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Sample records for alk inhibitor crizotinib

  1. The ALK inhibitor ceritinib overcomes crizotinib resistance in non-small cell lung cancer

    PubMed Central

    Lee, Christian C.; Gainor, Justin F.; Crystal, Adam S.; Michellys, Pierre-Yves; Awad, Mark M.; Yanagitani, Noriko; Kim, Sungjoon; Pferdekamper, AnneMarie C.; Li, Jie; Kasibhatla, Shailaja; Sun, Frank; Sun, Xiuying; Hua, Su; McNamara, Peter; Mahmood, Sidra; Lockerman, Elizabeth L.; Fujita, Naoya; Nishio, Makoto; Harris, Jennifer L.; Shaw, Alice T.; Engelman, Jeffrey A.

    2014-01-01

    Non-small cell lung cancers (NSCLC) harboring anaplastic lymphoma kinase (ALK) gene rearrangements invariably develop resistance to the ALK tyrosine kinase inhibitor (TKI) crizotinib. Herein, we report the first preclinical evaluation of the next-generation ALK TKI, ceritinib (LDK378) in the setting of crizotinib resistance. Interrogation of in vitro and in vivo models of acquired resistance to crizotinib, including cell lines established from biopsies of crizotinib-resistant NSCLC patients revealed that ceritinib potently overcomes crizotinib resistance mutations. In particular, ceritinib effectively inhibits ALK harboring L1196M, G1269A, I1171T and S1206Y mutations, and a co-crystal of ceritinib bound to ALK provides structural bases for this increased potency. However, we observed that ceritinib did not overcome two crizotinib-resistant ALK mutations, G1202R and F1174C, and one of these mutations were identified in 5 out of 11 biopsies from patients with acquired resistance to ceritinib. Altogether our results demonstrate that ceritinib can overcome crizotinib resistance, consistent with clinical data showing marked efficacy of ceritinib in patients with crizotinib-resistant disease. PMID:24675041

  2. The ALK/ROS1 inhibitor PF-06463922 overcomes primary resistance to crizotinib in ALK-driven neuroblastoma

    PubMed Central

    Infarinato, Nicole R.; Park, Jin H.; Krytska, Kateryna; Ryles, Hannah T.; Sano, Renata; Szigety, Katherine M.; Li, Yimei; Zou, Helen Y.; Lee, Nathan V.; Smeal, Tod; Lemmon, Mark A.; Mossé, Yael P.

    2015-01-01

    Neuroblastomas (NBs) harboring activating point mutations in Anaplastic Lymphoma Kinase (ALK) are differentially sensitive to the ALK inhibitor crizotinib, with certain mutations conferring intrinsic crizotinib resistance. To overcome this clinical obstacle, our goal was to identify inhibitors with improved potency that can target intractable ALK variants such as F1174L. We find that PF-06463922 has high potency across ALK variants, and inhibits ALK more effectively than crizotinib in vitro. Most importantly, PF-06463922 induces complete tumor regression in both crizotinib-resistant and sensitive xenograft mouse models of NB, as well as in PDXs harboring the crizotinib-resistant F1174L or F1245C mutations. These studies demonstrate that PF-06463922 has the potential to overcome crizotinib resistance, and exerts unprecedented activity as a single targeted agent against F1174L and F1245C ALK-mutated xenograft tumors, while also inducing responses in a R1275Q xenograft model. Taken together, these results provide the rationale to move PF-06463922 into clinical trials for treatment of patients with ALK-mutated NB. PMID:26554404

  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. 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

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. Patterns of response to crizotinib in recurrent glioblastoma according to ALK and MET molecular profile in two patients.

    PubMed

    Le Rhun, Emilie; Chamberlain, Marc C; Zairi, Fahed; Delmaire, Christine; Idbaih, Ahmed; Renaud, Florence; Maurage, Claude Alain; Grégoire, Valérie

    2015-01-01

    Two patients with an unmethylated MGMT promoter and IDH1 (R132H) wild-type recurrent glioblastoma were treated with crizotinib. Prolonged stabilization of the disease (17 months) was achieved in the first case. Interestingly, anaplastic lymphoma kinase (ALK) expression and c-MET protein overexpression was observed. Conversely, no response to crizotinib was obtained in the second case with MET protein overexpression and c-MET amplification but no ALK expression or ALK gene amplification. These case studies suggest that novel targeted ALK inhibitors may provide relevant clinical benefit in selected cases in which driver mutations are demonstrable. PMID:26498130

  10. Co-clinical trials demonstrate superiority of crizotinib to chemotherapy in ALK-rearranged non-small cell lung cancer and predict strategies to overcome resistance

    PubMed Central

    Tupper, Tanya; Cheng, Katherine; Wang, Yuchuan; Tan, Xiaohong; Altabef, Abigail; Woo, Sue-Ann; Chen, Liang; Reibel, Jacob B.; Janne, Pasi A.; Sharpless, Norman E.; Engelman, Jeffrey A.; Shapiro, Geoffrey I.; Kung, Andrew L.; Wong, Kwok-Kin

    2014-01-01

    Purpose To extend the results of a phase III trial in non-small cell lung cancer patients with adenocarcinomas harboring EML4-ALK fusion. Experimental Design we performed a co-clinical trial in a mouse model comparing the ALK inhibitor crizotinib to the standard-of-care cytotoxic agents docetaxel or pemetrexed. Results Concordant with the clinical outcome in humans, crizotinib produced a substantially higher response rate compared to chemotherapy, associated with significantly longer progression-free survival. Overall survival was also prolonged in crizotinib- compared to chemotherapy-treated mice. Pemetrexed produced superior overall survival compared to docetaxel, suggesting that this agent may be the preferred chemotherapy in the ALK population. Additionally, in the EML4-ALK-driven mouse lung adenocarcinoma model, HSP90 inhibition can overcome both primary and acquired crizotinib resistance. Furthermore, HSP90 inhibition, as well as the second-generation ALK inhibitor TAE684, demonstrated activity in newly developed lung adenocarcinoma models driven by crizotinib-insensitive EML4-ALK L1196M or F1174L. Conclusions Our findings suggest that crizotinib is superior to standard chemotherapy in ALK inhibitor-naïve disease and support further clinical investigation of HSP90 inhibitors and second-generation ALK inhibitors in tumors with primary or acquired crizotinib resistance. PMID:24327273

  11. The neuroblastoma associated F1174L ALK mutation causes resistance to an ALK kinase inhibitor in ALK translocated cancers

    PubMed Central

    Sasaki, Takaaki; Okuda, Katsuhiro; Zheng, Wei; Butrynski, James; Capelletti, Marzia; Wang, Liping; Gray, Nathanael S.; Wilner, Keith; Christensen, James G.; Demetri, George; Shapiro, Geoffrey I.; Rodig, Scott J.; Eck, Michael J.; Jänne, Pasi A.

    2011-01-01

    The ALK kinase inhibitor crizotinib (PF-02341066) is clinically effective in patients with ALK-translocated cancers, but its efficacy will ultimately be limited by acquired drug resistance. Here we report the identification of a secondary mutation in ALK, F1174L, as one cause of crizotinib resistance in a patient with an inflammatory myofibroblastic tumor (IMT) harbouring a RANBP2-ALK translocation who progressed while crizotinib therapy. When present in cis with an ALK translocation, this mutation (also detected in neuroblastomas) causes an increase in ALK phosphorylation, cell growth and downstream signaling. Furthermore, the F1174L mutation inhibits crizotinib mediated downregulation of ALK signaling and blocks apoptosis in RANBP2-ALK Ba/F3 cells. A chemically distinct ALK inhibitor, TAE684, or the HSP90 inhibitor 17-AAG are both effective in models harbouring the F1174L ALK mutation. Our findings highlight the importance of studying drug resistance mechanisms in order to develop effective clinical treatments for patients with ALK-translocated cancers. PMID:21030459

  12. 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

  13. 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

  14. Mechanisms of Acquired Resistance to ALK Inhibitors and the Rationale for Treating ALK-positive Lung Cancer.

    PubMed

    Isozaki, Hideko; Takigawa, Nagio; Kiura, Katsuyuki

    2015-01-01

    The discovery of an echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene led to improved clinical outcomes in patients with lung cancer after the development of the first ALK-targeting agent, crizotinib. Some second-generation ALK tyrosine kinase inhibitors (TKIs), which might be more potent than crizotinib or effective on crizotinib-resistant patients, have been developed. Although these ALK-TKIs show an excellent response initially, most patients eventually acquire resistance. Therefore, careful consideration of the resistance mechanisms might lead to superior therapeutic strategies. Here, we summarize the history of ALK-TKIs and their underlying resistance mechanisms in both the preclinical and clinical settings. In addition, we discuss potential future treatment strategies in ALK-TKI-naïve and -resistant patients with lung cancer harboring the EML4-ALK fusion gene. PMID:25941796

  15. Mechanisms of Acquired Resistance to ALK Inhibitors and the Rationale for Treating ALK-positive Lung Cancer

    PubMed Central

    Isozaki, Hideko; Takigawa, Nagio; Kiura, Katsuyuki

    2015-01-01

    The discovery of an echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene led to improved clinical outcomes in patients with lung cancer after the development of the first ALK-targeting agent, crizotinib. Some second-generation ALK tyrosine kinase inhibitors (TKIs), which might be more potent than crizotinib or effective on crizotinib-resistant patients, have been developed. Although these ALK-TKIs show an excellent response initially, most patients eventually acquire resistance. Therefore, careful consideration of the resistance mechanisms might lead to superior therapeutic strategies. Here, we summarize the history of ALK-TKIs and their underlying resistance mechanisms in both the preclinical and clinical settings. In addition, we discuss potential future treatment strategies in ALK-TKI-naïve and -resistant patients with lung cancer harboring the EML4-ALK fusion gene. PMID:25941796

  16. Moving molecularly directed therapies to the first-line in ALK-positive lung cancer: crizotinib is just the beginning.

    PubMed

    Klempner, Samuel J; Raufi, Alexander; Ou, Sai-Hong Ignatius

    2015-10-01

    The increasing appreciation of oncogenic driver alterations in non-small cell lung cancer (NSCLC) has resulted in a rapid expansion of therapeutic compounds. Epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) alterations are the prototypical examples and have driven the paradigm shift in NSCLC management. Early phase studies in previously treated ALK+ patients demonstrated activity and recently Solomon et al. confirmed the superiority of crizotinib over chemotherapy in first line treatment. The phase III PROFILE 1014 represents the culmination of the rapid development of crizotinib and provides lessons for future generation ALK inhibitors and other molecularly directed therapies in NSCLC. Important considerations for second and third generation inhibitors include the ability to overcome known resistance mechanisms, CNS activity, improvement in side effect profile, and safety in possible combination strategies. PMID:26629439

  17. Two novel ALK mutations mediate acquired resistance to the next generation ALK inhibitor alectinib

    PubMed Central

    Katayama, Ryohei; Friboulet, Luc; Koike, Sumie; Lockerman, Elizabeth L.; Khan, Tahsin M.; Gainor, Justin F.; Iafrate, A. John; Takeuchi, Kengo; Taiji, Makoto; Okuno, Yasushi; Fujita, Naoya; Engelman, Jeffrey A.; Shaw, Alice T.

    2014-01-01

    Purpose The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is a standard therapy for patients with ALK-rearranged NSCLC. Several next-generation ALK-TKIs have entered the clinic and have shown promising activity in crizotinib-resistant patients. As patients still relapse even on these next-generation ALK-TKIs, we examined mechanisms of resistance to the next-generation ALK-TKI alectinib and potential strategies to overcome this resistance. Experimental Design We established a cell line model of alectinib resistance, and analyzed a resistant tumor specimen from a patient who had relapsed on alectinib. We developed Ba/F3 models harboring alectinib-resistant ALK mutations and evaluated the potency of other next-generation ALK-TKIs in these models. We tested the antitumor activity of the next-generation ALK-TKI ceritinib in the patient with acquired resistance to alectinib. To elucidate structure-activity-relationships of ALK mutations, we performed computational thermodynamic simulation with MP-CAFEE. Results We identified a novel V1180L gatekeeper mutation from the cell line model and a second novel I1171T mutation from the patient who developed resistance to alectinib. Both ALK mutations conferred resistance to alectinib as well as to crizotinib, but were sensitive to ceritinib and other next-generation ALK-TKIs. Treatment of the patient with ceritinib led to a marked response. Thermodynamics simulation suggests that both mutations lead to distinct structural alterations that decrease the binding affinity with alectinib. Conclusions We have identified two novel ALK mutations arising after alectinib exposure which are sensitive to other next generation ALK-TKIs. The ability of ceritinib to overcome alectinib-resistance mutations suggests a potential role for sequential therapy with multiple next-generation ALK-TKIs. PMID:25228534

  18. A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors

    PubMed Central

    Sasaki, Takaaki; Koivunen, Jussi; Ogino, Atsuko; Yanagita, Masahiko; Nikiforow, Sarah; Zheng, Wei; Lathan, Christopher; Marcoux, J. Paul; Du, Jinyan; Okuda, Katsuhiro; Capelletti, Marzia; Shimamura, Takeshi; Ercan, Dalia; Stumpfova, Magda; Xiao, Yun; Weremowicz, Stanislawa; Butaney, Mohit; Heon, Stephanie; Wilner, Keith; Christensen, James G.; Eck, Michel J.; Wong, Kwok-Kin; Lindeman, Neal; Gray, Nathanael S.; Rodig, Scott J.; Jänne, Pasi A.

    2011-01-01

    Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs), including crizotinib, are effective treatments in preclinical models and in cancer patients with ALK-translocated cancers. However, their efficacy will ultimately be limited by the development of acquired drug resistance. Here we report two mechanisms of ALK TKI resistance identified from, a crizotinib treated non-small cell lung cancer (NSCLC) patient and in a cell line generated from the resistant tumor (DFCI076), and from studying a resistant version of the ALK TKI (TAE684) sensitive H3122 cell line. The crizotinib resistant DFCI076 cell line, harboured a unique L1152R ALK secondary mutation, and was also resistant to the structurally unrelated ALK TKI TAE684. Although the DFCI076 cell line was still partially dependent on ALK for survival, it also contained concurrent co-activation of epidermal growth factor receptor (EGFR) signalling. In contrast, the TAE684 resistant (TR3) H3122 cell line did not contain an ALK secondary mutation but instead harboured co-activation of EGFR signalling. Dual inhibition of both ALK and EGFR was the most effective therapeutic strategy for the DFCI076 and H3122 TR3 cell lines. We further identified a subset (3/50; 6%) of treatment naïve NSCLC patients with ALK rearrangements that also had concurrent EGFR activating mutations. Our studies identify resistance mechanisms to ALK TKIs mediated by both ALK and by a bypass signalling pathway mediated by EGFR. These mechanisms can occur independently, or in the same cancer, suggesting that the combination of both ALK and EGFR inhibitors may represent an effective therapy for these subsets of NSCLC patients. PMID:21791641

  19. ALK and ROS1 as targeted therapy paradigms and clinical implications to overcome crizotinib resistance

    PubMed Central

    Li, Nan; Zhang, Yong; Jing, Pengyu; Chang, Ning; Wu, Jianxiong; Ren, Xinling; Zhang, Jian

    2016-01-01

    During the past decade, more than 10 targetable oncogenic driver genes have been validated in non-small cell lung cancer (NSCLC). Anaplastic lymphoma kinase (ALK) and ROS1 kinase are two new driver genes implicated in ALK- and ROS1-rearranged NSCLC. Inhibition of ALK and ROS1 by crizotinib has been reported to be highly effective and well tolerated in these patients. However, resistance to crizotinib emerges years after treatment, and increasing efforts have been made to overcome this issue. Here, we review the biology of ALK and ROS1 and their roles in cancer progression. We also summarize the ongoing and completed clinical trials validating ALK and ROS1 as targets for cancer treatment. In the last section of the review, we will discuss the molecular mechanisms of crizotinib resistance and focus approaches to overcome it. This review describes an exciting new area of research and may provide new insights for targeted cancer therapies. PMID:26802023

  20. 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

  1. Effectiveness of crizotinib in a patient with ALK IHC-positive/FISH-negative metastatic lung adenocarcinoma.

    PubMed

    Rosoux, A; Pauwels, P; Duplaquet, F; D'Haene, N; Weynand, B; Delos, M; Menon, R; Heukamp, L C; Thunnissen, E; Ocak, S

    2016-08-01

    We report a case of crizotinib effectiveness in a heavily pretreated patient with a metastatic NSCLC initially considered IHC-positive and FISH-negative for ALK rearrangement. After repeated analyses of tumor samples, borderline ALK FISH-positivity (18.5% positive cells) was demonstrated. PMID:27393517

  2. Transformation to SCLC after Treatment with the ALK Inhibitor Alectinib.

    PubMed

    Fujita, Shiro; Masago, Katsuhiro; Katakami, Nobuyuki; Yatabe, Yasushi

    2016-06-01

    We report an anaplastic lymphoma receptor tyrosine kinase gene (ALK)-positive patient who showed a paradoxical response to the ALK inhibitor alectinib; the primary lesion increased in size, whereas other metastatic lesions decreased markedly. A biopsy of the primary lesion confirmed an ALK rearrangement; however, the tumor had transformed histologically into small cell lung cancer. The lack of reports of small cell lung cancer transformation in ALK-positive patients implies that this outcome was unusual; this patient was treated with alectinib, which is more selective and has a greater inhibitory effect than crizotinib. This case may reveal resistance mechanisms that differ according to the agent used for treatment. PMID:26751586

  3. ALK-rearranged non-small cell lung cancers: how best to optimize the safety of crizotinib in clinical practice?

    PubMed

    Girard, Nicolas; Audigier-Valette, Clarisse; Cortot, Alexis B; Mennecier, Bertrand; Debieuvre, Didier; Planchard, David; Zalcman, Gérard; Moro-Sibilot, Denis; Cadranel, Jacques; Barlési, Fabrice

    2015-02-01

    Crizotinib (XALKORI™, Pfizer) is a tyrosine kinase inhibitor targeting ALK, MET and ROS1, currently approved for the treatment of adults with ALK-rearranged non-small-cell lung cancer. Optimizing the management of frequent crizotinib-related adverse events is crucial to ensure its continuous administration and reproduce the response and survival rates reported in clinical trials. Here, we propose some practical measures, which are mostly derived from the recommendations given to the investigators of the PROFILE 1001, 1005, 1007 and 1014 trials and are based on experience and scientific findings regarding the management of these disorders. While visual disturbances or bradycardia are frequent but benign, the severity of the cardiac and hepatic adverse events requires special attention potential to QT interval prolongations and to the monitoring of electrolyte levels and liver function, taking into account potential drug-drug interactions. PMID:25413260

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

    PubMed

    Mussat, Elodie; Giraud, Violaine; Julie, Catherine; Chinet, Thierry; Leprieur, Etienne Giroux

    2016-03-01

    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

  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. Native and rearranged ALK copy number and rearranged cell count in NSCLC: Implications for ALK inhibitor therapy

    PubMed Central

    Camidge, D. Ross; Skokan, Margaret; Kiatsimkul, Porntip; Helfrich, Barbara; Lu, Xian; Barón, Anna E.; Schulte, Nathan; Maxson, DeLee; Aisner, Dara L.; Franklin, Wilbur A.; Doebele, Robert C.; Varella-Garcia, Marileila

    2013-01-01

    Background Anaplastic Lymphoma Kinase positive (ALK+) non-small cell lung cancer (NSCLC) responds to ALK inhibitors. Clinically, ≥ 15% cells showing rearrangements by break-apart FISH classify tumors as positive. Increases in native and rearranged ALK copy number also occur. Methods 1426 NSCLC clinical specimens (174 ALK+ and 1252 ALK negative), and 24 ALK negative NSCLC cell lines were investigated. ALK copy number and genomic status were assessed by FISH. Results Clinical specimens with 0–9%, 10–15%, 16–30%, 31–50% and >50% of ALK+ cells were found in 79.3%, 8.5%, 1.4%, 2.7% and 8.1% of cases, respectively. Increased native ALK copy number (≥3 copies/cell in ≥40% cells) was detected in 19% of ALK+ and 62% of ALK negative tumors. In ALK negative tumors, abundant focal amplification of native ALK was rare (0.8%). Other atypical patterns occurred in ~6% of tumors. Mean native ALK copy number ranged from 2.1–6.9 in cell lines and was not correlated with crizotinib sensitivity (IC50s 0.34–2.8 uM) (r=0.279, p=0.1764). Neither native, nor rearranged ALK copy number, nor percentage cells positive correlated with extra-central nervous system progression free survivalin ALK+ patients on crizotinib. Conclusions 8.5% of cases are below the established positivity threshold by ≤5%. Further investigation of ALK by other diagnostic techniques in such cases may be warranted. Native ALK copy number increases alone are not associated with sensitivity to ALK inhibition in vitro. However, rare complex patterns of increased native ALK in patients should be studied further as atypical rearrangements contained within these may otherwise be missed. PMID:24022839

  8. Durable brain response with pulse-dose crizotinib and ceritinib in ALK-positive non-small cell lung cancer compared with brain radiotherapy.

    PubMed

    Dudnik, Elizabeth; Siegal, Tali; Zach, Leor; Allen, Aaron M; Flex, Dov; Yust-Katz, Shlomit; Limon, Dror; Hirsch, Fred R; Peled, Nir

    2016-04-01

    Crizotinib achieves excellent systemic control in anaplastic lymphoma kinase-rearranged (ALK+) non-small cell lung cancer (NSCLC); however, central nervous system (CNS) metastases frequently occur as an early event. Whole brain irradiation, the standard treatment, results in neurocognitive impairment. We present a case series of three ALK+ NSCLC patients with progressing CNS metastases who were treated with pulse-dose crizotinib followed by ceritinib. Three ALK+ NSCLC patients treated between 2011 and 2014 (two males, two never smokers, age range 20-54years, all echinoderm microtubule-associated protein-like 4/ALK rearrangement), were diagnosed with progressing cerebral disease while receiving crizotinib. Clinico-pathological characteristics, treatments, and outcomes were analyzed. In two patients the progression was limited to the CNS, and radiological evidence of leptomeningeal spread was present in one patient. Sequential use of crizotinib 500mg administered once daily (pulse-dose) followed by ceritinib on progression achieved control of the disease in the CNS for over 18 months and over 7 months in Patient 1 and Patient 2, respectively. This strategy provided durable CNS control after whole-brain radiotherapy failure in Patient 1, and allowed the whole-brain radiotherapy to be deferred in Patient 2. Limited CNS progression was documented in Patient 3 while he was on standard-dose/pulse-dose crizotinib for 15months; durable (over 7 months) complete remission was achieved with stereotactic radiotherapy and ceritinib. Manipulating the crizotinib schedule in ALK+ NSCLC patients with CNS metastases and using a novel ALK-inhibitor at the time of further progression may provide durable CNS control and allow brain radiotherapy to be deferred. PMID:26677785

  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. Lung adenocarcinoma harboring concomitant SPTBN1-ALK fusion, c-Met overexpression, and HER-2 amplification with inherent resistance to crizotinib, chemotherapy, and radiotherapy.

    PubMed

    Gu, Fei-Fei; Zhang, Yong; Liu, Yang-Yang; Hong, Xiao-Hua; Liang, Jin-Yan; Tong, Fan; Yang, Jing-Song; Liu, Li

    2016-01-01

    Crizotinib is a multi-targeted tyrosine kinase inhibitor (TKI) with activity against mesenchymal-epithelial transition factor (MET) and anaplastic lymphoma kinase (ALK). However, the concomitant oncogenic drivers may affect the sensitivity of crizotinib. Herein, we present a 69-year-old never-smoker Chinese male with advanced lung adenocarcinoma harboring concomitant spectrin beta non-erythrocytic 1 (SPTBN1)-ALK fusion, c-Met overexpression, and human epidermal growth factor receptor-2 (HER-2) amplification with inherent resistance to crizotinib, chemotherapy, and radiotherapy. Although the patient received timely and comprehensive treatment, the overall survival was only 8 months. Therefore, c-Met overexpression, HER-2 gene amplification, and SPTBN1-ALK gene fusion can coexist in lung adenocarcinoma and may become a potential biomarker of cancer refractory to crizotinib, chemotherapy, and radiotherapy as well as of a relatively poor prognosis. In addition, the novel SPTBN1-ALK fusion gene may become a potential target for anti-tumor therapy. PMID:27496196

  11. 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.

  12. [Modalities of use of ceritinib (Zykadia™), a 2nd generation ALK inhibitor, in advanced stage non-small cell lung cancer].

    PubMed

    Giroux Leprieur, Etienne; Fallet, Vincent; Wislez, Marie

    2015-12-01

    Around 4% of advanced non-small cell lung cancers (NSCLC) harbor a ALK rearrangement, with high sensitivity to ALK inhibitor as crizotinib. However, the vast majority of these tumors end with a tumor progression after several months of treatment with crizotinib. Ceritinib is a 2nd generation ALK inhibitor, which showed high efficiency in NSCLC with ALK rearrangement. Results from phase I trial showed a response rate at 58% in these tumors, with a similar rate for previously crizotinib-treated patients or crizotinib-naïve patients. Moreover, cerebral responses were observed with ceritinib. Preliminary date from a phase 2 trial confirmed these results. These promising results allowed a European marketing authorization (autorisation de mise sur le marché [AMM]) since May 2015 for the treatment of advanced NSCLC with ALK rearrangement and resistance or intolerance to crizotinib. PMID:26597476

  13. 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

  14. New therapeutic strategies in neuroblastoma: combined targeting of a novel tyrosine kinase inhibitor and liposomal siRNAs against ALK

    PubMed Central

    Di Paolo, Daniela; Yang, D.; Pastorino, Fabio; Emionite, Laura; Cilli, Michele; Daga, Antonio; Destefanis, Elisa; Di Fiore, Annarita; Piaggio, Francesca; Brignole, Chiara; Xu, Xiaobao; Liang, Chris; Gibbons, James

    2015-01-01

    Many different aberrations in the Anaplastic Lymphoma Kinase (ALK) were found to be oncogenic drivers in several cancers including neuroblastoma (NB), therefore ALK is now considered a critical player in NB oncogenesis and a promising therapeutic target. The ALK-inhibitor crizotinib has a limited activity against the various ALK mutations identified in NB patients. We tested: the activity of the novel ALK-inhibitor X-396 administered alone or in combination with Targeted Liposomes carrying ALK-siRNAs (TL[ALK-siRNA]) that are active irrespective of ALK gene mutational status; the pharmacokinetic profiles and the biodistribution of X-396; the efficacy of X-396 versus crizotinib treatment in NB xenografts; whether the combination of X-396 with the TL[ALK-siRNA] could promote long-term survival in NB mouse models. X-396 revealed good bioavailability, moderate half-life, high mean plasma and tumor concentrations. X-396 was more effective than crizotinib in inhibiting in vitro cell proliferation of NB cells and in reducing tumor volume in subcutaneous NB models in a dose-dependent manner. In orthotopic NB xenografts, X-396 significantly increased life span independently of the ALK mutation status. In combination studies, all effects were significantly improved in the mice treated with TL[ALK-siRNA] and X-396 compared to mice receiving the single agents. Our findings provide a rational basis to design innovative molecular-based treatment combinations for clinical application in ALK-driven NB tumors. PMID:26299615

  15. 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

  16. ALK inhibitors in non-small cell lung cancer: the latest evidence and developments

    PubMed Central

    Sullivan, Ivana; Planchard, David

    2016-01-01

    The treatment of patients with advanced non-small cell lung cancer (NSCLC) harbouring chromosomal rearrangements of ALK (anaplastic lymphoma kinase) was revolutionized by crizotinib, a small molecule inhibitor of ALK, ROS1 and MET. Unfortunately, the disease progressed within the first 12 months in most of the patients because of the development of crizotinib resistance in the majority of patients and the emergence of acquired resistance mutations in most of them. Many of them had been reported even before its approval leading to the rapid development of second-generation ALK inhibitors for crizotinib-resistant NSCLC. In the last few years, novel potent ALK inhibitors with promising results and a good toxicity profile have become available: ceritinib (LDK378), alectinib (RG7853/AF-802/RO5424802/CH5424802), brigatinib (AP26113), entrectinib (RXDX-101, NMS-E628), PF-06463922, ASP3026, TSR-011, X-376/X-396 and CEP-28122/CEP-37440. Moreover, HSP90 (90 kDa heat shock protein) inhibitors have demonstrated clinical activity in patients with ALK+ NSCLC. This review focuses on the molecular and clinical properties of this new generation of ALK inhibitors under development in the clinic. PMID:26753004

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

    PubMed Central

    Manshouri, Roxsan; Shi, Ping; Amin, Hesham M.

    2014-01-01

    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. PMID:25026277

  18. Non-Small Cell Lung Cancer Cells Acquire Resistance to the ALK Inhibitor Alectinib by Activating Alternative Receptor Tyrosine Kinases.

    PubMed

    Isozaki, Hideko; Ichihara, Eiki; Takigawa, Nagio; Ohashi, Kadoaki; Ochi, Nobuaki; Yasugi, Masayuki; Ninomiya, Takashi; Yamane, Hiromichi; Hotta, Katsuyuki; Sakai, Katsuya; Matsumoto, Kunio; Hosokawa, Shinobu; Bessho, Akihiro; Sendo, Toshiaki; Tanimoto, Mitsune; Kiura, Katsuyuki

    2016-03-15

    Crizotinib is the standard of care for advanced non-small cell lung cancer (NSCLC) patients harboring the anaplastic lymphoma kinase (ALK) fusion gene, but resistance invariably develops. Unlike crizotinib, alectinib is a selective ALK tyrosine kinase inhibitor (TKI) with more potent antitumor effects and a favorable toxicity profile, even in crizotinib-resistant cases. However, acquired resistance to alectinib, as for other TKIs, remains a limitation of its efficacy. Therefore, we investigated the mechanisms by which human NSCLC cells acquire resistance to alectinib. We established two alectinib-resistant cell lines that did not harbor the secondary ALK mutations frequently occurring in crizotinib-resistant cells. One cell line lost the EML4-ALK fusion gene, but exhibited increased activation of insulin-like growth factor-1 receptor (IGF1R) and human epidermal growth factor receptor 3 (HER3), and overexpressed the HER3 ligand neuregulin 1. Accordingly, pharmacologic inhibition of IGF1R and HER3 signaling overcame resistance to alectinib in this cell line. The second alectinib-resistant cell line displayed stimulated HGF autocrine signaling that promoted MET activation and remained sensitive to crizotinib treatment. Taken together, our findings reveal two novel mechanisms underlying alectinib resistance that are caused by the activation of alternative tyrosine kinase receptors rather than by secondary ALK mutations. These studies may guide the development of comprehensive treatment strategies that take into consideration the various approaches ALK-positive lung tumors use to withstand therapeutic insult. PMID:26719536

  19. Crizotinib

    MedlinePlus

    Crizotinib comes as a capsule to take by mouth. It is usually taken with or without food ... medications such as itraconaozole (Sporanox), ketoconazole (Nizoral), and voriconazole (Vfend); chloroquine (Aralen); chlorpromazine (Thorazine); citalopram (Celexa); cyclosporine ( ...

  20. Alectinib: a novel second generation anaplastic lymphoma kinase (ALK) inhibitor for overcoming clinically-acquired resistance

    PubMed Central

    Song, Zilan; Wang, Meining; Zhang, Ao

    2015-01-01

    The development of inhibitors for the tyrosine anaplastic lymphoma kinase (ALK) has advanced rapidly, driven by biology and medicinal chemistry. The first generation ALK inhibitor crizotinib was granted US FDA approval with only four years of preclinical and clinical testing. Although this drug offers significant clinical benefit to the ALK-positive patients, resistance has been developed through a variety of mechanisms. In addition to ceritinib, alectinib is another second-generation ALK inhibitor launched in 2014 in Japan. This drug has a unique chemical structure bearing a 5H-benzo[b]carbazol-11(6H)-one structural scaffold with an IC50 value of 1.9 nmol/L, and is highly potent against ALK bearing the gatekeeper mutation L1196M with an IC50 of 1.56 nmol/L. In the clinic, alectinib is highly efficacious in treatment of ALK-positive non-small cell lung cancer (NSCLC), and retains potency to combat crizotinib-resistant ALK mutations L1196M, F1174L, R1275Q and C1156Y. PMID:26579422

  1. 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

  2. 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

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

    2016-05-01

    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

  3. 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

  4. 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

  5. ALK inhibitor resistance in ALK(F1174L)-driven neuroblastoma is associated with AXL activation and induction of EMT.

    PubMed

    Debruyne, D N; Bhatnagar, N; Sharma, B; Luther, W; Moore, N F; Cheung, N-K; Gray, N S; George, R E

    2016-07-14

    The crizotinib-resistant ALK(F1174L) mutation arises de novo in neuroblastoma (NB) and is acquired in ALK translocation-driven cancers, lending impetus to the development of novel anaplastic lymphoma kinase (ALK) inhibitors with different modes of action. The diaminopyrimidine TAE684 and its derivative ceritinib (LDK378), which are structurally distinct from crizotinib, are active against NB cells expressing ALK(F1174L). Here we demonstrate acquired resistance to TAE684 and LDK378 in ALK(F1174L)-driven human NB cells that is linked to overexpression and activation of the AXL tyrosine kinase and epithelial-to-mesenchymal transition (EMT). AXL phosphorylation conferred TAE684 resistance to NB cells through upregulated extracellular signal-regulated kinase (ERK) signaling. Inhibition of AXL partly rescued TAE684 resistance, resensitizing these cells to this compound. AXL activation in resistant cells was mediated through increased expression of the active form of its ligand, GAS6, that also served to stabilize the AXL protein. Although ectopic expression of AXL and TWIST2 individually in TAE684-sensitive parental cells led to the elevated expression of mesenchymal markers and invasive capacity, only AXL overexpression induced resistance to TAE684 as well. TAE684-resistant cells showed greater sensitivity to HSP90 inhibition than did their parental counterparts, with downregulation of AXL and AXL-mediated ERK signaling. Our studies indicate that aberrant AXL signaling and development of an EMT phenotype underlie resistance of ALK(F1174L)-driven NB cells to TAE684 and its derivatives. We suggest that the combination of ALK and AXL or HSP90 inhibitors be considered to delay the emergence of such resistance. PMID:26616860

  6. 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

  7. Alectinib for choroidal metastasis in a patient with crizotinib-resistant ALK rearranged positive non-small cell lung cancer.

