FISH on 34 of 34 samples (5 positive and 29 negative). Two cases with negative IHC and borderline-positive FFPE-FISH (15% and 18%, respectively) were reclassified as concordant based on negative matched ThinPrep-FISH and clinical data consistent with ALK-negative status. Overall, after including ThinPrep-FISH and amending the false-positive FFPE-FISH results, IHC demonstrated 100% sensitivity and specificity (95% CI, 0.86 to 1.00 and 0.97 to 1.00, respectively) for ALK detection on 249 dual-informative NSCLC samples. IHC was informative on significantly more samples than FFPE-FISH, revealing additional ALK-positive cases. The high concordance with FISH warrants IHC's routine use as the initial component of an algorithmic approach to clinical ALK testing in NSCLC, followed by reflex FISH confirmation of IHC-positive cases. PMID:23499337

  14. Successful alectinib treatment after crizotinib-induced interstitial lung disease.

    PubMed

    Fujiuchi, Satoru; Fujita, Yuka; Sasaki, Takaaki; Ohsaki, Yoshinobu

    2016-05-01

    A 70-year-old woman with lung adenocarcinoma, harbouring anaplastic lymphoma kinase gene rearrangement, was treated with crizotinib as third-line chemotherapy. After 2 months, crizotinib was discontinued because of the development of crizotinib-induced interstitial lung disease (ILD). Steroid treatment was then introduced and tapered off. Following complete resolution of the interstitial shadow, cytotoxic chemotherapy was initiated, and continued for over 2 years, until new intrapulmonary lesions developed. Although there was a risk of drug-induced interstitial pneumonia, alectinib was initiated as the fifth-line therapy, without steroid supplementation, as there was no alternative treatment. No recurrence of ILD was noted at 10 months. To our knowledge, this is the first report of successful alectinib treatment after the development of crizotinib-induced ILD without the use of prednisolone. PMID:27516885

  15. Profile of Ventana ALK (D5F3) companion diagnostic assay for non-small-cell lung carcinomas.

    PubMed

    Conde, Esther; Hernandez, Susana; Prieto, Mario; Martinez, Rebeca; Lopez-Rios, Fernando

    2016-06-01

    The development of several ALK inhibitors means that the importance of accurately identifying ALK-positive lung cancer has never been greater. Therefore, it is crucial that ALK testing assays become more standardized. The aim of this review is to comment on the recently FDA-approved VENTANA ALK (D5F3) Companion Diagnostic (CDx) Assay. This kit provides high sensitivity and specificity for the detection of ALK rearrangements and seamless integration into the laboratory workflow, with a fully automated analytical phase and fast interpretation. The use of controls increases the sensitivity and specificity and a dichotomous scoring approach enhances reproducibility.

  16. Novel CAD-ALK gene rearrangement is drugable by entrectinib in colorectal cancer

    PubMed Central

    Amatu, Alessio; Somaschini, Alessio; Cerea, Giulio; Bosotti, Roberta; Valtorta, Emanuele; Buonandi, Pasquale; Marrapese, Giovanna; Veronese, Silvio; Luo, David; Hornby, Zachary; Multani, Pratik; Murphy, Danielle; Shoemaker, Robert; Lauricella, Calogero; Giannetta, Laura; Maiolani, Martina; Vanzulli, Angelo; Ardini, Elena; Galvani, Arturo; Isacchi, Antonella; Sartore-Bianchi, Andrea; Siena, Salvatore

    2015-01-01

    Background: Activated anaplastic lymphoma kinase (ALK) gene fusions are recurrent events in a small fraction of colorectal cancers (CRCs), although these events have not yet been exploited as in other malignancies. Methods: We detected ALK protein expression by immunohistochemistry and gene rearrangements by fluorescence in situ hybridisation in the ALKA-372-001 phase I study of the pan-Trk, ROS1, and ALK inhibitor entrectinib. One out of 487 CRCs showed ALK positivity with a peculiar pattern that prompted further characterisation by targeted sequencing using anchored multiplex PCR. Results: A novel ALK fusion with the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene (CAD-ALK fusion gene) was identified. It resulted from inversion within chromosome 2 and the fusion of exons 1–35 of CAD with exons 20–29 of ALK. After failure of previous standard therapies, treatment of this patient with the ALK inhibitor entrectinib resulted in a durable objective tumour response. Conclusions: We describe the novel CAD-ALK rearrangement as an oncogene and provide the first evidence of its drugability as a new molecular target in CRC. PMID:26633560

  17. ALK-positive inflammatory myofibroblastic tumor of the abdomen with widespread microscopic multifocality.

    PubMed

    Lorenzi, Luisa; Cigognetti, Marta; Medicina, Daniela; Pellegrini, Vilma; Balzarini, Piera; Cestari, Renzo; Facchetti, Fabio

    2014-10-01

    Inflammatory myofibroblastic tumor (IMT) is a locally aggressive neoplasm, most frequently occurring in the abdominal cavity as multiple recurrent nodules. We report a case of IMT in a 24-year-old male presenting as multiple nodules involving the omentum, the liver, and the colon. Spindle tumor cells expressed ALK with a cytoplasmic granular distribution, the CLTC-ALK fusion gene was demonstrated by reverse-transcriptase polymerase chain reaction analysis, and break-apart fluorescence in situ hybridization (FISH) probes for the ALK gene showed a pathological pattern (single red signal associated with 1/2 normal fused signals) highly suggestive for combined gene fusion and deletion. To reduce the surgically unresectable liver mass, the patient was treated with crizotinib, and after 4 months of treatment the disease was defined stable according to RECIST criteria. Interestingly, ALK and FISH/FICTION analysis revealed that tumor cells were widely dispersed as multiple microscopic foci or as single cells beneath the omental mesothelium. These findings indicate that IMT multifocality might result either from dissemination from the main tumor mass or development of multiple independent neoplastic foci; furthermore, they underline the need of omentectomy in abdominal IMT to obtain surgical radicality.

  18. Oncogenic kinase NPM/ALK induces expression of HIF1α mRNA.

    PubMed

    Marzec, M; Liu, X; Wong, W; Yang, Y; Pasha, T; Kantekure, K; Zhang, P; Woetmann, A; Cheng, M; Odum, N; Wasik, M A

    2011-03-17

    The mechanisms of malignant cell transformation mediated by the oncogenic anaplastic lymphoma kinase (ALK) tyrosine kinase remain only partially understood. In this study, we report that T-cell lymphoma (TCL) cells carrying the nucleophosmin (NPM)/ALK fusion protein (ALK+ TCL) strongly express hypoxia-induced factor 1α (HIF1α) mRNA, even under normoxic conditions, and markedly upregulate HIF1α protein expression under hypoxia. HIF1α expression is strictly dependent on the expression and enzymatic activity of NPM/ALK, as shown in BaF3 cells transfected with wild-type NPM/ALK and kinase-inactive NPM/ALK K210R mutant and by the inhibition of the NPM/ALK function in ALK+ TCL cells by a small-molecule ALK inhibitor. NPM/ALK induces HIF1α expression by upregulating its gene transcription through its key signal transmitter signal transducer and activator of transcription 3 (STAT3), which binds to the HIF1α gene promoter as shown by the chromatin immunoprecipitation assay and is required for HIF1α gene expression as demonstrated by its small interfering RNA-mediated depletion. In turn, depletion of HIF1α increases mammalian target of rapamycin complex 1 activation, cell growth and proliferation and decreases vascular endothelial growth factor synthesis. These results identify a novel cell-transforming property of NPM/ALK, namely its ability to induce the expression of HIF1α, a protein with an important role in carcinogenesis. These results also provide another rationale to therapeutically target NPM/ALK and STAT3 in ALK+ TCL.

  19. Co-targeting ALK and EGFR parallel signaling in oral squamous cell carcinoma.

    PubMed

    Gonzales, Cara B; De La Chapa, Jorge J; Saikumar, Pothana; Singha, Prajjal K; Dybdal-Hargreaves, Nicholas F; Chavez, Jeffery; Horning, Aaron M; Parra, Jamie; Kirma, Nameer B

    2016-08-01

    Squamous cell carcinoma (SCC) comprises 90% of all head and neck cancers and has a poor survival rate due to late-stage disease that is refractive to traditional therapies. Epidermal growth factor receptor (EGFR) is over-expressed in greater than 80% of head and neck SCC (HNSCC). However, EGFR targeted therapies yielded little to no efficacy in clinical trials. This study investigated the efficacy of co-targeting EGFR and the anaplastic lymphoma kinase (ALK) whose promoter is hypomethylated in late-stage oral SCC (OSCC). We observed increased ALK activity in late-stage human OSCC tumors and invasive OSCC cell lines. We also found that while ALK inhibition alone had little effect on proliferation, co-targeting ALK and EGFR significantly reduced OSCC cell proliferation in vitro. Further analysis showed significant efficacy of combined treatment in HSC3-derived xenografts resulting in a 30% decrease in tumor volumes by 14days (p<0.001). Western blot analysis showed that co-targeting ALK and EGFR significantly reduced EGFR phosphorylation (Y1148) in HSC3 cells but not Cal27 cells. ALK and EGFR downstream signaling interactions are also demonstrated by Western blot analysis in which lone EGFR and ALK inhibitors attenuated AKT activity whereas co-targeting ALK and EGFR completely abolished AKT activation. No effects were observed on ERK1/2 activation. STAT3 activity was significantly induced by lone ALK inhibition in HSC3 cells and to a lower extent in Cal27 cells. Together, these data illustrate that ALK inhibitors enhance anti-tumor activity of EGFR inhibitors in susceptible tumors that display increased ALK expression, most likely through abolition of AKT activation. PMID:27424178

  20. The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase is enhanced by NPM-ALK: new insights in ALK-mediated pathogenesis and the treatment of ALCL

    PubMed Central

    Boccalatte, Francesco E.; Voena, Claudia; Riganti, Chiara; Bosia, Amalia; D'Amico, Lucia; Riera, Ludovica; Cheng, Mangeng; Ruggeri, Bruce; Jensen, Ole N.; Goss, Valerie L.; Lee, Kimberly; Nardone, Julie; Rush, John; Polakiewicz, Roberto D.; Comb, Michael J.; Chiarle, Roberto

    2009-01-01

    Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)–ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy. PMID:18845790

  1. Oncogenic tyrosine kinase NPM-ALK induces expression of the growth-promoting receptor ICOS.

    PubMed

    Zhang, Qian; Wang, Hongyi; Kantekure, Kanchan; Paterson, Jennifer C; Liu, Xiaobin; Schaffer, Andras; Paulos, Chrystal; Milone, Michael C; Odum, Niels; Turner, Suzanne; Marafioti, Teresa; Wasik, Mariusz A

    2011-09-15

    Here we report that T-cell lymphoma cells carrying the NPM-ALK fusion protein (ALK(+) TCL) frequently express the cell-stimulatory receptor ICOS. ICOS expression in ALK(+) TCL is moderate and strictly dependent on the expression and enzymatic activity of NPM-ALK. NPM-ALK induces ICOS expression via STAT3, which triggers the transcriptional activity of the ICOS gene promoter. In addition, STAT3 suppresses the expression of miR-219 that, in turn, selectively inhibits ICOS expression. ALK(+) TCL cell lines display extensive DNA methylation of the CpG island located within intron 1, the putative enhancer region, of the ICOS gene, whereas cutaneous T-cell lymphoma cell lines, which strongly express ICOS, show no methylation of the island. Treatment of the ALK(+) TCL cell lines with DNA methyltransferase inhibitor reversed the CpG island methylation and augmented the expression of ICOS mRNA and protein. Stimulation of the ICOS receptor with anti-ICOS antibody or ICOS ligand-expressing B cells markedly enhanced proliferation of the ALK(+) TCL cells. These results demonstrate that NPM-ALK, acting through STAT3 as the gene transcriptional activator, induces the expression of ICOS, a cell growth promoting receptor. These data also show that the DNA methylation status of the intronic CpG island affects transcriptional activity of the ICOS gene and, consequently, modulates the concentration of the expressed ICOS protein.

  2. Excess of NPM-ALK oncogenic signaling promotes cellular apoptosis and drug dependency.

    PubMed

    Ceccon, M; Merlo, M E Boggio; Mologni, L; Poggio, T; Varesio, L M; Menotti, M; Bombelli, S; Rigolio, R; Manazza, A D; Di Giacomo, F; Ambrogio, C; Giudici, G; Casati, C; Mastini, C; Compagno, M; Turner, S D; Gambacorti-Passerini, C; Chiarle, R; Voena, C

    2016-07-21

    Most of the anaplastic large-cell lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK (nucleophosmin-anaplastic lymphoma kinase). NPM-ALK-deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines, NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive because of heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or relocalization of NPM-ALK to the cytoplasm by NPM genetic knockout or knockdown caused ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) increased phosphorylation and cell death through the engagement of an ATM/Chk2- and γH2AX (phosphorylated H2A histone family member X)-mediated DNA-damage response. Remarkably, human NPM-ALK-amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A 'drug holiday' where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification.

  3. Alternative transcription initiation leads to expression of a novel ALK isoform in cancer

    PubMed Central

    Wiesner, Thomas; Lee, William; Obenauf, Anna C.; Ran, Leili; Murali, Rajmohan; Zhang, Qi Fan; Wong, Elissa W. P.; Hu, Wenhuo; Scott, Sasinya N.; Shah, Ronak H.; Landa, Iñigo; Button, Julia; Lailler, Nathalie; Sboner, Andrea; Gao, Dong; Murphy, Devan A.; Cao, Zhen; Shukla, Shipra; Hollmann, Travis J.; Wang, Lu; Borsu, Laetitia; Merghoub, Taha; Schwartz, Gary K.; Postow, Michael A.; Ariyan, Charlotte E.; Fagin, James A.; Zheng, Deyou; Ladanyi, Marc; Busam, Klaus J.; Berger, Michael F.; Chen, Yu; Chi, Ping

    2016-01-01

    Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ~ 11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALKATI. In ALKATI-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites1. ALKATI is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALKATI transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALKATI stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALKATI, suggesting that patients with ALKATI-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation. PMID:26444240

  4. NPM-ALK signals through glycogen synthase kinase 3β to promote oncogenesis.

    PubMed

    McDonnell, S R P; Hwang, S R; Basrur, V; Conlon, K P; Fermin, D; Wey, E; Murga-Zamalloa, C; Zeng, Z; Zu, Y; Elenitoba-Johnson, K S J; Lim, M S

    2012-08-01

    Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. In 70-80% of cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK fusion protein. NPM-ALK is a chimeric tyrosine kinase, which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3β as a signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the serine-9 phosphorylation of GSK3β. Expression of NPM-ALK in 293T cells led to an increase of pS(9)-GSK3β (glycogen synthase kinase 3 beta) compared with kinase-defective K210R mutant NPM-ALK, but did not affect total GSK3β levels. Phosphorylation of pS(9)-GSK3β by NPM-ALK was mediated by the PI3K/AKT signaling pathway. ALK inhibition resulted in degradation of GSK3β substrates Mcl-1 and CDC25A, which was recovered upon chemical inhibition of the proteasome (MG132). Furthermore, the degradation of Mcl-1 was recoverable with inhibition of GSK3β. ALK inhibition also resulted in decreased cell viability, which was rescued by GSK3β inhibition. Furthermore, stable knockdown of GSK3β conferred resistance to the growth inhibitory effects of ALK inhibition using viability and colony formation assays. pS(9)-GSK3β and CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies, and showed a significant correlation (P<0.001). Conversely, ALK-ALCL tissue biopsies did not show significant correlation of pS(9)-GSK3β and CDC25A expression (P<0.2). Our results demonstrate that NPM-ALK regulates the phosphorylation of S(9)-GSK3β by PI3K/AKT. The subsequent inhibition of

  5. Inhibition of Rac controls NPM-ALK-dependent lymphoma development and dissemination.

    PubMed

    Colomba, A; Giuriato, S; Dejean, E; Thornber, K; Delsol, G; Tronchère, H; Meggetto, F; Payrastre, B; Gaits-Iacovoni, F

    2011-06-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a tyrosine kinase oncogene responsible for the pathogenesis of the majority of human ALK-positive lymphomas. We recently reported that it activated the Rac1 GTPase in anaplastic large-cell lymphoma (ALCL), leading to Rac-dependent formation of active invadopodia required for invasiveness. Herein, we went further into the study of this pathway and used the inhibitor of Rac, NSC23766, to validate its potential as a molecular target in ALCL in vitro and in vivo in a xenograft model and in a conditional model of NPM-ALK transgenic mice. Our data demonstrate that Rac regulates important effectors of NPM-ALK-induced transformation such as Erk1/2, p38 and Akt. Moreover, inhibition of Rac signaling abrogates NPM-ALK-elicited disease progression and metastasis in mice, highlighting the potential of small GTPases and their regulators as additional therapic targets in lymphomas.

  6. Precision medicine in NSCLC and pathology: how does ALK fit in the pathway?

    PubMed

    Kerr, K M; López-Ríos, F

    2016-09-01

    The evolution of personalised medicine in lung cancer has dramatically impacted diagnostic pathology. Current challenges centre on the growing demands placed on small tissue samples by molecular diagnostic techniques. In this review, expert recommendations are provided regarding successful identification of anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC). Steps to correctly process and conserve tumour tissue during diagnostic testing are essential to ensure tissue availability. For example, storing extra tissue sections ready for molecular diagnostic steps allows faster testing and preserves tissue. Fluorescence in situ hybridisation (FISH) is commonly used to detect ALK rearrangements, with most laboratories favouring screening by immunohistochemistry followed by a confirmatory FISH assay. Reverse transcription-polymerase chain reaction can also identify ALK fusion gene mRNA transcripts but can be limited by the quality of RNA and the risk that rare fusion variants may not be captured. Next-generation sequencing (NGS) technology has recently provided an alternative method for detecting ALK rearrangements. While current experience is limited, NGS is set to become the most efficient approach as an increasing number of genetic abnormalities is required to be tested. Upfront, reflex testing for ALK gene rearrangement should become routine as ALK tyrosine kinase inhibitor therapy moves into the first-line setting. Guidelines recommend that EGFR and ALK tests are carried out in parallel on all confirmed and potential adenocarcinomas, and this is more efficient in terms of tissue usage and testing turnaround time for both of these actionable gene alterations. The practice of sequential testing is not recommended. Identification of ALK rearrangements is now essential for the diagnosis of NSCLC, underpinned by the benefits of ALK inhibitors. As scientific understanding and diagnostic technology develops, ALK testing will continue to be an

  7. A novel Patient Derived Tumorgraft model with TRAF1-ALK Anaplastic Large Cell Lymphoma translocation

    PubMed Central

    Abate, Francesco; Todaro, Maria; van der Krogt, Jo-Anne; Boi, Michela; Landra, Indira; Machiorlatti, Rodolfo; Tabbo’, Fabrizio; Messana, Katia; Barreca, Antonella; Novero, Domenico; Gaudiano, Marcello; Aliberti, Sabrina; Di Giacomo, Filomena; Tousseyn, Thomas; Lasorsa, Elena; Crescenzo, Ramona; Bessone, Luca; Ficarra, Elisa; Acquaviva, Andrea; Rinaldi, Andrea; Ponzoni, Maurilio; Longo, Dario Livio; Aime, Silvio; Cheng, Mangeng; Ruggeri, Bruce; Piccaluga, Pier Paolo; Pileri, Stefano; Tiacci, Enrico; Falini, Brunangelo; Pera-Gresely, Benet; Cerchietti, Leandro; Iqbal, Javeed; Chan, Wing C; Shultz, Leonard D.; Kwee, Ivo; Piva, Roberto; Wlodarska, Iwona; Rabadan, Raul; Bertoni, Francesco; Inghirami, Giorgio

    2016-01-01

    Although Anaplastic Large Cell Lymphomas (ALCL) carrying Anaplastic Lymphoma Kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human Patient Derived Tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and of NFkB pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells lacking PRDM1/Blimp-1 and with c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to down-regulation of p50/p52 and lymphoma growth inhibition. Moreover a NFkB gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Moreover, a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, but the disease could not be eradicated. These data indicate that the activation of NFkB signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable to validate the role of druggable molecules, predict therapeutic responses and are helpful tools for the implementation of patient specific therapies. PMID:25533804

  8. Detection of EML4-ALK fusion gene in Chinese non-small cell lung cancer by using a sensitive quantitative real-time reverse transcriptase PCR technique.

    PubMed

    Fu, Sha; Wang, Fang; Shao, Qiong; Zhang, Xu; Duan, Li-Ping; Zhang, Xiao; Zhang, Li; Shao, Jian-Yong

    2015-04-01

    Anaplastic lymphoma kinase (ALK) rearrangement is present in approximately 5% of lung adenocarcinoma. Clinical trials on ALK inhibitor phase I to III have shown an interesting disease control rate and acceptable tolerability in ALK rearrangement patients. In clinical application, the precise diagnostic strategy for identifying ALK rearrangements remains to be determined. In this study, ALK rearrangement was screened by using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), direct sequencing, 2 fluorescence in situ hybridization (FISH) assays, and immunohistochemistry in 173 lung adenocarcinomas. We identified 18 cases (10.4%) with EML4-ALK fusion-positive by qRT-PCR, and all were positive for EML4-ALK fusion gene validated by direct sequencing. The result was consistent with that of other methods. Furthermore, of the 18 EML4-ALK fusion-positive cases, 16 (9.2%) were positive by using EML4-ALK fusion probe FISH, and 15 (8.7%) were positive by using ALK break-apart probe FISH and immunohistochemistry staining. Of the 18 ALK fusion-positive lung adenocarcinomas, 8 cases (44.4%) were histologically diagnosed as subtypes of cribriform adenocarcinoma, 7 cases (38.9%) as cribriform adenocarcinoma mixed with papillary and/or mucinous pattern, 2 cases (11.1%) as papillary adenocarcinoma, and 1 case (5.6%) as mucinous adenocarcinoma. In the present study, the ALK rearrangement frequency detected by qRT-PCR in Chinese NSCLC patients was higher than that in the western populations. QRT-PCR is a rapid, sensitive technology that could be used as a screening tool for identifying EML4-ALK fusion-positive NSCLC patients who would be sensitive for receiving ALK inhibitor therapy.

  9. Oncogenic kinase NPM/ALK induces through STAT3 expression of immunosuppressive protein CD274 (PD-L1, B7-H1).

    PubMed

    Marzec, Michal; Zhang, Qian; Goradia, Ami; Raghunath, Puthiyaveettil N; Liu, Xiaobin; Paessler, Michele; Wang, Hong Yi; Wysocka, Maria; Cheng, Mangeng; Ruggeri, Bruce A; Wasik, Mariusz A

    2008-12-30

    The mechanisms of malignant cell transformation caused by the oncogenic, chimeric nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) remain only partially understood, with most of the previous studies focusing mainly on the impact of NPM/ALK on cell survival and proliferation. Here we report that the NPM/ALK-carrying T cell lymphoma (ALK+TCL) cells strongly express the immunosuppressive cell-surface protein CD274 (PD-L1, B7-H1), as determined on the mRNA and protein level. The CD274 expression is strictly dependent on the expression and enzymatic activity of NPM/ALK, as demonstrated by inhibition of the NPM/ALK function in ALK+TCL cells by the small molecule ALK inhibitor CEP-14083 and by documenting CD274 expression in IL-3-depleted BaF3 cells transfected with the wild-type NPM/ALK, but not the kinase-inactive NPM/ALK K210R mutant or empty vector alone. NPM/ALK induces CD274 expression by activating its key signal transmitter, transcription factor STAT3. STAT3 binds to the CD274 gene promoter in vitro and in vivo, as shown in the gel electromobility shift and chromatin immunoprecipitation assays, and is required for the PD-L1 gene expression, as demonstrated by siRNA-mediated STAT3 depletion. These findings identify an additional cell-transforming property of NPM/ALK and describe a direct link between an oncoprotein and an immunosuppressive cell-surface protein. These results also provide an additional rationale to therapeutically target NPM/ALK and STAT3 in ALK+TCL. Finally, they suggest that future immunotherapeutic protocols for this type of lymphoma may need to include the inhibition of NPM/ALK and STAT3 to achieve optimal clinical efficacy.

  10. Oncogenic tyrosine kinase NPM/ALK induces activation of the rapamycin-sensitive mTOR signaling pathway.

    PubMed

    Marzec, M; Kasprzycka, M; Liu, X; El-Salem, M; Halasa, K; Raghunath, P N; Bucki, R; Wlodarski, P; Wasik, M A

    2007-08-16

    The mechanisms of cell transformation mediated by the nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) tyrosine kinase are only partially understood. Here, we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma display persistent activation of mammalian target of rapamycin (mTOR) as determined by phosphorylation of mTOR targets S6rp and 4E-binding protein 1 (4E-BP1). The mTOR activation is serum growth factor-independent but nutrient-dependent. It is also dependent on the expression and enzymatic activity of NPM/ALK as demonstrated by cell transfection with wild-type and functionally deficient NPM/ALK, small interfering RNA (siRNA)-mediated NPM/ALK depletion and kinase activity suppression using the inhibitor WHI-P154. The NPM/ALK-induced mTOR activation is transduced through the mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway and, to a much lesser degree, through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Accordingly, whereas the low-dose PI3K inhibitor wortmannin and Akt inhibitor III profoundly inhibited Akt phosphorylation, they had a very modest effect on S6rp and 4E-BP1 phosphorylation. In turn, MEK inhibitors U0126 and PD98059 and siRNA-mediated depletion of either ERK1 or ERK2 inhibited S6rp phosphorylation much more effectively. Finally, the mTOR inhibitor rapamycin markedly decreased proliferation and increased the apoptotic rate of ALK+TCL cells. These findings identify mTOR as a novel key target of NPM/ALK and suggest that mTOR inhibitors may prove effective in therapy of ALK-induced malignancies.

  11. TKI sensitivity patterns of novel kinase-domain mutations suggest therapeutic opportunities for patients with resistant ALK+ tumors

    PubMed Central

    Rajan, Soumya S.; Gokhale, Vijay; Groysman, Matthew J.; Pongtornpipat, Praechompoo; Tapia, Edgar O.; Wang, Mengdie; Schatz, Jonathan H.

    2016-01-01

    The anaplastic lymphoma kinase (ALK) protein drives tumorigenesis in subsets of several tumors through chromosomal rearrangements that express and activate its C-terminal kinase domain. In addition, germline predisposition alleles and acquired mutations are found in the full-length protein in the pediatric tumor neuroblastoma. ALK-specific tyrosine kinase inhibitors (TKIs) have become important new drugs for ALK-driven lung cancer, but acquired resistance via multiple mechanisms including kinase-domain mutations eventually develops, limiting median progression-free survival to less than a year. Here we assess the impact of several kinase-domain mutations that arose during TKI resistance selections of ALK+ anaplastic large-cell lymphoma (ALCL) cell lines. These include novel variants with respect to ALK-fusion cancers, R1192P and T1151M, and with respect to ALCL, F1174L and I1171S. We assess the effects of these mutations on the activity of six clinical inhibitors in independent systems engineered to depend on either the ALCL fusion kinase NPM-ALK or the lung-cancer fusion kinase EML4-ALK. Our results inform treatment strategies with a likelihood of bypassing mutations when detected in resistant patient samples and highlight differences between the effects of particular mutations on the two ALK fusions. PMID:27009859

  12. PDGFR blockade is a rational and effective therapy for NPM-ALK-driven lymphomas.

    PubMed

    Laimer, Daniela; Dolznig, Helmut; Kollmann, Karoline; Vesely, Paul W; Schlederer, Michaela; Merkel, Olaf; Schiefer, Ana-Iris; Hassler, Melanie R; Heider, Susi; Amenitsch, Lena; Thallinger, Christiane; Staber, Philipp B; Simonitsch-Klupp, Ingrid; Artaker, Matthias; Lagger, Sabine; Turner, Suzanne D; Pileri, Stefano; Piccaluga, Pier Paolo; Valent, Peter; Messana, Katia; Landra, Indira; Weichhart, Thomas; Knapp, Sylvia; Shehata, Medhat; Todaro, Maria; Sexl, Veronika; Höfler, Gerald; Piva, Roberto; Medico, Enzo; Ruggeri, Bruce A; Cheng, Mangeng; Eferl, Robert; Egger, Gerda; Penninger, Josef M; Jaeger, Ulrich; Moriggl, Richard; Inghirami, Giorgio; Kenner, Lukas

    2012-11-01

    Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin's lymphoma found in children and young adults. ALCLs frequently carry a chromosomal translocation that results in expression of the oncoprotein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). The key molecular downstream events required for NPM-ALK-triggered lymphoma growth have been only partly unveiled. Here we show that the activator protein 1 family members JUN and JUNB promote lymphoma development and tumor dissemination through transcriptional regulation of platelet-derived growth factor receptor-β (PDGFRB) in a mouse model of NPM-ALK-triggered lymphomagenesis. Therapeutic inhibition of PDGFRB markedly prolonged survival of NPM-ALK transgenic mice and increased the efficacy of an ALK-specific inhibitor in transplanted NPM-ALK tumors. Notably, inhibition of PDGFRA and PDGFRB in a patient with refractory late-stage NPM-ALK(+) ALCL resulted in rapid, complete and sustained remission. Together, our data identify PDGFRB as a previously unknown JUN and JUNB target that could be a highly effective therapy for ALCL.

  13. Rapamycin reverses NPM-ALK-induced glucocorticoid resistance in lymphoid tumor cells by inhibiting mTOR signaling pathway, enhancing G1 cell cycle arrest and apoptosis.

    PubMed

    Gu, L; Gao, J; Li, Q; Zhu, Y P; Jia, C S; Fu, R Y; Chen, Y; Liao, Q K; Ma, Z

    2008-11-01

    The anaplastic lymphoma kinase (ALK) is an oncogene product involved in hematopoietic and non-hematopoietic malignancies. Recent studies have demonstrated that nucleophosmin (NPM)-ALK, originated from the fusion of NPM and ALK genes, causes cell transformation through diverse mechanisms. Here, we show a novel mechanism by which NPM-ALK transforms lymphoid tumor cells to become resistant to glucocorticoid (GC) or dexamethasone (Dex) treatment. Transformed BaF3 cells by NPM-ALK were much more resistant to Dex compared with their parental cells, and concurrently had a constitutive activation of mammalian target of rapamycin (mTOR) signaling, as evidenced by hyperphosphorylation of its downstream effectors, p70 S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The mTOR inhibitor rapamycin suppressed activation of p70S6K in BaF3/NPM-ALK cells and reversed GC resistance by synergistically inhibiting mTOR signaling pathway, enhancing cell cycle arrest at G(1) phase and promoting apoptotic cell death. In conclusion, our data indicate that the ALK fusion kinase, NPM-ALK, induces GC resistance by activating mTOR signaling, and addition of mTOR inhibitors to the chemotherapeutic regimen of ALK+ lymphomas may improve the prognosis.

  14. Establishment of a novel model of chondrogenesis using murine embryonic stem cells carrying fibrodysplasia ossificans progressiva-associated mutant ALK2.

    PubMed

    Fujimoto, Mai; Ohte, Satoshi; Shin, Masashi; Yoneyama, Katsumi; Osawa, Kenji; Miyamoto, Arei; Tsukamoto, Sho; Mizuta, Takato; Kokabu, Shoichiro; Machiya, Aiko; Okuda, Akihiko; Suda, Naoto; Katagiri, Takenobu

    2014-12-12

    Fibrodysplasia ossificans progressiva (FOP) is a genetic disorder characterized by heterotopic endochondral ossification in soft tissue. A mutation in the bone morphogenetic protein (BMP) receptor ALK2, R206H, has been identified in patients with typical FOP. In the present study, we established murine embryonic stem (ES) cells that express wild-type human ALK2 or typical mutant human ALK2 [ALK2(R206H)] under the control of the Tet-Off system. Although wild-type ALK2 and mutant ALK2(R206H) were expressed in response to a withdrawal of doxycycline (Dox), BMP signaling was activated only in the mutant ALK2(R206H)-expressing cells without the addition of exogenous BMPs. The Dox-dependent induction of BMP signaling was blocked by a specific kinase inhibitor of the BMP receptor. The mutant ALK2(R206H)-carrying cells showed Dox-regulated chondrogenesis in vitro, which occurred in co-operation with transforming growth factor-β1 (TGF-β1). Overall, our ES cells are useful for studying the molecular mechanisms of heterotopic ossification in FOP in vitro and for developing novel inhibitors of chondrogenesis induced by mutant ALK2(R206H) associated with FOP.

  15. Malignant transformation of CD4+ T lymphocytes mediated by oncogenic kinase NPM/ALK recapitulates IL-2-induced cell signaling and gene expression reprogramming.

    PubMed

    Marzec, Michal; Halasa, Krzysztof; Liu, Xiaobin; Wang, Hong Y; Cheng, Mangeng; Baldwin, Donald; Tobias, John W; Schuster, Stephen J; Woetmann, Anders; Zhang, Qian; Turner, Suzanne D; Ødum, Niels; Wasik, Mariusz A

    2013-12-15

    Anaplastic lymphoma kinase (ALK), physiologically expressed only by nervous system cells, displays a remarkable capacity to transform CD4(+) T lymphocytes and other types of nonneural cells. In this study, we report that activity of nucleophosmin (NPM)/ALK chimeric protein, the dominant form of ALK expressed in T cell lymphomas (TCLs), closely resembles cell activation induced by IL-2, the key cytokine supporting growth and survival of normal CD4(+) T lymphocytes. Direct comparison of gene expression by ALK(+) TCL cells treated with an ALK inhibitor and IL-2-dependent ALK(-) TCL cells stimulated with the cytokine revealed a very similar, albeit inverse, gene-regulation pattern. Depending on the analysis method, up to 67% of the affected genes were modulated in common by NPM/ALK and IL-2. Based on the gene expression patterns, Jak/STAT- and IL-2-signaling pathways topped the list of pathways identified as affected by both IL-2 and NPM/ALK. The expression dependence on NPM/ALK and IL-2 of the five selected genes-CD25 (IL-2Rα), Egr-1, Fosl-1, SOCS3, and Irf-4-was confirmed at the protein level. In both ALK(+) TCL and IL-2-stimulated ALK(-) TCL cells, CD25, SOCS3, and Irf-4 genes were activated predominantly by the STAT5 and STAT3 transcription factors, whereas transcription of Egr-1 and Fosl-1 was induced by the MEK-ERK pathway. Finally, we found that Egr-1, a protein not associated previously with either IL-2 or ALK, contributes to the cell proliferation. These findings indicate that NPM/ALK transforms the target CD4(+) T lymphocytes, at least in part, by using the pre-existing, IL-2-dependent signaling pathways.

  16. Targeting tumour vasculature by inhibiting activin receptor-like kinase (ALK)1 function.

    PubMed

    de Vinuesa, Amaya García; Bocci, Matteo; Pietras, Kristian; Ten Dijke, Peter

    2016-08-15

    Angiogenesis is a hallmark of cancer and is now a validated therapeutic target in the clinical setting. Despite the initial success, anti-angiogenic compounds impinging on the vascular endothelial growth factor (VEGF) pathway display limited survival benefits in patients and resistance often develops due to activation of alternative pathways. Thus, finding and validating new targets is highly warranted. Activin receptor-like kinase (ALK)1 is a transforming growth factor beta (TGF-β) type I receptor predominantly expressed in actively proliferating endothelial cells (ECs). ALK1 has been shown to play a pivotal role in regulating angiogenesis by binding to bone morphogenetic protein (BMP)9 and 10. Two main pharmacological inhibitors, an ALK1-Fc fusion protein (Dalantercept/ACE-041) and a fully human antibody against the extracellular domain of ALK1 (PF-03446962) are currently under clinical development. Herein, we briefly recapitulate the role of ALK1 in blood vessel formation and the current status of the preclinical and clinical studies on inhibition of ALK1 signalling as an anti-angiogenic strategy. Future directions in terms of new combination regimens will also be presented. PMID:27528762

  17. Endoglin and Alk5 regulate epithelial-mesenchymal transformation during cardiac valve formation

    PubMed Central

    Mercado-Pimentel, Melania E.; Hubbard, Antony D.; Runyan, Raymond B.

    2007-01-01

    Endoglin is an accessory receptor for TGFß and can associate with Alk5 or Alk2. Although prior studies indicated that endoglin and Alk5 were not directly involved in epithelial-mesenchymal transformation (EMT) in the heart, the expression pattern of endoglin prompted a re-examination. We here show that loss of endoglin expression mediated by either antisense DNA or siRNA results in a direct perturbation of EMT and reduced expression of EMT markers including slug, runx2, RhoA, and latrophilin-2. An examination of BrdU incorporation shows that, while endoglin regulates proliferation at an early stage, reduced endothelial cell proliferation does not account for the loss of mesenchyme. As Alk5 interacts with endoglin, we utilized siRNA and a specific inhibitor, HTS466284 (HTS), to perturb this receptor as well. Alk5 inhibition produced similar effects to inhibition of endoglin. There was a reduction in mesenchymal cell formation and loss of EMT marker expression similar to that seen with endoglin. Alk5 kinase inhibition produced a similar loss of EMT marker expression but showed a contrasting upregulation of the proliferation and remodeling markers, Cyclin B2 and ß-catenin. Alk5 and endoglin both mediate endothelial cell proliferation in younger explants but, by stage 16, loss of endoglin no longer alters proliferation rates. These data show that both Alk5 and endoglin are directly involved in the process of EMT, that they interact with both TGFß-regulated activation and invasion pathways and that the roles of these receptors change during cardiac development. PMID:17250821

  18. Crizotinib Improves Progression-Free Survival in Some Patients with Advanced Lung Cancer

    MedlinePlus

    ... Prevention Lung Cancer Screening Research Crizotinib Improves Progression-Free Survival in Some Patients with Advanced Lung Cancer ( ... starting treatment without their disease getting worse (progression-free survival), as assessed by radiologic review. Results Progression- ...

  19. Oncogenic tyrosine kinase NPM/ALK induces activation of the MEK/ERK signaling pathway independently of c-Raf.

    PubMed

    Marzec, M; Kasprzycka, M; Liu, X; Raghunath, P N; Wlodarski, P; Wasik, M A

    2007-02-01

    The mechanisms of cell transformation mediated by the highly oncogenic, chimeric NPM/ALK tyrosine kinase remain only partially understood. Here we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma (ALK+ TCL) display phosphorylation of the extracellular signal-regulated protein kinase (ERK) 1/2 complex. Transfection of BaF3 cells with NPM/ALK induces phosphorylation of EKR1/2 and of its direct activator mitogen-induced extracellular kinase (MEK) 1/2. Depletion of NPM/ALK by small interfering RNA (siRNA) or its inhibition by WHI-154 abrogates the MEK1/2 and ERK1/2 phosphorylation. The NPM/ALK-induced MEK/ERK activation is independent of c-Raf as evidenced by the lack of MEK1/2 and ERK1/2 phosphorylation upon c-Raf inactivation by two different inhibitors, RI and ZM336372, and by its siRNA-mediated depletion. In contrast, ERK1/2 activation is strictly MEK1/2 dependent as shown by suppression of the ERK1/2 phosphorylation by the MEK1/2 inhibitor U0126. The U0126-mediated inhibition of ERK1/2 activation impaired proliferation and viability of the ALK+ TCL cells and expression of antiapoptotic factor Bcl-xL and cell cycle-promoting CDK4 and phospho-RB. Finally, siRNA-mediated depletion of both ERK1 and ERK2 inhibited cell proliferation, whereas depletion of ERK 1 (but not ERK2) markedly increased cell apoptosis. These findings identify MEK/ERK as a new signaling pathway activated by NPM/ALK and indicate that the pathway represents a novel therapeutic target in the ALK-induced malignancies.

  20. Clinical Utility of Circulating Tumor Cells in ALK-Positive Non-Small-Cell Lung Cancer

    PubMed Central

    Faugeroux, Vincent; Pailler, Emma; Auger, Nathalie; Taylor, Melissa; Farace, Françoise

    2014-01-01

    The advent of rationally targeted therapies such as small-molecule tyrosine kinase inhibitors (TKIs) has considerably transformed the therapeutic management of a subset of patients with non-small-cell lung cancer (NSCLC) harboring defined molecular abnormalities. When such genetic molecular alterations are detected the use of specific TKI has demonstrated better results (overall response rate, progression free survival) compared to systemic therapy. However, the detection of such molecular abnormalities is complicated by the difficulty in obtaining sufficient tumor material, in terms of quantity and quality, from a biopsy. Here, we described how circulating tumor cells (CTCs) can have a clinical utility in anaplastic lymphoma kinase (ALK) positive NSCLC patients to diagnose ALK-EML4 gene rearrangement and to guide therapeutic management of these patients. The ability to detect genetic abnormalities such ALK rearrangement in CTCs shows that these cells could offer new perspectives both for the diagnosis and the monitoring of ALK-positive patients eligible for treatment with ALK inhibitors. PMID:25414829

  1. Clinical Utility of Circulating Tumor Cells in ALK-Positive Non-Small-Cell Lung Cancer.

    PubMed

    Faugeroux, Vincent; Pailler, Emma; Auger, Nathalie; Taylor, Melissa; Farace, Françoise

    2014-01-01

    The advent of rationally targeted therapies such as small-molecule tyrosine kinase inhibitors (TKIs) has considerably transformed the therapeutic management of a subset of patients with non-small-cell lung cancer (NSCLC) harboring defined molecular abnormalities. When such genetic molecular alterations are detected the use of specific TKI has demonstrated better results (overall response rate, progression free survival) compared to systemic therapy. However, the detection of such molecular abnormalities is complicated by the difficulty in obtaining sufficient tumor material, in terms of quantity and quality, from a biopsy. Here, we described how circulating tumor cells (CTCs) can have a clinical utility in anaplastic lymphoma kinase (ALK) positive NSCLC patients to diagnose ALK-EML4 gene rearrangement and to guide therapeutic management of these patients. The ability to detect genetic abnormalities such ALK rearrangement in CTCs shows that these cells could offer new perspectives both for the diagnosis and the monitoring of ALK-positive patients eligible for treatment with ALK inhibitors. PMID:25414829

  2. Lumican binds ALK5 to promote epithelium wound healing.

    PubMed

    Yamanaka, Osamu; Yuan, Yong; Coulson-Thomas, Vivien Jane; Gesteira, Tarsis Ferreira; Call, Mindy K; Zhang, Yujin; Zhang, Jianhua; Chang, Shao-Hsuan; Xie, Changchun; Liu, Chia-Yang; Saika, Shizuya; Jester, James V; Kao, Winston W-Y

    2013-01-01

    Lumican (Lum), a small leucine-rich proteoglycan (SLRP) family member, has multiple matricellular functions both as an extracellular matrix component and as a matrikine regulating cell proliferation, gene expression and wound healing. To date, no cell surface receptor has been identified to mediate the matrikine functions of Lum. This study aimed to identify a perspective receptor that mediates Lum effects on promoting wound healing. Transforming growth factor-β receptor 1 (ALK5) was identified as a potential Lum-interacting protein through in silico molecular docking and molecular dynamics. This finding was verified by biochemical pull-down assays. Moreover, the Lum function on wound healing was abrogated by an ALK5-specific chemical inhibitor as well as by ALK5 shRNAi. Finally, we demonstrated that eukaryote-specific post-translational modifications are not required for the wound healing activity of Lum, as recombinant GST-Lum fusion proteins purified from E. coli and a chemically synthesized LumC13 peptide (the last C-terminal 13 amino acids of Lum) have similar effects on wound healing in vitro and in vivo.

  3. Reactive oxygen species and lipoxygenases regulate the oncogenicity of NPM-ALK-positive anaplastic large cell lymphomas.

    PubMed

    Thornber, K; Colomba, A; Ceccato, L; Delsol, G; Payrastre, B; Gaits-Iacovoni, F

    2009-07-23

    The chimera nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), the tyrosine kinase activity of which is constitutively upregulated, is the causative agent of 75% of the anaplastic large-cell lymphomas (ALCLs). We have demonstrated that NPM-ALK induces the production of reactive oxygen species (ROS) by a pathway involving the arachidonic acid-metabolizing enzymes of the lipoxygenase (LOX) family. The use of the LOX inhibitor nordihydroguaiaretic acid (NDGA) and of the anti-oxidant N-acetylcysteine (NAC) demonstrated that ROS are important in maintaining the ALK kinase active. Consistent with this, NDGA treatment resulted in the inhibition of key pathways, such as Akt, signal transducer and activator of transcription factor 3 (STAT3) and extracellular signal-regulated kinase (ERK), which are involved in NPM-ALK antiapoptotic and pro-mitogenic functions. Conversely, the stress-activated kinase p38, described in some instances as a mediator of apoptosis, was activated. Interestingly, 5-LOX, an isoform involved in many cancers, was found to be activated in NPM-ALK(+) cells. Functional studies have shown that transforming properties, namely proliferation and resistance to apoptosis, were abrogated by treatment with either NDGA or the 5-LOX inhibitor (N-(3-phenoxycinnamyl)-acetohydroxamic acid) (BW A4C). Together, these data point to the ROS/LOX pathway as a potential new target for therapy in NPM-ALK-positive tumors.

  4. Alk5 inhibition increases delivery of macromolecular and protein-bound contrast agents to tumors

    PubMed Central

    Daldrup-Link, Heike E.; Mohanty, Suchismita; Ansari, Celina; Ito, Ken; Hong, Su Hyun; Hoffmann, Matthias; Pisani, Laura; Boudreau, Nancy; Gambhir, Sanjiv Sam; Coussens, Lisa M.

    2016-01-01

    Limited transendothelial permeability across tumor microvessels represents a significant bottleneck in the development of tumor-specific diagnostic agents and theranostic drugs. Here, we show an approach to increase transendothelial permeability of macromolecular and nanoparticle-based contrast agents via inhibition of the type I TGF-β receptor, activin-like kinase 5 (Alk5), in tumors. Alk5 inhibition significantly increased tumor contrast agent delivery and enhancement on imaging studies, while healthy organs remained relatively unaffected. Imaging data correlated with significantly decreased tumor interstitial fluid pressure, while tumor vascular density remained unchanged. This immediately clinically translatable concept involving Alk5 inhibitor pretreatment prior to an imaging study could be leveraged for improved tumor delivery of macromolecular and nanoparticle-based imaging probes and, thereby, facilitate development of more sensitive imaging tests for cancer diagnosis, enhanced tumor characterization, and personalized, image-guided therapies. PMID:27182558

  5. Serine phosphorylation of NPM-ALK, which is dependent on the auto-activation of the kinase activation loop, contributes to its oncogenic potential.

    PubMed

    Wang, Peng; Wu, Fang; Zhang, Jingdong; McMullen, Todd; Young, Leah C; Ingham, Robert J; Li, Liang; Lai, Raymond

    2011-02-01

    It is well established that the tumorigenic potential of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), an oncogenic tyrosine kinase, is dependent on its tyrosine phosphorylation. Using tandem affinity purification-mass spectrometry, we found evidence of phosphorylation of three serine residues of NPM-ALK (Serine¹³⁵, Serine¹⁶⁴ and Serine⁴⁹⁷) ectopically expressed in GP293 cells. Using a specific anti-phosphoserine antibody and immunoprecipitation, we confirmed the presence of serine phosphorylation of NPM-ALK in all three NPM-ALK-expressing cell lines examined. Similar to the tyrosine phosphorylation, phosphorylation of these serine residues was dependent on the activation status of the kinase activation loop of ALK. All of these three serine residues are biologically important as mutation of any one of these residues resulted in a significant reduction in the tumorigenicity of NPM-ALK (assessed by cell viability and clonogenic assay), which correlated with a substantial reduction in the phosphorylation of extracellular signal-regulated kinase 1/2, c-jun N-terminal kinase and signal transducer and activator of transcription 6. Serine phosphorylation of NPM-ALK appears to be regulated by multiple serine kinases since it was markedly reduced by pharmacologic inhibitors for glycogen synthase kinase-3, casein kinase I or mitogen-activated protein kinases. In summary, our study is the first to identify serine phosphorylation of NPM-ALK and to provide evidence that it enhances the tumorigenic potential of this oncogenic protein.

  6. High MET amplification level as a resistance mechanism to osimertinib (AZD9291) in a patient that symptomatically responded to crizotinib treatment post-osimertinib progression.

    PubMed

    Ou, Sai-Hong Ignatius; Agarwal, Nikita; Ali, Siraj M

    2016-08-01

    Third-generation EGFR TKI has been approved in the US and EU for the treatment of EGFR mutant T790M+ NSCLC patients that are resistant to first- or second generation EGFR TKIs. Here we report a patient who developed resistance to osimertinib after a confirmed partial response for 9 months. Pre-osimertinib and post-osimertinib tumor biopsy revealed the emergence of high level of MET amplification (30 copies) post osimertinib treatment. Patient was treated with single agent crizotinib, a known MET inhibitor, with transient symptomatic benefit. MET amplification is one potential resistance mechanism to osimertinib and combination of osimertinib and a MET inhibitor should be investigated post-osimertinib progression in EGFR mutant T790M+ NSCLC patients whose harbored acquired MET amplification. PMID:27393507

  7. Overcoming acquired resistance to kinase inhibition: the cases of EGFR, ALK and BRAF.

    PubMed

    Giroux, Simon

    2013-01-15

    In the past decade, several kinase inhibitors have been approved based on their clinical benefit for cancer patients. Unfortunately, in many cases, patients develop resistance to these agents via secondary mutations and alternative mechanisms. This review will focus on the cases of acquired resistance to EGFR and ALK inhibitors for non-small cell lung cancer patients and BRAF inhibitors for melanoma patients. I will overview the main causes of acquired resistance, and explore the chemical scaffolds as well as combination of drugs, used to tackle these major causes of resistance. PMID:23245516

  8. SUMOylation Confers Posttranslational Stability on NPM-ALK Oncogenic Protein.

    PubMed

    Vishwamitra, Deeksha; Curry, Choladda V; Shi, Ping; Alkan, Serhan; Amin, Hesham M

    2015-09-01

    Nucleophosmin-anaplastic lymphoma kinase-expressing (NPM-ALK+) T-cell lymphoma is an aggressive form of cancer that commonly affects children and adolescents. The expression of NPM-ALK chimeric oncogene results from the chromosomal translocation t(2;5)(p23;q35) that causes the fusion of the ALK and NPM genes. This translocation generates the NPM-ALK protein tyrosine kinase that forms the constitutively activated NPM-ALK/NPM-ALK homodimers. In addition, NPM-ALK is structurally associated with wild-type NPM to form NPM/NPM-ALK heterodimers, which can translocate to the nucleus. The mechanisms that sustain the stability of NPM-ALK are not fully understood. SUMOylation is a posttranslational modification that is characterized by the reversible conjugation of small ubiquitin-like modifiers (SUMOs) with target proteins. SUMO competes with ubiquitin for substrate binding and therefore, SUMOylation is believed to protect target proteins from proteasomal degradation. Moreover, SUMOylation contributes to the subcellular distribution of target proteins. Herein, we found that the SUMOylation pathway is deregulated in NPM-ALK+ T-cell lymphoma cell lines and primary lymphoma tumors from patients. We also identified Lys24 and Lys32 within the NPM domain as the sites where NPM-ALK conjugates with SUMO-1 and SUMO-3. Importantly, antagonizing SUMOylation by the SENP1 protease decreased the accumulation of NPM-ALK and suppressed lymphoma cell viability, proliferation, and anchorage-independent colony formation. One possible mechanism for the SENP1-mediated decrease in NPM-ALK levels was the increase in NPM-ALK association with ubiquitin, which facilitates its degradation. Our findings propose a model in which aberrancies in SUMOylation contribute to the pathogenesis of NPM-ALK+ T-cell lymphoma. Unraveling such pathogenic mechanisms may lead to devising novel strategies to eliminate this aggressive neoplasm.

  9. Structure of the bone morphogenetic protein receptor ALK2 and implications for fibrodysplasia ossificans progressiva.

    PubMed

    Chaikuad, Apirat; Alfano, Ivan; Kerr, Georgina; Sanvitale, Caroline E; Boergermann, Jan H; Triffitt, James T; von Delft, Frank; Knapp, Stefan; Knaus, Petra; Bullock, Alex N

    2012-10-26

    Bone morphogenetic protein (BMP) receptor kinases are tightly regulated to control development and tissue homeostasis. Mutant receptor kinase domains escape regulation leading to severely degenerative diseases and represent an important therapeutic target. Fibrodysplasia ossificans progressiva (FOP) is a rare but devastating disorder of extraskeletal bone formation. FOP-associated mutations in the BMP receptor ALK2 reduce binding of the inhibitor FKBP12 and promote leaky signaling in the absence of ligand. To establish structural mechanisms of receptor regulation and to address the effects of FOP mutation, we determined the crystal structure of the cytoplasmic domain of ALK2 in complex with the inhibitors FKBP12 and dorsomorphin. FOP mutations break critical interactions that stabilize the inactive state of the kinase, thereby facilitating structural rearrangements that diminish FKBP12 binding and promote the correct positioning of the glycine-serine-rich loop and αC helix for kinase activation. The balance of these effects accounts for the comparable activity of R206H and L196P. Kinase activation in the clinically benign mutant L196P is far weaker than R206H but yields equivalent signals due to the stronger interaction of FKBP12 with R206H. The presented ALK2 structure offers a valuable template for the further design of specific inhibitors of BMP signaling.

  10. A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD.

    PubMed

    Alseth, Ingrun; Rognes, Torbjørn; Lindbäck, Toril; Solberg, Inger; Robertsen, Kristin; Kristiansen, Knut Ivan; Mainieri, Davide; Lillehagen, Lucy; Kolstø, Anne-Brit; Bjørås, Magnar

    2006-03-01

    Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3-methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E. coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E. coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N-alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B. cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily.

  11. A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD

    PubMed Central

    Alseth, Ingrun; Rognes, Torbjørn; Lindbäck, Toril; Solberg, Inger; Robertsen, Kristin; Kristiansen, Knut Ivan; Mainieri, Davide; Lillehagen, Lucy; Kolstø, Anne-Brit; Bjørås, Magnar

    2006-01-01

    Summary Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3-methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E. coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E. coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N-alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B. cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily. PMID:16468998

  12. A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD.

    PubMed

    Alseth, Ingrun; Rognes, Torbjørn; Lindbäck, Toril; Solberg, Inger; Robertsen, Kristin; Kristiansen, Knut Ivan; Mainieri, Davide; Lillehagen, Lucy; Kolstø, Anne-Brit; Bjørås, Magnar

    2006-03-01

    Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3-methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E. coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E. coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N-alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B. cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily. PMID:16468998

  13. Hyperactivation of Alk induces neonatal lethality in knock-in AlkF1178L mice

    PubMed Central

    Lopez-Delisle, Lucille; Pierre-Eugène, Cécile; Bloch-Gallego, Evelyne; Birling, Marie-Christine; Duband, Jean-Loup; Durand, Estelle; Bourgeois, Thomas; Matrot, Boris; Sorg, Tania; Huerre, Michel; Meziane, Hamid; Roux, Michel J.; Champy, Marie-France; Gallego, Jorge; Delattre, Olivier; Janoueix-Lerosey, Isabelle

    2014-01-01

    The ALK (Anaplastic Lymphoma Kinase) gene encodes a tyrosine kinase receptor preferentially expressed in the central and peripheral nervous systems. A syndromic presentation associating congenital neuroblastoma with severe encephalopathy and an abnormal shape of the brainstem has been described in patients harbouring de novo germline F1174V and F1245V ALK mutations. Here, we investigated the phenotype of knock-in (KI) mice bearing the AlkF1178L mutation (F1174L in human). Although heterozygous KI mice did not reproduce the severe breathing and feeding difficulties observed in human patients, behavioral tests documented a reduced activity during dark phases and an increased anxiety of mutated mice. Matings of heterozygotes yielded the expected proportions of wild-type, heterozygotes and homozygotes at birth but a high neonatal lethality was noticed for homozygotes. We documented Alk expression in several motor nuclei of the brainstem involved in the control of sucking and swallowing. Evaluation of basic physiological functions 12 hours after birth revealed slightly more apneas but a dramatic reduced milk intake for homozygotes compared to control littermates. Overall, our data demonstrate that Alk activation above a critical threshold is not compatible with survival in mice, in agreement with the extremely severe phenotype of patients carrying aggressive de novo ALK germline mutations. PMID:24811761

  14. Combinatorial delivery of Crizotinib-Palbociclib-Sildenafil using TPGS-PLA micelles for improved cancer treatment.

    PubMed

    de Melo-Diogo, Duarte; Gaspar, Vítor M; Costa, Elisabete C; Moreira, André F; Oppolzer, David; Gallardo, Eugénia; Correia, Ilídio J

    2014-11-01

    The co-delivery of multiple chemotherapeutics by micellar delivery systems is a valuable approach to improve cancer treatment since various disease hallmarks can be targeted simultaneously. However, the delivery of multiple drugs requires a nanocarrier structure that can encapsulate various bioactive molecules. In this study, we evaluate the simultaneous encapsulation of a novel triple drug combination in D-α-tocopheryl polyethylene glycol 1000 succinate-poly(lactic acid) (TPGS-PLA) amphiphilic micelles for cancer therapy. The drug mixture involves two anti-tumoral drugs, Crizotinib and Palbociclib combined with Sildenafil, a compound that is capable of increasing drug accumulation in the intracellular compartment. Such combination aims to achieve an enhanced cytotoxic effect in cancer cells. Our results demonstrated that TPGS-PLA copolymers self-assembled into stable nanosized micelles (158.3nm) capable of co-encapsulating the three drugs with high loading efficiency. Triple drug loaded TPGS-PLA micelles were internalized in A549 non-small lung cancer cells and exhibited an improved cytotoxic effect in comparison with single (Crizotinib) or dual (Crizotinib-Palbociclib) drug loaded micelles, indicating the therapeutic potential of the triple co-delivery strategy. These findings demonstrate that TPGS-PLA micelles are suitable carriers for multiple drug delivery and also that this particular drug combination may have potential to improve cancer treatment. PMID:25308930

  15. Combinatorial delivery of Crizotinib-Palbociclib-Sildenafil using TPGS-PLA micelles for improved cancer treatment.

    PubMed

    de Melo-Diogo, Duarte; Gaspar, Vítor M; Costa, Elisabete C; Moreira, André F; Oppolzer, David; Gallardo, Eugénia; Correia, Ilídio J

    2014-11-01

    The co-delivery of multiple chemotherapeutics by micellar delivery systems is a valuable approach to improve cancer treatment since various disease hallmarks can be targeted simultaneously. However, the delivery of multiple drugs requires a nanocarrier structure that can encapsulate various bioactive molecules. In this study, we evaluate the simultaneous encapsulation of a novel triple drug combination in D-α-tocopheryl polyethylene glycol 1000 succinate-poly(lactic acid) (TPGS-PLA) amphiphilic micelles for cancer therapy. The drug mixture involves two anti-tumoral drugs, Crizotinib and Palbociclib combined with Sildenafil, a compound that is capable of increasing drug accumulation in the intracellular compartment. Such combination aims to achieve an enhanced cytotoxic effect in cancer cells. Our results demonstrated that TPGS-PLA copolymers self-assembled into stable nanosized micelles (158.3nm) capable of co-encapsulating the three drugs with high loading efficiency. Triple drug loaded TPGS-PLA micelles were internalized in A549 non-small lung cancer cells and exhibited an improved cytotoxic effect in comparison with single (Crizotinib) or dual (Crizotinib-Palbociclib) drug loaded micelles, indicating the therapeutic potential of the triple co-delivery strategy. These findings demonstrate that TPGS-PLA micelles are suitable carriers for multiple drug delivery and also that this particular drug combination may have potential to improve cancer treatment.