    PubMed

    Okuma, Yusuke; Tanaka, Yuichiro; Kamei, Tina; Hosomi, Yukio; Okamura, Tatsuru

    2015-01-01

    Choroidal metastasis is rare in cancer patients. Small molecules of molecular targeted agents for lung cancer with actionable mutations were reported to be palliated for symptoms caused by choroidal metastasis. Visual disturbance by choroidal metastasis significantly decreases quality of life during the patient's remaining lifespan; therefore, radiotherapy or laser photocoagulation is proposed with consensus. However, improvement in survival with matched molecular targeted agents for oncogenic driver mutations reminds us to also be concerned with late treatment toxicities. A 30-year-old female patient previously treated with crizotinib harboring ALK rearranged non-small cell lung cancer complained of visual disturbance, fever, and bone pains undergoing anti-PD-1 antibody treatment. A decreased proportion of ALK fusion was demonstrated by fluorescence in situ hybridization in liver metastasis compared to the primary site in a chemo-naïve state. She was diagnosed with low vision, choroidal metastasis and retinal detachment. Therefore, she started alectinib treatment and both her ocular and systemic symptoms were palliated in a week. Later, she temporarily discontinued alectinib because of skin rash although the choroidal metastasis and retinal detachment resolved and she regained low vision completely at 2 weeks. She obtained partial response with alectinib for more than 5 months after recovering from skin rash. PMID:26082648

  8. Treatment of ALK-Rearranged Non-Small Cell Lung Cancer: Recent Progress and Future Directions.

    PubMed

    Cameron, Laird; Solomon, Benjamin

    2015-07-01

    Rearrangements of the anaplastic lymphoma kinase (ALK) gene originally discovered nearly 20 years ago in the context of anaplastic large cell lymphoma were identified as oncogenic drivers in a subset of non-small cell lung cancers (NSCLCs) in 2007. These ALK gene rearrangements are present in 3-5 % of NSCLC patients, typically younger, never or light smokers with adenocarcinomas. Crizotinib is a first-in-class ALK tyrosine kinase inhibitor with significant activity in ALK-positive NSCLC that received accelerated US Food and Drug Administration approval for treatment of ALK-positive NSCLC in 2011, just 4 years after identification of ALK rearrangements in this setting. Subsequently, two phase III trials have shown crizotinib to have a tolerable toxicity profile and to be superior to standard chemotherapy for the first- or second-line treatment of advanced ALK-positive lung cancer and numerous countries have approved its use. Despite initial responses, acquired resistance to crizotinib invariably leads to disease progression. Mechanisms of resistance have been described to include ALK tyrosine kinase mutations, activation of bypass signalling pathways and pharmacokinetic failure of crizotinib. Several next-generation ALK inhibitors, including ceritinib and alectinib, are in clinical development and show efficacy in both the crizotinib naïve and crizotinib refractory settings. Ongoing clinical trials will identify the optimal strategy to incorporate these novel agents in the treatment of patients with ALK-positive NSCLC. PMID:26076736

  9. 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

  10. 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

  11. Ability of the Met Kinase Inhibitor Crizotinib and New Generation EGFR Inhibitors to Overcome Resistance to EGFR Inhibitors

    PubMed Central

    Nanjo, Shigeki; Yamada, Tadaaki; Nishihara, Hiroshi; Takeuchi, Shinji; Sano, Takako; Nakagawa, Takayuki; Ishikawa, Daisuke; Zhao, Lu; Ebi, Hiromichi; Yasumoto, Kazuo; Matsumoto, Kunio; Yano, Seiji

    2013-01-01

    Purpose Although EGF receptor tyrosine kinase inhibitors (EGFR-TKI) have shown dramatic effects against EGFR mutant lung cancer, patients ultimately develop resistance by multiple mechanisms. We therefore assessed the ability of combined treatment with the Met inhibitor crizotinib and new generation EGFR-TKIs to overcome resistance to first-generation EGFR-TKIs. Experimental Design Lung cancer cell lines made resistant to EGFR-TKIs by the gatekeeper EGFR-T790M mutation, Met amplification, and HGF overexpression and mice with tumors induced by these cells were treated with crizotinib and a new generation EGFR-TKI. Results The new generation EGFR-TKI inhibited the growth of lung cancer cells containing the gatekeeper EGFR-T790M mutation, but did not inhibit the growth of cells with Met amplification or HGF overexpression. In contrast, combined therapy with crizotinib plus afatinib or WZ4002 was effective against all three types of cells, inhibiting EGFR and Met phosphorylation and their downstream molecules. Crizotinib combined with afatinib or WZ4002 potently inhibited the growth of mouse tumors induced by these lung cancer cell lines. However, the combination of high dose crizotinib and afatinib, but not WZ4002, triggered severe adverse events. Conclusions Our results suggest that the dual blockade of mutant EGFR and Met by crizotinib and a new generation EGFR-TKI may be promising for overcoming resistance to reversible EGFR-TKIs but careful assessment is warranted clinically. PMID:24386407

  12. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer

    PubMed Central

    Koivunen, Jussi P.; Mermel, Craig; Zejnullahu, Kreshnik; Murphy, Carly; Lifshits, Eugene; Holmes, Alison J.; Choi, Hwan Geun; Kim, Jhingook; Chiang, Derek; Thomas, Roman; Lee, Jinseon; Richards, William G.; Sugarbaker, David J.; Ducko, Christopher; Lindeman, Neal; Marcoux, J. Paul; Engelman, Jeffrey A.; Gray, Nathanael S.; Lee, Charles; Meyerson, Matthew; Jänne, Pasi A.

    2011-01-01

    Purpose The EML4-ALK fusion gene has been detected in ~7% of Japanese non-small cell lung cancers (NSCLC). We determined the frequency of EML4-ALK in Caucasian NSCLCs and in NSCLC cell lines. We also determined whether TAE684, a specific ALK kinase inhibitor, would inhibit the growth of EML4-ALK containing cell lines in vitro and in vivo. Experimental Design We screened 305 primary NSCLCs (both US (n=138) and Korean (n=167) patients) and 83 NSCLC cell lines using RT-PCR and by exon array analyses. We evaluated the efficacy of TAE684 against NSCLC cell lines in vitro and in vivo. Results We detected 4 different variants, including two novel variants, of EML4-ALK using RT-PCR in 8/305 tumors (3%) and in 3/83 (3.6%) NSCLC cell lines. All EML4-ALK containing tumors and cell lines were adenocarcinomas. EML4-ALK was detected more frequently in NSCLC patients who were never or light (< 10 pack years) cigarette smokers compared to current/former smokers (6% vs. 1%; p=0.049). TAE684 inhibited the growth of 1 of 3 (H3122) EML4-ALK containing cell lines in vitro and in vivo, inhibited Akt phosphorylation and caused apoptosis. In another EML4-ALK cell line, DFCI032, TAE684 was ineffective due to co-activation of EGFR and ERBB2. The combination of TAE684 and CL-387,785 (EGFR/ERBB2 kinase inhibitor), inhibited growth and Akt phosphorylation and led to apoptosis in the DFCI032 cell line. Conclusions EML4-ALK is found in the minority of NSCLCs. ALK kinase inhibitors alone or in combination may nevertheless be clinically effective treatments for NSCLC patients whose tumors contain EML4-ALK. PMID:18594010

  13. 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

  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. Personalized treatment options for ALK-positive metastatic non-small-cell lung cancer: potential role for Ceritinib

    PubMed Central

    El-Osta, Hazem; Shackelford, Rodney

    2015-01-01

    The fusion of echinoderm microtubule-associated protein-like 4 with the anaplastic lymphoma kinase (EML4-ALK) is found in 3%–7% of non-small-cell lung cancer (NSCLC) cases and confers sensitivity to crizotinib, the first United States Food and Drug Administration (FDA)-approved ALK inhibitor drug. Although crizotinib has an excellent initial therapeutic effect, acquired resistance to this drug invariably develops within the first year of treatment. Resistance may involve secondary gatekeeper mutations within the ALK gene interfering with crizotinib–ALK interactions, or compensatory activation of aberrant bypass signaling pathways. New therapeutic strategies to overcome crizotinib resistance are needed. Ceritinib, a second-generation ALK inhibitor, overcomes several crizotinib-resistant ALK mutations and has demonstrated efficacy against tumor growth in several in vitro and in vivo preclinical models of crizotinib resistance. Notably, the dose-escalation Phase I ASCEND-1 trial has shown a marked activity of ceritinib in both crizotinib-naïve and crizotinib-resistant ALK-rearranged lung cancer. The overall response rate was 58% in a subgroup of patients with ALK-rearranged late-stage NSCLC. Drug discontinuation rate due to toxicity was 10%. The standard dose was established at 750 mg daily. This paper outlines the pathogenesis and treatment of ALK-positive lung cancer, focuses on the preclinical and clinical results surrounding the accelerated FDA approval of ceritinib for the treatment of ALK-positive metastatic NSCLC patients who have progressed on/or are crizotinib intolerant, and discusses the potential efforts seeking to maximize ceritinib efficacy and expand its usage to other indications in cancer therapy. PMID:26622190

  16. Complete and Repeated Response of a Metastatic ALK-rearranged Inflammatory Myofibroblastic Tumor to Crizotinib in a Teenage Girl.

    PubMed

    Gaudichon, Jérémie; Jeanne-Pasquier, Corinne; Deparis, Marianna; Veyssière, Alexis; Heyndrickx, Maxime; Minckes, Odile; Orbach, Daniel

    2016-05-01

    Inflammatory myofibroblastic tumors (IMT) are rare tumors in children and young adults, considered by the World Health Organization to be intermediate malignancies and rarely metastasizing, with the presence of an anaplastic lymphoma kinase rearrangement in about 50% of the cases. We report the case of a teenager who presented with a metastatic aggressive IMT that was life-threatening despite multiple treatments, and which responded repeatedly to anaplastic lymphoma kinase-targeted crizotinib therapy. Crizotinib induced drastic primary tumor regression, which was sufficient to allow surgical resection and to control distant disease. This case shows that crizotinib is a promising therapy in IMT, even in adolescents and young adults. PMID:26808369

  17. 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. PMID:26517679

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26517679

  19. 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

  20. 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

  1. ALK inhibitor resistance in ALKF1174L-driven neuroblastoma is associated with AXL activation and induction of EMT

    PubMed Central

    Debruyne, D N; Bhatnagar, N; Sharma, B; Luther, W; Moore, N F; Cheung, N-K; Gray, N S; George, R E

    2016-01-01

    The crizotinib-resistant ALKF1174L mutation arises de novo in neuroblastoma (NB) and is acquired in ALK translocation-driven cancers, lending impetus to the development of novel anaplastic lymphoma kinase (ALK) inhibitors with different modes of action. The diaminopyrimidine TAE684 and its derivative ceritinib (LDK378), which are structurally distinct from crizotinib, are active against NB cells expressing ALKF1174L. Here we demonstrate acquired resistance to TAE684 and LDK378 in ALKF1174L-driven human NB cells that is linked to overexpression and activation of the AXL tyrosine kinase and epithelial-to-mesenchymal transition (EMT). AXL phosphorylation conferred TAE684 resistance to NB cells through upregulated extracellular signal-regulated kinase (ERK) signaling. Inhibition of AXL partly rescued TAE684 resistance, resensitizing these cells to this compound. AXL activation in resistant cells was mediated through increased expression of the active form of its ligand, GAS6, that also served to stabilize the AXL protein. Although ectopic expression of AXL and TWIST2 individually in TAE684-sensitive parental cells led to the elevated expression of mesenchymal markers and invasive capacity, only AXL overexpression induced resistance to TAE684 as well. TAE684-resistant cells showed greater sensitivity to HSP90 inhibition than did their parental counterparts, with downregulation of AXL and AXL-mediated ERK signaling. Our studies indicate that aberrant AXL signaling and development of an EMT phenotype underlie resistance of ALKF1174L-driven NB cells to TAE684 and its derivatives. We suggest that the combination of ALK and AXL or HSP90 inhibitors be considered to delay the emergence of such resistance. PMID:26616860

  2. 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

  3. 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

  4. 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.

  5. Anaplastic Lymphoma Kinase (ALK) Signaling in Lung Cancer.

    PubMed

    Ou, Sai-Hong Ignatius; Shirai, Keisuke

    2016-01-01

    Chromosomal rearrangement in the anaplastic lymphoma kinase (ALK) gene was identified as an oncogenic driver in non-small cell lung cancer (NSCLC) in 2007. A multi-targeted ALK/ROS1/MET inhibitor, crizotinib, targeting this activated tyrosine kinase has led to significant clinical benefit including tumor shrinkage and prolonged survival without disease progression and has been approved by US FDA since 2011 for the treatment of advanced ALK-rearranged NSCLC (Ou et al. Oncologist 17:1351-1375, 2012). Knowledge gained from treating ALK-rearranged NSCLC patients including the presenting clinicopathologic characteristics, methods of detecting ALK-rearranged NSCLC, pattern of relapse and acquired resistance mechanisms while on crizotinib, and the clinical activities of more potent ALK inhibitors has led us to a detailed and ever expanding knowledge of the ALK signaling pathway in lung cancer but also raising many more questions that remained to be answered in the future. This book chapter will provide a concise summary of the importance of ALK signaling pathway in lung cancer. Understanding the ALK signaling pathway in lung cancer will likely provide the roadmap to the management of major epithelial malignancies driven by receptor tyrosine kinase rearrangement. PMID:26667344

  6. Treatment for ALK-mutated non-small-cell lung cancer: a new miracle in the research race.

    PubMed

    de Castro-Carpeño, J; Perona, R; Belda-Iniesta, C

    2011-11-01

    The discovery of anaplastic lymphoma kinase (ALK) rearrangements in a subset of patients with nonsmall- cell lung cancer (NSCLC) and its potential blockage by specific inhibitors such as crizotinib has been one of the latest advances in the treatment of this disease. In this article, we will review the most important clinical aspects of ALK alterations in NSCLC patients and the pending questions to answer: the most effective means of diagnosing ALK-rearranged NSCLC, and efficacy, toxicity profile and potential mechanisms of resistance to crizotinib. PMID:22082640

  7. Resistance mechanisms after tyrosine kinase inhibitors afatinib and crizotinib in non-small cell lung cancer, a review of the literature.

    PubMed

    van der Wekken, A J; Saber, A; Hiltermann, T J N; Kok, K; van den Berg, A; Groen, H J M

    2016-04-01

    Targeted treatment of advanced non-small cell lung cancer patients with afatinib in EGFR mutation or crizotinib in ALK break positive patients results in profound tumor responses but inevitably induces resistance. In this review we present currently known resistance mechanisms for afatinib and crizotinib two recently approved drugs. Resistance mechanisms identified for afatinib include c-MET amplification and the V843I EGFR mutation. Expression of FGFR1, increased IL6R/JAK/STAT signaling, enhanced interference with aerobic glycolysis and autophagy are associated with resistance to afatinib. Most common resistance mechanisms for ALK break positive cases are gatekeeper mutations in the ALK gene. Also activation of the EGFR pathway, KRAS mutations, the autophagy pathway and epithelial mesenchymal transition (EMT), have been associated with resistance. Many of the proposed resistance mechanisms need to be functionally studied to proof a causative relationship with resistance. PMID:26852079

  8. Ceritinib for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer.

    PubMed

    Landi, Lorenza; Cappuzzo, Federico

    2016-02-01

    Non-small cell lung cancer (NSCLC) represents the paradigm of personalized treatment of human cancer. Several oncogenic druggable alterations have been so far identified, with anaplastic lymphoma kinase (ALK) gene rearrangements representing one of the newest and most appealing. Crizotinib is now recognized as the standard of care in ALK-positive NSCLC due to the positive results of recently published trials. Unfortunately, resistance inevitably occurs within the first year of treatment. Overcoming resistance is the major challenge in clinical oncology, and novel potent ALK inhibitors are currently under evaluation, including ceritinib. Ceritinib is an oral, potent, second-generation ALK inhibitor demonstrating activity in patients who develop resistance to crizotinib. Recent data also suggested efficacy in ALK-inhibitor-naive population, thus supporting investigation of the drug in front-line setting. PMID:26582431

  9. Cabozantinib overcomes crizotinib resistance in ROS1 fusion positive cancer

    PubMed Central

    Katayama, Ryohei; Kobayashi, Yuka; Friboulet, Luc; Lockerman, Elizabeth. L.; Koike, Sumie; Shaw, Alice T.; Engelman, Jeffrey A.; Fujita, Naoya

    2014-01-01

    Purpose ROS1 rearrangement leads to constitutive ROS1 activation with potent transforming activity. In an ongoing phase 1 trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib shows remarkable initial responses in patients with non-small cell lung cancer (NSCLC) harboring ROS1 fusions; however, cancers eventually develop crizotinib resistance due to acquired mutations such as G2032R in ROS1. Thus, understanding the crizotinib resistance mechanisms in ROS1 rearranged NSCLC and identification of therapeutic strategies to overcome the resistance are required. Experimental Design The sensitivity of CD74-ROS1-transformed Ba/F3 cells to multiple ALK inhibitors was examined. Acquired ROS1 inhibitor resistant mutations in CD74-ROS1 fusion were screened by N-ethyl-N-nitrosourea mutagenesis with Ba/F3 cells. To overcome the resistance mutation, we performed high throughput drug screening with small molecular inhibitors and anticancer drugs used in clinical practice or being currently tested in clinical trials. The effect of the identified drug was assessed in the CD74-ROS1 mutant Ba/F3 cells and crizotinib resistant patient-derived cancer cells (MGH047) harboring G2032R mutated CD74-ROS1. Results We identified multiple novel crizotinib resistance mutations in the ROS1 kinase domain including the G2032R mutation. As the result of high-throughput drug screening, we found that the cMET/RET/VEGFR inhibitor cabozantinib (XL184) effectively inhibited the survival of CD74-ROS1-WT and resistant mutants harboring Ba/F3 and MGH047 cells. Furthermore, cabozantinib could overcome all the resistance by all newly identified secondary mutations. Conclusions We developed a comprehensive model of acquired resistance to ROS1 inhibitors in NSCLC with ROS1 rearrangement and identified cabozantinib as a therapeutic strategy to overcome the resistance. PMID:25351743

  10. Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications.

    PubMed

    Ardini, Elena; Menichincheri, Maria; Banfi, Patrizia; Bosotti, Roberta; De Ponti, Cristina; Pulci, Romana; Ballinari, Dario; Ciomei, Marina; Texido, Gemma; Degrassi, Anna; Avanzi, Nilla; Amboldi, Nadia; Saccardo, Maria Beatrice; Casero, Daniele; Orsini, Paolo; Bandiera, Tiziano; Mologni, Luca; Anderson, David; Wei, Ge; Harris, Jason; Vernier, Jean-Michel; Li, Gang; Felder, Eduard; Donati, Daniele; Isacchi, Antonella; Pesenti, Enrico; Magnaghi, Paola; Galvani, Arturo

    2016-04-01

    Activated ALK and ROS1 tyrosine kinases, resulting from chromosomal rearrangements, occur in a subset of non-small cell lung cancers (NSCLC) as well as other tumor types and their oncogenic relevance as actionable targets has been demonstrated by the efficacy of selective kinase inhibitors such as crizotinib, ceritinib, and alectinib. More recently, low-frequency rearrangements of TRK kinases have been described in NSCLC, colorectal carcinoma, glioblastoma, and Spitzoid melanoma. Entrectinib, whose discovery and preclinical characterization are reported herein, is a novel, potent inhibitor of ALK, ROS1, and, importantly, of TRK family kinases, which shows promise for therapy of tumors bearing oncogenic forms of these proteins. Proliferation profiling against over 200 human tumor cell lines revealed that entrectinib is exquisitely potent in vitro against lines that are dependent on the drug's pharmacologic targets. Oral administration of entrectinib to tumor-bearing mice induced regression in relevant human xenograft tumors, including the TRKA-dependent colorectal carcinoma KM12, ROS1-driven tumors, and several ALK-dependent models of different tissue origins, including a model of brain-localized lung cancer metastasis. Entrectinib is currently showing great promise in phase I/II clinical trials, including the first documented objective responses to a TRK inhibitor in colorectal carcinoma and in NSCLC. The drug is, thus, potentially suited to the therapy of several molecularly defined cancer settings, especially that of TRK-dependent tumors, for which no approved drugs are currently available. Mol Cancer Ther; 15(4); 628-39. ©2016 AACR. PMID:26939704

  11. A combination of tyrosine kinase inhibitors, crizotinib and dasatinib for the treatment of glioblastoma multiforme

    PubMed Central

    Nehoff, Hayley; Parayath, Neha N.; McConnell, Melanie J.; Taurin, Sebastien; Greish, Khaled

    2015-01-01

    Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Despite the advances in surgery, radiotherapy and chemotherapy, patient survival averages only 14.6 months. In most GBM tumors, tyrosine kinases show increased activity and/or expression and actively contribute to the development, recurrence and onset of treatment resistance; making their inhibition an appealing therapeutic strategy. We compared the cytotoxicity of 12 tyrosine kinase inhibitors in vitro. A combination of crizotinib and dasatinib emerged as the most cytotoxic across established and primary human GBM cell lines. The combination treatment induced apoptotic cell death and polyploidy. Furthermore, the combination treatment led to the altered expression and localization of several tyrosine kinase receptors such as Met and EGFR and downstream effectors as such as SRC. Furthermore, the combination treatment reduced the migration and invasion of GBM cells and prevented endothelial cell tube formation in vitro. Overall, our study demonstrated the broad specificity of a combination of crizotinib and dasatinib across multiple GBM cell lines. These findings provide insight into the development of alternative therapy for the treatment of GBM. PMID:26517812

  12. Extraordinary response to crizotinib in a woman with squamous cell lung cancer after two courses of failed chemotherapy

    PubMed Central

    2014-01-01

    Background The discovery of the fusion gene echinodermmicro tubule associated proteinlike 4-anaplastic lymphoma kinase, EML4-ALK, in patients with non-small-cell lung cancer has led to the remarkable development of anaplastic lymphoma kinase inhibitors, such as crizotinib. Consequently, the clinical outcomes of these patients have improved dramatically. Herein, we report the case of a woman with ALK gene translocation-squamous cell lung cancer who experienced a remarkable tumor response to crizotinib after two courses of failed chemotherapy. Case presentation A 55-year-old Chinese woman was diagnosed with cervical lymph node metastatic squamous carcinoma. Chest computed tomography scan showed the primary tumor in the lower lobe of the right lung. The patient had received two successive courses of first-line chemotherapy without tumor response. Tumor cells were negative for wild-type of epidermal growth factor receptor/K-RAS variants; thus, she was not eligible for tyrosine kinase inhibitor therapy. Unfortunately, increased levels of interleukin-6 and carcinoembryonic antigen, and computed tomography scan results indicated cancer progression. Once crizotinib was approved by the China Food and Drug Administration and the ALK gene translocation was identified in tumor cells by fluorescent in situ hybridization, the patient commenced treatment with crizotinib. Remarkably, the tumor response to crizotinib was classified as partial response after only 26 days of treatment commencement. The partial response status has been maintained to date (23 weeks). Conclusion Considering this remarkable response to crizotinib, we can safely conclude that patients with squamous cell lung cancer should have the option of undergoing ALK testing to determine if there is indication for crizotinib treatment even after they have failed chemotherapy. PMID:24885608

  13. 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

  14. 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

  15. 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

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

    PubMed

    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

    2016-06-01

    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

  17. Peptides derived from the dependence receptor ALK are proapoptotic for ALK-positive tumors

    PubMed Central

    Aubry, A; Galiacy, S; Ceccato, L; Marchand, C; Tricoire, C; Lopez, F; Bremner, R; Racaud-Sultan, C; Monsarrat, B; Malecaze, F; Allouche, M

    2015-01-01

    ALK is a receptor tyrosine kinase with an oncogenic role in various types of human malignancies. Despite constitutive activation of the kinase through gene alterations, such as chromosomal translocation, gene amplification or mutation, treatments with kinase inhibitors invariably lead to the development of resistance. Aiming to develop new tools for ALK targeting, we took advantage of our previous demonstration identifying ALK as a dependence receptor, implying that in the absence of ligand the kinase-inactive ALK triggers or enhances apoptosis. Here, we synthesized peptides mimicking the proapoptotic domain of ALK and investigated their biological effects on tumor cells. We found that an ALK-derived peptide of 36 amino acids (P36) was cytotoxic for ALK-positive anaplastic large-cell lymphoma and neuroblastoma cell lines. In contrast, ALK-negative tumor cells and normal peripheral blood mononuclear cells were insensitive to P36. The cytotoxic effect was due to caspase-dependent apoptosis and required N-myristoylation of the peptide. Two P36-derived shorter peptides as well as a cyclic peptide also induced apoptosis. Surface plasmon resonance and mass spectrometry analysis of P36-interacting proteins from two responsive cell lines, Cost lymphoma and SH-SY5Y neuroblastoma, uncovered partners that could involve p53-dependent signaling and pre-mRNA splicing. Furthermore, siRNA-mediated knockdown of p53 rescued these cells from P36-induced apoptosis. Finally, we observed that a treatment combining P36 with the ALK-specific inhibitor crizotinib resulted in additive cytotoxicity. Therefore, ALK-derived peptides could represent a novel targeted therapy for ALK-positive tumors. PMID:25950466

  18. 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).

  19. 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

  20. 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.

  1. Quantification and pharmacokinetics of crizotinib in rats by liquid chromatography-tandem mass spectrometry.

    PubMed

    Qiu, Feng; Gu, Yanan; Wang, Tingting; Gao, Yingying; Li, Xiao; Gao, Xiangyu; Cheng, Shan

    2016-06-01

    Crizotinib is a small molecule inhibitor of anaplastic lymphoma kinase (ALK) and can be used to treat ALK-positive nonsmall-cell lung cancer. A rapid and simple high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of crizotinib in rat plasma using a chemical synthetic compound buspirone as the internal standard (IS). The plasma samples were pretreated by a simple protein precipitation with methanol-acetonitrile (1:1, v/v). Chromatographic separation was successfully achieved on an Agilent Zorbax XDB C18 column (2.1 × 50 mm, 3.5 µm). The gradient elution system was composed of 0.1% formic acid aqueous solution and 0.1% formic acid in methanol solution. The flow rate was set at 0.50 mL/min. The multiple reaction monitoring was based on the transitions of m/z = 450.3 → 177.1 for crizotinib and 386.2 → 122.2 for buspirone (IS). The assay was successfully validated to demonstrate the selectivity, matrix effect, linearity, lower limit of quantification, accuracy, precision, recovery and stability according to the international guidelines. The lower limit of quantification was 1.00 ng/mL in 50 μL of rat plasma. This LC-MS/MS assay was successfully applied to the quantification and pharmacokinetic study of crizotinib in rats after intravenous and oral administration of crizotinib. The oral absolute bioavailability of crizotinib in rats was 68.6 ± 9.63%. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26467669

  2. 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

  3. Testing for anaplastic lymphoma kinase rearrangement to target crizotinib therapy: oncology, pathology and health economic perspectives.

    PubMed

    Lee, James A; Bubendorf, Lukas; Stahel, Rolf; Peters, Solange

    2013-05-01

    Crizotinib is a first-in-class oral anaplastic lymphoma kinase (ALK) inhibitor targeting ALK-rearranged non-small-cell lung cancer. The therapy was approved by the US FDA in August 2011 and received conditional marketing approval by the European Commission in October 2012 for advanced non-small-cell lung cancer. A break-apart FISH-based assay was jointly approved with crizotinib by the FDA. This assay and an immunohistochemistry assay that uses a D5F3 rabbit monoclonal primary antibody were also approved for marketing in Europe in October 2012. While ALK rearrangement has relatively low prevalence, a clinical benefit is exhibited in more than 85% of patients with median progression-free survival of 8-10 months. In this article, the authors summarize the therapy and alternative test strategies for identifying patients who are likely to respond to therapy, including key issues for effective and efficient testing. The key economic considerations regarding the joint companion diagnostic and therapy are also presented. Given the observed clinical benefit and relatively high cost of crizotinib therapy, companion diagnostics should be evaluated relative to response to therapy versus correlation alone whenever possible, and both high inter-rater reliability and external quality assessment programs are warranted. PMID:23617353

  4. 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

  5. 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.

  6. An orally available tyrosine kinase ALK and RET dual inhibitor bearing the tetracyclic benzo[b]carbazolone core.

    PubMed

    Song, Zilan; Xia, Zongjun; Ji, Yinchun; Xing, Li; Gao, Yinglei; Ai, Jing; Geng, Meiyu; Zhang, Ao

    2016-08-01

    Our early structure-activity relationship study has identified benzo[b]carbazolone 6 as a high potency orally bioavailable ALK inhibitor. Further lead profiling disclosed that 6 is active against both ALK resistant and hot spot-activating mutants, and is also highly potent against RET kinase. Tumor stasis and partial tumor regression were achieved with 6 in both NIH/3T3-EML4-ALK and NIH/3T3-EML4-ALK L1196M xenograft models. Based on the optimal in vitro and in vivo antitumor efficacy, compound 6 is now being profiled further in our preclinical settings as a new orally available ALK/RET dual inhibitor. PMID:27131066

  7. Drastic initial response and subsequent response to two ALK inhibitors in a patient with a highly aggressive ALK-rearranged inflammatory myofibroblastic tumor arising in the pleural cavity.

    PubMed

    Ono, Akira; Murakami, Haruyasu; Serizawa, Masakuni; Wakuda, Kazushige; Kenmotsu, Hirotsugu; Naito, Tateaki; Taira, Tetsuhiko; Koh, Yasuhiro; Ohde, Yasuhisa; Nakajima, Takashi; Endo, Masahiro; Takahashi, Toshiaki

    2016-09-01

    A 57-year-old male current smoker was diagnosed with an aggressive variant of ALK-rearranged inflammatory myofibroblastic tumor (IMT) arising in the pleural cavity. First line treatment with ASP3026 was initiated at a dose of 125mg once daily. A follow-up CT scan revealed drastic regression of the pleural lesion. After disease progression with ASP3026 treatment, LDK378 (ceritinib) was initiated at a dose of 750mg once daily. A follow-up CT scan revealed a second drastic regression of the pleural lesion. Furthermore, it is noteworthy that this case represents the use of serum hyaluronan levels to assist in monitoring of treatment efficacy in an IMT. Herein, we present the first case of a patient with a highly aggressive ALK-rearranged IMT arising in the pleural cavity, who showed both initial and subsequent drastic response to two ALK inhibitors while being monitored for serum hyaluronan. PMID:27565932

  8. Identification of the transforming STRN-ALK fusion as a potential therapeutic target in the aggressive forms of thyroid cancer

    PubMed Central

    Kelly, Lindsey M.; Barila, Guillermo; Liu, Pengyuan; Evdokimova, Viktoria N.; Trivedi, Sumita; Panebianco, Federica; Gandhi, Manoj; Carty, Sally E.; Hodak, Steven P.; Luo, Jianhua; Dacic, Sanja; Yu, Yan P.; Nikiforova, Marina N.; Ferris, Robert L.; Altschuler, Daniel L.; Nikiforov, Yuri E.

    2014-01-01

    Thyroid cancer is a common endocrine malignancy that encompasses well-differentiated as well as dedifferentiated cancer types. The latter tumors have high mortality and lack effective therapies. Using a paired-end RNA-sequencing approach, we report the discovery of rearrangements involving the anaplastic lymphoma kinase (ALK) gene in thyroid cancer. The most common of these involves a fusion between ALK and the striatin (STRN) gene, which is the result of a complex rearrangement involving the short arm of chromosome 2. STRN-ALK leads to constitutive activation of ALK kinase via dimerization mediated by the coiled-coil domain of STRN and to a kinase-dependent, thyroid-stimulating hormone–independent proliferation of thyroid cells. Moreover, expression of STRN-ALK transforms cells in vitro and induces tumor formation in nude mice. The kinase activity of STRN-ALK and the ALK-induced cell growth can be blocked by the ALK inhibitors crizotinib and TAE684. In addition to well-differentiated papillary cancer, STRN-ALK was found with a higher prevalence in poorly differentiated and anaplastic thyroid cancers, and it did not overlap with other known driver mutations in these tumors. Our data demonstrate that STRN-ALK fusion occurs in a subset of patients with highly aggressive types of thyroid cancer and provide initial evidence suggesting that it may represent a therapeutic target for these patients. PMID:24613930

  9. Identification of novel inhibitors of the transforming growth factor beta1 (TGF-beta1) type 1 receptor (ALK5).

    PubMed

    Callahan, James F; Burgess, Joelle L; Fornwald, James A; Gaster, Laramie M; Harling, John D; Harrington, Frank P; Heer, Jag; Kwon, Chet; Lehr, Ruth; Mathur, A; Olson, Barbara A; Weinstock, Joseph; Laping, Nicholas J

    2002-02-28

    Screening of our internal compound collection for inhibitors of the transforming growth factor beta1 (TGF-beta1) type I receptor (ALK5) identified several hits. Optimization of the dihydropyrroloimidazole hit 2 by introduction of a 2-pyridine and 3,4-methylenedioxyphenyl group gave 7, a selective ALK5 inhibitor. With this information, optimization of the triarylimidazole hit 8 gave the selective inhibitor 14, which inhibits TGF-beta1-induced fibronectin mRNA formation while displaying no measurable cytotoxicity in the 48 h XTT assay. PMID:11855979

  10. Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors

    PubMed Central

    Lindeman, Neal I.; Cagle, Philip T.; Beasley, Mary Beth; Chitale, Dhananjay Arun; Dacic, Sanja; Giaccone, Giuseppe; Jenkins, Robert Brian; Kwiatkowski, David J.; Saldivar, Juan-Sebastian; Squire, Jeremy; Thunnissen, Erik; Ladanyi, Marc

    2014-01-01

    Objective To establish evidence-based recommendations for the molecular analysis of lung cancers that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. Participants Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies. Evidence Three unbiased literature searches of electronic databases were performed to capture articles published from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation. Consensus Process Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4). Conclusions The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert

  11. Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors

    PubMed Central

    Lindeman, Neal I.; Cagle, Philip T.; Beasley, Mary Beth; Chitale, Dhananjay Arun; Dacic, Sanja; Giaccone, Giuseppe; Jenkins, Robert Brian; Kwiatkowski, David J.; Saldivar, Juan-Sebastian; Squire, Jeremy; Thunnissen, Erik; Ladanyi, Marc

    2014-01-01

    Objective To establish evidence-based recommendations for the molecular analysis of lung cancers that are that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. Participants Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies. Evidence Three unbiased literature searches of electronic databases were performed to capture articles published published from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation. Consensus Process Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4). Conclusions The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed

  12. 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

  13. Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors

    PubMed Central

    Davare, Monika A.; Vellore, Nadeem A.; Wagner, Jacob P.; Eide, Christopher A.; Goodman, James R.; Drilon, Alexander; Deininger, Michael W.; O’Hare, Thomas; Druker, Brian J.

    2015-01-01

    Oncogenic ROS1 fusion proteins are molecular drivers in multiple malignancies, including a subset of non-small cell lung cancer (NSCLC). The phylogenetic proximity of the ROS1 and anaplastic lymphoma kinase (ALK) catalytic domains led to the clinical repurposing of the Food and Drug Administration (FDA)-approved ALK inhibitor crizotinib as a ROS1 inhibitor. Despite the antitumor activity of crizotinib observed in both ROS1- and ALK-rearranged NSCLC patients, resistance due to acquisition of ROS1 or ALK kinase domain mutations has been observed clinically, spurring the development of second-generation inhibitors. Here, we profile the sensitivity and selectivity of seven ROS1 and/or ALK inhibitors at various levels of clinical development. In contrast to crizotinib’s dual ROS1/ALK activity, cabozantinib (XL-184) and its structural analog foretinib (XL-880) demonstrate a striking selectivity for ROS1 over ALK. Molecular dynamics simulation studies reveal structural features that distinguish the ROS1 and ALK kinase domains and contribute to differences in binding site and kinase selectivity of the inhibitors tested. Cell-based resistance profiling studies demonstrate that the ROS1-selective inhibitors retain efficacy against the recently reported CD74-ROS1G2032R mutant whereas the dual ROS1/ALK inhibitors are ineffective. Taken together, inhibitor profiling and stringent characterization of the structure–function differences between the ROS1 and ALK kinase domains will facilitate future rational drug design for ROS1- and ALK-driven NSCLC and other malignancies. PMID:26372962

  14. 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

  15. 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. PMID:25576649

  16. Discovery of Clinical Candidate CEP-37440, a Selective Inhibitor of Focal Adhesion Kinase (FAK) and Anaplastic Lymphoma Kinase (ALK).

    PubMed

    Ott, Gregory R; Cheng, Mangeng; Learn, Keith S; Wagner, Jason; Gingrich, Diane E; Lisko, Joseph G; Curry, Matthew; Mesaros, Eugen F; Ghose, Arup K; Quail, Matthew R; Wan, Weihua; Lu, Lihui; Dobrzanski, Pawel; Albom, Mark S; Angeles, Thelma S; Wells-Knecht, Kevin; Huang, Zeqi; Aimone, Lisa D; Bruckheimer, Elizabeth; Anderson, Nathan; Friedman, Jay; Fernandez, Sandra V; Ator, Mark A; Ruggeri, Bruce A; Dorsey, Bruce D

    2016-08-25

    Analogues structurally related to anaplastic lymphoma kinase (ALK) inhibitor 1 were optimized for metabolic stability. The results from this endeavor not only led to improved metabolic stability, pharmacokinetic parameters, and in vitro activity against clinically derived resistance mutations but also led to the incorporation of activity for focal adhesion kinase (FAK). FAK activation, via amplification and/or overexpression, is characteristic of multiple invasive solid tumors and metastasis. The discovery of the clinical stage, dual FAK/ALK inhibitor 27b, including details surrounding SAR, in vitro/in vivo pharmacology, and pharmacokinetics, is reported herein. PMID:27527804

  17. Crizotinib in advanced, chemoresistant anaplastic lymphoma kinase-positive lymphoma patients.

    PubMed

    Gambacorti Passerini, Carlo; Farina, Francesca; Stasia, Alessandra; Redaelli, Sara; Ceccon, Monica; Mologni, Luca; Messa, Cristina; Guerra, Luca; Giudici, Giovanni; Sala, Elena; Mussolin, Lara; Deeren, Dries; King, Michael H; Steurer, Michael; Ordemann, Rainer; Cohen, Amos M; Grube, Matthias; Bernard, Lea; Chiriano, Gianpaolo; Antolini, Laura; Piazza, Rocco

    2014-02-01

    Anaplastic lymphoma kinase (ALK)-positive lymphomas respond to chemotherapy, but relapses, which bear a poor prognosis, occur. Crizotinib inhibits ALK in vitro and in vivo and was administered as monotherapy to 11 ALK+ lymphoma patients who were resistant/refractory to cytotoxic therapy. The overall response rate was 10 of 11 (90.9%; 95% confidence interval [CI] = 58.7% to 99.8%). Disease status at the latest follow-up is as follows: four patients are in complete response (CR) (months >21, >30, >35, >40) under continuous crizotinib administration; 4 patients had progression of disease (months 1, 2, 2, 2); 1 patient obtained CR on crizotinib, received an allogeneic bone marrow transplant, and is in CR; 2 patients (treated before and/or after allogeneic bone marrow transplant) obtained and are still in CR but they have stopped crizotinib. Overall and progression-free survival rates at 2 years are 72.7% (95% CI = 39.1% to 94.0%) and 63.7% (95% CI = 30.8% to 89.1%), respectively. ALK mutations conferring resistance to crizotinib in vitro could be identified in relapsed patients. Crizotinib exerted a potent antitumor activity with durable responses in advanced, heavily pretreated ALK+ lymphoma patients, with a benign safety profile. PMID:24491302

  18. Complex renal cysts associated with crizotinib treatment

    PubMed Central

    Schnell, Patrick; Bartlett, Cynthia H; Solomon, Benjamin J; Tassell, Vanessa; Shaw, Alice T; de Pas, Tommaso; Lee, Soo-Hyun; Lee, Geon Kook; Tanaka, Kaoru; Tan, Weiwei; Tang, Yiyun; Wilner, Keith D; Safferman, Allan; Han, Ji-Youn

    2015-01-01

    An apparent causal association between crizotinib treatment and renal cyst development emerged during clinical trials in anaplastic lymphoma kinase (ALK)-positive non–small cell lung cancer (NSCLC). Serious adverse event (SAE) reports of renal cysts from a safety database of 1375 patients from four clinical trials were reviewed. A blinded, retrospective, independent radiologic review (IRR) was performed using scans from patients on study for ≥6 months in three clinical trials; risk factors for renal cyst development were assessed. Among 17 patients with renal cysts reported as SAEs, evidence of invasion into adjacent structures was noted in seven patients, with no evidence of malignancy found. These patients generally did not require dose reductions, none required permanent crizotinib discontinuation due to this AE, and most continued treatment with clinical benefit. In the blinded IRR, among 255 crizotinib-treated patients, 22%, 3%, and 2% had preexisting simple cysts, complex cysts, or both, respectively. At the 6-month tumor assessment, 9% of all patients had acquired new cysts, and 2% of patients with preexisting cysts had developed new cysts and enlargements (>50%) of preexisting simple cysts. Asians appeared to have an increased risk of developing new cysts on treatment; Koreans in particular had 5.18 times higher odds of developing cysts than non-Asians (95% confidence interval, 1.51–17.78; P = 0.05). Crizotinib treatment appears to be associated with an increased risk of development and progression of renal cysts in patients with ALK-positive NSCLC. While close monitoring is recommended, dosing modification was not generally necessary, allowing patients to remain on crizotinib treatment. PMID:25756473

  19. Histologic subtypes, immunohistochemistry, FISH or molecular screening for the accurate diagnosis of ALK-rearrangement in lung cancer: a comprehensive study of Caucasian non-smokers.

    PubMed

    Just, Pierre-Alexandre; Cazes, Aurélie; Audebourg, Anne; Cessot, Anatole; Pallier, Karine; Danel, Claire; Vacher-Lavenu, Marie-Cécile; Laurent-Puig, Pierre; Terris, Benoît; Blons, Hélène

    2012-06-01

    EML4-ALK adenocarcinomas constitute a new molecular subgroup of lung tumours that respond very well to crizotinib, an ALK inhibitor. However, the diagnosis of ALK rearrangement in lung cancer is challenging. The aim of this study was to compare the diagnostic accuracy of five different methods in a series of 20 EGFR(wt/wt) lung adenocarcinomas from non- or light- smokers. Multiplex RT-PCR was considered as gold standard and identified four ALK-rearranged tumours among the 20 tested tumours. qRT-PCR got an interpretability rate of 100% and accurately typed all 20 tumours. qRT-PCR from corresponding formalin-fixed paraffin-embedded (FFPE) specimens got an interpretability rate of 65%. Out of the four previously identified ALK-rearranged cases, three were interpretable and two were retrieved using FFPE qRT-PCR. ALK break-apart FISH got an interpretability rate of 60% and accurately typed all of the twelve remaining cases. Anti-ALK immunohistochemistry (IHC) accurately typed all twenty tumours using a cut-off value of strong staining of 100% tumour cells. The 16 non ALK-rearranged tumours got no/light staining in 13 cases, and a moderate staining of 80-100% tumour cells in 3 cases. We then analysed four solid signet-ring lung adenocarcinomas. FFPE qRT-PCR, FISH and immunohistochemistry were concordant in three cases, with positive and negative results in respectively one and two cases. The fourth case, which was positive by FISH and immunohistochemistry but negative by RT-PCR, was shown to have a non-EML4-ALK ALK-rearrangement. As various factors such as RNA quality, fixation quality and type of ALK rearrangement may impede ALK screening, we propose a combined FISH/molecular biology diagnostic algorithm in which anti-ALK immunohistochemistry is used as a pre-screening step. PMID:22153831

  20. EML4-ALK translocation is associated with early onset of disease and other clinicopathological features in Chinese female never-smokers with non-small-cell lung cancer

    PubMed Central

    REN, WEIHONG; ZHANG, BO; MA, JIE; LI, WENCAI; LAN, JIANYUN; MEN, HUI; ZHANG, QINXIAN

    2015-01-01

    Non-small-cell lung cancer (NSCLC) with echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) translocation is resistant to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib, but responds to the ALK-TKI crizotinib. Characterization of EML4-ALK translocation may provide invaluable information to facilitate disease diagnosis and improve the outcome of customized treatment. Although the occurrence of EML4-ALK translocation is likely to be affected by the smoking habits and gender of patients, the translocation has not been characterized extensively in female never-smokers with NSCLC. Therefore, 280 female never-smokers that were diagnosed with NSCLC were enrolled in the present study, and characteristics of EML4-ALK translocation, including the frequency, were determined in these NSCLC patients. EML4-ALK fusion variants were detected using Multiplex one-step reverse transcription-polymerase chain reaction and subsequently confirmed by DNA sequencing and Vysis ALK Break Apart fluorescence in situ hybridization analysis. The EML4-ALK fusion variants were detected in 21 carcinoma tissue specimens, accounting for 7.5% of the enrolled patients. Out of these patients with EML4-ALK fusion variants, EML4-ALK fusion variant 1 was identified in 12 patients, indicating that variant 1 is the most common type of EML4-ALK fusion gene in the present cohort of patients. ALK mRNA was aberrantly expressed in all the tissues with EML4-ALK translocation, but not in the carcinoma tissues without EML4-ALK translocation. In addition, the EML4-ALK translocation was more frequently found in younger patients. The median age of patients with EML4-ALK translocation was 50.95±2.29 years, which was significantly younger (P<0.01) than the median age of the patients without EML4-ALK translocation (57.15±0.56). The EML4-ALK translocation was detected exclusively in undifferentiated tumors that were graded as

  1. Importance of protein flexibility in ranking inhibitor affinities: modeling the binding mechanisms of piperidine carboxamides as Type I1/2 ALK inhibitors.

    PubMed

    Kong, Xiaotian; Pan, Peichen; Li, Dan; Tian, Sheng; Li, Youyong; Hou, Tingjun

    2015-02-28

    Anaplastic lymphoma kinase (ALK) has gained increased attention as an attractive therapeutic target for the treatment of various cancers, especially non-small-cell lung cancer (NSCLC). Recently, piperidine carboxamides were reported as Type I1/2 inhibitors of ALK, which occupy both the ATP binding site and the back ATP hydrophobic cavity in DFG-in conformation. Due to the dynamic behavior of ALK in the binding of Type I1/2 inhibitors, the accurate predictions of the binding structures and relative binding potencies of these inhibitors are quite challenging. In this study, different modeling techniques, including molecular docking, ensemble docking based on multiple receptor conformations, molecular dynamics simulations and free energy calculations, were utilized to explore the binding mechanisms of piperidine carboxamides. Our predictions show that the conventional docking protocols are not sufficient to predict the relative binding potencies of the studied inhibitors with high accuracy, but incorporating protein flexibility before or after docking is quite effective to improve the prediction accuracy. Notably, the binding free energies predicted by MM/GBSA or MM/PBSA based on the MD simulations for the docked poses give the highest correlation with the experimental data, highlighting the importance of the inclusion of receptor flexibility for the accurate predictions of the binding potencies for Type I1/2 inhibitors of ALK. Furthermore, the comprehensive analysis of several pairs of representative inhibitors demonstrates the importance of hydrophobic interactions in improving the binding affinities of the inhibitors with the hot-spot residues surrounding the binding pocket. This work is expected to provide valuable clues for further rational design of novel and potent Type I1/2 ALK inhibitors. PMID:25644934

  2. Understanding the Interplay between Expression, Mutation and Activity of ALK Receptor in Rhabdomyosarcoma Cells for Clinical Application of Small-Molecule Inhibitors

    PubMed Central

    Peron, Marica; Lovisa, Federica; Poli, Elena; Basso, Giuseppe; Bonvini, Paolo

    2015-01-01

    Background Receptor tyrosine kinases (RTKs) have a central role in cancer initiation and progression, since changes in their expression and activity potentially results in cell transformation. This concept is essential from a therapeutic standpoint, as clinical evidence indicates that tumours carrying deregulated RTKs are particularly susceptible to their activity but also to their inhibition. Rhabdomyosarcoma (RMS) is an aggressive childhood cancer where emerging therapies rely on the use kinase inhibitors, and among druggable kinases ALK represents a potential therapeutic target to commit efforts against. However, the functional relevance of ALK in RMS is not known, likewise the multi-component deregulated RTK profile to which ALK belongs. Methods In this study we used RMS cell lines representative of the alveolar and embrional histotype and looked at ALK intracellular localization, activity and cell signalling. Results We found that ALK was properly located at the plasma membrane of RMS cells, though in an unphosphorylated and inactive state due to intracellular tyrosine phosphatases (PTPases) activity. Indeed, increase of ALK phosphorylation was observed upon PTPase inhibition, as well as after ligand binding or protein overexpression. In these conditions, ALK signalling proceeded through the MAPK/ERK and PI3K/AKT pathways, and it was susceptible to ATP-competitive inhibitors exposure. However, drug-induced growth inhibition, cell cycle arrest and apoptosis did not correlate with ALK expression only, but relied also on the expression of other RTKs with akin drug binding affinity. Indeed, analysis of baseline and inducible RTK phosphorylation confirmed that RMS cells were susceptible to ALK kinase inhibitors even in the absence of the primary intended target, due to the presence of compensatory RTKs signalling pathways. Conclusions These data, hence, provided evidences of a potentially active role of ALK in RMS cells, but also suggest caution in considering ALK

  3. 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

  4. Prospective screening for ALK: clinical features and outcome according to ALK status.

    PubMed

    Fallet, Vincent; Cadranel, Jacques; Doubre, Hélène; Toper, Cécile; Monnet, Isabelle; Chinet, Thierry; Oliviero, Gérard; Foulon, Guillaume; De Cremoux, Hubert; Vieira, Thibault; Antoine, Martine; Wislez, Marie

    2014-05-01

    The aim of this study was to analyse the clinico-pathological characteristics and outcomes of a cohort of French patients who were prospectively screened for Anaplastic Lymphoma Kinase (ALK) rearrangement. One hundred and sixteen consecutive patients screened for ALK rearrangement to be recruited into a crizotinib registration trial were included from eight French centres. ALK rearrangement was detected by fluorescence in situ hybridization. Seventeen patients (14.6%) were positive for ALK. ALK+ patients were younger (p = 0.049) and more likely to be males (p=0.032), non- or light-smokers (p = 0.048) and without underlying respiratory disease (p=0.025) compared to ALK- patients. Thyroid-transcription factor-1 expression was present in all ALK+ tumours. ALK+ tumours tended to have lymph node and brain metastases. In multivariate analyses, gender, smoking history and N stage were independently associated with ALK status. Median overall survival (OS) was not reached for ALK+ patients and was significantly longer than for ALK- patients (hazard ratio for death for ALK- patients 2.98; 95% CI [1.29-6.90], p=0.01). French ALK+ patients present a specific phenotype. ALK rearrangement should be determined to improve OS with an effective targeted therapy. PMID:24589437

  5. Factors associated with sinus bradycardia during crizotinib treatment: a retrospective analysis of two large-scale multinational trials (PROFILE 1005 and 1007).

    PubMed

    Ou, Sai-Hong Ignatius; Tang, Yiyun; Polli, Anna; Wilner, Keith D; Schnell, Patrick

    2016-04-01

    Decreases in heart rate (HR) have been described in patients receiving crizotinib. We performed a large retrospective analysis of HR changes during crizotinib therapy. HRs from vital-sign data for patients with anaplastic lymphoma kinase (ALK)-positive nonsmall cell lung cancer enrolled in PROFILE 1005 and the crizotinib arm of PROFILE 1007 were analyzed. Sinus bradycardia (SB) was defined as HR <60 beats per minute (bpm). Magnitude and timing of HR changes were assessed. Potential risk factors for SB were investigated by logistic regression analysis. Progression-free survival (PFS) was evaluated according to HR decrease by <20 versus ≥20 bpm within the first 50 days of starting treatment. For the 1053 patients analyzed, the mean maximum postbaseline HR decrease was 25 bpm (standard deviation 15.8). Overall, 441 patients (41.9%) had at least one episode of postbaseline SB. The mean precrizotinib treatment HR was significantly lower among patients with versus without postbaseline SB (82.2 bpm vs. 92.6 bpm). The likelihood of experiencing SB was statistically significantly higher among patients with a precrizotinib treatment HR <70 bpm. PFS was comparable among patients with or without HR decrease of ≥20 bpm within the first 50 days of starting crizotinib. Decrease in HR is very common among patients on crizotinib. The likelihood of experiencing SB was statistically significantly higher among patients with a precrizotinib treatment HR <70 bpm. This is the first large-scale report investigating the association between treatment with a tyrosine kinase inhibitor and the development of bradycardia. HRs should be closely monitored during crizotinib treatment. PMID:26823131

  6. Biomarkers that currently affect clinical practice: EGFR, ALK, MET, KRAS

    PubMed Central

    Vincent, M.D.; Kuruvilla, M.S.; Leighl, N.B.; Kamel–Reid, S.

    2012-01-01

    New drugs such as pemetrexed, the epidermal growth factor receptor (egfr) tyrosine kinase inhibitors, and the Alk inhibitor crizotinib have recently enabled progress in the management of advanced non-small-cell lung cancer (nsclc). More drugs, especially Met inhibitors, will follow. However, the benefits of these agents are not uniform across the spectrum of nsclc, and optimizing their utility requires some degree of subgrouping of nsclc by the presence or absence of certain biomarkers. The biomarkers of current or imminent value are EGFR and KRAS mutational status, ALK rearrangements, and MET immunohistochemistry. As a predictor of benefit for anti-egfr monoclonal antibodies, EGFR immunohistochemistry is also of potential interest. Some of the foregoing biomarkers (EGFR, ALK, MET) are direct drivers of the malignant phenotype. As such, they are, quite rationally, the direct targets of inhibitory drugs. However, KRAS, while definitely a driver, has resisted attempts at direct pharmacologic manipulation, and its main value might lie in its role as part of an efficient testing algorithm, because KRAS mutations appear to exclude EGFR and ALK mutations. The indirect value of KRAS in determining sensitivity to other targeted agents or to pemetrexed remains controversial. The other biomarkers (EGFR, ALK, MET) may also have indirect value as predictors of sensitivity to chemotherapy in general, to pemetrexed specifically, and to radiotherapy and molecularly targeted agents. These biomarkers have all enabled the co-development of new drugs with companion diagnostics, and they illustrate the paradigm that will govern progress in oncology in the immediate future. However, in nsclc, the acquisition of sufficient biopsy material remains a stubborn obstacle to the evolution of novel targeted therapies. PMID:22787409

  7. Progress of EGFR-TKI and ALK/ROS1 inhibitors in advanced non-small cell lung cancer

    PubMed Central

    Ge, Liangqing; Shi, Ruizheng

    2015-01-01

    To discuss the mechanism and clinical application of EGFR-TKI and ALK/ROS1 inhibitors in non-small cell lung cancer (NSCLC), we reviewed recent available data mainly from PubMed. We found that chemotherapy, progression-free survival (PFS), objective response rate (ORR), and quality of life of patients with advanced NSCLC can be greatly improved in these drugs medication compared with conventional chemotherapy. Though many questions like resistance to EGFR-TKI and ALK/ROS1 inhibitors exist, molecular targeted therapy is an important therapeutic method for the management of NSCLC. The role of molecule targeted therapy in the initiation and development of NSCLC deserves further study. PMID:26379824

  8. 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

  9. [Renal cysts - A novel complication of crizotinib treatment for lung cancer].

    PubMed

    CsToth, I; Meert, A-P; Sculier, J-P; Berghmans, T

    2015-11-01

    We report the case of a woman with an ALK positive lung adenocarcinoma, who developed bilateral complex renal cysts 17 months after the introduction of treatment with crizotinib. Clinical investigation led to the conclusion that the cysts were due to anticancer drug. Regression of the renal cysts was observed one month after cessation of the crizotinib. This case illustrates that specific and little known toxicities can occur with these novel molecules which have entered use for the management of lung cancer. PMID:26033699

  10. Identification of Driving ALK Fusion Genes and Genomic Landscape of Medullary Thyroid Cancer

    PubMed Central

    Yun, Jae Won; Lee, Hyun-Woo; Kim, DeokGeun; Ji, Yongick; Kim, Duk-Hwan; Park, Woong-Yang; Shin, Hyun-Tae; Kim, Kyoung-Mee; Ahn, Myung-Ju; Park, Keunchil; Sun, Jong-Mu

    2015-01-01

    The genetic landscape of medullary thyroid cancer (MTC) is not yet fully understood, although some oncogenic mutations have been identified. To explore genetic profiles of MTCs, formalin-fixed, paraffin-embedded tumor tissues from MTC patients were assayed on the Ion AmpliSeq Cancer Panel v2. Eighty-four sporadic MTC samples and 36 paired normal thyroid tissues were successfully sequenced. We discovered 101 hotspot mutations in 18 genes in the 84 MTC tissue samples. The most common mutation was in the ret proto-oncogene, which occurred in 47 cases followed by mutations in genes encoding Harvey rat sarcoma viral oncogene homolog (N = 14), serine/threonine kinase 11 (N = 11), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (N = 6), mutL homolog 1 (N = 4), Kiesten rat sarcoma viral oncogene homolog (N = 3) and MET proto-oncogene (N = 3). We also evaluated anaplastic lymphoma kinase (ALK) rearrangement by immunohistochemistry and break-apart fluorescence in situ hybridization (FISH). Two of 98 screened cases were positive for ALK FISH. To identify the genomic breakpoint and 5’ fusion partner of ALK, customized targeted cancer panel sequencing was performed using DNA from tumor samples of the two patients. Glutamine:fructose-6-phosphate transaminase 1 (GFPT1)-ALK and echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusions were identified. Additional PCR analysis, followed by Sanger sequencing, confirmed the GFPT1-ALK fusion, indicating that the fusion is a result of intra-chromosomal translocation or deletion. Notably, a metastatic MTC case harboring the EML4-ALK fusion showed a dramatic response to an ALK inhibitor, crizotinib. In conclusion, we found several genetic mutations in MTC and are the first to identify ALK fusions in MTC. Our results suggest that the EML4-ALK fusion in MTC may be a potential driver mutation and a valid target of ALK inhibitors. Furthermore, the GFPT1-ALK fusion may be a potential candidate for molecular target

  11. Detection of EML4-ALK in Lung Adenocarcinoma Using Pleural Effusion with FISH, IHC, and RT-PCR Methods

    PubMed Central

    Zhou, Xiaodie; Song, Yong; Zhou, Xiaojun; Yu, Like; Wang, Jiandong

    2015-01-01

    Anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) gene rearrangements occur in approximately 5% of non-small-cell lung cancers (NSCLC), leading to the overexpression of anaplastic lymphoma kinase and predicting a response to the targeted inhibitor, crizotinib. Malignant pleural effusion occurs in most patients with advanced lung cancer, especially adenocarcinoma, and tissue samples are not always available from these patients. We attempted to clarify the feasibility of detecting the EML4-ALK fusion gene in pleural effusion cells using different methods. We obtained 66 samples of pleural effusion from NSCLC patients. The pleural effusion fluid was centrifuged, and the cellular components obtained were formalin fixed and paraffin embedded. The EML4-ALK fusion gene status was determined with fluorescent in situ hybridization (FISH), reverse transcription—polymerase chain reaction (RT-PCR), and immunohistochemistry (IHC). EML4-ALK was detected in three of 66 patient samples (4.5%) with RT-PCR. When the RT-PCR data were used as the standard, one false positive and one false negative samples were identified with IHC; and one false negative sample was identified with FISH. These results suggest that a block of pleural effusion cells can be used to detect the EML4-ALK fusion gene. IHC had good sensitivity, but low specificity. FISH had low sensitivity, but high specificity. RT-PCR is a good candidate method for detecting EML4-ALK in blocks of pleural effusion cells from lung cancer patients. PMID:25785456

  12. Inhibition of TGF-β signaling by an ALK5 inhibitor protects rats from dimethylnitrosamine-induced liver fibrosis

    PubMed Central

    de Gouville, Anne-Charlotte; Boullay, Valerie; Krysa, Gael; Pilot, Julia; Brusq, Jean-Marie; Loriolle, Florence; Gauthier, Jean-Michel; Papworth, Stephen A; Laroze, Alain; Gellibert, Françoise; Huet, Stephane

    2005-01-01

    Chronic liver disease is characterized by an exacerbated accumulation of matrix, causing progressive fibrosis, which may lead to cirrhosis. Transforming growth factor beta (TGF-β), a well-known profibrotic cytokine, transduces its signal through the ALK5 ser/thr kinase receptor, and increases transcription of different genes including PAI-1 and collagens. The identification of GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine), an ALK5 inhibitor, allowed us to evaluate the therapeutic potential of inhibiting TGF-β pathway in different models of liver disease. A cellular assay was used to identify GW6604 as a TGF-β signaling pathway inhibitor. This ALK5 inhibitor was then tested in a model of liver hepatectomy in TGF-β-overexpressing transgenic mice, in an acute model of liver disease and in a chronic model of dimethylnitrosamine (DMN)-induced liver fibrosis. In vitro, GW6604 inhibited autophosphorylation of ALK5 with an IC50 of 140 nM and in a cellular assay inhibited TGF-β-induced transcription of PAI-1 (IC50: 500 nM). In vivo, GW6604 (40 mg kg−1 p.o.) increased liver regeneration in TGF-β-overexpressing mice, which had undergone partial hepatectomy. In an acute model of liver disease, GW6604 reduced by 80% the expression of collagen IA1. In a chronic model of DMN-induced fibrosis where DMN was administered for 6 weeks and GW6604 dosed for the last 3 weeks (80 mg kg−1 p.o., b.i.d.), mortality was prevented and DMN-induced elevations of mRNA encoding for collagen IA1, IA2, III, TIMP-1 and TGF-β were reduced by 50–75%. Inhibition of matrix genes overexpression was accompanied by reduced matrix deposition and reduction in liver function deterioration, as assessed by bilirubin and liver enzyme levels. Our results suggest that inhibition of ALK5 could be an attractive new approach to treatment of liver fibrotic diseases by both preventing matrix deposition and promoting hepatocyte regeneration. PMID:15723089

  13. Effects of meal type on the oral bioavailability of the ALK inhibitor ceritinib in healthy adult subjects.

    PubMed

    Lau, Yvonne Y; Gu, Wen; Lin, Tiffany; Song, Dongweon; Yu, Richard; Scott, Jeffrey W

    2016-05-01

    Ceritinib is a potent inhibitor of anaplastic lymphoma kinase (ALK), which has shown acceptable safety and substantial antitumor activity in ALK-positive non-small cell lung cancer (NSCLC) patients. Two food-effect studies were conducted in healthy adults to investigate the influence of food on the oral bioavailability of ceritinib: a study with low- or high-fat meals at 500 mg and a study with a light snack at 750 mg. Compared with the fasted state, AUC0-∞ (90%CI) of ceritinib was increased by 58% (34%, 86%) after the intake of a low-fat meal, by 73% (46%, 105%) after the intake of a high-fat meal, and by 54% (19%, 99%) after the intake of a light snack. Safety assessments also suggested that food may improve gastrointestinal (GI) tolerability after a single ceritinib dose. Based on the pharmacokinetic results, it is essential to avoid any type of meal during dosing of ceritinib because the intake of food may increase the occurrence of exposure-dependent, non-GI toxicities at the labeled dose of 750 mg daily during fasting. A randomized trial is ongoing to determine an alternative way to give ceritinib (450 mg or 600 mg with food) that may enhance GI tolerability in ALK-positive NSCLC patients. PMID:26272586