  16. Silibinin suppresses NPM-ALK, potently induces apoptosis and enhances chemosensitivity in ALK-positive anaplastic large cell lymphoma.

    PubMed

    Molavi, Ommoleila; Samadi, Nasser; Wu, Chengsheng; Lavasanifar, Afsaneh; Lai, Raymond

    2016-05-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), an oncogenic fusion protein carrying constitutively active tyrosine kinase, is known to be central to the pathogenesis of ALK-positive anaplastic large cell lymphoma (ALK+ALCL). Here, it is reported that silibinin, a non-toxic naturally-occurring compound, potently suppressed NPM-ALK and effectively inhibited the growth and soft agar colony formation of ALK+ALCL cells. By western blots, it was found that silibinin efficiently suppressed the phosphorylation/activation of NPM-ALK and its key substrates/downstream mediators (including STAT3, MEK/ERK and Akt) in a time- and dose-dependent manner. Correlating with these observations, silibinin suppressed the expression of Bcl-2, survivin and JunB, all of which are found to be upregulated by NPM-ALK and pathogenetically important in ALK+ALCL. Lastly, silibinin augmented the chemosensitivity of ALK+ALCL cells to doxorubicin, particularly the small cell sub-set expressing the transcriptional activity of Sox2, an embryonic stem cell marker. To conclude, the findings suggest that silibinin might be useful in treating ALK+ALCL.

  17. Structural insights into the ATP binding pocket of the anaplastic lymphoma kinase by site-directed mutagenesis, inhibitor binding analysis, and homology modeling.

    PubMed

    Gunby, Rosalind H; Ahmed, Shaheen; Sottocornola, Roberta; Gasser, Marc; Redaelli, Sara; Mologni, Luca; Tartari, Carmen J; Belloni, Valentina; Gambacorti-Passerini, Carlo; Scapozza, Leonardo

    2006-09-21

    Anaplastic lymphoma kinase (ALK) is a valid target for anticancer therapy; however, potent ALK inhibitors suitable for clinical use are lacking. Because the majority of described kinase inhibitors bind in the ATP pocket of the kinase domain, we have characterized this pocket in ALK using site-directed mutagenesis, inhibition studies, and molecular modeling. Mutation of the gatekeeper residue, a key structural determinant influencing inhibitor binding, rendered the fusion protein, NPM/ALK, sensitive to inhibition by SKI-606 in the nanomolar range, while PD173955 inhibited the NPM/ALK mutant at micromolar concentrations. In contrast, both wild type and mutant NPM/ALK were insensitive to imatinib. Computer modeling indicated that docking solutions obtained with a homology model representing the intermediate conformation of the ALK kinase domain reflected closely experimental data. The good agreement between experimental and virtual results indicate that the ALK molecular models described here are useful tools for the rational design of ALK selective inhibitors. In addition, 4-phenylamino-quinoline compounds may have potential as templates for ALK inhibitors. PMID:16970400

  18. Inhibitors

    MedlinePlus

    ... Community Counts Blood Safety Inhibitors Articles & Key Findings Free Materials Videos Starting the Conversation Playing it Safe A Look at Hemophilia Joint Range of Motion My Story Links to Other Websites ...

  19. Prognostic significance of NPM-ALK fusion transcript overexpression in ALK-positive anaplastic large-cell lymphoma.

    PubMed

    Li, Chunmei; Takino, Hisashi; Eimoto, Tadaaki; Ishida, Takashi; Inagaki, Atsushi; Ueda, Ryuzo; Suzuki, Ritsuro; Yoshino, Tadashi; Nakagawa, Atsuko; Nakamura, Shigeo; Inagaki, Hiroshi

    2007-06-01

    In anaplastic large-cell lymphomas positive for anaplastic lymphoma kinase (ALK) protein, the ALK gene is most commonly fused to the NPM gene, and less commonly to TPM3, TFG, ATIC, and other rare genes. Although this lymphoma is generally associated with a favorable clinical outcome, 25% of the patients die of the disease within 5 years. In this study, we developed three assays, all of which can be used with archival formalin-fixed, paraffin-embedded tissues: (1) a sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay for various X-ALK fusion genes, (2) a 5' rapid amplification of cDNA ends (RACE) assay to identify unknown fusion partners, and (3) a real-time RT-PCR assay to quantify the amount of the NPM-ALK fusion transcript. In 26 cases of ALK(+) anaplastic large-cell lymphoma, the RT-PCR assay showed that the ALK was fused to NPM in 21 cases, to TPM3 in three, and to TFG in one. The 5' RACE assay detected ATIC-ALK fusion in the remaining case. The real-time quantitative RT-PCR assay showed that the NPM-ALK transcript was over expressed in four of 20 quantifiable cases. Patients with NPM-ALK overexpression showed a significantly unfavorable overall survival compared with those with a low expression of this transcript. The RT-PCR and 5' RACE assays developed here may be useful for identification of known and unknown gene partners fused to the ALK gene. Overexpression of the NPM-ALK fusion transcript may be associated with a poor prognosis of the patients with ALK(+) anaplastic large-cell lymphomas.

  20. Insertion element analysis and mapping of the Pseudomonas plasmid alk regulon.

    PubMed Central

    Fennewald, M; Benson, S; Oppici, M; Shapiro, J

    1979-01-01

    We characterized and mapped new mutations of the alk (alkane utilization) genes found on Pseudomonas plasmids of the Inc P-2 group. These mutations were isolated after (i) nitrosoguanidine mutagenesis, (ii) transposition of the Tn7 trimethoprim and streptomycin resistance determinant, and (iii) reversion of polarity effects of alk::Tn7 insertion mutations. Our results indicate the existence of two alk loci not previously described--alkD, whose product is required for synthesis of membrane alkane-oxidizing activities, and alkE, whose product is required for synthesis of inducible membrane alcohol dehydrogenase activity. Polarity of alk::Tn7 insertion mutations indicates the existence of an alkBAE operon. Mapping of alk loci by transduction in P. aeruginosa shows that there are at least three alk clusters in the CAM-OCT plasmid--alkRD, containing regulatory genes; alkBAE, containing genes for specific biochemical activities; and alkC, containing one or more genes needed for normal synthesis of membrane alcohol dehydrogenase. The alkRD and alkBAE clusters are linked but separated by about 42 kilobases. The alkC cluster is not linked to either of the other two alk regions. Altogether, these results indicate a complex genetic control of the alkane utilization phenotype in P. putida and P. aeruginosa involving at least six separate genes. Images PMID:479111

  1. Non-small Cell Lung Cancer with Concomitant EGFR, KRAS, and ALK Mutation: Clinicopathologic Features of 12 Cases

    PubMed Central

    Lee, Taebum; Lee, Boram; Choi, Yoon-La; Han, Joungho; Ahn, Myung-Ju; Um, Sang-Won

    2016-01-01

    Background: Although epidermal growth factor receptor (EGFR), v-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS), and anaplastic lymphoma kinase (ALK) mutations in non-small cell lung cancer (NSCLC) were thought to be mutually exclusive, some tumors harbor concomitant mutations. Discovering a driver mutation on the basis of morphologic features and therapeutic responses with mutation analysis can be used to understand pathogenesis and predict resistance in targeted therapy. Methods: In 6,637 patients with NSCLC, 12 patients who had concomitant mutations were selected and clinicopathologic features were reviewed. Clinical characteristics included sex, age, smoking history, previous treatment, and targeted therapy with response and disease-free survival. Histologic features included dominant patterns, nuclear and cytoplasmic features. Results: All patients were diagnosed with adenocarcinoma and had an EGFR mutation. Six patients had concomitant KRAS mutations and the other six had KRAS mutations. Five of six EGFR-KRAS mutation patients showed papillary and acinar histologic patterns with hobnail cells. Three of six received EGFR tyrosine kinase inhibitor (TKI) and showed partial response for 7–29 months. All six EGFR-ALK mutation patients showed solid or cribriform patterns and three had signet ring cells. Five of six EGFR-ALK mutation patients received EGFR TKI and/or ALK inhibitor and four showed partial response or stable disease, except for one patient who had acquired an EGFR mutation. Conclusions: EGFR and ALK mutations play an important role as driver mutations in double mutated NSCLC, and morphologic analysis can be used to predict treatment response. PMID:27086595

  2. Anaplastic lymphoma kinase: role in cancer pathogenesis and small-molecule inhibitor development for therapy

    PubMed Central

    Webb, Thomas R; Slavish, Jake; George, Rani E; Look, A Thomas; Xue, Liquan; Jiang, Qin; Cui, Xiaoli; Rentrop, Walter B; Morris, Stephan W

    2009-01-01

    Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase in the insulin receptor superfamily, was initially identified in constitutively activated oncogenic fusion forms – the most common being nucleophosmin-ALK – in anaplastic large-cell lymphomas, and subsequent studies have identified ALK fusions in diffuse large B-cell lymphomas, systemic histiocytosis, inflammatory myofibroblastic tumors, esophageal squamous cell carcinomas and non-small-cell lung carcinomas. More recently, genomic DNA amplification and protein overexpression, as well as activating point mutations, of ALK have been described in neuroblastomas. In addition to those cancers for which a causative role for aberrant ALK activity is well validated, more circumstantial links implicate the full-length, normal ALK receptor in the genesis of other malignancies – including glioblastoma and breast cancer – via a mechanism of receptor activation involving autocrine and/or paracrine growth loops with the reported ALK ligands, pleiotrophin and midkine. This review summarizes normal ALK biology, the confirmed and putative roles of ALK in the development of human cancers and efforts to target ALK using small-molecule kinase inhibitors. PMID:19275511

  3. NPM-ALK up-regulates iNOS expression through a STAT3/microRNA-26a-dependent mechanism.

    PubMed

    Zhu, Haifeng; Vishwamitra, Deeksha; Curry, Choladda V; Manshouri, Roxsan; Diao, Lixia; Khan, Aarish; Amin, Hesham M

    2013-05-01

    NPM-ALK chimeric oncogene is aberrantly expressed in an aggressive subset of T-cell lymphomas that frequently occurs in children and young adults. The mechanisms underlying the oncogenic effects of NPM-ALK are not completely elucidated. Inducible nitric oxide synthase (iNOS) promotes the survival and maintains the malignant phenotype of cancer cells by generating NO, a highly active free radical. We tested the hypothesis that iNOS is deregulated in NPM-ALK(+) T-cell lymphoma and promotes the survival of this lymphoma. In line with this possibility, an iNOS inhibitor and NO scavenger decreased the viability, adhesion, and migration of NPM-ALK(+) T-cell lymphoma cells, and an NO donor reversed these effects. Moreover, the NO donor salvaged the viability of lymphoma cells treated with ALK inhibitors. In further support of an important role of iNOS, we found iNOS protein to be highly expressed in NPM-ALK(+) T-cell lymphoma cell lines and in 79% of primary tumours but not in human T lymphocytes. Although expression of iNOS mRNA was identified in NPM-ALK(+) T-cell lymphoma cell lines and tumours, iNOS mRNA was remarkably elevated in T lymphocytes, suggesting post-transcriptional regulation. Consistently, we found that miR-26a contains potential binding sites and interacts with the 3'-UTR of iNOS. In addition, miR-26a was significantly decreased in NPM-ALK(+) T-cell lymphoma cell lines and tumours compared with T lymphocytes and reactive lymph nodes. Restoration of miR-26a in lymphoma cells abrogated iNOS protein expression and decreased NO production and cell viability, adhesion, and migration. Importantly, the effects of miR-26a were substantially attenuated when the NO donor was simultaneously used to treat lymphoma cells. Our investigation of the mechanisms underlying the decrease in miR-26a in this lymphoma revealed novel evidence that STAT3, a major downstream substrate of NPM-ALK tyrosine kinase activity, suppresses MIR26A1 gene expression.

  4. Midkine and Alk signaling in sympathetic neuron proliferation and neuroblastoma predisposition.

    PubMed

    Reiff, Tobias; Huber, Leslie; Kramer, Marco; Delattre, Olivier; Janoueix-Lerosey, Isabelle; Rohrer, Hermann

    2011-11-01

    Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and arises from cells of the developing sympathoadrenergic lineage. Activating mutations in the gene encoding the ALK tyrosine kinase receptor predispose for NB. Here, we focus on the normal function of Alk signaling in the control of sympathetic neuron proliferation, as well as on the effects of mutant ALK. Forced expression of wild-type ALK and NB-related constitutively active ALK mutants in cultures of proliferating immature sympathetic neurons results in a strong proliferation increase, whereas Alk knockdown and pharmacological inhibition of Alk activity decrease proliferation. Alk activation upregulates NMyc and trkB and maintains Alk expression by an autoregulatory mechanism involving Hand2. The Alk-ligand Midkine (Mk) is expressed in immature sympathetic neurons and in vivo inhibition of Alk signaling by virus-mediated shRNA knockdown of Alk and Mk leads to strongly reduced sympathetic neuron proliferation. Taken together, these results demonstrate that the extent and timing of sympathetic neurogenesis is controlled by Mk/Alk signaling. The predisposition for NB caused by activating ALK mutations may thus be explained by aberrations of normal neurogenesis, i.e. elevated and sustained Alk signaling and increased NMyc expression. PMID:21989914

  5. Regioselective alkane hydroxylation with a mutant AlkB enzyme

    DOEpatents

    Koch, Daniel J.; Arnold, Frances H.

    2012-11-13

    AlkB from Pseudomonas putida was engineered using in-vivo directed evolution to hydroxylate small chain alkanes. Mutant AlkB-BMO1 hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. Mutant AlkB-BMO2 similarly hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. These biocatalysts are highly active for small chain alkane substrates and their regioselectivity is retained in whole-cell biotransformations.

  6. TGF-ß induces Lysyl hydroxylase 2b in human synovial osteoarthritic fibroblasts through ALK5 signaling.

    PubMed

    Remst, Dennis F G; Blaney Davidson, Esmeralda N; Vitters, Elly L; Bank, Ruud A; van den Berg, Wim B; van der Kraan, Peter M

    2014-01-01

    Lysyl hydroxylase 2b (LH2b) is known to increase pyridinoline cross-links, making collagen less susceptible to enzymatic degradation. Previously, we observed a relationship between LH2b and osteoarthritis-related fibrosis in murine knee joint. For this study, we investigate if transforming growth factor-beta (TGF-ß) and connective tissue growth factor (CTGF) regulate procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (gene encoding LH2b) and LH2b expression differently in osteoarthritic human synovial fibroblasts (hSF). Furthermore, we investigate via which TGF-ß route (Smad2/3P or Smad1/5/8P) LH2b is regulated, to explore options to inhibit LH2b during fibrosis. To answer these questions, fibroblasts were isolated from knee joints of osteoarthritis patients. The hSF were stimulated with TGF-ß with or without a kinase inhibitor of ALK4/5/7 (SB-505124) or ALK1/2/3/6 (dorsomorphin). TGF-ß, CTGF, constitutively active (ca)ALK1 and caALK5 were adenovirally overexpressed in hSF. The gene expression levels of PLOD1/2/3, CTGF and COL1A1 were analyzed with Q-PCR. LH2 protein levels were determined with western blot. As expected, TGF-ß induced PLOD2/LH2 expression in hSF, whereas CTGF did not. PLOD1 and PLOD3 were not affected by either TGF-ß or CTGF. SB-505124 prevented the induction of TGF-ß-induced PLOD2, CTGF and COL1A1. Surprisingly, dorsomorphin completely blocked the induction of CTGF and COL1A1, whereas TGF-ß-induced PLOD2 was only slightly reduced. Overexpression of caALK5 in osteoarthritic hSF significantly induced PLOD2/LH2 expression, whereas caALK1 had no effect. We showed, in osteoarthritic hSF, that TGF-ß induced PLOD2/LH2 via ALK5 Smad2/3P. This elevation of LH2b in osteoarthritic hSF makes LH2b an interesting target to interfere with osteoarthritis-related persistent fibrosis.

  7. Unique substrate specificity of anaplastic lymphoma kinase (ALK): development of phosphoacceptor peptides for the assay of ALK activity.

    PubMed

    Donella-Deana, Arianna; Marin, Oriano; Cesaro, Luca; Gunby, Rosalind H; Ferrarese, Anna; Coluccia, Addolorata M L; Tartari, Carmen J; Mologni, Luca; Scapozza, Leonardo; Gambacorti-Passerini, Carlo; Pinna, Lorenzo A

    2005-06-14

    The anaplastic lymphoma kinase (ALK), whose constitutively active fusion proteins are responsible for 5-10% of non-Hodgkin's lymphomas, shares with the other members of the insulin receptor kinase (IRK) subfamily an activation loop (A-loop) with the triple tyrosine motif Y-x-x-x-Y-Y. However, the amino acid sequence of the ALK A-loop differs significantly from the sequences of both the IRK A-loop and the consensus A-loop for this kinase subfamily. A major difference is the presence of a unique "RAS" triplet between the first and second tyrosines of the ALK A-loop, which in IRK is replaced by "ETD". Here we show that a peptide reproducing the A-loop of ALK is readily phosphorylated by ALK, while a homologous IRK A-loop peptide is not unless its "ETD" triplet is substituted by "RAS". Phosphorylation occurs almost exclusively at the first tyrosine of the Y-x-x-x-Y-Y motif, as judged by Edman analysis of the phosphoradiolabeled product. Consequently, a peptide in which the first tyrosine had been replaced by phenylalanine (FYY) was almost unaffected by ALK. In contrast, a peptide in which the second and third tyrosines had been replaced by phenylalanine (YFF) was phosphorylated more rapidly than the parent peptide (YYY). A number of substitutions in the YFF peptide outlined the importance of Ile and Arg at positions n - 1 and n + 6 in addition to the central triplet, to ensure efficient phosphorylation by ALK. Such a peculiar substrate specificity allows the specific monitoring of ALK activity in crude extracts of NPM-ALK positive cells, using the YFF peptide, which is only marginally phosphorylated by a number of other tyrosine kinases. PMID:15938644

  8. Stereotactic Radiation Therapy can Safely and Durably Control Sites of Extra-Central Nervous System Oligoprogressive Disease in Anaplastic Lymphoma Kinase-Positive Lung Cancer Patients Receiving Crizotinib

    SciTech Connect

    Gan, Gregory N.; Weickhardt, Andrew J.; Scheier, Benjamin; Doebele, Robert C.; Gaspar, Laurie E.; Kavanagh, Brian D.; Camidge, D. Ross

    2014-03-15

    Purpose: To analyze the durability and toxicity of radiotherapeutic local ablative therapy (LAT) applied to extra-central nervous system (eCNS) disease progression in anaplastic lymphoma kinase-positive non-small cell lung cancer (NSCLC) patients. Methods and Materials: Anaplastic lymphoma kinase-positive NSCLC patients receiving crizotinib and manifesting ≤4 discrete sites of eCNS progression were classified as having oligoprogressive disease (OPD). If subsequent progression met OPD criteria, additional courses of LAT were considered. Crizotinib was continued until eCNS progression was beyond OPD criteria or otherwise not suitable for further LAT. Results: Of 38 patients, 33 progressed while taking crizotinib. Of these, 14 had eCNS progression meeting OPD criteria suitable for radiotherapeutic LAT. Patients with eCNS OPD received 1-3 courses of LAT with radiation therapy. The 6- and 12-month actuarial local lesion control rates with radiation therapy were 100% and 86%, respectively. The 12-month local lesion control rate with single-fraction equivalent dose >25 Gy versus ≤25 Gy was 100% versus 60% (P=.01). No acute or late grade >2 radiation therapy-related toxicities were observed. Median overall time taking crizotinib among those treated with LAT versus those who progressed but were not suitable for LAT was 28 versus 10.1 months, respectively. Patients continuing to take crizotinib for >12 months versus ≤12 months had a 2-year overall survival rate of 72% versus 12%, respectively (P<.0001). Conclusions: Local ablative therapy safely and durably eradicated sites of individual lesion progression in anaplastic lymphoma kinase-positive NSCLC patients receiving crizotinib. A dose–response relationship for local lesion control was observed. The suppression of OPD by LAT in patients taking crizotinib allowed an extended duration of exposure to crizotinib, which was associated with longer overall survival.

  9. BMP4 and BMP7 Suppress StAR and Progesterone Production via ALK3 and SMAD1/5/8-SMAD4 in Human Granulosa-Lutein Cells.

    PubMed

    Zhang, Han; Klausen, Christian; Zhu, Hua; Chang, Hsun-Ming; Leung, Peter C K

    2015-11-01

    Adequate production of progesterone by the corpus luteum is critical to the successful establishment of pregnancy. In animal models, bone morphogenetic protein (BMP) 4 and BMP7 have been shown to suppress either basal or gonadotropin-induced progesterone production, depending on the species examined. However, the effects of BMP4 and BMP7 on progesterone production in human granulosa cells are unknown. In the present study, we used immortalized (SVOG) and primary human granulosa-lutein cells to investigate the effects of BMP4 and BMP7 on steroidogenic acute regulatory protein (StAR) expression and progesterone production and to examine the underlying molecular mechanism. Treatment of primary and immortalized human granulosa cells with recombinant BMP4 or BMP7 decreased StAR expression and progesterone accumulation. In SVOG cells, the suppressive effects of BMP4 and BMP7 on StAR expression were blocked by pretreatment with inhibitors of activin receptor-like kinase (ALK)2/3/6 (dorsomorphin) or ALK2/3 (DMH1) but not ALK4/5/7 (SB-431542). Moreover, small interfering RNA-mediated depletion of ALK3, but not ALK2 or ALK6, reversed the effects of BMP4 and BMP7 on StAR expression. Likewise, BMP4- and BMP7-induced phosphorylation of SMAD 1/5/8 was reversed by treatment with DMH1 or small interfering RNA targeting ALK3. Knockdown of SMAD4, the essential common SMAD for BMP/TGF-β signaling, abolished the effects of BMP4 and BMP7 on StAR expression. Our results suggest that BMP4 and BMP7 down-regulate StAR and progesterone production via ALK3 and SMAD1/5/8-SMAD4 signaling in human granulosa-lutein cells.

  10. Identification of a novel crosstalk between casein kinase 2α and NPM-ALK in ALK-positive anaplastic large cell lymphoma.

    PubMed

    Armanious, Hanan; Gelebart, Pascal; Anand, Mona; Lai, Raymond

    2013-02-01

    It was previously reported that β-catenin contributes to the tumorigenesis of ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL), and the oncogenic effects of β-catenin in these tumors are promoted by NPM-ALK, an abnormal fusion protein characteristic of ALK(+)ALCL. In this study, we hypothesized that NPM-ALK promotes the oncogenic activity of β-catenin via its functional interactions with the Wnt canonical pathway (WCP). To test this hypothesis, we examined if NPM-ALK modulates the gene expression of various members in the WCP. Using a Wnt pathway-specific oligonucleotide array and Western blots, we found that the expression of casein kinase 2α (CK2α) was substantially downregulated in ALK(+)ALCL cells in response to siRNA knockdown of NPM-ALK. CK2α is biologically important in ALK(+)ALCL, as its inhibition using 4,5,6,7-tetrabromobenzotriazole or siRNA resulted in a significant decrease in cell growth and a substantial decrease in the β-catenin protein level. Furthermore, CK2α co-immunoprecipitated with NPM-ALK and regulated its level of serine phosphorylation, a feature previously shown to correlate with the oncogenic potential of this fusion protein. To conclude, this study has revealed a novel crosstalk between NPM-ALK and CK2α, and our data supports the model that these two molecules work synergistically to promote the tumorigenicity of these lymphomas.

  11. The proteomic signature of NPM/ALK reveals deregulation of multiple cellular pathways.

    PubMed

    Lim, Megan S; Carlson, Mary L; Crockett, David K; Fillmore, G Chris; Abbott, David R; Elenitoba-Johnson, Olaotan F; Tripp, Sheryl R; Rassidakis, George Z; Medeiros, L Jeffrey; Szankasi, Philippe; Elenitoba-Johnson, Kojo S J

    2009-08-20

    Constitutive expression of the chimeric NPM/ALK fusion protein encoded by the t(2;5)(p32;q35) is a key oncogenic event in the pathogenesis of most anaplastic large cell lymphomas (ALCLs). The proteomic network alterations produced by this aberration remain largely uncharacterized. Using a mass spectrometry (MS)-driven approach to identify changes in protein expression caused by the NPM/ALK fusion, we identified diverse NPM/ALK-induced changes affecting cell proliferation, ribosome synthesis, survival, apoptosis evasion, angiogenesis, and cytoarchitectural organization. MS-based findings were confirmed using Western blotting and/or immunostaining of NPM/ALK-transfected cells and ALK-deregulated lymphomas. A subset of the proteins distinguished NPM/ALK-positive ALCLs from NPM/ALK-negative ALCLs and Hodgkin lymphoma. The multiple NPM/ALK-deregulated pathways identified by MS analysis also predicted novel biologic effects of NPM/ALK expression. In this regard, we showed loss of cell adhesion as a consequence of NPM/ALK expression in a kinase-dependent manner, and sensitivity of NPM/ALK-positive ALCLs to inhibition of the RAS, p42/44ERK, and FRAP/mTOR signaling pathways. These findings reveal that the NPM/ALK alteration affects diverse cellular pathways, and provide novel insights into NPM/ALK-positive ALCL pathobiology. Our studies carry important implications for the use of MS-driven approaches for the elucidation of neoplastic pathobiology, the identification of novel diagnostic biomarkers, and pathogenetically relevant therapeutic targets.

  12. Alk2 regulates early chondrogenic fate in fibrodysplasia ossificans progressiva heterotopic endochondral ossification.

    PubMed

    Culbert, Andria L; Chakkalakal, Salin A; Theosmy, Edwin G; Brennan, Tracy A; Kaplan, Frederick S; Shore, Eileen M

    2014-05-01

    Bone morphogenetic protein (BMP) signaling is a critical regulator of cartilage differentiation and endochondral ossification. Gain-of-function mutations in ALK2, a type I BMP receptor, cause the debilitating disorder fibrodysplasia ossificans progressiva (FOP) and result in progressive heterotopic (extraskeletal) endochondral ossification within soft connective tissues. Here, we used murine mesenchymal progenitor cells to investigate the contribution of Alk2 during chondrogenic differentiation and heterotopic endochondral ossification (HEO). Alk2(R206H/+) (gain-of-function), Alk2(CKO) (loss-of-function), and wild-type mouse embryonic fibroblasts were evaluated for chondrogenic potential. Chondrogenic differentiation was accelerated in Alk2(R206H/+) cells, due in part to enhanced sensitivity to BMP ligand. In vivo, Alk2(R206H/+) cells initiated robust HEO and recruited wild-type cell contribution. Despite expression of other type I BMP receptors (Alk3 and Alk6), chondrogenesis of Alk2(CKO) cells was severely impaired by absence of Alk2 during early differentiation. Alk2 is therefore a direct regulator of cartilage formation and mediates chondrogenic commitment of progenitor cells. These data establish that at least one effect of ALK2 gain-of-function mutations in FOP patients is enhanced chondrogenic differentiation which supports formation of heterotopic endochondral bone. This establishes ALK2 as a plausible therapeutic target during early chondrogenic stages of lesion formation for preventing heterotopic bone formation in FOP and other conditions.

  13. Antibody targeting of anaplastic lymphoma kinase induces cytotoxicity of human neuroblastoma.

    PubMed

    Carpenter, E L; Haglund, E A; Mace, E M; Deng, D; Martinez, D; Wood, A C; Chow, A K; Weiser, D A; Belcastro, L T; Winter, C; Bresler, S C; Vigny, M; Mazot, P; Asgharzadeh, S; Seeger, R C; Zhao, H; Guo, R; Christensen, J G; Orange, J S; Pawel, B R; Lemmon, M A; Mossé, Y P

    2012-11-15

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in neuroblastoma, a devastating pediatric cancer of the sympathetic nervous system. Germline and somatically acquired ALK aberrations induce increased autophosphorylation, constitutive ALK activation and increased downstream signaling. Thus, ALK is a tractable therapeutic target in neuroblastoma, likely to be susceptible to both small-molecule tyrosine kinase inhibitors and therapeutic antibodies-as has been shown for other receptor tyrosine kinases in malignancies such as breast and lung cancer. Small-molecule inhibitors of ALK are currently being studied in the clinic, but common ALK mutations in neuroblastoma appear to show de novo insensitivity, arguing that complementary therapeutic approaches must be developed. We therefore hypothesized that antibody targeting of ALK may be a relevant strategy for the majority of neuroblastoma patients likely to have ALK-positive tumors. We show here that an antagonistic ALK antibody inhibits cell growth and induces in vitro antibody-dependent cellular cytotoxicity of human neuroblastoma-derived cell lines. Cytotoxicity was induced in cell lines harboring either wild type or mutated forms of ALK. Treatment of neuroblastoma cells with the dual Met/ALK inhibitor crizotinib sensitized cells to antibody-induced growth inhibition by promoting cell surface accumulation of ALK and thus increasing the accessibility of antigen for antibody binding. These data support the concept of ALK-targeted immunotherapy as a highly promising therapeutic strategy for neuroblastomas with mutated or wild-type ALK.

  14. Partial Response to First-Line Crizotinib in an Elderly Male Patient with ROS1 Translocation-Positive Lung Cancer.

    PubMed

    Overbeck, Tobias R; Schmitz, Katja; Engelke, Christoph; Sahlmann, Carsten-Oliver; Hugo, Sara; Kellner, Laura; Trümper, Lorenz; Schildhaus, Hans-Ulrich

    2016-01-01

    We report on a 90-year-old male patient with a ROS1-translocated adenocarcinoma of the lung who was treated with crizotinib as first-line therapy. After 11 months of treatment, we noticed complete metabolic response as measured by (18)F-FDG-PET/CT scan and a partial response according to RECIST criteria. This patient indicates that ROS1 translocations are not restricted to young age, female gender and low stage. Furthermore, this case illustrates exemplarily that crizotinib therapy is effective and manageable even as first-line treatment in elderly patients with comorbidities. Based on our findings, we recommend to include elderly patients with advanced pulmonary adenocarcinomas in molecular screening approaches for ROS1 translocations.

  15. Partial Response to First-Line Crizotinib in an Elderly Male Patient with ROS1 Translocation-Positive Lung Cancer

    PubMed Central

    Overbeck, Tobias R.; Schmitz, Katja; Engelke, Christoph; Sahlmann, Carsten-Oliver; Hugo, Sara; Kellner, Laura; Trümper, Lorenz; Schildhaus, Hans-Ulrich

    2016-01-01

    We report on a 90-year-old male patient with a ROS1-translocated adenocarcinoma of the lung who was treated with crizotinib as first-line therapy. After 11 months of treatment, we noticed complete metabolic response as measured by 18F-FDG-PET/CT scan and a partial response according to RECIST criteria. This patient indicates that ROS1 translocations are not restricted to young age, female gender and low stage. Furthermore, this case illustrates exemplarily that crizotinib therapy is effective and manageable even as first-line treatment in elderly patients with comorbidities. Based on our findings, we recommend to include elderly patients with advanced pulmonary adenocarcinomas in molecular screening approaches for ROS1 translocations. PMID:27065847

  16. Uterine ALK3 is essential during the window of implantation.

    PubMed

    Monsivais, Diana; Clementi, Caterina; Peng, Jia; Titus, Mary M; Barrish, James P; Creighton, Chad J; Lydon, John P; DeMayo, Francesco J; Matzuk, Martin M

    2016-01-19

    The window of implantation is defined by the inhibition of uterine epithelial proliferation, structural epithelial cell remodeling, and attenuated estrogen (E2) response. These changes occur via paracrine signaling between the uterine epithelium and stroma. Because implantation defects are a major cause of infertility in women, identifying these signaling pathways will improve infertility interventions. Bone morphogenetic proteins (BMPs) are TGF-β family members that regulate the postimplantation and midgestation stages of pregnancy. In this study, we discovered that signaling via activin-like kinase 3 (ALK3/BMPR1A), a BMP type 1 receptor, is necessary for blastocyst attachment. Conditional knockout (cKO) of ALK3 in the uterus was obtained by producing Alk3(flox) (/flox)-Pgr-cre-positive females. Alk3 cKO mice are sterile and have defects in the luminal uterine epithelium, including increased microvilli density and maintenance of apical cell polarity. Moreover, Alk3 cKO mice exhibit an elevated uterine E2 response and unopposed epithelial cell proliferation during the window of implantation. We determined that dual transcriptional regulation of Kruppel-like factor 15 (Klf15), by both the transforming growth factor β (TGF-β) transcription factor SMAD family member 4 (SMAD4) and progesterone receptor (PR), is necessary to inhibit uterine epithelial cell proliferation, a key step for embryo implantation. Our findings present a convergence of BMP and steroid hormone signaling pathways in the regulation of uterine receptivity. PMID:26721398

  17. Timely topic: anaplastic lymphoma kinase (ALK) spreads its influence.

    PubMed

    Cheuk, W; Chan, J K

    2001-02-01

    Anaplastic lymphoma kinase (ALK) is normally not expressed in human tissues except selected sites in the nervous system. Its expression and constitutive activation as a result of a chromosomal translocation involving 2p23 plays a pivotal role in the genesis of anaplastic large cell lymphoma. ALK expression has been instrumental in defining a homogeneous subset from the category of anaplastic large cell lymphoma, characterised by occurrence in young patients, primary systemic presentation, favorable prognosis, a broad morphological spectrum, nuclear and/or cytoplasmic immunostaining for ALK protein, and a number of possible fusion partner genes such as NPM, ATIC, TFG, TPM3 and CLTCL. Recently ALK has been implicated in the genesis of another tumour type, the inflammatory myofibroblastic tumours. The ALK-positive examples occur in children and young adults, involving a variety of sites, such as the abdomen, mesentery, liver, bladder, mediastinum, lung and bone. The partner genes identified in some cases are TPM3 (tropomyosin 3) and TPM4 (tropomyosin 4). These molecular findings also further support the neoplastic nature of at least a subset of inflammatory myofibroblastic tumours.

  18. Targeted Therapies in Non-Small Cell Lung Cancer-Beyond EGFR and ALK.

    PubMed

    Rothschild, Sacha I

    2015-01-01

    Systemic therapy for non-small cell lung cancer (NSCLC) has undergone a dramatic paradigm shift over the past decade. Advances in our understanding of the underlying biology of NSCLC have revealed distinct molecular subtypes. A substantial proportion of NSCLC depends on oncogenic molecular aberrations (so-called "driver mutations") for their malignant phenotype. Personalized therapy encompasses the strategy of matching these subtypes with effective targeted therapies. EGFR mutations and ALK translocation are the most effectively targeted oncogenes in NSCLC. EGFR mutations and ALK gene rearrangements are successfully being targeted with specific tyrosine kinase inhibitors. The number of molecular subgroups of NSCLC continues to grow. The scope of this review is to discuss recent data on novel molecular targets as ROS1, BRAF, KRAS, HER2, c-MET, RET, PIK3CA, FGFR1 and DDR2. Thereby the review will focus on therapeutic strategies targeting these aberrations. Moreover, the emerging challenge of acquired resistance to initially effective therapies will be discussed. PMID:26018876

  19. NPM-ALK and the JunB transcription factor regulate the expression of cytotoxic molecules in ALK-positive, anaplastic large cell lymphoma.

    PubMed

    Pearson, Joel D; Lee, Jason K H; Bacani, Julinor T C; Lai, Raymond; Ingham, Robert J

    2011-01-30

    Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is an aggressive non-Hodgkin lymphoma of T/null immunophenotype that is most prevalent in children and young adults. The normal cellular counterpart of this malignancy is presumed to be the cytotoxic T lymphocyte (CTL), and this presumption is partly based on the observation that these tumour cells often express cytotoxic granules containing Granzyme B (GzB) and Perforin. Chromosomal translocations involving the gene encoding for the ALK tyrosine kinase are also characteristic of ALK+ ALCL, and the resulting fusion proteins (e.g. NPM-ALK) initiate signalling events important in ALK+ ALCL pathogenesis. These events include the elevated expression of JunB; an AP-1 family transcription factor that promotes ALK+ ALCL proliferation. In this report we demonstrate that JunB is a direct transcriptional activator of GzB and that GzB transcription is also promoted by NPM-ALK. We found that Perforin expression was not regulated by JunB, but was promoted by NPM-ALK in some cell lines and inhibited by it in others. In conclusion, our study makes the novel observation that signalling through NPM-ALK and JunB affect the expression of cytotoxic molecules in ALK+ ALCL. Moreover, these findings demonstrate the expression of GzB and Perforin in this lymphoma is not solely due its presumed CTL origin, but that oncogenic signalling is actively influencing the expression of these proteins.

  20. STAT1 is phosphorylated and downregulated by the oncogenic tyrosine kinase NPM-ALK in ALK-positive anaplastic large-cell lymphoma.

    PubMed

    Wu, Chengsheng; Molavi, Ommoleila; Zhang, Haifeng; Gupta, Nidhi; Alshareef, Abdulraheem; Bone, Kathleen M; Gopal, Keshav; Wu, Fang; Lewis, Jamie T; Douglas, Donna N; Kneteman, Norman M; Lai, Raymond

    2015-07-16

    The tumorigenicity of most cases of ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Because it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ ALCL, thereby leaving the STAT3 signaling unchecked. Compared with normal T cells, ALK+ ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ ALCL cells, because it effectively upregulated interferon-γ, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 signaling by decreasing STAT3 transcriptional activity/DNA binding and its homodimerization. The importance of the STAT1/STAT3 functional interaction was further highlighted by the observation that short interfering RNA knockdown of STAT1 significantly decreased apoptosis induced by STAT3 inhibition. Thus, STAT1 is a tumor suppressor in ALK+ ALCL. Phosphorylation and downregulation of STAT1 by NPM-ALK represent other mechanisms by which this oncogenic tyrosine kinase promotes tumorigenesis.

  1. SHP1 tyrosine phosphatase negatively regulates NPM-ALK tyrosine kinase signaling.

    PubMed

    Honorat, Jean-François; Ragab, Ashraf; Lamant, Laurence; Delsol, Georges; Ragab-Thomas, Jeannie

    2006-05-15

    Anaplastic large-cell lymphoma (ALCL) is frequently associated with the 2;5 translocation and expresses the NPM-ALK fusion protein, which possesses a constitutive tyrosine kinase activity. We analyzed SHP1 tyrosine phosphatase expression and activity in 3 ALK-positive ALCL cell lines (Karpas 299, Cost, and SU-DHL1) and in lymph node biopsies (n = 40). We found an inverse correlation between the level of NPM-ALK phosphorylation and SHP1 phosphatase activity. Pull-down and coimmunoprecipitation experiments demonstrated a SHP1/NPM-ALK association. Furthermore, confocal microscopy performed on ALCL cell lines and biopsy specimens showed the colocalization of the 2 proteins in cytoplasmic bodies containing Y664-phosphorylated NPM-ALK. Dephosphorylation of NPM-ALK by SHP1 demonstrated that NPM-ALK was a SHP1 substrate. Downregulation of SHP1 expression by RNAi in Karpas cells led to hyperphosphorylation of NPM-ALK, STAT3 activation, and increase in cell proliferation. Furthermore, SHP1 overexpression in 3T3 fibroblasts stably expressing NPM-ALK led to the decrease of NPM-ALK phosphorylation, lower cell proliferation, and tumor progression in nude mice. These findings show that SHP1 is a negative regulator of NPM-ALK signaling. The use of tissue microarrays revealed that 50% of ALK-positive ALCLs were positive for SHP1. Our results suggest that SHP1 could be a critical enzyme in ALCL biology and a potential therapeutic target.

  2. 40 CFR 721.435 - Alkylphenylpolyether-alk-a-nol-a-mines (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Alkylphenylpolyether-alk-a-nol-a-mines... Specific Chemical Substances § 721.435 Alkylphenylpolyether-alk-a-nol-a-mines (generic). (a) Chemical... as alkylphenylpolyether-alk-a-nol-a-mines (PMNs P-97-880/881/882) are subject to reporting under...

  3. 40 CFR 721.435 - Alkylphenylpolyether-alk-a-nol-a-mines (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Alkylphenylpolyether-alk-a-nol-a-mines... Specific Chemical Substances § 721.435 Alkylphenylpolyether-alk-a-nol-a-mines (generic). (a) Chemical... as alkylphenylpolyether-alk-a-nol-a-mines (PMNs P-97-880/881/882) are subject to reporting under...

  4. Analysis of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-reactive CD8(+) T cell responses in children with NPM-ALK(+) anaplastic large cell lymphoma.

    PubMed

    K Singh, V; Werner, S; Hackstein, H; Lennerz, V; Reiter, A; Wölfel, T; Damm-Welk, C; Woessmann, W

    2016-10-01

    Cellular immune responses against the oncoantigen anaplastic lymphoma kinase (ALK) in patients with ALK-positive anaplastic large cell lymphoma (ALCL) have been detected using peptide-based approaches in individuals preselected for human leucocyte antigen (HLA)-A*02:01. In this study, we aimed to evaluate nucleophosmin (NPM)-ALK-specific CD8(+) T cell responses in ALCL patients ensuring endogenous peptide processing of ALK antigens and avoiding HLA preselection. We also examined the HLA class I restriction of ALK-specific CD8(+) T cells. Autologous dendritic cells (DCs) transfected with in-vitro-transcribed RNA (IVT-RNA) encoding NPM-ALK were used as antigen-presenting cells for T cell stimulation. Responder T lymphocytes were tested in interferon-gamma enzyme-linked immunospot (ELISPOT) assays with NPM-ALK-transfected autologous DCs as well as CV-1 in Origin with SV40 genes (COS-7) cells co-transfected with genes encoding the patients' HLA class I alleles and with NPM-ALK encoding cDNA to verify responses and define the HLA restrictions of specific T cell responses. NPM-ALK-specific CD8(+) T cell responses were detected in three of five ALK-positive ALCL patients tested between 1 and 13 years after diagnosis. The three patients had also maintained anti-ALK antibody responses. No reactivity was detected in samples from five healthy donors. The NPM-ALK-specific CD8(+) T cell responses were restricted by HLA-C-alleles (C*06:02 and C*12:02) in all three cases. This approach allowed for the detection of NPM-ALK-reactive T cells, irrespective of the individual HLA status, up to 9 years after ALCL diagnosis.

  5. ALK positivity on pleuroscopic pleural biopsy in lung adenocarcinoma.

    PubMed

    Vaidya, Preyas J; Kate, Arvind H; Mehta, Deval; Dhabar, Boman N; Chhajed, Prashant N

    2016-01-01

    Lung cancer is the leading cause of cancer deaths worldwide, and around 75% to 80% of lung cancers are detected in advanced stage. Multiple genetic mutations are identified and reported in adenocarcinoma of the lung. Various pulmonary samples can be tested for molecular mutations in lung cancer. However, feasibility of molecular profiling of pleuroscopic pleural biopsies in lung adenocarcinoma is not reported. We describe a case of advanced adenocarcinoma of lung with positive anaplastic lymphoma tyrosine kinase mutation on pleuroscopic pleural biopsy and improved with oral crizotinib. The current case highlights the feasibility of pleuroscopy.-guided pleural biopsies in molecular profiling of lung adenocarcinoma. PMID:27461706

  6. SUMOylation Confers Posttranslational Stability on NPM-ALK Oncogenic Protein1

    PubMed Central

    Vishwamitra, Deeksha; Curry, Choladda V.; Shi, Ping; Alkan, Serhan; Amin, Hesham M.

    2015-01-01

    Nucleophosmin-anaplastic lymphoma kinase–expressing (NPM-ALK+) T-cell lymphoma is an aggressive form of cancer that commonly affects children and adolescents. The expression of NPM-ALK chimeric oncogene results from the chromosomal translocation t(2;5)(p23;q35) that causes the fusion of the ALK and NPM genes. This translocation generates the NPM-ALK protein tyrosine kinase that forms the constitutively activated NPM-ALK/NPM-ALK homodimers. In addition, NPM-ALK is structurally associated with wild-type NPM to form NPM/NPM-ALK heterodimers, which can translocate to the nucleus. The mechanisms that sustain the stability of NPM-ALK are not fully understood. SUMOylation is a posttranslational modification that is characterized by the reversible conjugation of small ubiquitin-like modifiers (SUMOs) with target proteins. SUMO competes with ubiquitin for substrate binding and therefore, SUMOylation is believed to protect target proteins from proteasomal degradation. Moreover, SUMOylation contributes to the subcellular distribution of target proteins. Herein, we found that the SUMOylation pathway is deregulated in NPM-ALK+ T-cell lymphoma cell lines and primary lymphoma tumors from patients. We also identified Lys24 and Lys32 within the NPM domain as the sites where NPM-ALK conjugates with SUMO-1 and SUMO-3. Importantly, antagonizing SUMOylation by the SENP1 protease decreased the accumulation of NPM-ALK and suppressed lymphoma cell viability, proliferation, and anchorage-independent colony formation. One possible mechanism for the SENP1-mediated decrease in NPM-ALK levels was the increase in NPM-ALK association with ubiquitin, which facilitates its degradation. Our findings propose a model in which aberrancies in SUMOylation contribute to the pathogenesis of NPM-ALK+ T-cell lymphoma. Unraveling such pathogenic mechanisms may lead to devising novel strategies to eliminate this aggressive neoplasm. PMID:26476082

  7. A novel crosstalk between Alk7 and cGMP signaling differentially regulates brown adipocyte function

    PubMed Central

    Balkow, Aileen; Jagow, Johanna; Haas, Bodo; Siegel, Franziska; Kilić, Ana; Pfeifer, Alexander

    2015-01-01

    Objective Obesity is an enormous burden for patients and health systems world-wide. Brown adipose tissue dissipates energy in response to cold and has been shown to be metabolically active in human adults. The type I transforming growth factor β (TGFβ) receptor Activin receptor-like kinase 7 (Alk7) is highly expressed in adipose tissues and is down-regulated in obese patients. Here, we studied the function of Alk7 in brown adipocytes. Methods Using pharmacological and genetic tools, Alk7 signaling pathway and its effects were studied in murine brown adipocytes. Brown adipocyte differentiation and activation was analyzed. Results Alk7 is highly upregulated during differentiation of brown adipocytes. Interestingly, Alk7 expression is increased by cGMP/protein kinase G (PKG) signaling, which enhances brown adipocyte differentiation. Activin AB effectively activates Alk7 and SMAD3 signaling. Activation of Alk7 in brown preadipocytes suppresses the master adipogenic transcription factor PPARγ and differentiation. Stimulation of Alk7 during late differentiation of brown adipocytes reduces lipid content and adipogenic marker expression but enhances UCP1 expression. Conclusions We found a so far unknown crosstalk between cGMP and Alk7 signaling pathways. Tight regulation of Alk7 is required for efficient differentiation of brown adipocytes. Alk7 has differential effects on adipogenic differentiation and the development of the thermogenic program in brown adipocytes. PMID:26266090

  8. IGF-IR tyrosine kinase interacts with NPM-ALK oncogene to induce survival of T-cell ALK+ anaplastic large-cell lymphoma cells.

    PubMed

    Shi, Ping; Lai, Raymond; Lin, Quan; Iqbal, Abid S; Young, Leah C; Kwak, Larry W; Ford, Richard J; Amin, Hesham M

    2009-07-01

    Type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase plays important roles in the pathogenesis of several malignancies. Although it promotes the growth of stimulated hematopoietic cells, a direct role of IGF-IR in malignant lymphoma has not been identified. Anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma (ALK(+) ALCL) is a unique type of T-cell lymphoma. Approximately 85% of ALK(+) ALCL cases harbor the translocation t(2;5)(p23;q35), which generates the chimeric oncogene NPM-ALK. In the present study, we explored a possible role of IGF-IR in ALK(+) ALCL. Our results demonstrate that IGF-IR and IGF-I are widely expressed in ALK(+) ALCL cell lines and primary tumors. Importantly, we identified novel reciprocal functional interactions between IGF-IR and NPM-ALK. Antagonism of IGF-IR decreased the viability, induced apoptosis and cell-cycle arrest, and decreased proliferation and colony formation of ALK(+) ALCL cell lines. These effects could be explained by alterations of cell survival regulatory proteins downstream of IGF-IR signaling. Our findings improve current understanding of the biology of IGF-IR and NPM-ALK and have significant therapeutic implications as they identify IGF-IR signaling as a potential therapeutic target in ALK(+) ALCL and possibly other types of malignant lymphoma.

  9. IGF-IR tyrosine kinase interacts with NPM-ALK oncogene to induce survival of T-cell ALK+ anaplastic large-cell lymphoma cells

    PubMed Central

    Shi, Ping; Lai, Raymond; Lin, Quan; Iqbal, Abid S.; Young, Leah C.; Kwak, Larry W.; Ford, Richard J.

    2009-01-01

    Type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase plays important roles in the pathogenesis of several malignancies. Although it promotes the growth of stimulated hematopoietic cells, a direct role of IGF-IR in malignant lymphoma has not been identified. Anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma (ALK+ ALCL) is a unique type of T-cell lymphoma. Approximately 85% of ALK+ ALCL cases harbor the translocation t(2;5)(p23;q35), which generates the chimeric oncogene NPM-ALK. In the present study, we explored a possible role of IGF-IR in ALK+ ALCL. Our results demonstrate that IGF-IR and IGF-I are widely expressed in ALK+ ALCL cell lines and primary tumors. Importantly, we identified novel reciprocal functional interactions between IGF-IR and NPM-ALK. Antagonism of IGF-IR decreased the viability, induced apoptosis and cell-cycle arrest, and decreased proliferation and colony formation of ALK+ ALCL cell lines. These effects could be explained by alterations of cell survival regulatory proteins downstream of IGF-IR signaling. Our findings improve current understanding of the biology of IGF-IR and NPM-ALK and have significant therapeutic implications as they identify IGF-IR signaling as a potential therapeutic target in ALK+ ALCL and possibly other types of malignant lymphoma. PMID:19423729

  10. Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity

    PubMed Central

    Alsamarah, Abdelaziz; LaCuran, Alecander E.; Oelschlaeger, Peter; Hao, Jijun; Luo, Yun

    2015-01-01

    Abnormal alteration of bone morphogenetic protein (BMP) signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI) to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2) tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD) simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5) or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2), as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189) will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling. PMID:26133550

  11. Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity.

    PubMed

    Alsamarah, Abdelaziz; LaCuran, Alecander E; Oelschlaeger, Peter; Hao, Jijun; Luo, Yun

    2015-01-01

    Abnormal alteration of bone morphogenetic protein (BMP) signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI) to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2) tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD) simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5) or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2), as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189) will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling. PMID:26133550

  12. Activin Receptor-Like Kinase Receptors ALK5 and ALK1 Are Both Required for TGFβ-Induced Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    de Kroon, Laurie M. G.; Narcisi, Roberto; Blaney Davidson, Esmeralda N.; Cleary, Mairéad A.; van Beuningen, Henk M.; Koevoet, Wendy J. L. M.; van Osch, Gerjo J. V. M.; van der Kraan, Peter M.

    2015-01-01

    Introduction Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor β (TGFβ) is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK) receptors ALK5 and ALK1. Since the specific role of these two TGFβ receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs. Materials & Methods ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFβ1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca) receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1. Results ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFβ receptors did not clearly associate with chondrogenic induction. TGFβ increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFβ. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFβ-induced chondrogenesis. Conclusion ALK5 as well as ALK1 are required for TGFβ-induced chondrogenic differentiation of BMSCs, and TGFβ not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by

  13. Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes.

    PubMed

    van Beilen, J B; Panke, S; Lucchini, S; Franchini, A G; Röthlisberger, M; Witholt, B

    2001-06-01

    The Pseudomonas putida GPo1 (commonly known as Pseudomonas oleovorans GPo1) alkBFGHJKL and alkST gene clusters, which encode proteins involved in the conversion of n-alkanes to fatty acids, are located end to end on the OCT plasmid, separated by 9.7 kb of DNA. This DNA segment encodes, amongst others, a methyl-accepting transducer protein (AlkN) that may be involved in chemotaxis to alkanes. In P. putida P1, the alkBFGHJKL and alkST gene clusters are flanked by almost identical copies of the insertion sequence ISPpu4, constituting a class 1 transposon. Other insertion sequences flank and interrupt the alk genes in both strains. Apart from the coding regions of the GPo1 and P1 alk genes (80-92% sequence identity), only the alkB and alkS promoter regions are conserved. Competition experiments suggest that highly conserved inverted repeats in the alkB and alkS promoter regions bind ALKS: PMID:11390693

  14. Impact of the putative cancer stem cell markers and growth factor receptor expression on the sensitivity of ovarian cancer cells to treatment with various forms of small molecule tyrosine kinase inhibitors and cytotoxic drugs

    PubMed Central

    Puvanenthiran, Soozana; Essapen, Sharadah; Seddon, Alan M.; Modjtahedi, Helmout

    2016-01-01

    Increased expression and activation of human epidermal growth factor receptor (EGFR) and HER-2 have been reported in numerous cancers. The aim of this study was to determine the sensitivity of a large panel of human ovarian cancer cell lines (OCCLs) to treatment with various forms of small molecule tyrosine kinase inhibitors (TKIs) and cytotoxic drugs. The aim was to see if there was any association between the protein expression of various biomarkers including three putative ovarian cancer stem cell (CSC) markers (CD24, CD44, CD117/c-Kit), P-glycoprotein (P-gp), and HER family members and response to treatment with these agents. The sensitivity of 10 ovarian tumour cell lines to the treatment with various forms of HER TKIs (gefitinib, erlotinib, lapatinib, sapitinib, afatinib, canertinib, neratinib), as well as other TKIs (dasatinib, imatinib, NVP-AEW541, crizotinib) and cytotoxic agents (paclitaxel, cisplatin and doxorubicin), as single agents or in combination, was determined by SRB assay. The effect on these agents on the cell cycle distribution, and downstream signaling molecules and tumour migration were determined using flow cytometry, western blotting, and the IncuCyte Clear View cell migration assay respectively. Of the HER inhibitors, the irreversible pan-TKIs (canertinib, neratinib and afatinib) were the most effective TKIs for inhibiting the growth of all ovarian cancer cells, and for blocking the phosphorylation of EGFR, HER-2, AKT and MAPK in SKOV3 cells. Interestingly, while the majority of cancer cells were highly sensitive to treatment with dasatinib, they were relatively resistant to treatment with imatinib (i.e., IC50 >10 μM). Of the cytotoxic agents, paclitaxel was the most effective for inhibiting the growth of OCCLs, and of various combinations of these drugs, only treatment with a combination of NVP-AEW541 and paclitaxel produced a synergistic or additive anti-proliferative effect in all three cell lines examined (i.e., SKOV3, Caov3, ES2

  15. Alk7 Depleted Mice Exhibit Prolonged Cardiac Repolarization and Are Predisposed to Ventricular Arrhythmia

    PubMed Central

    Ying, Shaozhen; Cao, Hong; Hu, He; Wang, Xin; Tang, Yanhong; Huang, Congxin

    2016-01-01

    We aimed to investigate the role of activin receptor-like kinase (ALK7) in regulating cardiac electrophysiology. Here, we showed that Alk7-/- mice exhibited prolonged QT intervals in telemetry ECG recordings. Furthermore, Langendorff-perfused Alk7-/- hearts had significantly longer action potential duration (APD) and greater incidence of ventricular arrhythmia (AV) induced by burst pacing. Using whole-cell patch clamp, we found that the densities of repolarizing K+ currents Ito and IK1 were profoundly reduced in Alk7-/- ventricular cardiomyocytes. Mechanistically, the expression of Kv4.2 (a major subunit of Ito carrying channel) and KCHIP2 (a key accessory subunit of Ito carrying channel), was markedly decreased in Alk7-/- hearts. These findings suggest that endogenous expression of ALK7 is necessary to maintain repolarizing K+ currents in ventricular cardiomyocytes, and finally prevent action potential prolongation and ventricular arrhythmia. PMID:26882027

  16. New strategies in neuroblastoma: Therapeutic targeting of MYCN and ALK.

    PubMed

    Barone, Giuseppe; Anderson, John; Pearson, Andrew D J; Petrie, Kevin; Chesler, Louis

    2013-11-01

    Clinical outcome remains poor in patients with high-risk neuroblastoma, in which chemoresistant relapse is common following high-intensity conventional multimodal therapy. Novel treatment approaches are required. Although recent genomic profiling initiatives have not revealed a high frequency of mutations in any significant number of therapeutically targeted genes, two exceptions, amplification of the MYCN oncogene and somatically acquired tyrosine kinase domain point mutations in anaplastic lymphoma kinase (ALK), present exciting possibilities for targeted therapy. In contrast with the situation with ALK, in which a robust pipeline of pharmacologic agents is available from early clinical use in adult malignancy, therapeutic targeting of MYCN (and MYC oncoproteins in general) represents a significant medicinal chemistry challenge that has remained unsolved for two decades. We review the latest approaches envisioned for blockade of ALK activity in neuroblastoma, present a classification of potential approaches for therapeutic targeting of MYCN, and discuss how recent developments in targeting of MYC proteins seem to make therapeutic inhibition of MYCN a reality in the clinic.

  17. A "liaison dangereuse" between AUF1/hnRNPD and the oncogenic tyrosine kinase NPM-ALK.

    PubMed

    Fawal, Mohamad; Armstrong, Florence; Ollier, Severine; Dupont, Henri; Touriol, Christian; Monsarrat, Bernard; Delsol, Georges; Payrastre, Bernard; Morello, Dominique

    2006-10-15

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a chimeric protein expressed in a subset of cases of anaplastic large cell lymphoma (ALCL) for which constitutive expression represents a key oncogenic event. The ALK signaling pathway is complex and probably involves functional redundancy between various signaling substrates of ALK. Despite numerous studies on signaling mediators, the molecular mechanisms contributing to the distinct oncogenic features of NPM-ALK remain incompletely understood. The search for additional interacting partners of NPM-ALK led to the discovery of AUF1/hnRNPD, a protein implicated in AU-rich element (ARE)-directed mRNA decay. AUF1 was immunoprecipitated with ALK both in ALCL-derived cells and in NIH3T3 cells stably expressing NPM-ALK or other X-ALK fusion proteins. AUF1 and NPM-ALK were found concentrated in the same cytoplasmic foci, whose formation required NPM-ALK tyrosine kinase activity. AUF1 was phosphorylated by ALK in vitro and was hyperphosphorylated in NPM-ALK-expressing cells. Its hyperphosphorylation was correlated with increased stability of several AUF1 target mRNAs encoding key regulators of cell proliferation and with increased cell survival after transcriptional arrest. Thus, AUF1 could function in a novel pathway mediating the oncogenic effects of NPM-ALK. Our data establish an important link between oncogenic kinases and mRNA turnover, which could constitute a critical aspect of tumorigenesis.

  18. Extended Survival and Prognostic Factors for Patients With ALK-Rearranged Non–Small-Cell Lung Cancer and Brain Metastasis

    PubMed Central

    Johung, Kimberly L.; Yeh, Norman; Desai, Neil B.; Williams, Terence M.; Lautenschlaeger, Tim; Arvold, Nils D.; Ning, Matthew S.; Attia, Albert; Lovly, Christine M.; Goldberg, Sarah; Beal, Kathryn; Yu, James B.; Kavanagh, Brian D.; Chiang, Veronica L.; Camidge, D. Ross

    2016-01-01

    Purpose We performed a multi-institutional study to identify prognostic factors and determine outcomes for patients with ALK-rearranged non–small-cell lung cancer (NSCLC) and brain metastasis. Patients and Methods A total of 90 patients with brain metastases from ALK-rearranged NSCLC were identified from six institutions; 84 of 90 patients received radiotherapy to the brain (stereotactic radiosurgery [SRS] or whole-brain radiotherapy [WBRT]), and 86 of 90 received tyrosine kinase inhibitor (TKI) therapy. Estimates for overall (OS) and intracranial progression-free survival were determined and clinical prognostic factors were identified by Cox proportional hazards modeling. Results Median OS after development of brain metastases was 49.5 months (95% CI, 29.0 months to not reached), and median intracranial progression-free survival was 11.9 months (95% CI, 10.1 to 18.2 months). Forty-five percent of patients with follow-up had progressive brain metastases at death, and repeated interventions for brain metastases were common. Absence of extracranial metastases, Karnofsky performance score ≥ 90, and no history of TKIs before development of brain metastases were associated with improved survival (P = .003, < .001, and < .001, respectively), whereas a single brain metastasis or initial treatment with SRS versus WBRT were not (P = .633 and .666, respectively). Prognostic factors significant by multivariable analysis were used to describe four patient groups with 2-year OS estimates of 33%, 59%, 76%, and 100%, respectively (P < .001). Conclusion Patients with brain metastases from ALK-rearranged NSCLC treated with radiotherapy (SRS and/or WBRT) and TKIs have prolonged survival, suggesting that interventions to control intracranial disease are critical. The refinement of prognosis for this molecular subtype of NSCLC identifies a population of patients likely to benefit from first-line SRS, close CNS observation, and treatment of emergent CNS disease. PMID:26438117

  19. BMP15 suppresses progesterone production by down-regulating StAR via ALK3 in human granulosa cells.

    PubMed

    Chang, Hsun-Ming; Cheng, Jung-Chien; Klausen, Christian; Leung, Peter C K

    2013-12-01

    In addition to somatic cell-derived growth factors, oocyte-derived growth differentiation factor (GDF)9 and bone morphogenetic protein (BMP)15 play essential roles in female fertility. However, few studies have investigated their effects on human ovarian steroidogenesis, and fewer still have examined their differential effects or underlying molecular determinants. In the present study, we used immortalized human granulosa cells (SVOG) and human granulosa cell tumor cells (KGN) to compare the effects of GDF9 and BMP15 on steroidogenic enzyme expression and investigate potential mechanisms of action. In SVOG cells, neither GDF9 nor BMP15 affects the mRNA levels of P450 side-chain cleavage enzyme or 3β-hydroxysteroid dehydrogenase. However, treatment with BMP15, but not GDF9, significantly decreases steroidogenic acute regulatory protein (StAR) mRNA and protein levels as well as progesterone production. These suppressive effects, along with the induction of Sma and Mad-related protein (SMAD)1/5/8 phosphorylation, are attenuated by cotreatment with 2 different BMP type I receptor inhibitors (dorsomorphin and DMH-1). Furthermore, depletion of activin receptor-like kinase (ALK)3 using small interfering RNA reverses the effects of BMP15 on SMAD1/5/8 phosphorylation and StAR expression. Similarly, knockdown of ALK3 abolishes BMP15-induced SMAD1/5/8 phosphorylation in KGN cells. These results provide evidence that oocyte-derived BMP15 down-regulates StAR expression and decreases progesterone production in human granulosa cells, likely via ALK3-mediated SMAD1/5/8 signaling. Our findings suggest that oocyte may play a critical role in the regulation of progesterone to prevent premature luteinization during the late stage of follicle development.

  20. BMP-9 regulates the osteoblastic differentiation and calcification of vascular smooth muscle cells through an ALK1 mediated pathway.

    PubMed

    Zhu, Dongxing; Mackenzie, Neil Charles Wallace; Shanahan, Catherine M; Shroff, Rukshana C; Farquharson, Colin; MacRae, Vicky Elizabeth

    2015-01-01

    The process of vascular calcification shares many similarities with that of physiological skeletal mineralization, and involves the deposition of hydroxyapatite crystals in arteries. However, the cellular mechanisms responsible have yet to be fully explained. Bone morphogenetic protein (BMP-9) has been shown to exert direct effects on both bone development and vascular function. In the present study, we have investigated the role of BMP-9 in vascular smooth muscle cell (VSMC) calcification. Vessel calcification in chronic kidney disease (CKD) begins pre-dialysis, with factors specific to the dialysis milieu triggering accelerated calcification. Intriguingly, BMP-9 was markedly elevated in serum from CKD children on dialysis. Furthermore, in vitro studies revealed that BMP-9 treatment causes a significant increase in VSMC calcium content, alkaline phosphatase (ALP) activity and mRNA expression of osteogenic markers. BMP-9-induced calcium deposition was significantly reduced following treatment with the ALP inhibitor 2,5-Dimethoxy-N-(quinolin-3-yl) benzenesulfonamide confirming the mediatory role of ALP in this process. The inhibition of ALK1 signalling using a soluble chimeric protein significantly reduced calcium deposition and ALP activity, confirming that BMP-9 is a physiological ALK1 ligand. Signal transduction studies revealed that BMP-9 induced Smad2, Smad3 and Smad1/5/8 phosphorylation. As these Smad proteins directly bind to Smad4 to activate target genes, siRNA studies were subsequently undertaken to examine the functional role of Smad4 in VSMC calcification. Smad4-siRNA transfection induced a significant reduction in ALP activity and calcium deposition. These novel data demonstrate that BMP-9 induces VSMC osteogenic differentiation and calcification via ALK1, Smad and ALP dependent mechanisms. This may identify new potential therapeutic strategies for clinical intervention.

  1. GDF-15 inhibits integrin activation and mouse neutrophil recruitment through the ALK-5/TGF-βRII heterodimer.

    PubMed

    Artz, Annette; Butz, Stefan; Vestweber, Dietmar

    2016-07-28

    Growth differentiation factor 15 (GDF-15) is the first cytokine known to counteract chemokine-induced activation of leukocyte integrins. We showed recently that this activity dampens neutrophil recruitment into inflamed tissue and is required for survival of myocardial infarction in mice. The receptor responsible for this GDF-15-triggered anti-inflammatory mechanism on myeloid cells is not known. Here, we identify this receptor as transforming growth factor β receptor I (TGF-βRI) (activin receptor-like kinase 5 [ALK-5]) and TGF-β receptor II (TGF-βRII). We show that interference with these receptors by small-molecule inhibitors, antibodies, or small interfering RNA, blocked the GDF-15 effect on leukocyte integrin activation. Likewise, gene inactivation of each of the 2 receptors in neutrophils isolated from conditional gene-deficient mice abolished the inhibitory effect of GDF-15 on CXCL1-induced β2-integrin activation and neutrophil diapedesis. Rapid neutrophil arrest induced by CXCL1 in vivo was inhibited by GDF-15 in an ALK-5 and TGF-βRII dependent way. As for GDF-15 gene-deficient mice, we found that extravasation of neutrophils deficient for ALK-5 or TGF-βRII was strongly increased in the interleukin-1β inflamed cremaster. The inhibitory effects of GDF-15 on neutrophil integrin activation and in vivo neutrophil arrest were also found for TGF-β1. Mechanistically, GDF-15 and TGF-β1 interfered with integrin activation by inhibiting the activation of Ras-related protein 1 (Rap-1), an effect that depended on CalDAG- guanine nucleotide exchange factor 1 (GEF1) and cell division control protein 42 homolog. We conclude that both GDF-15 and TGF-β1 counteract chemokine-induced integrin activation on neutrophils via the ALK-5/TGF-βRII heterodimer. This represents a novel, rapid anti-inflammatory activity of the 2 TGF-β receptors and of TGF-β1. PMID:27235139

  2. RANBP2-ALK fusion combined with monosomy 7 in acute myelomonocytic leukemia.

    PubMed

    Lim, Ji-Hun; Jang, Seongsoo; Park, Chan-Jeoung; Cho, Young-Uk; Lee, Je-Hwan; Lee, Kyoo-Hyung; Lee, Jin-Ok; Shin, Jong-Yeon; Kim, Jong-Il; Huh, Jooryung; Seo, Eul-Ju

    2014-01-01

    Anaplastic lymphoma receptor tyrosine kinase (ALK) is located on chromosome 2p23; the chromosomal rearrangements of this gene are common genetic alterations, resulting in the creation of multiple fusion genes involved in tumorigenesis. However, the presence of an ALK fusion in myeloid malignancies is extremely rare. We report a case of acute myelomonocytic leukemia in a 31-year-old woman with an unusual rearrangement between RAN-binding protein 2 (RANBP2) and ALK and a karyotype of 45,XX,inv(2)(p23q21),-7[20]. We detected an ALK rearrangement using fluorescence in situ hybridization, identified the ALK fusion partner by using RNA transcriptome sequencing, and demonstrated the RANBP2-ALK fusion transcript by reverse transcriptase--PCR and Sanger sequencing. Immunohistochemistry for ALK showed strong staining of the nuclear membrane in leukemic cells. The patient had an unfavorable clinical course. Our results, together with a literature review, suggest the RANBP2-ALK fusion combined with monosomy 7 may be related to a unique clonal hematologic disorder of childhood and adolescence, characterized by myelomonocytic leukemia and a poor prognosis.

  3. Shear induced collateral artery growth modulated by endoglin but not by ALK1

    PubMed Central

    Seghers, Leonard; de Vries, Margreet R; Pardali, Evangelia; Hoefer, Imo E; Hierck, Beerend P; ten Dijke, Peter ten; Goumans, Marie Jose; Quax, Paul HA

    2012-01-01

    Transforming growth factor-beta (TGF-β) stimulates both ischaemia induced angiogenesis and shear stress induced arteriogenesis by signalling through different receptors. How these receptors are involved in both these processes of blood flow recovery is not entirely clear. In this study the role of TGF-β receptors 1 and endoglin is assessed in neovascularization in mice. Unilateral femoral artery ligation was performed in mice heterozygous for either endoglin or ALK1 and in littermate controls. Compared with littermate controls, blood flow recovery, monitored by laser Doppler perfusion imaging, was significantly hampered by maximal 40% in endoglin heterozygous mice and by maximal 49% in ALK1 heterozygous mice. Collateral artery size was significantly reduced in endoglin heterozygous mice compared with controls but not in ALK1 heterozygous mice. Capillary density in ischaemic calf muscles was unaffected, but capillaries from endoglin and ALK1 heterozygous mice were significantly larger when compared with controls. To provide mechanistic evidence for the differential role of endoglin and ALK1 in shear induced or ischaemia induced neovascularization, murine endothelial cells were exposed to shear stress in vitro. This induced increased levels of endoglin mRNA but not ALK1. In this study it is demonstrated that both endoglin and ALK1 facilitate blood flow recovery. Importantly, endoglin contributes to both shear induced collateral artery growth and to ischaemia induced angiogenesis, whereas ALK1 is only involved in ischaemia induced angiogenesis. PMID:22436015

  4. Lobatin B inhibits NPM/ALK and NF-κB attenuating anaplastic-large-cell-lymphomagenesis and lymphendothelial tumour intravasation.

    PubMed

    Kiss, Izabella; Unger, Christine; Huu, Chi Nguyen; Atanasov, Atanas Georgiev; Kramer, Nina; Chatruphonprasert, Waranya; Brenner, Stefan; McKinnon, Ruxandra; Peschel, Andrea; Vasas, Andrea; Lajter, Ildiko; Kain, Renate; Saiko, Philipp; Szekeres, Thomas; Kenner, Lukas; Hassler, Melanie R; Diaz, Rene; Frisch, Richard; Dirsch, Verena M; Jäger, Walter; de Martin, Rainer; Bochkov, Valery N; Passreiter, Claus M; Peter-Vörösmarty, Barbara; Mader, Robert M; Grusch, Michael; Dolznig, Helmut; Kopp, Brigitte; Zupko, Istvan; Hohmann, Judit; Krupitza, Georg

    2015-01-28

    An apolar extract of the traditional medicinal plant Neurolaena lobata inhibited the expression of the NPM/ALK chimera, which is causal for the majority of anaplastic large cell lymphomas (ALCLs). Therefore, an active principle of the extract, the furanoheliangolide sesquiterpene lactone lobatin B, was isolated and tested regarding the inhibition of ALCL expansion and tumour cell intravasation through the lymphendothelium. ALCL cell lines, HL-60 cells and PBMCs were treated with plant compounds and the ALK inhibitor TAE-684 to measure mitochondrial activity, proliferation and cell cycle progression and to correlate the results with protein- and mRNA-expression of selected gene products. Several endpoints indicative for cell death were analysed after lobatin B treatment. Tumour cell intravasation through lymphendothelial monolayers was measured and potential causal mechanisms were investigated analysing NF-κB- and cytochrome P450 activity, and 12(S)-HETE production. Lobatin B inhibited the expression of NPM/ALK, JunB and PDGF-Rβ, and attenuated proliferation of ALCL cells by arresting them in late M phase. Mitochondrial activity remained largely unaffected upon lobatin B treatment. Nevertheless, caspase 3 became activated in ALCL cells. Also HL-60 cell proliferation was attenuated whereas PBMCs of healthy donors were not affected by lobatin B. Additionally, tumour cell intravasation, which partly depends on NF-κB, was significantly suppressed by lobatin B most likely due to its NF-κB-inhibitory property. Lobatin B, which was isolated from a plant used in ethnomedicine, targets malignant cells by at least two properties: I) inhibition of NPM/ALK, thereby providing high specificity in combating this most prevalent fusion protein occurring in ALCL; II) inhibition of NF-κB, thereby not affecting normal cells with low constitutive NF-κB activity. This property also inhibits tumour cell intravasation into the lymphatic system and may provide an option to manage this

  5. Transforming growth factor-beta type I receptor/ALK5 contributes to doxazosin-induced apoptosis in H9C2 cells.

    PubMed

    Yang, Yi-Fan; Wu, Chau-Chung; Chen, Wen-Pin; Su, Ming-Jai

    2009-12-01

    The mechanism of doxazosin-induced apoptosis through alpha(1)-adrenoceptor-independent pathway has been reported in various types of cell models. However, the molecular events involved in this effect are still not fully discovered. In present study, we proposed that the transforming growth factor-beta type I receptor (TbetaRI/ALK5) may contribute to the doxazosin-induced apoptosis in H9C2 cardiomyoblasts. Via the detection of cell viability, apoptotic nuclei, and caspase-3 activity, we found that doxazosin induced concentration- and time-dependent apoptosis in H9C2 cells. The cell apoptosis induced by 30 muM doxazosin was exacerbated by the addition of 10 ng/ml transforming growth factor-beta1 (TGF-beta1). Doxazosin or TGF-beta1 alone respectively elevated p38 mitogen-activated protein kinases (MAPK) and Smad3 protein phosphorylation in H9C2 cells. However, the cotreatment of doxazosin and TGF-beta1 attenuated the TGF-beta1-induced Smad3 protein phosphorylation and increased doxazosin-induced p38 MAPK protein phosphorylation. Furthermore, inhibitors of TbetaRI/ALK5 (SB431542) and p38 MAPK (SB202190) or TbetaRI/ALK5 knockdown all dramatically reduced the doxazosin-induced apoptosis in H9C2 cells. In conclusion, our results demonstrated that TbetaRI/ALK5-p38 MAPK phosphorylation signaling pathway could contribute to doxazosin-induced cell apoptosis, which could be further enhanced by TGF-beta1 in association with attenuating Smad3 phosphorylation in H9C2 cells.

  6. USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling

    PubMed Central

    Herhaus, Lina; Al-Salihi, Mazin A.; Dingwell, Kevin S.; Cummins, Timothy D.; Wasmus, Lize; Vogt, Janis; Ewan, Richard; Bruce, David; Macartney, Thomas; Weidlich, Simone; Smith, James C.; Sapkota, Gopal P.

    2014-01-01

    Protein kinase ALK3/BMPR1A mediates bone morphogenetic protein (BMP) signalling through phosphorylation and activation of SMADs 1/5/8. SMAD6, a transcriptional target of BMP, negatively regulates the BMP pathway by recruiting E3 ubiquitin ligases and targeting ALK3 for ubiquitin-mediated degradation. Here, we identify a deubiquitylating enzyme USP15 as an interactor of SMAD6 and ALK3. We show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3. RNAi-mediated depletion of USP15 increases ALK3 K48-linked polyubiquitylation, and reduces both BMP-induced SMAD1 phosphorylation and transcription of BMP target genes. We also show that loss of USP15 expression from mouse myoblast cells inhibits BMP-induced osteoblast differentiation. Furthermore, USP15 modulates BMP-induced phosphorylation of SMAD1 and transcription during Xenopus embryogenesis. PMID:24850914

  7. USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling.

    PubMed

    Herhaus, Lina; Al-Salihi, Mazin A; Dingwell, Kevin S; Cummins, Timothy D; Wasmus, Lize; Vogt, Janis; Ewan, Richard; Bruce, David; Macartney, Thomas; Weidlich, Simone; Smith, James C; Sapkota, Gopal P

    2014-05-01

    Protein kinase ALK3/BMPR1A mediates bone morphogenetic protein (BMP) signalling through phosphorylation and activation of SMADs 1/5/8. SMAD6, a transcriptional target of BMP, negatively regulates the BMP pathway by recruiting E3 ubiquitin ligases and targeting ALK3 for ubiquitin-mediated degradation. Here, we identify a deubiquitylating enzyme USP15 as an interactor of SMAD6 and ALK3. We show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3. RNAi-mediated depletion of USP15 increases ALK3 K48-linked polyubiquitylation, and reduces both BMP-induced SMAD1 phosphorylation and transcription of BMP target genes. We also show that loss of USP15 expression from mouse myoblast cells inhibits BMP-induced osteoblast differentiation. Furthermore, USP15 modulates BMP-induced phosphorylation of SMAD1 and transcription during Xenopus embryogenesis. PMID:24850914

  8. VCL-ALK Renal Cell Carcinoma in Children With Sickle-cell Trait

    PubMed Central

    Smith, Nathaniel E.; Deyrup, Andrea T.; Marinño-Enriquez, Adrian; Fletcher, Jonathan A.; Bridge, Julia A.; Illei, Peter B.; Netto, George J.; Argani, Pedram

    2015-01-01

    We report the third case of a renal cell carcinoma bearing a fusion of the vinculin (VCL) and anaplastic lymphoma kinase (ALK) genes. Like the 2 other reported cases, this neoplasm occurred in a young patient (6 y old) with sickle-cell trait and demonstrated distinctive morphologic features including medullary epicenter, discohesive polygonal or spindle-shaped cells with prominent cytoplasmic vacuoles, and prominent lymphocytic infiltrate. The neoplastic cells demonstrated focal membranous labeling for ALK protein by immunohistochemistry, ALK gene rearrangement by fluorescence in situ hybridization, and a specific VCL-ALK gene fusion by reverse transcriptase polymerase chain reaction. VCL-ALK renal cell carcinoma may represent the eighth sickle-cell nephropathy. PMID:24698962

  9. NPM/ALK binds and phosphorylates the RNA/DNA-binding protein PSF in anaplastic large-cell lymphoma.

    PubMed

    Galietta, Annamaria; Gunby, Rosalind H; Redaelli, Sara; Stano, Paola; Carniti, Cristiana; Bachi, Angela; Tucker, Philip W; Tartari, Carmen J; Huang, Ching-Jung; Colombo, Emanuela; Pulford, Karen; Puttini, Miriam; Piazza, Rocco G; Ruchatz, Holger; Villa, Antonello; Donella-Deana, Arianna; Marin, Oriano; Perrotti, Danilo; Gambacorti-Passerini, Carlo

    2007-10-01

    The oncogenic fusion tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) induces cellular transformation in anaplastic large-cell lymphomas (ALCLs) carrying the t(2;5) chromosomal translocation. Protein-protein interactions involving NPM/ALK are important for the activation of downstream signaling pathways. This study was aimed at identifying novel NPM/ALK-binding proteins that might contribute to its oncogenic transformation. Using a proteomic approach, several RNA/DNA-binding proteins were found to coimmunoprecipitate with NPM/ALK, including the multifunctional polypyrimidine tract binding proteinassociated splicing factor (PSF). The interaction between NPM/ALK and PSF was dependent on an active ALK kinase domain and PSF was found to be tyrosine-phosphorylated in NPM/ALK-expressing cell lines and in primary ALK(+) ALCL samples. Furthermore, PSF was shown to be a direct substrate of purified ALK kinase domain in vitro, and PSF Tyr293 was identified as the site of phosphorylation. Y293F PSF was not phosphorylated by NPM/ALK and was not delocalized in NPM/ALK(+) cells. The expression of ALK fusion proteins induced delocalization of PSF from the nucleus to the cytoplasm and forced overexpression of PSF-inhibited proliferation and induced apoptosis in cells expressing NPM/ALK. PSF phosphorylation also increased its binding to RNA and decreased the PSF-mediated suppression of GAGE6 expression. These results identify PSF as a novel NPM/ALK-binding protein and substrate, and suggest that PSF function may be perturbed in NPM/ALK-transformed cells.

  10. NPM/ALK binds and phosphorylates the RNA/DNA-binding protein PSF in anaplastic large-cell lymphoma

    PubMed Central

    Gunby, Rosalind H.; Redaelli, Sara; Stano, Paola; Carniti, Cristiana; Bachi, Angela; Tucker, Philip W.; Tartari, Carmen J.; Huang, Ching-Jung; Colombo, Emanuela; Pulford, Karen; Puttini, Miriam; Piazza, Rocco G.; Ruchatz, Holger; Villa, Antonello; Donella-Deana, Arianna; Marin, Oriano; Perrotti, Danilo; Gambacorti-Passerini, Carlo

    2007-01-01

    The oncogenic fusion tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) induces cellular transformation in anaplastic large-cell lymphomas (ALCLs) carrying the t(2;5) chromosomal translocation. Protein-protein interactions involving NPM/ALK are important for the activation of downstream signaling pathways. This study was aimed at identifying novel NPM/ALK-binding proteins that might contribute to its oncogenic transformation. Using a proteomic approach, several RNA/DNA-binding proteins were found to coimmunoprecipitate with NPM/ALK, including the multifunctional polypyrimidine tract binding proteinassociated splicing factor (PSF). The interaction between NPM/ALK and PSF was dependent on an active ALK kinase domain and PSF was found to be tyrosine-phosphorylated in NPM/ALK-expressing cell lines and in primary ALK+ ALCL samples. Furthermore, PSF was shown to be a direct substrate of purified ALK kinase domain in vitro, and PSF Tyr293 was identified as the site of phosphorylation. Y293F PSF was not phosphorylated by NPM/ALK and was not delocalized in NPM/ALK+ cells. The expression of ALK fusion proteins induced delocalization of PSF from the nucleus to the cytoplasm and forced overexpression of PSF-inhibited proliferation and induced apoptosis in cells expressing NPM/ALK. PSF phosphorylation also increased its binding to RNA and decreased the PSF-mediated suppression of GAGE6 expression. These results identify PSF as a novel NPM/ALK-binding protein and substrate, and suggest that PSF function may be perturbed in NPM/ALK-transformed cells. PMID:17537995

  11. NPM/ALK binds and phosphorylates the RNA/DNA-binding protein PSF in anaplastic large-cell lymphoma.

    PubMed

    Galietta, Annamaria; Gunby, Rosalind H; Redaelli, Sara; Stano, Paola; Carniti, Cristiana; Bachi, Angela; Tucker, Philip W; Tartari, Carmen J; Huang, Ching-Jung; Colombo, Emanuela; Pulford, Karen; Puttini, Miriam; Piazza, Rocco G; Ruchatz, Holger; Villa, Antonello; Donella-Deana, Arianna; Marin, Oriano; Perrotti, Danilo; Gambacorti-Passerini, Carlo

    2007-10-01

    The oncogenic fusion tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) induces cellular transformation in anaplastic large-cell lymphomas (ALCLs) carrying the t(2;5) chromosomal translocation. Protein-protein interactions involving NPM/ALK are important for the activation of downstream signaling pathways. This study was aimed at identifying novel NPM/ALK-binding proteins that might contribute to its oncogenic transformation. Using a proteomic approach, several RNA/DNA-binding proteins were found to coimmunoprecipitate with NPM/ALK, including the multifunctional polypyrimidine tract binding proteinassociated splicing factor (PSF). The interaction between NPM/ALK and PSF was dependent on an active ALK kinase domain and PSF was found to be tyrosine-phosphorylated in NPM/ALK-expressing cell lines and in primary ALK(+) ALCL samples. Furthermore, PSF was shown to be a direct substrate of purified ALK kinase domain in vitro, and PSF Tyr293 was identified as the site of phosphorylation. Y293F PSF was not phosphorylated by NPM/ALK and was not delocalized in NPM/ALK(+) cells. The expression of ALK fusion proteins induced delocalization of PSF from the nucleus to the cytoplasm and forced overexpression of PSF-inhibited proliferation and induced apoptosis in cells expressing NPM/ALK. PSF phosphorylation also increased its binding to RNA and decreased the PSF-mediated suppression of GAGE6 expression. These results identify PSF as a novel NPM/ALK-binding protein and substrate, and suggest that PSF function may be perturbed in NPM/ALK-transformed cells. PMID:17537995

  12. Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

    SciTech Connect

    Amano, Yosuke; Ishikawa, Rie; Sakatani, Toshio; Ichinose, Junji; Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori; Nakajima, Jun; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2015-02-13

    Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation. - Highlights: • TPM3-ALK fusion protein dimerizes through the coiled-coil structure of TPM3. • Longer coiled-coil structure of TPM3 leads to higher TPM3-ALK dimer formation. • Presence of TPM3-ALK dimer leads to ALK, STAT3, and ERK1/2 phosphorylation. • Presence of TPM3-ALK leads to loss of contact inhibition. • BN-PAGE is a simple technique for visualizing oncogenic dimerization.

  13. [News about targeted therapies in non-small-cell lung cancer in 2015 (except immuno-therapy)].

    PubMed

    Hamard, Cécile; Ruppert, Anne-Marie; Lavole, Armelle; Rozensztajn, Nathalie; Antoine, Martine; Cadranel, Jacques; Wislez, Marie

    2016-01-01

    Recently, developments of therapies that target abnormally activated signaling pathways are increasing for patients with non-small cell lung cancer. EGFR mutations are found in about 10% of lung cancers, especially in adenocarcinoma, women and non-smokers. Three EGFR inhibitors (erlotinib, gefitinib and afatinib) received a European marketing authorization for up to first line treatment of EGFR mutated NSCLC. Effectiveness of EGFR inhibitors is higher than conventional chemotherapy. Third generation EGFR inhibitors (rociletinib, AZD9291) are effective for patients who develop a resistance mutation such as T790M resistance mutation; they obtained temporary authorization for use in France in 2015. The EML4-ALK translocation is found in about 5% of NSCLC and more particularly in adenocarcinoma of young non-smoking patients. Crizotinib is a new therapeutic standard in ALK translocated NSCLC in second line. Ceritinib is a 2nd generation ALK inhibitor which received a European marketing authorization for up to treatment of ALK translocated NSCLC after progression with crizotinib. INCA supports ACSé program evaluating the efficacy of crizotinib in NSCLC amplified for MET or translocated for ROS1 and ACSé program evaluating the efficacy of vemurafenib in tumors non melanoma mutated V600E BRAF. The role of other biomarkers such as KRAS, BRAF, HER2 and PI3KCA mutations remains to be defined in NSCLC. PMID:26775573

  14. Analysis of gene expression profile of TPM3-ALK positive anaplastic large cell lymphoma reveals overlapping and unique patterns with that of NPM-ALK positive anaplastic large cell lymphoma.

    PubMed

    Bohling, Sandra D; Jenson, Stephen D; Crockett, David K; Schumacher, Jonathan A; Elenitoba-Johnson, Kojo S J; Lim, Megan S

    2008-03-01

    Anaplastic large cell lymphoma (ALCL) comprises a group of non-Hodgkin lymphomas characterized by the expression of the CD30/Ki-1 antigen. A subset of ALCL is characterized by chromosomal translocations involving the anaplastic lymphoma kinase (ALK) gene on chromosome 2. While the most common translocation is the t(2;5)(p23;q35) involving the nucleophosmin (NPM) gene on chromosome 5, up to 12 other translocations partners of the ALK gene have been identified. One of these is the t(1;2)(q25;p23) which results in the formation of the chimeric protein TPM3-ALK. While several of the signaling pathways induced by NPM-ALK have been elucidated, those involved in ALCLs harboring TPM3-ALK are largely unknown. In order to investigate the expression profiles of ALCLs carrying the NPM-ALK and TPM3-ALK fusions, we carried out cDNA microarray analysis of two ALCL tissue samples, one expressing the NPM-ALK fusion protein and the other the TPM3-ALK fusion protein. RNA was extracted from snap-frozen tissues, labeled with fluorescent dyes and analyzed using cDNAs microarray containing approximately 9,200 genes and expressed sequence tags (ESTs). Quantitative fluorescence RT-PCR was performed to validate the cDNA microarray data on nine selected gene targets. Our results show a significant overlap of genes deregulated in the NPM-ALK and TPM-ALK positive lymphomas. These deregulated genes are involved in diverse cellular functions, such as cell cycle regulation, apoptosis, proliferation, and adhesion. Interestingly, a subset of the genes was distinct in their expression pattern in the two types of lymphomas. More importantly, many genes that were not previously associated with ALK positive lymphomas were identified. Our results demonstrate the overlapping and unique transcriptional patterns associated with the NPM-ALK and TPM3-ALK fusions in ALCL.

  15. Beyond ALK-RET, ROS1 and other oncogene fusions in lung cancer

    PubMed Central

    Nakaoku, Takashi; Tsuta, Koji; Tsuchihara, Katsuya; Matsumoto, Shingo; Yoh, Kiyotaka; Goto, Koichi

    2015-01-01

    Fusions of the RET and ROS1 protein tyrosine kinase oncogenes with several partner genes were recently identified as new targetable genetic aberrations in cases of non-small cell lung cancer (NSCLC) lacking activating EGFR, KRAS, ALK, BRAF, or HER2 oncogene aberrations. RET and ROS1 fusion-positive tumors are mainly observed in young, female, and/or never smoking patients. Studies based on in vitro and in vivo (i.e., mouse) models and studies of several fusion-positive patients indicate that inhibiting the kinase activity of the RET and ROS1 fusion proteins is a promising therapeutic strategy. Accordingly, there are several ongoing clinical trials aimed at examining the efficacy of tyrosine kinase inhibitors (TKIs) against RET and ROS1 proteins in patients with fusion-positive lung cancer. Other gene fusions (NTRK1, NRG1, and FGFR1/2/3) that are targetable by existing TKIs have also been identified in NSCLCs. Options for personalized lung cancer therapy will be increased with the help of multiplex diagnosis systems able to detect multiple druggable gene fusions. PMID:25870798

  16. Role of the TGF-β/Alk5 Signaling Pathway in Monocrotaline-induced Pulmonary Hypertension

    PubMed Central

    Zaiman, Ari L.; Podowski, Megan; Medicherla, Satya; Gordy, Kimberley; Xu, Fang; Zhen, Lijie; Shimoda, Larissa A.; Neptune, Enid; Higgins, Linda; Murphy, Alison; Chakravarty, Sarvajit; Protter, Andrew; Sehgal, Pravin B.; Champion, Hunter C.; Tuder, Rubin M.

    2008-01-01

    Rationale: Pulmonary arterial hypertension is a progressive disease characterized by an elevation in the mean pulmonary artery pressure leading to right heart failure and a significant risk of death. Alterations in two transforming growth factor (TGF) signaling pathways, bone morphogenetic protein receptor II and the TGF-β receptor I, Alk1, have been implicated in the pathogenesis of pulmonary hypertension (PH). However, the role of TGF-β family signaling in PH and pulmonary vascular remodeling remains unclear. Objectives: To determine whether inhibition of TGF-β signaling will attenuate and reverse monocrotaline-induced PH (MCT-PH). Methods: We have used an orally active small-molecule TGF-β receptor I inhibitor, SD-208, to determine the functional role of this pathway in MCT-PH. Measurements and Main Results: The development of MCT-PH was associated with increased vascular cell apoptosis, which paralleled TGF-β signaling as documented by psmad2 expression. Inhibition of TGF-β signaling with SD-208 significantly attenuated the development of the PH and reduced pulmonary vascular remodeling. These effects were associated with decreased early vascular cell apoptosis, adventitial cell proliferation, and matrix metalloproteinase expression. Inhibition of TGF-β signaling with SD-208 in established MCT-PH resulted in a small but significant improvement in hemodynamic parameters and medial remodeling. Conclusions: These findings provide evidence that increased TGF-β signaling participates in the pathogenesis of experimental severe PH. PMID:18202349

  17. De novo cerebrovascular malformation in the adult mouse after endothelial Alk1 deletion and angiogenic stimulation

    PubMed Central

    Chen, Wanqiu; Sun, Zhengda; Han, Zhenying; Jun, Kristine; Camus, Marine; Wankhede, Mamta; Mao, Lei; Arnold, Tom; Young, William L.; Su, Hua

    2014-01-01

    Background and Purpose In humans, activin receptor-like kinase 1 (Alk1) deficiency causes arteriovenous malformations (AVMs) in multiple organs, including the brain. Focal Alk1 pan-cellular deletion plus vascular endothelial growth factor (VEGF) stimulation induces brain AVMs (bAVMs) in the adult mouse. We hypothesized that deletion of Alk1 in endothelial cell (EC) alone plus focal VEGF stimulation is sufficient to induce bAVM in the adult mouse. Methods Focal angiogenesis was induced in the brain of eight-week-old Pdgfb-iCreER;Alk12f/2f mice by injection of adeno-associated viral vectors expressing VEGF (AAV-VEGF). Two weeks later, EC-Alk1 deletion was induced by tamoxifen (TM) treatment. Vascular morphology was analyzed, and EC proliferation and Dysplasia Index (number of vessels with diameter >15μm per 200 vessels) were quantified10 days after TM administration. Results Tangles of enlarged vessels resembling AVMs were present in the brain angiogenic region of TM-treated Pdgfb-iCreER;Alk12f/2f mice. Induced bAVMs were marked by increased Dysplasia Index (P<0.001), and EC proliferation clustered within the dysplastic vessels. AVMs were also detected around the ear tag-wound and in other organs. Conclusions Deletion of Alk1 in EC in adult mice leads to an increased local EC proliferation during brain angiogenesis and de novo bAVM. PMID:24457293

  18. The pathobiology of the oncogenic tyrosine kinase NPM-ALK: a brief update.

    PubMed

    Lai, Raymond; Ingham, Robert J

    2013-04-01

    Extensive research has been carried out in the past two decades to study the pathobiology of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), which is an oncogenic fusion protein found exclusively in a specific type of T-cell lymphoid malignancy, namely ALK-positive anaplastic large cell lymphoma. Results from these studies have provided highly useful insights into the mechanisms by which a constitutively tyrosine kinase, such as NPM-ALK, promotes tumorigenesis. Several previous publications have comprehensively summarized the advances in this field. In this review, we provide readers with a brief update on specific areas of NPM-ALK pathobiology. In the first part, the NPM-ALK/signal transducer and activator of transcription 3 (STAT3) signaling axis is discussed, with an emphasis on the existence of multiple biochemical defects that have been shown to amplify the oncogenic effects of this signaling axis. Specifically, findings regarding JAK3, SHP1 and the stimulatory effects of several cytokines including interleukin (IL)-9, IL-21 and IL-22 are summarized. New concepts stemming from recent observations regarding the functional interactions among the NPM-ALK/STAT3 axis, β catenin and glycogen synthase kinase 3β will be postulated. Lastly, new mechanisms by which the NPM-ALK/STAT3 axis promotes tumorigenesis, such as its modulations of Twist1, hypoxia-induced factor 1α, CD274, will be described. In the second part, we summarize recent data generated by mass spectrometry studies of NPM-ALK, and use MSH2 and heat shock proteins as examples to illustrate the use of mass spectrometry data in stimulating new research in this field. In the third part, the evolving field of microRNA in the context of NPM-ALK biology is discussed.

  19. Synthesis of an aryloxy oxo pyrimidinone library that displays ALK-selective inhibition.

    PubMed

    Slavish, P Jake; Price, Jeanine E; Jiang, Qin; Cui, Xiaoli; Morris, Stephan W; Webb, Thomas R

    2011-08-01

    We report the synthesis of a pyrimidinone library that targets anaplastic lymphoma kinase (ALK), an oncogenic receptor tyrosine kinase. This library was generated in three steps from a versatile commercially available starting material. Some compounds within this library showed single digit micromolar inhibition of ALK in vitro, while showing minimal inhibition of other homologous insulin receptor family kinases including the human insulin receptor kinase (IRK), at the highest concentrations investigated. We also present initial ALK structure-activity relationships for this library. PMID:21708465

  20. ALK-negative anaplastic large cell lymphoma is a genetically heterogeneous disease with widely disparate clinical outcomes

    PubMed Central

    Parrilla Castellar, Edgardo R.; Jaffe, Elaine S.; Said, Jonathan W.; Swerdlow, Steven H.; Ketterling, Rhett P.; Knudson, Ryan A.; Sidhu, Jagmohan S.; Hsi, Eric D.; Karikehalli, Shridevi; Jiang, Liuyan; Vasmatzis, George; Gibson, Sarah E.; Ondrejka, Sarah; Nicolae, Alina; Grogg, Karen L.; Allmer, Cristine; Ristow, Kay M.; Wilson, Wyndham H.; Macon, William R.; Law, Mark E.; Cerhan, James R.; Habermann, Thomas M.; Ansell, Stephen M.; Dogan, Ahmet; Maurer, Matthew J.

    2014-01-01

    Anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) is a CD30-positive T-cell non-Hodgkin lymphoma that morphologically resembles ALK-positive ALCL but lacks chromosomal rearrangements of the ALK gene. The genetic and clinical heterogeneity of ALK-negative ALCL has not been delineated. We performed immunohistochemistry and fluorescence in situ hybridization on 73 ALK-negative ALCLs and 32 ALK-positive ALCLs and evaluated the associations among pathology, genetics, and clinical outcome. Chromosomal rearrangements of DUSP22 and TP63 were identified in 30% and 8% of ALK-negative ALCLs, respectively. These rearrangements were mutually exclusive and were absent in ALK-positive ALCLs. Five-year overall survival rates were 85% for ALK-positive ALCLs, 90% for DUSP22-rearranged ALCLs, 17% for TP63-rearranged ALCLs, and 42% for cases lacking all 3 genetic markers (P < .0001). Hazard ratios for death in these 4 groups after adjusting for International Prognostic Index and age were 1.0 (reference group), 0.58, 8.63, and 4.16, respectively (P = 7.10 × 10−5). These results were similar when restricted to patients receiving anthracycline-based chemotherapy, as well as to patients not receiving stem cell transplantation. Thus, ALK-negative ALCL is a genetically heterogeneous disease with widely disparate outcomes following standard therapy. DUSP22 and TP63 rearrangements may serve as predictive biomarkers to help guide patient management. PMID:24894770

  1. Structure-function studies of an unusual 3-methyladenine DNA glycosylase II (AlkA) from Deinococcus radiodurans.

    PubMed

    Moe, Elin; Hall, David R; Leiros, Ingar; Monsen, Vivi Talstad; Timmins, Joanna; McSweeney, Sean

    2012-06-01

    3-Methyladenine DNA glycosylase II (AlkA) is a DNA-repair enzyme that removes alkylated bases in DNA via the base-excision repair (BER) pathway. The enzyme belongs to the helix-hairpin-helix (HhH) superfamily of DNA glycosylases and possesses broad substrate specificity. In the genome of Deinococcus radiodurans, two genes encoding putative AlkA have been identified (Dr_2074 and Dr_2584). Dr_2074 is a homologue of human AlkA (MPG or AAG) and Dr_2584 is a homologue of bacterial AlkAs. Here, the three-dimensional structure of Dr_2584 (DrAlkA2) is presented and compared with the previously determined structure of Escherichia coli AlkA (EcAlkA). The results show that the enzyme consists of two helical-bundle domains separated by a wide DNA-binding cleft and contains an HhH motif. Overall, the protein fold is similar to the two helical-bundle domains of EcAlkA, while the third N-terminal mixed α/β domain observed in EcAlkA is absent. Substrate-specificity analyses show that DrAlkA2, like EcAlkA, is able to remove both 3-methyladenine (3meA) and 7-methylguanine (7meG) from DNA; however, the enzyme possesses no activity towards 1,N(6)-ethenoadenine (ℇA) and hypoxanthine (Hx). In addition, it shows activity towards the AlkB dioxygenase substrates 3-methylcytosine (3meC) and 1-methyladenine (1meA). Thus, the enzyme seems to preferentially repair methylated bases with weakened N-glycosidic bonds; this is an unusual specificity for a bacterial AlkA protein and is probably dictated by a combination of the wide DNA-binding cleft and a highly accessible specificity pocket.

  2. Structure-function studies of an unusual 3-methyladenine DNA glycosylase II (AlkA) from Deinococcus radiodurans.

    PubMed

    Moe, Elin; Hall, David R; Leiros, Ingar; Monsen, Vivi Talstad; Timmins, Joanna; McSweeney, Sean

    2012-06-01

    3-Methyladenine DNA glycosylase II (AlkA) is a DNA-repair enzyme that removes alkylated bases in DNA via the base-excision repair (BER) pathway. The enzyme belongs to the helix-hairpin-helix (HhH) superfamily of DNA glycosylases and possesses broad substrate specificity. In the genome of Deinococcus radiodurans, two genes encoding putative AlkA have been identified (Dr_2074 and Dr_2584). Dr_2074 is a homologue of human AlkA (MPG or AAG) and Dr_2584 is a homologue of bacterial AlkAs. Here, the three-dimensional structure of Dr_2584 (DrAlkA2) is presented and compared with the previously determined structure of Escherichia coli AlkA (EcAlkA). The results show that the enzyme consists of two helical-bundle domains separated by a wide DNA-binding cleft and contains an HhH motif. Overall, the protein fold is similar to the two helical-bundle domains of EcAlkA, while the third N-terminal mixed α/β domain observed in EcAlkA is absent. Substrate-specificity analyses show that DrAlkA2, like EcAlkA, is able to remove both 3-methyladenine (3meA) and 7-methylguanine (7meG) from DNA; however, the enzyme possesses no activity towards 1,N(6)-ethenoadenine (ℇA) and hypoxanthine (Hx). In addition, it shows activity towards the AlkB dioxygenase substrates 3-methylcytosine (3meC) and 1-methyladenine (1meA). Thus, the enzyme seems to preferentially repair methylated bases with weakened N-glycosidic bonds; this is an unusual specificity for a bacterial AlkA protein and is probably dictated by a combination of the wide DNA-binding cleft and a highly accessible specificity pocket. PMID:22683793

  3. The bHLH transcription factor Hand is regulated by Alk in the Drosophila embryonic gut

    SciTech Connect

    Varshney, Gaurav K.; Palmer, Ruth H. . E-mail: Ruth.Palmer@ucmp.umu.se

    2006-12-29

    During embryonic development the midgut visceral muscle is formed by fusion of cells within the visceral mesoderm, a process initiated by the specification of a specialised cell type, the founder cell, within this tissue. Activation of the receptor tyrosine kinase Anaplastic lymphoma kinase (Alk) in the developing visceral muscle of Drosophila melanogaster initiates a signal transduction pathway required for muscle fusion. In this paper, we have investigated downstream components which are regulated by this novel signalling pathway. Here we show that Alk-mediated signal transduction drives the expression of the bHLH transcription factor Hand in vivo. Loss of Alk function results in a complete lack of Hand expression in this tissue, whereas Alk gain of function results in an expansion of Hand expression. Finally, we have investigated the process of muscle fusion in the gut of Hand mutant animals and can find no obvious defects in this process, suggesting that Hand is not critical for visceral muscle fusion per se.

  4. Fusion tyrosine kinase NPM-ALK Deregulates MSH2 and suppresses DNA mismatch repair function novel insights into a potent oncoprotein.

    PubMed

    Young, Leah C; Bone, Kathleen M; Wang, Peng; Wu, Fang; Adam, Benjamin A; Hegazy, Samar; Gelebart, Pascal; Holovati, Jelena; Li, Liang; Andrew, Susan E; Lai, Raymond

    2011-07-01

    The fusion tyrosine kinase NPM-ALK is central to the pathogenesis of ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL). We recently identified that MSH2, a key DNA mismatch repair (MMR) protein integral to the suppression of tumorigenesis, is an NPM-ALK-interacting protein. In this study, we found in vitro evidence that enforced expression of NPM-ALK in HEK293 cells suppressed MMR function. Correlating with these findings, six of nine ALK(+)ALCL tumors displayed evidence of microsatellite instability, as opposed to none of the eight normal DNA control samples (P = 0.007, Student's t-test). Using co-immunoprecipitation, we found that increasing levels of NPM-ALK expression in HEK293 cells resulted in decreased levels of MSH6 bound to MSH2, whereas MSH2·NPM-ALK binding was increased. The NPM-ALK·MSH2 interaction was dependent on the activation/autophosphorylation of NPM-ALK, and the Y191 residue of NPM-ALK was a crucial site for this interaction and NPM-ALK-mediated MMR suppression. MSH2 was found to be tyrosine phosphorylated in the presence of NPM-ALK. Finally, NPM-ALK impeded the expected DNA damage-induced translocation of MSH2 out of the cytoplasm. To conclude, our data support a model in which the suppression of MMR by NPM-ALK is attributed to its ability to interfere with normal MSH2 biochemistry and function.

  5. Use of minimal disseminated disease and immunity to NPM-ALK antigen to stratify ALK-positive ALCL patients with different prognosis.

    PubMed

    Mussolin, L; Damm-Welk, C; Pillon, M; Zimmermann, M; Franceschetto, G; Pulford, K; Reiter, A; Rosolen, A; Woessmann, W

    2013-02-01

    We studied the prognostic value of minimal disseminated disease (MDD) and anti-ALK immune response in children with NPM-ALK-positive anaplastic-large cell lymphoma (ALCL) and evaluated their potential for risk stratification. NPM-ALK transcripts were analyzed by RT-PCR in bone marrow/peripheral blood of 128 ALCL patients at diagnosis, whereas ALK antibody titers in plasma were assessed using an immunocytochemical approach. MDD was positive in 59% of patients and 96% showed an anti-ALK response. Using MDD and antibody titer results, patients could be divided into three biological risk groups (bRG) with different prognosis: high risk (bHR): MDD-positive and antibody titer ≤ 1/750, 26/128 (20%); low risk (bLR): MDD negative and antibody titer >1/750, 40/128 (31%); intermediate risk (bIR): all remaining patients, 62/128 (48%). Progression-free survival was 28% (s.e., 9%), 68% (s.e., 6%) and 93% (s.e., 4%) for bHR, bIR and bLR, respectively (P<0.0001). Survival was 71% (s.e., 9%), 83% (s.e., 5%) and 98% (s.e., 2%) for bHR, bIR and bLR (P=0.02). Only bHR and histology other than common type were predictive of higher risk of failure (hazard ratio 4.9 and 2.7, respectively) in multivariate analysis. Stratification of ALCL patients based on MDD and anti-ALK titer should be considered in future ALCL trials to optimize treatment.

  6. The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration.

    PubMed

    Voena, Claudia; Conte, Chiara; Ambrogio, Chiara; Boeri Erba, Elisabetta; Boccalatte, Francesco; Mohammed, Shabaz; Jensen, Ole N; Palestro, Giorgio; Inghirami, Giorgio; Chiarle, Roberto

    2007-05-01

    Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542 of Shp2 mainly stained ALK-positive cells. In ALCL cell lines, Shp2-constitutive phosphorylation was dependent on NPM-ALK, as it significantly decreased after short hairpin RNA (shRNA)-mediated NPM-ALK knock down. In addition, only the constitutively active NPM-ALK, but not the kinase dead NPM-ALK(K210R), formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416 in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration.

  7. Are alkane hydroxylase genes (alkB) relevant to assess petroleum bioremediation processes in chronically polluted coastal sediments?

    PubMed

    Paisse, Sandrine; Duran, Robert; Coulon, Frédéric; Goñi-Urriza, Marisol

    2011-11-01

    The diversity of alkB-related alkane hydroxylase sequences and the relationship between alkB gene expression and the hydrocarbon contamination level have been investigated in the chronically polluted Etang-de-Berre sediments. For this purpose, these sediments were maintained in microcosms and submitted to a controlled oil input miming an oil spill. New degenerated PCR primers targeting alkB-related alkane hydroxylase sequences were designed to explore the diversity and the expression of these genes using terminal restriction fragment length polymorphism fingerprinting and gene library analyses. Induction of alkB genes was detected immediately after oil addition and their expression detected only during 2 days, although the n-alkane degradation was observed throughout the 14 days of incubation. The alkB gene expression within triplicate microcosms was heterogeneous probably due to the low level of alkB transcripts. Moreover, the alkB gene expression of dominant OTUs has been observed in unoiled microcosms indicating that the expression of this gene cannot be directly related to the oil contamination. Although the dominant alkB genes and transcripts detected were closely related to the alkB of Marinobacter aquaeolei isolated from an oil-producing well, and to alkB genes related to the obligate alkanotroph Alcanivorax borkumensis, no clear relationship between the oil contamination and the expression of the alkB genes could be established. This finding suggests that in such coastal environments, alkB gene expression is not a function relevant enough to monitor bacterial response to oil contamination. PMID:21660544

  8. Crystal structure of EML1 reveals the basis for Hsp90 dependence of oncogenic EML4-ALK by disruption of an atypical β-propeller domain

    PubMed Central

    Richards, Mark W.; Law, Edward W. P.; Rennalls, La’Verne P.; Busacca, Sara; O’Regan, Laura; Fry, Andrew M.; Fennell, Dean A.; Bayliss, Richard

    2014-01-01

    Proteins of the echinoderm microtubule-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule network. They contain a unique hydrophobic EML protein (HELP) motif and a variable number of WD40 repeats. Recurrent gene rearrangements in nonsmall cell lung cancer fuse EML4 to anaplastic lymphoma kinase (ALK), causing expression of several fusion oncoprotein variants. We have determined a 2.6-Å crystal structure of the representative ∼70-kDa core of EML1, revealing an intimately associated pair of β-propellers, which we term a TAPE (tandem atypical propeller in EMLs) domain. One propeller is highly atypical, having a discontinuous subdomain unrelated to a WD40 motif in place of one of its blades. This unexpected feature shows how a propeller structure can be assembled from subdomains with distinct folds. The HELP motif is not an independent domain but forms part of the hydrophobic core that joins the two β-propellers. The TAPE domain binds α/β-tubulin via its conserved, concave surface, including part of the atypical blade. Mapping the characteristic breakpoints of each EML4-ALK variant onto our structure indicates that the EML4 TAPE domain is truncated in many variants in a manner likely to make the fusion protein structurally unstable. We found that the heat shock protein 90 (Hsp90) inhibitor ganetespib induced degradation of these variants whereas others lacking a partial TAPE domain were resistant in both overexpression models and patient-derived cell lines. The Hsp90-sensitive EML4-ALK variants are exceptions to the rule that oncogenic fusion proteins involve breakpoints in disordered regions of both partners. PMID:24706829

  9. Charge-transfer reaction of 2,3-dichloro-1,4-naphthoquinone with crizotinib: Spectrophotometric study, computational molecular modeling and use in development of microwell assay for crizotinib.

    PubMed

    Alzoman, Nourah Z; Alshehri, Jamilah M; Darwish, Ibrahim A; Khalil, Nasr Y; Abdel-Rahman, Hamdy M

    2015-01-01

    The reaction of 2,3-dichloro-1,4-naphthoquinone (DCNQ) with crizotinib (CZT; a novel drug used for treatment of non-small cell lung cancer) was investigated in different solvents of varying dielectric constants and polarity indexes. The reaction produced a red-colored product. Spectrophotometric investigations confirmed that the reaction proceeded through charge-transfer (CT) complex formation. The molar absorptivity of the complex was found to be linearly correlated with the dielectric constant and polarity index of the solvent; the correlation coefficients were 0.9567 and 0.9069, respectively. The stoichiometric ratio of DCNQ:CZT was found to be 2:1 and the association constant of the complex was found to be 1.07 × 10(2) l/mol. The kinetics of the reaction was studied; the order of the reaction, rate and rate constant were determined. Computational molecular modeling for the complex between DCNQ and CZT was conducted, the sites of interaction on CZT molecule were determined, and the mechanism of the reaction was postulated. The reaction was employed as a basis in the development of a novel 96-microwell assay for CZT in a linear range of 4-500 μg/ml. The assay limits of detection and quantitation were 2.06 and 6.23 μg/ml, respectively. The assay was validated as per the guidelines of the International Conference on Harmonization (ICH) and successfully applied to the analysis of CZT in its bulk and capsules with good accuracy and precision. The assay has high throughput and consumes a minimum volume of organic solvents thus it reduces the exposures of the analysts to the toxic effects of organic solvents, and significantly reduces the analysis cost.

  10. PI3 Kinase Pathway and MET Inhibition is Efficacious in Malignant Pleural Mesothelioma.

    PubMed

    Kanteti, Rajani; Riehm, Jacob J; Dhanasingh, Immanuel; Lennon, Frances E; Mirzapoiazova, Tamara; Mambetsariev, Bolot; Kindler, Hedy L; Salgia, Ravi

    2016-01-01

    Malignant pleural mesothelioma (MPM) is an aggressive cancer that is commonly associated with prior asbestos exposure. Receptor tyrosine kinases (RTKs) such as MET and its downstream target PI3K are overexpressed and activated in a majority of MPMs. Here, we studied the combinatorial therapeutic efficacy of the MET/ALK inhibitor crizotinib, with either a pan-class I PI3K inhibitor, BKM120, or with a PI3K/mTOR dual inhibitor, GDC-0980, in mesothelioma. Cell viability results showed that MPM cells were highly sensitive to crizotinib, BKM120 and GDC-0980 when used individually and their combination was more effective in suppressing growth. Treatment of MPM cells with these inhibitors also significantly decreased cell migration, and the combination of them was synergistic. Treatment with BKM120 alone or in combination with crizotinib induced G2-M arrest and apoptosis. Both crizotinib and BKM120 strongly inhibited the activity of MET and PI3K as evidenced by the decreased phosphorylation of MET, AKT and ribosomal S6 kinase. Using a PDX mouse model, we showed that a combination of crizotinib with BKM120 was highly synergetic in inhibiting MPM tumor growth. In conclusion our findings suggest that dual inhibition of PI3K and MET pathway is an effective strategy in treating MPM as compared to a single agent. PMID:27623107

  11. PI3 Kinase Pathway and MET Inhibition is Efficacious in Malignant Pleural Mesothelioma

    PubMed Central

    Kanteti, Rajani; Riehm, Jacob J.; Dhanasingh, Immanuel; Lennon, Frances E.; Mirzapoiazova, Tamara; Mambetsariev, Bolot; Kindler, Hedy L.; Salgia, Ravi

    2016-01-01

    Malignant pleural mesothelioma (MPM) is an aggressive cancer that is commonly associated with prior asbestos exposure. Receptor tyrosine kinases (RTKs) such as MET and its downstream target PI3K are overexpressed and activated in a majority of MPMs. Here, we studied the combinatorial therapeutic efficacy of the MET/ALK inhibitor crizotinib, with either a pan-class I PI3K inhibitor, BKM120, or with a PI3K/mTOR dual inhibitor, GDC-0980, in mesothelioma. Cell viability results showed that MPM cells were highly sensitive to crizotinib, BKM120 and GDC-0980 when used individually and their combination was more effective in suppressing growth. Treatment of MPM cells with these inhibitors also significantly decreased cell migration, and the combination of them was synergistic. Treatment with BKM120 alone or in combination with crizotinib induced G2-M arrest and apoptosis. Both crizotinib and BKM120 strongly inhibited the activity of MET and PI3K as evidenced by the decreased phosphorylation of MET, AKT and ribosomal S6 kinase. Using a PDX mouse model, we showed that a combination of crizotinib with BKM120 was highly synergetic in inhibiting MPM tumor growth. In conclusion our findings suggest that dual inhibition of PI3K and MET pathway is an effective strategy in treating MPM as compared to a single agent. PMID:27623107

  12. L- and S-endoglin differentially modulate TGFbeta1 signaling mediated by ALK1 and ALK5 in L6E9 myoblasts.

    PubMed

    Velasco, Soraya; Alvarez-Muñoz, Patricia; Pericacho, Miguel; Dijke, Peter Ten; Bernabéu, Carmelo; López-Novoa, José M; Rodríguez-Barbero, Alicia

    2008-03-15

    TGFbeta regulates cellular processes by binding to type I and type II TGFbeta receptors (TbetaRI and TbetaRII, respectively). In addition to these signaling receptors, endoglin is an accessory TGFbeta receptor that regulates TGFbeta signaling. Although there are two different alternatively spliced isoforms of endoglin, L-endoglin (L, long) and S-endoglin (S, short), little is known about the effects of S-endoglin isoform on TGFbeta signaling. Here, we have analyzed the TGFbeta1 signaling pathways and the effects of L- and S-endoglin in endoglin-deficient L6E9 cells. We found that TGFbeta activates two distinct TbetaRI-Smad signaling pathways: ALK1-Smad1-Id1 and ALK5-Smad2-PAI1, in these cells. Interestingly, L-endoglin enhanced the ALK1-Id1 pathway, while S-endoglin promoted the ALK5-PAI1 route. These effects on signaling are supported by biological effects on TGFbeta1-induced collagen I expression and inhibition of cell proliferation. Thus, while L-endoglin decreased TGFbeta1-induced collagen I and CTGF expression and increased TGFbeta1-induced proliferation, S-endoglin strongly increased TGFbeta1-induced collagen I and CTGF expression, and reduced TGFbeta1-induced cell proliferation.