  14. 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

  15. 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

  16. Genomic Aberrations in Crizotinib Resistant Lung Adenocarcinoma Samples Identified by Transcriptome Sequencing

    PubMed Central

    Saber, Ali; van der Wekken, Anthonie J.; Kok, Klaas; Terpstra, M. Martijn; Bosman, Lisette J.; Mastik, Mirjam F.; Timens, Wim; Schuuring, Ed; Hiltermann, T. Jeroen N.; Groen, Harry J. M.; van den Berg, Anke

    2016-01-01

    ALK-break positive non-small cell lung cancer (NSCLC) patients initially respond to crizotinib, but resistance occurs inevitably. In this study we aimed to identify fusion genes in crizotinib resistant tumor samples. Re-biopsies of three patients were subjected to paired-end RNA sequencing to identify fusion genes using deFuse and EricScript. The IGV browser was used to determine presence of known resistance-associated mutations. Sanger sequencing was used to validate fusion genes and digital droplet PCR to validate mutations. ALK fusion genes were detected in all three patients with EML4 being the fusion partner. One patient had no additional fusion genes. Another patient had one additional fusion gene, but without a predicted open reading frame (ORF). The third patient had three additional fusion genes, of which two were derived from the same chromosomal region as the EML4-ALK. A predicted ORF was identified only in the CLIP4-VSNL1 fusion product. The fusion genes validated in the post-treatment sample were also present in the biopsy before crizotinib. ALK mutations (p.C1156Y and p.G1269A) detected in the re-biopsies of two patients, were not detected in pre-treatment biopsies. In conclusion, fusion genes identified in our study are unlikely to be involved in crizotinib resistance based on presence in pre-treatment biopsies. The detection of ALK mutations in post-treatment tumor samples of two patients underlines their role in crizotinib resistance. PMID:27045755

  17. 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

  18. Enhanced Effectivity of an ALK5-Inhibitor after Cell-Specific Delivery to Hepatic Stellate Cells in Mice with Liver Injury

    PubMed Central

    van Beuge, Marike Marjolijn; Prakash, Jai; Lacombe, Marie; Post, Eduard; Reker-Smit, Catharina; Beljaars, Leonie; Poelstra, Klaas

    2013-01-01

    Transforming growth factor-β (TGF-β) is a major pro-fibrotic cytokine, causing the overproduction of extracellular matrix molecules in many fibrotic diseases. Inhibition of its type-I receptor (ALK5) has been shown to effectively inhibit fibrosis in animal models. However, apart from its pro-fibrotic effects, TGF-β also has a regulatory role in the immune system and influences tumorigenesis, which limits the use of inhibitors. We therefore explored the cell-specific delivery of an ALK5-inhibitor to hepatic stellate cells, a key cell in the development of liver fibrosis. We synthesized a conjugate of the ALK5-inhibitor LY-364947 coupled to mannose-6-phosphate human serum albumin (M6PHSA), which binds to the insulin-like growth factor II receptor on activated HSC. The effectivity of the conjugate was evaluated in primary HSC and in an acute liver injury model in mice. In vitro, the free drug and the conjugate significantly inhibited fibrotic markers in HSC. In hepatocytes, TGF-β-dependent signaling was inhibited by free drug, but not by the conjugate, thus showing its cell-specificity. In vivo, the conjugate localized in desmin-positive cells in the liver and not in hepatocytes or immune cells. In the acute liver injury model in mice, the conjugate reduced fibrogenic markers and collagen deposition more effectively than free drug. We conclude that we can specifically deliver an ALK5-inhibitor to HSC using the M6PHSA carrier and that this targeted drug reduces fibrogenic parameters in vivo, without affecting other cell-types. PMID:23441194

  19. Activated Alk triggers prolonged neurogenesis and Ret upregulation providing a therapeutic target in ALK-mutated neuroblastoma

    PubMed Central

    Cazes, Alex; Lopez-Delisle, Lucille; Tsarovina, Konstantina; Pierre-Eugène, Cécile; De Preter, Katleen; Peuchmaur, Michel; Nicolas, André; Provost, Claire; Louis-Brennetot, Caroline; Daveau, Romain; Kumps, Candy; Cascone, Ilaria; Schleiermacher, Gudrun; Prignon, Aurélie; Speleman, Frank; Rohrer, Hermann; Delattre, Olivier; Janoueix-Lerosey, Isabelle

    2014-01-01

    Activating mutations of the ALK (Anaplastic lymphoma Kinase) gene have been identified in sporadic and familial cases of neuroblastoma, a cancer of early childhood arising from the sympathetic nervous system (SNS). To decipher ALK function in neuroblastoma predisposition and oncogenesis, we have characterized knock-in (KI) mice bearing the two most frequent mutations observed in neuroblastoma patients. A dramatic enlargement of sympathetic ganglia is observed in AlkF1178L mice from embryonic to adult stages associated with an increased proliferation of sympathetic neuroblasts from E14.5 to birth. In a MYCN transgenic context, the F1178L mutation displays a higher oncogenic potential than the R1279Q mutation as evident from a shorter latency of tumor onset. We show that tumors expressing the R1279Q mutation are sensitive to ALK inhibition upon crizotinib treatment. Furthermore, our data provide evidence that activated ALK triggers RET upregulation in mouse sympathetic ganglia at birth as well as in murine and human neuroblastoma. Using vandetanib, we show that RET inhibition strongly impairs tumor growth in vivo in both MYCN/KI AlkR1279Q and MYCN/KI AlkF1178L mice. Altogether, our findings demonstrate the critical role of activated ALK in SNS development and pathogenesis and identify RET as a therapeutic target in ALK mutated neuroblastoma. PMID:24811913

  20. Crizotinib induces autophagy through inhibition of the STAT3 pathway in multiple lung cancer cell lines

    PubMed Central

    Deng, Danchen; Jiang, Liming; Jing, Zhao; Yao, Junlin; Li, Hongsen; Xie, Jiansheng; Wang, Zhanggui; Pan, Qin; Pan, Hongming; Huang, Wendong; Han, Weidong

    2015-01-01

    Autophagy is an evolutionarily conserved survival pathway in eukaryote and is frequently upregulated in cancer cells after chemotherapy or targeted therapy. Thus induction of autophagy has emerged as a drug resistance mechanism. In this study, we found that crizotinib induced a high level of autophagy in lung cancer cells through inhibition of STAT3. Ectopic expression of wild-type or constitutive activated STAT3 significantly suppressed the effect of crizotinib on autophagy. Interestingly, crizotinib-mediated inhibition of STAT3 is in a step-wise manner. Firstly it inhibited cytoplasmic STAT3, which leads to the phosphorylation of EIF2A, then inhibited nuclear STAT3, which leads to the downregulation of BCL-2. Cell death induced by crizotinib was greatly enhanced after the inhibition of autophagy by the pharmacological inhibitors or shRNAs against Beclin-1. Moreover, the autophagy inhibitor HCQ significantly augmented the anti-tumor effect of crizotinib in a mouse xenograft model. In conclusion, crizotinib can induce cytoprotective autophagy by suppression of STAT3 in lung cancer cells. Thus, autophagy inhibition represents a promising approach to improve the efficacy of crizotinib in the treatment of targeted lung cancer patients. PMID:26384345

  1. 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

  2. Influence of Chirality of Crizotinib on Its MTH1 Protein Inhibitory Activity: Insight from Molecular Dynamics Simulations and Binding Free Energy Calculations

    PubMed Central

    Niu, Yuzhen; Pan, Dabo; Shi, Danfeng; Bai, Qifeng; Liu, Huanxiang; Yao, Xiaojun

    2015-01-01

    As a promising target for the treatment of lung cancer, the MutT Homolog 1 (MTH1) protein can be inhibited by crizotinib. A recent work shows that the inhibitory potency of (S)-crizotinib against MTH1 is about 20 times over that of (R)-crizotinib. But the detailed molecular mechanism remains unclear. In this study, molecular dynamics (MD) simulations and free energy calculations were used to elucidate the mechanism about the effect of chirality of crizotinib on the inhibitory activity against MTH1. The binding free energy of (S)-crizotinib predicted by the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) and Adaptive biasing force (ABF) methodologies is much lower than that of (R)-crizotinib, which is consistent with the experimental data. The analysis of the individual energy terms suggests that the van der Waals interactions are important for distinguishing the binding of (S)-crizotinib and (R)-crizotinib. The binding free energy decomposition analysis illustrated that residues Tyr7, Phe27, Phe72 and Trp117 were important for the selective binding of (S)-crizotinib to MTH1. The adaptive biasing force (ABF) method was further employed to elucidate the unbinding process of (S)-crizotinib and (R)-crizotinib from the binding pocket of MTH1. ABF simulation results suggest that the reaction coordinates of the (S)-crizotinib from the binding pocket is different from (R)-crizotinib. The results from our study can reveal the details about the effect of chirality on the inhibition activity of crizotinib to MTH1 and provide valuable information for the design of more potent inhibitors. PMID:26677850

  3. SKI2162, an inhibitor of the TGF-β type I receptor (ALK5), inhibits radiation-induced fibrosis in mice.

    PubMed

    Park, Jin-hong; Ryu, Seung-Hee; Choi, Eun Kyung; Ahn, Seung Do; Park, Euisun; Choi, Kyung-Chul; Lee, Sang-wook

    2015-02-28

    Here we demonstrated that SKI2162, a small-molecule inhibitor of the TGF-β type I receptor (ALK5), prevented radiation-induced fibrosis (RIF) in mice. SKI2162 inhibited phosphorylation of Smad and induction of RIF-related genes in vitro. In RIF a mouse model, SKI2162 reduced late skin reactions and leg-contracture without jeopardizing the acute skin reaction. Irradiation of mouse tissue increased COL1A2 mRNA levels, and topical administration of SKI2162 significantly inhibited this effect. Thus, these findings support that SKI2162 has potential value as novel RIF-protective agent, and could be candidate for clinical trials. PMID:25686821

  4. Droplet Digital PCR for Absolute Quantification of EML4-ALK Gene Rearrangement in Lung Adenocarcinoma.

    PubMed

    Wang, Qiushi; Yang, Xin; He, Yong; Ma, Qiang; Lin, Li; Fu, Ping; Xiao, Hualiang

    2015-09-01

    Crizotinib treatment significantly prolongs progression-free survival, increases response rates, and improves the quality of life in patients with ALK-positive non-small-cell lung cancer. Droplet Digital PCR (ddPCR), a recently developed technique with high sensitivity and specificity, was used in this study to evaluate the association between the abundance of ALK rearrangements and crizotinib effectiveness. FFPE tissues were obtained from 103 consecutive patients with lung adenocarcinoma. Fluorescent in situ hybridization (FISH) and ddPCR were performed. The results revealed that 14 (13.6%) of the 103 patients were positive by dual-color, break-apart FISH. Three variants (1, 2, and 3) of the EML4-ALK gene rearrangements were detected. Thirteen of 14 ALK-positive cases identified by FISH were confirmed by ddPCR (four with variant 1, two with variant 2, and seven with variant 3). The case missed by ddPCR was identified as KIF5B-ALK gene rearrangement by PCR-based direct sequencing. Sixteen patients were detected with low copy numbers of EML4-ALK gene rearrangement, which failed to meet the positive cutoff point of FISH. Two of them responded well to crizotinib after unsuccessful chemotherapy. Our study indicates that ddPCR can be used as a molecular analytical tool to accurately measure the EML4-ALK rearrangement copy numbers in FFPE samples of lung adenocarcinoma patients. PMID:26142544

  5. Proactive management strategies for potential gastrointestinal adverse reactions with ceritinib in patients with advanced ALK-positive non-small-cell lung cancer

    PubMed Central

    Schaefer, Eric S; Baik, Christina

    2016-01-01

    Anaplastic lymphoma kinase (ALK) gene fusions occur in 3%–7% of non-small-cell lung cancer (NSCLC) cases. Ceritinib, a once-daily, oral ALK inhibitor, has activity against crizotinib-resistant and crizotinib-naïve NSCLC, including brain metastases. Ceritinib (Zykadia™) was granted accelerated approval by the US Food and Drug Administration in 2014 for treating crizotinib-resistant ALK-positive NSCLC. Adverse events (AEs), particularly gastrointestinal (GI) AEs, are commonly experienced at the recommended dose of 750 mg/d and ∼38% of patients require dose interruption or reduction for GI AEs. This case study details our experience with the use of proactive GI AE management regimens in patients treated with ceritinib (750 mg/d) across two study sites. Proactive Regimens A and B were implemented in patients with metastatic ALK-positive NSCLC treated with ceritinib to manage drug-related GI AEs. Regimen A comprised ondansetron and diphenoxylate/atropine or loperamide, taken 30 minutes prior to ceritinib dose. Regimen B included dicyclomine (taken with the first ceritinib dose), ondansetron (taken 30 minutes prior to ceritinib dose for the first seven doses), and loperamide (taken as needed with the onset of diarrhea). The proactive medications were tapered off depending on patient tolerability to ceritinib. Nine patient cases are presented. Starting Regimens A or B before the first dose of ceritinib, or as soon as GI symptoms were encountered, prevented the need for dose reduction due to GI toxicity in eight of the nine patients. Using these regimens, 78% of patients were able to remain on 750 mg/d fasting. Two patients received 23 months and 16 months of therapy and remain on ceritinib 750 mg/d and 600 mg/d, respectively. Although not currently recommended or implemented in clinical studies, based on the patients evaluated here, upfront or proactive treatment plans that address AEs early on can allow the majority of patients to remain on the approved 750 mg

  6. Proactive management strategies for potential gastrointestinal adverse reactions with ceritinib in patients with advanced ALK-positive non-small-cell lung cancer.

    PubMed

    Schaefer, Eric S; Baik, Christina

    2016-01-01

    Anaplastic lymphoma kinase (ALK) gene fusions occur in 3%-7% of non-small-cell lung cancer (NSCLC) cases. Ceritinib, a once-daily, oral ALK inhibitor, has activity against crizotinib-resistant and crizotinib-naïve NSCLC, including brain metastases. Ceritinib (Zykadia™) was granted accelerated approval by the US Food and Drug Administration in 2014 for treating crizotinib-resistant ALK-positive NSCLC. Adverse events (AEs), particularly gastrointestinal (GI) AEs, are commonly experienced at the recommended dose of 750 mg/d and ∼38% of patients require dose interruption or reduction for GI AEs. This case study details our experience with the use of proactive GI AE management regimens in patients treated with ceritinib (750 mg/d) across two study sites. Proactive Regimens A and B were implemented in patients with metastatic ALK-positive NSCLC treated with ceritinib to manage drug-related GI AEs. Regimen A comprised ondansetron and diphenoxylate/atropine or loperamide, taken 30 minutes prior to ceritinib dose. Regimen B included dicyclomine (taken with the first ceritinib dose), ondansetron (taken 30 minutes prior to ceritinib dose for the first seven doses), and loperamide (taken as needed with the onset of diarrhea). The proactive medications were tapered off depending on patient tolerability to ceritinib. Nine patient cases are presented. Starting Regimens A or B before the first dose of ceritinib, or as soon as GI symptoms were encountered, prevented the need for dose reduction due to GI toxicity in eight of the nine patients. Using these regimens, 78% of patients were able to remain on 750 mg/d fasting. Two patients received 23 months and 16 months of therapy and remain on ceritinib 750 mg/d and 600 mg/d, respectively. Although not currently recommended or implemented in clinical studies, based on the patients evaluated here, upfront or proactive treatment plans that address AEs early on can allow the majority of patients to remain on the approved 750 mg

  7. 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

  8. 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

  9. Design, synthesis and evaluation of small molecule imidazo[2,1-b][1,3,4]thiadiazoles as inhibitors of transforming growth factor-β type-I receptor kinase (ALK5).

    PubMed

    Patel, Harun M; Sing, Baljeet; Bhardwaj, Varun; Palkar, Mahesh; Shaikh, Mahamadhanif S; Rane, Rajesh; Alwan, Wesam S; Gadad, Andanappa K; Noolvi, Malleshappa N; Karpoormath, Rajshekhar

    2015-03-26

    A new series of imidazo[2,1-b][1,3,4]thiadiazoles 5(a-g), 6(a-g), 9(a-i) and 12(a-h) were synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin receptor-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and their TGF-β -induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. Compound 6d, 2-(5-((2-cyclopropyl-6-(4-fluorophenyl) imidazo [2,1-b][1,3,4]thiadiazol-5-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl) acetic acid, shows prominent ALK5 inhibition (IC50 = 0.0012 μM) and elective inhibition (91%) against the P38αkinase at10 μM. The binding mode of compound 6d by XP docking studies shows that it fits well into the active site cavity of ALK5 by forming broad and tight interactions. Lipinski's rule and in silico ADME pharmacokinetic parameters are within the acceptable range defined for human use thereby indicating their potential as a drug-like molecules. PMID:25234355

  10. Detection of tumor ALK status in neuroblastoma patients using peripheral blood.

    PubMed

    Combaret, Valérie; Iacono, Isabelle; Bellini, Angela; Bréjon, Stéphanie; Bernard, Virginie; Marabelle, Aurélien; Coze, Carole; Pierron, Gaelle; Lapouble, Eve; Schleiermacher, Gudrun; Blay, Jean Yves

    2015-04-01

    New protocols based on ALK-targeted therapy by crizotinib or other ALK-targeting molecules have opened for the treatment of patients with neuroblastoma (NB) if their tumors showed mutation and/or amplification of the ALK gene. However, tumor samples are not always available for analysis of ALK mutational status in particular at relapse. Here, we evaluated the ALK mutational status of NB samples by analysis of circulating DNA, using the droplet digital PCR (ddPCR) system. ddPCR assays was developed for the detection of ALK mutations at F1174 and R1275 hotspots found in NB tumors and was applied for the analysis of circulating DNA obtained from 200 μL of serum or plasma samples collected from 114 patients with NB. The mutations F1174L (exon 23 position 3520, T>C and position 3522, C>A) and the mutation R1275Q (exon 25 position 3824, G>A) were detected in circulating DNA. The sensitivity of our test was 100%, 85%, and 92%, respectively, and the specificity was 100%, 91%, and 98%, respectively. In conclusion, the assay that we have developed offers a reliable, noninvasive blood test to assess ALK mutational status at F1174 and R1275 hotspots and should help clinicians to identify patients showing an ALK mutation in particular when no tumor tissue is available. PMID:25653133

  11. ALK is a MYCN target gene and regulates cell migration and invasion in neuroblastoma.

    PubMed

    Hasan, Md Kamrul; Nafady, Asmaa; Takatori, Atsushi; Kishida, Satoshi; Ohira, Miki; Suenaga, Yusuke; Hossain, Shamim; Akter, Jesmin; Ogura, Atsushi; Nakamura, Yohko; Kadomatsu, Kenji; Nakagawara, Akira

    2013-01-01

    Human anaplastic lymphoma kinase (ALK) has been identified as an oncogene that is mutated or amplified in NBLs. To obtain a better understanding of the molecular events associated with ALK in the pathogenesis of NBL, it is necessary to clarify how ALK gene contributes to NBL progression. In the present study, we found that ALK expression was significantly high in NBL clinical samples with amplified MYCN (n = 126, P < 0.01) and in developing tumors of MYCN-transgenic mice. Indeed, promoter analysis revealed that ALK is a direct transcriptional target of MYCN. Overexpression and knockdown of ALK demonstrated its function in cell proliferation, migration and invasion. Moreover, treatment with an ALK inhibitor, TAE-684, efficiently suppressed such biological effects in MYCN amplified cells and tumor growth of the xenograft in mice. Our present findings explore the fundamental understanding of ALK in order to develop novel therapeutic tools by targeting ALK for aggressive NBL treatment. PMID:24356251

  12. Fluorescence In Situ Hybridization, Immunohistochemistry, and Next-Generation Sequencing for Detection of EML4-ALK Rearrangement in Lung Cancer

    PubMed Central

    Pekar-Zlotin, Marina; Hirsch, Fred R.; Soussan-Gutman, Lior; Ilouze, Maya; Dvir, Addie; Boyle, Theresa; Wynes, Murry; Miller, Vincent A.; Lipson, Doron; Palmer, Gary A.; Ali, Siraj M.; Dekel, Shlomi; Brenner, Ronen; Bunn, Paul A.

    2015-01-01

    Background. The U.S. Food and Drug Administration-approved method for detecting EML4-ALK rearrangement is fluorescence in situ hybridization (FISH); however, data supporting the use of immunohistochemistry (IHC) for that purpose are accumulating. Previous studies that compared FISH and IHC considered FISH the gold standard, but none compared data with the results of next-generation sequencing (NGS) analysis. Materials and Methods. We studied FISH and IHC (D5F3 antibody) systematically for EML4-ALK rearrangement in 51 lung adenocarcinoma patients, followed by NGS in case of discordance. Results. Of 51 patients, 4 were positive with FISH (7.8%), and 8 were positive with IHC (15.7%). Three were positive with both. NGS confirmed that four of the five patients who were positive with IHC and negative with FISH were positive for ALK. Two were treated by crizotinib, with progression-free survival of 18 and 6 months. Considering NGS as the most accurate test, the sensitivity and specificity were 42.9% and 97.7%, respectively, for FISH and 100% and 97.7%, respectively, for IHC. Conclusion. The FISH-based method of detecting EML4-ALK rearrangement in lung cancer may miss a significant number of patients who could benefit from targeted ALK therapy. Screening for EML4-ALK rearrangement by IHC should be strongly considered, and NGS is recommended in borderline cases. Two patients who were negative with FISH and positive with IHC were treated with crizotinib and responded to therapy. PMID:25721120

  13. Molecular mechanisms that underpin EML4-ALK driven cancers and their response to targeted drugs.

    PubMed

    Bayliss, Richard; Choi, Jene; Fennell, Dean A; Fry, Andrew M; Richards, Mark W

    2016-03-01

    A fusion between the EML4 (echinoderm microtubule-associated protein-like) and ALK (anaplastic lymphoma kinase) genes was identified in non-small cell lung cancer (NSCLC) in 2007 and there has been rapid progress in applying this knowledge to the benefit of patients. However, we have a poor understanding of EML4 and ALK biology and there are many challenges to devising the optimal strategy for treating EML4-ALK NSCLC patients. In this review, we describe the biology of EML4 and ALK, explain the main features of EML4-ALK fusion proteins and outline the therapies that target EML4-ALK. In particular, we highlight the recent advances in our understanding of the structures of EML proteins, describe the molecular mechanisms of resistance to ALK inhibitors and assess current thinking about combinations of ALK drugs with inhibitors that target other kinases or Hsp90. PMID:26755435

  14. 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

  15. 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

  16. 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

  17. Primary and Metastatic Cutaneous Melanomas Express ALK Through Alternative Transcriptional Initiation.

    PubMed

    Busam, Klaus J; Vilain, Ricardo E; Lum, Trina; Busam, Jonathan A; Hollmann, Travis J; Saw, Robyn P M; Coit, Daniel C; Scolyer, Richard A; Wiesner, Thomas

    2016-06-01

    A number of common driver mutations have been identified in melanoma, but other genetic or epigenetic aberrations are also likely to play a role in the pathogenesis of melanoma and present potential therapeutic targets. Translocations of the anaplastic lymphoma kinase (ALK), for example, have been reported in spitzoid melanocytic neoplasms leading to kinase-fusion proteins that result in immunohistochemically detectable ALK expression. In this study, we sought to determine whether ALK was also expressed in nonspitzoid primary and metastatic cutaneous melanomas. ALK immunohistochemistry was performed on 603 melanomas (303 primary and 300 metastatic tumors) from 600 patients. ALK immunohistochemistry expression was identified in 7 primary and 9 metastatic tumors. In 5 of 7 primary tumors and in 6 of 9 metastatic lesions, the majority of tumor cells were immunoreactive for ALK. In the other 2 primary and 3 metastatic lesions, positive staining was identified in less than half of the tumor cells. ALK positivity was found in the presence or absence of BRAF or NRAS mutations. In contrast to prior observations with ALK-positive Spitz tumors, none of the ALK-positive melanomas harbored a translocation. Instead, the ALK-positive melanomas predominantly expressed the recently described ALK isoform, ALK, which lacks the extracellular and transmembrane domains of wild-type ALK, consists primarily of the intracellular tyrosine kinase domain, and originates from an alternative transcriptional initiation site within the ALK gene. The findings are clinically relevant as patients with metastatic melanoma who have ALK expression may potentially benefit from treatment with ALK kinase inhibitors. PMID:26872010

  18. Intact or broken-apart RNA: an alternative concept for ALK fusion screening in non-small cell lung cancer (NSCLC).

    PubMed

    Kotoula, Vassiliki; Bobos, Mattheos; Vassilakopoulou, Maria; Tsolaki, Eleftheria; Chrisafi, Sofia; Psyrri, Amanda; Lazaridis, George; Papadopoulou, Kyriaki; Efstratiou, Ioannis; Michail-Strantzia, Catherine; Debelenko, Larisa V; Kosmidis, Paris; Fountzilas, George

    2015-01-01

    Anaplastic lymphoma kinase (ALK) break-apart fluorescent in situ hybridization (FISH) is currently used in diagnostics for the selection of non-small cell lung cancer (NSCLC) patients to receive crizotinib. We evaluated ALK status in NSCLC with a novel ALK mRNA test based on the break-apart FISH concept, which we called break-apart transcript (BAT) test. ALK5' and ALK3' transcript patterns were established with qPCR for ALK-expressing controls including fusion-negative neuroblastomas, as well as fusion-positive anaplastic large cell lymphomas and NSCLC. The BAT test was evaluated on 271 RNA samples from routinely processed paraffin NSCLC tissues. Test results were compared with ALK FISH (n=121), immunohistochemical (IHC) analysis (n=86), and automated quantitative analysis (AQUA, n=83). On the basis of the nonoverlapping ALK BAT patterns in ALK-expressing controls (P<0.0001), 8/174 adenocarcinomas (4.6%) among 259 informative NSCLC were predicted as fusion positive. Overall concordance for paired method results was high (94.1% to 98.8%) but mainly concerned negative prediction because of the limited availability of positive-matched cases. Tumors with 100% cytoplasmic IHC staining of any intensity (n=3) were positive for AQUA, FISH, and BAT test; tumors with lower IHC positivity and different staining patterns were AQUA-negative. Upon multiple reevaluations, ALK gene status was considered as originally misinterpreted by FISH in 3/121 cases (2.5%). Tumors with >4 ALK gene copies were associated with longer overall survival upon first-line chemotherapy. In conclusion, application of the ALK BAT test on routinely processed NSCLC tissues yields the same fusion partner independent information as ALK break-apart FISH but is more robust and cost-effective. The BAT concept may be considered for the development of further drug-predictive translocation tests. PMID:25153496

  19. Prospective and clinical validation of ALK immunohistochemistry: results from the phase I/II study of alectinib for ALK-positive lung cancer (AF-001JP study)

    PubMed Central

    Takeuchi, K.; Togashi, Y.; Kamihara, Y.; Fukuyama, T.; Yoshioka, H.; Inoue, A.; Katsuki, H.; Kiura, K.; Nakagawa, K.; Seto, T.; Maemondo, M.; Hida, T.; Harada, M.; Ohe, Y.; Nogami, N.; Yamamoto, N.; Nishio, M.; Tamura, T.

    2016-01-01

    Background Anaplastic lymphoma kinase (ALK) fusions need to be accurately and efficiently detected for ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) remains the reference test. Although increasing data are supporting that ALK immunohistochemistry (IHC) is highly concordant with FISH, IHC screening needed to be clinically and prospectively validated. Patients and methods In the AF-001JP trial for alectinib, 436 patients were screened for ALK fusions through IHC (n = 384) confirmed with FISH (n = 181), multiplex RT-PCR (n = 68), or both (n = 16). IHC results were scored with iScore. Result ALK fusion was positive in 137 patients and negative in 250 patients. Since the presence of cancer cells in the samples for RT-PCR was not confirmed, ALK fusion negativity could not be ascertained in 49 patients. IHC interpreted with iScore showed a 99.4% (173/174) concordance with FISH. All 41 patients who had iScore 3 and were enrolled in phase II showed at least 30% tumor reduction with 92.7% overall response rate. Two IHC-positive patients with an atypical FISH pattern responded to ALK inhibitor therapy. The reduction rate was not correlated with IHC staining intensity. Conclusions Our study showed (i) that when sufficiently sensitive and appropriately interpreted, IHC can be a stand-alone diagnostic for ALK inhibitor therapies; (ii) that when atypical FISH patterns are accompanied by IHC positivity, the patients should be considered as candidates for ALK inhibitor therapies, and (iii) that the expression level of ALK fusion is not related to the level of response to ALK inhibitors and is thus not required for patient selection. Registration number JapicCTI-101264 (This study is registered with the Japan Pharmaceutical Information Center). PMID:26487585

  20. A Screening Method for the ALK Fusion Gene in NSCLC.

    PubMed

    Murakami, Yoshiko; Mitsudomi, Tetsuya; Yatabe, Yasushi

    2012-01-01

    Lung cancer research has recently made significant progress in understanding the molecular pathogenesis of lung cancer and in developing treatments for it. Such achievements are directly utilized in clinical practice. Indeed, the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (ALK) fusion gene was first described in non-small cell lung cancer in 2007, and a molecularly targeted drug against the fusion was approved in 2011. However, lung cancer with the ALK fusion constitutes only a small fraction of lung cancers; therefore, efficient patient selection is crucial for successful treatment using the ALK inhibitor. Currently, RT-PCR, fluorescent in situ hybridization (FISH), and immunohistochemistry are commonly used to detect the ALK fusion. Although FISH is currently the gold standard technique, there are no perfect methods for detecting these genetic alterations. In this article, we discuss the advantages and disadvantages of each method and the possible criteria for selecting patients who are more likely to have the ALK fusion. If we can successfully screen patients, then ALK inhibitor treatment will be the best example of personalized therapy in terms of selecting patients with an uncommon genotype from a larger group with the same tumor phenotype. In other words, the personalized therapy may offer a new challenge for current clinical oncology. PMID:22655265

  1. A Screening Method for the ALK Fusion Gene in NSCLC

    PubMed Central

    Murakami, Yoshiko; Mitsudomi, Tetsuya; Yatabe, Yasushi

    2012-01-01

    Lung cancer research has recently made significant progress in understanding the molecular pathogenesis of lung cancer and in developing treatments for it. Such achievements are directly utilized in clinical practice. Indeed, the echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase (ALK) fusion gene was first described in non-small cell lung cancer in 2007, and a molecularly targeted drug against the fusion was approved in 2011. However, lung cancer with the ALK fusion constitutes only a small fraction of lung cancers; therefore, efficient patient selection is crucial for successful treatment using the ALK inhibitor. Currently, RT-PCR, fluorescent in situ hybridization (FISH), and immunohistochemistry are commonly used to detect the ALK fusion. Although FISH is currently the gold standard technique, there are no perfect methods for detecting these genetic alterations. In this article, we discuss the advantages and disadvantages of each method and the possible criteria for selecting patients who are more likely to have the ALK fusion. If we can successfully screen patients, then ALK inhibitor treatment will be the best example of personalized therapy in terms of selecting patients with an uncommon genotype from a larger group with the same tumor phenotype. In other words, the personalized therapy may offer a new challenge for current clinical oncology. PMID:22655265

  2. Regulation of Endothelial Barrier Function by TGF-β type I Receptor ALK5: Potential Role of Contractile Mechanisms and Heat Shock Protein 90

    PubMed Central

    Antonov, Alexander S.; Antonova, Galina N.; Fujii, Makiko; Dijke, Peter ten; Handa, Vaishali; Catravas, John D.; Verin, Alexander D.

    2013-01-01

    Multifunctional cytokine transforming growth factor-beta (TGF-β1) plays a critical role in the pathogenesis of acute lung inflammation by controlling endothelial monolayer permeability. TGF-β1 regulates endothelial cell (EC) functions via two distinct receptors, activin receptor-like kinase 1 (ALK1) and activin receptor-like kinase 5 (ALK5). The precise roles of ALK1 and ALK5 in the regulation of TGF-β1-induced lung endothelium dysfunction remain mostly unknown. We now report that adenoviral infection with constitutively active ALK5 (caALK5), but not caALK1, induces EC retraction and that this receptor predominantly controls EC permeability. We demonstrate that ubiquitinated ALK5 and phosphorylated heat shock protein 27 (phospho-Hsp27) specifically accumulate in the cytoskeleton fraction, which parallels with microtubule collapse, cortical actin disassembly and increased EC permeability. We have found that ALK1 and ALK5 interact with heat shock protein 90 (Hsp90). Moreover, the Hsp90 inhibitor radicicol (RA) prevents accumulation of ubiquitinated caALK5 and phospho-Hsp27 in the cytoskeletal fraction and restore the decreased EC permeability induced by caALK5. We hypothesize that specific translocation of ubiquitinated ALK5 receptor into the cytoskeleton compartment due to its lack of degradation is the mechanism that causes the divergence of caALK1 and caALK5 signaling. PMID:21465483

  3. Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells

    PubMed Central

    Sattu, Kamaraj; Hochgräfe, Falko; Wu, Jianmin; Umapathy, Ganesh; Schönherr, Christina; Ruuth, Kristina; Chand, Damini; Witek, Barbara; Fuchs, James; Li, Pui-Kai; Hugosson, Fredrik; Daly, Roger J; Palmer, Ruth H; Hallberg, Bengt

    2013-01-01

    Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin–ALK and echinoderm microtubule-associated protein-like 4–ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma. PMID:23889739

  4. 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

  5. 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

  6. 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

  7. 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. PMID:26657151

  8. 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

  9. Mouse models for ROS1-fusion-positive lung cancers and their application to the analysis of multikinase inhibitor efficiency.