  13. Brain metastasis of ALK positive anaplastic large cell lymphoma after a long-term disease free survival in an old adult

    PubMed Central

    Wang, Cai-Xia; Wang, Hai; Li, Jie; Ma, Heng-Hui; Yu, Bo; Shi, Shan-Shan; Zhou, Xiao-Jun; Shi, Qun-Li

    2014-01-01

    Anaplastic large cell lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma composed of CD30-positive cells and now recognized as three different entities: primary cutaneous ALCL, primary systemic anaplastic lymphoma kinase (ALK)-positive (ALK+) ALCL and primary ALK-negative (ALK-) ALCL. ALK+ ALCL is supposed to have a better prognosis than ALK- ALCL. It is rarely metastasized to other sites, especially to the central nervous system (CNS). Herein, we present a rare case of systemic ALK+ ALCL which metastasized to the brain after a long-term disease free survival in an adult. Neuroimaging revealed a well-enhanced mass in the left frontal lobe. And it was completely resected. The results of the pathological and immunohistochemical studies were consistent with the metastasized ALK+ ALCL. The clinical findings, pathologic characteristics and treatment are described. PMID:24696735

  14. Identification of multiple SNT-binding sites on NPM-ALK oncoprotein and their involvement in cell transformation.

    PubMed

    Chikamori, M; Fujimoto, J; Tokai-Nishizumi, N; Yamamoto, T

    2007-05-01

    The t(2;5) chromosomal translocation occurs in anaplastic large-cell lymphoma arising from activated T lymphocytes. This genomic rearrangement generates the nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) oncoprotein that is a chimeric protein consisting of parts of the nuclear protein NPM and ALK receptor protein-tyrosine kinase. We used yeast two-hybrid screening to identify an adaptor protein Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target (SNT)-2 as a new partner that interacted with the cytoplasmic domain of ALK. Immunoprecipitation assay revealed that SNT-1 and SNT-2 interacted with NPM-ALK and kinase-negative NPM-ALK mutant. Y156, Y567 and a 19-amino-acid sequence (aa 631-649) of NPM-ALK were essential for this interaction. The interaction through Y156 and Y567 was dependent on phosphorylation of these tyrosines, whereas the interaction through the 19-amino-acid sequence was independent of phosphorylation. NPM-ALK mutant protein mutated at these three binding sites showed significantly reduced transforming activity. This transformation-defective NPM-ALK mutant still interacted with signal transducing proteins such as phospholipase C-gamma and phosphatidylinositol 3-kinase, which were previously reported to be relevant to NPM-ALK-dependent tumorigenesis. These observations indicate that the three SNT-binding sites of NPM-ALK are important for its transforming activity. This raises a possibility that SNT family proteins play significant roles in cellular transformation triggered by NPM-ALK, which though remains to be verified.

  15. The role of AlkB protein in repair of 1,N⁶-ethenoadenine in Escherichia coli cells.

    PubMed

    Maciejewska, Agnieszka M; Sokołowska, Beata; Nowicki, Adam; Kuśmierek, Jarosław T

    2011-05-01

    Etheno (ε) DNA adducts, including 1,N(6)-ethenoadenine (εA), are formed by various bifunctional agents of exogenous and endogenous origin. The AT→TA transversion, the most frequent mutation provoked by the presence of εA in DNA, is very common in critical codons of the TP53 and RAS genes in tumours induced by exposure to carcinogenic vinyl compounds. Here, using a method that allows examination of the mutagenic potency of a metabolite of vinyl chloride, chloroacetaldehyde (CAA), but eliminates its cytotoxicity, we studied the participation of alkA, alkB and mug gene products in the repair of εA in Escherichia coli cells. The test system used comprised the pIF105 plasmid bearing the lactose operon of CC105 origin, which allowed monitoring of Lac(+) revertants that arose by AT→TA substitutions due to the modification of adenine by CAA. The plasmid was CAA-modified in vitro and replicated in E.coli of various genetic backgrounds (wt, alkA, alkB, mug, alkAalkB, alkAmug and alkBmug). To modify the levels of the AlkA and AlkB proteins, mutagenesis was studied in E.coli cells induced or not in adaptive response to alkylating agents. Considering the levels of CAA-induced Lac(+) revertants in strains harbouring the CAA-modified pIF105 plasmid and induced or not in adaptive response, we conclude that the AlkB dioxygenase plays a major role in decreasing the level of AT→TA mutations, thus in the repair of εA in E.coli cells. The observed differences of mutation frequencies in the various mutant strains assayed indicate that Mug glycosylase is also engaged in the repair of εA, whereas the role the AlkA glycosylase in this repair is negligible. PMID:21193516

  16. IL-2R common gamma-chain is epigenetically silenced by nucleophosphin-anaplastic lymphoma kinase (NPM-ALK) and acts as a tumor suppressor by targeting NPM-ALK.

    PubMed

    Zhang, Qian; Wang, Hong Yi; Liu, Xiaobin; Bhutani, Gauri; Kantekure, Kanchan; Wasik, Mariusz

    2011-07-19

    Anaplastic lymphoma kinase (ALK), physiologically expressed only by certain neural cells, becomes highly oncogenic, when aberrantly expressed in nonneural tissues as a fusion protein with nucleophosphin (NPM) and other partners. The reason why NPM-ALK succeeds in transforming specifically CD4(+) T lymphocytes remains unknown. The IL-2R common γ-chain (IL-2Rγ) is shared by receptors for several cytokines that play key roles in the maturation and growth of normal CD4(+) T lymphocytes and other immune cells. We show that IL-2Rγ expression is inhibited in T-cell lymphoma cells expressing NPM-ALK kinase as a result of DNA methylation of the IL-2Rγ gene promoter. IL-2Rγ promoter methylation is induced in malignant T cells by NPM-ALK. NPM-ALK acts through STAT3, a transcription factor that binds to the IL-2Rγ gene promoter and enhances binding of DNA methyltransferases (DNMTs) to the promoter. In addition, STAT3 suppresses expression of miR-21, which selectively inhibits DNMT1 mRNA expression. Reconstitution of IL-2Rγ expression leads to loss of the NPM-ALK protein and, consequently, apoptotic cell death of the lymphoma cells. These results demonstrate that the oncogenic tyrosine kinase NPM-ALK induces epigenetic silencing of the IL-2Rγ gene and that IL-2Rγ acts as a tumor suppressor by reciprocally inhibiting expression of NPM-ALK.

  17. The tyrosine 343 residue of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) is important for its interaction with SHP1, a cytoplasmic tyrosine phosphatase with tumor suppressor functions.

    PubMed

    Hegazy, Samar A; Wang, Peng; Anand, Mona; Ingham, Robert J; Gelebart, Pascal; Lai, Raymond

    2010-06-25

    The cytoplasmic tyrosine phosphatase SHP1 has been shown to inhibit the oncogenic fusion protein nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), and loss of SHP1 contributes to NPM-ALK-mediated tumorigenesis. In this study, we aimed to further understand how SHP1 interacts and regulates NPM-ALK. We employed an in vitro model in which GP293 cells were transfected with various combinations of NPM-ALK (or mutants) and SHP1 (or mutants) expression vectors. We found that SHP1 co-immunoprecipitated with NPM-ALK, but not the enzymatically inactive NPM-ALK(K210R) mutant, or the mutant in which all three functionally important tyrosine residues (namely, Tyr(338), Tyr(342), and Tyr(343)) in the kinase activation loop (KAL) of ALK were mutated. Interestingly, whereas mutation of Tyr(338) or Tyr(342) did not result in any substantial change in the NPM-ALK/SHP1 binding (assessed by co-immunoprecipitation), mutation of Tyr(343) abrogated this interaction. Furthermore, the NPM-ALK/SHP1 binding was readily detectable when each of the remaining 8 tyrosine residues known to be phosphorylated were mutated. Although the expression of SHP1 effectively reduced the level of tyrosine phosphorylation of NPM-ALK, it did not affect that of the NPM-ALK(Y343F) mutant. In soft agar clonogenic assay, SHP1 expression significantly reduced the tumorigenicity of NPM-ALK but not that of NPM-ALK(Y343F). In conclusion, we identified Tyr(343) of NPM-ALK as the crucial site for mediating the NPM-ALK/SHP1 interaction. Our results also support the notion that the tumor suppressor effects of SHP1 on NPM-ALK are dependent on its ability to bind to this oncogenic protein.

  18. Molecular characterization of WDCP, a novel fusion partner for the anaplastic lymphoma tyrosine kinase ALK

    PubMed Central

    YOKOYAMA, NORIKO; MILLER, W. TODD

    2015-01-01

    Anaplastic lymphoma kinase (ALK) is a member of the receptor tyrosine kinase superfamily. The ALK gene is a site of frequent mutation and chromosomal rearrangement in various types of human cancers. A novel chromosomal translocation was recently identified in human colorectal cancer between the ALK gene and chromosome 2, open reading frame 44 (C2orf44), a gene of unknown function. As a first step in understanding the oncogenic properties of this fusion protein, C2orf44 cDNA was cloned and the encoded protein was characterized, which was designated as WD repeat and coiled coil containing protein (WDCP). A C-terminal proline-rich segment in WDCP was shown to mediate binding to the Src homology 3 domain of the Src family kinase hematopoietic cell kinase (Hck). Co-expression with Hck lead to tyrosine phosphorylation of WDCP. Chromatographic fractionation of WDCP-containing lysates indicates that the protein exists as an oligomer in mammalian cells. These results suggest that, in the context of the ALK-C2orf44 gene fusion, WDCP imposes an oligomeric structure on ALK that results in constitutive kinase activation and signaling. PMID:25469238

  19. Targeting Oncogenic ALK and MET: A Promising Therapeutic Strategy for Glioblastoma

    PubMed Central

    Wallace, Gerald C; Dixon-Mah, Yaenette N; Vandergrift, W Alex; Ray, Swapan K; Haar, Catherine P; Mittendorf, Amber M; Patel, Sunil J; Banik, Naren L; Giglio, Pierre; Das, Arabinda

    2015-01-01

    Glioblastoma is the most common aggressive, highly glycolytic, and lethal brain tumor. In fact, it is among the most commonly diagnosed lethal malignancies, with thousands of new cases reported in the United States each year. Glioblastoma's lethality is derived from a number of factors including highly active pro-mitotic and pro-metastatic pathways. Two factors increasingly associated with the intracellular signaling and transcriptional machinery required for such changes are anaplastic lymphoma kinase (ALK) and the hepatocyte growth factor receptor (HGFR or, more commonly MET). Both receptors are members of the receptor tyrosine kinase (RTK) family, which has itself gained much attention for its role in modulating mitosis, migration, and survival in cancer cells. ALK was first described as a vital oncogene in lymphoma studies, but it has since been connected to many carcinomas, including non-small cell lung cancer and glioblastoma. As the receptor for HGF, MET has also been highly characterized and regulates numerous developmental and wound healing events which, when upregulated in cancer, can promote tumor progression. The wealth of information gathered over the last 30 years regarding these RTKs suggests three downstream cascades that depend upon activation of STAT3, Ras, and AKT. This review outlines the significance of ALK and MET as they relate to glioblastoma, explores the significance of STAT3, Ras, and AKT downstream of ALK/MET, and touches on the potential for new chemotherapeutics targeting ALK and MET to improve glioblastoma patient prognosis. PMID:23543207

  20. Albumin induces excitatory synaptogenesis through astrocytic TGF-β/ALK5 signaling in a model of acquired epilepsy following blood-brain barrier dysfunction.

    PubMed

    Weissberg, Itai; Wood, Lydia; Kamintsky, Lyn; Vazquez, Oscar; Milikovsky, Dan Z; Alexander, Allyson; Oppenheim, Hannah; Ardizzone, Carolyn; Becker, Albert; Frigerio, Federica; Vezzani, Annamaria; Buckwalter, Marion S; Huguenard, John R; Friedman, Alon; Kaufer, Daniela

    2015-06-01

    Post-injury epilepsy (PIE) is a common complication following brain insults, including ischemic, and traumatic brain injuries. At present, there are no means to identify the patients at risk to develop PIE or to prevent its development. Seizures can occur months or years after the insult, do not respond to anti-seizure medications in over third of the patients, and are often associated with significant neuropsychiatric morbidities. We have previously established the critical role of blood-brain barrier dysfunction in PIE, demonstrating that exposure of brain tissue to extravasated serum albumin induces activation of inflammatory transforming growth factor beta (TGF-β) signaling in astrocytes and eventually seizures. However, the link between the acute astrocytic inflammatory responses and reorganization of neural networks that underlie recurrent spontaneous seizures remains unknown. Here we demonstrate in vitro and in vivo that activation of the astrocytic ALK5/TGF-β-pathway induces excitatory, but not inhibitory, synaptogenesis that precedes the appearance of seizures. Moreover, we show that treatment with SJN2511, a specific ALK5/TGF-β inhibitor, prevents synaptogenesis and epilepsy. Our findings point to astrocyte-mediated synaptogenesis as a key epileptogenic process and highlight the manipulation of the TGF-β-pathway as a potential strategy for the prevention of PIE.

  1. Albumin induces excitatory synaptogenesis through astrocytic TGF-β/ALK5 signaling in a model of acquired epilepsy following blood-brain barrier dysfunction.

    PubMed

    Weissberg, Itai; Wood, Lydia; Kamintsky, Lyn; Vazquez, Oscar; Milikovsky, Dan Z; Alexander, Allyson; Oppenheim, Hannah; Ardizzone, Carolyn; Becker, Albert; Frigerio, Federica; Vezzani, Annamaria; Buckwalter, Marion S; Huguenard, John R; Friedman, Alon; Kaufer, Daniela

    2015-06-01

    Post-injury epilepsy (PIE) is a common complication following brain insults, including ischemic, and traumatic brain injuries. At present, there are no means to identify the patients at risk to develop PIE or to prevent its development. Seizures can occur months or years after the insult, do not respond to anti-seizure medications in over third of the patients, and are often associated with significant neuropsychiatric morbidities. We have previously established the critical role of blood-brain barrier dysfunction in PIE, demonstrating that exposure of brain tissue to extravasated serum albumin induces activation of inflammatory transforming growth factor beta (TGF-β) signaling in astrocytes and eventually seizures. However, the link between the acute astrocytic inflammatory responses and reorganization of neural networks that underlie recurrent spontaneous seizures remains unknown. Here we demonstrate in vitro and in vivo that activation of the astrocytic ALK5/TGF-β-pathway induces excitatory, but not inhibitory, synaptogenesis that precedes the appearance of seizures. Moreover, we show that treatment with SJN2511, a specific ALK5/TGF-β inhibitor, prevents synaptogenesis and epilepsy. Our findings point to astrocyte-mediated synaptogenesis as a key epileptogenic process and highlight the manipulation of the TGF-β-pathway as a potential strategy for the prevention of PIE. PMID:25836421

  2. The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond*

    PubMed Central

    Fedeles, Bogdan I.; Singh, Vipender; Delaney, James C.; Li, Deyu; Essigmann, John M.

    2015-01-01

    The AlkB family of Fe(II)- and α-ketoglutarate-dependent dioxygenases is a class of ubiquitous direct reversal DNA repair enzymes that remove alkyl adducts from nucleobases by oxidative dealkylation. The prototypical and homonymous family member is an Escherichia coli “adaptive response” protein that protects the bacterial genome against alkylation damage. AlkB has a wide variety of substrates, including monoalkyl and exocyclic bridged adducts. Nine mammalian AlkB homologs exist (ALKBH1–8, FTO), but only a subset functions as DNA/RNA repair enzymes. This minireview presents an overview of the AlkB proteins including recent data on homologs, structural features, substrate specificities, and experimental strategies for studying DNA repair by AlkB family proteins. PMID:26152727

  3. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

    PubMed Central

    Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø.; Rizzo, Carmelo J.; Guengerich, F. Peter; Tudek, Barbara

    2015-01-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno (ε)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ε-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ε-adducts, 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC) and 1,N2-ethenoguanine (1,N2-εG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ε-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed εA and εC from ds and ssDNA but were inactive toward 1,N2-εG. SC-1A repaired only εA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ε-adducts in dsDNA, while only εA and εC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only εC in ssDNA Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N2-εG and that ALKBH3 removes only εC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. PMID:25797601

  4. Chaperones as thermodynamic sensors of drug-target interactions reveal kinase inhibitor specificities in living cells.

    PubMed

    Taipale, Mikko; Krykbaeva, Irina; Whitesell, Luke; Santagata, Sandro; Zhang, Jianming; Liu, Qingsong; Gray, Nathanael S; Lindquist, Susan

    2013-07-01

    The interaction between the HSP90 chaperone and its client kinases is sensitive to the conformational status of the kinase, and stabilization of the kinase fold by small molecules strongly decreases chaperone interaction. Here we exploit this observation and assay small-molecule binding to kinases in living cells, using chaperones as 'thermodynamic sensors'. The method allows determination of target specificities of both ATP-competitive and allosteric inhibitors in the kinases' native cellular context in high throughput. We profile target specificities of 30 diverse kinase inhibitors against >300 kinases. Demonstrating the value of the assay, we identify ETV6-NTRK3 as a target of the FDA-approved drug crizotinib (Xalkori). Crizotinib inhibits proliferation of ETV6-NTRK3-dependent tumor cells with nanomolar potency and induces the regression of established tumor xenografts in mice. Finally, we show that our approach is applicable to other chaperone and target classes by assaying HSP70/steroid hormone receptor and CDC37/kinase interactions, suggesting that chaperone interactions will have broad application in detecting drug-target interactions in vivo.

  5. EML4-ALK induces epithelial–mesenchymal transition consistent with cancer stem cell properties in H1299 non-small cell lung cancer cells

    SciTech Connect

    Guo, Fuchun; Liu, Xiaoke Qing, Qin Sang, Yaxiong Feng, Chengjun Li, Xiaoyu Jiang, Li Su, Pei Wang, Yongsheng

    2015-04-10

    The echinoderm microtubule-associated protein-like 4(EML4) – anaplastic lymphoma kinase (ALK) fusion gene has been identified as a driver mutation in non-small-cell lung cancer (NSCLC). However, the role of EML4-ALK in malignant transformation is not entirely clear. Here, for the first time, we showed that H1299 NSCLC cells stably expressing EML4-ALK acquire EMT phenotype, associated with enhanced invasive migration and increased expression of EMT-inducing transcription factors. H1299-EML4-ALK cells also displayed cancer stem cell-like properties with a concomitant up-regulation of CD133 and enhanced ability of mammospheres formation. Moreover, we found that inhibition of ERK1/2 reversed EMT induced by EML4-ALK in H1299 cells. Taken together, these results suggested that EML4-ALK induced ERK activation is mechanistically associated with EMT phenotype. Thus, inhibition of ERK signaling pathway could be a potential strategy in treatment of NSCLC patients with EML4-ALK translocation. - Highlights: • EML4-ALK induced epithelial–mesenchymal transition in H1299 cells. • Expression of EML4-ALK promotes invasion and migration in vitro. • EML4-ALK enhanced sphere formation and stem cell-like properties in H1299 cells. • Blockage of ERK1/2 reverse Epithelial–Mesenchymal transition induced by EML4-ALK.

  6. ALK1 signalling analysis identifies angiogenesis related genes and reveals disparity between TGF-β and constitutively active receptor induced gene expression

    PubMed Central

    Lux, Andreas; Salway, Fiona; Dressman, Holly K; Kröner-Lux, Gabriele; Hafner, Mathias; Day, Philip JR; Marchuk, Douglas A; Garland, John

    2006-01-01

    Background TGF-β1 is an important angiogenic factor involved in the different aspects of angiogenesis and vessel maintenance. TGF-β signalling is mediated by the TβRII/ALK5 receptor complex activating the Smad2/Smad3 pathway. In endothelial cells TGF-β utilizes a second type I receptor, ALK1, activating the Smad1/Smad5 pathway. Consequently, a perturbance of ALK1, ALK5 or TβRII activity leads to vascular defects. Mutations in ALK1 cause the vascular disorder hereditary hemorrhagic telangiectasia (HHT). Methods The identification of ALK1 and not ALK5 regulated genes in endothelial cells, might help to better understand the development of HHT. Therefore, the human microvascular endothelial cell line HMEC-1 was infected with a recombinant constitutively active ALK1 adenovirus, and gene expression was studied by using gene arrays and quantitative real-time PCR analysis. Results After 24 hours, 34 genes were identified to be up-regulated by ALK1 signalling. Analysing ALK1 regulated gene expression after 4 hours revealed 13 genes to be up- and 2 to be down-regulated. Several of these genes, including IL-8, ET-1, ID1, HPTPη and TEAD4 are reported to be involved in angiogenesis. Evaluation of ALK1 regulated gene expression in different human endothelial cell types was not in complete agreement. Further on, disparity between constitutively active ALK1 and TGF-β1 induced gene expression in HMEC-1 cells and primary HUVECs was observed. Conclusion Gene array analysis identified 49 genes to be regulated by ALK1 signalling and at least 14 genes are reported to be involved in angiogenesis. There was substantial agreement between the gene array and quantitative real-time PCR data. The angiogenesis related genes might be potential HHT modifier genes. In addition, the results suggest endothelial cell type specific ALK1 and TGF-β signalling. PMID:16594992

  7. Effects of changes in intracellular iron pool on AlkB-dependent and AlkB-independent mechanisms protecting E.coli cells against mutagenic action of alkylating agent.

    PubMed

    Sikora, Anna; Maciejewska, Agnieszka M; Poznański, Jarosław; Pilżys, Tomasz; Marcinkowski, Michał; Dylewska, Małgorzata; Piwowarski, Jan; Jakubczak, Wioletta; Pawlak, Katarzyna; Grzesiuk, Elżbieta

    2015-08-01

    An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH(+) strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3 → Arg(+) reversion induction after λ>320 nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O2) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg(+) revertants in AB1157 alkB(+)hemH(-)/pMW1 strain was 40 and 26% reduced comparing to the alkB(+)hemH(-) and alkB(+)hemH(+)/pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ>320 nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg(+) reversion was observed in irradiated and MMS-treated hemH(-)alkB(-) cells comparing to the hemH(+)alkB(-) strain. Also, the level of Arg(+) revertants in the irradiated and MMS treated hemH(-) alkB(-) mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen species. According to our hypothesis it may be caused by non-enzymatic dealkylation of alkylated dNTPs in E. coli cells. In in vitro studies, the absence of AlkB protein in the presence of iron ions allowed etheno(ϵ) dATP and ϵdCTP to spontaneously convert to dAMP and dCMP, respectively. Thus, hemH(-) intra-cellular conditions may favor Fe-dependent dealkylation of modified dNTPs.

  8. Imaging Characteristics in ALK Fusion-Positive Lung Adenocarcinomas by Using HRCT

    PubMed Central

    Okumura, Sakae; Kuroda, Hiroaki; Uehara, Hirofumi; Mun, Mingyon; Takeuchi, Kengo; Nakagawa, Ken

    2014-01-01

    Objectives: We aimed to identify high-resolution computed tomography (HRCT) features useful to distinguish the anaplastic lymphoma kinase gene (ALK) fusion-positive and negative lung adenocarcinomas. Methods: We included 236 surgically resected adenocarcinoma lesions, which included 27 consecutive ALK fusion-positive (AP) lesions, 115 epidermal growth factor receptor mutation-positive lesions, and 94 double-negative lesions. HRCT parameters including size, air bronchograms, pleural indentation, spiculation, and tumor disappearance rate (TDR) were compared. In addition, prevalence of small lesions (≤20 mm) and solid lesions (TDR ≤20%) were compared. Results: AP lesions were significantly smaller and had lower TDR (%) than ALK fusion-negative (AN) lesions (tumor diameter: 20.7 mm ± 14.1 mm vs. 27.4 mm ± 13.8 mm, respectively, p <0.01; TDR: 22.8% ± 24.8% vs. 44.8% ± 33.2%, respectively, p <0.01). All AP lesions >20 mm (n = 7, 25.9%) showed a solid pattern. Among all small lesions, AP lesions had lower TDR and more frequent spiculation than AN lesions (p <0.01). Among solid lesions, AP lesions were smaller than AN lesions (p = 0.01). Conclusion: AP lung lesions were significantly smaller and had a lower TDR than AN lesions. Spiculation was more frequent in small lesions. Non-solid >20 mm lesions may be ALK fusion-negative. PMID:24899136

  9. Determining the contribution of NPM1 heterozygosity to NPM-ALK-induced lymphomagenesis.

    PubMed

    Mduff, Fiona K E; Hook, C Elizabeth; Tooze, Reuben M; Huntly, Brian J; Pandolfi, Pier Paolo; Turner, Suzanne D

    2011-09-01

    Heterozygous expression of Nucleophosmin (NPM1) predisposes to hematological malignancies in the mouse and cooperates with Myc in lymphomagenesis. NPM1 is therefore regarded as a haploinsufficient tumor suppressor. Heterozygous loss of NPM1 occurs as a result of the t(2;5), which generates the oncogenic fusion tyrosine kinase, NPM-anaplastic lymphoma kinase (ALK), a molecule underlying the pathogenesis of anaplastic large cell lymphoma (ALCL). Given the aforementioned role of NPM1 as a tumor suppressor, we hypothesized that NPM1 heterozygosity would cooperate with NPM-ALK in lymphomagenesis. In the event, we observed no difference in tumor latency, incidence or phenotype in NPM-ALK-transgenic mice heterozygous for NPM1 relative to transgenic mice expressing both NPM1 alleles. We propose that although the t(2;5) simultaneously reduces NPM1 allelic dosage and creates the NPM-ALK fusion protein, the two events do not cooperate in the pathogenesis of ALCL in our mouse model. These data indicate that a tumor-suppressive role for NPM1 may depend on cellular and/or genetic context.

  10. Atractylodin Inhibits Interleukin-6 by Blocking NPM-ALK Activation and MAPKs in HMC-1.

    PubMed

    Chae, Hee-Sung; Kim, Young-Mi; Chin, Young-Won

    2016-09-02

    Atractylodin is one of the major constituents of the rhizome of Atractylodes lancea, which is widely used in Korean traditional medicine as a remedy for the treatment of gastritis and gastric ulcers. Despite of a major constituent of widely used botanical to treat inflammatory responses little is known about anti-inflammatory effect of atractylodin in the human mast cell (HMC-1). Hence, we evaluated the effect of atractylodin on the release of IL-6, the involvement of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) and mitogen-activated protein kinases (MAPKs) in phorbol-12-myristate-13-acetate and A23187-induced HMC-1. In addition, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), phospholipase C (PLC) gamma 1, and AKT phosphorylation relevant to NPM-ALK signal pathway were assessed. IL-6 levels in the HMC-1 stimulated by phorbol-12-myristate-13-acetate and A23187 were apparently decreased by the treatment of atractylodin. Concurrently, atractylodin not only inhibited the phosphorylation of NPM-ALK, but also suppressed the phosphorylation of JAK2, STAT3, PLC gamma 1, and AKT. Furthermore, the activated mitogen-activated protein kinases (MAPKs) by phorbol-12-myristate-13-acetate and A23187 were inhibited by atractylodin. These results suggested that atractylodin might have a potential regulatory effect on inflammatory mediator expression through blockade of both the phosphorylation of MAPKs and the NPM-ALK signaling pathway.

  11. Clinicopathologic characteristics of EGFR, KRAS, and ALK alterations in 6,595 lung cancers

    PubMed Central

    Lee, Boram; Lee, Taebum; Lee, Se-Hoon; Choi, Yoon-La; Han, Joungho

    2016-01-01

    Background EGFR, KRAS, and ALK alterations are major genetic changes found in non-small cell lung cancers (NSCLCs). Testing advanced lung adenocarcinoma tumors for these three genes is now standard care. The purpose of this study was to investigate the clinicopathologic expression pattern of these three genes in East Asian NSCLC patients. Patients and methods We conducted a retrospective study of all patients tested for mutations of these three genes at a single institute in Korea between 2006 and 2014. Study data were extracted from electronic medical records. Univariate and multivariate logistic regression analyses were used to measure associations between clinicopathologic features and alterations of EGFR, KRAS, and ALK. Results We detected 12 EGFR-mutated tumors with additional mutations in KRAS (N=6, 0.1%) or ALK (N=6, 0.1%). General clinicopathologic characteristics of tumors with EGFR, KRAS, or ALK mutations were similar to previous reports. Patients having EGFR L858R point mutations were older than patients having EGFR exon 19 deletions. EGFR G719X point mutations were more common in men and smokers than exon 19 deletions or L858R point mutations. Tumors having KRAS G12C mutations were less often of mucinous type than those with G12D or G12V, mutations. Conclusions This is the largest three gene molecular epidemiology study in East Asian NSCLC patients. Each genetic alteration was associated with distinct clinicopathologic characteristics. Furthermore, different age and sex are associated with different subtypes of EGFR and KRAS mutations. PMID:26992209

  12. ClogP(alk): a method for predicting alkane/water partition coefficient.

    PubMed

    Kenny, Peter W; Montanari, Carlos A; Prokopczyk, Igor M

    2013-05-01

    Alkane/water partition coefficients (P(alk)) are less familiar to the molecular design community than their 1-octanol/water equivalents and access to both data and prediction tools is much more limited. A method for predicting alkane/water partition coefficient from molecular structure is introduced. The basis for the ClogP(alk) model is the strong (R² = 0.987) relationship between alkane/water partition coefficient and molecular surface area (MSA) that was observed for saturated hydrocarbons. The model treats a molecule as a perturbation of a saturated hydrocarbon molecule with the same MSA and uses increments defined for functional groups to quantify the extent to which logP(alk) is perturbed by the introduction each functional group. Interactions between functional groups, such as intramolecular hydrogen bonds are also parameterized within a perturbation framework. The functional groups and interactions between them are specified substructurally in a transparent and reproducible manner using SMARTS notation. The ClogP(alk) model was parameterized using data measured for structurally prototypical compounds that dominate the literature on alkane/water partition coefficients and then validated using an external test set of 100 alkane/water logP measurements, the majority of which were for drugs.

  13. Atractylodin Inhibits Interleukin-6 by Blocking NPM-ALK Activation and MAPKs in HMC-1.

    PubMed

    Chae, Hee-Sung; Kim, Young-Mi; Chin, Young-Won

    2016-01-01

    Atractylodin is one of the major constituents of the rhizome of Atractylodes lancea, which is widely used in Korean traditional medicine as a remedy for the treatment of gastritis and gastric ulcers. Despite of a major constituent of widely used botanical to treat inflammatory responses little is known about anti-inflammatory effect of atractylodin in the human mast cell (HMC-1). Hence, we evaluated the effect of atractylodin on the release of IL-6, the involvement of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) and mitogen-activated protein kinases (MAPKs) in phorbol-12-myristate-13-acetate and A23187-induced HMC-1. In addition, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), phospholipase C (PLC) gamma 1, and AKT phosphorylation relevant to NPM-ALK signal pathway were assessed. IL-6 levels in the HMC-1 stimulated by phorbol-12-myristate-13-acetate and A23187 were apparently decreased by the treatment of atractylodin. Concurrently, atractylodin not only inhibited the phosphorylation of NPM-ALK, but also suppressed the phosphorylation of JAK2, STAT3, PLC gamma 1, and AKT. Furthermore, the activated mitogen-activated protein kinases (MAPKs) by phorbol-12-myristate-13-acetate and A23187 were inhibited by atractylodin. These results suggested that atractylodin might have a potential regulatory effect on inflammatory mediator expression through blockade of both the phosphorylation of MAPKs and the NPM-ALK signaling pathway. PMID:27598116

  14. 40 CFR 721.435 - Alkylphenylpolyether-alk-a-nol-a-mines (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.435 Alkylphenylpolyether-alk-a-nol-a-mines (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substances identified...

  15. 40 CFR 721.435 - Alkylphenylpolyether-alk-a-nol-a-mines (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.435 Alkylphenylpolyether-alk-a-nol-a-mines (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substances identified...

  16. Detection of EML4-ALK fusion gene and features associated with EGFR mutations in Chinese patients with non-small-cell lung cancer

    PubMed Central

    Wen, Miaomiao; Wang, Xuejiao; Sun, Ying; Xia, Jinghua; Fan, Liangbo; Xing, Hao; Zhang, Zhipei; Li, Xiaofei

    2016-01-01

    Purpose Echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK) and epidermal growth factor receptor (EGFR) define specific molecular subsets of lung cancer with distinct clinical features. We aimed at revealing the clinical features of EML4-ALK fusion gene and EGFR mutation in non-small-cell lung cancer (NSCLC). Methods We enrolled 694 Chinese patients with NSCLC for analysis. EML4-ALK fusion gene was analyzed by real-time polymerase chain reaction, and EGFR mutations were analyzed by amplified refractory mutation system. Results Among the 694 patients, 60 (8.65%) patients had EML4-ALK fusions. In continuity correction χ2 test analysis, EML4-ALK fusion gene was correlated with sex, age, smoking status, and histology, but no significant association was observed between EML4-ALK fusion gene and clinical stage. A total of 147 (21.18%) patients had EGFR mutations. In concordance with previous reports, EGFR mutation was correlated with age, smoking status, histology, and clinical stage, whereas patient age was not significantly associated with EGFR mutation. Meanwhile, to our surprise, six (0.86%) patients had coexisting EML4-ALK fusions and EGFR mutations. Conclusion EML4-ALK fusion gene defines a new molecular subset in patients with NSCLC. Six patients who harbored both EML4-ALK fusion genes and EGFR mutations were identified in our study. The EGFR mutations and the EML4-ALK fusion genes are coexistent. PMID:27103824

  17. Molecular Characterization of Inflammatory Myofibroblastic Tumors with Frequent ALK and ROS1 Fusions and Rare Novel RET Gene Rearrangement

    PubMed Central

    Antonescu, Cristina R; Suurmeijer, Albert JH; Zhang, Lei; Sung, Yun-Shao; Jungbluth, Achim A; Travis, William D; Al-Ahmadie, Hikmat; Fletcher, Christopher DM; Alaggio, Rita

    2015-01-01

    Approximately 50% of conventional IMTs harbor ALK gene rearrangement and overexpress ALK. Recently gene fusions involving other kinases have been implicated in the pathogenesis of IMT, including ROS1 and in one patient PDGFRB. However, it remains uncertain if the emerging genotypes correlate with clinicopathologic characteristics of IMT. In this study we expand the molecular investigation of IMT in a large cohort of different clinical presentations and analyze for potential genotype-phenotype associations. Criteria for inclusion in the study were typical morphology and tissue availability for molecular studies. The lack of ALK immunoreactivity was not an excluding factor. As overlapping gene fusions involving actionable kinases are emerging in both IMT and lung cancer, we set out to evaluate abnormalities in ALK, ROS1, PDGFRB, NTRK1 and RET by FISH. Additionally, next generation paired-end RNA sequencing and FusionSeq algorithm was applied in 4 cases, which identified EML4-ALK fusions in 2 cases. Of the 62 IMTs (25 children and 37 adults), 35 (56%) showed ALK gene rearrangement. Of note, EML4-ALK inversion was noted in 7 (20%) cases, seen mainly in the lung and soft tissue of young children including 2 lesions from newborns. There were 6 (10%) ROS1 rearranged IMTs, all except one presenting in children, mainly in the lung and intra-abdominal and showed a distinctive fascicular growth of spindle cells with long cell processes, often positive for ROS1 IHC. Two of the cases showed TFG-ROS1 fusions. Interestingly, one adult IMT revealed a RET gene rearrangement, a previously unreported finding. Our results show that 42/62 (68%) of IMTs are characterized by kinase fusions, offering a rationale for targeted therapeutic strategies. Interestingly 90% of fusion negative IMT were seen in adults, while >90% of pediatric IMT showed gene rearrangements.EML4-ALK inversion and ROS1 fusions emerge as common fusion abnormalities in IMT, closely recapitulating the pattern seen in

  18. Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching

    SciTech Connect

    Holland, P.; Hollis, T

    2010-01-01

    In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions, but it also repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a 'searching' mode and 'repair' mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  19. Redundant and nonredundant roles for Cdc42 and Rac1 in lymphomas developed in NPM-ALK transgenic mice.

    PubMed

    Choudhari, Ramesh; Minero, Valerio Giacomo; Menotti, Matteo; Pulito, Roberta; Brakebusch, Cord; Compagno, Mara; Voena, Claudia; Ambrogio, Chiara; Chiarle, Roberto

    2016-03-10

    Increasing evidence suggests that Rho family GTPases could have a critical role in the biology of T-cell lymphoma. In ALK-rearranged anaplastic large cell lymphoma (ALCL), a specific subtype of T-cell lymphoma, the Rho family GTPases Cdc42 and Rac1 are activated by the ALK oncogenic activity. In vitro studies have shown that Cdc42 and Rac1 control rather similar phenotypes of ALCL biology such as the proliferation, survival, and migration of lymphoma cells. However, their role and possible redundancy in ALK-driven lymphoma development in vivo are still undetermined. We genetically deleted Cdc42 or Rac1 in a mouse model of ALK-rearranged ALCL to show that either Cdc42 or Rac1 deletion impaired lymphoma development, modified lymphoma morphology, actin filament distribution, and migration properties of lymphoma cells. Cdc42 or Rac1 deletion primarily affected survival rather than proliferation of lymphoma cells. Apoptosis of lymphoma cells was equally induced following Cdc42 or Rac1 deletion, was associated with upregulation of the proapoptotic molecule Bid, and was blocked by Bcl2 overexpression. Remarkably, Cdc42/Rac1 double deletion, but not Cdc42 or Rac1 single deletions, completely prevented NPM-ALK lymphoma dissemination in vivo. Thus, Cdc42 and Rac1 have nonredundant roles in controlling ALK-rearranged lymphoma survival and morphology but are redundant for lymphoma dissemination, suggesting that targeting both GTPases could represent a preferable therapeutic option for ALCL treatment.

  20. 5-(1,3-Benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazole derivatives as potent and selective transforming growth factor-β type I receptor inhibitors.

    PubMed

    Amada, Hideaki; Sekiguchi, Yoshinori; Ono, Naoya; Koami, Takeshi; Takayama, Tetsuo; Yabuuchi, Tetsuya; Katakai, Hironori; Ikeda, Akiko; Aoki, Mari; Naruse, Takumi; Wada, Reiko; Nozoe, Akiko; Sato, Masakazu

    2012-12-15

    A series of 5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazole derivatives was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and for their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. As a representative compound, 16i was a potent and selective ALK5 inhibitor, exhibiting a good enzyme inhibitory activity (IC(50) = 5.5 nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC(50) = 36 nM). Furthermore, the topical application of 3% 16i lotion significantly inhibited Smad2 phosphorylation in Mouse skin (90% inhibition compared with vehicle-treated animals).

  1. BMP type I receptor ALK2 is required for angiotensin II-induced cardiac hypertrophy.

    PubMed

    Shahid, Mohd; Spagnolli, Ester; Ernande, Laura; Thoonen, Robrecht; Kolodziej, Starsha A; Leyton, Patricio A; Cheng, Juan; Tainsh, Robert E T; Mayeur, Claire; Rhee, David K; Wu, Mei X; Scherrer-Crosbie, Marielle; Buys, Emmanuel S; Zapol, Warren M; Bloch, Kenneth D; Bloch, Donald B

    2016-04-15

    Bone morphogenetic protein (BMP) signaling contributes to the development of cardiac hypertrophy. However, the identity of the BMP type I receptor involved in cardiac hypertrophy and the underlying molecular mechanisms are poorly understood. By using quantitative PCR and immunoblotting, we demonstrated that BMP signaling increased during phenylephrine-induced hypertrophy in cultured neonatal rat cardiomyocytes (NRCs), as evidenced by increased phosphorylation of Smads 1 and 5 and induction of Id1 gene expression. Inhibition of BMP signaling with LDN193189 or noggin, and silencing of Smad 1 or 4 using small interfering RNA diminished the ability of phenylephrine to induce hypertrophy in NRCs. Conversely, activation of BMP signaling with BMP2 or BMP4 induced hypertrophy in NRCs. Luciferase reporter assay further showed that BMP2 or BMP4 treatment of NRCs repressed atrogin-1 gene expression concomitant with an increase in calcineurin protein levels and enhanced activity of nuclear factor of activated T cells, providing a mechanism by which BMP signaling contributes to cardiac hypertrophy. In a model of cardiac hypertrophy, C57BL/6 mice treated with angiotensin II (A2) had increased BMP signaling in the left ventricle. Treatment with LDN193189 attenuated A2-induced cardiac hypertrophy and collagen deposition in left ventricles. Cardiomyocyte-specific deletion of BMP type I receptor ALK2 (activin-like kinase 2), but not ALK1 or ALK3, inhibited BMP signaling and mitigated A2-induced cardiac hypertrophy and left ventricular fibrosis in mice. The results suggest that BMP signaling upregulates the calcineurin/nuclear factor of activated T cell pathway via BMP type I receptor ALK2, contributing to cardiac hypertrophy and fibrosis. PMID:26873969

  2. The potent oncogene NPM-ALK mediates malignant transformation of normal human CD4(+) T lymphocytes.

    PubMed

    Zhang, Qian; Wei, Fang; Wang, Hong Yi; Liu, Xiaobin; Roy, Darshan; Xiong, Qun-Bin; Jiang, Shuguang; Medvec, Andrew; Danet-Desnoyers, Gwenn; Watt, Christopher; Tomczak, Ewa; Kalos, Michael; Riley, James L; Wasik, Mariusz A

    2013-12-01

    With this study we have demonstrated that in vitro transduction of normal human CD4(+) T lymphocytes with NPM-ALK results in their malignant transformation. The transformed cells become immortalized and display morphology and immunophenotype characteristic of patient-derived anaplastic large-cell lymphomas. These unique features, which are strictly dependent on NPM-ALK activity and expression, include perpetual cell growth, proliferation, and survival; activation of the key signal transduction pathways STAT3 and mTORC1; and expression of CD30 (the hallmark of anaplastic large-cell lymphoma) and of immunosuppressive cytokine IL-10 and cell-surface protein PD-L1/CD274. Implantation of NPM-ALK-transformed CD4(+) T lymphocytes into immunodeficient mice resulted in formation of tumors indistinguishable from patients' anaplastic large-cell lymphomas. Our findings demonstrate that the key aspects of human carcinogenesis closely recapitulating the features of the native tumors can be faithfully reproduced in vitro when an appropriate oncogene is used to transform its natural target cells; this in turn points to the fundamental role in malignant cell transformation of potent oncogenes expressed in the relevant target cells. Such transformed cells should permit study of the early stages of carcinogenesis, and in particular the initial oncogene-host cell interactions. This experimental design could also be useful for studies of the effects of early therapeutic intervention and likely also the mechanisms of malignant progression.

  3. Regulation of the ALK1 ligands, BMP9 and BMP10.

    PubMed

    Li, Wei; Salmon, Richard M; Jiang, He; Morrell, Nicholas W

    2016-08-15

    Bone morphogenetic protein (BMP)9 and BMP10 are high affinity ligands for activin receptor-like kinase 1 (ALK1), a type I BMP receptor mainly expressed on vascular endothelial cells (ECs). ALK1-mediated BMP9/BMP10 signalling pathways have emerged as essential in EC biology and in angiogenesis. Several genetic mutations in the genes encoding the ligands and receptors of this pathway have been reported in two cardiovascular diseases, pulmonary arterial hypertension (PAH) and hereditary haemorrhagic telangiectasia (HHT). Administration of recombinant BMP9 reverses experimental PAH in preclinical rodent models. Dalantercept, an Fc-fusion protein of the extracellular domain of ALK1 and a ligand trap for BMP9 and BMP10, is in phase II clinical trials for anti-tumour angiogenesis. Understanding the regulation of BMP9 and BMP10, at both gene and protein levels, under physiological and pathological conditions, will reveal essential information and potential novel prognostic markers for the BMP9/BMP10-targeted therapies. PMID:27528761

  4. Quantitative determination of S-alk(en)ylcysteine-S-oxides by micellar electrokinetic capillary chromatography.

    PubMed

    Kubec, Roman; Dadáková, Eva

    2008-11-28

    A novel method for determination of S-alk(en)ylcysteine-S-oxides by capillary electrophoresis has been developed and validated. The method is based on extraction of these sulfur amino acids by methanol, their derivatization by fluorenylmethyl chloroformate and subsequent separation by micellar electrokinetic capillary chromatography. Main advantages of the new method are simplicity, sensitivity, high specificity and very low running costs, making it suitable for routine analysis of a large number of samples. Employing this method, the content of S-alk(en)ylcysteine-S-oxides was determined in 12 commonly consumed alliaceous and cruciferous vegetables (e.g. garlic, onion, leek, chive, cabbage, radish, cauliflower and broccoli). The total content of these amino acids in the Allium species evaluated varied between 0.59 and 12.3mg g(-1) fresh weight. Whereas alliin was found only in garlic, isoalliin was the major S-alk(en)ylcysteine-S-oxide in onion, leek, chive and shallot. On the other hand, the cruciferous species analyzed contained only methiin in the range of 0.06-2.45mg g(-1) fresh weight. PMID:18952220

  5. ALK5-dependent TGF-β signaling is a major determinant of late stage adult neurogenesis

    PubMed Central

    He, Yingbo; Zhang, Hui; Yung, Andrea; Villeda, Saul A; Jaeger, Philipp A; Olayiwola, Oluwatobi; Fainberg, Nina; Wyss-Coray, Tony

    2014-01-01

    The transforming growth factor-β (TGF-β) signaling pathway serves critical functions in central nervous system (CNS) development, but apart from its proposed neuroprotective actions, its physiological role in the adult brain is unclear. We observed a prominent activation of TGF-β signaling in the adult dentate gyrus and expression of downstream Smad proteins in this neurogenic zone. Consistent with a function of TGF-β signaling in adult neurogenesis, genetic deletion of the TGF-β receptor ALK5 reduced the number, migration, and dendritic arborization of newborn neurons. Conversely, constitutive activation of neuronal ALK5 in forebrain caused a striking increase in these aspects of neurogenesis and was associated with higher expression of c-fos in newborn neurons and with stronger memory function. Our findings describe a new and unexpected role for ALK5-dependent TGF-β signaling as a regulator of the late stages of adult hippocampal neurogenesis which may have implications for changes in neurogenesis during aging and disease. PMID:24859199

  6. Emerging Paradigms in the Development of Resistance to Tyrosine Kinase Inhibitors in Lung Cancer

    PubMed Central

    Gainor, Justin F.; Shaw, Alice T.

    2013-01-01

    The success of tyrosine kinase inhibitors (TKIs) in select patients with non–small-cell lung cancer (NSCLC) has transformed management of the disease, placing new emphasis on understanding the molecular characteristics of tumor specimens. It is now recognized that genetic alterations in the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) define two unique subtypes of NSCLC that are highly responsive to genotype-directed TKIs. Despite this initial sensitivity, however, the long-term effectiveness of such therapies is universally limited by the development of resistance. Identifying the mechanisms underlying this resistance is an area of intense, ongoing investigation. In this review, we provide an overview of recent experience in the field, focusing on results from preclinical resistance models and studies of patient-derived, TKI-resistant tumor specimens. Although diverse TKI resistance mechanisms have been identified within EGFR-mutant and ALK-positive patients, we highlight common principles of resistance shared between these groups. These include the development of secondary mutations in the kinase target, gene amplification of the primary oncogene, and upregulation of bypass signaling tracts. In EGFR-mutant and ALK-positive patients alike, acquired resistance may also be a dynamic and multifactorial process that may necessitate the use of treatment combinations. We believe that insights into the mechanisms of TKI resistance in patients with EGFR mutations or ALK rearrangements may inform the development of novel treatment strategies in NSCLC, which may also be generalizable to other kinase-driven malignancies. PMID:24101047

  7. Reduced mural cell coverage and impaired vessel integrity after angiogenic stimulation in the Alk1-deficient brain

    PubMed Central

    Chen, Wanqiu; Guo, Yi; Walker, Espen J.; Shen, Fanxia; Jun, Kristine; Oh, S. Paul; Degos, Vincent; Lawton, Michael T.; Tihan, Tarik; Davalos, Dimitrios; Akassoglou, Katerina; Nelson, Jeffrey; Pile-Spellman, John; Su, Hua; Young, William L.

    2013-01-01

    Objective Vessels in brain arteriovenous malformations (bAVM) are prone to rupture. The underlying pathogenesis is not clear. Hereditary hemorrhagic telangiectasia type 2 (HHT2) patients with activin receptor-like kinase 1 (Alk1) mutation have a higher incidence of bAVM than the general population. We tested the hypothesis that vascular endothelial growth factor (VEGF) impairs vascular integrity in the Alk1-deficient brain through reduction of mural cell-coverage. Methods and Results Adult Alk11f/2f mice (loxP sites flanking exons 4-6) and wild-type (WT) mice were injected with 2×107 PFU Ad-Cre and 2×109 genome copies of AAV-VEGF to induce focal homozygous Alk1 deletion (in Alk11f/2f mice) and angiogenesis. Brain vessels were analyzed eight weeks later. Compared to WT mice, the Alk1-deficient brain had more fibrin (99±30×103 pixels/mm2 vs. 40±13×103, P=0.001), iron deposition (508±506 pixels/mm2 vs. 6 ±49, P=0.04), and Iba1+ microglia/macrophage infiltration (888±420 Iba1+ cells/mm2 vs. 240±104 Iba1+, P=0.001) after VEGF stimulation. In the angiogenic foci, the Alk1-deficient brain had more α-SMA- vessels (52±9% vs. 12±7%, P<0.001), fewer vascular associated pericytes (503±179/mm2 vs. 931±115, P<0.001), and reduced PDGFR-β expression (26±9%, P<0.001). Conclusion Reduction of mural cell coverage in response to VEGF stimulation is a potential mechanism for the impairment of vessel wall integrity in HHT2-associated bAVM. PMID:23241407

  8. Concurrent progress of reprogramming and gene correction to overcome therapeutic limitation of mutant ALK2-iPSC

    PubMed Central

    Kim, Bu-Yeo; Jeong, SangKyun; Lee, Seo-Young; Lee, So Min; Gweon, Eun Jeong; Ahn, Hyunjun; Kim, Janghwan; Chung, Sun-Ku

    2016-01-01

    Fibrodysplasia ossificans progressiva (FOP) syndrome is caused by mutation of the gene ACVR1, encoding a constitutive active bone morphogenetic protein type I receptor (also called ALK2) to induce heterotopic ossification in the patient. To genetically correct it, we attempted to generate the mutant ALK2-iPSCs (mALK2-iPSCs) from FOP-human dermal fibroblasts. However, the mALK2 leads to inhibitory pluripotency maintenance, or impaired clonogenic potential after single-cell dissociation as an inevitable step, which applies gene-correction tools to induced pluripotent stem cells (iPSCs). Thus, current iPSC-based gene therapy approach reveals a limitation that is not readily applicable to iPSCs with ALK2 mutation. Here we developed a simplified one-step procedure by simultaneously introducing reprogramming and gene-editing components into human fibroblasts derived from patient with FOP syndrome, and genetically treated it. The mixtures of reprogramming and gene-editing components are composed of reprogramming episomal vectors, CRISPR/Cas9-expressing vectors and single-stranded oligodeoxynucleotide harboring normal base to correct ALK2 c.617G>A. The one-step-mediated ALK2 gene-corrected iPSCs restored global gene expression pattern, as well as mineralization to the extent of normal iPSCs. This procedure not only helps save time, labor and costs but also opens up a new paradigm that is beyond the current application of gene-editing methodologies, which is hampered by inhibitory pluripotency-maintenance requirements, or vulnerability of single-cell-dissociated iPSCs. PMID:27256111

  9. Clinical and Pathologic Findings of Spitz Nevi and Atypical Spitz Tumors with ALK Fusions

    PubMed Central

    Busam, Klaus J; Kutzner, Heinz; Cerroni, Lorenzo; Wiesner, Thomas

    2016-01-01

    Spitz tumors represent a group of melanocytic neoplasms that typically affects young individuals. Microscopically the lesions are composed of cytologically distinct spindle and epithelioid melanocytes, with a range in the architectural display or the cells, their nuclear features, and secondary epidermal or stromal changes. Recently, kinase fusions have been documented in a subset of Spitz tumors, but there is limited information on the clinical and pathologic features associated with those lesions. Here, we report a series of 17 patients (9 male, 8 female) with spitzoid neoplasms showing ALK fusions (5 Spitz nevi and 12 atypical Spitz tumors). The patients’ ages ranged from 2 years to 35 years (mean = 17; median = 16). Most lesions were located on the lower extremities and presented clinically as polypoid nodules. All tumors were compound melanocytic proliferations with a predominant intradermal growth. Tumor thickness ranged from 1.1 to 6 mm (mean = 2.9 mm; median = 2.5 mm). The most characteristic histopathologic feature of the tumors (seen in all but two lesions) was a plexiform dermal growth of intersecting fascicles of fusiform melanocytes. All but two tumors were amelanotic. All tumors were strongly immunoreactive for ALK. The ALK rearrangements were confirmed in all cases by fluorescence in situ hybridization (FISH) and the fusion partner was determined by quantitative polymerase chain reaction as TPM3 (tropomyosin 3) in 11 cases and DCTN1 (dynactin 1) in 6 cases. None of the eight tumors, which were analyzed by FISH for copy number changes of 6p, 6q, 9p, or 11q met criteria for melanoma. Two patients underwent a sentinel lymph node biopsy, and in both cases melanocytes nests were found in the subcapsular sinus of the node. Array comparative genomic hybridization of these two tumors revealed no chromosomal gains or losses. In conclusion, our study revealed that Spitz nevi/tumors with ALK rearrangement show a characteristic plexiform morphology and that

  10. ALK as a novel lymphoma-associated tumor antigen: identification of 2 HLA-A2.1-restricted CD8+ T-cell epitopes.

    PubMed

    Passoni, Lorena; Scardino, Antonio; Bertazzoli, Carla; Gallo, Barbara; Coluccia, Addolorata M L; Lemonnier, François A; Kosmatopoulos, Konstadinos; Gambacorti-Passerini, Carlo

    2002-03-15

    Oncogenic anaplastic lymphoma kinase (ALK) fusion proteins (NPM/ALK and associated variants) are expressed in about 60% of anaplastic large cell lymphomas (ALCLs) but are absent in normal tissues. In this study, we investigated whether ALK, which is expressed at high levels in lymphoma cells, could be a target for antigen-specific cell-mediated immunotherapy. A panel of ALK-derived peptides was tested for their binding affinity to HLA-A*0201 molecules. Binding peptides were assessed for their capacity to elicit a specific immune response mediated by cytotoxic T lymphocytes (CTLs) both in vivo, in HLA-A*0201 transgenic mice, and in vitro in the peripheral blood lymphocytes (PBLs) from healthy donors. Two HLA-A*0201-restricted CTL epitopes, p280-89 (SLAMLDLLHV) and p375-86 (GVLLWEIFSL), both located in the ALK kinase domain were identified. The p280-89- and p375-86-induced peptide-specific CTL lines were able to specifically release interferon-gamma (IFN-gamma) on stimulation with ALK peptide-pulsed autologous Epstein-Barr virus-transformed B cells (LCLs) or T2 cells. Anti-ALK CTLs lysed HLA-matched ALCL and neuroblastoma cell lines endogenously expressing ALK proteins. CTL activity was inhibited by anti-HLA-A2 monoclonal antibody CR11.351, consistent with a class I-restricted mechanism of cytotoxicity. These results show the existence of functional anti-ALK CTL precursors within the peripheral T-cell repertoire of healthy donors, clearly indicating ALK as a tumor antigen and ALK-derived peptides, p280-89 and p375-86, as suitable epitopes for the development of vaccination strategies.

  11. Rearranged Anaplastic Lymphoma Kinase (ALK) Gene in Adult-Onset Papillary Thyroid Cancer Amongst Atomic Bomb Survivors

    PubMed Central

    Mukai, Mayumi; Takahashi, Keiko; Hayashi, Yuzo; Nakachi, Kei; Kusunoki, Yoichiro

    2012-01-01

    Background We previously noted that among atomic bomb survivors (ABS), the relative frequency of cases of adult papillary thyroid cancer (PTC) with chromosomal rearrangements (mainly RET/PTC) was significantly greater in those with relatively higher radiation exposure than those with lower radiation exposure. In contrast, the frequency of PTC cases with point mutations (mainly BRAFV600E) was significantly lower in patients with relatively higher radiation exposure than those with lower radiation exposure. We also found that among ABS, the frequency of PTC cases with no detectable gene alterations in RET, neurotrophic tyrosine kinase receptor 1 (NTRK1), BRAF, or RAS was significantly higher in patients with relatively higher radiation exposure than those with lower radiation exposure. However, in ABS with PTC, the relationship between the presence of the anaplastic lymphoma kinase (ALK) gene fused with other gene partners and radiation exposure has received little study. In this study, we tested the hypothesis that the relative frequency of rearranged ALK in ABS with PTC, and with no detectable gene alterations in RET, NTRK1, BRAF, or RAS, would be greater in those having relatively higher radiation exposures. Methods The 105 subjects in the study were drawn from the Life Span Study cohort of ABS of Hiroshima and Nagasaki who were diagnosed with PTC between 1956 and 1993. Seventy-nine were exposed (>0 mGy), and 26 were not exposed to A-bomb radiation. In the 25 ABS with PTC, and with no detectable gene alterations in RET, NTRK1, BRAF, or RAS, we examined archival, formalin-fixed, paraffin-embedded PTC specimens for rearrangement of ALK using reverse transcription–polymerase chain reaction and 5′ rapid amplification of cDNA ends (5′ RACE). Results We found rearranged ALK in 10 of 19 radiation-exposed PTC cases, but none among 6 patients with PTC with no radiation exposure. In addition, solid/trabecular-like architecture in PTC was closely associated with ALK

  12. The NPM-ALK tyrosine kinase mimics TCR signalling pathways, inducing NFAT and AP-1 by RAS-dependent mechanisms.

    PubMed

    Turner, Suzanne D; Yeung, Debra; Hadfield, Kathryn; Cook, Simon J; Alexander, Denis R

    2007-04-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expression is associated with the lymphoid malignancy anaplastic large cell lymphoma (ALCL) and results from a t(2;5) chromosomal translocation. We show that NPM-ALK induces Ras activation and phosphorylation of the ERK MAP Kinase consistent with activation of the Ras-MAP Kinase pathway. Furthermore, we demonstrate that activation of Ras is necessary for inducing transcription via NFAT/AP-1 composite transcriptional binding sites. This activity is dependent on NPM-ALK forming complexes with proteins that bind to autophosphorylated tyrosine residues at positions 156, 567 and 664, associated with binding to IRS-1, Shc and PLCgamma, respectively. Specifically, NPM-ALK activates transcription from the TRE promoter element, an AP-1 binding region, an activity dependent on both Ras and Shc activity. Our results show that NPM-ALK mimics activated T-cell receptor signalling by inducing pathways associated with the activation of NFAT/AP-1 transcription factors that bind to promoter elements found in a broad array of cytokine genes.

  13. NPM-ALK oncogenic tyrosine kinase controls T-cell identity by transcriptional regulation and epigenetic silencing in lymphoma cells.

    PubMed

    Ambrogio, Chiara; Martinengo, Cinzia; Voena, Claudia; Tondat, Fabrizio; Riera, Ludovica; di Celle, Paola Francia; Inghirami, Giorgio; Chiarle, Roberto

    2009-11-15

    Transformed cells in lymphomas usually maintain the phenotype of the postulated normal lymphocyte from which they arise. By contrast, anaplastic large cell lymphoma (ALCL) is a T-cell lymphoma with aberrant phenotype because of the defective expression of the T-cell receptor and other T-cell-specific molecules for still undetermined mechanisms. The majority of ALCL carries the translocation t(2;5) that encodes for the oncogenic tyrosine kinase NPM-ALK, fundamental for survival, proliferation, and migration of transformed T cells. Here, we show that loss of T-cell-specific molecules in ALCL cases is broader than reported previously and involves most T-cell receptor-related signaling molecules, including CD3epsilon, ZAP70, LAT, and SLP76. We further show that NPM-ALK, but not the kinase-dead NPM-ALK(K210R), downregulated the expression of these molecules by a STAT3-mediated gene transcription regulation and/or epigenetic silencing because this downregulation was reverted by treating ALCL cells with 5-aza-2-deoxycytidine or by knocking down STAT3 through short hairpin RNA. Finally, NPM-ALK increased the methylation of ZAP70 intron 1-exon 2 boundary region, and both NPM-ALK and STAT3 regulated the expression levels of DNA methyltransferase 1 in transformed T cells. Thus, our data reveal that oncogene-deregulated tyrosine kinase activity controls the expression of molecules that determine T-cell identity and signaling.

  14. Biofortification of oilseed Brassica juncea with the anti-cancer compound glucoraphanin by suppressing GSL-ALK gene family

    PubMed Central

    Augustine, Rehna; Bisht, Naveen C.

    2015-01-01

    Glucosinolates are amino acids derived secondary metabolites, invariably present in Brassicales, which have huge health and agricultural benefits. Sulphoraphane, the breakdown product of glucosinolate glucoraphanin is known to posses anti-cancer properties. AOP (2-oxoglutarate-dependent dioxygenases) or GSL-ALK enzyme catalyzes the conversion of desirable glucoraphanin to deleterious gluconapin and progoitrin, which are present in very high amounts in most of the cultivable Brassica species including Brassica juncea. In this study we showed that B. juncea encodes four functional homologs of GSL-ALK gene and constitutive silencing of GSL-ALK homologs resulted in accumulation of glucoraphanin up to 43.11 μmoles g−1 DW in the seeds with a concomitant reduction in the anti-nutritional glucosinolates. Glucoraphanin content was found remarkably high in leaves as well as sprouts of the transgenic lines. Transcript quantification of high glucoraphanin lines confirmed significant down-regulation of GSL-ALK homologs. Growth and other seed quality parameters of the transgenic lines did not show drastic difference, compared to the untransformed control. High glucoraphanin lines also showed higher resistance towards stem rot pathogen Sclerotinia sclerotiorum. Our results suggest that metabolic engineering of GSL-ALK has huge potential for enriching glucoraphanin content, and improve the oil quality and vegetable value of Brassica crops. PMID:26657321

  15. GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium

    PubMed Central

    Luo, Qun; He, Ying; Hou, Deng-Yong; Zhang, Jian-Guo; Shen, Xian-Rong

    2015-01-01

    To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium. PMID:26413044

  16. Sensitivity Analysis of the NPM-ALK Signalling Network Reveals Important Pathways for Anaplastic Large Cell Lymphoma Combination Therapy

    PubMed Central

    Buetti-Dinh, Antoine; O’Hare, Thomas

    2016-01-01

    A large subset of anaplastic large cell lymphoma (ALCL) patients harbour a somatic aberration in which anaplastic lymphoma kinase (ALK) is fused to nucleophosmin (NPM) resulting in a constitutively active signalling fusion protein, NPM-ALK. We computationally simulated the signalling network which mediates pathological cell survival and proliferation through NPM-ALK to identify therapeutically targetable nodes through which it may be possible to regain control of the tumourigenic process. The simulations reveal the predominant role of the VAV1-CDC42 (cell division control protein 42) pathway in NPM-ALK-driven cellular proliferation and of the Ras / mitogen-activated ERK kinase (MEK) / extracellular signal-regulated kinase (ERK) cascade in controlling cell survival. Our results also highlight the importance of a group of interleukins together with the Janus kinase 3 (JAK3) / signal transducer and activator of transcription 3 (STAT3) signalling in the development of NPM-ALK derived ALCL. Depending on the activity of JAK3 and STAT3, the system may also be sensitive to activation of protein tyrosine phosphatase-1 (SHP1), which has an inhibitory effect on cell survival and proliferation. The identification of signalling pathways active in tumourigenic processes is of fundamental importance for effective therapies. The prediction of alternative pathways that circumvent classical therapeutic targets opens the way to preventive approaches for countering the emergence of cancer resistance. PMID:27669408

  17. Identification of a new subclass of ALK-negative ALCL expressing aberrant levels of ERBB4 transcripts.

    PubMed

    Scarfò, Irene; Pellegrino, Elisa; Mereu, Elisabetta; Kwee, Ivo; Agnelli, Luca; Bergaggio, Elisa; Garaffo, Giulia; Vitale, Nicoletta; Caputo, Manuel; Machiorlatti, Rodolfo; Circosta, Paola; Abate, Francesco; Barreca, Antonella; Novero, Domenico; Mathew, Susan; Rinaldi, Andrea; Tiacci, Enrico; Serra, Sara; Deaglio, Silvia; Neri, Antonino; Falini, Brunangelo; Rabadan, Raul; Bertoni, Francesco; Inghirami, Giorgio; Piva, Roberto

    2016-01-14

    Anaplastic large-cell lymphoma (ALCL) is a clinical and biological heterogeneous disease that includes systemic anaplastic lymphoma kinase (ALK)-positive and ALK-negative entities. To discover biomarkers and/or genes involved in ALK-negative ALCL pathogenesis, we applied the cancer outlier profile analysis algorithm to a gene expression profiling data set including 249 cases of T-cell non-Hodgkin lymphoma and normal T cells. Ectopic coexpression of ERBB4 and COL29A1 genes was detected in 24% of ALK-negative ALCL patients. RNA sequencing and 5' RNA ligase-mediated rapid amplification of complementary DNA ends identified 2 novel ERBB4-truncated transcripts displaying intronic transcription start sites. By luciferase assays, we defined that the expression of ERBB4-aberrant transcripts is promoted by endogenous intronic long terminal repeats. ERBB4 expression was confirmed at the protein level by western blot analysis and immunohistochemistry. Lastly, we demonstrated that ERBB4-truncated forms show oncogenic potentials and that ERBB4 pharmacologic inhibition partially controls ALCL cell growth and disease progression in an ERBB4-positive patient-derived tumorgraft model. In conclusion, we identified a new subclass of ALK-negative ALCL characterized by aberrant expression of ERBB4-truncated transcripts carrying intronic 5' untranslated regions. PMID:26463425

  18. Biofortification of oilseed Brassica juncea with the anti-cancer compound glucoraphanin by suppressing GSL-ALK gene family.

    PubMed

    Augustine, Rehna; Bisht, Naveen C

    2015-01-01

    Glucosinolates are amino acids derived secondary metabolites, invariably present in Brassicales, which have huge health and agricultural benefits. Sulphoraphane, the breakdown product of glucosinolate glucoraphanin is known to posses anti-cancer properties. AOP (2-oxoglutarate-dependent dioxygenases) or GSL-ALK enzyme catalyzes the conversion of desirable glucoraphanin to deleterious gluconapin and progoitrin, which are present in very high amounts in most of the cultivable Brassica species including Brassica juncea. In this study we showed that B. juncea encodes four functional homologs of GSL-ALK gene and constitutive silencing of GSL-ALK homologs resulted in accumulation of glucoraphanin up to 43.11 μmoles g(-1) DW in the seeds with a concomitant reduction in the anti-nutritional glucosinolates. Glucoraphanin content was found remarkably high in leaves as well as sprouts of the transgenic lines. Transcript quantification of high glucoraphanin lines confirmed significant down-regulation of GSL-ALK homologs. Growth and other seed quality parameters of the transgenic lines did not show drastic difference, compared to the untransformed control. High glucoraphanin lines also showed higher resistance towards stem rot pathogen Sclerotinia sclerotiorum. Our results suggest that metabolic engineering of GSL-ALK has huge potential for enriching glucoraphanin content, and improve the oil quality and vegetable value of Brassica crops. PMID:26657321

  19. Downregulation of NPM-ALK by siRNA causes anaplastic large cell lymphoma cell growth inhibition and augments the anti cancer effects of chemotherapy in vitro.

    PubMed

    Hsu, Faye Yuan-yi; Zhao, Yi; Anderson, W French; Johnston, Patrick B

    2007-06-01

    The fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), results from the chromosome translocation t(2;5)(p23;q25) and is present in 50-70 percent of anaplastic large-cell lymphomas (ALCLs). NPM-ALK is a constitutively activated kinase that transforms cells through stimulating several mitogenic signaling pathways. To examine if the NPM-ALK is a potential therapeutic target in ALCL, we used siRNA to specifically downregulate the expression of the NPM-ALK in ALCL cell lines. In this report, we demonstrated viability loss in t(2;5)-positive ALCL cell lines, SUDHL-1 and Karpas 299 cells, but not in lymphoma cell lines without the chromosome translocation, Jurkat and Granta 519 cells. Further study demonstrated that the downregulation of NPM-ALK resulted in decreased cell proliferation and increased cell apoptosis. When used in combination with chemotherapeutic agents, such as doxorubicin, the inhibition of the NPM-ALK augments the chemosensitivity of the tumor cells. These results revealed the importance of continuous expression of NPM-ALK in maintaining the growth of ALCL cells. Our data also suggested that the repression of the fusion gene might be a potential novel therapeutic strategy for NPM-ALK positive ALCLs.

  20. Involvement of Grb2 adaptor protein in nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-mediated signaling and anaplastic large cell lymphoma growth.

    PubMed

    Riera, Ludovica; Lasorsa, Elena; Ambrogio, Chiara; Surrenti, Nadia; Voena, Claudia; Chiarle, Roberto

    2010-08-20

    Most anaplastic large cell lymphomas (ALCL) express oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. Frequently ALCL carry the t(2;5) translocation, which fuses the ALK gene to the nucleophosmin (NPM1) gene. The transforming activity mediated by NPM-ALK fusion induces different pathways that control proliferation and survival of lymphoma cells. Grb2 is an adaptor protein thought to play an important role in ALK-mediated transformation, but its interaction with NPM-ALK, as well as its function in regulating ALCL signaling pathways and cell growth, has never been elucidated. Here we show that active NPM-ALK, but not a kinase-dead mutant, bound and induced Grb2 phosphorylation in tyrosine 160. An intact SH3 domain at the C terminus of Grb2 was required for Tyr(160) phosphorylation. Furthermore, Grb2 did not bind to a single region but rather to different regions of NPM-ALK, mainly Tyr(152-156), Tyr(567), and a proline-rich region, Pro(415-417). Finally, shRNA knockdown experiments showed that Grb2 regulates primarily the NPM-ALK-mediated phosphorylation of SHP2 and plays a key role in ALCL cell growth.

  1. Activation of Rac1 and the exchange factor Vav3 are involved in NPM-ALK signaling in anaplastic large cell lymphomas.

    PubMed

    Colomba, A; Courilleau, D; Ramel, D; Billadeau, D D; Espinos, E; Delsol, G; Payrastre, B; Gaits-Iacovoni, F

    2008-04-24

    The majority of anaplastic large cell lymphomas (ALCLs) express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein, which is oncogenic due to its constitutive tyrosine kinase activity. Transformation by NPM-ALK not only increases proliferation, but also modifies cell shape and motility in both lymphoid and fibroblastic cells. We report that the Rac1 GTPase, a known cytoskeletal regulator, is activated by NPM-ALK in ALCL cell lines (Karpas 299 and Cost) and transfected cells (lymphoid Ba/F3 cells, NIH-3T3 fibroblasts). We have identified Vav3 as one of the exchange factors involved in Rac1 activation. Stimulation of Vav3 and Rac1 by NPM-ALK is under the control of Src kinases. It involves formation of a signaling complex between NPM-ALK, pp60(c-src), Lyn and Vav3, in which Vav3 associates with tyrosine 343 of NPM-ALK via its SH2 domain. Moreover, Vav3 is phosphorylated in NPM-ALK positive biopsies from patients suffering from ALCL, demonstrating the pathological relevance of this observation. The use of Vav3-specific shRNA and a dominant negative Rac1 mutant demonstrates the central role of GTPases in NPM-ALK elicited motility and invasion.

  2. Expression of an alkane monooxygenase (alkB) gene and methyl tert-butyl ether co-metabolic oxidation in Pseudomonas citronellolis.

    PubMed

    Bravo, Ana Luisa; Sigala, Juan Carlos; Le Borgne, Sylvie; Morales, Marcia

    2015-04-01

    Pseudomonas citronellolis UAM-Ps1 co-metabolically transforms methyl tert-butyl ether (MTBE) to tert-butyl alcohol with n-pentane (2.6 mM), n-octane (1.5 mM) or dicyclopropylketone (DCPK) (4.4 mM), a gratuitous inducer of alkane hydroxylase (AlkB) activity. The reverse transcription quantitative real-time PCR was used to quantify the alkane monooxygenase (alkB) gene expression. The alkB gene was expressed in the presence of n-alkanes and DCPK and MTBE oxidation occurred only in cultures when alkB was transcribed. A correlation between the number of alkB transcripts and MTBE consumption was found (ΜΤΒΕ consumption in μmol = 1.44e(-13) x DNA copies, R(2) = 0.99) when MTBE (0.84 mM) was added. Furthermore, alkB was cloned and expressed into Escherichia coli and the recombinant AlkB had a molecular weight of 42 kDa. This is the first report where the expression of alkB is related to the co-metabolic oxidation of MTBE. PMID:25432418

  3. CHARACTERIZATION OF THE ALKANE-INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS: IDENTIFICATION OF A NEW P450 FAMILY

    EPA Science Inventory

    The P450alk gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. Structural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures ...

  4. Expression of an alkane monooxygenase (alkB) gene and methyl tert-butyl ether co-metabolic oxidation in Pseudomonas citronellolis.

    PubMed

    Bravo, Ana Luisa; Sigala, Juan Carlos; Le Borgne, Sylvie; Morales, Marcia

    2015-04-01

    Pseudomonas citronellolis UAM-Ps1 co-metabolically transforms methyl tert-butyl ether (MTBE) to tert-butyl alcohol with n-pentane (2.6 mM), n-octane (1.5 mM) or dicyclopropylketone (DCPK) (4.4 mM), a gratuitous inducer of alkane hydroxylase (AlkB) activity. The reverse transcription quantitative real-time PCR was used to quantify the alkane monooxygenase (alkB) gene expression. The alkB gene was expressed in the presence of n-alkanes and DCPK and MTBE oxidation occurred only in cultures when alkB was transcribed. A correlation between the number of alkB transcripts and MTBE consumption was found (ΜΤΒΕ consumption in μmol = 1.44e(-13) x DNA copies, R(2) = 0.99) when MTBE (0.84 mM) was added. Furthermore, alkB was cloned and expressed into Escherichia coli and the recombinant AlkB had a molecular weight of 42 kDa. This is the first report where the expression of alkB is related to the co-metabolic oxidation of MTBE.

  5. An enzyme-linked immunosorbent assay to screen for inhibitors of the oncogenic anaplastic lymphoma kinase.

    PubMed

    Gunby, Rosalind Helen; Tartari, Carmen Julia; Porchia, Francesca; Donella-Deana, Arianna; Scapozza, Leonardo; Gambacorti-Passerini, Carlo

    2005-07-01

    The discovery of novel anti-cancer drugs targeting anaplastic lymphoma kinase (ALK), an oncogenic tyrosine kinase, raises the need for in vitro assays suitable for screening compounds for ALK inhibition. To this aim we have developed and optimized an ALK-specific enzyme-linked immunosorbent assay that employs a novel ALK peptide substrate and purified ALK kinase domain. PMID:15996942

  6. Alectinib-Induced Alopecia in a Patient with Anaplastic Lymphoma Kinase-Positive Non-Small Cell Lung Cancer

    PubMed Central

    Koizumi, Tomonobu; Fukushima, Toshirou; Gomi, Daisuke; Kobayashi, Takashi; Sekiguchi, Nodoka; Sakamoto, Akiyuki; Sasaki, Shigeru; Mamiya, Keiko

    2016-01-01

    Alectinib, a novel alternative anaplastic lymphoma kinase (ALK) inhibitor, is highly effective against ALK-positive non-small cell lung cancer (NSCLC) and is well tolerated. Molecular targeted agents generally have little contribution to alopecia. We encountered a case of alopecia that developed gradually over 2 months after initiation of alectinib administration for the treatment of ALK-positive NSCLC. The patient had no history of alopecia in previous treatments of cisplatin + pemetrexed and crizotinib. The present case indicates that alopecia should be taken into consideration as toxicity during alectinib treatment, which could adversely affect the psychological and emotional condition and quality of life even in patients treated with specific molecular targeted agents. PMID:27194980

  7. ETV6-NTRK3 Is Expressed in a Subset of ALK-Negative Inflammatory Myofibroblastic Tumors.

    PubMed

    Alassiri, Ali H; Ali, Rola H; Shen, Yaoqing; Lum, Amy; Strahlendorf, Caron; Deyell, Rebecca; Rassekh, Rod; Sorensen, Poul H; Laskin, Janessa; Marra, Marco; Yip, Stephen; Lee, Cheng-Han; Ng, Tony L

    2016-08-01

    Inflammatory myofibroblastic tumor (IMT) is a genetically heterogenous tumor of the viscera and soft tissues, with multiple molecular features having been demonstrated in this tumor type. About 50% of cases harbor an anaplastic lymphoma kinase (ALK) gene rearrangement, and recent studies have described novel fusions involving the ROS1 and PDGFRβ genes in a subset of ALK-negative cases. However, the molecular features of the remaining subset of cases are not yet defined. We report a case of a large, highly aggressive IMT of the lung in a 17-year-old girl. This case was molecularly characterized through whole-genome and transcriptome sequencing. Subsequently, we investigated a cohort of 15 ALK-negative IMTs of various anatomic sites. All cases were screened using fluorescence in situ hybridization (FISH) for rearrangement of the ETV6 locus and with reverse transcription polymerase chain reaction (RT-PCR) for the ETV6-NTRK3 fusion transcript. Whole-genome and transcriptome sequencing revealed an ETV6-NTRK3 fusion transcript in our index case. This was confirmed by FISH studies for ETV6 gene rearrangement, as well as by RT-PCR. In addition, 2 additional cases in our cohort demonstrated ETV6 rearrangement by FISH. The presence of ETV6-NTRK3 fusion transcript was demonstrated by RT-PCR in one of these additional cases. In summary, we demonstrate the expression of the ETV6-NTRK3 fusion oncogene in a small subset of IMTs, lending further support to the role of oncogenic tyrosine kinases in the pathophysiology of this tumor type. Our data also further expand the growing spectrum of tumor types expressing the ETV6-NTRK3 fusion. PMID:27259007

  8. Chloroacetaldehyde-induced mutagenesis in Escherichia coli: the role of AlkB protein in repair of 3,N(4)-ethenocytosine and 3,N(4)-alpha-hydroxyethanocytosine.

    PubMed

    Maciejewska, Agnieszka M; Ruszel, Karol P; Nieminuszczy, Jadwiga; Lewicka, Joanna; Sokołowska, Beata; Grzesiuk, Elzbieta; Kuśmierek, Jarosław T

    2010-02-01

    Etheno (epsilon) adducts are formed in reaction of DNA bases with various environmental carcinogens and endogenously created products of lipid peroxidation. Chloroacetaldehyde (CAA), a metabolite of carcinogen vinyl chloride, is routinely used to generate epsilon-adducts. We studied the role of AlkB, along with AlkA and Mug proteins, all engaged in repair of epsilon-adducts, in CAA-induced mutagenesis. The test system used involved pIF102 and pIF104 plasmids bearing the lactose operon of CC102 or CC104 origin (Cupples and Miller (1989) [17]) which allowed to monitor Lac(+) revertants, the latter arose by GC-->AT or GC-->TA substitutions, respectively, as a result of modification of guanine and cytosine. The plasmids were CAA-damaged in vitro and replicated in Escherichia coli of various genetic backgrounds. To modify the levels of AlkA and AlkB proteins, mutagenesis was studied in E. coli cells induced or not in adaptive response. Formation of varepsilonC proceeds via a relatively stable intermediate, 3,N(4)-alpha-hydroxyethanocytosine (HEC), which allowed to compare repair of both adducts. The results indicate that all three genes, alkA, alkB and microg, are engaged in alleviation of CAA-induced mutagenesis. The frequency of mutation was higher in AlkA-, AlkB- and Mug-deficient strains in comparison to alkA(+), alkB(+), and microg(+) controls. Considering the levels of CAA-induced Lac(+) revertants in strains harboring the pIF plasmids and induced or not in adaptive response, we conclude that AlkB protein is engaged in the repair of epsilonC and HEC in vivo. Using the modified TTCTT 5-mers as substrates, we confirmed in vitro that AlkB protein repairs epsilonC and HEC although far less efficiently than the reference adduct 3-methylcytosine. The pH optimum for repair of HEC and epsilonC is significantly different from that for 3-methylcytosine. We propose that the protonated form of adduct interact in active site of AlkB protein. PMID:19941873

  9. The type I BMP receptor ACVR1/ALK2 is required for chondrogenesis during development

    PubMed Central

    Rigueur, Diana; Brugger, Sean; Anbarchian, Teni; Kim, Jong Kil; Lee, Yoo Jin; Lyons, Karen

    2014-01-01

    Bone morphogenetic proteins (BMPs) are crucial regulators of chondrogenesis. BMPs transduce their signals through three type I receptors: BMPR1A, BMPR1B, and ACVR1/ALK2. Fibrodysplasia ossificans progressiva (FOP), a rare disorder characterized by progressive ossification of connective tissue, is caused by an activating mutation in Acvr1 (the gene that encodes ACVR1/ALK2). However, there are few developmental defects associated with FOP. Thus, the role of ACVR1 in chondrogenesis during development is unknown. Here we report the phenotype of mice lacking ACVR1 in cartilage. Acvr1CKO mice are viable but exhibit defects in the development of cranial and axial structures. Mutants exhibit a shortened cranial base, and cervical vertebrae are hypoplastic. Acvr1CKO adult mice develop progressive kyphosis. These morphological defects were associated with decreased levels of Smad1/5 and p38 activation, and with reduced rates of chondrocyte proliferation in vertebral cartilage. We also tested whether ACVR1 exerts coordinated functions with BMPR1A and BMPR1B through analysis of double mutants. Acvr1/Bmpr1a and Acvr1/Bmpr1b mutant mice exhibited generalized perinatal lethal chondrodysplasia that was much more severe than in any of the corresponding mutant strains. These findings demonstrate that ACVR1 is required for chondrocyte proliferation and differentiation, particularly in craniofacial and axial elements, but exerts coordinated functions with both BMPR1A and BMPR1B throughout the developing endochondral skeleton. PMID:25413979

  10. Proteome-wide identification of novel binding partners to the oncogenic fusion gene protein, NPM-ALK, using tandem affinity purification and mass spectrometry.

    PubMed

    Wu, Fang; Wang, Peng; Young, Leah C; Lai, Raymond; Li, Liang

    2009-02-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), an oncogenic fusion gene protein that is characteristically found in a subset of anaplastic large cell lymphomas, promotes tumorigenesis through its functional and physical interactions with various biologically important proteins. The identification of these interacting proteins has proven to be useful to further our understanding of NPM-ALK-mediated tumorigenesis. For the first time, we performed a proteome-wide identification of NPM-ALK-binding proteins using tandem affinity purification and a highly sensitive mass spectrometric technique. Tandem affinity purification is a recently developed method that carries a lower background and higher sensitivity compared with the conventional immunoprecipitation-based protein purification protocols. The NPM-ALK gene was cloned into an HB-tagged vector and expressed in GP293 cells. Three independent experiments were performed and the reproducibility of the data was 68%. The vast majority of the previously reported NPM-ALK-binding proteins were detected. We also identified proteins that are involved in various cellular processes that were not previously described in association with NPM-ALK, such as MCM6 and MSH2 (DNA repair), Nup98 and importin 8 (subcellular protein transport), Stim1 (calcium signaling), 82Fip (RNA regulation), and BAG2 (proteosome degradation). We believe that these data highlight the functional diversity of NPM-ALK and provide new research directions for the study of the biology of this oncoprotein.

  11. Identification of C/EBPβ Target Genes in ALK+ Anaplastic Large Cell Lymphoma (ALCL) by Gene Expression Profiling and Chromatin Immunoprecipitation

    PubMed Central

    Bonzheim, Irina; Irmler, Martin; Klier-Richter, Margit; Steinhilber, Julia; Anastasov, Nataša; Schäfer, Sabine; Adam, Patrick; Beckers, Johannes; Raffeld, Mark; Fend, Falko; Quintanilla-Martinez, Leticia

    2013-01-01

    C/EBPβ (CCAAT enhancer binding protein) is a transcription factor that plays a crucial role in survival and transformation of ALK+ anaplastic large cell lymphoma (ALCL). The aim of this study was to identify the downstream targets of C/EBPβ responsible for ALK-mediated oncogenesis. C/EBPβ was knocked down in ALK+ ALCL cell lines with a C/EBPβ-shRNA, followed by gene expression profiling (GEP). GEP analysis revealed a reproducible signature of genes that were significantly regulated by C/EBPβ. Classification into biological categories revealed overrepresentation of genes involved in the immune response, apoptosis and cell proliferation. Transcriptional regulation by C/EBPβ was found in 6 of 11 (BCL2A1, G0S2, TRIB1, S100A9, DDX21 and DDIT4) genes investigated by chromatin immunoprecipitation. We demonstrated that BCL2A1, G0S2 and DDX21 play a crucial role in survival and proliferation of ALK+ ALCL cells. DDX21, a gene involved in rRNA biogenesis, was found differentially overexpressed in primary ALK+ ALCL cases. All three candidate genes were validated in primary ALCL cases by either immunohistochemistry or RT-qPCR. In conclusion, we identified and validated several key C/EBPβ-regulated genes with major impact on survival and cell growth in ALK+ ALCL, supporting the central role of C/EBPβ in ALK-mediated oncogenesis. PMID:23741337

  12. In situ detection of alkB2 gene involved in Alcanivorax borkumensis SK2(T) hydrocarbon biodegradation.

    PubMed

    Matturro, Bruna; Frascadore, Emanuela; Cappello, Simone; Genovese, Mariella; Rossetti, Simona

    2016-09-15

    This study aimed to develop a new assay based on the whole cell hybridization in order to monitor alkane hydroxylase genes (alkB system) of the marine bacterium Alcanivorax borkumensis SK2(T) commonly reported as the predominant microorganism responsible for the biodegradation of n-alkanes which are the major fraction of petroleum hydrocarbons. The assay based on the whole cell hybridization targeting alkB2 gene was successfully developed and calibrated on a pure culture of Alcanivorax borkumensis SK2(T) with a detection efficiency up to 80%. The approach was further successfully validated on hydrocarbon-contaminated seawater and provided cells abundance (6.74E+04alkB2-carryingcellsmL(-1)) higher of about one order of magnitude than those obtained by qPCR (4.96E+03alkB2genecopiesmL(-1)). This study highlights the validity of the assay for the detection at single cell level of key-functional genes involved in the biodegradation of n-alkanes. PMID:27315756

  13. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    EPA Science Inventory

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  14. Hereditary haemorrhagic telangiectasia: a questionnaire based study to delineate the different phenotypes caused by endoglin and ALK1 mutations

    PubMed Central

    Berg, J; Porteous, M; Reinhardt, D; Gallione, C; Holloway, S; Umasunthar, T; Lux, A; McKinnon, W; Marchuk, D; Guttmacher, A

    2003-01-01

    Background: Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia characterised by mucocutaneous telangiectasis, epistaxis, gastrointestinal haemorrhage, and arteriovenous malformations in the lung and brain. Causative mutations for HHT have been identified in two genes, endoglin and ALK1, which encode proteins involved in serine-threonine kinase signalling in the endothelial cell. Methods: A number of people affected with HHT had completed a postal questionnaire as part of an international study to delineate the HHT phenotype. We identified questionnaires completed by subjects in whom we had identified a mutation in endoglin or ALK1. Further questionnaires were sent to families with known mutations. Data were only included from questionnaires returned by people known to carry disease causing mutations. Results: Questionnaires were completed by 83 subjects with known mutations. Of these, 49 had endoglin mutations (HHT1) and 34 had ALK1 mutations (HHT2). Subjects with HHT1 reported an earlier onset of epistaxis (p=0.01) and telangiectasis (p=0.0001) than those with HHT2. Pulmonary arteriovenous malformations were only reported in the endoglin mutation group in our study (p<0.001). Conclusions: Our questionnaire based study provides evidence that the HHT phenotype caused by mutations in endoglin (HHT1) is distinct from, and more severe than, HHT caused by mutations in ALK1 (HHT2). This has significant implications for diagnosis, screening, and treatment in the two different forms of HHT, as well as for understanding the pathogenesis of the disease. PMID:12920067

  15. NPM-ALK mediates phosphorylation of MSH2 at tyrosine 238, creating a functional deficiency in MSH2 and the loss of mismatch repair.

    PubMed

    Bone, K M; Wang, P; Wu, F; Wu, C; Li, L; Bacani, J T; Andrew, S E; Lai, R

    2015-05-15

    The vast majority of anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ALCL) tumors express the characteristic oncogenic fusion protein NPM-ALK, which mediates tumorigenesis by exerting its constitutive tyrosine kinase activity on various substrates. We recently identified MSH2, a protein central to DNA mismatch repair (MMR), as a novel binding partner and phosphorylation substrate of NPM-ALK. Here, using liquid chromatography-mass spectrometry, we report for the first time that MSH2 is phosphorylated by NPM-ALK at a specific residue, tyrosine 238. Using GP293 cells transfected with NPM-ALK, we confirmed that the MSH2(Y238F) mutant is not tyrosine phosphorylated. Furthermore, transfection of MSH2(Y238F) into these cells substantially decreased the tyrosine phosphorylation of endogenous MSH2. Importantly, gene transfection of MSH2(Y238F) abrogated the binding of NPM-ALK with endogenous MSH2, re-established the dimerization of MSH2:MSH6 and restored the sensitivity to DNA mismatch-inducing drugs, indicative of MMR return. Parallel findings were observed in two ALK+ALCL cell lines, Karpas 299 and SUP-M2. In addition, we found that enforced expression of MSH2(Y238F) into ALK+ALCL cells alone was sufficient to induce spontaneous apoptosis. In conclusion, our findings have identified NPM-ALK-induced phosphorylation of MSH2 at Y238 as a crucial event in suppressing MMR. Our studies have provided novel insights into the mechanism by which oncogenic tyrosine kinases disrupt MMR.

  16. NPM-ALK oncogenic kinase promotes cell-cycle progression through activation of JNK/cJun signaling in anaplastic large-cell lymphoma.

    PubMed

    Leventaki, Vasiliki; Drakos, Elias; Medeiros, L Jeffrey; Lim, Megan S; Elenitoba-Johnson, Kojo S; Claret, Francois X; Rassidakis, George Z

    2007-09-01

    Anaplastic large-cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35), resulting in aberrant expression of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). We show that in 293T and Jurkat cells, forced expression of active NPM-ALK, but not kinase-dead mutant NPM-ALK (210K>R), induced JNK and cJun phosphorylation, and this was linked to a dramatic increase in AP-1 transcriptional activity. Conversely, inhibition of ALK activity in NPM-ALK(+) ALCL cells resulted in a concentration-dependent dephosphorylation of JNK and cJun and decreased AP-1 DNA-binding. In addition, JNK physically binds NPM-ALK and is highly activated in cultured and primary NPM-ALK(+) ALCL cells. cJun phosphorylation in NPM-ALK(+) ALCL cells is mediated by JNKs, as shown by selective knocking down of JNK1 and JNK2 genes using siRNA. Inhibition of JNK activity using SP600125 decreased cJun phosphorylation and AP-1 transcriptional activity and this was associated with decreased cell proliferation and G2/M cell-cycle arrest in a dose-dependent manner. Silencing of the cJun gene by siRNA led to a decreased S-phase cell-cycle fraction associated with upregulation of p21 and downregulation of cyclin D3 and cyclin A. Taken together, these findings reveal a novel function of NPM-ALK, phosphorylation and activation of JNK and cJun, which may contribute to uncontrolled cell-cycle progression and oncogenesis.

  17. Effects of different compost amendments on the abundance and composition of alkB harboring bacterial communities in a soil under industrial use contaminated with hydrocarbons.

    PubMed

    Wallisch, Stefanie; Gril, Tjasa; Dong, Xia; Welzl, Gerd; Bruns, Christian; Heath, Ester; Engel, Marion; Suhadolc, Marjetka; Schloter, Michael

    2014-01-01

    Alkane degrading microorganisms play an important role for the bioremediation of petrogenic contaminated environments. In this study, we investigated the effects of compost addition on the abundance and diversity of bacteria harboring the alkane monooxygenase gene (alkB) in an oil-contaminated soil originated from an industrial zone in Celje, Slovenia (Technosol). Soil without any amendments (control soil) and soil amended with two composts differing in their maturation stage and nutrient availability, were incubated under controlled conditions in a microcosm experiment and sampled after 0, 6, 12, and 36 weeks of incubation. As expected the addition of compost stimulated the degradation of alkanes in the investigated soil shortly after the addition. By using quantitative real-time PCR higher number of alkB genes were detected in soil samples amended with compost compared to the control soils. To get an insight into the composition of alkB harboring microbial communities, we performed next generation sequencing of amplicons of alkB gene fragment. Richness and diversity of alkB gene harboring prokaryotes was higher in soil mixed with compost compared to control soils with stronger effects of the less maturated, nutrient poor compost. The phylogenetic analysis of communities suggested that the addition of compost stimulated the abundance of alkB harboring Actinobacteria during the experiment independent from the maturation stage of the compost. AlkB harboring γ-proteobacteria like Shewanella or Hydrocarboniphaga as well as α-proteobacteria of the genus Agrobacterium responded also positively to the addition of compost to soil. The amendment of the less maturated, nutrient poor compost resulted in addition in a large increase of alkB harboring bacteria of the Cytophaga group (Microscilla) mainly at the early sampling time points. Our data indicates that compost amendments significantly change abundance and diversity pattern of alkB harboring microbes in Technosol and

  18. Effects of different compost amendments on the abundance and composition of alkB harboring bacterial communities in a soil under industrial use contaminated with hydrocarbons

    PubMed Central

    Wallisch, Stefanie; Gril, Tjasa; Dong, Xia; Welzl, Gerd; Bruns, Christian; Heath, Ester; Engel, Marion; Suhadolc, Marjetka; Schloter, Michael

    2014-01-01

    Alkane degrading microorganisms play an important role for the bioremediation of petrogenic contaminated environments. In this study, we investigated the effects of compost addition on the abundance and diversity of bacteria harboring the alkane monooxygenase gene (alkB) in an oil-contaminated soil originated from an industrial zone in Celje, Slovenia (Technosol). Soil without any amendments (control soil) and soil amended with two composts differing in their maturation stage and nutrient availability, were incubated under controlled conditions in a microcosm experiment and sampled after 0, 6, 12, and 36 weeks of incubation. As expected the addition of compost stimulated the degradation of alkanes in the investigated soil shortly after the addition. By using quantitative real-time PCR higher number of alkB genes were detected in soil samples amended with compost compared to the control soils. To get an insight into the composition of alkB harboring microbial communities, we performed next generation sequencing of amplicons of alkB gene fragment. Richness and diversity of alkB gene harboring prokaryotes was higher in soil mixed with compost compared to control soils with stronger effects of the less maturated, nutrient poor compost. The phylogenetic analysis of communities suggested that the addition of compost stimulated the abundance of alkB harboring Actinobacteria during the experiment independent from the maturation stage of the compost. AlkB harboring γ-proteobacteria like Shewanella or Hydrocarboniphaga as well as α-proteobacteria of the genus Agrobacterium responded also positively to the addition of compost to soil. The amendment of the less maturated, nutrient poor compost resulted in addition in a large increase of alkB harboring bacteria of the Cytophaga group (Microscilla) mainly at the early sampling time points. Our data indicates that compost amendments significantly change abundance and diversity pattern of alkB harboring microbes in Technosol and

  19. ALK-positive anaplastic large cell lymphoma limited to the skin: clinical, histopathological and molecular analysis of 6 pediatric cases. A report from the ALCL99 study.

    PubMed

    Oschlies, Ilske; Lisfeld, Jasmin; Lamant, Laurence; Nakazawa, Atsuko; d'Amore, Emanuele S G; Hansson, Ulrika; Hebeda, Konnie; Simonitsch-Klupp, Ingrid; Maldyk, Jadwiga; Müllauer, Leonhard; Tinguely, Marianne; Stücker, Markus; Ledeley, Marie-Cecile; Siebert, Reiner; Reiter, Alfred; Brugières, Laurence; Klapper, Wolfram; Woessmann, Wilhelm

    2013-01-01

    Anaplastic large cell lymphomas are peripheral T-cell lymphomas that are characterized by a proliferation of large anaplastic blasts expressing CD30. In children, systemic anaplastic large cell lymphomas often present at advanced clinical stage and harbor translocations involving the anaplastic lymphoma kinase (ALK) gene leading to the expression of chimeric anaplastic lymphoma kinase (ALK)-fusion proteins. Primary cutaneous anaplastic large cell lymphoma is regarded as an ALK-negative variant confined to the skin and is part of the spectrum of primary cutaneous CD30-positive T-cell lymphoproliferative disorders. Thirty-three of 487 pediatric patients registered within the Anaplastic Large Cell Lymphoma-99 trial (1999 to 2006) presented with a skin limited CD30-positive lympho-proliferative disorder. In 23 of the 33 patients, material for international histopathological review was available, and the cases were studied for histopathological, immunophenotypical and clinical features as well as for breaks within the ALK gene. Five of 23 cases and one additional case (identified after closure of the trial) expressed ALK-protein. Complete staging excluded any other organ involvement in all children. Expression of ALK proteins was demonstrated by immunohistochemistry in all cases and the presence of breaks of the ALK gene was genetically confirmed in 5 evaluable cases. The histopathological and clinical picture of these skin-restricted ALK-positive lymphomas was indistinguishable from that of cutaneous anaplastic large cell lymphoma. Five children presented with a single skin lesion that was completely resected in 4 and incompletely resected in one. Three of these patients received no further therapy, 2 additional local radiotherapy, and one chemotherapy. All children remain in complete remission with a median follow up of seven years (range 1-8 years). We present 6 pediatric cases of ALK-positive primary cutaneous anaplastic large cell lymphomas. After thorough

  20. Decoding Tumor Phenotypes for ALK, ROS1, and RET Fusions in Lung Adenocarcinoma Using a Radiomics Approach

    PubMed Central

    Yoon, Hyun Jung; Sohn, Insuk; Cho, Jong Ho; Lee, Ho Yun; Kim, Jae-Hun; Choi, Yoon-La; Kim, Hyeseung; Lee, Genehee; Lee, Kyung Soo; Kim, Jhingook

    2015-01-01

    Abstract Quantitative imaging using radiomics can capture distinct phenotypic differences between tumors and may have predictive power for certain phenotypes according to specific genetic mutations. We aimed to identify the clinicoradiologic predictors of tumors with ALK (anaplastic lymphoma kinase), ROS1 (c-ros oncogene 1), or RET (rearranged during transfection) fusions in patients with lung adenocarcinoma. A total of 539 pathologically confirmed lung adenocarcinomas were included in this retrospective study. The baseline clinicopathologic characteristics were retrieved from the patients’ medical records and the ALK/ROS1/RET fusion status was reviewed. Quantitative computed tomography (CT) and positron emission tomography imaging characteristics were evaluated using a radiomics approach. Significant features for the fusion-positive tumor prediction model were extracted from all of the clinicoradiologic features, and were used to calculate diagnostic performance for predicting 3 fusions’ positivity. The clinicoradiologic features were compared between ALK versus ROS1/RET fusion-positive tumors to identify the clinicoradiologic similarity between the 2 groups. The fusion-positive tumor prediction model was a combination of younger age, advanced tumor stage, solid tumor on CT, higher values for SUVmax and tumor mass, lower values for kurtosis and inverse variance on 3-voxel distance than those of fusion-negative tumors (sensitivity and specificity, 0.73 and 0.70, respectively). ALK fusion-positive tumors were significantly different in tumor stage, central location, SUVmax, homogeneity on 1-, 2-, and 3-voxel distances, and sum mean on 2-voxel distance compared with ROS1/RET fusion-positive tumors. ALK/ROS1/RET fusion-positive lung adenocarcinomas possess certain clinical and imaging features that enable good discrimination of fusion-positive from fusion-negative lung adenocarcinomas. PMID:26469915

  1. Intrinsic susceptibility MRI identifies tumors with ALKF1174L mutation in genetically-engineered murine models of high-risk neuroblastoma.

    PubMed

    Jamin, Yann; Glass, Laura; Hallsworth, Albert; George, Rani; Koh, Dow-Mu; Pearson, Andrew D J; Chesler, Louis; Robinson, Simon P

    2014-01-01

    The early identification of children presenting ALK(F1174L)-mutated neuroblastoma, which are associated with resistance to the promising ALK inhibitor crizotinib and a marked poorer prognosis, has become a clinical priority. In comparing the radiology of the novel Th-ALK(F1174L)/Th-MYCN and the well-established Th-MYCN genetically-engineered murine models of neuroblastoma using MRI, we have identified a marked ALK(F1174L)-driven vascular phenotype. We demonstrate that quantitation of the transverse relaxation rate R2* (s(-1)) using intrinsic susceptibility-MRI under baseline conditions and during hyperoxia, can robustly discriminate this differential vascular phenotype, and identify MYCN-driven tumors harboring the ALK(F1174L) mutation with high specificity and selectivity. Intrinsic susceptibility-MRI could thus potentially provide a non-invasive and clinically-exploitable method to help identifying children with MYCN-driven neuroblastoma harboring the ALK(F1174L) mutation at the time of diagnosis. PMID:24667968

  2. Proteasome inhibitors.

    PubMed

    Teicher, Beverly A; Tomaszewski, Joseph E

    2015-07-01

    Proteasome inhibitors have a 20 year history in cancer therapy. The first proteasome inhibitor, bortezomib (Velcade, PS-341), a break-through multiple myeloma treatment, moved rapidly through development from bench in 1994 to first approval in 2003. Bortezomib is a reversible boronic acid inhibitor of the chymotrypsin-like activity of the proteasome. Next generation proteasome inhibitors include carfilzomib and oprozomib which are irreversible epoxyketone proteasome inhibitors; and ixazomib and delanzomib which are reversible boronic acid proteasome inhibitors. Two proteasome inhibitors, bortezomib and carfilzomib are FDA approved drugs and ixazomib and oprozomib are in late stage clinical trials. All of the agents are potent cytotoxics. The disease focus for all the proteasome inhibitors is multiple myeloma. This focus arose from clinical observations made in bortezomib early clinical trials. Later preclinical studies confirmed that multiple myeloma cells were indeed more sensitive to proteasome inhibitors than other tumor cell types. The discovery and development of the proteasome inhibitor class of anticancer agents has progressed through a classic route of serendipity and scientific investigation. These agents are continuing to have a major impact in their treatment of hematologic malignancies and are beginning to be explored as potential treatment agent for non-cancer indications. PMID:25935605

  3. Platelet Inhibitors.

    PubMed

    Shifrin, Megan M; Widmar, S Brian

    2016-03-01

    Antithrombotic medications have become standard of care for management of acute coronary syndrome. Platelet adhesion, activation, and aggregation are essential components of platelet function; platelet-inhibiting medications interfere with these components and reduce incidence of thrombosis. Active bleeding is a contraindication for administration of platelet inhibitors. There is currently no reversal agent for platelet inhibitors, although platelet transfusion may be used to correct active bleeding after administration of platelet inhibitors. PMID:26897422

  4. The Drosophila Midkine/Pleiotrophin Homologues Miple1 and Miple2 Affect Adult Lifespan but Are Dispensable for Alk Signaling during Embryonic Gut Formation

    PubMed Central

    Hugosson, Fredrik; Sjögren, Camilla; Birve, Anna; Hedlund, Ludmilla; Eriksson, Therese; Palmer, Ruth H.

    2014-01-01

    Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo. PMID:25380037

  5. Amplified expression of the tag+ and alkA+ genes in Escherichia coli: identification of gene products and effects on alkylation resistance.

    PubMed Central

    Kaasen, I; Evensen, G; Seeberg, E

    1986-01-01

    We have constructed plasmids which overproduce the tag and alkA gene products of Escherichia coli, i.e., 3-methyladenine DNA glycosylases I and II. The tag and alkA gene products were identified radiochemically in maxi- or minicells as polypeptides of 21 and 30 kilodaltons, respectively, which are consistent with the gel filtration molecular weights of the enzyme activities, thus confirming the identity of the cloned genes. High expression of the tag+-coded glycosylase almost completely suppressed the alkylation sensitivity of alkA mutants, indicating that high levels of 3-methyladenine DNA glycosylase I will eliminate the need for 3-methyladenine DNA glycosylase II in repair of alkylated DNA. Furthermore, overproduction of the alkA+-coded glycosylase greatly sensitizes wild-type cells to alkylation, suggesting that only a limited expression of this enzyme will allow efficient DNA repair. Images PMID:3536857

  6. Imaging Characteristics of Driver Mutations in EGFR, KRAS, and ALK among Treatment-Naïve Patients with Advanced Lung Adenocarcinoma

    PubMed Central

    Park, Jangchul; Kobayashi, Yoshihisa; Urayama, Kevin Y.; Yamaura, Hidekazu; Yatabe, Yasushi; Hida, Toyoaki

    2016-01-01

    This study aimed to identify the computed tomography characteristics of treatment-naïve patients with lung adenocarcinoma and known driver mutations in EGFR, KRAS, or ALK. Patients with advanced lung adenocarcinoma (stage IIIB–IV) and known mutations in EGFR, KRAS, or ALK were assessed. The radiological findings for the main tumor and intra-thoracic status were retrospectively analyzed in each group, and the groups’ characteristics were compared. We identified 265 treatment-naïve patients with non-small-cell carcinoma, who had EGFR mutations (n = 159), KRAS mutations (n = 55), or ALK rearrangements (n = 51). Among the three groups, we evaluated only patients with stage IIIB–IV lung adenocarcinoma who had EGFR mutations (n = 126), KRAS mutations (n = 35), or ALK rearrangements (n = 47). We found that ground-glass opacity at the main tumor was significantly more common among EGFR-positive patients, compared to ALK-positive patients (p = 0.009). Lymphadenopathy was significantly more common among ALK-positive patients, compared to EGFR-positive patients (p = 0.003). Extranodal invasion was significantly more common among ALK-positive patients, compared to EGFR-positive patients and KRAS-positive patients (p = 0.001 and p = 0.049, respectively). Lymphangitis was significantly more common among ALK-positive patients, compared to EGFR-positive patients (p = 0.049). Pleural effusion was significantly less common among KRAS-positive patients, compared to EGFR-positive patients and ALK-positive patients (p = 0.046 and p = 0.026, respectively). Lung metastases were significantly more common among EGFR-positive patients, compared to KRAS-positive patients and ALK-positive patients (p = 0.007 and p = 0.04, respectively). In conclusion, EGFR mutations were associated with ground-glass opacity, KRAS-positive tumors were generally solid and less likely to metastasize to the lung and pleura, and ALK-positive tumors tended to present with lymphadenopathy, extranodal

  7. Exocyclic carbons adjacent to the N6 of adenine are targets for oxidation by the Escherichia coli adaptive response protein AlkB.

    PubMed

    Li, Deyu; Delaney, James C; Page, Charlotte M; Yang, Xuedong; Chen, Alvin S; Wong, Cintyu; Drennan, Catherine L; Essigmann, John M

    2012-05-30

    The DNA and RNA repair protein AlkB removes alkyl groups from nucleic acids by a unique iron- and α-ketoglutarate-dependent oxidation strategy. When alkylated adenines are used as AlkB targets, earlier work suggests that the initial target of oxidation can be the alkyl carbon adjacent to N1. Such may be the case with ethano-adenine (EA), a DNA adduct formed by an important anticancer drug, BCNU, whereby an initial oxidation would occur at the carbon adjacent to N1. In a previous study, several intermediates were observed suggesting a pathway involving adduct restructuring to a form that would not hinder replication, which would match biological data showing that AlkB almost completely reverses EA toxicity in vivo. The present study uses more sensitive spectroscopic methodology to reveal the complete conversion of EA to adenine; the nature of observed additional putative intermediates indicates that AlkB conducts a second oxidation event in order to release the two-carbon unit completely. The second oxidation event occurs at the exocyclic carbon adjacent to the N(6) atom of adenine. The observation of oxidation of a carbon at N(6) in EA prompted us to evaluate N(6)-methyladenine (m6A), an important epigenetic signal for DNA replication and many other cellular processes, as an AlkB substrate in DNA. Here we show that m6A is indeed a substrate for AlkB and that it is converted to adenine via its 6-hydroxymethyl derivative. The observation that AlkB can demethylate m6A in vitro suggests a role for AlkB in regulation of important cellular functions in vivo. PMID:22512456

  8. A large-scale cross-sectional study of ALK rearrangements and EGFR mutations in non-small-cell lung cancer in Chinese Han population.

    PubMed

    Hong, Shaodong; Fang, Wenfeng; Hu, Zhihuang; Zhou, Ting; Yan, Yue; Qin, Tao; Tang, Yanna; Ma, Yuxiang; Zhao, Yuanyuan; Xue, Cong; Huang, Yan; Zhao, Hongyun; Zhang, Li

    2014-01-01

    The predictive power of age at diagnosis and smoking history for ALK rearrangements and EGFR mutations in non-small-cell lung cancer (NSCLC) remains not fully understood. In this cross-sectional study, 1160 NSCLC patients were prospectively enrolled and genotyped for EML4-ALK rearrangements and EGFR mutations. Multivariate logistic regression analysis was performed to explore the association between clinicopathological features and these two genetic aberrations. Receiver operating characteristic (ROC) curves methodology was applied to evaluate the predictive value. We showed that younger age at diagnosis was the only independent variable associated with EML4-ALK rearrangements (odds ratio (OR) per 5 years' increment, 0.68; p < 0.001), while lower tobacco exposure (OR per 5 pack-years' increment, 0.88; p < 0.001), adenocarcinoma (OR, 6.61; p < 0.001), and moderate to high differentiation (OR, 2.05; p < 0.001) were independently associated with EGFR mutations. Age at diagnosis was a very strong predictor of ALK rearrangements but poorly predicted EGFR mutations, while smoking pack-years may predict the presence of EGFR mutations and ALK rearrangements but with rather limited power. These findings should assist clinicians in assessing the likelihood of EML4-ALK rearrangements and EGFR mutations and understanding their biological implications in NSCLC.

  9. Integrated phosphoproteomic and metabolomic profiling reveals NPM-ALK-mediated phosphorylation of PKM2 and metabolic reprogramming in anaplastic large cell lymphoma.

    PubMed

    McDonnell, Scott R P; Hwang, Steven R; Rolland, Delphine; Murga-Zamalloa, Carlos; Basrur, Venkatesha; Conlon, Kevin P; Fermin, Damian; Wolfe, Thomas; Raskind, Alexander; Ruan, Chunhai; Jiang, Jian-Kang; Thomas, Craig J; Hogaboam, Cory M; Burant, Charles F; Elenitoba-Johnson, Kojo S J; Lim, Megan S

    2013-08-01

    The mechanisms underlying the pathogenesis of the constitutively active tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressing anaplastic large cell lymphoma are not completely understood. Here we show using an integrated phosphoproteomic and metabolomic strategy that NPM-ALK induces a metabolic shift toward aerobic glycolysis, increased lactate production, and biomass production. The metabolic shift is mediated through the anaplastic lymphoma kinase (ALK) phosphorylation of the tumor-specific isoform of pyruvate kinase (PKM2) at Y105, resulting in decreased enzymatic activity. Small molecule activation of PKM2 or expression of Y105F PKM2 mutant leads to reversal of the metabolic switch with increased oxidative phosphorylation and reduced lactate production coincident with increased cell death, decreased colony formation, and reduced tumor growth in an in vivo xenograft model. This study provides comprehensive profiling of the phosphoproteomic and metabolomic consequences of NPM-ALK expression and reveals a novel role of ALK in the regulation of multiple components of cellular metabolism. Our studies show that PKM2 is a novel substrate of ALK and plays a critical role in mediating the metabolic shift toward biomass production and tumorigenesis.

  10. In contrast to agonist monoclonal antibodies, both C-terminal truncated form and full length form of Pleiotrophin failed to activate vertebrate ALK (anaplastic lymphoma kinase)?

    PubMed

    Mathivet, Thomas; Mazot, Pierre; Vigny, Marc

    2007-12-01

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed during development in specific regions of the central and peripheral nervous system. ALK expression persists at a lower level in the adult brain. Thus, it might play an important role in both the normal development and function of the nervous system. The nature of the cognate ligand of this receptor in vertebrates is still a matter of debate. Pleiotrophin and midkine have been proposed as ligands of ALK but several independent studies do not confirm this hypothesis. Interestingly, a recent study proposed that a C-terminal truncated form of Pleiotrophin (Pleiotrophin.15) and not the full length form (Pleiotrophin.18) promotes glioblastoma proliferation in an ALK-dependent fashion. These data were obviously a strong basis to conciliate the conflicting results so far reported in the literature. In the present study, we first purified to homogeneity the two forms of Pleiotrophin secreted by HEK 293 cells. In contrast to agonist monoclonal antibodies, both Pleiotrophin.15 and Pleiotrophin.18 failed to activate ALK in neuroblastoma and glioblastoma cells expressing this receptor. Thus, for our point of view, ALK is still an orphan receptor in vertebrates.

  11. Evidence that the lung adenocarcinoma EML4-ALK fusion gene is not caused by exposure to secondhand tobacco smoke during childhood

    PubMed Central

    Jen, Jin; Yi, Eunhee S.; Olivo-Marston, Susan; Yang, Ping; Harris, Curtis C.

    2014-01-01

    Background The EML4-ALK fusion gene is more frequently found in younger, never smoking, lung cancer patients. Meanwhile, never smokers exposed to secondhand tobacco smoke (SHS) during childhood are diagnosed at a younger age compared with never smoking lung cancer patients that are not exposed. We therefore hypothesized that SHS, which can induce DNA damage, is associated with the EML4-ALK fusion gene. Methods We compared the frequency of the EML4-ALK fusion gene among 197 never smoker lung cancer patients with and without a history of exposure to SHS during childhood at Mayo Clinic. Results The EML4-ALK fusion gene was detected in 33% of cases from never smokers with a history of SHS exposure during childhood, while 47% of never smoking lung cancer cases without a history of childhood SHS exposure tested positive for the fusion gene. Conclusions The EML4-ALK fusion gene is not enriched in tumors from individuals exposed to SHS during childhood. Impact These data suggest that childhood exposure to SHS is not a significant etiologic cause of the EML4-ALK fusion gene in lung cancer. PMID:24755712

  12. Corrosion inhibitor

    SciTech Connect

    Wisotsky, M.J.; Metro, S.J.