    PubMed

    Inoue, Maki; Toki, Hideaki; Matsui, Junko; Togashi, Yuki; Dobashi, Akito; Fukumura, Ryutaro; Gondo, Yoichi; Minowa, Osamu; Tanaka, Norio; Mori, Seiichi; Takeuchi, Kengo; Noda, Tetsuo

    2016-05-01

    ROS1-fusion genes, resulting from chromosomal rearrangement, have been reported in 1-2% of human non-small cell lung cancer cases. More than 10 distinct ROS1-fusion genes, including break-point variants, have been identified to date. In this study, to investigate the in vivo oncogenic activities of one of the most frequently detected fusions, CD74-ROS1, as well as another SDC4-ROS1 fusion that has also been reported in several studies, we generated transgenic (TG) mouse strains that express either of the two ROS1-fusion genes specifically in lung alveolar type II cells. Mice in all TG lines developed tumorigenic nodules in the lung, and a few strains of both TG mouse lines demonstrated early-onset nodule development (multiple tumor lesions present in the lung at 2-4 weeks after birth); therefore, these two strains were selected for further investigation. Tumors developed progressively in the untreated TG mice of both lines, whereas those receiving oral administration of an ALK/MET/ROS1 inhibitor, crizotinib, and an ALK/ROS1 inhibitor, ASP3026, showed marked reduction in the tumor burden. Collectively, these data suggest that each of these two ROS1-fusion genes acts as a driver for the pathogenesis of lung adenocarcinoma in vivo The TG mice developed in this study are expected to serve as valuable tools for exploring novel therapeutic agents against ROS1-fusion-positive lung cancer. PMID:26964870

  10. 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

  11. 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

  12. Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage.

    PubMed

    Li, Qi; Huang, Yue; Liu, Xichun; Gan, Jianhua; Chen, Hao; Yang, Cai-Guang

    2016-05-20

    The AlkB repair enzymes, including Escherichia coli AlkB and two human homologues, ALKBH2 and ALKBH3, are iron(II)- and 2-oxoglutarate-dependent dioxygenases that efficiently repair N(1)-methyladenine and N(3)-methylcytosine methylated DNA damages. The development of small molecule inhibitors of these enzymes has seen less success. Here we have characterized a previously discovered natural product rhein and tested its ability to inhibit AlkB repair enzymes in vitro and to sensitize cells to methyl methane sulfonate that mainly produces N(1)-methyladenine and N(3)-methylcytosine lesions. Our investigation of the mechanism of rhein inhibition reveals that rhein binds to AlkB repair enzymes in vitro and promotes thermal stability in vivo In addition, we have determined a new structural complex of rhein bound to AlkB, which shows that rhein binds to a different part of the active site in AlkB than it binds to in fat mass and obesity-associated protein (FTO). With the support of these observations, we put forth the hypothesis that AlkB repair enzymes would be effective pharmacological targets for cancer treatment. PMID:27015802

  13. In silico studies on the interaction between bioactive ligands and ALK5, a biological target related to the cancer treatment.

    PubMed

    Almeida, Michell O; Trossini, Gustavo H G; Maltarollo, Vinícius G; Silva, Danielle da C; Honorio, Kathia M

    2016-09-01

    Studies have showed that there are many biological targets related to the cancer treatment, for example, TGF type I receptor (TGF-βRI or ALK5). The ALK5 inhibition is a strategy to treat some types of cancer, such as breast, lung, and pancreas. Here, we performed CoMFA and CoMSIA studies for 70 ligands with ALK5 inhibition. The internal validation for both models (q(2)LOO = 0.887 and 0.822, respectively) showed their robustness, while the external validations showed their predictive power (CoMFA: r(2)test = 0.998; CoMSIA: r(2)test = 0.975). After all validations, CoMFA and CoMSIA maps indicated physicochemical evidences on the main factors involved in the interaction between bioactive ligands and ALK5. Therefore, these results suggest molecular modifications to design new ALK5 inhibitors. PMID:26524124

  14. ALK ambiguous-positive non-small cell lung cancers are tumors challenged by diagnostic and therapeutic issues.

    PubMed

    Uguen, Arnaud; Andrieu-Key, Sophie; Vergne, Florence; Descourt, Renaud; Quéré, Gilles; Quintin-Roué, Isabelle; Key, Stéphane; Guéguen, Paul; Talagas, Matthieu; De Braekeleer, Marc; Marcorelles, Pascale

    2016-09-01

    Searching for ALK rearrangements using the approved fluorescent in situ hybridization (FISH) test and complementary immunohistochemistry (IHC) has become the rule to treat patients with advanced non‑small cell lung cancer (NSCLC) with anti‑ALK targeted therapy. The concordance between the two techniques is reported to be strong but imperfect. We report our experience with cases of ALK‑rearranged lung adenocarcinomas pointing out particularly ambiguous cases. FISH and IHC data on ALK but also c‑MET IHC as well as EGFR and KRAS mutation screening are considered, together with response to crizotinib treatment. We classified the 55 FISH ALK‑rearranged tumors into two groups according to the FISH and IHC results: a concordant FISH+IHC+ group (31 tumors) and an ambiguous group (24 tumors). These tumors were considered as 'ambiguous' ALK‑positive due to negative (21 tumors) or non‑contributive (3 tumors) IHC. In addition, the percentage of FISH-positive nuclei was between 15 and 20% in 17 tumors belonging to one or the other group (now called borderline tumors). We discuss the accuracy of the different tests with intent to determine whether ambiguous and borderline tumors are real positive ALK‑rearranged tumors. To conclude, ambiguous ALK‑positive lung cancers are challenging tumors with diagnosis and therapeutic issues that can justify parallel FISH, IHC and molecular screening strategy. PMID:27460205

  15. A novel patient-derived tumorgraft model with TRAF1-ALK anaplastic large-cell lymphoma translocation.

    PubMed

    Abate, F; Todaro, M; van der Krogt, J-A; Boi, M; Landra, I; Machiorlatti, R; Tabbò, F; Messana, K; Abele, C; Barreca, A; Novero, D; Gaudiano, M; Aliberti, S; Di Giacomo, F; Tousseyn, T; Lasorsa, E; Crescenzo, R; Bessone, L; Ficarra, E; Acquaviva, A; Rinaldi, A; Ponzoni, M; Longo, D L; Aime, S; Cheng, M; Ruggeri, B; Piccaluga, P P; Pileri, S; Tiacci, E; Falini, B; Pera-Gresely, B; Cerchietti, L; Iqbal, J; Chan, W C; Shultz, L D; Kwee, I; Piva, R; Wlodarska, I; Rabadan, R; Bertoni, F; Inghirami, G

    2015-06-01

    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 nuclear factor kB (NFkB) pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells, lacking PRDM1/Blimp1 and carrying c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to the downregulation of p50/p52 and lymphoma growth inhibition. Moreover, a NFkB gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Although a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, nevertheless 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 tools to validate the role of druggable molecules, predict therapeutic responses and implement patient specific therapies. PMID:25533804

  16. Adipocyte ALK7 links nutrient overload to catecholamine resistance in obesity

    PubMed Central

    Guo, Tingqing; Marmol, Patricia; Moliner, Annalena; Björnholm, Marie; Zhang, Chao; Shokat, Kevan M; Ibanez, Carlos F

    2014-01-01

    Obesity is associated with blunted β-adrenoreceptor (β-AR)-mediated lipolysis and lipid oxidation in adipose tissue, but the mechanisms linking nutrient overload to catecholamine resistance are poorly understood. We report that targeted disruption of TGF-β superfamily receptor ALK7 alleviates diet-induced catecholamine resistance in adipose tissue, thereby reducing obesity in mice. Global and fat-specific Alk7 knock-out enhanced adipose β-AR expression, β-adrenergic signaling, mitochondrial biogenesis, lipid oxidation, and lipolysis under a high fat diet, leading to elevated energy expenditure, decreased fat mass, and resistance to diet-induced obesity. Conversely, activation of ALK7 reduced β-AR-mediated signaling and lipolysis cell-autonomously in both mouse and human adipocytes. Acute inhibition of ALK7 in adult mice by a chemical-genetic approach reduced diet-induced weight gain, fat accumulation, and adipocyte size, and enhanced adipocyte lipolysis and β-adrenergic signaling. We propose that ALK7 signaling contributes to diet-induced catecholamine resistance in adipose tissue, and suggest that ALK7 inhibitors may have therapeutic value in human obesity. DOI: http://dx.doi.org/10.7554/eLife.03245.001 PMID:25161195

  17. 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

  18. 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

  19. Increases in Serum CYFRA21-1 Concentration during Successful Treatment with Crizotinib

    PubMed Central

    Kanaji, Nobuhiro; Tadokoro, Akira; Watanabe, Naoki; Inoue, Takuya; Ishii, Tomoya; Dobashi, Hiroaki; Bandoh, Shuji

    2014-01-01

    Case series Patient: — Final Diagnosis: — Symptoms: — Medication: — Clinical Procedure: — Specialty: — Objective: Unexpected drug reaction Background: Increases in tumor marker concentrations usually suggest disease progression. Cases Report: We here describe on 3 patients with non-small cell lung cancer whose serum concentrations of CYFRA21-1 increased in spite of successful treatment with crizotinib. Discontinuation of crizotinib resulted in a rapid decrease in serum CYFRA21-1 concentrations in all cases. In 1 patient with progressive disease, in spite of increasing the dose of crizotinib, CYFRA21-1 concentrations decreased. Conclusions: Crizotinib can induce increases in CYFRA21-1 concentration without disease progression. Pulmonologists and oncologists should be aware of this novel phenomenon, and focus on interpretation of CYFRA21-1 concentrations in monitoring tumor response to crizotinib treatment. PMID:25380070

  20. Off Target Effects of c-MET Inhibitors on Thyroid Cancer Cells

    PubMed Central

    Zhou, Yan; Zhao, Conghui; Gery, Sigal; Braunstein, Glenn D.; Okamoto, Ryoko; Alvarez, Rocio; Miles, Steven A.; Doan, Ngan B.; Said, Jonathan W.; Gu, Jiang; Koeffler, H. Phillip

    2013-01-01

    Aberrantly activated c-MET signaling occurs in several cancers, promoting the development of c-MET inhibitors. In this study, we found that eight of 8 thyroid cancer cell lines (including six anaplastic thyroid cell lines) have prominent expression of c-MET protein. Fifty percent of the thyroid cancer cell lines (four of 8) were growth-inhibited by two small molecule c-MET inhibitors (Tivantinib and Crizotinib), associated with apoptosis and G2/M cell cycle arrest. However, Crizotinib did not inhibit 50% proliferation of thyroid cancer cells (SW1736 and TL3) at a concentration at which the drug completely inhibited ligand-stimulated c-MET phosphorylation. On the other hand, Tivantinib was less potent than Crizotinib at inhibiting c-MET phosphorylation, but was more potent than Crizotinib at decreasing cell growth. Suppressing c-MET protein expression and phosphorylation using siRNA targeting c-MET did not induce cell cycle arrest and apoptosis. Taken together, Tivantinib and Crizotinib have off target(s) activity, contributing to their anti-tumor activity. In vivo study showed that Crizotinib markedly inhibited the growth of thyroid cancer cells (SW1736) in immunodeficient mice. In summary, c-MET inhibitors (Tivantinib and Crizotinib) suppress the growth of aggressive thyroid cancer cells, and this potential therapeutic benefit results from their non-MET-targeting effects. PMID:24170771

  1. 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

  2. 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

  3. ALK-positive large B-cell lymphoma: identification of EML4-ALK and a review of the literature focusing on the ALK immunohistochemical staining pattern.

    PubMed

    Sakamoto, Kana; Nakasone, Hideki; Togashi, Yuki; Sakata, Seiji; Tsuyama, Naoko; Baba, Satoko; Dobashi, Akito; Asaka, Reimi; Tsai, Chien-Chen; Chuang, Shih-Sung; Izutsu, Koji; Kanda, Yoshinobu; Takeuchi, Kengo

    2016-04-01

    Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK+LBCL) is a rare, aggressive B-cell lymphoma with ALK fusion genes. Histopathologically, the ALK immunohistochemical staining pattern is suggestive of the fusion partner of ALK. Here, we examined an ALK+LBCL case showing a unique diffuse cytoplasmic ALK staining pattern and identified EML4-ALK, which has not previously been reported in ALK+LBCL. Furthermore, to clarify whether the prognosis differs depending on the staining pattern, we reviewed 112 previously reported cases, and analyzed immunohistochemical markers and clinical data stratified by the staining pattern. We found that ALK staining can be classified into a granular cytoplasmic staining (GCS) or a non-GCS patterns. Sixty-four adult cases for which both the ALK staining pattern and survival time were reported were further analyzed for survival trends. The non-GCS pattern was significantly associated with inferior overall survival (P = 0.031). This difference remained significant after adjusting for age and clinical stage (hazard ratio 5.08, 95 % CI 1.88-13.7, P = 0.0013). Given that the ALK immunohistochemical staining pattern is associated with the ALK fusion partner, the present results suggest that the prognosis for ALK+LBCL differs depending on the ALK fusion partner. PMID:26781614

  4. P-glycoprotein Mediates Ceritinib Resistance in Anaplastic Lymphoma Kinase-rearranged Non-small Cell Lung Cancer

    PubMed Central

    Katayama, Ryohei; Sakashita, Takuya; Yanagitani, Noriko; Ninomiya, Hironori; Horiike, Atsushi; Friboulet, Luc; Gainor, Justin F.; Motoi, Noriko; Dobashi, Akito; Sakata, Seiji; Tambo, Yuichi; Kitazono, Satoru; Sato, Shigeo; Koike, Sumie; John Iafrate, A.; Mino-Kenudson, Mari; Ishikawa, Yuichi; Shaw, Alice T.; Engelman, Jeffrey A.; Takeuchi, Kengo; Nishio, Makoto; Fujita, Naoya

    2015-01-01

    The anaplastic lymphoma kinase (ALK) fusion oncogene is observed in 3%–5% of non-small cell lung cancer (NSCLC). Crizotinib and ceritinib, a next-generation ALK tyrosine kinase inhibitor (TKI) active against crizotinib-refractory patients, are clinically available for the treatment of ALK-rearranged NSCLC patients, and multiple next-generation ALK-TKIs are currently under clinical evaluation. These ALK-TKIs exhibit robust clinical activity in ALK-rearranged NSCLC patients; however, the emergence of ALK-TKI resistance restricts the therapeutic effect. To date, various secondary mutations or bypass pathway activation-mediated resistance have been identified, but large parts of the resistance mechanism are yet to be identified. Here, we report the discovery of p-glycoprotein (P-gp/ABCB1) overexpression as a ceritinib resistance mechanism in ALK-rearranged NSCLC patients. P-gp exported ceritinib and its overexpression conferred ceritinib and crizotinib resistance, but not to PF-06463922 or alectinib, which are next-generation ALK inhibitors. Knockdown of ABCB1 or P-gp inhibitors sensitizes the patient-derived cancer cells to ceritinib, in vitro and in vivo. P-gp overexpression was identified in three out of 11 cases with in ALK-rearranged crizotinib or ceritinib resistant NSCLC patients. Our study suggests that alectinib, PF-06463922, or P-gp inhibitor with ceritinib could overcome the ceritinib or crizotinib resistance mediated by P-gp overexpression. PMID:26870817

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

    PubMed Central

    Daldrup-Link, Heike E.; Mohanty, Suchismita; Ansari, Celina; Lenkov, Olga; Shaw, Aubie; 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

  6. 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- ...

  7. Phosphorylation of ΔNp63α via a Novel TGFβ/ALK5 Signaling Mechanism Mediates the Anti-Clonogenic Effects of TGFβ

    PubMed Central

    Cherukuri, Pratima; DeCastro, Andrew J.; Balboni, Amanda L.; Downey, Sondra L.; Liu, Jennifer Y.; Hutchinson, Justine A.; DiRenzo, James

    2012-01-01

    Genetic analysis of TP63 implicates ΔNp63 isoforms in preservation of replicative capacity and cellular lifespan within adult stem cells. ΔNp63α is also an oncogene and survival factor that mediates therapeutic resistance in squamous carcinomas. These diverse activities are the result of genetic and functional interactions between TP63 and an array of morphogenic and morphostatic signals that govern tissue and tumor stasis, mitotic polarity, and cell fate; however the cellular signals that account for specific functions of TP63 are incompletely understood. To address this we sought to identify signaling pathways that regulate expression, stability or activity of ΔNp63α. An siRNA-based screen of the human kinome identified the Type 1 TGFβ receptor, ALK5, as the kinase required for phosphorylation of ΔNp63α at Serine 66/68 (S66/68). This activity is TGFβ-dependent and sensitive to either ALK5-directed siRNA or the ALK5 kinase inhibitor A83-01. Mechanistic studies support a model in which ALK5 is proteolytically cleaved at the internal juxtamembrane region resulting in the translocation of the C-terminal ALK5-intracellular kinase domain (ALK5IKD). In this study, we demonstrate that ALK5-mediated phosphorylation of ΔNp63α is required for the anti-clonogenic effects of TGFΒ and ectopic expression of ALK5IKD mimics these effects. Finally, we present evidence that ultraviolet irradiation-mediated phosphorylation of ΔNp63α is sensitive to ALK5 inhibitors. These findings identify a non-canonical TGFβ-signaling pathway that mediates the anti-clonogenic effects of TGFβ and the effects of cellular stress via ΔNp63α phosphorylation. PMID:23166821

  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. PMID:26476082

  9. 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

  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. PMID:16468998

  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. 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

  13. Comprehensive histologic analysis of ALK-rearranged lung carcinomas.

    PubMed

    Yoshida, Akihiko; Tsuta, Koji; Nakamura, Harumi; Kohno, Takashi; Takahashi, Fumiaki; Asamura, Hisao; Sekine, Ikuo; Fukayama, Masashi; Shibata, Tatsuhiro; Furuta, Koh; Tsuda, Hitoshi

    2011-08-01

    A subset (1% to 5%) of non-small-cell lung carcinomas harbors the EML4-ALK fusion gene. Data from previous studies on the histomorphology of ALK-rearranged lung cancer are inconsistent, and the specific histologic parameters that characterize this subset and how accurately such parameters predict underlying ALK abnormality remain uncertain. To answer these questions, we performed a comprehensive histologic analysis of 54 surgically resected, extensively sampled ALK-rearranged lung carcinomas and compared them with 100 consecutive resections of ALK-wild-type lung cancers. All 54 cases showed at least a focal adenocarcinoma component, and 3 and 2 cases had additional squamous and sarcomatoid differentiation, respectively. Solid or acinar growth pattern, cribriform structure, presence of mucous cells (signet-ring cells or goblet cells), abundant extracellular mucus, lack of lepidic growth, and lack of significant nuclear pleomorphism were more common in ALK-positive cancers. Two recognizable constellations of findings, a solid signet-ring cell pattern and a mucinous cribriform pattern, were present at least focally in the majority (78%) of ALK-positive tumors, but were rare (1%) in ALK-negative tumors. Multivariate analysis showed that a combination of these 2 patterns was the most powerful histologic indicator of ALK rearrangement. Characteristic histologies were present both in primary sites and in metastases. Thus, histologic findings may help to identify cases for ALK testing. However, none of the histologic parameters were completely sensitive or specific to ALK rearrangement, and histomorphology should not replace confirmatory molecular or immunohistochemical studies. ALK-positive cancers commonly showed coexpression of thyroid transcription factor-1 and p63, and its significance is currently unclear. PMID:21753699

  14. Diagnostic Assays for Identification of Anaplastic Lymphoma Kinase–Positive Non–Small Cell Lung Cancer

    PubMed Central

    Weickhardt, Andrew J.; Aisner, Dara L.; Franklin, Wilbur A.; Varella-Garcia, Marileila; Doebele, Robert C.; Camidge, D. Ross

    2014-01-01

    In series dominated by adenocarcinoma histology, approximately 5% of non–small cell lung cancers (NSCLCs) harbor an anaplastic lymphoma kinase (ALK) gene rearrangement. Crizotinib, a tyrosine kinase inhibitor with significant activity against ALK, has demonstrated high response rates and prolonged progression-free survival in ALK-positive patients enrolled in phase 1/2 clinical trials. In 2011, crizotinib received accelerated approval from the US Food and Drug Administration (FDA) for the treatment of proven ALK-positive NSCLC using an FDA-approved diagnostic test. Currently, only break-apart fluorescence in situ hybridization testing is FDA approved as a companion diagnostic for crizotinib; however, many other assays are available or in development. In the current review, the authors summarize the diagnostic tests available, or likely to become available, that could be used to identify patients with ALK-positive NSCLC, highlighting the pros and cons of each. PMID:23280244

  15. Therapeutic Targeting of Anaplastic Lymphoma Kinase in Lung Cancer: A Paradigm for Precision Cancer Medicine

    PubMed Central

    Katayama, Ryohei; Lovly, Christine M.; Shaw, Alice T.

    2015-01-01

    The anaplastic lymphoma kinase (ALK) receptor tyrosine kinase was initially discovered as a component of the fusion protein nucleophosmin (NPM)-ALK in anaplastic large-cell lymphoma (ALCL). Genomic alterations in ALK, including rearrangements, point mutations, and genomic amplification, have now been identified in several malignancies, including lymphoma, non-small cell lung cancer (NSCLC), neuroblastoma, inflammatory myofibroblastic tumor, and others. Importantly, ALK serves as a validated therapeutic target in these diseases. Several ALK tyrosine kinase inhibitors (TKIs), including crizotinib, ceritinib and alectinib, have been developed, and some of them have already been approved for clinical use. These ALK inhibitors have all shown remarkable clinical outcomes in ALK-rearranged NSCLC. Unfortunately, as is the case for other kinase inhibitors in clinical use, sensitive tumors inevitably relapse due to acquired resistance. This review focuses on the discovery, function, and therapeutic targeting of ALK, with a particular focus on ALK-rearranged NSCLC. PMID:25979929

  16. Alk1 and Alk5 inhibition by Nrp1 controls vascular sprouting downstream of Notch

    PubMed Central

    Aspalter, Irene Maria; Gordon, Emma; Dubrac, Alexandre; Ragab, Anan; Narloch, Jarek; Vizán, Pedro; Geudens, Ilse; Collins, Russell Thomas; Franco, Claudio Areias; Abrahams, Cristina Luna; Thurston, Gavin; Fruttiger, Marcus; Rosewell, Ian; Eichmann, Anne; Gerhardt, Holger

    2015-01-01

    Sprouting angiogenesis drives blood vessel growth in healthy and diseased tissues. Vegf and Dll4/Notch signalling cooperate in a negative feedback loop that specifies endothelial tip and stalk cells to ensure adequate vessel branching and function. Current concepts posit that endothelial cells default to the tip-cell phenotype when Notch is inactive. Here we identify instead that the stalk-cell phenotype needs to be actively repressed to allow tip-cell formation. We show this is a key endothelial function of neuropilin-1 (Nrp1), which suppresses the stalk-cell phenotype by limiting Smad2/3 activation through Alk1 and Alk5. Notch downregulates Nrp1, thus relieving the inhibition of Alk1 and Alk5, thereby driving stalk-cell behaviour. Conceptually, our work shows that the heterogeneity between neighbouring endothelial cells established by the lateral feedback loop of Dll4/Notch utilizes Nrp1 levels as the pivot, which in turn establishes differential responsiveness to TGF-β/BMP signalling. PMID:26081042

  17. 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

  18. SEC31A-ALK Fusion Gene in Lung Adenocarcinoma.

    PubMed

    Kim, Ryong Nam; Choi, Yoon-La; Lee, Mi-Sook; Lira, Maruja E; Mao, Mao; Mann, Derrick; Stahl, Joshua; Licon, Abel; Choi, So Jung; Van Vrancken, Michael; Han, Joungho; Wlodarska, Iwona; Kim, Jhingook

    2016-01-01

    Anaplastic lymphoma kinase (ALK) fusion is a common mechanism underlying pathogenesis of non-small cell lung carcinoma (NSCLC) where these rearrangements represent important diagnostic and therapeutic targets. In this study, we found a new ALK fusion gene, SEC31A-ALK, in lung carcinoma from a 53-year-old Korean man. The conjoined region in the fusion transcript was generated by the fusion of SEC31A exon 21 and ALK exon 20 by genomic rearrangement, which contributed to generation of an intact, in-frame open reading frame. SEC31A-ALK encodes a predicted fusion protein of 1,438 amino acids comprising the WD40 domain of SEC31A at the N-terminus and ALK kinase domain at the C-terminus. Fluorescence in situ hybridization studies suggested that SEC31A-ALK was generated by an unbalanced genomic rearrangement associated with loss of the 3'-end of SEC31A. This is the first report of SEC31A-ALK fusion transcript in clinical NSCLC, which could be a novel diagnostic and therapeutic target for patients with NSCLC. PMID:25715771

  19. 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

  20. Wild-type ALK and activating ALK-R1275Q and ALK-F1174L mutations upregulate Myc and initiate tumor formation in murine neural crest progenitor cells

    PubMed Central

    Montavon, Gisèle; Jauquier, Nicolas; Coulon, Aurélie; Peuchmaur, Michel; Flahaut, Marjorie; Bourloud, Katia Balmas; Yan, Pu; Delattre, Olivier; Sommer, Lukas; Joseph, Jean-Marc; Janoueix-Lerosey, Isabelle; Gross, Nicole; Mühlethaler-Mottet, Annick

    2014-01-01

    The anaplastic lymphoma kinase (ALK) gene is overexpressed, mutated or amplified in most neuroblastoma (NB), a pediatric neural crest-derived embryonal tumor. The two most frequent mutations, ALK-F1174L and ALK-R1275Q, contribute to NB tumorigenesis in mouse models, and cooperate with MYCN in the oncogenic process. However, the precise role of activating ALK mutations or ALK-wt overexpression in NB tumor initiation needs further clarification. Human ALK-wt, ALK-F1174L, or ALK-R1275Q were stably expressed in murine neural crest progenitor cells (NCPC), MONC-1 or JoMa1, immortalized with v-Myc or Tamoxifen-inducible Myc-ERT, respectively. While orthotopic implantations of MONC-1 parental cells in nude mice generated various tumor types, such as NB, osteo/chondrosarcoma, and undifferentiated tumors, due to v-Myc oncogenic activity, MONC-1-ALK-F1174L cells only produced undifferentiated tumors. Furthermore, our data represent the first demonstration of ALK-wt transforming capacity, as ALK-wt expression in JoMa1 cells, likewise ALK-F1174L, or ALK-R1275Q, in absence of exogenous Myc-ERT activity, was sufficient to induce the formation of aggressive and undifferentiated neural crest cell-derived tumors, but not to drive NB development. Interestingly, JoMa1-ALK tumors and their derived cell lines upregulated Myc endogenous expression, resulting from ALK activation, and both ALK and Myc activities were necessary to confer tumorigenic properties on tumor-derived JoMa1 cells in vitro. PMID:24947326

  1. 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 ...

  2. 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.

  3. 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. PMID:24192939

  4. Alk1 controls arterial endothelial cell migration in lumenized vessels.

    PubMed

    Rochon, Elizabeth R; Menon, Prahlad G; Roman, Beth L

    2016-07-15

    Heterozygous loss of the arterial-specific TGFβ type I receptor, activin receptor-like kinase 1 (ALK1; ACVRL1), causes hereditary hemorrhagic telangiectasia (HHT). HHT is characterized by development of fragile, direct connections between arteries and veins, or arteriovenous malformations (AVMs). However, how decreased ALK1 signaling leads to AVMs is unknown. To understand the cellular mis-steps that cause AVMs, we assessed endothelial cell behavior in alk1-deficient zebrafish embryos, which develop cranial AVMs. Our data demonstrate that alk1 loss has no effect on arterial endothelial cell proliferation but alters arterial endothelial cell migration within lumenized vessels. In wild-type embryos, alk1-positive cranial arterial endothelial cells generally migrate towards the heart, against the direction of blood flow, with some cells incorporating into endocardium. In alk1-deficient embryos, migration against flow is dampened and migration in the direction of flow is enhanced. Altered migration results in decreased endothelial cell number in arterial segments proximal to the heart and increased endothelial cell number in arterial segments distal to the heart. We speculate that the consequent increase in distal arterial caliber and hemodynamic load precipitates the flow-dependent development of downstream AVMs. PMID:27287800

  5. Management of anaplastic lymphoma kinase positive orbito-conjunctival inflammatory myofibroblastic tumor with crizotinib.

    PubMed

    Kiratli, Hayyam; Uzun, Salih; Varan, Ali; Akyüz, Canan; Orhan, Diclehan

    2016-06-01

    Inflammatory myofibroblastic tumor (IMT) is a distinct mesenchymal neoplasm of myofibroblastic spindle cells associated with an inflammatory infiltrate formed by lymphocytes, eosinophils, and plasma cells in a myxoid or collagenous stroma. This tumor has a predilection for children and young adults and most commonly occurs in the lungs, retroperitoneum, abdomen, and pelvis. Ocular and orbital involvement is exceedingly rare. We describe a case of IMT in a 7-year-old girl involving the cornea, conjunctiva, and the anterior orbit treated with crizotinib, resulting in complete tumor remission. PMID:27312965

  6. Identification of ALK as the Major Familial Neuroblastoma Predisposition Gene

    PubMed Central

    Mossë, Yalë P; Laudenslager, Marci; Longo, Luca; Cole, Kristina A; Wood, Andrew; Attiyeh, Edward F; Laquaglia, Michael J; Sennett, Rachel; Lynch, Jill E; Perri, Patrizia; Laureys, Geneviève; Speleman, Frank; Hakonarson, Hakon; Torkamani, Ali; Schork, Nicholas J; Brodeur, Garrett M; Tonini, Gian Paolo; Rappaport, Eric; Devoto, Marcella; Maris, John M

    2009-01-01

    SUMMARY Survival rates for the childhood cancer neuroblastoma have not substantively improved despite dramatic escalation in chemotherapy intensity. Like most human cancers, this embryonal malignancy can be inherited, but the genetic etiology of familial and sporadically occurring neuroblastoma was largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase gene (ALK) explain the majority of hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at the short arm of chromosome 2 (maximum nonparametric LOD=4.23 at rs1344063) using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate missense mutations in the tyrosine kinase domain of ALK (G1128A, R1192P and R1275Q) that segregated with the disease in eight separate families. Examination of 491 sporadically occurring human neuroblastoma samples showed that the ALK locus was gained in 22.8%, and highly amplified in an additional 3.3%, and that these aberrations were highly associated with death from disease (P=0.0003). Resequencing of 194 high-risk neuroblastoma samples showed somatically acquired mutations within the tyrosine kinase domain in 12.4%. Nine of the ten mutations map to critical regions of the kinase domain and were predicted to be oncogenic drivers with high probability. Mutations resulted in constitutive phosphorylation consistent with activation, and targeted knockdown of ALK mRNA resulted in profound growth inhibition of 4 of 4 cell lines harboring mutant or amplified ALK, as well as 2 of 6 wild type for ALK. Our results demonstrate that heritable mutations of ALK are the major cause of familial neuroblastoma, and that germline or acquired activation of this cell surface kinase is a tractable therapeutic target for this lethal pediatric malignancy. PMID:18724359

  7. A never-smoker lung adenocarcinoma patient with a MET exon 14 mutation (D1028N) and a rapid partial response after crizotinib.