    1989-10-31

    A corrosion inhibitor for use in synthetic ester lubricating oils is disclosed. It comprises an effective amount of: at least one aromatic amide; and at least one hydroxy substituted aromatic compound. The corrosion inhibitor thus formed is particularly useful in synthetic ester turbo lubricating oils.

  13. Diagnostic and therapeutic issues for patients with advanced non‑small cell lung cancer harboring anaplastic lymphoma kinase rearrangement: European vs. US perspective (review).

    PubMed

    Di Maio, Massimo; De Marinis, Filippo; Hirsch, Fred R; Gridelli, Cesare

    2014-08-01

    The recent availability of crizotinib in clinical practice, for the treatment of patients with advanced non-small cell lung cancer (NSCLC) selected by the presence of anaplastic lymphoma kinase (ALK) rearrangement, has relevant implications for both the diagnostic phase and the treatment choices. In the United States, crizotinib was approved by the Food and Drug Administration (FDA) in 2011 for patients with ALK positivity detected by FDA-approved companion diagnostic test. As of January, 2014, the only FDA-approved diagnostic test is Vysis ALK Break-Apart FISH Probe Kit. In Europe, European Medicines Agency (EMA) approved crizotinib for ALK-positive patients in 2012, without specifying the type of test used for determining the positivity. FISH remains the reference technique for ALK determination, but, if fully validated, immunohistochemistry could challenge the current ALK screening practice. Given the robust evidence of activity of crizotinib in ALK-positive patients both pretreated and chemotherapy-naïve, and the favourable tolerability profile of the drug, many oncologists would prefer to administer the drug as early as possible. This is technically feasible in the United States, where crizotinib was approved well before the availability of the results of the randomized phase III trial comparing the drug with standard second-line chemotherapy, and the use of crizotinib in ALK-positive patients is not restricted to a specific line of treatment. On the contrary, in Europe, differently from the FDA decision, crizotinib cannot be used in chemotherapy-naïve patients. In both realities, a deeper knowledge of mechanisms of resistance, the role of repeated biopsies, the treatment strategy for patients experiencing disease progression with crizotinib, the choice of the best chemotherapy regimen are challenging topics for the management of ALK-positive patients in clinical practice.

  14. The oncoprotein NPM-ALK of anaplastic large-cell lymphoma induces JUNB transcription via ERK1/2 and JunB translation via mTOR signaling.

    PubMed

    Staber, Philipp B; Vesely, Paul; Haq, Naznin; Ott, Rene G; Funato, Kotaro; Bambach, Isabella; Fuchs, Claudia; Schauer, Silvia; Linkesch, Werner; Hrzenjak, Andelko; Dirks, Wilhelm G; Sexl, Veronika; Bergler, Helmut; Kadin, Marshall E; Sternberg, David W; Kenner, Lukas; Hoefler, Gerald

    2007-11-01

    Anaplastic large cell lymphomas (ALCLs) are highly proliferating tumors that commonly express the AP-1 transcription factor JunB. ALK fusions occur in approximately 50% of ALCLs, and among these, 80% have the t(2;5) translocation with NPM-ALK expression. We report greater activity of JunB in NPM-ALK-positive than in NPM-ALK-negative ALCLs. Specific knockdown of JUNB mRNA using small interfering RNA and small hairpin RNA in NPM-ALK-expressing cells decreases cellular proliferation as evidenced by a reduced cell count in the G2/M phase of the cell cycle. Expression of NPM-ALK results in ERK1/2 activation and transcriptional up-regulation of JUNB. Both NPM-ALK-positive and -negative ALCL tumors demonstrate active ERK1/2 signaling. In contrast to NPM-ALK-negative ALCL, the mTOR pathway is active in NPM-ALK-positive lymphomas. Pharmacological inhibition of mTOR in NPM-ALK-positive cells down-regulates JunB protein levels by shifting JUNB mRNA translation from large polysomes to monosomes and ribonucleic particles (RNPs), and decreases cellular proliferation. Thus, JunB is a critical target of mTOR and is translationally regulated in NPM-ALK-positive lymphomas. This is the first study demonstrating translational control of AP-1 transcription factors in human neoplasia. In conjunction with NPM-ALK, JunB enhances cell cycle progression and may therefore represent a therapeutic target.

  15. Enzymological and Structural Studies of the Mechanism of Promiscuous Substrate Recognition by the Oxidative DNA Repair Enzyme AlkB

    SciTech Connect

    Yu, B.; Hunt, J

    2009-01-01

    Promiscuous substrate recognition, the ability to catalyze transformations of chemically diverse compounds, is an evolutionarily advantageous, but poorly understood phenomenon. The promiscuity of DNA repair enzymes is particularly important, because it enables diverse kinds of damage to different nucleotide bases to be repaired in a metabolically parsimonious manner. We present enzymological and crystallographic studies of the mechanisms underlying promiscuous substrate recognition by Escherichia coli AlkB, a DNA repair enzyme that removes methyl adducts and some larger alkylation lesions from endocyclic positions on purine and pyrimidine bases. In vitro Michaelis-Menten analyses on a series of alkylated bases show high activity in repairing N1-methyladenine (m1A) and N3-methylcytosine (m3C), comparatively low activity in repairing 1,N6-ethenoadenine, and no detectable activity in repairing N1-methylguanine or N3-methylthymine. AlkB has a substantially higher kcat and Km for m3C compared with m1A. Therefore, the enzyme maintains similar net activity on the chemically distinct substrates by increasing the turnover rate of the substrate with nominally lower affinity. Cocrystal structures provide insight into the structural basis of this 'kcat/Km compensation,' which makes a significant contribution to promiscuous substrate recognition by AlkB. In analyzing a large ensemble of crystal structures solved in the course of these studies, we observed 2 discrete global conformations of AlkB differing in the accessibility of a tunnel hypothesized to control diffusion of the O2 substrate into the active site. Steric interactions between a series of protein loops control this conformational transition and present a plausible mechanism for preventing O2 binding before nucleotide substrate binding.

  16. Circulating tumour DNA profiling reveals heterogeneity of EGFR inhibitor resistance mechanisms in lung cancer patients.

    PubMed

    Chabon, Jacob J; Simmons, Andrew D; Lovejoy, Alexander F; Esfahani, Mohammad S; Newman, Aaron M; Haringsma, Henry J; Kurtz, David M; Stehr, Henning; Scherer, Florian; Karlovich, Chris A; Harding, Thomas C; Durkin, Kathleen A; Otterson, Gregory A; Purcell, W Thomas; Camidge, D Ross; Goldman, Jonathan W; Sequist, Lecia V; Piotrowska, Zofia; Wakelee, Heather A; Neal, Joel W; Alizadeh, Ash A; Diehn, Maximilian

    2016-01-01

    Circulating tumour DNA (ctDNA) analysis facilitates studies of tumour heterogeneity. Here we employ CAPP-Seq ctDNA analysis to study resistance mechanisms in 43 non-small cell lung cancer (NSCLC) patients treated with the third-generation epidermal growth factor receptor (EGFR) inhibitor rociletinib. We observe multiple resistance mechanisms in 46% of patients after treatment with first-line inhibitors, indicating frequent intra-patient heterogeneity. Rociletinib resistance recurrently involves MET, EGFR, PIK3CA, ERRB2, KRAS and RB1. We describe a novel EGFR L798I mutation and find that EGFR C797S, which arises in ∼33% of patients after osimertinib treatment, occurs in <3% after rociletinib. Increased MET copy number is the most frequent rociletinib resistance mechanism in this cohort and patients with multiple pre-existing mechanisms (T790M and MET) experience inferior responses. Similarly, rociletinib-resistant xenografts develop MET amplification that can be overcome with the MET inhibitor crizotinib. These results underscore the importance of tumour heterogeneity in NSCLC and the utility of ctDNA-based resistance mechanism assessment. PMID:27283993

  17. Circulating tumour DNA profiling reveals heterogeneity of EGFR inhibitor resistance mechanisms in lung cancer patients

    PubMed Central

    Chabon, Jacob J.; Simmons, Andrew D.; Lovejoy, Alexander F.; Esfahani, Mohammad S.; Newman, Aaron M.; Haringsma, Henry J.; Kurtz, David M.; Stehr, Henning; Scherer, Florian; Karlovich, Chris A.; Harding, Thomas C.; Durkin, Kathleen A.; Otterson, Gregory A.; Purcell, W. Thomas; Camidge, D. Ross; Goldman, Jonathan W.; Sequist, Lecia V.; Piotrowska, Zofia; Wakelee, Heather A.; Neal, Joel W.; Alizadeh, Ash A.; Diehn, Maximilian

    2016-01-01

    Circulating tumour DNA (ctDNA) analysis facilitates studies of tumour heterogeneity. Here we employ CAPP-Seq ctDNA analysis to study resistance mechanisms in 43 non-small cell lung cancer (NSCLC) patients treated with the third-generation epidermal growth factor receptor (EGFR) inhibitor rociletinib. We observe multiple resistance mechanisms in 46% of patients after treatment with first-line inhibitors, indicating frequent intra-patient heterogeneity. Rociletinib resistance recurrently involves MET, EGFR, PIK3CA, ERRB2, KRAS and RB1. We describe a novel EGFR L798I mutation and find that EGFR C797S, which arises in ∼33% of patients after osimertinib treatment, occurs in <3% after rociletinib. Increased MET copy number is the most frequent rociletinib resistance mechanism in this cohort and patients with multiple pre-existing mechanisms (T790M and MET) experience inferior responses. Similarly, rociletinib-resistant xenografts develop MET amplification that can be overcome with the MET inhibitor crizotinib. These results underscore the importance of tumour heterogeneity in NSCLC and the utility of ctDNA-based resistance mechanism assessment. PMID:27283993

  18. Reducing progoitrin and enriching glucoraphanin in Brassica napus seeds through silencing of the GSL-ALK gene family.

    PubMed

    Liu, Zheng; Hirani, Arvind H; McVetty, Peter B E; Daayf, Fouad; Quiros, Carlos F; Li, Genyi

    2012-05-01

    The hydrolytic products of glucosinolates in brassica crops are bioactive compounds. Some glucosinolate derivatives such as oxazolidine-2-thione from progoitrin in brassica oilseed meal are toxic and detrimental to animals, but some isothiocyanates such as sulforaphane are potent anti-carcinogens that have preventive effects on several human cancers. In most B. rapa, B. napus and B. juncea vegetables and oilseeds, there is no or only trace amount of glucoraphanin that is the precursor to sulforaphane. In this paper, RNA interference (RNAi) of the GSL-ALK gene family was used to down-regulate the expression of GSL-ALK genes in B. napus. The detrimental glucosinolate progoitrin was reduced by 65 %, and the beneficial glucosinolate glucoraphanin was increased to a relatively high concentration (42.6 μmol g(-1) seed) in seeds of B. napus transgenic plants through silencing of the GSL-ALK gene family. Therefore, there is potential application of the new germplasm with reduced detrimental glucosinolates and increased beneficial glucosinolates for producing improved brassica vegetables.

  19. Expression of growth differentiation factor 9 (GDF9), ALK5, and claudin-11 in adult alpaca testis.

    PubMed

    Guo, Qing Yun; Gao, Zhen Zhen; Zhao, Li; He, Jun Ping; Dong, Cheng Sheng

    2013-01-01

    Growth differentiation factor 9 (GDF9) is an oocyte-derived factor critical for folliculogenesis. Recently, in vitro data showed that GDF9 inhibited the localization of tight junction (TJ) proteins, suggesting that GDF9 could potentially regulate spermatogenesis in vivo, via inhibition of Sertoli cell TJ function. The purpose of the present study was to determine the expression and localization of GDF9, its receptor, ALK5, and its latent target protein, claudin-11 (one of TJ proteins) in adult alpaca testis using Western blot and immunohistochemistry. Western blotting results demonstrated that GDF9, ALK5 and claudin-11 were expressed in the adult alpaca testis. Immunohistochemistry revealed that GDF9 was expressed stage-specifically in the cytoplasm of pachytene spermatocytes and round spermatids of the adult alpaca seminiferous epithelium. Type I receptor, ALK5 was mainly localized in the cytoplasm of round spermatids and Leydig cells, and to a lesser extent in the cytoplasm of pachytene spermatocytes and Sertoli cells. Its latent target protein, claudin-11, was perpendicular or parallel to the basal lamina in the basal part of Sertoli cells. These results indicated that GDF9, as a paracrine and autocrine growth factor derived from round spermatids and pachytene spermatocytes, is involved in regulating spermatogenesis via action on germ cells or somatic cells (i.e. Leydig cells, Sertoli cells).

  20. Fluorescence in situ hybridization analysis of the ALK gene in 2,045 non-small cell lung cancer patients from North-Western Spain (Galicia)

    PubMed Central

    Sánchez-Ares, María; Cameselle-Teijeiro, José M.; Vázquez-Estévez, Sergio; Lázaro-Quintela, Martín; Vázquez-Boquete, Ángel; Afonso-Afonso, Francisco J.; Casal-Rubio, Joaquín; González-Piñeiro, Ana L.; Rico-Rodríguez, Yolanda; Fírvida-Pérez, José L.; Ruíz-Bañobre, Juan; Couso, Elena; Santomé, Lucía; Pérez-Becerra, Raquel; García-Campelo, Rosario; Amenedo, Margarita; Azpitarte-Raposeiras, Cristina; Antúnez, José; Abdulkader, Ihab

    2016-01-01

    Identification of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements is a standard diagnostic test in patients with advanced non-small cell lung cancer (NSCLC). The current study describes the experience of ALK rearrangement detection of a referral center in the public health care system of Galicia in North-Western Spain. The fluorescence in situ hybridization (FISH) patterns of the ALK gene and the clinical and pathological features of these patients are reported. This study is also of interest for comparative purposes due to the relative geographical isolation of the area, which could have contributed to particular genetic features. A total of 2,045 tissue samples from NSCLC patients were collected between October 2010 and July 2015 and tested for ALK rearrangements by FISH. Examination of 1,686 paraffin-embedded tissue specimens and 395 cytological samples (306 cell block preparations and 53 cytological smears) was conducted, and any associations between the FISH results and clinicopathological features were assessed. The rate of successful evaluation was marginally higher in tissue samples than in cytological samples (92.9% vs. 84.1%); this difference was not significant. ALK rearrangements were identified in 82 patients(4%): 65 (79.3%) in tissue specimens, 15 (18.3%) in cell block samples and 2 (2.4%) in cytological smears. This genetic translocation appeared to be associated with a non-smoking history, younger age, female gender, stage IV and adenocarcinoma histological type. The findings demonstrate that ALK evaluation by FISH is feasible in tissue and cytological samples. The clinical and pathological features of the ALK-positive series of patients are similar to those previously reported in the literature. PMID:27446444

  1. Structural basis for specificity of TGF[beta] family receptor small molecule inhibitors

    SciTech Connect

    Ogunjimi, Abiodun A.; Zeqiraj, Elton; Ceccarelli, Derek F.; Sicheri, Frank; Wrana, Jeffrey L.; David, Laurent

    2012-07-24

    Transforming growth factor-{beta} (TGF{beta}) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGF{beta}/Activin pathway receptors, but needs improvement of its EC{sub 50} (EC{sub 50} = 1 {mu}M) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGF{beta}/Activin pathway but not the closely related receptors from the bone morphogenic proteins (BMP) pathway. To understand the mechanisms of this selectivity, we solved the crystal structure of the TGF{beta} type I receptor (T{beta}RI) kinase domain in complex with SB431542. We mutated T{beta}RI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated T{beta}RI. We discovered that a Ser280Thr mutation yielded a T{beta}RI variant that was resistant to SB431542 inhibition. Furthermore, the corresponding Thr283Ser mutation in ALK2 yielded a BMP receptor sensitive to SB431542. This demonstrated that Ser280 is the key determinant of selectivity for SB431542. This work provides a framework for optimising the SB431542 scaffold to more potent and selective inhibitors of the TGF{beta}/Activin pathway.

  2. Development of treatment strategies for advanced neuroblastoma.

    PubMed

    Hara, Junichi

    2012-06-01

    Neuroblastoma is the most common cancer in childhood. The majority of patients with neuroblastoma are assigned to the high-risk group based on age at diagnosis, stage, histology, MYCN status, and DNA ploidy. Their prognosis remains unsatisfactory; the 5-year event-free survival (EFS) rate is generally 40 %. During the past 20 years, much effort has been made to reinforce chemotherapy, including the introduction of high-dose chemotherapy with autologous stem cell rescue, resulting in a 5-year EFS rate of around 30 %. Subsequently, maintenance therapy aimed at eradicating residual tumors after induction and consolidation therapies was introduced, consisting of differentiation-inducing agents, retinoids, and immunotherapy using anti-GD2 antibodies combined with cytokines. However, such additional treatment provided benefit to only 10-20 % of patients, while the prognosis of about half the patients remains poor. Currently, novel targeted agents are under development. Among them, anaplastic lymphoma kinase (ALK) inhibitors and aurora kinase A inhibitors are promising. ALK somatic mutation or gene amplification predisposing neuroblastoma development occurs in up to 15 % of neuroblastomas. Crizotinib is a dual-specific inhibitor of ALK/Met and inhibits proliferation of neuroblastoma cells harboring R1275Q-mutated ALK or amplified wild-type ALK, but not cells harboring F1174L. Instead, cells with F1174L are sensitive to another small molecule ALK inhibitor, TAE684. Aurora kinase A plays a pivotal role in centrosome maturation and spindle formation during mitosis. MLN8237 (alisertib) is a small molecule inhibitor of aurora kinase A that is currently in early-phase clinical testing. Future treatment will be individually planned, adapting targeted agents based on personal biological tumor characteristics.

  3. Functional characterization of the kinase activation loop in nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) using tandem affinity purification and liquid chromatography-mass spectrometry.

    PubMed

    Wang, Peng; Wu, Fang; Ma, Yupo; Li, Liang; Lai, Raymond; Young, Leah C

    2010-01-01

    Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of >or=1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK.

  4. Early assessment of minimal residual disease identifies patients at very high relapse risk in NPM-ALK-positive anaplastic large-cell lymphoma.

    PubMed

    Damm-Welk, Christine; Mussolin, Lara; Zimmermann, Martin; Pillon, Marta; Klapper, Wolfram; Oschlies, Ilske; d'Amore, Emanuele S G; Reiter, Alfred; Woessmann, Wilhelm; Rosolen, Angelo

    2014-01-16

    Detection of minimal disseminated disease (MDD) at diagnosis correlates with relapse risk in children with anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALCL). We investigated whether minimal residual disease (MRD) positivity by qualitative reverse-transcriptase polymerase chain reaction (RT-PCR) for Nucleophosmin (NPM)-ALK during treatment identifies patients at the highest relapse risk. Blood and/or bone marrow of 180 patients with NPM-ALK-positive ALCL treated with Berlin-Frankfurt-Münster-type protocols were screened for NPM-ALK transcripts at diagnosis; 103 were found to be MDD-positive. MRD before the second therapy course could be evaluated in 52 MDD-positive patients. MRD positivity correlated with uncommon histology. The cumulative incidence of relapses (CIR) of 26 MDD-positive/MRD-positive patients (81% ± 8%) was significantly higher than the CIR of 26 MDD-positive/MRD-negative (31% ± 9%) and 77 MDD-negative patients (15% ± 5%) (P < .001). Five-year survival of MDD-negative and MDD-positive/MRD-negative patients was 91% ± 3% and 92% ± 5%, respectively, compared with 65% ± 9% of MDD-positive/MRD-positive patients (P < .001). Early evaluation of MRD in NPM-ALK-positive ALCL identifies patients with a very high relapse risk and inferior survival.

  5. Repair of DNA Alkylation Damage by the Escherichia coli Adaptive Response Protein AlkB as Studied by ESI-TOF Mass Spectrometry

    PubMed Central

    Li, Deyu; Delaney, James C.; Page, Charlotte M.; Chen, Alvin S.; Wong, Cintyu; Drennan, Catherine L.; Essigmann, John M.

    2010-01-01

    DNA alkylation can cause mutations, epigenetic changes, and even cell death. All living organisms have evolved enzymatic and non-enzymatic strategies for repairing such alkylation damage. AlkB, one of the Escherichia coli adaptive response proteins, uses an α-ketoglutarate/Fe(II)-dependent mechanism that, by chemical oxidation, removes a variety of alkyl lesions from DNA, thus affording protection of the genome against alkylation. In an effort to understand the range of acceptable substrates for AlkB, the enzyme was incubated with chemically synthesized oligonucleotides containing alkyl lesions, and the reaction products were analyzed by electrospray ionization time-of-flight (ESI-TOF) mass spectrometry. Consistent with the literature, but studied comparatively here for the first time, it was found that 1-methyladenine, 1,N 6-ethenoadenine, 3-methylcytosine, and 3-ethylcytosine were completely transformed by AlkB, while 1-methylguanine and 3-methylthymine were partially repaired. The repair intermediates (epoxide and possibly glycol) of 3,N 4-ethenocytosine are reported for the first time. It is also demonstrated that O 6-methylguanine and 5-methylcytosine are refractory to AlkB, lending support to the hypothesis that AlkB repairs only alkyl lesions attached to the nitrogen atoms of the nucleobase. ESI-TOF mass spectrometry is shown to be a sensitive and efficient tool for probing the comparative substrate specificities of DNA repair proteins in vitro. PMID:21048928

  6. Functional Characterization of the Kinase Activation Loop in Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Using Tandem Affinity Purification and Liquid Chromatography-Mass Spectrometry*

    PubMed Central

    Wang, Peng; Wu, Fang; Ma, Yupo; Li, Liang; Lai, Raymond; Young, Leah C.

    2010-01-01

    Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of ≥1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK. PMID:19887368

  7. ALK5-mediated transforming growth factor β signaling in neural crest cells controls craniofacial muscle development via tissue-tissue interactions.

    PubMed

    Han, Arum; Zhao, Hu; Li, Jingyuan; Pelikan, Richard; Chai, Yang

    2014-08-01

    The development of the craniofacial muscles requires reciprocal interactions with surrounding craniofacial tissues that originate from cranial neural crest cells (CNCCs). However, the molecular mechanism involved in the tissue-tissue interactions between CNCCs and muscle progenitors during craniofacial muscle development is largely unknown. In the current study, we address how CNCCs regulate the development of the tongue and other craniofacial muscles using Wnt1-Cre; Alk5(fl/fl) mice, in which loss of Alk5 in CNCCs results in severely disrupted muscle formation. We found that Bmp4 is responsible for reduced proliferation of the myogenic progenitor cells in Wnt1-Cre; Alk5(fl/fl) mice during early myogenesis. In addition, Fgf4 and Fgf6 ligands were reduced in Wnt1-Cre; Alk5(fl/fl) mice and are critical for differentiation of the myogenic cells. Addition of Bmp4 or Fgf ligands rescues the proliferation and differentiation defects in the craniofacial muscles of Alk5 mutant mice in vitro. Taken together, our results indicate that CNCCs play critical roles in controlling craniofacial myogenic proliferation and differentiation through tissue-tissue interactions.

  8. A Catalytic Role for C-H/π Interactions in Base Excision Repair by Bacillus cereus DNA Glycosylase AlkD.

    PubMed

    Parsons, Zachary D; Bland, Joshua M; Mullins, Elwood A; Eichman, Brandt F

    2016-09-14

    DNA glycosylases protect genomic integrity by locating and excising aberrant nucleobases. Substrate recognition and excision usually take place in an extrahelical conformation, which is often stabilized by π-stacking interactions between the lesion nucleobase and aromatic side chains in the glycosylase active site. Bacillus cereus AlkD is the only DNA glycosylase known to catalyze base excision without extruding the damaged nucleotide from the DNA helix. Instead of contacting the nucleobase itself, the AlkD active site interacts with the lesion deoxyribose through a series of C-H/π interactions. These interactions are ubiquitous in protein structures, but evidence for their catalytic significance in enzymology is lacking. Here, we show that the C-H/π interactions between AlkD and the lesion deoxyribose participate in catalysis of glycosidic bond cleavage. This is the first demonstration of a catalytic role for C-H/π interactions as intermolecular forces important to DNA repair. PMID:27571247

  9. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions

    NASA Astrophysics Data System (ADS)

    Mullins, Elwood A.; Shi, Rongxin; Parsons, Zachary D.; Yuen, Philip K.; David, Sheila S.; Igarashi, Yasuhiro; Eichman, Brandt F.

    2015-11-01

    Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge-dipole and CH-π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.

  10. Bioactive S-alk(en)yl cysteine sulfoxide metabolites in the genus Allium: the chemistry of potential therapeutic agents.

    PubMed

    Rose, Peter; Whiteman, Matt; Moore, Philip K; Zhu, Yi Zhun

    2005-06-01

    S-Alk(en)yl cysteine sulfoxides are odourless, non-protein sulfur amino acids typically found in members of the family Alliaceae and are the precursors to the lachrymatory and flavour compounds found in the agronomically important genus Allium. Traditionally, Allium species, particularly the onion (Allium cepa) and garlic (A. sativum), have been used for centuries in European, Asian and American folk medicines for the treatment of numerous human pathologies, however it is only recently that any significant progress has been made in determining their mechanisms of action. Indeed, our understanding of the role of Allium species in human health undoubtedly comes from the combination of several academic disciplines including botany, biochemistry and nutrition. During tissue damage, S-alk(en)yl cysteine sulfoxides are converted to their respective thiosulfinates or propanethial-S-oxide by the action of the enzyme alliinase (EC 4.4.1.4). Depending on the Allium species, and under differing conditions, thiosulfinates can decompose to form additional sulfur constituents including diallyl, methyl allyl, and diethyl mono-, di-, tri-, tetra-, penta-, and hexasulfides, the vinyldithiins and (E)- and (Z)-ajoene. Recent reports have shown onion and garlic extracts, along with several principal sulfur constituents, can induce phase II detoxification enzymes like glutathione-S-transferases (EC 2.5.1.18) and quinone reductase (QR) NAD(P)H: (quinine acceptor) oxidoreductase (EC 1.6.99.2) in mammalian tissues, as well as also influencing cell cycle arrest and apoptosis in numerous in vitro cancer cell models. Moreover, studies are also beginning to highlight a role of Allium-derived sulfur compounds in cardiovascular protection. In this review, we discuss the chemical diversity of S-alk(en)yl cysteine sulfoxide metabolites in the context of their biochemical and pharmacological mechanisms.

  11. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions.

    PubMed

    Mullins, Elwood A; Shi, Rongxin; Parsons, Zachary D; Yuen, Philip K; David, Sheila S; Igarashi, Yasuhiro; Eichman, Brandt F

    2015-11-12

    Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge-dipole and CH-π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision. PMID:26524531

  12. Mechanism of repair of acrolein- and malondialdehyde-derived exocyclic guanine adducts by the α-ketoglutarate/Fe(II) dioxygenase AlkB.

    PubMed

    Singh, Vipender; Fedeles, Bogdan I; Li, Deyu; Delaney, James C; Kozekov, Ivan D; Kozekova, Albena; Marnett, Lawrence J; Rizzo, Carmelo J; Essigmann, John M

    2014-09-15

    The structurally related exocyclic guanine adducts α-hydroxypropano-dG (α-OH-PdG), γ-hydroxypropano-dG (γ-OH-PdG), and M1dG are formed when DNA is exposed to the reactive aldehydes acrolein and malondialdehyde (MDA). These lesions are believed to form the basis for the observed cytotoxicity and mutagenicity of acrolein and MDA. In an effort to understand the enzymatic pathways and chemical mechanisms that are involved in the repair of acrolein- and MDA-induced DNA damage, we investigated the ability of the DNA repair enzyme AlkB, an α-ketoglutarate/Fe(II) dependent dioxygenase, to process α-OH-PdG, γ-OH-PdG, and M1dG in both single- and double-stranded DNA contexts. By monitoring the repair reactions using quadrupole time-of-flight (Q-TOF) mass spectrometry, it was established that AlkB can oxidatively dealkylate γ-OH-PdG most efficiently, followed by M1dG and α-OH-PdG. The AlkB repair mechanism involved multiple intermediates and complex, overlapping repair pathways. For example, the three exocyclic guanine adducts were shown to be in equilibrium with open-ring aldehydic forms, which were trapped using (pentafluorobenzyl)hydroxylamine (PFBHA) or NaBH4. AlkB repaired the trapped open-ring form of γ-OH-PdG but not the trapped open-ring of α-OH-PdG. Taken together, this study provides a detailed mechanism by which three-carbon bridge exocyclic guanine adducts can be processed by AlkB and suggests an important role for the AlkB family of dioxygenases in protecting against the deleterious biological consequences of acrolein and MDA.

  13. Management of Brain Metastases in ALK-Positive Non-Small-Cell Lung Cancer.

    PubMed

    Rusthoven, Chad G; Doebele, Robert C

    2016-08-20

    The Oncology Grand Rounds series is designed to place original reports published in the Journal into clinical context. A case presentation is followed by a description of diagnostic and management challenges, a review of the relevant literature, and a summary of the authors' suggested management approaches. The goal of this series is to help readers better understand how to apply the results of key studies, including those published in Journal of Clinical Oncology, to patients seen in their own clinical practice.A 54-year-old man with a former 15-pack-year smoking history presents with cough and dyspnea. Initial work-up with imaging demonstrates a right suprahilar mass measuring 4.7 cm as well as several enlarged hilar and ipsilateral mediastinal lymph nodes. Bronchoscopy with biopsy reveals adenocarcinoma consistent with a lung primary. Staging with positron emission tomography/computed tomography (PET/CT) reidentifies the primary mass and lymph nodes and shows several PET-avid bone metastases. Brain magnetic resonance imaging (MRI) demonstrates a 1.6-cm right parietal mass with mild vasogenic edema and four additional brain metastases measuring 4 to 9 mm in size. Molecular testing is positive for an anaplastic lymphoma kinase (ALK) gene rearrangement using fluorescence in situ hybridization and negative for EGFR, ROS1, RET, BRAF, KRAS, and other oncogenes. The patient denies any neurologic symptoms and has no significant findings on neurologic exam. He is referred to you for management options for newly diagnosed stage IV (T2aN2M1b) lung adenocarcinoma. PMID:27298405

  14. ALK evaluation in the world of multiplex testing: Network Genomic Medicine (NGM): the Cologne model for implementing personalised oncology.

    PubMed

    Heydt, C; Kostenko, A; Merkelbach-Bruse, S; Wolf, J; Büttner, R

    2016-09-01

    Comprehensive molecular genotyping of lung cancers has become a key requirement for guiding therapeutic decisions. As a paradigm model of implementing next-generation comprehensive diagnostics, Network Genomic Medicine (NGM) has established central diagnostic and clinical trial platforms for centralised testing and decentralised personalised treatment in clinical practice. Here, we describe the structures of the NGM network and give a summary of technologies to identify patients with anaplastic lymphoma kinase (ALK) fusion-positive lung adenocarcinomas. As unifying test platforms will become increasingly important for delivering reliable, quick and affordable tests, the NGM diagnostic platform is currently implementing a comprehensive hybrid capture-based parallel sequencing pan-cancer assay.

  15. ALK evaluation in the world of multiplex testing: Network Genomic Medicine (NGM): the Cologne model for implementing personalised oncology.

    PubMed

    Heydt, C; Kostenko, A; Merkelbach-Bruse, S; Wolf, J; Büttner, R

    2016-09-01

    Comprehensive molecular genotyping of lung cancers has become a key requirement for guiding therapeutic decisions. As a paradigm model of implementing next-generation comprehensive diagnostics, Network Genomic Medicine (NGM) has established central diagnostic and clinical trial platforms for centralised testing and decentralised personalised treatment in clinical practice. Here, we describe the structures of the NGM network and give a summary of technologies to identify patients with anaplastic lymphoma kinase (ALK) fusion-positive lung adenocarcinomas. As unifying test platforms will become increasingly important for delivering reliable, quick and affordable tests, the NGM diagnostic platform is currently implementing a comprehensive hybrid capture-based parallel sequencing pan-cancer assay. PMID:27573753

  16. n-Alkane and clofibrate, a peroxisome proliferator, activate transcription of ALK2 gene encoding cytochrome P450alk2 through distinct cis-acting promoter elements in Candida maltosa

    SciTech Connect

    Kogure, Takahisa; Takagi, Masamichi; Ohta, Akinori . E-mail: aaohta@mail.ecc.u-tokyo.ac.jp

    2005-04-01

    The ALK2 gene, encoding one of the n-alkane-hydroxylating cytochromes P450 in Candida maltosa, is induced by n-alkanes and a peroxisome proliferator, clofibrate. Deletion analysis of this gene's promoter revealed two cis-acting elements-an n-alkane-responsive element (ARE2) and a clofibrate-responsive element (CRE2)-that partly overlap in sequence but have distinct functions. ARE2-mediated activation responded to n-alkanes but not to clofibrate and was repressed by glucose. CRE2-mediated activation responded to polyunsaturated fatty acids and steroid hormones as well as to peroxisome proliferators but not to n-alkanes, and it was not repressed by glucose. Both elements mediated activation by oleic acid. Mutational analysis demonstrated that three CCG sequences in CRE2 were critical to the activation by clofibrate as well as to the in vitro binding of a specific protein to this element. These findings suggest that ALK2 is induced by peroxisome proliferators and steroid hormones through a specific CRE2-mediated regulatory mechanism.

  17. Kinase inhibitor profiling reveals unexpected opportunities to inhibit disease-associated mutant kinases

    PubMed Central

    Duong-Ly, Krisna C.; Devarajan, Karthik; Liang, Shuguang; Horiuchi, Kurumi Y.; Wang, Yuren; Ma, Haiching; Peterson, Jeffrey R.

    2016-01-01

    Summary Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant, mutant kinases. The results revealed lead compounds with activity against clinically important mutant kinases including ALK, LRRK2, RET, and EGFR as well as unexpected opportunities for repurposing FDA-approved kinase inhibitors as leads for additional indications. Furthermore, using T674I PDGFRα as an example, we show how single-dose screening data can provide predictive structure-activity data to guide subsequent inhibitor optimization. This study provides a resource for the development of inhibitors against numerous disease-associated mutant kinases and illustrates the potential of unbiased profiling as an approach to compound-centric inhibitor development. PMID:26776524

  18. Quantum mechanics/molecular mechanics study on the oxygen binding and substrate hydroxylation step in AlkB repair enzymes.

    PubMed

    Quesne, Matthew G; Latifi, Reza; Gonzalez-Ovalle, Luis E; Kumar, Devesh; de Visser, Sam P

    2014-01-01

    AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N(1) -methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)-oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate-determining hydrogen-atom abstraction on competitive σ- and π-pathways on a quintet spin-state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen-bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained.

  19. ARM-Seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments

    PubMed Central

    Cozen, Aaron E.; Quartley, Erin; Holmes, Andrew D.; Robinson, Eva H.; Phizicky, Eric M.; Lowe, Todd M.

    2015-01-01

    High throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA Methylation sequencing (ARM-Seq) which uses pre-treatment with Escherichia coli AlkB to demethylate 1-methyladenosine, 3-methylcytidine, and 1-methylguanosine, all commonly found in transfer RNAs. Comparative methylation analysis using ARM-Seq provides the first detailed, transcriptome-scale map of these modifications, and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs. ARM-Seq demonstrates that tRNA-derived small RNAs accurately recapitulate the m1A modification state for well-characterized yeast tRNAs, and generates new predictions for a large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs. Thus, ARM-Seq provides broad utility for identifying previously overlooked methyl-modified RNAs, can efficiently monitor methylation state, and may reveal new roles for tRNA-derived RNAs as biomarkers or signaling molecules. PMID:26237225

  20. Monitoring the alkane monooxygenase gene alkB in different soil interfaces during plant litter degradation of C3 and C4 plants

    NASA Astrophysics Data System (ADS)

    Schulz, S.; Munch, J. C.; Schloter, M.

    2009-04-01

    Hydrocarbons like n-alkanes are ubiquitous in the environment as a result of anthropogenic contamination (e.g. oil spills) as well as a part of an ecosystem's biomass. For example n-alkanes become released during plant litter degradation; consequently they become a high abundant carbon source for microorganism. One possibility for the prokaryotic hydrocarbon metabolisation is an aerobic degradation pathway where the initial step is catalysed by the membrane bound alkane monooxygenase alkB. We analysed the influence of alkanes on the abundance of the alkB gene in different interfaces of the litter-soil system during the degradation of maize and pea litter. Therefore soil samples of a sandy and a loamy soil have been incubated with straw of maize and pea plants up to 30 weeks with constant soil moisture and temperature. Using quantitative real-time PCR we were able to monitor the changes of the abundance and the expression rates of alkB. In our experiments we focused on the straw layer, the litter/soil interface and the soil 1 cm below this interface (bulk soil). Our results clearly demonstrate time and space dependent abundance patterns of alkB genes and transcripts in the different layers studied, which are additionally shaped by the soil type used.

  1. MPN- and Real-Time-Based PCR Methods for the Quantification of Alkane Monooxygenase Homologous Genes (alkB) in Environmental Samples

    NASA Astrophysics Data System (ADS)

    Pérez-de-Mora, Alfredo; Schulz, Stephan; Schloter, Michael

    Hydrocarbons are major contaminants of soil ecosystems as a result of uncontrolled oil spills and wastes disposal into the environment. Ecological risk assessment and remediation of affected sites is often constrained due to lack of suitable prognostic and diagnostic tools that provide information of abiotic-biotic interactions occurring between contaminants and biological targets. Therefore, the identification and quantification of genes involved in the degradation of hydrocarbons may play a crucial role for evaluating the natural attenuation potential of contaminated sites and the development of successful bioremediation strategies. Besides other gene clusters, the alk operon has been identified as a major player for alkane degradation in different soils. An oxygenase gene (alkB) codes for the initial step of the degradation of aliphatic alkanes under aerobic conditions. In this work, we present an MPN- and a real-time PCR method for the quantification of the bacterial gene alkB (coding for rubredoxin-dependent alkane monooxygenase) in environmental samples. Both approaches enable a rapid culture-independent screening of the alkB gene in the environment, which can be used to assess the intrinsic natural attenuation potential of a site or to follow up the on-going progress of bioremediation assays.

  2. Case report: A unique pediatric case of a primary CD8 expressing ALK-1 positive anaplastic large cell lymphoma of skeletal muscle.

    PubMed

    Gaiser, Timo; Geissinger, Eva; Schattenberg, Torsten; Scharf, Hanns-Peter; Dürken, Matthias; Dinter, Dietmar; Rosenwald, Andreas; Marx, Alexander

    2012-01-01

    Primary involvement of skeletal muscle is a very rare event in ALK-1 positive anaplastic large cell lymphoma (ALCL). We describe a case of a 10-year old boy presenting with a three week history of pain and a palpable firm swelling at the dorsal aspect of the left thigh. Histological examination of the lesion revealed a tumoral and diffuse polymorphic infiltration of the muscle by large lymphoid cells. Tumor cells displayed eccentric, lobulated "horse shoe" or "kidney-shape" nuclei. The cells showed immunohistochemical positivity for CD30, ALK-1, CD2, CD3, CD7, CD8, and Perforin. Fluorescence in situ hybridization analysis revealed a characteristic rearrangement of the ALK-1 gene in 2p23 leading to the diagnosis of ALK-1 positive ALCL. Chemotherapy according to the ALCL-99-NHL-BFM protocol was initiated and resulted in a complete remission after two cycles. This case illustrates the unusual presentation of a pediatric ALCL in soft tissue with a good response to chemotherapy.

  3. Pleiotrophin promotes vascular abnormalization in gliomas and correlates with poor survival in patients with astrocytomas.

    PubMed

    Zhang, Lei; Kundu, Soumi; Feenstra, Tjerk; Li, Xiujuan; Jin, Chuan; Laaniste, Liisi; El Hassan, Tamador Elsir Abu; Ohlin, K Elisabet; Yu, Di; Olofsson, Tommie; Olsson, Anna-Karin; Pontén, Fredrik; Magnusson, Peetra U; Nilsson, Karin Forsberg; Essand, Magnus; Smits, Anja; Dieterich, Lothar C; Dimberg, Anna

    2015-12-01

    Glioblastomas are aggressive astrocytomas characterized by endothelial cell proliferation and abnormal vasculature, which can cause brain edema and increase patient morbidity. We identified the heparin-binding cytokine pleiotrophin as a driver of vascular abnormalization in glioma. Pleiotrophin abundance was greater in high-grade human astrocytomas and correlated with poor survival. Anaplastic lymphoma kinase (ALK), which is a receptor that is activated by pleiotrophin, was present in mural cells associated with abnormal vessels. Orthotopically implanted gliomas formed from GL261 cells that were engineered to produce pleiotrophin showed increased microvessel density and enhanced tumor growth compared with gliomas formed from control GL261 cells. The survival of mice with pleiotrophin-producing gliomas was shorter than that of mice with gliomas that did not produce pleiotrophin. Vessels in pleiotrophin-producing gliomas were poorly perfused and abnormal, a phenotype that was associated with increased deposition of vascular endothelial growth factor (VEGF) in direct proximity to the vasculature. The growth of pleiotrophin-producing GL261 gliomas was inhibited by treatment with the ALK inhibitor crizotinib, the ALK inhibitor ceritinib, or the VEGF receptor inhibitor cediranib, whereas control GL261 tumors did not respond to either inhibitor. Our findings link pleiotrophin abundance in gliomas with survival in humans and mice, and show that pleiotrophin promotes glioma progression through increased VEGF deposition and vascular abnormalization. PMID:26645582

  4. Interleukin-9 (IL-9) and NPM-ALK each generate mast cell hyperplasia as single 'hit' and cooperate in producing a mastocytosis-like disease in mice.

    PubMed

    Merz, Hartmut; Kaehler, Christian; Hoefig, Kai P; Branke, Biggi; Uckert, Wolfgang; Nadrowitz, Roger; Cerny-Reiterer, Sabine; Herrmann, Harald; Feller, Alfred C; Valent, Peter

    2010-06-01

    Mast cell neoplasms are characterized by abnormal growth and focal accumulation of mast cells (MC) in one or more organs. Although several cytokines, including stem cell factor (SCF) and interleukin-9 (IL-9) have been implicated in growth of normal MC, little is known about pro-oncogenic molecules and conditions triggering differentiation and growth of MC far enough to lead to the histopathological picture of overt mastocytosis. The anaplastic lymphoma kinase (ALK) has recently been implicated in growth of neoplastic cells in malignant lymphomas. Here, we describe that transplantation of NPM-ALK-transplanted mouse bone marrow progenitors into lethally irradiated IL-9 transgenic mice not only results in lymphoma-formation, but also in the development of a neoplastic disease exhibiting histopathological features of systemic mastocytosis, including multifocal dense MC-infiltrates, occasionally with devastating growth in visceral organs. Transplantation of NPM-ALK-transduced progenitors into normal mice or maintenance of IL-9-transgenic mice without NPM-ALK each resulted in MC hyperplasia, but not in mastocytosis. Neoplastic MC in mice not only displayed IL-9, but also the IL-9 receptor, and the same was found to hold true for human neoplastic MC. Together, our data show that neoplastic MC express IL-9 receptors, that IL-9 and NPM-ALK upregulate MC-production in vivo, and that both'hits' act in concert to induce a mastocytosis-like disease in mice. These data may have pathogenetic and clinical implications and fit well with the observation that neoplastic MC in advanced SM strongly express NPM and multiple "lymphoid" antigens including CD25 and CD30.

  5. Interleukin-9 (IL-9) and NPM-ALK each generate mast cell hyperplasia as single 'hit' and cooperate in producing a mastocytosis-like disease in mice.

    PubMed

    Merz, Hartmut; Kaehler, Christian; Hoefig, Kai P; Branke, Biggi; Uckert, Wolfgang; Nadrowitz, Roger; Cerny-Reiterer, Sabine; Herrmann, Harald; Feller, Alfred C; Valent, Peter

    2010-06-01

    Mast cell neoplasms are characterized by abnormal growth and focal accumulation of mast cells (MC) in one or more organs. Although several cytokines, including stem cell factor (SCF) and interleukin-9 (IL-9) have been implicated in growth of normal MC, little is known about pro-oncogenic molecules and conditions triggering differentiation and growth of MC far enough to lead to the histopathological picture of overt mastocytosis. The anaplastic lymphoma kinase (ALK) has recently been implicated in growth of neoplastic cells in malignant lymphomas. Here, we describe that transplantation of NPM-ALK-transplanted mouse bone marrow progenitors into lethally irradiated IL-9 transgenic mice not only results in lymphoma-formation, but also in the development of a neoplastic disease exhibiting histopathological features of systemic mastocytosis, including multifocal dense MC-infiltrates, occasionally with devastating growth in visceral organs. Transplantation of NPM-ALK-transduced progenitors into normal mice or maintenance of IL-9-transgenic mice without NPM-ALK each resulted in MC hyperplasia, but not in mastocytosis. Neoplastic MC in mice not only displayed IL-9, but also the IL-9 receptor, and the same was found to hold true for human neoplastic MC. Together, our data show that neoplastic MC express IL-9 receptors, that IL-9 and NPM-ALK upregulate MC-production in vivo, and that both'hits' act in concert to induce a mastocytosis-like disease in mice. These data may have pathogenetic and clinical implications and fit well with the observation that neoplastic MC in advanced SM strongly express NPM and multiple "lymphoid" antigens including CD25 and CD30. PMID:21297223

  6. Co-active receptor tyrosine kinases mitigate the effect of FGFR inhibitors in FGFR1-amplified lung cancers with low FGFR1 protein expression.

    PubMed

    Kotani, H; Ebi, H; Kitai, H; Nanjo, S; Kita, K; Huynh, T G; Ooi, A; Faber, A C; Mino-Kenudson, M; Yano, S

    2016-07-01

    Targeted therapies are effective in subsets of lung cancers with EGFR mutations and anaplastic lymphoma kinase (ALK) translocations. Large-scale genomics have recently expanded the lung cancer landscape with FGFR1 amplification found in 10-20% of squamous cell carcinomas (SCCs). However, the response rates have been low for biomarker-directed fibroblast growth factor receptor (FGFR) inhibitor therapy in SCC, which contrasts to the relatively high rates of response seen in EGFR mutant and ALK-translocated lung cancers treated with epidermal growth factor receptor (EGFR) inhibitors and ALK inhibitors, respectively. In order to better understand the low response rates of FGFR1-amplified lung cancers to FGFR inhibitors, relationships between gene copy number, mRNA expression and protein expression of FGFR1 were assessed in cell lines, tumor specimens and data from The Cancer Genome Atlas. The importance of these factors for the sensitivity to FGFR inhibitors was determined by analyzing drug screen data and conducting in vitro and in vivo experiments. We report that there was a discrepancy between FGFR1 amplification level and FGFR1 protein expression in a number of these cell lines, and the cancers with unexpectedly low FGFR1 expression were uniformly resistant to the different FGFR inhibitors. Further interrogation of the receptor tyrosine kinase activity in these discordant cell lines revealed co-activation of HER2 and platelet-derived growth factor receptor-α (PDGFRα) caused by gene amplification or ligand overexpression maintained phosphoinositide 3-kinase (PI3K) and MEK/ERK signaling even in the presence of FGFR inhibitor. Accordingly, co-inhibition of FGFR1 and HER2 or PDGFRα led to enhanced drug responses. In contrast, FGFR1-amplified high FGFR1 protein-expressing lung cancers are sensitive to FGFR inhibitor monotherapy by downregulating ERK signaling. Addition of a PI3K inhibitor to these high FGFR1 protein-expressing cancers further sensitized them to FGFR

  7. Specific activin receptor-like kinase 3 inhibitors enhance liver regeneration.

    PubMed

    Tsugawa, Daisuke; Oya, Yuki; Masuzaki, Ryota; Ray, Kevin; Engers, Darren W; Dib, Martin; Do, Nhue; Kuramitsu, Kaori; Ho, Karen; Frist, Audrey; Yu, Paul B; Bloch, Kenneth D; Lindsley, Craig W; Hopkins, Corey R; Hong, Charles C; Karp, Seth J

    2014-12-01

    Pharmacologic agents to enhance liver regeneration after injury would have wide therapeutic application. Based on previous work suggesting inhibition of bone morphogenetic protein (BMP) signaling stimulates liver regeneration, we tested known and novel BMP inhibitors for their ability to accelerate regeneration in a partial hepatectomy (PH) model. Compounds were produced based on the 3,6-disubstituted pyrazolo[1,5-a] pyrimidine core of the BMP antagonist dorsomorphin and evaluated for their ability to inhibit BMP signaling and enhance liver regeneration. Antagonists of the BMP receptor activin receptor-like kinase 3 (ALK3), including LDN-193189 (LDN; 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), DMH2 (4-(2-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; VU0364849), and the novel compound VU0465350 (7-(4-isopropoxyphenyl)-3-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine; VU5350), blocked SMAD phosphorylation in vitro and in vivo, and enhanced liver regeneration after PH. In contrast, an antagonist of the BMP receptor ALK2, VU0469381 (5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone; 1LWY), did not affect liver regeneration. LDN did not affect liver synthetic or metabolic function. Mechanistically, LDN increased serum interleukin-6 levels and signal transducer and activator of transcription 3 phosphorylation in the liver, and modulated other factors known to be important for liver regeneration, including suppressor of cytokine signaling 3 and p53. These findings suggest that inhibition of ALK3 may be part of a therapeutic strategy for treating human liver disease.

  8. Specific activin receptor-like kinase 3 inhibitors enhance liver regeneration.

    PubMed

    Tsugawa, Daisuke; Oya, Yuki; Masuzaki, Ryota; Ray, Kevin; Engers, Darren W; Dib, Martin; Do, Nhue; Kuramitsu, Kaori; Ho, Karen; Frist, Audrey; Yu, Paul B; Bloch, Kenneth D; Lindsley, Craig W; Hopkins, Corey R; Hong, Charles C; Karp, Seth J

    2014-12-01

    Pharmacologic agents to enhance liver regeneration after injury would have wide therapeutic application. Based on previous work suggesting inhibition of bone morphogenetic protein (BMP) signaling stimulates liver regeneration, we tested known and novel BMP inhibitors for their ability to accelerate regeneration in a partial hepatectomy (PH) model. Compounds were produced based on the 3,6-disubstituted pyrazolo[1,5-a] pyrimidine core of the BMP antagonist dorsomorphin and evaluated for their ability to inhibit BMP signaling and enhance liver regeneration. Antagonists of the BMP receptor activin receptor-like kinase 3 (ALK3), including LDN-193189 (LDN; 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), DMH2 (4-(2-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; VU0364849), and the novel compound VU0465350 (7-(4-isopropoxyphenyl)-3-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine; VU5350), blocked SMAD phosphorylation in vitro and in vivo, and enhanced liver regeneration after PH. In contrast, an antagonist of the BMP receptor ALK2, VU0469381 (5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone; 1LWY), did not affect liver regeneration. LDN did not affect liver synthetic or metabolic function. Mechanistically, LDN increased serum interleukin-6 levels and signal transducer and activator of transcription 3 phosphorylation in the liver, and modulated other factors known to be important for liver regeneration, including suppressor of cytokine signaling 3 and p53. These findings suggest that inhibition of ALK3 may be part of a therapeutic strategy for treating human liver disease. PMID:25271257

  9. Fasting potentiates the anticancer activity of tyrosine kinase inhibitors by strengthening MAPK signaling inhibition.

    PubMed

    Caffa, Irene; D'Agostino, Vito; Damonte, Patrizia; Soncini, Debora; Cea, Michele; Monacelli, Fiammetta; Odetti, Patrizio; Ballestrero, Alberto; Provenzani, Alessandro; Longo, Valter D; Nencioni, Alessio

    2015-05-20

    Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use.

  10. Quantitative PCR detection of NPM/ALK fusion gene and CD30 gene expression in patients with anaplastic large cell lymphoma--residual disease monitoring and a correlation with the disease status.

    PubMed

    Kalinova, Marketa; Krskova, Lenka; Brizova, Helena; Kabickova, Edita; Kepak, Tomas; Kodet, Roman

    2008-01-01

    Anaplastic large cell lymphoma (ALCL) represents a heterogeneous group of malignant lymphoproliferative diseases with a consistent expression of the cytokine receptor CD30. ALCL is frequently associated with a NPM/ALK fusion gene which is found in up to 75% of pediatric ALCLs. Real-time quantitative RT-PCR (RQ-RT-PCR) of NPM/ALK and CD30 gene expression was employed to analyze minimal residual disease (MRD) in 10 patients with NPM/ALK positive ALCL in 79 follow-up bone marrow (BM) and/or peripheral blood (PB) samples. In all BM samples from relapses and/or closely before a relapse, BM samples revealed NPM/ALK and CD30 positivity in at least one of the iliac BM trephines. Five out of nine relapses were preceded or were accompanied by minimally half log increased NPM/ALK levels in the BM. We found that RQ-RT-PCR of the CD30 expression is not suitable for MRD detection--only two relapses were accompanied by an increase of the CD30 level above a level which was detected in BM/PB samples from healthy individuals. RQ-RT-PCR of NPM/ALK expression is a promising and rapid approach for monitoring MRD.

  11. HDAC Inhibitors.

    PubMed

    Olzscha, Heidi; Bekheet, Mina E; Sheikh, Semira; La Thangue, Nicholas B

    2016-01-01

    Lysine acetylation in proteins is one of the most abundant posttranslational modifications in eukaryotic cells. The dynamic homeostasis of lysine acetylation and deacetylation is dictated by the action of histone acetyltransferases (HAT) and histone deacetylases (HDAC). Important substrates for HATs and HDACs are histones, where lysine acetylation generally leads to an open and transcriptionally active chromatin conformation. Histone deacetylation forces the compaction of the chromatin with subsequent inhibition of transcription and reduced gene expression. Unbalanced HAT and HDAC activity, and therefore aberrant histone acetylation, has been shown to be involved in tumorigenesis and progression of malignancy in different types of cancer. Therefore, the development of HDAC inhibitors (HDIs) as therapeutic agents against cancer is of great interest. However, treatment with HDIs can also affect the acetylation status of many other non-histone proteins which play a role in different pathways including angiogenesis, cell cycle progression, autophagy and apoptosis. These effects have led HDIs to become anticancer agents, which can initiate apoptosis in tumor cells. Hematological malignancies in particular are responsive to HDIs, and four HDIs have already been approved as anticancer agents. There is a strong interest in finding adequate biomarkers to predict the response to HDI treatment. This chapter provides information on how to assess HDAC activity in vitro and determine the potency of HDIs on different HDACs. It also gives information on how to analyze cellular markers following HDI treatment and to analyze tissue biopsies from HDI-treated patients. Finally, a protocol is provided on how to detect HDI sensitivity determinants in human cells, based on a pRetroSuper shRNA screen upon HDI treatment. PMID:27246222

  12. In Vitro Drug Sensitivity Tests to Predict Molecular Target Drug Responses in Surgically Resected Lung Cancer

    PubMed Central

    Miyazaki, Ryohei; Anayama, Takashi; Hirohashi, Kentaro; Okada, Hironobu; Kume, Motohiko; Orihashi, Kazumasa

    2016-01-01

    Background Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and anaplastic lymphoma kinase (ALK) inhibitors have dramatically changed the strategy of medical treatment of lung cancer. Patients should be screened for the presence of the EGFR mutation or echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene prior to chemotherapy to predict their clinical response. The succinate dehydrogenase inhibition (SDI) test and collagen gel droplet embedded culture drug sensitivity test (CD-DST) are established in vitro drug sensitivity tests, which may predict the sensitivity of patients to cytotoxic anticancer drugs. We applied in vitro drug sensitivity tests for cyclopedic prediction of clinical responses to different molecular targeting drugs. Methods The growth inhibitory effects of erlotinib and crizotinib were confirmed for lung cancer cell lines using SDI and CD-DST. The sensitivity of 35 cases of surgically resected lung cancer to erlotinib was examined using SDI or CD-DST, and compared with EGFR mutation status. Results HCC827 (Exon19: E746-A750 del) and H3122 (EML4-ALK) cells were inhibited by lower concentrations of erlotinib and crizotinib, respectively than A549, H460, and H1975 (L858R+T790M) cells were. The viability of the surgically resected lung cancer was 60.0 ± 9.8 and 86.8 ± 13.9% in EGFR-mutants vs. wild types in the SDI (p = 0.0003). The cell viability was 33.5 ± 21.2 and 79.0 ± 18.6% in EGFR mutants vs. wild-type cases (p = 0.026) in CD-DST. Conclusions In vitro drug sensitivity evaluated by either SDI or CD-DST correlated with EGFR gene status. Therefore, SDI and CD-DST may be useful predictors of potential clinical responses to the molecular anticancer drugs, cyclopedically. PMID:27070423

  13. The atomic resolution structure of human AlkB homolog 7 (ALKBH7), a key protein for programmed necrosis and fat metabolism.

    PubMed

    Wang, Guoqiang; He, Qingzhong; Feng, Chong; Liu, Yang; Deng, Zengqin; Qi, Xiaoxuan; Wu, Wei; Mei, Pinchao; Chen, Zhongzhou

    2014-10-01

    ALKBH7 is the mitochondrial AlkB family member that is required for alkylation- and oxidation-induced programmed necrosis. In contrast to the protective role of other AlkB family members after suffering alkylation-induced DNA damage, ALKBH7 triggers the collapse of mitochondrial membrane potential and promotes cell death. Moreover, genetic ablation of mouse Alkbh7 dramatically increases body weight and fat mass. Here, we present crystal structures of human ALKBH7 in complex with Mn(II) and α-ketoglutarate at 1.35 Å or N-oxalylglycine at 2.0 Å resolution. ALKBH7 possesses the conserved double-stranded β-helix fold that coordinates a catalytically active iron by a conserved HX(D/E) … Xn … H motif. Self-hydroxylation of Leu-110 was observed, indicating that ALKBH7 has the potential to catalyze hydroxylation of its substrate. Unlike other AlkB family members whose substrates are DNA or RNA, ALKBH7 is devoid of the "nucleotide recognition lid" which is essential for binding nucleobases, and thus exhibits a solvent-exposed active site; two loops between β-strands β6 and β7 and between β9 and β10 create a special outer wall of the minor β-sheet of the double-stranded β-helix and form a negatively charged groove. These distinct features suggest that ALKBH7 may act on protein substrate rather than nucleic acids. Taken together, our findings provide a structural basis for understanding the distinct function of ALKBH7 in the AlkB family and offer a foundation for drug design in treating cell death-related diseases and metabolic diseases.

  14. Quantitative determination of allergenic 5-alk(en)ylresorcinols in mango (Mangifera indica L.) peel, pulp, and fruit products by high-performance liquid chromatography.

    PubMed

    Knödler, Matthias; Reisenhauer, Katharina; Schieber, Andreas; Carle, Reinhold

    2009-05-13

    Despite a number of serious case reports of mango dermatitis, no attempts at the identification and quantification of allergenic 5-alk(en)ylresorcinols in mango fruits have so far been made. Therefore, total alk(en)ylresorcinol content and relative homologue composition in 13 mango peel samples and 7 samples of mango pulp were determined by HPLC and LC-MS/MS analyses. Furthermore, mango puree and nectar prepared on pilot plant scale were also analyzed and compared with commercially available thermally preserved products. Depending on cultivar, alk(en)ylresorcinol contents ranged from 79.3 to 1850.5 mg/kg of dry matter (DM) in mango peels and from 4.9 to 187.3 mg/kg of DM in samples of mango pulp. The profile of alk(en)ylresorcinols was found to be highly characteristic, with an average homologue composition of C15:0 (6.1%), C15:1 (1.7%), C17:0 (1.1%), C17:1 (52.5%), C17:2 (33.4%), C17:3 (2.4%), C19:1 (2.1%), and C19:2 (0.8%). Mango puree samples prepared from peeled and unpeeled fruits revealed contents of 3.8 and 12.3 mg/kg of fresh weight, respectively. Content and homologue composition were not significantly affected during puree processing and thermal preservation. In nectar samples prepared from peeled and unpeeled fruits, contents of 1.4 and 4.6 mg/L, respectively, were found.

  15. Measuring mass transfer processes of octane with the help of an alkSalkB::gfp-tagged Escherichia coli.

    PubMed

    Jaspers, M C; Meier, C; Zehnder, A J; Harms, H; van der Meer, J R

    2001-08-01

    Diffusion of octane from oily droplets in different microscale settings was measured using Escherichia coli expressing the stable green fluorescent protein (GFP) from the alkB promoter of Pseudomonas oleovorans. GFP fluorescence intensities were determined quantitatively at the single-cell level after 1.0 or 2.5 h incubation and compared with different calibration series using known concentrations of octane. By immobilizing the E. coli sensor cells on the bottom glass plate of a microscope flow chamber, it was possible to monitor the diffusion process for octane in aqueous solution as a function of time and distance from non-aqueous phase droplets of octane alone or oily octane mixtures. When a gas phase was included in the flow chambers, octane transport could be demonstrated from the oily mixtures to the cells through both gas and liquid phase. Assays of non-immobilized sensor cells in microdroplets in the presence or absence of soil particles incubated with octane through the vapour phase revealed a slight reduction in the total amount of induced E. coli cells in the presence of soil. Our results indicate the power of using GFP-marked single-cell biosensors in determining microscale bioavailability of organic pollutants. PMID:11578312

  16. Proton pump inhibitors

    MedlinePlus

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This ...

  17. Prognostic significance of circulating tumor cells in bone marrow or peripheral blood as detected by qualitative and quantitative PCR in pediatric NPM-ALK-positive anaplastic large-cell lymphoma.

    PubMed

    Damm-Welk, Christine; Busch, Kerstin; Burkhardt, Birgit; Schieferstein, Jutta; Viehmann, Susanne; Oschlies, Ilske; Klapper, Wolfram; Zimmermann, Martin; Harbott, Jochen; Reiter, Alfred; Woessmann, Willi

    2007-07-15

    Clinical and histopathological characteristics have limited prognostic value for children with anaplastic large-cell lymphoma (ALCL). We evaluated the presence, extent, and prognostic impact of circulating tumor cells in bone marrow (BM) and peripheral blood (PB) of children and adolescents with NPM-ALK-positive ALCL at diagnosis using qualitative and quantitative polymerase chain reaction (PCR) for NPM-ALK. Numbers of NPM-ALK transcripts were normalized to 10(4) copies ABL (NCNs). BM was analyzed from 80 patients and PB from 52. BM was positive for NPM-ALK in 47.5% of patients, and positivity was significantly correlated with clinical stage, mediastinal or visceral involvement, microscopic BM involvement, and histologic subtype. Qualitative and quantitative PCR results in BM and PB strongly correlated. BM PCR was associated with the cumulative incidence of relapses (CI-Rs): CI-R was 50% +/- 10% for 38 PCR-positive and 15% +/- 7% for 42 PCR-negative patients (P < .001). Sixteen patients with more than 10 NCNs NPM-ALK in BM had a CI-R of 71% +/- 14% compared with a CI-R of 18% +/- 6% for 59 patients with 10 or fewer NCNs (P < .001). PB PCR results led to a similar grouping. Thus, quantitative PCR in BM or PB allows identification of 20% of patients experiencing 60% of all relapses with an event-free survival of 20%.

  18. The lymphoma-associated NPM-ALK oncogene elicits a p16INK4a/pRb-dependent tumor-suppressive pathway.

    PubMed

    Martinelli, Paola; Bonetti, Paola; Sironi, Cristina; Pruneri, Giancarlo; Fumagalli, Caterina; Raviele, Paola Rafaniello; Volorio, Sara; Pileri, Stefano; Chiarle, Roberto; McDuff, Fiona Kate Elizabeth; Tusi, Betsabeh Khoramian; Turner, Suzanne D; Inghirami, Giorgio; Pelicci, Pier Giuseppe; Colombo, Emanuela

    2011-06-16

    Oncogene-induced senescence (OIS) is a barrier for tumor development. Oncogene-dependent DNA damage and activation of the ARF/p53 pathway play a central role in OIS and, accordingly, ARF and p53 are frequently mutated in human cancer. A number of leukemia/lymphoma-initiating oncogenes, however, inhibit ARF/p53 and only infrequently select for ARF or p53 mutations, suggesting the involvement of other tumor-suppressive pathways. We report that NPM-ALK, the initiating oncogene of anaplastic large cell lymphomas (ALCLs), induces DNA damage and irreversibly arrests the cell cycle of primary fibroblasts and hematopoietic progenitors. This effect is associated with inhibition of p53 and is caused by activation of the p16INK4a/pRb tumor-suppressive pathway. Analysis of NPM-ALK lymphomagenesis in transgenic mice showed p16INK4a-dependent accumulation of senescent cells in premalignant lesions and decreased tumor latency in the absence of p16INK4a. Accordingly, human ALCLs showed no expression of either p16INK4a or pRb. Up-regulation of the histone-demethylase Jmjd3 and de-methylation at the p16INK4a promoter contributed to the effect of NPM-ALK on p16INK4a, which was transcriptionally regulated. These data demonstrate that p16INK4a/pRb may function as an alternative pathway of oncogene-induced senescence, and suggest that the reactivation of p16INK4a expression might be a novel strategy to restore the senescence program in some tumors.

  19. miR-135b mediates NPM-ALK-driven oncogenicity and renders IL-17-producing immunophenotype to anaplastic large cell lymphoma.

    PubMed

    Matsuyama, Hironori; Suzuki, Hiroshi I; Nishimori, Hikaru; Noguchi, Masaaki; Yao, Takashi; Komatsu, Norio; Mano, Hiroyuki; Sugimoto, Koichi; Miyazono, Kohei

    2011-12-22

    Many transformed lymphoma cells show immune-phenotypes resembling the corresponding normal lymphocytes; thus, they provide a guide for proper diagnosis and present promising routes to improve their pathophysiologic understanding and to identify novel therapeutic targets. However, the underlying molecular mechanism(s) of these aberrant immune-phenotypes is largely unknown. Here, we report that microRNA-135b (miR-135b) mediates nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-driven oncogenicity and empowers IL-17-producing immunophenotype in anaplastic large cell lymphoma (ALCL). NPM-ALK oncogene strongly promoted the expression of miR-135b and its host gene LEMD1 through activation of signal transducer and activator of transcription (STAT) 3. In turn, elevated miR-135b targeted FOXO1 in ALCL cells. miR-135b introduction also decreased chemosensitivity in Jurkat cells, suggesting its contribution to oncogenic activities of NPM-ALK. Interestingly, miR-135b suppressed T-helper (Th) 2 master regulators STAT6 and GATA3, and miR-135b blockade attenuated IL-17 production and paracrine inflammatory response by ALCL cells, indicating that miR-135b-mediated Th2 suppression may lead to the skewing to ALCL immunophenotype overlapping with Th17 cells. Furthermore, antisense-based miR-135b inhibition reduced tumor angiogenesis and growth in vivo, demonstrating significance of this "Th17 mimic" pathway as a therapeutic target. These results collectively illuminated unique contribution of oncogenic kinase-linked microRNA to tumorigenesis through modulation of tumor immune-phenotype and microenvironment.

  20. Plant litter and soil type drive abundance, activity and community structure of alkB harbouring microbes in different soil compartments

    PubMed Central

    Schulz, Stephan; Giebler, Julia; Chatzinotas, Antonis; Wick, Lukas Y; Fetzer, Ingo; Welzl, Gerhard; Harms, Hauke; Schloter, Michael

    2012-01-01

    Alkanes are major constituents of plant-derived waxy materials. In this study, we investigated the abundance, community structure and activity of bacteria harbouring the alkane monooxygenase gene alkB, which catalyses a major step in the pathway of aerobic alkane degradation in the litter layer, the litter–soil interface and in bulk soil at three time points during the degradation of maize and pea plant litter (2, 8 and 30 weeks) to improve our understanding about drivers for microbial performance in different soil compartments. Soil cores of different soil textures (sandy and silty) were taken from an agricultural field and incubated at constant laboratory conditions. The abundance of alkB genes and transcripts (by qPCR) as well as the community structure (by terminal restriction fragment polymorphism fingerprinting) were measured in combination with the concentrations and composition of alkanes. The results obtained indicate a clear response pattern of all investigated biotic and abiotic parameters depending on the applied litter material, the type of soil used, the time point of sampling and the soil compartment studied. As expected the distribution of alkanes of different chain length formed a steep gradient from the litter layer to the bulk soil. Mainly in the two upper soil compartments community structure and abundance patterns of alkB were driven by the applied litter type and its degradation. Surprisingly, the differences between the compartments in one soil were more pronounced than the differences between similar compartments in the two soils studied. This indicates the necessity for analysing processes in different soil compartments to improve our mechanistic understanding of the dynamics of distinct functional groups of microbes. PMID:22402403

  1. Sustained co-cultivation with human placenta-derived MSCs enhances ALK5/Smad3 signaling in human breast epithelial cells, leading to EMT and differentiation.

    PubMed

    Yoo, Young A; Kang, Myoung Hee; Kim, Byung Soo; Kim, Jun Suk; Seo, Jae Hong

    2009-06-01

    The interaction between mammary epithelial cells and their surrounding microenvironment are important in the development of the mammary gland. Thus, mesenchymal stem cells (MSCs), which retain pluripotency for various mesenchymal lineages, may provide a permissive environment for the morphologic alteration and differentiation of mammary epithelial cells. To this end, we investigated whether the interactions between mammary epithelial cells and human placenta-derived MSCs (hPMSC) affect the morphology, proliferation, and differentiation of epithelial cells in a co-culture system. We show that after co-culture with hPMSCs, human mammary epithelial cell lines (MCF-10F and HEMC) underwent significant morphologic alterations and a dramatic increase in ductal-alveolar branching, which was accompanied by a decrease or loss of the epithelial marker E-cadherin and a gain of the mesenchymal markers, alpha-SMA and vimentin. MCF-10F and HEMC proliferation was also inhibited in the presence of hPMSCs, and this retardation in growth was due to cell cycle arrest. Furthermore, in MCF-10F and HMEC cells, hPMSCs induced the production of lipid droplets, milk fat globule protein, and milk protein lactoferrin, which are markers of functional mammary differentiation. We also noticed an elevation in ALK5 and phosphorylated Smad3 protein levels upon hPMSC co-culture. Strikingly, the changes in morphology, proliferation, and differentiation were reversed by treatment with ALK5 or Smad3 knockdown in MCF-10F/hPMSC co-cultures. Collectively, our findings suggest that co-cultivation with hPMSCs leads to epithelial to mesenchymal transition (EMT) and differentiation of human breast epithelial cells through the ALK5/Smad3 signaling pathway. PMID:19375841

  2. The anti-fibrotic effect of inhibition of TGFβ-ALK5 signalling in experimental pulmonary fibrosis in mice is attenuated in the presence of concurrent γ-herpesvirus infection.

    PubMed

    Smoktunowicz, Natalia; Alexander, Robert E; Franklin, Linda; Williams, Andrew E; Holman, Beverley; Mercer, Paul F; Jarai, Gabor; Scotton, Chris J; Chambers, Rachel C

    2015-09-01

    TGFβ-ALK5 pro-fibrotic signalling and herpesvirus infections have been implicated in the pathogenesis and exacerbation of pulmonary fibrosis. In this study we addressed the role of TGFβ-ALK5 signalling during the progression of fibrosis in a two-hit mouse model of murine γ-herpesvirus 68 (MHV-68) infection on the background of pre-existing bleomycin-induced pulmonary fibrosis. Assessment of total lung collagen levels in combination with ex vivo micro-computed tomography (µCT) analysis of whole lungs demonstrated that MHV-68 infection did not enhance lung collagen deposition in this two-hit model but led to a persistent and exacerbated inflammatory response. Moreover, µCT reconstruction and analysis of the two-hit model revealed distinguishing features of diffuse ground-glass opacities and consolidation superimposed on pre-existing fibrosis that were reminiscent of those observed in acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF). Virally-infected murine fibrotic lungs further displayed evidence of extensive inflammatory cell infiltration and increased levels of CCL2, TNFα, IL-1β and IL-10. Blockade of TGFβ-ALK5 signalling attenuated lung collagen accumulation in bleomycin-alone injured mice, but this anti-fibrotic effect was reduced in the presence of concomitant viral infection. In contrast, inhibition of TGFβ-ALK5 signalling in virally-infected fibrotic lungs was associated with reduced inflammatory cell aggregates and increased levels of the antiviral cytokine IFNγ. These data reveal newly identified intricacies for the TGFβ-ALK5 signalling axis in experimental lung fibrosis, with different outcomes in response to ALK5 inhibition depending on the presence of viral infection. These findings raise important considerations for the targeting of TGFβ signalling responses in the context of pulmonary fibrosis.

  3. Breast implant-associated ALK-negative anaplastic large cell lymphoma: a case report and discussion of possible pathogenesis

    PubMed Central

    George, Eva V; Pharm, John; Houston, Courtney; Al-Quran, Semar; Brian, Grey; Dong, Huijia; Hai, Wang; Reeves, Westley; Yang, Li-Jun

    2013-01-01

    Breast implant associated anaplastic large cell lymphoma (BIA-ALCL) is a recently recognized clinical entity, with only 39 well-documented cases reported worldwide, including 3 fatalities. Because of its rarity, the clinical and pathologic features of this malignancy have yet to be fully defined. Moreover, the pathogenesis of ALCL in association with textured silicone gel breast implants is poorly understood. Here we report a case of BIA-ALCL arising in a 67-year-old woman with a mastectomy due to breast cancer followed by implantation of textured silicone gel breast prosthesis. The patient presented with breast enlargement and tenderness 8 years following reconstructive surgery. MRI revealed a fluid collection surrounding the affected breast implant. Pathologic examination confirmed the presence of malignant ALCL T cells that were CD30+, CD8+, CD15+, HLA-DR+, CD25+ ALK- and p53. A diagnosis of indolent BIA-ALCL was made since tumor cells were not found outside of the capsule. Interestingly, an extensive mixed lymphocytic infiltrate and ectopic lymphoid tissue (lymphoid neogenesis) adjacent to the fibrous implant capsule were present. The patient was treated with capsulectomy and implantation of new breast prostheses. Six months later, the patient was found to have BIA-ALCL involvement of an axillary lymph node with cytogenetic evolutionof the tumor. To our knowledge, this is the sixth reported case of aggressive BIA-ALCL. Unique features of this case include the association with lymphoid neogenesis and the in vivo cytogenetic progression of the tumor. This case provides insight into the potential role of chronic inflammation and genetic instability in the pathogenesis of BIA-ALCL. PMID:23923082

  4. Breast implant-associated ALK-negative anaplastic large cell lymphoma: a case report and discussion of possible pathogenesis.

    PubMed

    George, Eva V; Pharm, John; Houston, Courtney; Al-Quran, Semar; Brian, Grey; Dong, Huijia; Hai, Wang; Reeves, Westley; Yang, Li-Jun

    2013-01-01

    Breast implant associated anaplastic large cell lymphoma (BIA-ALCL) is a recently recognized clinical entity, with only 39 well-documented cases reported worldwide, including 3 fatalities. Because of its rarity, the clinical and pathologic features of this malignancy have yet to be fully defined. Moreover, the pathogenesis of ALCL in association with textured silicone gel breast implants is poorly understood. Here we report a case of BIA-ALCL arising in a 67-year-old woman with a mastectomy due to breast cancer followed by implantation of textured silicone gel breast prosthesis. The patient presented with breast enlargement and tenderness 8 years following reconstructive surgery. MRI revealed a fluid collection surrounding the affected breast implant. Pathologic examination confirmed the presence of malignant ALCL T cells that were CD30+, CD8+, CD15+, HLA-DR+, CD25+ ALK- and p53. A diagnosis of indolent BIA-ALCL was made since tumor cells were not found outside of the capsule. Interestingly, an extensive mixed lymphocytic infiltrate and ectopic lymphoid tissue (lymphoid neogenesis) adjacent to the fibrous implant capsule were present. The patient was treated with capsulectomy and implantation of new breast prostheses. Six months later, the patient was found to have BIA-ALCL involvement of an axillary lymph node with cytogenetic evolution of the tumor. To our knowledge, this is the sixth reported case of aggressive BIA-ALCL. Unique features of this case include the association with lymphoid neogenesis and the in vivo cytogenetic progression of the tumor. This case provides insight into the potential role of chronic inflammation and genetic instability in the pathogenesis of BIA-ALCL. PMID:23923082

  5. Alterations in genes other than EGFR/ALK/ROS1 in non-small cell lung cancer: trials and treatment options

    PubMed Central

    Desai, Arpita; Menon, Smitha P.; Dy, Grace K.

    2016-01-01

    During the last decade, we have seen tremendous progress in the therapy of lung cancer. Discovery of actionable mutations in EGFR and translocations in ALK and ROS1 have identified subsets of patients with excellent tumor response to oral targeted agents with manageable side effects. In this review, we highlight treatment options including corresponding clinical trials for oncogenic alterations affecting the receptor tyrosine kinases MET, FGFR, NTRK, RET, HER2, HER3, and HER4 as well as components of the RAS-RAF-MEK signaling pathway. PMID:27144064

  6. Molecular Pathways: Resistance to Kinase Inhibitors and Implications for Therapeutic Strategies

    PubMed Central

    Lovly, Christine M.; Shaw, Alice T.

    2014-01-01

    The development of targeted therapies has revolutionized the treatment of cancer patients. The identification of ‘druggable’ oncogenic kinases and the creation of small molecule inhibitors designed to specifically target these mutant kinases has become an important therapeutic paradigm across several different malignancies. Often these inhibitors induce dramatic clinical responses in molecularly defined cohorts. However, resistance to such targeted therapies is an inevitable consequence of this therapeutic approach. Resistance can be either primary (de novo) or acquired. Mechanisms leading to primary resistance may be categorized as tumor intrinsic factors or as patient/drug specific factors. Acquired resistance may be mediated by target gene modification, activation of ‘bypass tracks’ which serve as compensatory signaling loops, or histological transformation. This brief review is a snapshot of the complex problem of therapeutic resistance, with a focus on resistance to kinase inhibitors in EGFR mutant and ALK rearranged non-small cell lung cancer, BRAF mutant melanoma, and BCR-ABL positive chronic myeloid leukemia. We will describe specific mechanisms of primary and acquired resistance and then review emerging strategies to delay or overcome drug resistance. PMID:24789032

  7. Occurrence of alk(en)ylresorcinols in the fruits of two mango (Mangifera indica L.) cultivars during on-tree maturation and postharvest storage.

    PubMed

    Kienzle, Stefanie; Carle, Reinhold; Sruamsiri, Pittaya; Tosta, Carola; Neidhart, Sybille

    2014-01-01

    Regarding their relevance for the fungal resistance of mangoes in long supply chains, the alk(en)ylresorcinols (AR) were quantitated in peel and mesocarp throughout storage (27 days, 14 °C, ethylene absorption). The 12 'Chok Anan' and 11 'Nam Dokmai #4' lots picked between 83 and 115 days after full bloom (DAFB) had different harvest maturity indices. The development of dry matter and fruit growth indicated physiological maturity ∼100 DAFB. During storage, all fruits ripened slowly, mostly until over-ripeness and visible decay. The total AR contents always ranged at 73 ± 4.5 and 6.4 ± 0.7 mg hg(-1) of 'Chok Anan' and 'Nam Dokmai #4' peel dry weight, respectively, but only at 6.7 ± 0.7 and 0.9 ± 0.1 mg hg(-1) for the corresponding mesocarp (P ≤ 0.05). These narrow concentration ranges were contradictory to the decreasing fungal resistance. Accordingly, the alk(en)ylresorcinols have not been a deciding factor for the fungal resistance.

  8. Novel corrosion inhibitor technology

    SciTech Connect

    Van de Ven, P.; Fritz, P.; Pellet, R.

    1999-11-01

    A novel, patented corrosion inhibitor technology has been identified for use in heat transfer applications such as automotive and heavy-duty coolant. The new technology is based on a low-toxic, virtually depletion-free carboxylic acid corrosion inhibitor package that performs equally well in mono ethylene glycol and in less toxic propylene glycol coolants. An aqueous inhibitor concentrate is available to provide corrosion protection where freezing protection is not an issue. In the present paper, this inhibitor package is evaluated in the different base fluids: mono ethylene glycol, mono propylene glycol and water. Results are obtained in both standardized and specific corrosion tests as well as in selected field trials. These results indicate that the inhibitor package remains effective and retains the benefits previously identified in automotive engine coolant applications: excellent corrosion protection under localized conditions, general corrosion conditions as well as at high temperature.

  9. Pathway modulators and inhibitors.

    PubMed

    Smith, John A

    2009-07-01

    Inhibitors of specific cellular pathways are useful for investigating the roles of proteins of unknown function, and for selectively inhibiting a protein in complex pathways to uncover its relationships to other proteins in this and other interacting pathways. This appendix provides links to Web sites that describe cellular processes and pathways along with the various classes of inhibitors, numerous references, downloadable diagrams, and technical tips.

  10. Update on TNF Inhibitors.

    PubMed

    Kerdel, Francisco A

    2016-06-01

    The introduction of tumor necrosis factor (TNF)-α inhibitors dramatically improved the management of psoriasis. Some newer or investigational biologics with different mechanisms of action have demonstrated noninferiority or superiority to etanercept, the first self-injectable anti-TNF-α agent to become available in the United States. Nonetheless, TNF-α inhibitors are likely to remain a mainstay of therapy for many years.

  11. Synthetic inhibitors of elastase.

    PubMed

    Edwards, P D; Bernstein, P R

    1994-03-01

    For more than two decades investigators around the world, in both academic and industrial institutions, have been developing inhibitors of human neutrophil elastase. A number of very elegant and insightful strategies have been reported. In the case of reversible peptidic inhibitors, this has resulted in the identification of some extremely potent compounds with dissociation constants in the 10(-11) M range. This is quite an accomplishment considering that these low molecular-weight inhibitors are only tri- and tetrapeptides. In the case of the heterocyclic-based inhibitors, the challenge of balancing the heterocycle's inherent reactivity and aqueous stability with the stability of the enzyme-inhibitor adduct has been meet by either using a latent, reactive functionality which is only activated within the enzyme, or by incorporating features which selectively obstruct deacylation but have little effect on the enzyme acylation step. The underlying goal of this research has been the identification of agents to treat diseases associated with HNE. Several animal models have been developed for evaluating the in vivo activity of elastase inhibitors, and compounds have been shown to be effective in all of these models by the intravenous, intratrachael or oral routes of administration. However, only a very small percentage of compounds have possessed all the necessary properties, including lack of toxicity, for progression into the clinic. The peptidyl TFMK ICI 200,880 (25-12) has many of the desired characteristics of a drug to treat the diseases associated with HNE: chemical stability, in vitro and in vivo activity, a long duration of action, and adequate metabolic stability. Currently ICI 200,880 is the only low molecular-weight HNE inhibitor known to be undergoing clinical trials, and may be the compound which finally demonstrates the clinical utility of a synthetic HNE inhibitor. PMID:8189835

  12. Linking off-target kinase pharmacology to the differential cellular effects observed among PARP inhibitors.

    PubMed

    Antolín, Albert A; Mestres, Jordi

    2014-05-30

    PARP inhibitors hold promise as a novel class of targeted anticancer drugs. However, their true mechanism of action is still not well understood following recent reports that show marked differences in cellular effects. Here, we demonstrate that three PARP drug candidates, namely, rucaparib, veliparib, and olaparib, have a clearly different in vitro affinity profile across a panel of diverse kinases selected using a computational approach that relates proteins by ligand similarity. In this respect, rucaparib inhibits nine kinases with micromolar affinity, including PIM1, PIM2, PRKD2, DYRK1A, CDK1, CDK9, HIPK2, CK2, and ALK. In contrast, olaparib does not inhibit any of the sixteen kinases tested. In between, veliparib inhibits only two, namely, PIM1 and CDK9. The differential kinase pharmacology observed among PARP inhibitors provides a plausible explanation to their different cellular effects and offers unexplored opportunities for this drug class, but alerts also on the risk associated to transferring directly both preclinical and clinical outcomes from one PARP drug candidate to another. PMID:24632590

  13. Structural Distinction of Diacyl-, Alkylacyl, and Alk-1-Enylacyl Glycerophosphocholines as [M - 15]- Ions by Multiple-Stage Linear Ion-Trap Mass Spectrometry with Electrospray Ionization

    NASA Astrophysics Data System (ADS)

    Hsu, Fong-Fu; Lodhi, Irfan J.; Turk, John; Semenkovich, Clay F.

    2014-08-01

    We describe a linear ion-trap (LIT) multiple-stage (MSn) mass spectrometric approach towards differentiation of alkylacyl, alk-1-enylacyl- and diacyl-glycerophoscholines (PCs) as the [M - 15]- ions desorbed by electrospray ionization (ESI) in the negative-ion mode. The MS4 mass spectra of the [M - 15 - R2'CH = CO]- ions originated from the three PC subfamilies are readily distinguishable, resulting in unambiguous distinction of the lipid classes. This method is applied to two alkyl ether rich PC mixtures isolated from murine bone marrow neutrophils and kidney, respectively, to explore its utility in the characterization of complex PC mixture of biological origin, resulting in the realization of the detailed structures of the PC species, including various classes and many minor isobaric isomers.

  14. Structure of the E. coli DNA Glycosylase AlkA Bound to the Ends of Duplex DNA: A System for the Structure Determination of Lesion-Containing DNA

    SciTech Connect

    Bowman, B.R.; Lee, S.; Wang, S.; Verdine, G.L.

    2008-10-24

    The constant attack on DNA by endogenous and exogenous agents gives rise to nucleobase modifications that cause mutations, which can lead to cancer. Visualizing the effects of these lesions on the structure of duplex DNA is key to understanding their biologic consequences. The most definitive method of obtaining such structures, X-ray crystallography, is troublesome to employ owing to the difficulty of obtaining diffraction-quality crystals of DNA. Here, we present a crystallization system that uses a protein, the DNA glycosylase AlkA, as a scaffold to mediate the crystallization of lesion-containing duplex DNA. We demonstrate the use of this system to facilitate the rapid structure determination of DNA containing the lesion 8-oxoguanine in several different sequence contexts, and also deoxyinosine and 1,N{sup 6}-ethenoadenine, each stabilized as the corresponding 2{prime}-flouro analog. The structures of 8-oxoguanine provide a correct atomic-level view of this important endogenous lesion in DNA.

  15. Sarcomatoid variant of ALK- anaplastic large cell lymphoma involving multiple lymph nodes and both lungs with production of proinflammatory cytokines: report of a case and review of literature

    PubMed Central

    Yu, Lu; Yan, Lin Li; Yang, Shou Jing

    2014-01-01

    Sarcomatoid variant of anaplastic large cell lymphoma (ALCL) is one of the rarest histologic variants of ALCL that consists of large, bizarre, often spindle-shaped, neoplastic cells resembling a soft tissue sarcoma. We report here such a case of ALCL with both pulmonary and multiple nodal involvement in a 47-year-old woman who initially presented with fever, cough, sputum, itching skin, and weight loss. The initial transbronchial lung biopsy showed discohesive pleomorphic malignant cells in a strong inflammatory milieu reminiscent of inflammatory malignant fibrous histiocytoma (MFH). Subsequent cervical lymph node biopsy revealed a spindle cell sarcoma predominantly composed of plump spindle and oval neoplastic cells in interweaving fascicles, with sparse inflammatory infiltrates, resembling pleomorphic-storiform type of MFH. However, these tumor cells in the lung and node lesions revealed essentially similar immunohistochemical features that were positive for CD30, EMA, TIA-1, granzyme B, and fascin, but negative for anaplastic lymphoma kinase (ALK), and T- or B-lineage-specific marker. The spindled cells stains diffuse strong positive for smooth muscle actin (SMA), along with vimentin. Further studies showed that the tumor produced large quantities of the proinflammatory cytokines interleukin-2 (IL-2), IL-6, and IL-8, which we believe may contribute to the pathogenesis of sarcomatoid transformation of this tumor, and was associated with the patient’s inflammatory symptoms. To the best of our knowledge, this is the first reported case of sarcomatoid variant of ALK-negative ALCL with null cell phenotype and in situ production of proinflammatory cytokines presenting as multiple nodes and pulmonary involvement. PMID:25197351

  16. Parallel and Competitive Pathways for Substrate Desaturation, Hydroxylation and Radical Rearrangement by the Non-heme Diiron Hydroxylase AlkB

    PubMed Central

    Cooper, Harriet L. R.; Mishra, Girish; Huang, Xiongyi; Pender-Cudlip, Marilla; Austin, Rachel N.; Shanklin, John; Groves, John T.

    2012-01-01

    A purified and highly active form of the non-heme diiron hydroxylase AlkB was investigated using the diagnostic probe substrate norcarane. The reaction afforded C2 (26%) and C3 (43%) hydroxylation and desaturation products (31%). Initial C-H cleavage at C2 led to 7% C2 hydroxylation and 19% 3-hydroxymethylcyclohexene, a rearrangement product characteristic of a radical rearrangement pathway. A deuterated substrate analog, 3,3,4,4-norcarane-d4, afforded drastically reduced amounts of C3 alcohol (8%) and desaturation products (5%), while the radical rearranged alcohol was now the major product (65%). This change in product ratios indicates a large kinetic hydrogen isotope effect of ~20 for both the C-H hydroxylation at C3 and the desaturation pathway, with all of the desaturation originating via hydrogen abstraction at C3 and not C2. The data indicates that AlkB reacts with norcarane via initial C-H hydrogen abstraction from C2 or C3 and that the three pathways, C3 hydroxylation, C3 desaturation and C2 hydroxylation/radical rearrangement, are parallel and competitive. Thus, the incipient radical at C3 either reacts with the iron-oxo center to form an alcohol or proceeds along the desaturation pathway via a second H-abstraction to afford both 2-norcarene and 3-norcarene. Subsequent reactions of these norcarenes lead to detectable amounts of hydroxylation products and toluene. By contrast, the 2-norcaranyl radical intermediate leads to C2 hydroxylation and the diagnostic radical rearrangement, but this radical apparently does not afford desaturation products. The results indicate that C-H hydroxylation and desaturation follow analogous stepwise reaction channels via carbon radicals that diverge at the product-forming step. PMID:23157204

  17. Remission of ALK-negative primary pulmonary inflammatory myofibroblastic tumor on treatment with clarithromycin: A case report and review of the literature

    PubMed Central

    WATANABE, HIDEHIRO; URUMA, TOMONORI; TAZAKI, GEN; TAJIRI, TAKUMA; KIKUCHI, RYOTA; ITOH, MASAYUKI; AOSHIBA, KAZUTETSU; NAKAMURA, HIROYUKI

    2016-01-01

    Inflammatory myofibroblastic tumors (IMTs) belong to an intermediate group of soft-tissue tumors, they are relatively rare but exhibit a wide range of pathologies, from benign to malignant. At present, no standard treatment has been established, however, it is known to be important to determine the grade of malignancy of the tumor, prior to treatment. The present study reports a 73-year-old female patient with no clinical manifestations, who, when examined radiographically at a health check exhibited bilateral thoracic infiltrative shadows and nodular shadows by chest CT. A metastatic tumor or an organizing pneumonia were suspected. Blood examination showed no abnormal findings, and a pathological diagnosis of IMT was given from the histological findings of the tissue extracted by video-assisted thoracic surgery. Histological analysis established the lack of expression of anaplastic lymphoma kinase (ALK1) and immunoglobulin subtype G4 (IgG4). Alteration of the radiological shadows was observed over several weeks, and after concluding that chronic inflammation was worsening the patient's condition, clarithromycin was administered as a long-term macrolide therapy. The IMT decreased in size, and eight months later it had almost resolved. The patient was last reported to be maintaining a stable condition with no relapse. Some IMT cases have malignant pathology, and should be carefully followed-up. However, in the present case, where the IMT is both ALK1-negative and IgG4-negative, its biological immune responsiveness appears to differ from positive cases, and an inflammatory response was predominant. Clarithromycin, has immunomodulatory and anti-inflammatory effects and appeared to be effective in treating the IMT of the patient in the present study. PMID:26998073

  18. Inhibitors for the bacterial ectonucleotidase Lp1NTPDase from Legionella pneumophila.

    PubMed

    Fiene, Amelie; Baqi, Younis; Malik, Enas M; Newton, Patrice; Li, Wenjin; Lee, Sang-Yong; Hartland, Elizabeth L; Müller, Christa E

    2016-09-15

    Legionella pneumophila is an aerobic, Gram-negative bacterium of the genus Legionella, which constitutes the major causative agent of Legionnaires' disease. Recently a nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila was identified and termed Lp1NTPDase; it was found to be a structural and functional homolog of mammalian NTPDases catalyzing the hydrolysis of ATP to ADP and ADP to AMP. Its activity is believed to contribute to the virulence of Legionella pneumophila. Therefore Lp1NTPDase inhibitors are considered as novel antibacterial drugs. However, only weakly potent compounds are available so far. In the present study, a capillary electrophoresis (CE)-based enzyme assay for monitoring the Lp1NTPDase activity was established. The enzymatic reaction was performed in a test tube followed by separation of substrate and products by CE and subsequent quantification by UV analysis. After kinetic characterization of the enzyme, a series of 1-amino-4-ar(alk)ylamino-2-sulfoanthraquinone derivatives structurally related to the anthraquinone dye Reactive Blue 2, a non-selective ecto-NTPDase inhibitor, was investigated for inhibitory activity on Lp1NTPDase using the CE-based enzyme assay. Derivatives bearing a large lipophilic substituent (e.g., fused aromatic rings) in the 4-position of the 1-amino-2-sulfoanthraquinone showed the highest inhibitory activity. Compounds with IC50 values in the low micromolar range were identified. The most potent inhibitor was 1-amino-4-[phenanthrene-9-yl-amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (28, PSB-16131), with an IC50-value of 4.24μM. It represents the most potent Lp1NTPDase inhibitor described to date. These findings may serve as a starting point for further optimization. Lp1NTPDase inhibition provides a novel approach for the (immuno)therapy of Legionella infections. PMID:27522579

  19. Small-molecule caspase inhibitors

    NASA Astrophysics Data System (ADS)

    Zhenodarova, S. M.

    2010-02-01

    The review considers low-molecular weight inhibitors of caspases, cysteine proteases being key contributors to apoptosis (programmed cell death). The inhibitors with aspartic acid residues or various heterocyclic systems (both synthetic and natural) are covered. Their possible mechanisms of action are discussed. Data on inhibitor structure-activity relationship studies are systematically surveyed. The interactions of the non-peptide fragments of an inhibitor with the enzymes are examined. Examples of the use of some inhibitors for apoptosis suppression are provided.

  20. Natural inhibitors of thrombin.

    PubMed

    Huntington, James A

    2014-04-01

    The serine protease thrombin is the effector enzyme of blood coagulation. It has many activities critical for the formation of stable clots, including cleavage of fibrinogen to fibrin, activation of platelets and conversion of procofactors to active cofactors. Thrombin carries-out its multiple functions by utilising three special features: a deep active site cleft and two anion binding exosites (exosite I and II). Similarly, thrombin inhibitors have evolved to exploit the unique features of thrombin to achieve rapid and specific inactivation of thrombin. Exogenous thrombin inhibitors come from several different protein families and are generally found in the saliva of haematophagous animals (blood suckers) as part of an anticoagulant cocktail that allows them to feed. Crystal structures of several of these inhibitors reveal how peptides and proteins can be targeted to thrombin in different and interesting ways. Thrombin activity must also be regulated by endogenous inhibitors so that thrombi do not occlude blood flow and cause thrombosis. A single protein family, the serpins, provides all four of the endogenous thrombin inhibitors found in man. The crystal structures of these serpins bound to thrombin have been solved, revealing a similar exosite-dependence on complex formation. In addition to forming the recognition complex, serpins destroy the structure of thrombin, allowing them to be released from cofactors and substrates for clearance. This review examines how the special features of thrombin have been exploited by evolution to achieve inhibition of the ultimate coagulation protease.

  1. SGLT2 inhibitors.

    PubMed

    Dardi, I; Kouvatsos, T; Jabbour, S A

    2016-02-01

    Diabetes mellitus is a serious health issue and an economic burden, rising in epidemic proportions over the last few decades worldwide. Although several treatment options are available, only half of the global diabetic population achieves the recommended or individualized glycemic targets. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic agents with a novel insulin-independent action. SGLT2 is a transporter found in the proximal renal tubules, responsible for the reabsorption of most of the glucose filtered by the kidney. Inhibition of SGLT2 lowers the blood glucose level by promoting the urinary excretion of excess glucose. Due to their insulin-independent action, SGLT2 inhibitors can be used with any degree of beta-cell dysfunction or insulin resistance, related to a very low risk of hypoglycemia. In addition to improving glycemic control, SGLT2 inhibitors have been associated with a reduction in weight and blood pressure when used as monotherapy or in combination with other antidiabetic agents in patients with type 2 diabetes mellitus (T2DM). Treatment with SGLT2 inhibitors is usually well tolerated; however, they have been associated with an increased incidence of urinary tract and genital infections, although these infections are usually mild and easy to treat. SGLT2 inhibitors are a promising new option in the armamentarium of drugs for patients with T2DM. PMID:26362302

  2. Acquired resistance of pancreatic cancer cells to treatment with gemcitabine and HER-inhibitors is accompanied by increased sensitivity to STAT3 inhibition

    PubMed Central

    IOANNOU, NIKOLAOS; SEDDON, ALAN M.; DALGLEISH, ANGUS; MACKINTOSH, DAVID; SOLCA, FLAVIO; MODJTAHEDI, HELMOUT

    2016-01-01

    . In addition, treatment with a combination of afatinib with either c-MET inhibitor Crizotinib or Stattic resulted in an additive or synergistic growth inhibition in all three variants. Our results suggest that activation of STAT3 may play an important role in the acquisition of resistance to gemcitabine and HER inhibitors in pancreatic cancer and warrant further studies on the therapeutic potential of STAT3 inhibitors in such a setting. PMID:26781210

  3. Cholinesterase inhibitors from botanicals

    PubMed Central

    Ahmed, Faiyaz; Ghalib, Raza Murad; Sasikala, P.; Ahmed, K. K. Mueen

    2013-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease, wherein a progressive loss of cholinergic synapses occurs in hippocampus and neocortex. Decreased concentration of the neurotransmitter, acetylcholine (ACh), appears to be critical element in the development of dementia, and the most appropriate therapeutic approach to treat AD and other form of dementia is to restore acetylcholine levels by inhibiting both major form of cholinesterase: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Consequently, researches have focused their attention towards finding cholinesterase inhibitors from natural products. A large number of such inhibitors have been isolated from medicinal plants. This review presents a comprehensive account of the advances in field of cholinesterase inhibitor phytoconstituents. The structures of some important phytoconstituents (collected through www.Chemspider.com) are also presented and the scope for future research is discussed. PMID:24347920

  4. Cholinesterase inhibitors from botanicals.

    PubMed

    Ahmed, Faiyaz; Ghalib, Raza Murad; Sasikala, P; Ahmed, K K Mueen

    2013-07-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease, wherein a progressive loss of cholinergic synapses occurs in hippocampus and neocortex. Decreased concentration of the neurotransmitter, acetylcholine (ACh), appears to be critical element in the development of dementia, and the most appropriate therapeutic approach to treat AD and other form of dementia is to restore acetylcholine levels by inhibiting both major form of cholinesterase: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Consequently, researches have focused their attention towards finding cholinesterase inhibitors from natural products. A large number of such inhibitors have been isolated from medicinal plants. This review presents a comprehensive account of the advances in field of cholinesterase inhibitor phytoconstituents. The structures of some important phytoconstituents (collected through www.Chemspider.com) are also presented and the scope for future research is discussed. PMID:24347920

  5. Phosphodiesterase-5 inhibitors.

    PubMed

    Cockrill, Barbara A; Waxman, Aaron B

    2013-01-01

    Nitric oxide (NO) signaling plays a key role in modulating vascular tone and remodeling in the pulmonary circulation. The guanylate cyclase/cyclic guanylate monophosphate-signaling pathway primarily mediates nitric oxide signaling. This pathway is critical in normal regulation of the pulmonary vasculature, and is an important target for therapy in patients with pulmonary hypertension. In the pulmonary vasculature, degradation of cGMP is primarily regulated by PDE-5, and inhibition of this enzyme has important effects on pulmonary vasculature smooth muscle tone. Large randomized placebo-controlled trials of PDE-5 inhibitors demonstrated improved exercise capacity, hemodynamics and quality of life in adult patients with PAH. This chapter will discuss the mechanisms of NO signaling in the vasculature, characteristics of the PDE5-inhibitors approved for treatment of PH, and review available data on the use of phosphodiesterase inhibitors in PH. PMID:24092343

  6. Origin and palaeoenvironmental significance of C25 and C27n-alk-1-enes in a 25,000-year lake-sedimentary record from equatorial East Africa

    NASA Astrophysics Data System (ADS)

    van Bree, L. G. J.; Rijpstra, W. I. C.; Cocquyt, C.; Al-Dhabi, N. A.; Verschuren, D.; Sinninghe Damsté, J. S.; de Leeuw, J. W.

    2014-11-01

    We studied the distribution of long-chain alkenes (n-C23 to n-C31) in well-dated sediments from Lake Challa, a deep crater lake near Mt. Kilimanjaro in equatorial East Africa, to reveal signatures of palaeo-environmental and palaeo-climatic changes affecting the production of these compounds during the last 25 kyr. The apolar fractions of organic sediment extracts dated to the last 16 kyr showed an unusual dominance of δ13C-depleted n-C25:1 and n-C27:1 alk-1-enes. These alkenes were not detected in soil and litter from near the shoreline and from the inner rim of the crater, pointing to an autochthonous, aquatic source. Analysis of suspended particulate matter indicated that the n-alk-1-enes are produced in the well-oxygenated upper 30 m of the water column, indicating a phytoplanktonic origin. Sedimenting particles collected monthly from December 2006 to November 2007 showed increased fluxes of n-alk-1-enes following the locally prominent short rain season in November-December. Green algae and/or cyanobacteria were identified as candidate sources of these alkenes. Production of the n-C25:1 and n-C27:1 alkenes in Lake Challa was much reduced during the Last Glacial Maximum and early late-glacial period, suggesting a temperature or CO2 effect on habitat suitability. We explored the potential of n-alk-1-ene accumulation rates, and of a derived Alkene Index [n-C27:1]/([n-C25:1] + [n-C27:1]), to record longer-term climatic changes. The Alkene Index record of Lake Challa over the past 25 kyr shows clear periodicity with a dominant frequency of ∼2.3 kyr, potentially indicative of monsoon variability directly or indirectly forced by variation in solar radiation.

  7. Pectin methylesterase inhibitor.

    PubMed

    Giovane, A; Servillo, L; Balestrieri, C; Raiola, A; D'Avino, R; Tamburrini, M; Ciardiello, M A; Camardella, L

    2004-02-12

    Pectin methylesterase (PME) is the first enzyme acting on pectin, a major component of plant cell wall. PME action produces pectin with different structural and functional properties, having an important role in plant physiology. Regulation of plant PME activity is obtained by the differential expression of several isoforms in different tissues and developmental stages and by subtle modifications of cell wall local pH. Inhibitory activities from various plant sources have also been reported. A proteinaceous inhibitor of PME (PMEI) has been purified from kiwi fruit. The kiwi PMEI is active against plant PMEs, forming a 1:1 non-covalent complex. The polypeptide chain comprises 152 amino acid residues and contains five Cys residues, four of which are connected by disulfide bridges, first to second and third to fourth. The sequence shows significant similarity with the N-terminal pro-peptides of plant PME, and with plant invertase inhibitors. In particular, the four Cys residues involved in disulfide bridges are conserved. On the basis of amino acid sequence similarity and Cys residues conservation, a large protein family including PMEI, invertase inhibitors and related proteins of unknown function has been identified. The presence of at least two sequences in the Arabidopsis genome having high similarity with kiwi PMEI suggests the ubiquitous presence of this inhibitor. PMEI has an interest in food industry as inhibitor of endogenous PME, responsible for phase separation and cloud loss in fruit juice manufacturing. Affinity chromatography on resin-bound PMEI can also be used to concentrate and detect residual PME activity in fruit and vegetable products.

  8. Acyclic peptide inhibitors of amylases.

    PubMed

    Pohl, Nicola

    2005-12-01

    In this issue of Chemistry and Biology, a library screening approach reveals a linear octapeptide inhibitor of alpha-amylases reached by de novo design . The selected molecule shares characteristics with naturally occurring protein inhibitors -- a result that suggests general rules for the design of peptide-based amylase inhibitors may be achievable.

  9. [JAK2 inhibitors].

    PubMed

    Hernández Boluda, Juan Carlos; Gómez, Montse; Pérez, Ariadna

    2016-07-15

    Pharmacological inhibition of the kinase activity of JAK proteins can interfere with the signaling of immunomodulatory cytokines and block the constitutive activation of the JAK-STAT pathway that characterizes certain malignancies, including chronic myeloproliferative neoplasms. JAK inhibitors may, therefore, be useful to treat malignancies as well as inflammatory or immune disorders. Currently, the most significant advances have been made in the treatment of myelofibrosis, where these drugs may lead to a remarkable improvement in the control of hyperproliferative manifestations. However, available data suggest that this treatment is not curative of myelofibrosis. In general, JAK2 inhibition induces cytopaenias, with this being considered a class side-effect. By contrast, the extrahaematologic toxicity profile varies significantly among the different JAK inhibitors. At present, there are several clinical trials evaluating the combination of ruxolitinib with other drugs, in order to improve its therapeutic activity as well as reducing haematologic toxicity. PMID:27033437

  10. Coagulation inhibitors in inflammation.

    PubMed

    Esmon, C T

    2005-04-01

    Coagulation is triggered by inflammatory mediators in a number of ways. However, to prevent unwanted clot formation, several natural anticoagulant mechanisms exist, such as the antithrombin-heparin mechanism, the tissue factor pathway inhibitor mechanism and the protein C anticoagulant pathway. This review examines the ways in which these pathways are down-regulated by inflammation, thus limiting clot formation and decreasing the natural anti-inflammatory mechanisms that these pathways possess. PMID:15787615

  11. Discovery of 7-methoxy-6-[4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-5-yl]-1,3-benzothiazole (TASP0382088): a potent and selective transforming growth factor-β type I receptor inhibitor as a topical drug for alopecia.

    PubMed

    Amada, Hideaki; Asanuma, Hajime; Koami, Takeshi; Okada, Atsushi; Endo, Mayumi; Ueda, Yasuji; Naruse, Takumi; Ikeda, Akiko

    2013-01-01

    7-Methoxy-6-[4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-5-yl]-1,3-benzothiazole 11 (TASP0382088) was synthesized and evaluated as transforming growth factor-β (TGF-β) type I receptor (also known as activin receptor-like kinase 5 or ALK5) inhibitor. Compound 11, a potent and selective ALK5 inhibitor, exhibited good enzyme inhibitory activity (IC50=4.8 nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC50=17 nM). The introduction of a methoxy group to the benzothiazole ring in 1 and the break up of the planarity between the imidazole ring and the thiazole ring improved the solubility in the lotion base of 11. Furthermore, the topical application of 3% 11 lotion significantly inhibited Smad2 phosphorylation in mouse skin at 8 h after application (71% inhibition, compared with vehicle-treated animals).

  12. Neutrophil Elastase Inhibitors

    PubMed Central

    Groutas, William C.; Dou, Dengfeng; Alliston, Kevin R.

    2011-01-01

    Introduction Chronic obstructive pulmonary disease (COPD) constitutes a worldwide health problem. There is currently an urgent and unmet need for the development of small molecule therapeutics capable of blocking and/or reversing the progression of the disorder. Recent studies have greatly illuminated our understanding of the multiple pathogenic processes associated with COPD. Of paramount importance is the key role played by proteases, oxidative stress, apoptosis, and inflammation. Insights gained from these studies have made possible the exploration of new therapeutic approaches. Areas covered An overview of major developments in COPD research with emphasis on low molecular weight neutrophil elastase inhibitors is described in this review. Expert opinion Great strides have been made toward our understanding of the biochemical and cellular events associated with COPD. However, our knowledge regarding the inter-relationships among the multiple pathogenic mechanisms and their mediators involved is till limited. The problem is further compounded by the unavailability of suitable validated biomarkers for assessing the efficacy of potential therapeutic interventions. The complexity of COPD suggests that effective therapeutic interventions may require the administration of more than one agent such as, for instance, an HNE or MMP-12 inhibitor with an anti-inflammatory agent such as a phosphodiesterase-4 inhibitor, or a dual function agent capable of disrupting the cycle of proteolysis, apoptosis, inflammation and oxidative stress PMID:21235378

  13. Mutant form C115H of Clostridium sporogenes methionine γ-lyase efficiently cleaves S-Alk(en)yl-l-cysteine sulfoxides to antibacterial thiosulfinates.

    PubMed

    Kulikova, Vitalia V; Anufrieva, Natalya V; Revtovich, Svetlana V; Chernov, Alexander S; Telegin, Georgii B; Morozova, Elena A; Demidkina, Tatyana V

    2016-10-01

    Pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) catalyzes the β-elimination reaction of S-alk(en)yl-l-cysteine sulfoxides to thiosulfinates, which possess antimicrobial activity. Partial inactivation of the enzyme in the course of the reaction occurs due to oxidation of active site cysteine 115 conserved in bacterial MGLs. In this work, the C115H mutant form of Clostridium sporogenes MGL was prepared and the steady-state kinetic parameters of the enzyme were determined. The substitution results in an increase in the catalytic efficiency of the mutant form towards S-substituted l-cysteine sulfoxides compared to the wild type enzyme. We used a sulfoxide/enzyme system to generate antibacterial activity in situ. Two-component systems composed of the mutant enzyme and three S-substituted l-cysteine sulfoxides were demonstrated to be effective against Gram-positive and Gram-negative bacteria and three clinical isolates from mice. © 2016 IUBMB Life, 68(10):830-835, 2016. PMID:27647488

  14. Alk-1-enylacyl, alkylacyl, and diacyl subclasses of native ethanolamine and choline glycerophospholipids can be quantified directly by phosphorus-31 NMR in solution.

    PubMed

    Malewicz, B; Baumann, W J

    1996-11-01

    We show that phosphorus-31 nuclear magnetic resonance spectroscopy can be used to distinguish and to quantify the alk-1-enylacyl, alkylacyl, and diacyl glycerophosphoethanolamine (GPE) subclasses, and the respective glycerophosphocholine (GPC) subclasses, in their native form without prior degradation or derivatization, provided the phospholipids are observed in the nonaggregated state. Monomeric phospholipid distribution is ascertained by recording the spectra, after removal of metal ions, on CDCl3/CD3OD/D2O (50:50:15, by vol) solutions. The utility of this approach is exemplified for the ethanolamine glycerophospholipids (EPL) from bovine brain and the choline glycerophospholipids (CPL) from bovine heart. Sharp and well-resolved resonances are obtained for alkylacylGPE (+0.395 ppm; re 1% H3PO4), alkenylacylGPE (+0.353 ppm), and diacylGPE (+0.315 ppm), and for alkylacylGPC (-0.383 ppm), alkenyl-acylGPC (-0.436 ppm) and diacylGPC (-0.451 ppm). Integrated peak areas are shown to closely correlate with dose. Accurate quantitation of EPL and CPL subclasses at submicromolar levels can further be facilitated by use of synthetic dialkylGPE (+0.602 ppm) and dialkylGPC (-0.196 ppm) as internal standards. The method is simple, rapid, sensitive and reproducible, and permits the complete resolution and direct quantitation of all ethanolamine and choline glycerophospholipid subclasses quite independent of fatty chain length and degree of unsaturation. PMID:8934452

  15. Mutant form C115H of Clostridium sporogenes methionine γ-lyase efficiently cleaves S-Alk(en)yl-l-cysteine sulfoxides to antibacterial thiosulfinates.

    PubMed

    Kulikova, Vitalia V; Anufrieva, Natalya V; Revtovich, Svetlana V; Chernov, Alexander S; Telegin, Georgii B; Morozova, Elena A; Demidkina, Tatyana V

    2016-10-01

    Pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) catalyzes the β-elimination reaction of S-alk(en)yl-l-cysteine sulfoxides to thiosulfinates, which possess antimicrobial activity. Partial inactivation of the enzyme in the course of the reaction occurs due to oxidation of active site cysteine 115 conserved in bacterial MGLs. In this work, the C115H mutant form of Clostridium sporogenes MGL was prepared and the steady-state kinetic parameters of the enzyme were determined. The substitution results in an increase in the catalytic efficiency of the mutant form towards S-substituted l-cysteine sulfoxides compared to the wild type enzyme. We used a sulfoxide/enzyme system to generate antibacterial activity in situ. Two-component systems composed of the mutant enzyme and three S-substituted l-cysteine sulfoxides were demonstrated to be effective against Gram-positive and Gram-negative bacteria and three clinical isolates from mice. © 2016 IUBMB Life, 68(10):830-835, 2016.

  16. Integrating In Silico Prediction Methods, Molecular Docking, and Molecular Dynamics Simulation to Predict the Impact of ALK Missense Mutations in Structural Perspective

    PubMed Central

    Priya Doss, C. George; Chen, Luonan

    2014-01-01

    Over the past decade, advancements in next generation sequencing technology have placed personalized genomic medicine upon horizon. Understanding the likelihood of disease causing mutations in complex diseases as pathogenic or neutral remains as a major task and even impossible in the structural context because of its time consuming and expensive experiments. Among the various diseases causing mutations, single nucleotide polymorphisms (SNPs) play a vital role in defining individual's susceptibility to disease and drug response. Understanding the genotype-phenotype relationship through SNPs is the first and most important step in drug research and development. Detailed understanding of the effect of SNPs on patient drug response is a key factor in the establishment of personalized medicine. In this paper, we represent a computational pipeline in anaplastic lymphoma kinase (ALK) for SNP-centred study by the application of in silico prediction methods, molecular docking, and molecular dynamics simulation approaches. Combination of computational methods provides a way in understanding the impact of deleterious mutations in altering the protein drug targets and eventually leading to variable patient's drug response. We hope this rapid and cost effective pipeline will also serve as a bridge to connect the clinicians and in silico resources in tailoring treatments to the patients' specific genotype. PMID:25054154

  17. Improving the bioavailability and anticancer effect of the PCA-1/ALKBH3 inhibitor HUHS015 using sodium salt.

    PubMed

    Mabuchi, Miyuki; Shimizu, Tadashi; Ueda, Masahiro; Sasakawa, Yuka; Nakao, Syuhei; Ueda, Yuko; Kawamura, Akio; Tsujikawa, Kazutake; Tanaka, Akito

    2015-01-01

    Prostate cancer antigen (PCA)-1/AlkB homologue 3 (ALKBH3) has been identified as a clinically significant factor and siRNA of PCA-1 inhibits DU145 proliferation both in vitro and in vivo. HUHS015 ( 1: ), a previous reported PCA-1 small-molecule inhibitor, was also effective without any obvious side-effects or toxicity. The potency of HUHS015, however, is not satisfying. We thought the reason is poor solubility of HUHS015 because insoluble material remained at the injection site after subcutaneous administration. To improve this inhibitor's solubility, we prepared various salts of HUHS015 and examined their solubility, which resulted in the selection of HUHS015 sodium salt ( 2: ) for further studies in vivo. Next, we compared the pharmacokinetics of 1: and 2: via several administration routes. We observed significant improvements in the pharmacokinetic parameters. For example, subcutaneous administration of 2: increased the area under the curve (AUC)0-24 by 8-fold compared to 1 and increased the suppressive effect on the proliferation of DU145 cells in a xenograft model.