    PubMed

    Mahjoubi, Linda; Gazzah, Anas; Besse, Benjamin; Lacroix, Ludovic; Soria, Jean-Charles

    2016-06-01

    During the past decade, the treatment of lung adenocarcinomas has been revolutionized with novel molecular targeted therapies. We describe a case of clinical activity of crizotinib in a female patient with a lung adenocarcinoma displaying a MET exon 14 donor splice site mutation (D1028N) detected using next generation sequencing. Within 5 weeks of crizotinib therapy, a partial response was observed in this 67 year-old woman. Further clinical trials of crizotinib are needed for non-small cell lung cancer exhibiting MET mutations. PMID:26892698

  8. Uterine ALK3 is essential during the window of implantation

    PubMed Central

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

    2016-01-01

    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 Alk3flox/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

  9. 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

  10. A Rare Case of Pleomorphic Carcinoma of the Lung Harboring an Anaplastic Lymphoma Kinase (ALK) Rearrangement.

    PubMed

    Shiroyama, Takayuki; Tanaka, Ayako; Tamiya, Motohiro; Hamaguchi, Masanari; Osa, Akio; Takeoka, Sawa; Tani, Eriko; Azuma, Yuichiro; Morishita, Naoko; Suzuki, Hidekazu; Okamoto, Norio; Kimura, Kenji; Kadota, Yoshihisa; Kawahara, Kunimitsu; Hirashima, Tomonori; Kawase, Ichiro

    2015-01-01

    Molecular testing for anomalies, such as epidermal growth factor receptor mutations and anaplastic lymphoma kinase (ALK) rearrangement, is part of the current standard of care for non-small cell lung cancer, particularly adenocarcinoma. ALK rearrangement occurs most frequently in adenocarcinoma cells and rarely in non-adenocarcinoma cells. We herein report a rare case of pleomorphic lung carcinoma with ALK rearrangement in both its adenocarcinoma and spindle cell components. This case suggests the possibility of ALK rearrangement in pleomorphic carcinoma. PMID:26521903

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

    PubMed Central

    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

  12. 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

  13. 40 CFR 721.435 - Alkylphenylpolyether-alk-a-nol-a-mines (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 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...

  14. 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...

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

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 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...

  16. 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...

  17. Activated ALK Collaborates with MYCN in Neuroblastoma Pathogenesis

    PubMed Central

    Zhu, Shizhen; Lee, Jeong-Soo; Guo, Feng; Shin, Jimann; Perez-Atayde, Antonio R.; Kutok, Jeffery L.; Rodig, Scott J.; Neuberg, Donna S.; Helman, Daniel; Feng, Hui; Stewart, Rodney A.; Wang, Wenchao; George, Rani E.; Kanki, John P.; Look, A. Thomas

    2012-01-01

    SUMMARY Amplification of the MYCN oncogene in childhood neuroblastoma is often accompanied by mutational activation of ALK (anaplastic lymphoma kinase), suggesting their pathogenic cooperation. We generated a transgenic zebrafish model of neuroblastoma in which MYCN-induced tumors arise from a subpopulation of neuroblasts that migrate into the adrenal medulla analogue following organogenesis. Coexpression of activated ALK with MYCN in this model triples the disease penetrance and markedly accelerates tumor onset. MYCN overexpression induces adrenal sympathetic neuroblast hyperplasia, blocks chromaffin cell differentiation, and ultimately triggers a developmentally-timed apoptotic response in the hyperplastic sympathoadrenal cells. Coexpression of activated ALK with MYCN provides prosurvival signals that block this apoptotic response and allow continued expansion and oncogenic transformation of hyperplastic neuroblasts, thus promoting progression to neuroblastoma. PMID:22439933

  18. Combination of conventional immunohistochemistry and qRT-PCR to detect ALK rearrangement

    PubMed Central

    2014-01-01

    Background Compared with FISH and qRT-PCR analyses, immunohistochemistry (IHC) is the preferred screening test in most pathology practices for ALK-rearrangement detection. With 100% sensitivity and 98% specificity, the VENTANA ALK (D5F3) IHC assay has been approved in the EU and some Asian countries for ALK-rearrangement detection. However, an automated Ventana IHC platform is not available in most pathology labs. In this study, we evaluated the applicability of conventional IHC with D5F3 antibody in routine pathological practice and proposed detection methods and procedures that ensure that patients with ALK+ are not missed. Methods FISH and IHC analyses were performed on 297 lung adenocarcinoma cases. VENTANA IHC and qRT-PCR assay were applied to evaluate ALK-fusion status in the discordant cases of FISH and IHC. The association of ALK+ with clinicopathological characteristics was statistically analyzed. Results IHC had 100% sensitivity and 81.8% specificity for detecting ALK+. Eight ALK-expressed cases were ALK-, five of which had ALK fusion detected by qRT-PCR analysis. Three of these five cases showed ALK expression using VENTANA IHC assay. ALK+ was associated with younger age and lymph node metastasis in this Chinese lung adenocarcinoma patient cohort. Conclusions The advantages of low cost and 100% sensitivity allow conventional IHC to serve as a robust diagnostic tool for screening patients with ALK+, especially in pathology labs without a VENTANA IHC platform. For cases in which ALK is weakly expressed, qRT-PCR is necessary as a diagnostic test for ALK-fusion detection. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2269448351088278. PMID:24422905

  19. 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.

  20. Rapid and dramatic response to alectinib in an anaplastic lymphoma kinase rearranged non-small-cell lung cancer patient who is critically ill.

    PubMed

    Yoshida, Tatsuya; Hida, Toyoaki; Yatabe, Yasushi

    2016-07-01

    Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) have shown promising clinical activity in the treatment of non-small-cell lung cancer (NSCLC) that harbors ALK rearrangement. The next-generation ALK-TKI, alectinib, has been reported to have potent efficacy in ALK-positive NSCLC patients including on mutations that confer resistance to crizotinib, which was the first ALK-TKI approved for ALK-positive NSCLC. The efficacy and safety of ALK-TKIs, including crizotinib and alectinib, as the first-line treatment in critically ill patients is unclear. We report one ALK-positive NSCLC patient with poor performance status (PS) and disseminated intravascular coagulation because of respiratory failure and multiple metastases, and experienced the rapid and dramatic response to alectinib without adverse events that can lead to discontinuation and dose reduction of the drug. After a couple of months of treatment with alectinib, radiological review indicated a complete response. The present case is the first reported case of rapid and marked response to alectinib in ALK-positive NSCLC patients who had poor PS and severe organ dysfunction, such as disseminated intravascular coagulation. Further investigation of the safety and efficacy of ALK-TKI for ALK-positive NSCLC patients who are critically ill is warranted. PMID:26938871

  1. Design and synthesis of novel selective anaplastic lymphoma kinase inhibitors.

    PubMed

    Michellys, Pierre-Yves; Chen, Bei; Jiang, Tao; Jin, Yunho; Lu, Wenshuo; Marsilje, Thomas H; Pei, Wei; Uno, Tetsuo; Zhu, Xuefeng; Wu, Baogen; Nguyen, Truc Ngoc; Bursulaya, Badry; Lee, Christian; Li, Nanxin; Kim, Sungjoon; Tuntland, Tove; Liu, Bo; Sun, Frank; Steffy, Auzon; Hood, Tami

    2016-02-01

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. Expression of ALK in normal human tissues is only found in a subset of neural cells, however it is involved in the genesis of several cancers through genetic aberrations involving translocation of the kinase domain with multiple fusion partners (e.g., NPM-ALK in anaplastic large cell lymphoma ALCL or EML4-ALK in non-small cell lung cancer) or activating mutations in the full-length receptor resulting in ligand-independent constitutive activation (e.g., neuroblastoma). Here we are reporting the discovery of novel and selective anaplastic lymphoma kinase inhibitors from specific modifications of the 2,4-diaminopyridine core present in TAE684 and LDK378. Synthesis, structure activity relationships (SAR), absorption, distribution, metabolism, and excretion (ADME) profile, and in vivo efficacy in a mouse xenograft model of anaplastic large cell lymphoma are described. PMID:26750252

  2. 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

  3. Charge-transfer reaction of 1,4-benzoquinone with crizotinib: spectrophotometric study, computational molecular modeling and use in development of microwell assay for crizotinib.

    PubMed

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

    2014-10-15

    The reaction of 1,4-benzoquinone (BQ) 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 resulted in the formation of 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 0.9425 and 0.8340, respectively. The stoichiometric ratio of BQ:CZT was found to be 2:1 and the association constant of the complex was found to be 0.26×10(3)lmol(-1). 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 BQ 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. The assay limits of detection and quantitation were 5.2 and 15.6μgml(-1), 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 minimum volume of organic solvent thus it reduces the exposures of the analysts to the toxic effects of organic solvents, and significantly reduces the analysis cost. PMID:24835938

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

    The reaction of 1,4-benzoquinone (BQ) 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 resulted in the formation of 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 0.9425 and 0.8340, respectively. The stoichiometric ratio of BQ:CZT was found to be 2:1 and the association constant of the complex was found to be 0.26 × 103 l mol-1. 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 BQ 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. The assay limits of detection and quantitation were 5.2 and 15.6 μg ml-1, 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 minimum volume of organic solvent thus it reduces the exposures of the analysts to the toxic effects of organic solvents, and significantly reduces the analysis cost.

  5. The therapeutic potential of p53 reactivation by nutlin-3a in ALK+ anaplastic large cell lymphoma with wild-type or mutated p53.

    PubMed

    Drakos, E; Atsaves, V; Schlette, E; Li, J; Papanastasi, I; Rassidakis, G Z; Medeiros, L J

    2009-12-01

    p53 is expressed frequently, but is rarely mutated in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) tumours. Nutlin-3a is a recently developed small molecule that targets Mdm2, a critical negative regulator of p53, and disrupts the p53-Mdm2 interaction resulting in p53 stabilization and activation. We show that nutlin-3a activates p53 in ALK+ ALCL cells carrying a wild type (wt) or mutated but partially functional p53 gene resulting in p53-dependent cell-cycle arrest and apoptosis. Cell-cycle arrest was associated with upregulation of the cyclin-dependent kinase inhibitor p21. Nutlin-3a-induced apoptotic cell death was accompanied by Bax and Puma upregulation, downregulation of Bcl-xl, survivin, and caspase-3 cleavage, and this was reduced when p53-dependent transactivation activity was inhibited by pifithrin-alpha, or when pifithrin-mu was used to inhibit direct p53 targeting of mitochondria. Nutlin-3a sensitized the activation of the extrinsic apoptotic pathway in wt-p53 ALK+ ALCL cells, in part, through upregulation of DR-5 and downregulation of c-Flip(S/L), and was synergistic with TRAIL in cell death induction. In addition, nutlin-3a treatment enhanced doxorubicin cytotoxicity against ALK+ ALCL cells harbouring mt p53, and this was associated with p73 upregulation. These data suggest that disruption of the p53-mdm2 interaction by nutlin-3a offers a novel therapeutic approach for ALK+ ALCL patients. PMID:19741726

  6. Viral AlkB proteins repair RNA damage by oxidative demethylation

    PubMed Central

    van den Born, Erwin; Omelchenko, Marina V.; Bekkelund, Anders; Leihne, Vibeke; Koonin, Eugene V.; Dolja, Valerian V.; Falnes, Pål Ø.

    2008-01-01

    Bacterial and mammalian AlkB proteins are iron(II)- and 2-oxoglutarate-dependent dioxygenases that reverse methylation damage, such as 1-methyladenine and 3-methylcytosine, in RNA and DNA. An AlkB-domain is encoded by the genome of numerous single-stranded, plant-infecting RNA viruses, the majority of which belong to the Flexiviridae family. Our phylogenetic analysis of AlkB sequences suggests that a single plant virus might have acquired AlkB relatively recently, followed by horizontal dissemination among other viruses via recombination. Here, we describe the first functional characterization of AlkB proteins from three plant viruses. The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro. Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity. Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant. PMID:18718927

  7. Dependence-induced ethanol drinking and GABA neurotransmission are altered in Alk deficient mice.

    PubMed

    Schweitzer, Paul; Cates-Gatto, Chelsea; Varodayan, Florence P; Nadav, Tali; Roberto, Marisa; Lasek, Amy W; Roberts, Amanda J

    2016-08-01

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is expressed in the brain and implicated in alcohol abuse in humans and behavioral responses to ethanol in mice. Previous studies have shown an association of human ALK with acute responses to alcohol and alcohol dependence. In addition, Alk knockout (Alk -/-) mice consume more ethanol in a binge-drinking test and show increased sensitivity to ethanol sedation. However, the function of ALK in excessive drinking following the establishment of ethanol dependence has not been examined. In this study, we tested Alk -/- mice for dependence-induced drinking using the chronic intermittent ethanol-two bottle choice drinking (CIE-2BC) protocol. We found that Alk -/- mice initially consume more ethanol prior to CIE exposure, but do not escalate ethanol consumption after exposure, suggesting that ALK may promote the escalation of drinking after ethanol dependence. To determine the mechanism(s) responsible for this behavioral phenotype we used an electrophysiological approach to examine GABA neurotransmission in the central nucleus of the amygdala (CeA), a brain region that regulates alcohol consumption and shows increased GABA signaling after chronic ethanol exposure. GABA transmission in ethanol-naïve Alk -/- mice was enhanced at baseline and potentiated in response to acute ethanol application when compared to wild-type (Alk +/+) mice. Moreover, basal GABA transmission was not elevated by CIE exposure in Alk -/- mice as it was in Alk +/+ mice. These data suggest that ALK plays a role in dependence-induced drinking and the regulation of presynaptic GABA release in the CeA. PMID:26946429

  8. 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

  9. 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

  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. 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

  12. ALK Signaling and Target Therapy in Anaplastic Large Cell Lymphoma

    PubMed Central

    Tabbó, Fabrizio; Barreca, Antonella; Piva, Roberto; Inghirami, Giorgio

    2012-01-01

    The discovery by Morris et al. (1994) of the genes contributing to the t(2;5)(p23;q35) translocation has laid the foundation for a molecular based recognition of anaplastic large cell lymphoma and highlighted the need for a further stratification of T-cell neoplasia. Likewise the detection of anaplastic lymphoma kinase (ALK) genetic lesions among many human cancers has defined unique subsets of cancer patients, providing new opportunities for innovative therapeutic interventions. The objective of this review is to appraise the molecular mechanisms driving ALK-mediated transformation, and to maintain the neoplastic phenotype. The understanding of these events will allow the design and implementation of novel tailored strategies for a well-defined subset of cancer patients. PMID:22649787

  13. Is ALK-gene rearrangement overlooked in primary gastrointestinal T-cell lymphomas? About two cases.

    PubMed

    Mneimneh, Wadad S; Vyas, Shikhar Gautam; Cheng, Liang; Cummings, Oscar W; Czader, Magdalena

    2015-12-01

    A 41-year-old male patient with a history of ankylosing spondylitis and Crohn disease, treated with immunomodulators and disease-modifying drugs, was diagnosed with a primary intestinal T-cell lymphoma that followed a 7.5-year-course. This transmural proliferation lacked cytological characteristics of anaplastic large cell lymphoma (ALCL), and was CD8-positive, and CD30- and anaplastic lymphoma kinase (ALK)-negative by immunohistochemistry (IHC). However, ALK-gene rearrangement (ALK-gr) was detected by fluorescence in situ hybridization (FISH) in both initial and persistent disease. The possibility of indolent T-cell lymphoproliferative disease of the gastrointestinal tract with atypical features (transmural involvement) related to ALK-gr was suggested. A previous case of aggressive 'enteropathy-associated ALCL' in the context of celiac disease was recently reported, which also lacked anaplastic morphology, and where CD30 and ALK expression was incidentally demonstrated by IHC, and ALK-gr subsequently confirmed by FISH. These two recent cases represent two distinct rare entities pertaining to the group of primary intestinal T-cell lymphomas, and they both show unexpected ALK-gr. This suggests that ALK-gr has been overlooked in the group of primary intestinal T-cell lymphomas. Performing IHC and FISH tests for ALK-gr in primary gastrointestinal T-cell lymphomas might be of importance, particularly with the advancement of targeted therapy that could impact treatment and prognosis. PMID:26531107

  14. Competitive, uncompetitive, and mixed inhibitors of the alkaline phosphatase activity associated with the isolated brush border membrane of the tapeworm Hymenolepis diminuta.

    PubMed

    Pappas, P W; Leiby, D A

    1989-06-01

    Several compounds were tested as inhibitors of the alkaline phosphatase (AlkPase) activity associated with the isolated brush border membrane of the tapeworm, Hymenolepis diminuta. Molybdate, arsenate, arsenite and beta-glycerophosphate (BGP) were competitive inhibitors of the hydrolysis of p-nitrophenyl phosphate, while levamisole and clorsulon were uncompetitive and mixed inhibitors, respectively. Molybdate was also a competitive inhibitor of the hydrolysis of BGP and 5'-adenosine monophosphate, and levamisole was an uncompetitive inhibitor of BGP hydrolysis. The apparent inhibitor constants (Ki') for molybdate and levamisole were virtually identical regardless of the substrate, and these data support the hypothesis that the AlkPase activity is represented by a single membrane-bound enzyme with low substrate specificity. Quinacrine, Hg2+, and ethylenediaminetetraacetate were also potent inhibitors of the AlkPase activity, but the mechanisms by which these latter three inhibitors function were not clear. PMID:2768348

  15. The Use of a Combination of alkB Primers to Better Characterize the Distribution of Alkane-Degrading Bacteria

    PubMed Central

    Jurelevicius, Diogo; Alvarez, Vanessa Marques; Peixoto, Raquel; Rosado, Alexandre S.; Seldin, Lucy

    2013-01-01

    The alkane monooxygenase AlkB, which is encoded by the alkB gene, is a key enzyme involved in bacterial alkane degradation. To study the alkB gene within bacterial communities, researchers need to be aware of the variations in alkB nucleotide sequences; a failure to consider the sequence variations results in the low representation of the diversity and richness of alkane-degrading bacteria. To minimize this shortcoming, the use of a combination of three alkB-targeting primers to enhance the detection of the alkB gene in previously isolated alkane-degrading bacteria was proposed. Using this approach, alkB-related PCR products were detected in 79% of the strains tested. Furthermore, the chosen set of primers was used to study alkB richness and diversity in different soils sampled in Carmópolis, Brazil and King George Island, Antarctica. The DNA extracted from the different soils was PCR amplified with each set of alkB-targeting primers, and clone libraries were constructed, sequenced and analyzed. A total of 255 alkB phylotypes were detected. Venn diagram analyses revealed that only low numbers of alkB phylotypes were shared among the different libraries derived from each primer pair. Therefore, the combination of three alkB-targeting primers enhanced the richness of alkB phylotypes detected in the different soils by 45% to 139%, when compared to the use of a single alkB-targeting primer. In addition, a dendrogram analysis and beta diversity comparison of the alkB composition showed that each of the sampling sites studied had a particular set of alkane-degrading bacteria. The use of a combination of alkB primers was an efficient strategy for enhancing the detection of the alkB gene in cultivable bacteria and for better characterizing the distribution of alkane-degrading bacteria in different soil environments. PMID:23825163

  16. ALK-positive inflammatory myofibroblastic tumor harboring ALK gene rearrangement, occurring after allogeneic stem cell transplant in an adult male.

    PubMed

    Vroobel, Katherine; Judson, Ian; Dainton, Melissa; McCormick, Alison; Fisher, Cyril; Thway, Khin

    2016-08-01

    Inflammatory myofibroblastic tumor arose as a defined neoplasm from the disparate group of tumors (both neoplastic and inflammatory) originally described as inflammatory pseudotumors. The morphologic features are well described, and 50-60% of cases are associated with fusions of the anaplastic lymphoma kinase (ALK) gene. We describe an inflammatory myofibroblastic tumor in the lower abdominal wall of an adult male, which occurred 88days after he received an allogeneic stem cell transplant for T-lymphoblastic lymphoma, and which was positive for ALK immunohistochemistry and showed ALK gene rearrangement by fluorescence in situ hybridization. Two other cases are reported in the post-stem cell transplant setting, but both occurred in children and did not have molecular analysis performed. The etiology remains unclear, but may be due to immune dysregulation caused by any combination of prior chemotherapy, radiotherapy and immune suppression. These neoplasms should be considered as a rare consequence of allogeneic stem cell transplantation and referral to a specialist sarcoma center for further management may be required. PMID:27155927

  17. 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. PMID:25297851

  18. Akt shows variable sensitivity to an Hsp90 inhibitor depending on cell context

    SciTech Connect

    Theodoraki, Maria A.; Kunjappu, Mary; Sternberg, David W.; Caplan, Avrom J.

    2007-11-01

    Hsp90 inhibitors are currently in clinical trials for cancer therapy based on their ability to promote proteasomal degradation of oncogenic protein kinases and nuclear receptors. Results from recent studies suggest that cancer cells are more sensitive to these inhibitors than cells from healthy tissues. We analyzed an immortalized cell line Ba/F3 for sensitivity to the Hsp90 inhibitor geldanamycin in the absence and presence of the oncogenic tyrosine fusion kinase NPM-ALK expressed from a retroviral vector. Our results showed that NPM-ALK expression makes Akt and Cdk4 more resistant to degradation in the presence of geldanamycin, and there was a slightly reduced amount of apoptosis. The mechanism underlying the effect of NPM-ALK on Akt stability was probed by comparison of the turnover of the kinase after translation inhibition and geldanamycin treatment. We observed that Akt was degraded more rapidly in the presence of GA than upon translation inhibition without NPM-ALK expression. This suggests that NPM-ALK protects the mature kinase. Furthermore, Akt failed to bind to the Cdc37 chaperone in cells expressing NPM-ALK, which also correlates with increased Akt stability.

  19. Defective processing of methylated single-stranded DNA by E. coli alkB mutants

    PubMed Central

    Dinglay, Suneet; Trewick, Sarah C.; Lindahl, Tomas; Sedgwick, Barbara

    2000-01-01

    Escherichia coli alkB mutants are very sensitive to DNA methylating agents. Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog. Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants. No such decrease occurred when using methylated λ phage or M13 duplex DNA. These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA. Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA. The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agent N-methyl-N-nitrosourea or by γ irradiation. Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant. A recA mutation caused a small but additive defect. Thus, AlkB functions in a novel pathway independent of these activities. We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions. Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells. PMID:10950872

  20. 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

  1. 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

  2. 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

  3. 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

  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. 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.

  6. Spitz Tumors: Comparison of Histological Features in Relationship to Immunohistochemical Staining for ALK and NTRK1.

    PubMed

    Kiuru, Maija; Jungbluth, Achim; Kutzner, Heinz; Wiesner, Thomas; Busam, Klaus J

    2016-05-01

    Spitz tumors are a group of melanocytic neoplasms with distinct morphological features that tend to affect young individuals. Distinguishing benign from malignant Spitz tumors can be challenging, but cytogenetic and molecular tests have contributed to improvements in diagnostic accuracy. Spitz tumors harbor diverse genetic alterations, including mutations in HRAS, loss of BAP1, or kinase fusions in ROS1, NTRK1, ALK, BRAF, and RET genes. Limited data exist on the correlation between histopathological features and kinase fusions. Here, we describe the histopathological features of 105 Spitz tumors (Spitz nevi and atypical Spitz tumors), comparing lesions according to their immunoreactivity for ALK or NTRK1. Intersecting fascicular growth of fusiform melanocytes was seen in all but one ALK-positive tumor (27 of 28 or 96.4%), whereas it was infrequent in NTRK1-positive tumors (5 of 20 or 25.0%) and tumors negative for both ALK and NTRK1 (96.4% vs 25.0% vs 8.7%, P < .0027). There was a trend toward ALK-positive tumors being amelanotic compared with NTRK1-positive tumors and combined ALK-/NTRK1-negative tumors (89.3% vs 45% vs 47.4%, respectively, P = .1023) and lacking epithelioid cell morphology (0% vs 45.0% vs 41%, respectively, P = .6985). In conclusion, this study confirms that although not specific, the growth pattern of intersecting fascicles of amelanotic fusiform melanocytes is strongly associated with ALK expression. PMID:26873340

  7. Alk Is a Transcriptional Target of LMO4 and ERα that Promotes Cocaine Sensitization and Reward

    PubMed Central

    Lasek, Amy W.; Gesch, Julie; Giorgetti, Francesco; Kharazia, Viktor; Heberlein, Ulrike

    2011-01-01

    Previously, we showed that the mouse LIM-domain only 4 (Lmo4) gene, which encodes a protein containing two zinc-finger LIM domains that interact with various DNA-binding transcription factors, attenuates behavioral sensitivity to repeated cocaine administration. Here we show that transcription of anaplastic lymphoma kinase (Alk) is repressed by LMO4 in the striatum and that Alk promotes the development of cocaine sensitization and conditioned place preference, a measure of cocaine reward. Since LMO4 is known to interact with estrogen receptor α (ERα) at the promoters of target genes, we investigated whether Alk expression might be controlled by a similar mechanism. We found that LMO4 and ERα are associated with the Alk promoter by chromatin immunoprecipitation and that Alk is an estrogen-responsive gene in the striatum. Moreover, we show that ERα knockout mice exhibit enhanced cocaine sensitization and conditioned place preference and an increase in Alk expression in the nucleus accumbens. These data define a novel regulatory network involved in behavioral responses to cocaine. Interestingly, sex differences in several behavioral responses to cocaine in humans and rodents have been described and estrogen is thought to mediate some of these differences. Our data suggest that estrogen regulation of Alk may be one mechanism responsible for sexually dimorphic responses to cocaine. PMID:21976498

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

    PubMed

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

    2015-04-01

    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

  9. 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

  10. Identification of anaplastic lymphoma kinase as a potential therapeutic target in Basal Cell Carcinoma

    PubMed Central

    Wang, Claire Q.F.; Suárez-Fariñas, Mayte; Gonzalez, Juana; Shah, Kejal R.; Chen, Jie; Coats, Israel; Felsen, Diane; Carucci, John A.; Krueger, James G.

    2013-01-01

    The pathogenesis of BCC is associated with sonic hedgehog (SHH) signaling. Vismodegib, a smoothened inhibitor that targets this pathway, is now in clinical use for advanced BCC patients, but its efficacy is limited. Therefore, new therapeutic options for this cancer are required. We studied gene expression profiling of BCC tumour tissues coupled with laser capture microdissection to identify tumour specific receptor tyrosine kinase expression that can be targeted by small molecule inhibitors. We found a >250 fold increase (FDR<10−4) of the oncogene, anaplastic lymphoma kinase (ALK) as well as its ligands, pleiotrophin and midkine in BCC compared to microdissected normal epidermis. qRT-PCR confirmed increased expression of ALK (p<0.05). Stronger expression of phosphorylated ALK in BCC tumour nests than normal skin was observed by immunohistochemistry. Crizotinib, an FDA-approved ALK inhibitor, reduced keratinocyte proliferation in culture, whereas a c-Met inhibitor did not. Crizotinib significantly reduced the expression of GLI1 and CCND2 (members of SHH-pathway) mRNA by approximately 60% and 20%, respectively (p<0.01). Our data suggest that ALK may increase GLI1 expression in parallel with the conventional SHH-pathway and promote keratinocyte proliferation. Hence, an ALK inhibitor alone or in combination with targeting SHH-pathway molecules may be a potential treatment for BCC patients. PMID:24163262

  11. Initial Diagnosis of ALK-Positive Non-Small-Cell Lung Cancer Based on Analysis of ALK Status Utilizing Droplet Digital PCR.

    PubMed

    Lund, H Louise; Hughesman, Curtis B; Fakhfakh, Kareem; McNeil, Kelly; Clemens, Shahira; Hocken, Kimberly; Pettersson, Ryan; Karsan, Aly; Foster, Leonard J; Haynes, Charles

    2016-05-01

    We describe a novel droplet digital PCR (ddPCR) assay capable of detecting genomic alterations associated with inversion translocations. It is applied here to detection of rearrangements in the anaplastic lymphoma kinase (ALK) gene associated with ALK-positive non-small-cell lung cancer (NSCLC). NSCLC patients may carry a nonreciprocal translocation on human chromosome 2, in which synchronized double stranded breaks (DSB) within the echinoderm microtubule-associated protein-like 4 (EML4) gene and ALK lead to an inversion of genetic material that forms the non-natural gene fusion EML4-ALK encoding a constitutively active tyrosine kinase that is associated with 3 to 7% of all NSCLCs. Detection of ALK rearrangements is currently achieved in clinics through direct visualization via a fluorescent in situ hybridization (FISH) assay, which can detect those rearrangements to a limit of detection (LOD) of ca. 15%. We show that the ddPCR assay presented here provides a LOD of 0.25% at lower cost and with faster turnaround times. PMID:27043019

  12. [STAT3 inhibitor].

    PubMed

    Kitamura, Toshio

    2011-01-01

    Clinical efficacies of various molecular-targeted drugs have been recently demonstrated. Most of these drugs are kinase inhibitors. A most successful drug Glivec is an inhibitor of Bcr-Abl fusion kinase, derived from a well-known causative chromosome translocation of chronic myeloid leukemia(CML). Although other kinase inhibitors have also proved to be useful in the therapy of malignant diseases including an ALK inhibitor for lung carcinomas, a general problem of kinase inhibitors is their lowspecificities. Therefore, the complication of these drugs must be overcome. Recently, trials to develop moleculartargeted therapy whose targets are molecules other than kinases have also been promising. Among molecular targets, STAT3 has attracted a great deal of researchers' attention because it is constitutively activated in most malignant tumors and plays important roles in carcinogenesis. This article summarizes the current situation and problems to be solved with STAT3 inhibitors as well as our recent findings on the molecular mechanisms of STAT3 activation. PMID:21368456

  13. 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.

  14. [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

  15. Adaptive Response Enzyme AlkB Preferentially Repairs 1-Methylguanine and 3-Methylthymine Adducts in Double-Stranded DNA.

    PubMed

    Chen, Fangyi; Tang, Qi; Bian, Ke; Humulock, Zachary T; Yang, Xuedong; Jost, Marco; Drennan, Catherine L; Essigmann, John M; Li, Deyu

    2016-04-18

    The AlkB protein is a repair enzyme that uses an α-ketoglutarate/Fe(II)-dependent mechanism to repair alkyl DNA adducts. AlkB has been reported to repair highly susceptible substrates, such as 1-methyladenine and 3-methylcytosine, more efficiently in ss-DNA than in ds-DNA. Here, we tested the repair of weaker AlkB substrates 1-methylguanine and 3-methylthymine and found that AlkB prefers to repair them in ds-DNA. We also discovered that AlkB and its human homologues, ABH2 and ABH3, are able to repair the aforementioned adducts when the adduct is present in a mismatched base pair. These observations demonstrate the strong adaptability of AlkB toward repairing various adducts in different environments. PMID:26919079

  16. Anterograde Jelly belly ligand to Alk receptor signaling at developing synapses is regulated by Mind the gap

    PubMed Central

    Rohrbough, Jeffrey; Broadie, Kendal

    2010-01-01

    Bidirectional trans-synaptic signals induce synaptogenesis and regulate subsequent synaptic maturation. Presynaptically secreted Mind the gap (Mtg) molds the synaptic cleft extracellular matrix, leading us to hypothesize that Mtg functions to generate the intercellular environment required for efficient signaling. We show in Drosophila that secreted Jelly belly (Jeb) and its receptor tyrosine kinase Anaplastic lymphoma kinase (Alk) are localized to developing synapses. Jeb localizes to punctate aggregates in central synaptic neuropil and neuromuscular junction (NMJ) presynaptic terminals. Secreted Jeb and Mtg accumulate and colocalize extracellularly in surrounding synaptic boutons. Alk concentrates in postsynaptic domains, consistent with an anterograde, trans-synaptic Jeb-Alk signaling pathway at developing synapses. Jeb synaptic expression is increased in Alk mutants, consistent with a requirement for Alk receptor function in Jeb uptake. In mtg null mutants, Alk NMJ synaptic levels are reduced and Jeb expression is dramatically increased. NMJ synapse morphology and molecular assembly appear largely normal in jeb and Alk mutants, but larvae exhibit greatly reduced movement, suggesting impaired functional synaptic development. jeb mutant movement is significantly rescued by neuronal Jeb expression. jeb and Alk mutants display normal NMJ postsynaptic responses, but a near loss of patterned, activity-dependent NMJ transmission driven by central excitatory output. We conclude that Jeb-Alk expression and anterograde trans-synaptic signaling are modulated by Mtg and play a key role in establishing functional synaptic connectivity in the developing motor circuit. PMID:20876658

  17. 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

  18. High concordance of ALK rearrangement between primary tumor and paired metastatic lymph node in patients with lung adenocarcinoma

    PubMed Central

    Hou, Likun; Ren, Shengxiang; Su, Bo; Zhang, Liping; Wu, Wei; Zhang, Wei; Dong, Zhengwei; Huang, Yan

    2016-01-01

    Background Lung cancer has heterogeneous features. It remains unclear whether ALK rearrangement was distributed heterogeneously in tumor from different anatomic sites. To address this issue, we investigate the concordance of ALK rearrangement between primary tumors and paired metastatic lymph nodes in pulmonary adenocarcinoma patients. Methods From Sep 2013 to May 2014, resectable lung adenocarcinoma patients with EGFR wildtype and paired metastatic lymph nodes from Tongji University affiliated Shanghai pulmonary hospital were selected into this study. An auto-mated Ventana ALK with clone D5F3 antibody immunohistochemistry (IHC) and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detected ALK rearrangement. Discordant cases between IHC and RT-PCR were further validated by fluorescence in situ hybridization (FISH). Results A total of 101 patients were enrolled into this study with a median age of 60 years old (range, 35–78 years). ALK rearrangement was found in 20 primary lesions, while in 18 paired metastatic lymph nodes. ALK rearrangement was more frequently happened in younger (P<0.001), Nonsmokers (P=0.012), high-stage disease (P=0.021) and predominantly solid growth pattern (P=0.024). The concordance rate between primary tumor and paired metastatic lymph nodes was 98%. Two patients with ALK rearrangement on primary tumor didn’t show ALK gene fusion on paired metastatic lymph nodes. Sixty-eight cases had more than two stations of metastatic lymph nodes. ALK rearrangement in the different station of metastatic lymph nodes of the same patient was consistent. Conclusions High concordant rate of ALK rearrangement between primary tumors and paired metastatic lymph nodes were found in this study. The authors concluded that specimens from metastatic lesions and primary tumors are equally suitable for detection ALK rearrangement. PMID:27293826

  19. 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

  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. PMID:25836421

  1. 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

  2. Occurrence of diverse alkane hydroxylase alkB genes in indigenous oil-degrading bacteria of Baltic Sea surface water.

    PubMed

    Viggor, Signe; Jõesaar, Merike; Vedler, Eve; Kiiker, Riinu; Pärnpuu, Liis; Heinaru, Ain

    2015-12-30

    Formation of specific oil degrading bacterial communities in diesel fuel, crude oil, heptane and hexadecane supplemented microcosms of the Baltic Sea surface water samples was revealed. The 475 sequences from constructed alkane hydroxylase alkB gene clone libraries were grouped into 30 OPFs. The two largest groups were most similar to Pedobacter sp. (245 from 475) and Limnobacter sp. (112 from 475) alkB gene sequences. From 56 alkane-degrading bacterial strains 41 belonged to the Pseudomonas spp. and 8 to the Rhodococcus spp. having redundant alkB genes. Together 68 alkB gene sequences were identified. These genes grouped into 20 OPFs, half of them being specific only to the isolated strains. Altogether 543 diverse alkB genes were characterized in the brackish Baltic Sea water; some of them representing novel lineages having very low sequence identities with corresponding genes of the reference strains. PMID:26541986

  3. Two Cases of Renal Cell Carcinoma Harboring a Novel STRN-ALK Fusion Gene.

    PubMed

    Kusano, Hironori; Togashi, Yuki; Akiba, Jun; Moriya, Fukuko; Baba, Katsuyoshi; Matsuzaki, Naomi; Yuba, Yoshiaki; Shiraishi, Yusuke; Kanamaru, Hiroshi; Kuroda, Naoto; Sakata, Seiji; Takeuchi, Kengo; Yano, Hirohisa

    2016-06-01

    Anaplastic lymphoma kinase (ALK) translocation renal cell carcinomas (RCCs) have been reported by several independent groups in recent times. The clinical behavior and histopathologic characteristics of these carcinomas are not fully understood because of the paucity of cases reported. Here, we describe 2 cases of RCC harboring a novel striatin (STRN)-ALK fusion. The first case was a 33-year-old woman with no sickle cell trait who underwent nephrectomy for right renal mass and had late recurrence in para-aortic lymph nodes twice 10 and 12 years after initial surgery. After the second recurrence, she was carefully observed without any treatment. Twenty-six years after the initial nephrectomy, the second para-aortic lymphadenectomy was performed, and gastrectomy was performed for newly developed primary gastric cancer. The resected para-aortic lymph nodes were largely replaced by metastatic carcinoma. The second case was a 38-year-old man with no sickle cell trait who underwent cytoreductive nephrectomy followed by sunitinib therapy for metastatic RCC. In both cases, the tumor showed solid, papillary, tubular, and mucinous cribriform structures. Psammoma bodies were occasionally seen in the stroma. Tumor cells had a large nucleus and prominent nucleoli with predominantly eosinophilic cytoplasm. Rhabdoid cells and signet-ring cells were also observed. Intracytoplasmic mucin deposition and background mucinous stroma were confirmed. In the second case, tumor necrosis was seen in some areas. Tumor cells exhibited diffuse positive staining for ALK in both cases. ALK translocation was confirmed by fluorescent in situ hybridization, and further gene analysis revealed a STRN-ALK fusion. These cases provide great insights into ALK translocation RCCs. PMID:26848800

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

    PubMed

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

    2015-06-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-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 ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) 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 ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC 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,N(2)-ɛG and that ALKBH3 removes only ɛC 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

  5. Next-Generation Sequencing Identifies Deregulation of MicroRNAs Involved in Both Innate and Adaptive Immune Response in ALK+ ALCL

    PubMed Central

    Steinhilber, Julia; Bonin, Michael; Walter, Michael; Fend, Falko; Bonzheim, Irina; Quintanilla-Martinez, Leticia

    2015-01-01

    Anaplastic large cell lymphoma (ALCL) is divided into two systemic diseases according to the expression of the anaplastic lymphoma kinase (ALK). We investigated the differential expression of miRNAs between ALK+ ALCL, ALK- ALCL cells and normal T-cells using next generation sequencing (NGS). In addition, a C/EBPβ-dependent miRNA profile was generated. The data were validated in primary ALCL cases. NGS identified 106 miRNAs significantly differentially expressed between ALK+ and ALK- ALCL and 228 between ALK+ ALCL and normal T-cells. We identified a signature of 56 miRNAs distinguishing ALK+ ALCL, ALK- ALCL and T-cells. The top candidates significant differentially expressed between ALK+ and ALK- ALCL included 5 upregulated miRNAs: miR-340, miR-203, miR-135b, miR-182, miR-183; and 7 downregulated: miR-196b, miR-155, miR-146a, miR-424, miR-503, miR-424*, miR-542-3p. The miR-17-92 cluster was also upregulated in ALK+ cells. Additionally, we identified a signature of 3 miRNAs significantly regulated by the transcription factor C/EBPβ, which is specifically overexpressed in ALK+ ALCL, including the miR-181 family. Of interest, miR-181a, which regulates T-cell differentiation and modulates TCR signalling strength, was significantly downregulated in ALK+ ALCL cases. In summary, our data reveal a miRNA signature linking ALK+ ALCL to a deregulated immune response and may reflect the abnormal TCR antigen expression known in ALK+ ALCL. PMID:25688981

  6. 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.

  7. Augmentor α and β (FAM150) are ligands of the receptor tyrosine kinases ALK and LTK: Hierarchy and specificity of ligand–receptor interactions

    PubMed Central

    Reshetnyak, Andrey V.; Murray, Phillip B.; Shi, Xiarong; Mo, Elizabeth S.; Mohanty, Jyotidarsini; Tome, Francisco; Bai, Hanwen; Gunel, Murat; Lax, Irit; Schlessinger, Joseph

    2015-01-01

    Receptor tyrosine kinases (RTKs) are a class of cell surface receptors that, upon ligand binding, stimulate a variety of critical cellular functions. The orphan receptor anaplastic lymphoma kinase (ALK) is one of very few RTKs that remain without a firmly established protein ligand. Here we present a novel cytokine, FAM150B, which we propose naming augmentor-α (AUG-α), as a ligand for ALK. AUG-α binds ALK with high affinity and activates ALK in cells with subnanomolar potency. Detailed binding experiments using cells expressing ALK or the related receptor leukocyte tyrosine kinase (LTK) demonstrate that AUG-α binds and robustly activates both ALK and LTK. We show that the previously established LTK ligand FAM150A (AUG-β) is specific for LTK and only weakly binds to ALK. Furthermore, expression of AUG-α stimulates transformation of NIH/3T3 cells expressing ALK, induces IL-3 independent growth of Ba/F3 cells expressing ALK, and is expressed in neuroblastoma, a cancer partly driven by ALK. These experiments reveal the hierarchy and specificity of two cytokines as ligands for ALK and LTK and set the stage for elucidating their roles in development and disease states. PMID:26630010

  8. 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

  9. Presence of anaplastic lymphoma kinase in inflammatory breast cancer.

    PubMed

    Robertson, Fredika M; Petricoin Iii, Emanuel F; Van Laere, Steven J; Bertucci, Francois; Chu, Khoi; Fernandez, Sandra V; Mu, Zhaomei; Alpaugh, Katherine; Pei, Jianming; Circo, Rita; Wulfkuhle, Julia; Ye, Zaiming; Boley, Kimberly M; Liu, Hui; Moraes, Ricardo; Zhang, Xuejun; Demaria, Ruggero; Barsky, Sanford H; Sun, Guoxian; Cristofanilli, Massimo

    2013-01-01

    Although Inflammatory Breast Cancer (IBC) is recognized as the most metastatic variant of locally advanced breast cancer, the molecular basis for the distinct clinical presentation and accelerated program of metastasis of IBC is unknown. Reverse phase protein arrays revealed activation of the receptor tyrosine kinase, anaplastic lymphoma kinase (ALK) and biochemically-linked downstream signaling molecules including JAK1/STAT3, AKT, mTor, PDK1, and AMPKβ in pre-clinical models of IBC. To evaluate the clinical relevance of ALK in IBC, analysis of 25 IBC patient tumors using the FDA approved diagnostic test for ALK genetic abnormalities was performed. These studies revealed that 20/25 (80%) had either increased ALK copy number, low level ALK gene amplification, or ALK gene expression, with a prevalence of ALK alterations in basal-like IBC. One of 25 patients was identified as having an EML4-ALK translocation. The generality of gains in ALK copy number in basal-like breast tumors with IBC characteristics was demonstrated by analysis of 479 breast tumors using the TGCA data-base and our newly developed 79 IBC-like gene signature. The small molecule dual tyrosine kinase cMET/ALK inhibitor, Crizotinib (PF-02341066/Xalkori®, Pfizer Inc), induced both cytotoxicity (IC50 = 0.89 μM) and apoptosis, with abrogation of pALK signaling in IBC tumor cells and in FC-IBC01 tumor xenograft model, a new IBC model derived from pleural effusion cells isolated from an ALK(+) IBC patient. Based on these studies, IBC patients are currently being evaluated for the presence of ALK genetic abnormalities and when eligible, are being enrolled into clinical trials evaluating ALK targeted therapeutics. PMID:24102046

  10. 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 Central

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

    2014-01-01

    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 × 102 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. PMID:25685046

  11. 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. PMID:25685046

  12. Stimulation of the midkine/ALK axis renders glioma cells resistant to cannabinoid antitumoral action.

    PubMed

    Lorente, M; Torres, S; Salazar, M; Carracedo, A; Hernández-Tiedra, S; Rodríguez-Fornés, F; García-Taboada, E; Meléndez, B; Mollejo, M; Campos-Martín, Y; Lakatosh, S A; Barcia, J; Guzmán, M; Velasco, G

    2011-06-01

    Identifying the molecular mechanisms responsible for the resistance of gliomas to anticancer treatments is an issue of great therapeutic interest. Δ(9)-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer, including glioma, an effect that relies, at least in part, on the stimulation of autophagy-mediated apoptosis in tumor cells. Here, by analyzing the gene expression profile of a large series of human glioma cells with different sensitivity to cannabinoid action, we have identified a subset of genes specifically associated to THC resistance. One of these genes, namely that encoding the growth factor midkine (Mdk), is directly involved in the resistance of glioma cells to cannabinoid treatment. We also show that Mdk mediates its protective effect via the anaplastic lymphoma kinase (ALK) receptor and that Mdk signaling through ALK interferes with cannabinoid-induced autophagic cell death. Furthermore, in vivo Mdk silencing or ALK pharmacological inhibition sensitizes cannabinod-resistant tumors to THC antitumoral action. Altogether, our findings identify Mdk as a pivotal factor involved in the resistance of glioma cells to THC pro-autophagic and antitumoral action, and suggest that selective targeting of the Mdk/ALK axis could help to improve the efficacy of antitumoral therapies for gliomas. PMID:21233844

  13. Economic Analysis of Alternative Strategies for Detection of ALK Rearrangements in Non Small Cell Lung Cancer.

    PubMed

    Doshi, Shivang; Ray, David; Stein, Karen; Zhang, Jie; Koduru, Prasad; Fogt, Franz; Wellman, Axel; Wat, Ricky; Mathews, Charles

    2016-01-01

    Identification of alterations in ALK gene and development of ALK-directed therapies have increased the need for accurate and efficient detection methodologies. To date, research has focused on the concordance between the two most commonly used technologies, fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC). However, inter-test concordance reflects only one, albeit important, aspect of the diagnostic process; laboratories, hospitals, and payors must understand the cost and workflow of ALK rearrangement detection strategies. Through literature review combined with interviews of pathologists and laboratory directors in the U.S. and Europe, a cost-impact model was developed that compared four alternative testing strategies-IHC only, FISH only, IHC pre-screen followed by FISH confirmation, and parallel testing by both IHC and FISH. Interviews were focused on costs of reagents, consumables, equipment, and personnel. The resulting model showed that testing by IHC alone cost less ($90.07 in the U.S., $68.69 in Europe) than either independent or parallel testing by both FISH and IHC ($441.85 in the U.S. and $279.46 in Europe). The strategies differed in cost of execution, turnaround time, reimbursement, and number of positive results detected, suggesting that laboratories must weigh the costs and the clinical benefit of available ALK testing strategies. PMID:26838801

  14. 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

  15. Economic Analysis of Alternative Strategies for Detection of ALK Rearrangements in Non Small Cell Lung Cancer

    PubMed Central

    Doshi, Shivang; Ray, David; Stein, Karen; Zhang, Jie; Koduru, Prasad; Fogt, Franz; Wellman, Axel; Wat, Ricky; Mathews, Charles

    2016-01-01

    Identification of alterations in ALK gene and development of ALK-directed therapies have increased the need for accurate and efficient detection methodologies. To date, research has focused on the concordance between the two most commonly used technologies, fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC). However, inter-test concordance reflects only one, albeit important, aspect of the diagnostic process; laboratories, hospitals, and payors must understand the cost and workflow of ALK rearrangement detection strategies. Through literature review combined with interviews of pathologists and laboratory directors in the U.S. and Europe, a cost-impact model was developed that compared four alternative testing strategies—IHC only, FISH only, IHC pre-screen followed by FISH confirmation, and parallel testing by both IHC and FISH. Interviews were focused on costs of reagents, consumables, equipment, and personnel. The resulting model showed that testing by IHC alone cost less ($90.07 in the U.S., $68.69 in Europe) than either independent or parallel testing by both FISH and IHC ($441.85 in the U.S. and $279.46 in Europe). The strategies differed in cost of execution, turnaround time, reimbursement, and number of positive results detected, suggesting that laboratories must weigh the costs and the clinical benefit of available ALK testing strategies. PMID:26838801

  16. 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 d

  17. Intratumor Heterogeneity of ALK-Rearrangements and Homogeneity of EGFR-Mutations in Mixed Lung Adenocarcinoma

    PubMed Central

    Marino, Federica Zito; Liguori, Giuseppina; Aquino, Gabriella; La Mantia, Elvira; Bosari, Silvano; Ferrero, Stefano; Rosso, Lorenzo; Gaudioso, Gabriella; De Rosa, Nicla; Scrima, Marianna; Martucci, Nicola; La Rocca, Antonello; Normanno, Nicola; Morabito, Alessandro; Rocco, Gaetano; Botti, Gerardo; Franco, Renato

    2015-01-01

    Background Non Small Cell Lung Cancer is a highly heterogeneous tumor. Histologic intratumor heterogeneity could be ‘major’, characterized by a single tumor showing two different histologic types, and ‘minor’, due to at least 2 different growth patterns in the same tumor. Therefore, a morphological heterogeneity could reflect an intratumor molecular heterogeneity. To date, few data are reported in literature about molecular features of the mixed adenocarcinoma. The aim of our study was to assess EGFR-mutations and ALK-rearrangements in different intratumor subtypes and/or growth patterns in a series of mixed adenocarcinomas and adenosquamous carcinomas. Methods 590 Non Small Cell Lung Carcinomas tumor samples were revised in order to select mixed adenocarcinomas with available tumor components. Finally, only 105 mixed adenocarcinomas and 17 adenosquamous carcinomas were included in the study for further analyses. Two TMAs were built selecting the different intratumor histotypes. ALK-rearrangements were detected through FISH and IHC, and EGFR-mutations were detected through IHC and confirmed by RT-PCR. Results 10/122 cases were ALK-rearranged and 7 from those 10 showing an intratumor heterogeneity of the rearrangements. 12/122 cases were EGFR-mutated, uniformly expressing the EGFR-mutated protein in all histologic components. Conclusion Our data suggests that EGFR-mutations is generally homogeneously expressed. On the contrary, ALK-rearrangement showed an intratumor heterogeneity in both mixed adenocarcinomas and adenosquamous carcinomas. The intratumor heterogeneity of ALK-rearrangements could lead to a possible impact on the therapeutic responses and the disease outcomes. PMID:26422230

  18. Growth differentiation factor 11 signals through the transforming growth factor-beta receptor ALK5 to regionalize the anterior-posterior axis.

    PubMed

    Andersson, Olov; Reissmann, Eva; Ibáñez, Carlos F

    2006-08-01

    Growth differentiation factor 11 (GDF11) contributes to regionalize the mouse embryo along its anterior-posterior axis by regulating the expression of Hox genes. The identity of the receptors that mediate GDF11 signalling during embryogenesis remains unclear. Here, we show that GDF11 can interact with type I receptors ALK4, ALK5 and ALK7, but predominantly uses ALK4 and ALK5 to activate a Smad3-dependent reporter gene. Alk5 mutant embryos showed malformations in anterior-posterior patterning, including the lack of expression of the posterior determinant Hoxc10, that resemble defects found in Gdf11-null mutants. A heterozygous mutation in Alk5, but not in Alk4 or Alk7, potentiated Gdf11(-/-)-like phenotypes in vertebral, kidney and palate development in an Acvr2b(-/-) background, indicating a genetic interaction between the two receptor genes. Thus, the transforming growth factor-beta (TGF-beta) receptor ALK5, which until now has only been associated with the biological functions of TGF-beta1 to TGF-beta3 proteins, mediates GDF11 signalling during embryogenesis. PMID:16845371

  19. 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

  20. Response to MET inhibitors in patients with stage IV lung adenocarcinomas harboring MET mutations causing exon 14 skipping

    PubMed Central

    Paik, Paul K.; Drilon, Alexander; Fan, Pang-Dian; Yu, Helena; Rekhtman, Natasha; Ginsberg, Michelle S.; Borsu, Laetitia; Schultz, Nikolaus; Berger, Michael F.; Rudin, Charles M.; Ladanyi, Marc

    2015-01-01

    Mutations in the MET exon 14 RNA splice acceptor and donor sites, which lead to exon skipping, deletion of the juxtamembrane domain containing the Cbl E3-ubiquitin ligase binding site, and decreased turnover of the resultant aberrant MET protein, were previously reported to be oncogenic in preclinical models. We now report responses to the MET inhibitors crizotinib and cabozantinib in four patients with stage IV lung adenocarcinomas harboring mutations leading to MET exon 14 skipping, highlighting a new therapeutic strategy for the 4% of lung adenocarcinoma patients whose tumors harbor this previously underappreciated genetic alteration. PMID:25971939

  1. 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.

  2. Novel Alkane Hydroxylase Gene (alkB) Diversity in Sediments Associated with Hydrocarbon Seeps in the Timor Sea, Australia▿

    PubMed Central

    Wasmund, Kenneth; Burns, Kathryn A.; Kurtböke, D. Ipek; Bourne, David G.

    2009-01-01

    Hydrocarbon seeps provide inputs of petroleum hydrocarbons to widespread areas of the Timor Sea. Alkanes constitute the largest proportion of chemical components found in crude oils, and therefore genes involved in the biodegradation of these compounds may act as bioindicators for this ecosystem's response to seepage. To assess alkane biodegradation potential, the diversity and distribution of alkane hydroxylase (alkB) genes in sediments of the Timor Sea were studied. Deduced AlkB protein sequences derived from clone libraries identified sequences only distantly related to previously identified AlkB sequences, suggesting that the Timor Sea maybe a rich reservoir for novel alkane hydroxylase enzymes. Most sequences clustered with AlkB sequences previously identified from marine Gammaproteobacteria though protein sequence identities averaged only 73% (with a range of 60% to 94% sequence identities). AlkB sequence diversity was lower in deep water (>400 m) samples off the continental slope than in shallow water (<100 m) samples on the continental shelf but not significantly different in response to levels of alkanes. Real-time PCR assays targeting Timor Sea alkB genes were designed and used to quantify alkB gene targets. No correlation was found between gene copy numbers and levels of hydrocarbons measured in sediments using sensitive gas chromatography-mass spectrometry techniques, probably due to the very low levels of hydrocarbons found in most sediment samples. Interestingly, however, copy numbers of alkB genes increased substantially in sediments exposed directly to active seepage even though only low or undetectable concentrations of hydrocarbons were measured in these sediments in complementary geochemical analyses due to efficient biodegradation. PMID:19820158

  3. 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

  4. Nonvolatile S-alk(en)ylthio-L-cysteine derivatives in fresh onion (Allium cepa L. cultivar).

    PubMed

    Starkenmann, Christian; Niclass, Yvan; Troccaz, Myriam

    2011-09-14

    The L-cysteine derivatives (R)-2-amino-3-(methyldisulfanyl)propanoic acid (S-methylthio-L-cysteine), (R)-2-amino-3-(propyldisulfanyl)propanoic acid (S-propylthio-L-cysteine), (R)-2-amino-3-(1-propenyldisulfanyl)propanoic acid (S-(1-propenylthio)-L-cysteine), and (R)-2-amino-3-(2-propenyldisulfanyl)propanoic acid (S-allylthio-L-cysteine) were prepared from 3-[(methoxycarbonyl)dithio]-L-alanine, obtained from the reaction of L-cysteine with methoxycarbonylsulfenyl chloride. The occurrence of these S-(+)-alk(en)ylthio-L-cysteine derivatives in onion (Allium cepa L.) was proven by using UPLC-MS-ESI(+) in SRM mode. Their concentrations in fresh onion were estimated to be 0.19 mg/kg S-methylthio-L-cysteine, 0.01 mg/kg S-propylthio-L-cysteine, and 0.56 mg/kg (S-(1-propenyllthio)-L-cysteine, concentrations that are about 3000 times lower than that of isoalliin (S-(1-propenyl-S-oxo-L-cysteine). These compounds were treated with Fusobacterium nucleatum, a microorganism responsible for the formation of mouth malodor. These L-cysteine disulfides were demonstrated to predominantly produce tri- and tetrasulfides. Isoalliin is almost entirely consumed by the plant enzyme alliin lyase (EC 4.4.1.4 S-alk(en)yl-S-oxo-L-cysteine lyase) in a few seconds, but it is not transformed by F. nucleatum. This example of flavor modulation shows that the plant produces different precursors, leading to the formation of the same types of volatile sulfur compounds. Whereas the plant enzyme efficiently transforms S-alk(en)yl-S-oxo-L-cysteine, mouth bacteria are responsible for the transformation of S-alk(en)ylthio-L-cysteine. PMID:21854077

  5. Relationship between lipophilicity and inhibitory activity against cancer cell growth of nine kinds of alk(en)yl trisulfides with different side chains.

    PubMed

    Iitsuka, Yuji; Tanaka, Yuki; Hosono-Fukao, Tomomi; Hosono, Takashi; Seki, Taiichiro; Ariga, Toyohiko

    2010-01-01

    Of the compounds contained in or derived from garlic (Allium sativum L.), alk(en)yl sulfides are known to be responsible for most of the physiological or neutraceutical functions of garlic. We previously found that diallyl trisulfide (DATS) is a potent inhibitor of tubulin polymerization and cancer cell growth, and an effective stimulator of the hepatic detoxification system. Here, we synthesized nine trisulfides having different aliphatic side chains, and determined their log P, a parameter for lipophilicity of nonionized solutes, and inhibitory activities, IC50 (microM), toward cancer cell growth. The log P values of these trisulfides ranged from the lowest, 2.72, for dimethyl trisulfide, to the highest, 7.62, for dipentyl trisulfide. The relationship between the IC50 and log P of the nine trisulfides was parabolic in nature, in which dibutenyl- and dipropyl-compounds, determined to have a log P of approximately 5, were located at the minimum point of the parabola, indicating the maximum potency. The reason why DATS, having a log P of about 4, was excessively stronger than diethyl trisulfide, with a similar log P, is not fully understood; but perhaps it can be explained by a higher reactivity of the diallyl compound in nucleophilic substitution. The compounds with 3-carbon chains were stronger in terms of growth inhibition than the others; but weaker compounds, those with 4- or 5-carbon chains, showed higher activity if a double bond was introduced into them to reduce their log P to the effective range. In this study, we confirmed the superiority of trisulfides with 3-carbon chains [i.e., DATS and dipropyl trisulfide (DPTS)]. In addition, we observed for the first time that dibutenyl trisulfide, a compound not found in garlic, is one of the potent structures among alk(en)yl trisulfides. PMID:20939433

  6. 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

  7. 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

  8. 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

  9. Structure of Escherichia coli AlkA in Complex with Undamaged DNA

    SciTech Connect

    Bowman, Brian R.; Lee, Seongmin; Wang, Shuyu; Verdine, Gregory L

    2010-11-22

    Because DNA damage is so rare, DNA glycosylases interact for the most part with undamaged DNA. Whereas the structural basis for recognition of DNA lesions by glycosylases has been studied extensively, less is known about the nature of the interaction between these proteins and undamaged DNA. Here we report the crystal structures of the DNA glycosylase AlkA in complex with undamaged DNA. The structures revealed a recognition mode in which the DNA is nearly straight, with no amino acid side chains inserted into the duplex, and the target base pair is fully intrahelical. A comparison of the present structures with that of AlkA recognizing an extrahelical lesion revealed conformational changes in both the DNA and protein as the glycosylase transitions from the interrogation of undamaged DNA to catalysis of nucleobase excision. Modeling studies with the cytotoxic lesion 3-methyladenine and accompanying biochemical experiments suggested that AlkA actively interrogates the minor groove of the DNA while probing for the presence of lesions.

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

    PubMed

    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

  11. 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

  12. 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

  13. 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

  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. Tmem100, an ALK1 receptor signaling-dependent gene essential for arterial endothelium differentiation and vascular morphogenesis

    PubMed Central

    Somekawa, Satoshi; Imagawa, Keiichi; Hayashi, Hisaki; Sakabe, Masahide; Ioka, Tomoko; Sato, Genki E.; Inada, Ken; Iwamoto, Takaaki; Mori, Toshio; Uemura, Shiro; Nakagawa, Osamu; Saito, Yoshihiko

    2012-01-01

    Members of the transforming growth factor-β superfamily play essential roles in various aspects of embryonic development and physiological organ function. Among them, bone morphogenetic protein (BMP) 9 and BMP10 regulate embryonic vascular development by activating their endothelial receptor ALK1 (activin receptor-like kinase 1, also called Acvrl1). ALK1-mediated intracellular signaling is implicated in the etiologies of human diseases, but their downstream functional proteins are largely unknown. In this study, we identified Tmem100, a gene encoding a previously uncharacterized intracellular transmembrane protein, to be an embryonic endothelium-enriched gene activated by BMP9 and BMP10 through the ALK1 receptor. Tmem100 null mice showed embryonic lethality due to impaired differentiation of arterial endothelium and defects of vascular morphogenesis, which phenocopied most of the vascular abnormalities observed with the Acvrl1/Alk1 deficiency. The activity of Notch- and Akt-mediated signaling, which is essential for vascular development, was down-regulated in Tmem100 null mice. Cre-mediated deletion of Tmem100 in endothelial cells was sufficient to recapitulate the null phenotypes. These data indicated that TMEM100 may play indispensable roles downstream of BMP9/BMP10-ALK1 signaling during endothelial differentiation and vascular morphogenesis. PMID:22783020

  16. 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

  17. 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

  18. CHARACTERIZATION OF THE ALKANE-INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS: IDENTIFICATION OF A NEW P450 GENE FAMILY

    EPA Science Inventory

    The P450ALK gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. tructural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures a...

  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. PMID:17612934

  20. 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 ...

  1. 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

  2. Metformin and salinomycin as the best combination for the eradication of NSCLC monolayer cells and their alveospheres (cancer stem cells) irrespective of EGFR, KRAS, EML4/ALK and LKB1 status

    PubMed Central

    Xiao, Zhiguang; Sperl, Bianca; Ullrich, Axel; Knyazev, Pjotr

    2014-01-01

    The presence of cancer stem cells (CSCs) is linked to preexisting or acquired drug resistance and tumor relapse. Therefore, targeting both differentiated tumor cells and CSCs was suggested as an effective approach for non-small cell lung cancer (NSCLC) treatment. After screening of chemotherapeutic agents, tyrosine kinase inhibitors (TKIs) or monoclonal antibody in combination with the putative stem cell killer Salinomycin (SAL), we found Metformin (METF), which modestly exerted a growth inhibitory effect on monolayer cells and alveospheres/CSCs of 5 NSCLC cell lines regardless of their EGFR, KRAS, EML4/ALK and LKB1 status, interacted synergistically with SAL to effectively promote cell death. Inhibition of EGFR (AKT, ERK1/2) and mTOR (p70 s6k) signaling with the combination of METF and SAL can be augmented beyond that achieved using each agent individually. Phospho-kinase assay further suggested the multiple roles of this combination in reducing oncogenic effects of modules, such as ß-catenin, Src family kinases (Src, Lyn, Yes), Chk-2 and FAK. Remarkably, significant reduction of sphere formation was seen under combinatorial treatment in all investigated NSCLC cell lines. In conclusion, METF in combination with SAL could be a promising treatment option for patients with advanced NSCLC irrespective of their EGFR, KRAS, EML4/ALK and LKB1 status. PMID:25375092

  3. Metformin and salinomycin as the best combination for the eradication of NSCLC monolayer cells and their alveospheres (cancer stem cells) irrespective of EGFR, KRAS, EML4/ALK and LKB1 status.

    PubMed

    Xiao, Zhiguang; Sperl, Bianca; Ullrich, Axel; Knyazev, Pjotr

    2014-12-30

    The presence of cancer stem cells (CSCs) is linked to preexisting or acquired drug resistance and tumor relapse. Therefore, targeting both differentiated tumor cells and CSCs was suggested as an effective approach for non-small cell lung cancer (NSCLC) treatment. After screening of chemotherapeutic agents, tyrosine kinase inhibitors (TKIs) or monoclonal antibody in combination with the putative stem cell killer Salinomycin (SAL), we found Metformin (METF), which modestly exerted a growth inhibitory effect on monolayer cells and alveospheres/CSCs of 5 NSCLC cell lines regardless of their EGFR, KRAS, EML4/ALK and LKB1 status, interacted synergistically with SAL to effectively promote cell death. Inhibition of EGFR (AKT, ERK1/2) and mTOR (p70 s6k) signaling with the combination of METF and SAL can be augmented beyond that achieved using each agent individually. Phospho-kinase assay further suggested the multiple roles of this combination in reducing oncogenic effects of modules, such as ß-catenin, Src family kinases (Src, Lyn, Yes), Chk-2 and FAK. Remarkably, significant reduction of sphere formation was seen under combinatorial treatment in all investigated NSCLC cell lines. In conclusion, METF in combination with SAL could be a promising treatment option for patients with advanced NSCLC irrespective of their EGFR, KRAS, EML4/ALK and LKB1 status. PMID:25375092

  4. Comparison of CRP and ALK-P serum levels in prediction of preterm delivery

    PubMed Central

    Shahshahan, Zahra; Iravani, Hoda

    2016-01-01

    Background: Preterm birth, defined as birth occurring before 37 weeks of gestation, is a common complication of pregnancy and may lead to death or long-term disability in newborns. Accurate diagnosis is, therefore, crucial for identifying those women undergoing preterm labor who are at greatest risk of preterm delivery. This may allow transport to a regional obstetrical center and permit time for corticosteroid therapy. Recent study recommends several markers such as CRP (C-reactive protein) and ALK-P (alkaline phosphatase) to predict preterm delivery. Materials and Methods: We select a total of 300 pregnant women that had symptoms of premature birth. All of them were under treatment with tocolytic and serum sample were taken to assess the level of CRP-ALKp. Cervix length and the time of response to tocolytic were measured. 110 pregnant of them had preterm labor. 110 patient that had a term labor selected as a control group. Results: Qualitative evaluation of efficacy CRP level on preterm delivery showed a significant relationship with 27 as a cut of point of CRP (P < 0.00001 –OR = 7.5). Investigate of effect of ALK-P level on preterm delivery refers to a significant relationship with 399 as a cut of point of ALKP (P < 0.00001 –OR = 5). Inquire of efficacy of CRP level and ALK-P level on preterm delivery demonstrate a significant relationship (P < 0.0001 1OR = 9). Conclusions: Maternal concentrations of CRP and ALKP and cervix length can be used as appropriate biomarker for predicting preterm labor and response to tocolytic therapy in pregnant women. PMID:26962519

  5. 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

  6. A novel molecular variant of the chimeric NPM-ALK transcript in Ki-1 positive large cell lymphoma

    SciTech Connect

    Ladanyi, M.