  18. Protein farnesyltransferase inhibitors.

    PubMed

    Ayral-Kaloustian, Semiramis; Salaski, Edward J

    2002-05-01

    Specific mutations in the ras gene impair the guanosine triphophatase (GTPase) activity of Ras proteins, which play a fundamental role in the signaling cascade, leading to uninterrupted growth signals and to the transformation of normal cells into malignant phenotypes. It has been shown that normal cells transfected with mutant ras gene become cancerous and that unfarnesylated, cytosolic mutant Ras protein does not anchor onto cell membranes and cannot induce this transformation. Posttranslational modification and plasma membrane association of mutant Ras is necessary for this transforming activity. Since its identification, the enzyme protein farnesyltransferase (FTase) that catalyzes the first and essential step of the three Ras-processing steps has emerged as the most promising target for therapeutic intervention. FTase has been implicated as a potential target in inhibiting the prenylation of a variety of proteins, thus in controlling varied disease states (e.g. cancer, neurofibromatosis, restenosis, viral hepatitis, bone resorption, parasitic infections, corneal inflammations, and diabetes) associated with prenyl modifications of Ras and other proteins. Furthermore, it has been suggested that FTase inhibitors indirectly help in inhibiting tumors via suppression of angiogenesis and induction of apoptosis. Major milestones have been achieved with small-molecule FTase inhibitors that show efficacy without toxicity in vitro, as well as in mouse models bearing ras-dependent tumors. With the determination of the crystal structure of mammalian FTase, existent leads have been fine-tuned and new potent molecules of diverse structural classes have been designed. A few of these molecules are currently in the clinic, with at least three drug candidates in Phase II studies and one in Phase III. This article will review the progress that has been reported with FTase inhibitors in drug discovery and in the clinic. PMID:12733981

  19. Synthesis of Lysine Methyltransferase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  20. High performance oilfield scale inhibitors

    SciTech Connect

    Duccini, Y.; Dufour, A.; Hann, W.M.; Sanders, T.W.; Weinstein, B.

    1997-08-01

    Sea water often reacts with the formation water in offshore fields to produce barium, calcium and strontium sulfate deposits that hinder oil production. Newer fields often have more difficult to control scale problems than older ones, and current technology scale inhibitors are not able to control the deposits as well as needed. In addition, ever more stringent regulations designed to minimize the impact of inhibitors on the environment are being enacted. Three new inhibitors are presented that overcome many of the problems of older technology scale inhibitors.

  1. Osteocompatibility of Biofilm Inhibitors

    PubMed Central

    Rawson, Monica; Haggard, Warren; Jennings, Jessica A

    2014-01-01

    The demand for infection prevention therapies has led to the discovery of several biofilm inhibitors. These inhibiting signals are released by bacteria, fungi, or marine organisms to signal biofilm dispersal or disruption in Gram-positive, Gram-negative, and fungal microorganisms. The purpose of this study was to test the biocompatibility of five different naturally-produced biofilm chemical dispersal and inhibition signals with osteoblast-like cells: D-amino acids (D-AA), lysostaphin (LS), farnesol, cis-2-decenoic acid (C2DA), and desformyl flustrabromine (dFBr). In this preliminary study, compatibility of these anti-biofilm agents with differentiating osteoblasts was examined over a 21 days period at levels above and below concentrations active against bacterial biofilm. Anti-biofilm compounds listed above were serially diluted in osteogenic media and added to cultures of MC3T3 cells. Cell viability and cytotoxicity, after exposure to each anti-biofilm agent, were measured using a DNA assay. Differentiation characteristics of osteoblasts were determined qualitatively by observing staining of mineral deposits and quantitatively with an alkaline phosphatase assay. D-AA, LS, and C2DA were all biocompatible within the reported biofilm inhibitory concentration ranges and supported osteoblast differentiation. Farnesol and dFBr induced cytotoxic responses within the reported biofilm inhibitory concentration range and low doses of dFBr were found to inhibit osteoblast differentiation. At high concentrations, such as those that may be present after local delivery, many of these biofilm inhibitors can have effects on cellular viability and osteoblast function. Concentrations at which negative effects on osteoblasts occur should serve as upper limits for delivery to orthopaedic trauma sites and guide development of these potential therapeutics for orthopaedics. PMID:25505496

  2. Osteocompatibility of biofilm inhibitors.

    PubMed

    Rawson, Monica; Haggard, Warren; Jennings, Jessica A

    2014-01-01

    The demand for infection prevention therapies has led to the discovery of several biofilm inhibitors. These inhibiting signals are released by bacteria, fungi, or marine organisms to signal biofilm dispersal or disruption in Gram-positive, Gram-negative, and fungal microorganisms. The purpose of this study was to test the biocompatibility of five different naturally-produced biofilm chemical dispersal and inhibition signals with osteoblast-like cells: D-amino acids (D-AA), lysostaphin (LS), farnesol, cis-2-decenoic acid (C2DA), and desformyl flustrabromine (dFBr). In this preliminary study, compatibility of these anti-biofilm agents with differentiating osteoblasts was examined over a 21 days period at levels above and below concentrations active against bacterial biofilm. Anti-biofilm compounds listed above were serially diluted in osteogenic media and added to cultures of MC3T3 cells. Cell viability and cytotoxicity, after exposure to each anti-biofilm agent, were measured using a DNA assay. Differentiation characteristics of osteoblasts were determined qualitatively by observing staining of mineral deposits and quantitatively with an alkaline phosphatase assay. D-AA, LS, and C2DA were all biocompatible within the reported biofilm inhibitory concentration ranges and supported osteoblast differentiation. Farnesol and dFBr induced cytotoxic responses within the reported biofilm inhibitory concentration range and low doses of dFBr were found to inhibit osteoblast differentiation. At high concentrations, such as those that may be present after local delivery, many of these biofilm inhibitors can have effects on cellular viability and osteoblast function. Concentrations at which negative effects on osteoblasts occur should serve as upper limits for delivery to orthopaedic trauma sites and guide development of these potential therapeutics for orthopaedics. PMID:25505496

  3. Biological abatement of cellulase inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bio-abatement uses a fungus to metabolize and remove fermentation inhibitors. To determine whether bio-abatement could alleviate enzyme inhibitor effects observed in biomass liquors after pretreatment, corn stover at 10% (w/v) solids was pretreated with either dilute acid or liquid hot water. The ...

  4. Anthranilamide inhibitors of factor Xa.

    PubMed

    Mendel, David; Marquart, Angela L; Joseph, Sajan; Waid, Philip; Yee, Ying K; Tebbe, Anne Louise; Ratz, Andrew M; Herron, David K; Goodson, Theodore; Masters, John J; Franciskovich, Jeffry B; Tinsley, Jennifer M; Wiley, Michael R; Weir, Leonard C; Kyle, Jeffrey A; Klimkowski, Valentine J; Smith, Gerald F; Towner, Richard D; Froelich, Larry L; Buben, John; Craft, Trelia J

    2007-09-01

    SAR about the B-ring of a series of N(2)-aroyl anthranilamide factor Xa (fXa) inhibitors is described. B-ring o-aminoalkylether and B-ring p-amine probes of the S1' and S4 sites, respectively, afforded picomolar fXa inhibitors that performed well in in vitro anticoagulation assays.

  5. Proteinaceous alpha-amylase inhibitors.

    PubMed

    Svensson, Birte; Fukuda, Kenji; Nielsen, Peter K; Bønsager, Birgit C

    2004-02-12

    Proteins that inhibit alpha-amylases have been isolated from plants and microorganisms. These inhibitors can have natural roles in the control of endogenous alpha-amylase activity or in defence against pathogens and pests; certain inhibitors are reported to be antinutritional factors. The alpha-amylase inhibitors belong to seven different protein structural families, most of which also contain evolutionary related proteins without inhibitory activity. Two families include bifunctional inhibitors acting both on alpha-amylases and proteases. High-resolution structures are available of target alpha-amylases in complex with inhibitors from five families. These structures indicate major diversity but also some similarity in the structural basis of alpha-amylase inhibition. Mutational analysis of the mechanism of inhibition was performed in a few cases and various protein engineering and biotechnological approaches have been outlined for exploitation of the inhibitory function. PMID:14871655

  6. Authentic HIV-1 integrase inhibitors

    PubMed Central

    Liao, Chenzhong; Marchand, Christophe; Burke, Terrence R; Pommier, Yves; Nicklaus, Marc C

    2010-01-01

    HIV-1 integrase (IN) is indispensable for HIV-1 replication and has become a validated target for developing anti-AIDS agents. In two decades of development of IN inhibition-based anti-HIV therapeutics, a significant number of compounds were identified as IN inhibitors, but only some of them showed antiviral activity. This article reviews a number of patented HIV-1 IN inhibitors, especially those that possess high selectivity for the strand transfer reaction. These compounds generally have a polar coplanar moiety, which is assumed to chelate two magnesium ions in the binding site. Resistance to those compounds, when given to patients, can develop as a result of IN mutations. We refer to those compounds as authentic IN inhibitors. Continued drug development has so far delivered one authentic IN inhibitor to the market (raltegravir in 2007). Current and future attention will be focused on the development of novel authentic IN inhibitors with the goal of overcoming viral resistance. PMID:21426159

  7. Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor.

    PubMed Central

    Boudier, C; Bieth, J G

    1994-01-01

    N-chlorosuccinimide oxidizes one of the methionine residues of mucus proteinase inhibitor with a second-order rate constant of 1.5 M-1.s-1. Cyanogen bromide cleavage and NH2-terminal sequencing show that the modified residue is methionine-73, the P'1 component of the inhibitor's active centre. Oxidation of the inhibitor decreases its neutrophil elastase inhibitory capacity but does not fully abolish it. The kinetic parameters describing the elastase-oxidized inhibitor interaction are: association rate constant kass. = 2.6 x 10(5) M-1.s-1, dissociation rate constant kdiss. = 2.9 x 10(-3) s-1 and equilibrium dissociation constant Ki = 1.1 x 10(-8) M. Comparison with the native inhibitor indicates that oxidation decreases kass. by a factor of 18.8 and increases kdiss. by a factor of 6.4, and therefore leads to a 120-fold increase in Ki. Yet, the oxidized inhibitor may still act as a potent elastase inhibitor in the upper respiratory tract where its concentration is 500-fold higher than Ki, i.e. where the elastase inhibition is pseudo-irreversible. Experiments in vitro with fibrous human lung elastin, the most important natural substrate of elastase, support this view: 1.35 microM elastase is fully inhibited by 5-6 microM oxidized inhibitor whether the enzyme-inhibitor complex is formed in the presence or absence of elastin and whether elastase is pre-adsorbed on elastin or not. PMID:7945266

  8. Flavivirus Entry Inhibitors.

    PubMed

    Wang, Qing-Yin; Shi, Pei-Yong

    2015-09-11

    Many flaviviruses are significant human pathogens that are transmitted by mosquitoes and ticks. Although effective vaccines are available for yellow fever virus, Japanese encephalitic virus, and tick-borne encephalitis virus, these and other flaviviruses still cause thousands of human deaths and millions of illnesses each year. No clinically approved antiviral therapy is available for flavivirus treatment. To meet this unmet medical need, industry and academia have taken multiple approaches to develop antiflavivirus therapy, among which targeting viral entry has been actively pursued in the past decade. Here we review the current knowledge of flavivirus entry and its use for small molecule drug discovery. Inhibitors of two major steps of flaviviral entry have been reported: (i) molecules that block virus-receptor interaction; (ii) compounds that prevent conformational change of viral envelope protein during virus-host membrane fusion. We also discuss the advantages and disadvantages of targeting viral entry for treatment of flavivirus infection as compared to targeting viral replication proteins. PMID:27617926

  9. Small molecules inhibitors of plasminogen activator inhibitor-1 - an overview.

    PubMed

    Rouch, Anne; Vanucci-Bacqué, Corinne; Bedos-Belval, Florence; Baltas, Michel

    2015-03-01

    PAI-1, a glycoprotein from the serpin family and the main inhibitor of tPA and uPA, plays an essential role in the regulation of intra and extravascular fibrinolysis by inhibiting the formation of plasmin from plasminogen. PAI-1 is also involved in pathological processes such as thromboembolic diseases, atherosclerosis, fibrosis and cancer. The inhibition of PAI-1 activity by small organic molecules has been observed in vitro and with some in vivo models. Based on these findings, PAI-1 appears as a potential therapeutic target for several pathological conditions. Over the past decades, many efforts have therefore been devoted to developing PAI-1 inhibitors. This article provides an overview of the publishing activity on small organic molecules used as PAI-1 inhibitors. The chemical synthesis of the most potent inhibitors as well as their biological and biochemical evaluations is also presented.

  10. Pleiotrophin induces neurite outgrowth and up-regulates growth-associated protein (GAP)-43 mRNA through the ALK/GSK3beta/beta-catenin signaling in developing mouse neurons.

    PubMed

    Yanagisawa, Hiroko; Komuta, Yukari; Kawano, Hitoshi; Toyoda, Masashi; Sango, Kazunori

    2010-01-01

    Pleiotrophin (PTN) is highly expressed in the nervous system during embryogenesis; however, little is known about its functional role in neural development. By using whole mount in situ hybridization, we observed that the expression pattern of PTN was similar to that of Wnt3a; PTN mRNA was abundant in the nervous tissue along the dorsal midline and in the forelimb and hindlimb buds of embryonic mice (E8.5-E12.5). Treatment with recombinant PTN (100ng/ml) induced phosphorylation of glycogen synthase kinase 3beta (GSK3beta), nuclear localization of beta-catenin and up-regulation of growth-associated protein (GAP)-43 mRNA in cultured embryonic mouse (E14.5) neurons. Furthermore, recombinant PTN enhanced neurite outgrowth from cortical explants embedded in Matrigel. These PTN-induced biochemical changes and neurite outgrowth were attenuated by the co-treatment with anti-anaplastic lymphoma kinase (ALK) antibodies, but not with anti-protein tyrosine phosphatase (PTP)zeta antibodies. These findings imply that ALK is involved in the PTN signaling on neural development.

  11. Synthetic conversion of ACAT inhibitor to acetylcholinesterase inhibitor.

    PubMed

    Obata, R; Sunazuka, T; Otoguro, K; Tomoda, H; Harigaya, Y; Omura, S

    2000-06-19

    Natural product acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor pyripyropene A was synthetically converted to acetylcholinesterase (AChE) inhibitor via heterolitic cleavage of the 2-pyrone ring, followed by gamma-acylation/cyclization with several aroyl chlorides. The 4-pyridyl analogue selectively showed AChE inhibitory activity (IC50 7.9 microM) and no ACAT inhibitory activity IC50 = >1000 microM. PMID:10890154

  12. Glycosylasparaginase inhibition studies: competitive inhibitors, transition state mimics, noncompetitive inhibitors.

    PubMed

    Risley, J M; Huang, D H; Kaylor, J J; Malik, J J; Xia, Y Q

    2001-01-01

    Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond between asparagine and N-acetylglucosamine in the catabolism of N-linked glycoproteins. Previously only three competitive inhibitors, one noncompetitive inhibitor, and one irreversible inhibitor of glycosylasparaginase activity had been reported. Using human glycosylasparaginase from human amniotic fluid, L-aspartic acid and four of its analogues, where the alpha-amino group was substituted with a chloro, bromo, methyl or hydrogen, were competitive inhibitors having Ki values between 0.6-7.7 mM. These results provide supporting evidence for a proposed intramolecular autoproteolytic activation reaction. A proposed phosphono transition state mimic and a sulfo transition state mimic were competitive inhibitors with Ki values 0.9 mM and 1.4 mM, respectively. These results support a mechanism for the enzyme-catalyzed reaction involving formation of a tetrahedral high-energy intermediate. Three analogues of the natural substrate were noncompetitive inhibitors with Ki values between 0.56-0.75 mM, indicating the presence of a second binding site that may recognize (substituted)acetamido groups.

  13. Selective Inhibitors of Protein Methyltransferases

    PubMed Central

    2015-01-01

    Mounting evidence suggests that protein methyltransferases (PMTs), which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and human diseases. In particular, PMTs have been recognized as major players in regulating gene expression and chromatin state. PMTs are divided into two categories: protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs). There has been a steadily growing interest in these enzymes as potential therapeutic targets and therefore discovery of PMT inhibitors has also been pursued increasingly over the past decade. Here, we present a perspective on selective, small-molecule inhibitors of PMTs with an emphasis on their discovery, characterization, and applicability as chemical tools for deciphering the target PMTs’ physiological functions and involvement in human diseases. We highlight the current state of PMT inhibitors and discuss future directions and opportunities for PMT inhibitor discovery. PMID:25406853

  14. Synthesis of lysine methyltransferase inhibitors

    PubMed Central

    Hui, Chunngai; Ye, Tao

    2015-01-01

    Lysine methyltransferase which catalyze methylation of histone and non-histone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery. PMID:26258118

  15. [Pharmacology of bone resorption inhibitor].

    PubMed

    Menuki, Kunitaka; Sakai, Akinori

    2015-10-01

    Currently, bone resorption inhibitor is mainly used for osteoporosis. A number of these agents have been developed. These pharmacological action are various. Bisphosphonate inhibit functions of the osteoclasts by inducing apoptosis. On the one hand, RANK-ligand inhibitor and selective estrogen receptor modulator inhibit formation of osteoclasts. It is important to understand these pharmacological action for the selection of the appropriate medicine. PMID:26529923

  16. Thermoreversible gel for delivery of activin receptor-like kinase 5 inhibitor SB-505124 for glaucoma filtration surgery.

    PubMed

    Sutariya, Vijaykumar; Miladore, Nicholas; Geldenhuys, Werner; Bhatia, Deepak; Wehrung, Daniel; Nakamura, Hiroshi

    2013-01-01

    The purpose of this study is to investigate a thermoreversible gel using Pluronic F-127 to deliver an activin receptor-like kinase 5 (ALK-5) inhibitor SB-505124 in glaucoma filtration surgery (GFS). The gel was characterized for in vitro drug release and viscosity studies. Cytotoxicity of Pluronic F-127 was examined by MTT assay using cultured rabbit subconjunctival fibroblasts. In addition, Pluronic F-127 gel (18% w/v) containing 5 mg of SB-505124 was applied at the surgical site in an in vivo rabbit GFS model. In the in vitro viscosity study, the gel showed a change in viscosity (from 1000 cps to 45,000 cps) from low temperature (10°C) to body temperature (37°C). The in vitro drug release study demonstrated 100% drug release within 12 h. The gel did not show cytotoxicity to the cultured rabbit subconjunctival cells by MTT assay. In the in vivo rabbit GFS model, the drug was successfully delivered by injection and no severe post-surgical complications were observed. A thermoreversible gel system with SB-505124 was successfully prepared and delivered for the rabbit GFS model, and it may provide a novel delivery system in GFS.

  17. Peptidomimetic inhibitors of HIV protease.

    PubMed

    Randolph, John T; DeGoey, David A

    2004-01-01

    There are currently (July, 2002) six protease inhibitors approved for the treatment of HIV infection, each of which can be classified as peptidomimetic in structure. These agents, when used in combination with other antiretroviral agents, produce a sustained decrease in viral load, often to levels below the limits of quantifiable detection, and a significant reconstitution of the immune system. Therapeutic regimens containing one or more HIV protease inhibitors thus provide a highly effective method for disease management. The important role of protease inhibitors in HIV therapy, combined with numerous challenges remaining in HIV treatment, have resulted in a continued effort both to optimize regimens using the existing agents and to identify new protease inhibitors that may provide unique properties. This review will provide an overview of the discovery and clinical trials of the currently approved HIV protease inhibitors, followed by an examination of important aspects of therapy, such as pharmacokinetic enhancement, resistance and side effects. A description of new peptidomimetic compounds currently being investigated in the clinic and in preclinical discovery will follow. PMID:15193140

  18. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted. PMID:27048482

  19. Evolutionary families of peptidase inhibitors.

    PubMed Central

    Rawlings, Neil D; Tolle, Dominic P; Barrett, Alan J

    2004-01-01

    The proteins that inhibit peptidases are of great importance in medicine and biotechnology, but there has never been a comprehensive system of classification for them. Some of the terminology currently in use is potentially confusing. In the hope of facilitating the exchange, storage and retrieval of information about this important group of proteins, we now describe a system wherein the inhibitor units of the peptidase inhibitors are assigned to 48 families on the basis of similarities detectable at the level of amino acid sequence. Then, on the basis of three-dimensional structures, 31 of the families are assigned to 26 clans. A simple system of nomenclature is introduced for reference to each clan, family and inhibitor. We briefly discuss the specificities and mechanisms of the interactions of the inhibitors in the various families with their target enzymes. The system of families and clans of inhibitors described has been implemented in the MEROPS peptidase database (http://merops.sanger.ac.uk/), and this will provide a mechanism for updating it as new information becomes available. PMID:14705960

  20. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted.

  1. Positron emitter labeled enzyme inhibitors

    SciTech Connect

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-04-03

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  2. Electrochemical studies of corrosion inhibitors

    NASA Technical Reports Server (NTRS)

    Danford, M. D.

    1990-01-01

    The effect of single salts, as well as multicomponent mixtures, on corrosion inhibition was studied for type 1010 steel; for 5052, 1100, and 2219-T87 aluminum alloys; and for copper. Molybdate-containing inhibitors exhibit an immediate, positive effect for steel corrosion, but an incubation period may be required for aluminum before the effect of a given inhibitor can be determined. The absence of oxygen was found to provide a positive effect (smaller corrosion rate) for steel and copper, but a negative effect for aluminum. This is attributed to the two possible mechanisms by which aluminum can oxidize. Corrosion inhibition is generally similar for oxygen-rich and oxygen-free environments. The results show that the electrochemical method is an effective means of screening inhibitors for the corrosion of single metals, with caution to be exercised in the case of aluminum.

  3. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    1987-05-22

    This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

  4. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, Joanna S.; MacGregor, Robert R.; Wolf, Alfred P.; Langstrom, Bengt

    1990-01-01

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  5. Corrosion inhibitors from expired drugs.

    PubMed

    Vaszilcsin, Nicolae; Ordodi, Valentin; Borza, Alexandra

    2012-07-15

    This paper presents a method of expired or unused drugs valorization as corrosion inhibitors for metals in various media. Cyclic voltammograms were drawn on platinum in order to assess the stability of pharmaceutically active substances from drugs at the metal-corrosive environment interface. Tafel slope method was used to determine corrosion rates of steel in the absence and presence of inhibitors. Expired Carbamazepine and Paracetamol tablets were used to obtain corrosion inhibitors. For the former, the corrosion inhibition of carbon steel in 0.1 mol L(-1) sulfuric acid solution was about 90%, whereas for the latter, the corrosion inhibition efficiency of the same material in the 0.25 mol L(-1) acetic acid-0.25 mol L(-1) sodium acetate buffer solution was about 85%.

  6. An environmentally friendly scale inhibitor

    SciTech Connect

    Dobbs, J.B.; Brown, J.M.

    1999-11-01

    This paper describes a method of inhibiting the formation of scales such as barium and strontium sulfate in low pH aqueous systems, and calcium carbonate in systems containing high concentrations of dissolved iron. The solution, chemically, involves treating the aqueous system with an inhibitor designed to replace organic-phosphonates. Typical low pH aqueous systems where the inhibitor is particularly useful are oilfield produced-water, resin bed water softeners that form scale during low pH, acid regeneration operations. Downhole applications are recommended where high concentrations of dissolved iron are present in the produced water. This new approach to inhibition replaces typical organic phosphonates and polymers with a non-toxic, biodegradable scale inhibitor that performs in harsh environments.

  7. Diverse inhibitors of aflatoxin biosynthesis.

    PubMed

    Holmes, Robert A; Boston, Rebecca S; Payne, Gary A

    2008-03-01

    Pre-harvest and post-harvest contamination of maize, peanuts, cotton, and tree nuts by members of the genus Aspergillus and subsequent contamination with the mycotoxin aflatoxin pose a widespread food safety problem for which effective and inexpensive control strategies are lacking. Since the discovery of aflatoxin as a potently carcinogenic food contaminant, extensive research has been focused on identifying compounds that inhibit its biosynthesis. Numerous diverse compounds and extracts containing activity inhibitory to aflatoxin biosynthesis have been reported. Only recently, however, have tools been available to investigate the molecular mechanisms by which these inhibitors affect aflatoxin biosynthesis. Many inhibitors are plant-derived and a few may be amenable to pathway engineering for tissue-specific expression in susceptible host plants as a defense against aflatoxin contamination. Other compounds show promise as protectants during crop storage. Finally, inhibitors with different modes of action could be used in comparative transcriptional and metabolomic profiling experiments to identify regulatory networks controlling aflatoxin biosynthesis.

  8. Nonnucleoside inhibitors of adenosine kinase.

    PubMed

    Gomtsyan, Arthur; Lee, Chih-Hung

    2004-01-01

    Adenosine (ADO) is an endogenous inhibitory neuromodulator that increases nociceptive thresholds in response to tissue trauma and inflammation. Adenosine kinase (AK) is a key intracellular enzyme regulating intra- and extracellular concentrations of ADO. AK inhibition selectively amplifies extracellular ADO levels at cell and tissue sites where accelerated release of ADO occurs. AK inhibitors have been shown to provide effective antinociceptive, antiinflammatory and anticonvulsant activity in animal models, thus suggesting their potential therapeutic utility for pain, inflammation, epilepsy and possibly other central and peripheral nervous system diseases associated with cellular trauma and inflammation. This beneficial outcome may potentially lack nonspecific effects associated with the systemic administration of ADO receptor agonists. Until recently all of the reported AK inhibitors contained adenosine-like structural motif. The present review will discuss design, synthesis and analgesic and antiinflammatory properties of the novel nonnucleoside AK inhibitors that do not have close structural resemblance with the natural substrate ADO. Two classes of the nonnucleoside AK inhibitors are built on pyridopyrimidine and alkynylpyrimidine cores.

  9. C1-inhibitor and transplantation.

    PubMed

    Kirschfink, Michael

    2002-09-01

    Excessive activation of the protein cascade systems has been associated with post-transplantation inflammatory disorders. There is increasing evidence that complement not only significantly contributes to ischemia/reperfusion injury upon cold storage of the organ but also, although to a different degree, to allograft rejection. Complement activation is most fulminant in hyperacute rejection but seems also to contribute to acute transplant rejection. Therapeutic substitution of appropriate regulators, therefore, appears to be a reasonable approach to reduce undesirable inflammatory reactions in the grafted organ. C1-inhibitor, a multifunctional regulator of the various kinin-generating cascade systems (for review see: E. Hack, chapter in this issue), is frequently reduced in patients suffering from severe inflammatory disorders. Studies applying pathophysiologically relevant animal models of allo- and xenotransplantation as well as promising first clinical results from successful allotransplantation now provide evidence that C1-inhibitor may also serve as an effective means to protect the grafted organ against inflammatory tissue injury. In xenotransplantation, complement inhibition by specific regulators such as C1-inhibitor may help to overcome hyperacute graft rejection. After a brief introduction on the significance of complement to allo- and xenotransplantation the following review will focus on the impact of C1-inhibitor treatment on transplantation-associated inflammatory disorders, where complement contributes to the pathogenesis.

  10. Bivalent Inhibitors of Protein Kinases

    PubMed Central

    Gower, Carrie M.; Chang, Matthew E. K.; Maly, Dustin J.

    2015-01-01

    Protein kinases are key players in a large number of cellular signaling pathways. Dysregulated kinase activity has been implicated in a number of diseases, and members of this enzyme family are of therapeutic interest. However, due to the fact that most inhibitors interact with the highly conserved ATP-binding sites of kinases, it is a significant challenge to develop pharmacological agents that target only one of the greater than 500 kinases present in humans. A potential solution to this problem is the development of bisubstrate and bivalent kinase inhibitors, in which an active site-directed moiety is tethered to another ligand that targets a location outside of the ATP-binding cleft. Because kinase signaling specificity is modulated by regions outside of the ATP-binding site, strategies that exploit these interactions have the potential to provide reagents with high target selectivity. This review highlights examples of kinase interaction sites that can potentially be exploited by bisubstrate and bivalent inhibitors. Furthermore, an overview of efforts to target these interactions with bisubstrate and bivalent inhibitors is provided. Finally, several examples of the successful application of these reagents in a cellular setting are described. PMID:24564382

  11. PDE-5 inhibitors: clinical points.

    PubMed

    Doumas, Michael; Lazaridis, Antonios; Katsiki, Niki; Athyros, Vasilios

    2015-01-01

    Erectile dysfunction is usually of vascular origin and is frequently encountered in men with cardiovascular disease. The introduction of phosphodiesterase-5 inhibitors has revolutionized the management of patients with erectile dysfunction. Currently available phosphodiesterase-5 inhibitors have distinct pharmacokinetic and pharmacodynamic properties, thus permitting for tailoring sexual therapy according to patient characteristics and needs. Phosphodiesterase-5 inhibitors possess vasorelaxing properties and exert systemic hemodynamic effects, which need to be taken into account when other cardiovascular drugs are co-administered. Special caution is needed with alpha-blockers, while the co-administration with nitrates is contra-indicated due to the risk of life-threatening hypotension. This review presents the advent of sexual therapy, describes the mechanism of action and the specific characteristics of commercially available phosphodiesterase-5 inhibitors, summarizes the efficacy and safety of these drugs with special emphasis on the cardiovascular system, and discusses the clinical criteria used for the selection of each drug for the individual patient. PMID:25392015

  12. Acetylcholinesterase Inhibitors: Pharmacology and Toxicology

    PubMed Central

    Čolović, Mirjana B; Krstić, Danijela Z; Lazarević-Pašti, Tamara D; Bondžić, Aleksandra M; Vasić, Vesna M

    2013-01-01

    Acetylcholinesterase is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation, induced by various inhibitors, leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. This review presents an overview of toxicology and pharmacology of reversible and irreversible acetylcholinesterase inactivating compounds. In the case of reversible inhibitors being commonly applied in neurodegenerative disorders treatment, special attention is paid to currently approved drugs (donepezil, rivastigmine and galantamine) in the pharmacotherapy of Alzheimer’s disease, and toxic carbamates used as pesticides. Subsequently, mechanism of irreversible acetylcholinesterase inhibition induced by organophosphorus compounds (insecticides and nerve agents), and their specific and nonspecific toxic effects are described, as well as irreversible inhibitors having pharmacological implementation. In addition, the pharmacological treatment of intoxication caused by organophosphates is presented, with emphasis on oxime reactivators of the inhibited enzyme activity administering as causal drugs after the poisoning. Besides, organophosphorus and carbamate insecticides can be detoxified in mammals through enzymatic hydrolysis before they reach targets in the nervous system. Carboxylesterases most effectively decompose carbamates, whereas the most successful route of organophosphates detoxification is their degradation by corresponding phosphotriesterases. PMID:24179466

  13. Biocatalysts with enhanced inhibitor tolerance

    DOEpatents

    Yang, Shihui; Linger, Jeffrey; Franden, Mary Ann; Pienkos, Philip T.; Zhang, Min

    2015-12-08

    Disclosed herein are biocatalysts for the production of biofuels, including microorganisms that contain genetic modifications conferring tolerance to growth and fermentation inhibitors found in many cellulosic feedstocks. Methods of converting cellulose-containing materials to fuels and chemicals, as well as methods of fermenting sugars to fuels and chemicals, using these biocatalysts are also disclosed.

  14. Systemic and CNS activity of the RET inhibitor vandetanib combined with the mTOR inhibitor everolimus in KIF5B-RET re-arranged non-small cell lung cancer with brain metastases.

    PubMed

    Subbiah, Vivek; Berry, Jenny; Roxas, Michael; Guha-Thakurta, Nandita; Subbiah, Ishwaria Mohan; Ali, Siraj M; McMahon, Caitlin; Miller, Vincent; Cascone, Tina; Pai, Shobha; Tang, Zhenya; Heymach, John V

    2015-07-01

    In-frame fusion KIF5B (the-kinesin-family-5B-gene)-RET transcripts have been characterized in 1-2% of non-small cell lung cancers and are known oncogenic drivers. The RET tyrosine kinase inhibitor, vandetanib, suppresses fusion-induced, anchorage-independent growth activity. In vitro studies have shown that vandetanib is a high-affinity substrate of breast cancer resistance protein (Bcrp1/Abcg2) but is not transported by P-glycoprotein (P-gp), limiting its blood-brain barrier penetration. A co-administration strategy to enhance the brain accumulation of vandetanib by modulating P-gp/Abcb1- and Bcrp1/Abcg2-mediated efflux with mTOR inhibitors, specifically everolimus, was shown to increase the blood-brain barrier penetration. We report the first bench-to-bedside evidence that RET inhibitor combined with an mTOR inhibitor is active against brain-metastatic RET-rearranged lung cancer and the first evidence of blood-brain barrier penetration. A 74-year-old female with progressive adenocarcinoma of the lung (wild-type EGFR and no ALK rearrangement) presented for therapy options. A deletion of 5'RET was revealed by FISH assay, indicating RET-gene rearrangement. Because of progressive disease in the brain, she was enrolled in a clinical trial with vandetanib and everolimus (NCT01582191). Comprehensive genomic profiling revealed fusion of KIF5B (the-kinesin-family-5B-gene) and RET, in addition to AKT2 gene amplification. After two cycles of therapy a repeat MRI brain showed a decrease in the intracranial disease burden and PET/CT showed systemic response as well. Interestingly, AKT2 amplification seen is a critical component of the PI3K/mTOR pathway, alterations of which has been associated with both de novo and acquired resistance to targeted therapy. The addition of everolimus may have both overcome the AKT2 amplification to produce a response in addition to its direct effects on the RET gene. Our case report forms the first evidence of blood-brain barrier penetration by

  15. Systemic and CNS activity of the RET inhibitor vandetanib combined with the mTOR inhibitor everolimus in KIF5B-RET re-arranged non-small cell lung cancer with brain metastases.

    PubMed

    Subbiah, Vivek; Berry, Jenny; Roxas, Michael; Guha-Thakurta, Nandita; Subbiah, Ishwaria Mohan; Ali, Siraj M; McMahon, Caitlin; Miller, Vincent; Cascone, Tina; Pai, Shobha; Tang, Zhenya; Heymach, John V

    2015-07-01

    In-frame fusion KIF5B (the-kinesin-family-5B-gene)-RET transcripts have been characterized in 1-2% of non-small cell lung cancers and are known oncogenic drivers. The RET tyrosine kinase inhibitor, vandetanib, suppresses fusion-induced, anchorage-independent growth activity. In vitro studies have shown that vandetanib is a high-affinity substrate of breast cancer resistance protein (Bcrp1/Abcg2) but is not transported by P-glycoprotein (P-gp), limiting its blood-brain barrier penetration. A co-administration strategy to enhance the brain accumulation of vandetanib by modulating P-gp/Abcb1- and Bcrp1/Abcg2-mediated efflux with mTOR inhibitors, specifically everolimus, was shown to increase the blood-brain barrier penetration. We report the first bench-to-bedside evidence that RET inhibitor combined with an mTOR inhibitor is active against brain-metastatic RET-rearranged lung cancer and the first evidence of blood-brain barrier penetration. A 74-year-old female with progressive adenocarcinoma of the lung (wild-type EGFR and no ALK rearrangement) presented for therapy options. A deletion of 5'RET was revealed by FISH assay, indicating RET-gene rearrangement. Because of progressive disease in the brain, she was enrolled in a clinical trial with vandetanib and everolimus (NCT01582191). Comprehensive genomic profiling revealed fusion of KIF5B (the-kinesin-family-5B-gene) and RET, in addition to AKT2 gene amplification. After two cycles of therapy a repeat MRI brain showed a decrease in the intracranial disease burden and PET/CT showed systemic response as well. Interestingly, AKT2 amplification seen is a critical component of the PI3K/mTOR pathway, alterations of which has been associated with both de novo and acquired resistance to targeted therapy. The addition of everolimus may have both overcome the AKT2 amplification to produce a response in addition to its direct effects on the RET gene. Our case report forms the first evidence of blood-brain barrier penetration by

  16. Proton pump inhibitor-induced hypomagnesemic hypoparathyroidism.

    PubMed

    Swaminathan, Krishnan

    2015-01-01

    Proton pump inhibitors are the one of the most widely used drugs in the world. Hypomagnesemic hypoparathyroidism has been reported with different proton pump inhibitors with prolonged oral use. We report the first reported case of possible such effect with intravenous preparation of proton pump inhibitor. This case report raises awareness among physicians worldwide of this often unknown association, as life-threatening cardiac and neuromuscular complications can arise with unrecognized hypocalcemia and hypomagnesemia with proton pump inhibitors.

  17. KID, a Kinase Inhibitor Database project.

    PubMed

    Collin, O; Meijer, L

    1999-01-01

    The Kinase Inhibitor Database is a small specialized database dedicated to the gathering of information on protein kinase inhibitors. The database is accessible through the World Wide Web system and gives access to structural and bibliographic information on protein kinase inhibitors. The data in the database will be collected and submitted by researchers working in the kinase inhibitor field. The submitted data will be checked by the curator of the database before entry.

  18. Salicylanilide inhibitors of Toxoplasma gondii.

    PubMed

    Fomovska, Alina; Wood, Richard D; Mui, Ernest; Dubey, Jitenter P; Ferreira, Leandra R; Hickman, Mark R; Lee, Patricia J; Leed, Susan E; Auschwitz, Jennifer M; Welsh, William J; Sommerville, Caroline; Woods, Stuart; Roberts, Craig; McLeod, Rima

    2012-10-11

    Toxoplasma gondii (T. gondii) is an apicomplexan parasite that can cause eye disease, brain disease, and death, especially in congenitally infected and immune-compromised people. Novel medicines effective against both active and latent forms of the parasite are greatly needed. The current study focused on the discovery of such medicines by exploring a family of potential inhibitors whose antiapicomplexan activity has not been previously reported. Initial screening efforts revealed that niclosamide, a drug approved for anthelmintic use, possessed promising activity in vitro against T. gondii. This observation inspired the evaluation of the activity of a series of salicylanilides and derivatives. Several inhibitors with activities in the nanomolar range with no appreciable in vitro toxicity to human cells were identified. An initial structure-activity relationship was explored. Four compounds were selected for evaluation in an in vivo model of infection, and two derivatives with potentially enhanced pharmacological parameters demonstrated the best activity profiles.

  19. Salicylanilide Inhibitors of Toxoplasma gondii

    PubMed Central

    Fomovska, Alina; Wood, Richard D.; Mui, Ernest; Dubey, Jitenter P.; Ferriera, Leandra R.; Hickman, Mark R.; Lee, Patricia J.; Leed, Susan E.; Auschwitz, Jennifer M.; Welsh, William J.; Sommerville, Caroline; Woods, Stuart; Roberts, Craig; McLeod, Rima

    2012-01-01

    Toxoplasma gondii(T. gondii) is an apicomplexan parasite that can cause eye disease, brain disease, and death, especially in congenitally infected and immune-compromised people. Novel medicines effective against both active and latent forms of the parasite are greatly needed. The current study focused on the discovery of such medicines by exploring a family of potential inhibitors whose anti-apicomplexan activity has not been previously reported. Initial screening efforts revealed that niclosamide, a drug approved for anthelmintic use, possessed promising activity in vitro against T. gondii. This observation inspired the evaluation of the activity of a series of salicylanilides and derivatives. Several inhibitors with activities in the nanomolar range with no appreciable in vitro toxicity to human cells were identified. An initial structure-activity relationship was explored. Four compounds were selected for evaluation in an in vivo model of infection, and two derivatives with potentially enhanced pharmacological parameters demonstrated the best activity profiles. PMID:22970937

  20. Macrocyclic compounds as corrosion inhibitors

    SciTech Connect

    Quraishi, M.A.; Rawat, J.; Ajmal, M.

    1998-12-01

    The influence of three macrocyclic compounds on corrosion of mild steel (MS) in hydrochloric acid (HCl) was investigated using weight loss, potentiodynamic polarization, alternating current (AC) impedance, and hydrogen permeation techniques. All the investigated compounds showed significant efficiencies and reduced permeation of hydrogen through MS in HCl. Inhibition efficiency (IE) varied with the nature and concentrations of the inhibitors, temperature, and concentrations of the acid solutions. The addition of iodide ions (I{sup {minus}}) increased IE of all the tested compounds as a result of the synergistic effect. Potentiodynamic polarization results revealed that macrocyclic compounds acted as mixed inhibitors in 1 M HCl to 5 M HCl. Adsorption on the metal surface obeyed Temkin`s adsorption isotherm. Auger electron spectroscopy (AES) of the polished MS surface, exposed with tetraphenyldithia-octaazacyclotetradeca-hexaene (PTAT) proved adsorption of this compound on the surface through nitrogen and sulfur atoms.

  1. [Proteasome inhibitors in cancer therapy].

    PubMed

    Romaniuk, Wioletta; Ołdziej, Agnieszka Ewa; Zińczuk, Justyna; Kłoczko, Janusz

    2015-01-01

    Proteasomes are multisubunit enzyme complexes. They contain three enzymatic active sites which are termed chymotrypsin-like, trypsin-like, and caspase-like. The elementary function of the proteasomes is degradation of damaged proteins. Proteasome inhibition leads to accumulation of damaged protein, which leads to caspase activation and cell death. This relationship is used in cancer therapy. Bortezomib is the first proteasome inhibitor approved by the US Food and Drug Administration for the treatment of relapsed/refractory multiple myeloma. Carfilzomib belongs to the second generation of drugs, which was approved by the US FDA in 2012. Currently in the study phase there are four new inhibitors: ixazomib (MLN9780/MLN2238), delanzomib (CEP-18770), oprozomib (ONX0912/PR-047) and marizomib (NPI-0052). PMID:27259216

  2. Aromatase Inhibitors and Other Compounds for Lowering Breast Cancer Risk

    MedlinePlus

    ... References Aromatase inhibitors and other compounds for lowering breast cancer risk Aromatase inhibitors (drugs that lower estrogen levels) ... day. Can aromatase inhibitors lower the risk of breast cancer? Aromatase inhibitors are used mainly to treat hormone ...

  3. Conformation-specific inhibitors of Raf kinases.

    PubMed

    Wang, Xiaolun; Schleicher, Kristin

    2013-01-01

    Since the discovery linking B-Raf mutations to human tumors in 2002, significant advances in the development of Raf inhibitors have been made, leading to the recent approval of two Raf inhibitor drugs. This chapter includes a brief introduction to B-Raf as a validated target and focuses on the three different binding modes observed with Raf small-molecule inhibitors. These various binding modes lock the Raf kinase in different conformations that impact the toxicity profiles of the inhibitors. Possible solutions to mitigate the side effects caused by inhibitor-induced dimerization are also discussed.

  4. Thioredoxin Reductase and its Inhibitors

    PubMed Central

    Saccoccia, Fulvio; Angelucci, Francesco; Boumis, Giovanna; Carotti, Daniela; Desiato, Gianni; Miele, Adriana E; Bellelli, Andrea

    2014-01-01

    Thioredoxin plays a crucial role in a wide number of physiological processes, which span from reduction of nucleotides to deoxyriboucleotides to the detoxification from xenobiotics, oxidants and radicals. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin NADPH Reductase (TrxR). In view of its indirect involvement in the above mentioned physio/pathological processes, inhibition of TrxR is an important clinical goal. As a general rule, the affinities and mechanisms of binding of TrxR inhibitors to the target enzyme are known with scarce precision and conflicting results abound in the literature. A relevant analysis of published results as well as the experimental procedures is therefore needed, also in view of the critical interest of TrxR inhibitors. We review the inhibitors of TrxR and related flavoreductases and the classical treatment of reversible, competitive, non competitive and uncompetitive inhibition with respect to TrxR, and in some cases we are able to reconcile contradictory results generated by oversimplified data analysis. PMID:24875642

  5. Carbonic anhydrase inhibitors drug design.

    PubMed

    McKenna, Robert; Supuran, Claudiu T

    2014-01-01

    Inhibition of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) has pharmacologic applications in the field of antiglaucoma, anticonvulsant, antiobesity, and anticancer agents but is also emerging for designing anti-infectives (antifungal and antibacterial agents) with a novel mechanism of action. As a consequence, the drug design of CA inhibitors (CAIs) is a very dynamic field. Sulfonamides and their isosteres (sulfamates/sulfamides) constitute the main class of CAIs which bind to the metal ion in the enzyme active site. Recently the dithiocarbamates, possessing a similar mechanism of action, were reported as a new class of inhibitors. Other families of CAIs possess a distinct mechanism of action: phenols, polyamines, some carboxylates, and sulfocoumarins anchor to the zinc-coordinated water molecule. Coumarins and five/six-membered lactones are prodrug inhibitors, binding in hydrolyzed form at the entrance of the active site cavity. Novel drug design strategies have been reported principally based on the tail approach for obtaining all these types of CAIs, which exploit more external binding regions within the enzyme active site (in addition to coordination to the metal ion), leading thus to isoform-selective compounds. Sugar-based tails as well as click chemistry were the most fruitful developments of the tail approach. Promising compounds that inhibit CAs from bacterial and fungal pathogens, of the dithiocarbamate, phenol and carboxylate types have also been reported. PMID:24146385

  6. ANTIDEPRESSANT ACTIONS OF HDAC INHIBITORS

    PubMed Central

    Covington, Herbert E.; Maze, Ian; LaPlant, Quincey C.; Vialou, Vincent F.; Yoshinori, Ohnishi N.; Berton, Olivier; Fass, Dan M.; Renthal, William; Rush, Augustus J.; Wu, Emma Y.; Ghose, Subroto; Krishnan, Vaishnav; Russo, Scott J.; Tamminga, Carol; Haggarty, Stephen J.; Nestler, Eric J.

    2009-01-01

    Persistent symptoms of depression suggest the involvement of stable molecular adaptations in brain, which may be reflected at the level of chromatin remodeling. We find that chronic social defeat stress in mice causes a transient decrease, followed by a persistent increase, in levels of acetylated histone H3 in the nucleus accumbens, an important limbic brain region. This persistent increase in H3 acetylation is associated with decreased levels of histone deacetylase 2 (HDAC2) in the nucleus accumbens. Similar effects were observed in the nucleus accumbens of depressed humans studied postmortem. These changes in H3 acetylation and HDAC2 expression mediate long-lasting positive neuronal adaptations, since infusion of HDAC inhibitors into the nucleus accumbens, which increases histone acetylation, exerts robust antidepressant-like effects in the social defeat paradigm and other behavioral assays. HDAC inhibitor (MS-275) infusion also reverses the effects of chronic defeat stress on global patterns of gene expression in the nucleus accumbens, as determined by microarray analysis, with striking similarities to the effects of the standard antidepressant, fluoxetine. Stress-regulated genes whose expression is normalized selectively by MS-275 may provide promising targets for the future development of novel antidepressant treatments. Together, these findings provide new insight into the underlying molecular mechanisms of depression and antidepressant action, and support the antidepressant potential of HDAC inhibitors and perhaps other agents that act at the level of chromatin structure. PMID:19759294

  7. Targeted therapies in development for non-small cell lung cancer.

    PubMed

    Reungwetwattana, Thanyanan; Dy, Grace Kho

    2013-01-01

    The iterative discovery in various malignancies during the past decades that a number of aberrant tumorigenic processes and signal transduction pathways are mediated by "druggable" protein kinases has led to a revolutionary change in drug development. In non-small cell lung cancer (NSCLC), the ErbB family of receptors (e.g., EGFR [epidermal growth factor receptor], HER2 [human epidermal growth factor receptor 2]), RAS (rat sarcoma gene), BRAF (v-raf murine sarcoma viral oncogene homolog B1), MAPK (mitogen-activated protein kinase) c-MET (c-mesenchymal-epithelial transition), FGFR (fibroblast growth factor receptor), DDR2 (discoidin domain receptor 2), PIK3CA (phosphatidylinositol-4,5-bisphosphate3-kinase, catalytic subunit alpha)), PTEN (phosphatase and tensin homolog), AKT (protein kinase B), ALK (anaplastic lym phoma kinase), RET (rearranged during transfection), ROS1 (reactive oxygen species 1) and EPH (erythropoietin-producing hepatoma) are key targets of various agents currently in clinical development. These oncogenic targets exert their selective growth advantage through various intercommunicating pathways, such as through RAS/RAF/MEK, phosphoinositide 3-kinase/AKT/mammalian target of rapamycin and SRC-signal transduction and transcription signaling. The recent clinical studies, EGFR tyrosine kinase inhibitors and crizotinib were considered as strongly effective targeted therapies in metastatic NSCLC. Currently, five molecular targeted agents were approved for treatment of advanced NSCLC: Gefitinib, erlotinib and afatinib for positive EGFR mutation, crizotinib for positive echinoderm microtubule-associated protein-like 4 (EML4)-ALK translocation and bevacizumab. Moreover, oncogenic mutant proteins are subject to regulation by protein trafficking pathways, specifically through the heat shock protein 90 system. Drug combinations affecting various nodes in these signaling and intracellular processes are predicted and demonstrated to be synergistic and

  8. Targeted therapies in development for non-small cell lung cancer

    PubMed Central

    Reungwetwattana, Thanyanan; Dy, Grace Kho

    2013-01-01

    The iterative discovery in various malignancies during the past decades that a number of aberrant tumorigenic processes and signal transduction pathways are mediated by “druggable” protein kinases has led to a revolutionary change in drug development. In non-small cell lung cancer (NSCLC), the ErbB family of receptors (e.g., EGFR [epidermal growth factor receptor], HER2 [human epidermal growth factor receptor 2]), RAS (rat sarcoma gene), BRAF (v-raf murine sarcoma viral oncogene homolog B1), MAPK (mitogen-activated protein kinase) c-MET (c-mesenchymal-epithelial transition), FGFR (fibroblast growth factor receptor), DDR2 (discoidin domain receptor 2), PIK3CA (phosphatidylinositol-4,5-bisphosphate3-kinase, catalytic subunit alpha)), PTEN (phosphatase and tensin homolog), AKT (protein kinase B), ALK (anaplastic lym phoma kinase), RET (rearranged during transfection), ROS1 (reactive oxygen species 1) and EPH (erythropoietin-producing hepatoma) are key targets of various agents currently in clinical development. These oncogenic targets exert their selective growth advantage through various intercommunicating pathways, such as through RAS/RAF/MEK, phosphoinositide 3-kinase/AKT/mammalian target of rapamycin and SRC-signal transduction and transcription signaling. The recent clinical studies, EGFR tyrosine kinase inhibitors and crizotinib were considered as strongly effective targeted therapies in metastatic NSCLC. Currently, five molecular targeted agents were approved for treatment of advanced NSCLC: Gefitinib, erlotinib and afatinib for positive EGFR mutation, crizotinib for positive echinoderm microtubule-associated protein-like 4 (EML4)-ALK translocation and bevacizumab. Moreover, oncogenic mutant proteins are subject to regulation by protein trafficking pathways, specifically through the heat shock protein 90 system. Drug combinations affecting various nodes in these signaling and intracellular processes are predicted and demonstrated to be synergistic and

  9. Investigating the selectivity of metalloenzyme inhibitors.

    PubMed

    Day, Joshua A; Cohen, Seth M

    2013-10-24

    The inhibitory activity of a broad group of known metalloenzyme inhibitors against a panel of metalloenzymes was evaluated. Clinically approved inhibitors were selected as well as several other reported metalloprotein inhibitors in order to represent a broad range of metal binding groups (MBGs), including hydroxamic acid, carboxylate, hydroxypyridinonate, thiol, and N-hydroxyurea functional groups. A panel of metalloenzymes, including carbonic anhydrase (hCAII), several matrix metalloproteinases (MMPs), angiotensin converting enzyme (ACE), histone deacetylase (HDAC-2), and tyrosinase (TY), was selected based on their clinical importance for a range of pathologies. In addition, each inhibitor was evaluated for its ability to remove Fe(3+) from holo-transferrin to gauge the ability of the inhibitors to access Fe(3+) from a primary transport protein. The results show that the metalloenzyme inhibitors are quite selective for their intended targets, suggesting that despite their ability to bind metal ions, metalloprotein inhibitors are not prone to widespread off-target enzyme inhibition activity.

  10. The burden of inhibitors in haemophilia patients.

    PubMed

    Walsh, Christopher E; Jiménez-Yuste, Víctor; Auerswald, Guenter; Grancha, Salvador

    2016-08-31

    The burden of disease in haemophilia patients has wide ranging implications for the family and to society. There is evidence that having a current inhibitor increases the risk of morbidity and mortality. Morbidity is increased by the inability to treat adequately and its consequent disabilities, which then equates to a poor quality of life compared with non-inhibitor patients. The societal cost of care, or `burden of inhibitors', increases with the ongoing presence of an inhibitor. Therefore, it is clear that successful eradication of inhibitors by immune tolerance induction (ITI) is the single most important milestone one can achieve in an inhibitor patient. The type of factor VIII (FVIII) product used in ITI regimens varies worldwide. Despite ongoing debate, there is in vitro and retrospective clinical evidence to support the use of plasma-derived VWF-containing FVIII concentrates in ITI regimens in order to achieve early and high inhibitor eradication success rates. PMID:27528280

  11. Investigating the Selectivity of Metalloenzyme Inhibitors

    PubMed Central

    Day, Joshua A.; Cohen, Seth M.

    2013-01-01

    The inhibitory activity of a broad group of known metalloenzyme inhibitors against a panel of metalloenzymes was evaluated. Clinically approved inhibitors were selected as well as several other reported metalloprotein inhibitors, in order to represent a broad range of metal binding groups (MBGs), including hydroxamic acid, carboxylate, hydroxypyridinonate, thiol, and N-hydroxyurea functional groups. A panel of metalloenzymes, including carbonic anhydrase (hCAII), several matrix metalloproteinases (MMPs), angiotensin converting enzyme (ACE), histone deacetylase (HDAC-2), and tyrosinase (TY) was selected based on their clinical importance for a range of pathologies. In addition, each inhibitor was evaluated for its ability to remove Fe3+ from holo-transferrin to gauge the ability of the inhibitors to access Fe3+ from a primary transport protein. The results show that the metalloenzyme inhibitors are quite selective for their intended targets, suggesting that despite their ability to bind metal ions, metalloprotein inhibitors are not prone to widespread off-target enzyme inhibition activity. PMID:24074025

  12. Tissue inhibitor of metalloproteinase-3 is up-regulated by transforming growth factor-beta1 in vitro and expressed in fibroblastic foci in vivo in idiopathic pulmonary fibrosis.

    PubMed

    García-Alvarez, Jorge; Ramirez, Remedios; Checa, Marco; Nuttall, Robert K; Sampieri, Clara L; Edwards, Dylan R; Selman, Moisés; Pardo, Annie

    2006-05-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by fibroblast expansion and extracellular matrix accumulation. However, the mechanisms involved in matrix remodeling have not been elucidated. In this study, the authors aimed to evaluate the expression of the tissue inhibitors of matrix metalloproteinases (TIMPs) in human fibroblasts and whole tissues from IPF and normal lungs. They also determined the role of mitogen-activated protein kinase (MAPK) in TIMP3 expression. TIMP1, TIMP2, and TIMP3 were highly expressed in lung fibroblasts. Transforming growth factor (TGF)-beta1, a profibrotic mediator, induced strong up-regulation of TIMP3 at the mRNA and protein levels. The authors examined whether the MAPK pathway was involved in TGF-beta1-induced TIMP3 expression. TGF-beta1 induced the phosphorylation of p38 and extracellular signal-regulated kinase (ERK)1/2. Biochemical blockade of p38 by SB203580, but not of the ERK MAPK pathway, inhibited the effect of this factor. The effect was also blocked by the tyrosine kinase inhibitor genistein and by antagonizing TGF-beta1 receptor type I (activin-linked kinase [ALK5]). In IPF tissues TIMP3 gene expression was significantly increased and the protein was localized to fibroblastic foci and extracellular matrix. Our findings suggest that TGF-beta1-induced TIMP3 may be an important mediator in lung fibrogenesis.

  13. Non-Invasive Methods to Monitor Mechanisms of Resistance to Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer: Where Do We Stand?

    PubMed Central

    Ulivi, Paola

    2016-01-01

    The induction of resistance mechanisms represents an important problem for the targeted therapy of patients with non-small-cell lung cancer (NSCLC). The best-known resistance mechanism induced during treatment with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) is EGFR T790M mutation for which specific drugs are have been developed. However, other molecular alterations have also been reported as induced resistance mechanisms to EGFR-TKIs. Similarly, there is growing evidence of acquired resistance mechanisms to anaplastic lymphoma kinase (ALK)-TKI treatment. A better understanding of these acquired resistance mechanisms is essential in clinical practice as patients could be treated with specific drugs that are active against the induced alterations. The use of free circulating tumor nucleic acids or circulating tumor cells (CTCs) enables resistance mechanisms to be characterized in a non-invasive manner and reduces the need for tumor re-biopsy. This review discusses the main resistance mechanisms to TKIs and provides a comprehensive overview of innovative strategies to evaluate known resistance mechanisms in free circulating nucleic acids or CTCs and potential future orientations for these non-invasive approaches. PMID:27455248

  14. An novel inhibitor of TGF-β type I receptor, IN-1130, blocks breast cancer lung metastasis through inhibition of epithelial-mesenchymal transition.

    PubMed

    Park, Chul-Yong; Min, Kyung Nan; Son, Jee-Yeon; Park, So-Yeon; Nam, Jeong-Seok; Kim, Dae-Kee; Sheen, Yhun Yhong

    2014-08-28

    TGF-β signaling plays an important role in breast cancer progression and metastasis. Epithelial-mesenchymal transition (EMT) is an important step in the progression of solid tumors to metastatic disease. We previously reported that IN-1130, a novel transforming growth factor-β type I receptor kinase (ALK5) inhibitor, suppressed renal fibrosis in obstructive nephropathy (Moon et al., 2006). Here, we show that IN-1130 suppressed EMT and the lung metastasis of mammary tumors in mouse models. Treating human and mouse cell lines with IN-1130 inhibited TGF-β-mediated transcriptional activation, the phosphorylation and nuclear translocation of Smad2, and TGF-β-induced-EMT, which induces morphological changes in epithelial cells. Additionally, we demonstrated that IN-1130 blocked TGF-β-induced 4T1 mammary cancer cell migration and invasion. The TGF-β-mediated increase in matrix metalloproteinase (MMP)-2 and MMP-9 expression was restored by IN-1130 co-treatment with TGF-β in human epithelial cells and in 4T1 cells. Furthermore, we found that lung metastasis from primary breast cancer was inhibited by IN-1130 in both 4T1-xenografted BALB/c mice and MMTV/c-Neu transgenic mice without any change in prima