    1994-09-01

    The breakpoints of the t(2;5)(p23;q35), reported to be present in 30-40% of Ki-1 positive large cell lymphoma (Ki-1 LCL), have recently been cloned. They involve a novel tyrosine kinase gene, ALK, at 2p23 and the nucleophosmin gene, NPM, at 5q35. Reverse transcriptase polymerase chain reaction (RT-PCR) using NPM and ALK primers detects a consistent fusion product in Ki-1 cases with the translocation. We performed NPM-ALK RT-PCR on 16 cases of Ki-1 LCL, of which 13 had informative cytogenetics. Amplifiable template was confirmed in all samples by RT-PCR using {beta}-actin primers. Ten cases showed the expected 177 base pair (bp) RT-PCR product indicative of the translocation, including all 6 cases with cytogenetic evidence of the t(2;5) and 3 cases without 5q35 abnormalities. One additional case, which had uninformative cytogenetics, showed only a novel 144 bp RT-PCR product. Sequencing of this PCR product showed an in-frame junction of NPM to ALK, 20 bp distal to the usual NPM junction site and 53 bp distal to usual ALK junction site. The predicted chimeric protein is thus shorter by 11 amino acids, but the putative ALK catalytic domain remains intact. This case was a histologically typical anaplastic Ki-1 LCL which showed a clonal rearrangement of the T-cell receptor {beta} gene. The functional significance of this molecular variant and its relation to the exon structure of both genes require further study. The overall incidence of the translocation in this series, about 70%, is higher than suspected from cytogenetic analysis alone.

  7. Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma.

    PubMed

    Patwardhan, Parag P; Ivy, Kathryn S; Musi, Elgilda; de Stanchina, Elisa; Schwartz, Gary K

    2016-01-26

    Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10-18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma. PMID:26675259

  8. Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma

    PubMed Central

    Musi, Elgilda; de Stanchina, Elisa; Schwartz, Gary K.

    2016-01-01

    Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10–18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma. PMID:26675259

  9. ALK-positive anaplastic large cell lymphoma with soft tissue involvement in a young woman

    PubMed Central

    Gao, Kehai; Li, Hongtao; Huang, Caihong; Li, Huazhuang; Fang, Jun; Tian, Chen

    2016-01-01

    Introduction Anaplastic large cell lymphoma (ALCL) is a type of non-Hodgkin lymphoma that has strong expression of CD30. ALCL can sometimes involve the bone marrow, and in advanced stages, it can produce destructive extranodal lesions. But anaplastic large cell lymphoma kinase (ALK)+ ALCL with soft tissue involvement is very rare. Case report A 35-year-old woman presented with waist pain for over 1 month. The biopsy of soft tissue lesions showed that these cells were positive for ALK-1, CD30, TIA-1, GranzymeB, CD4, CD8, and Ki67 (90%+) and negative for CD3, CD5, CD20, CD10, cytokeratin (CK), TdT, HMB-45, epithelial membrane antigen (EMA), and pan-CK, which identified ALCL. After six cycles of Hyper-CVAD/MA regimen, she achieved partial remission. Three months later, she died due to disease progression. Conclusion This case illustrates the unusual presentation of ALCL in soft tissue with a bad response to chemotherapy. Because of the tendency for rapid progression, ALCL in young adults with extra-nodal lesions are often treated with high-grade chemotherapy, such as Hyper-CVAD/MA. PMID:27445489

  10. 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

  11. 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

  12. 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 gtll library. solation of the gene has been identified on the basis of its inducibility and partial DNA sequence. ranscripts of this gene were indu...

  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. 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

  15. Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid

    PubMed Central

    Terato, Hiroaki; Masaoka, Aya; Asagoshi, Kenjiro; Honsho, Akiko; Ohyama, Yoshihiko; Suzuki, Toshinori; Yamada, Masaki; Makino, Keisuke; Yamamoto, Kazuo; Ide, Hiroshi

    2002-01-01

    Nitrosation of guanine in DNA by nitrogen oxides such as nitric oxide (NO) and nitrous acid leads to formation of xanthine (Xan) and oxanine (Oxa), potentially cytotoxic and mutagenic lesions. In the present study, we have examined the repair capacity of DNA N-glycosylases from Escherichia coli for Xan and Oxa. The nicking assay with the defined substrates containing Xan and Oxa revealed that AlkA [in combination with endonuclease (Endo) IV] and Endo VIII recognized Xan in the tested enzymes. The activity (Vmax/Km) of AlkA for Xan was 5-fold lower than that for 7-methylguanine, and that of Endo VIII was 50-fold lower than that for thymine glycol. The activity of AlkA and Endo VIII for Xan was further substantiated by the release of [3H]Xan from the substrate. The treatment of E.coli with N-methyl-N′-nitro-N-nitrosoguanidine increased the Xan-excising activity in the cell extract from alkA+ but not alkA– strains. The alkA and nei (the Endo VIII gene) double mutant, but not the single mutants, exhibited increased sensitivity to nitrous acid relative to the wild type strain. AlkA and Endo VIII also exhibited excision activity for Oxa, but the activity was much lower than that for Xan. PMID:12434002

  16. Expression levels of ROS1/ALK/c-MET and therapeutic efficacy of cetuximab plus chemotherapy in advanced biliary tract cancer

    PubMed Central

    Chiang, Nai-Jung; Hsu, Chiun; Chen, Jen-Shi; Tsou, Hsiao-Hui; Shen, Ying-Ying; Chao, Yee; Chen, Ming-Huang; Yeh, Ta-Sen; Shan, Yan-Shen; Huang, Shiu-Feng; Chen, Li-Tzong

    2016-01-01

    Aberrant expression of ROS1, ALK or c-MET (RAM) is implicated in carcinogenesis and cancer drug resistance. We retrospectively evaluated the effect of RAM expression on outcomes for advanced biliary tract cancer patients, who were treated with gemcitabine plus oxaliplatin (GEMOX), with or without cetuximab, in a randomized phase II trial. RAM expression levels on archived tissue sections were scored using immunohistochemistry (IHC). Of 110 tumors with IHC staining for all three markers, 18 were RAMhigh (IHC intensity 3+ for any markers). Ninety-two tumors were RAMlow (IHC intensity <3+ for all markers). All RAMhigh tumors were intra-hepatic cholangiocarcinomas (IHCC). Of the patients with IHCC (n = 80), median overall survival (OS) of RAMhigh group was inferior to that of the RAMlow group (5.7 vs. 11.7 months, p = 0.021). In multivariate analysis RAMhigh remained an independently adverse prognostic factor, with a hazard ratio of 2.01 (p = 0.039). In the RAMlow group, GEMOX treatment with cetuximab significantly improved the disease control rate (68% vs. 41%, p = 0.044), median progression-free survival (7.3 vs. 4.9 months, p = 0.026), and marginally prolonged median OS (14.1 vs 9.6 months, p = 0.056), compared to GEMOX treatment alone. Future trials of anti-EGFR inhibitors for IHCC may consider RAM expression as a patient stratification factor. PMID:27136744

  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 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

  18. NPM-ALK mediates phosphorylation of MSH2 at tyrosine 238, creating a functional deficiency in MSH2 and the loss of mismatch repair

    PubMed Central

    Bone, K M; Wang, P; Wu, F; Wu, C; Li, L; Bacani, J T; Andrew, S E; Lai, R

    2015-01-01

    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 MSH2Y238F mutant is not tyrosine phosphorylated. Furthermore, transfection of MSH2Y238F into these cells substantially decreased the tyrosine phosphorylation of endogenous MSH2. Importantly, gene transfection of MSH2Y238F 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 MSH2Y238F 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. PMID:25978431

  19. 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

  20. Alk3 Mediated Bmp Signaling Controls the Contribution of Epicardially Derived Cells to the Tissues of the Atrioventricular Junction

    PubMed Central

    Lockhart, Marie M.; Boukens, Bastiaan J. D.; Phelps, Aimee L.; Brown, Christina-Lin M.; Toomer, Katelynn A.; Burns, Tara A.; Mukherjee, Rupak D.; Norris, Russell A.; Trusk, Thomas C.; van den Hoff, Maurice J.B.; Wessels, Andy

    2014-01-01

    Recent studies using mouse models for cell fate tracing of epicardial derived cells (EPDCs) have demonstrated that at the atrioventricular (AV) junction EPDCs contribute to the mesenchyme of the AV sulcus, the annulus fibrosus, and the parietal leaflets of the AV valves. There is little insight, however, into the mechanisms that govern the contribution of EPDCs to these tissues. While it has been demonstrated that bone morphogenetic protein (Bmp) signaling is required for AV cushion formation, its role in regulating EPDC contribution to the AV junction remains unexplored. To determine the role of Bmp signaling in the contribution of EPDCs to the AV junction, the Bmp receptor activin-like kinase 3 (Alk3; or Bmpr1a) was conditionally deleted in the epicardium and EPDCs using the mWt1/IRES/GFP-Cre (Wt1Cre) mouse. Embryonic Wt1Cre;Alk3fl/fl specimens showed a significantly smaller AV sulcus and a severely underdeveloped annulus fibrosus. Electrophysiological analysis of adult Wt1Cre;Alk3fl/fl mice showed, unexpectedly, no ventricular pre-excitation. Cell fate tracing revealed a significant decrease in the number of EPDCs within the parietal leaflets of the AV valves. Postnatal Wt1Cre;Alk3fl/fl specimens showed myxomatous changes in the leaflets of the mitral valve. Together these observations indicate that Alk3 mediated Bmp signaling is important in the cascade of events that regulate the contribution of EPDCs to the AV sulcus, annulus fibrosus, and the parietal leaflets of the AV valves. Furthermore, this study shows that EPDCs do not only play a critical role in early developmental events at the AV junction, but that they also are important in the normal maturation of the AV valves. PMID:25300579

  1. 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

  2. A heterochronic genetic change from an EGFR mutation to an ALK rearrangement in a patient with lung adenocarcinoma: a case report.

    PubMed

    Shiroyama, Takayuki; Tamiya, Motohiro; Hayama, Manabu; Nishihara, Takashi; Nishida, Takuji; Tanaka, Ayako; Morishita, Naoko; Suzuki, Hidekazu; Okamoto, Norio; Kawahara, Kunimitsu; Hirashima, Tomonori

    2016-05-01

    An 87-year-old man with postoperative recurrent lung adenocarcinoma was treated with gefitinib. At the beginning of treatment, surgically resected archived tumor tissue revealed a deletion in exon 19 of the EGFR gene, but no ALK gene rearrangement. After gefitinib treatment for 9 months, the disease progressed. Bronchoscopic rebiopsy was used to evaluate resistance mechanisms, and revealed lung adenocarcinoma with wild-type EGFR genes and a newly emerged ALK gene rearrangement. Treatment was switched to alectinib and a partial response was achieved within 4 weeks. This is a rare case of heterochronic genetic change to an ALK gene rearrangement in EGFR mutant lung adenocarcinoma. PMID:27162697

  3. A heterochronic genetic change from an EGFR mutation to an ALK rearrangement in a patient with lung adenocarcinoma: a case report

    PubMed Central

    Tamiya, Motohiro; Hayama, Manabu; Nishihara, Takashi; Nishida, Takuji; Tanaka, Ayako; Morishita, Naoko; Suzuki, Hidekazu; Okamoto, Norio; Kawahara, Kunimitsu; Hirashima, Tomonori

    2016-01-01

    An 87-year-old man with postoperative recurrent lung adenocarcinoma was treated with gefitinib. At the beginning of treatment, surgically resected archived tumor tissue revealed a deletion in exon 19 of the EGFR gene, but no ALK gene rearrangement. After gefitinib treatment for 9 months, the disease progressed. Bronchoscopic rebiopsy was used to evaluate resistance mechanisms, and revealed lung adenocarcinoma with wild-type EGFR genes and a newly emerged ALK gene rearrangement. Treatment was switched to alectinib and a partial response was achieved within 4 weeks. This is a rare case of heterochronic genetic change to an ALK gene rearrangement in EGFR mutant lung adenocarcinoma. PMID:27162697

  4. 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

  5. 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

  6. Endocytic deficiency induced by ITSN-1s knockdown alters the Smad2/3-Erk1/2 signaling balance downstream of Alk5

    PubMed Central

    Bardita, Cristina; Predescu, Dan N.; Sha, Fei; Patel, Monal; Balaji, Ganesh; Predescu, Sanda A.

    2015-01-01

    Recently, we demonstrated in cultured endothelial cells and in vivo that deficiency of an isoform of intersectin-1, ITSN-1s, impairs caveolae and clathrin-mediated endocytosis and functionally upregulates compensatory pathways and their morphological carriers (i.e. enlarged endocytic structures, membranous rings or tubules) that are normally underrepresented. We now show that these endocytic structures internalize the broadly expressed transforming growth factor β receptor I (TGFβ-RI or TGFBR1), also known as Alk5, leading to its ubiquitylation and degradation. Moreover, the apoptotic or activated vascular cells of the ITSN-1s-knockdown mice release Alk5-bearing microparticles to the systemic circulation. These interact with and transfer Alk5 to endocytosis-deficient endothelial cells, resulting in lung endothelial cell survival and phenotypic alteration towards proliferation through activation of Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively). We also show that non-productive assembly of the Alk5–Smad–SARA (Smad anchor for receptor activation, also known as ZFYVE9) signaling complex and preferential formation of the Alk5–mSos–Grb2 complex account for Erk1/2 activation downstream of Alk5 and proliferation of pulmonary endothelial cells. Taken together, our studies demonstrate a functional relationship between the intercellular transfer of Alk5 by microparticles and endothelial cell survival and proliferation, and define a novel molecular mechanism for TGFβ and Alk5-dependent Erk1/2MAPK signaling that is significant for proliferative signaling and abnormal growth. PMID:25720380

  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. Endocytic deficiency induced by ITSN-1s knockdown alters the Smad2/3-Erk1/2 signaling balance downstream of Alk5.

    PubMed

    Bardita, Cristina; Predescu, Dan N; Sha, Fei; Patel, Monal; Balaji, Ganesh; Predescu, Sanda A

    2015-04-15

    Recently, we demonstrated in cultured endothelial cells and in vivo that deficiency of an isoform of intersectin-1, ITSN-1s, impairs caveolae and clathrin-mediated endocytosis and functionally upregulates compensatory pathways and their morphological carriers (i.e. enlarged endocytic structures, membranous rings or tubules) that are normally underrepresented. We now show that these endocytic structures internalize the broadly expressed transforming growth factor β receptor I (TGFβ-RI or TGFBR1), also known as Alk5, leading to its ubiquitylation and degradation. Moreover, the apoptotic or activated vascular cells of the ITSN-1s-knockdown mice release Alk5-bearing microparticles to the systemic circulation. These interact with and transfer Alk5 to endocytosis-deficient endothelial cells, resulting in lung endothelial cell survival and phenotypic alteration towards proliferation through activation of Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively). We also show that non-productive assembly of the Alk5-Smad-SARA (Smad anchor for receptor activation, also known as ZFYVE9) signaling complex and preferential formation of the Alk5-mSos-Grb2 complex account for Erk1/2 activation downstream of Alk5 and proliferation of pulmonary endothelial cells. Taken together, our studies demonstrate a functional relationship between the intercellular transfer of Alk5 by microparticles and endothelial cell survival and proliferation, and define a novel molecular mechanism for TGFβ and Alk5-dependent Erk1/2(MAPK) signaling that is significant for proliferative signaling and abnormal growth. PMID:25720380

  9. 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

  10. 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.

  11. 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

  12. Management and future directions in non-small cell lung cancer with known activating mutations.

    PubMed

    Gerber, David E; Gandhi, Leena; Costa, Daniel B

    2014-01-01

    Lung cancer accounts for a quarter of all cancer deaths. Non-small cell lung cancer (NSCLC) is currently segregated by the presence of actionable driver oncogenes. This review will provide an overview of molecular subsets of lung cancer, including descriptions of the defining oncogenes (EGFR, ALK, KRAS, ROS1, RET, BRAF, ERBB2, NTRK1, FGFR, among others) and how these predict for response to small molecule tyrosine kinase inhibitors (TKIs) that are either clinically available or in clinical trial development for advanced NSCLC. Particular focus will be placed on subsets with EGFR mutated and ALK rearranged NSCLC. Somatic TKI-sensitizing EGFR mutations (such as exon 19 deletions and L858R substitutions) are the most robust predictive biomarker for symptom improvement, radiographic response, and increment in progression-free survival (PFS) when EGFR TKIs (gefitinib, erlotinib, and afatinib) are used for patients with advanced NSCLC. However, the palliative benefits that EGFR TKIs afford are limited by multiple biologic mechanisms of tumor adaptation/resistance (such as the EGFR-T790M mutation and oncogene bypass tracks), and future efforts toward delaying, preventing, and treating resistance are underway. Similar to EGFR mutations, ALK rearrangements exemplify an oncogene-driven NSCLC that can be effectively palliated with a precision TKI therapy (the multitargeted ALK/MET/ROS1 TKI crizotinib). When resistance to first-line crizotinib therapy occurs, multiple second generation ALK TKIs have demonstrated impressive rates of disease control in clinical trials, and these may modify long-term outcomes for patients with ALK-positive NSCLC. The development of TKIs for other oncogene-driven NSCLCs may expand the portfolio of precision therapies for this recalcitrant cancer. PMID:24857124

  13. Targeted Therapies for Lung Cancer.

    PubMed

    Stinchcombe, Thomas E

    2016-01-01

    Targeted therapies have become standard therapies for patients with non-small cell lung cancer (NSCLC). A phase III trial of carboplatin and paclitaxel with and without bevacizumab in patients with advanced NSCLC with non-squamous histology demonstrated a statistically significant improvement in efficacy. In patients with NSCLC with an activating epidermal growth factor receptor (EGFR) mutation (defined as exon 19 deletion and exon 21 L858R point mutation), phase III trials of EGFR tyrosine kinase inhibitors (TKI) compared to platinum-based chemotherapy have demonstrated superior efficacy in the first-line setting. In patients with NSCLC with anaplastic lymphoma kinase (ALK) rearrangements, phase III trials of crizotinib have demonstrated superior efficacy compared to platinum-pemetrexed in the first-line setting and standard chemotherapy in the second-line setting. A second-generation ALK inhibitor, ceritinib, is available for patients who have progressed after or were intolerant of crizotinib. Crizotinib has also demonstrated activity on patients with ROS1 rearrangements, and BRAF inhibitors (dabrafenib, vemurafenib) have demonstrated activity in patients with NSCLC with BRAF V600E mutation. The oncogenic mutations that are susceptible to targeted therapy are mainly found in non-squamous NSCLC. The development of targeted therapy in patients with squamous NSCLC has been more challenging due to the genomic complexity observed in the squamous histology and the low prevalence of EGFR, ALK, and ROS1 molecular alterations. A phase III trial of cisplatin and gemcitabine with and without necitumumab in patients with advanced NSCLC with squamous histology demonstrated a statistically significant improvement in progression-free and overall survival. PMID:27535394

  14. 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

  15. 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

  16. Expression levels of ROS1/ALK/c-MET and therapeutic efficacy of cetuximab plus chemotherapy in advanced biliary tract cancer.

    PubMed

    Chiang, Nai-Jung; Hsu, Chiun; Chen, Jen-Shi; Tsou, Hsiao-Hui; Shen, Ying-Ying; Chao, Yee; Chen, Ming-Huang; Yeh, Ta-Sen; Shan, Yan-Shen; Huang, Shiu-Feng; Chen, Li-Tzong

    2016-01-01

    Aberrant expression of ROS1, ALK or c-MET (RAM) is implicated in carcinogenesis and cancer drug resistance. We retrospectively evaluated the effect of RAM expression on outcomes for advanced biliary tract cancer patients, who were treated with gemcitabine plus oxaliplatin (GEMOX), with or without cetuximab, in a randomized phase II trial. RAM expression levels on archived tissue sections were scored using immunohistochemistry (IHC). Of 110 tumors with IHC staining for all three markers, 18 were RAM(high) (IHC intensity 3+ for any markers). Ninety-two tumors were RAM(low) (IHC intensity <3+ for all markers). All RAM(high) tumors were intra-hepatic cholangiocarcinomas (IHCC). Of the patients with IHCC (n = 80), median overall survival (OS) of RAM(high) group was inferior to that of the RAM(low) group (5.7 vs. 11.7 months, p = 0.021). In multivariate analysis RAM(high) remained an independently adverse prognostic factor, with a hazard ratio of 2.01 (p = 0.039). In the RAM(low) group, GEMOX treatment with cetuximab significantly improved the disease control rate (68% vs. 41%, p = 0.044), median progression-free survival (7.3 vs. 4.9 months, p = 0.026), and marginally prolonged median OS (14.1 vs 9.6 months, p = 0.056), compared to GEMOX treatment alone. Future trials of anti-EGFR inhibitors for IHCC may consider RAM expression as a patient stratification factor. PMID:27136744

  17. ALK5-Mediated Transforming Growth Factor β Signaling in Neural Crest Cells Controls Craniofacial Muscle Development via Tissue-Tissue Interactions

    PubMed Central

    Han, Arum; Zhao, Hu; Li, Jingyuan; Pelikan, Richard

    2014-01-01

    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; Alk5fl/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; Alk5fl/fl mice during early myogenesis. In addition, Fgf4 and Fgf6 ligands were reduced in Wnt1-Cre; Alk5fl/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. PMID:24912677

  18. Protein Dynamics Control the Progression and Efficiency of the Catalytic Reaction Cycle of the Escherichia coli DNA-Repair Enzyme AlkB*

    PubMed Central

    Ergel, Burçe; Gill, Michelle L.; Brown, Lewis; Yu, Bomina; Palmer, Arthur G.; Hunt, John F.

    2014-01-01

    A central goal of enzymology is to understand the physicochemical mechanisms that enable proteins to catalyze complex chemical reactions with high efficiency. Recent methodological advances enable the contribution of protein dynamics to enzyme efficiency to be explored more deeply. Here, we utilize enzymological and biophysical studies, including NMR measurements of conformational dynamics, to develop a quantitative mechanistic scheme for the DNA repair enzyme AlkB. Like other iron/2-oxoglutarate-dependent dioxygenases, AlkB employs a two-step mechanism in which oxidation of 2-oxoglutarate generates a highly reactive enzyme-bound oxyferryl intermediate that, in the case of AlkB, slowly hydroxylates an alkylated nucleobase. Our results demonstrate that a microsecond-to-millisecond time scale conformational transition facilitates the proper sequential order of substrate binding to AlkB. Mutations altering the dynamics of this transition allow generation of the oxyferryl intermediate but promote its premature quenching by solvent, which uncouples 2-oxoglutarate turnover from nucleobase oxidation. Therefore, efficient catalysis by AlkB depends upon the dynamics of a specific conformational transition, establishing another paradigm for the control of enzyme function by protein dynamics. PMID:25043760

  19. 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

  20. 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

  1. Automated brightfield break-apart in situ hybridization (ba-ISH) application: ALK and MALT1 genes as models.

    PubMed

    Nitta, Hiroaki; Zhang, Wenjun; Kelly, Brian D; Miller, Melanie; Pestic-Dragovich, Lidija; Bieniarz, Christopher; Vasicek, Thomas J; Marafioti, Teresa; Rimsza, Lisa; Grogan, Thomas M

    2010-12-01

    Cancer diagnosis can be a complex process, which takes consideration of histopathological, clinical, immunophenotypic, and genetic features. Since non-random chromosomal translocations are specifically involved in the development of various cancers, the detection of these gene aberrations becomes increasingly important. In recent years, break-apart (or split-signal) fluorescence in situ hybridization (FISH) has emerged as an advantageous technique to detect gene translocations on tissue sections. However, FISH assays are technically challenging and require specialized fluorescence microscopes. Furthermore, the FISH signal is not stable for long term archiving due to photo bleaching. Our objective was to demonstrate the feasibility of brightfield break-apart in situ hybridization (ba-ISH) for anaplastic lymphoma kinase (ALK) and mucosa-associated lymphoid tissue translocation protein 1 (MALT1) genes as models. ALK or MALT1 break-apart probes were labeled with digoxigenin (DIG) or 2,4-dinitrophenyl (DNP) on both sides of a known gene breakpoint region and the hybridization sites were visualized with the combination of alkaline phosphatase (AP)-based blue and red detection. Therefore, normal genes are detected as purple dots by mixing blue and red colors while translocated genes are detected as isolated blue or red dots. Formalin-fixed, paraffin-embedded tonsil was used as control for the co-localized 5' and 3' probes. Gene translocations of ALK or MALT1 were detected as separate blue and red dots on ALCL and MALT lymphoma cases. Thus, ISH analyses of gene translocations can be conducted with a regular light microscope and the long term archiving of break-apart ISH slides can be achieved. PMID:20621192

  2. Alternative Pathway for the Reaction Catalyzed by DNA Dealkylase AlkB from Ab Initio QM/MM Calculations

    PubMed Central

    2015-01-01

    AlkB is the title enzyme of a family of DNA dealkylases that catalyze the direct oxidative dealkylation of nucleobases. The conventional mechanism for the dealkylation of N1-methyl adenine (1-meA) catalyzed by AlkB after the formation of FeIV–oxo is comprised by a reorientation of the oxo moiety, hydrogen abstraction, OH rebound from the Fe atom to the methyl adduct, and the dissociation of the resulting methoxide to obtain the repaired adenine base and formaldehyde. An alternative pathway with hydroxide as a ligand bound to the iron atom is proposed and investigated by QM/MM simulations. The results show OH– has a small impact on the barriers for the hydrogen abstraction and OH rebound steps. The effects of the enzyme and the OH– ligand on the hydrogen abstraction by the FeIV–oxo moiety are discussed in detail. The new OH rebound step is coupled with a proton transfer to the OH– ligand and results in a novel zwitterion intermediate. This zwitterion structure can also be characterized as Fe–O–C complex and facilitates the formation of formaldehyde. In contrast, for the pathway with H2O bound to iron, the hydroxyl product of the OH rebound step first needs to unbind from the metal center before transferring a proton to Glu136 or other residue/substrate. The consistency between our theoretical results and experimental findings is discussed. This study provides new insights into the oxidative repair mechanism of DNA repair by nonheme FeII and α-ketoglutarate (α-KG) dependent dioxygenases and a possible explanation for the substrate preference of AlkB. PMID:25400523

  3. 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

  4. 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.

  5. 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

  6. Detection of rearrangement of anaplastic lymphoma kinase (ALK) and mutation of epidermal growth factor receptor (EGFR) in primary pulmonary lymphoepithelioma-like carcinoma

    PubMed Central

    Wang, Liang; Lin, Yongbin; Cai, Qingqing; Long, Hao; Zhang, Yu; Rong, Tiehua

    2015-01-01

    Background Primary pulmonary lymphoepithelioma-like carcinoma (LELC) is a distinct rare subtype of lung cancer. The prevalence of anaplastic lymphoma kinase (ALK) rearrangement and epidermal growth factor receptor (EGFR) mutation in primary pulmonary LELC had not been thoroughly investigated. Methods We investigated a cohort of 42 patients with primary pulmonary LELC and genotyped for ALK rearrangement and EGFR mutation. ALK rearrangement was detected by fluorescence in situ hybridization (FISH). EGFR mutational analysis of exons 18 through 21 was analyzed by TaqMan real-time polymerase chain reaction (PCR). Results Epstein-Barr virus-encoded RNAs (EBERs) showed positive signals in all 42 patients. By immunohistochemistry staining, all patients demonstrated positive expression of CK5/6 and P63, but almost all patients were negative for TTF-1 (34/34, 100%) or CK7 (34/35, 97.1%). None of the 42 patients had ALK rearrangement. Of 42 patients tested, only one patient (2.4%) harbored L858R mutation and gefitinib was applied to this case, however no objective response was observed and the progression free survival (PFS) time was only 1 month. Conclusions Primary pulmonary LELC is a unique histological subtype of lung cancer. ALK rearrangement and EGFR mutation are lack and they may not be the oncogenic driver gene in pulmonary LELC. Future efforts should be made to explore other oncogenic driver gene to guide targeted therapy in this rare disease to determine the optimal treatment. Keywords Pulmonary lymphoepithelioma-like carcinoma (LELC); anaplastic lymphoma kinase (ALK); epidermal growth factor receptor (EGFR); targeted therapy; Epstein-Barr virus (EBV) PMID:26543602

  7. 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.

  8. Structural Basis for Specificity of TGFβ Family Receptor Small Molecule Inhibitors

    PubMed Central

    Ogunjimi, Abiodun A.; Zeqiraj, Elton; Ceccarelli, Derek F.; Sicheri, Frank; Wrana, Jeffrey L.; David, Laurent

    2015-01-01

    Transforming growth factor-β (TGFβ) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGFβ/Activin pathway receptors, but needs improvement of its EC50 (EC50 = 1 μM) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGFβ/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 TGFβ type I receptor (TβRI) kinase domain in complex with SB431542. We mutated TβRI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated TβRI. We discovered that a Ser280Thr mutation yielded a TβRI 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 optimizing the SB431542 scaffold to more potent and selective inhibitors of the TGFβ/Activin pathway. PMID:21983015

  9. 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

  10. 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

  11. 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.

  12. CFTR Inhibitors

    PubMed Central

    Verkman, Alan S.; Synder, David; Tradtrantip, Lukmanee; Thiagarajah, Jay R.; Anderson, Marc O.

    2014-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated Cl− channel whose major function is to facilitate epithelial fluid secretion. Loss-of-function mutations in CFTR cause the genetic disease cystic fibrosis. CFTR is required for transepithelial fluid transport in certain secretory diarrheas, such as cholera, and for cyst expansion in autosomal dominant polycystic kidney disease. High-throughput screening has yielded CFTR inhibitors of the thiazolidinone, glycine hydrazide and quinoxalinedione chemical classes. The glycine hydrazides target the extracellular CFTR pore, whereas the thiazolidinones and quinoxalinediones act at the cytoplasmic surface. These inhibitors have been widely used in cystic fibrosis research to study CFTR function at the cell and organ levels. The most potent CFTR inhibitor has IC50 of approximately 4 nM. Studies in animal models support the development of CFTR inhibitors for antisecretory therapy of enterotoxin-mediated diarrheas and polycystic kidney disease. PMID:23331030

  13. 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

  14. Investigations of Activated ACVR1/ALK2, a Bone Morphogenetic Protein Type I Receptor, That Causes Fibrodysplasia Ossificans Progressiva

    PubMed Central

    Kaplan, Frederick S.; Seemann, Petra; Haupt, Julia; Xu, Meiqi; Lounev, Vitali Y.; Mullins, Mary; Shore, Eileen M.

    2016-01-01

    Bone morphogenetic protein (BMP) type I receptors are serine-threonine kinase transmembrane signal transduction proteins that regulate a vast array of ligand-dependent cell-fate decisions with temporal and spatial fidelity during development and postnatal life. A recent discovery identified a recurrent activating heterozygous missense mutation in a BMP type I receptor [Activin receptor IA/activin-like kinase 2 (ACVR1; also known as ALK2)] in patients with the disabling genetic disorder fibrodysplasia ossificans progressiva (FOP). Individuals with FOP experience episodes of tissue metamorphosis that convert soft connective tissue such as skeletal muscle into a highly ramified and disabling second skeleton of heterotopic bone. The single nucleotide ACVR1/ALK2 mutation that causes FOP is one of the most specific disease-causing mutations in the human genome and to date the only known inherited activating mutation of a BMP receptor that causes a human disease. Thus, the study of FOP provides the basis for understanding the clinically relevant effects of activating mutations in the BMP signaling pathway. Here we briefly review methodologies that we have applied to studying activated BMP signaling in FOP. PMID:21036241

  15. Review of the current targeted therapies for non-small-cell lung cancer.

    PubMed

    Nguyen, Kim-Son H; Neal, Joel W; Wakelee, Heather

    2014-10-10

    The last decade has witnessed the development of oncogene-directed targeted therapies that have significantly changed the treatment of non-small-cell lung cancer (NSCLC). In this paper we review the data demonstrating efficacy of gefitinib, erlotinib, and afatinib, which target the epidermal growth factor receptor (EGFR), and crizotinib which targets anaplastic lymphoma kinase (ALK). We discuss the challenge of acquired resistance to these small-molecular tyrosine kinase inhibitors and review promising agents which may overcome resistance, including the EGFR T790M-targeted agents CO-1686 and AZD9291, and the ALK-targeted agents ceritinib (LDK378), AP26113, alectinib (CH/RO5424802), and others. Emerging therapies directed against other driver oncogenes in NSCLC including ROS1, HER2, and BRAF are covered as well. The identification of specific molecular targets in a significant fraction of NSCLC has led to the personalized deployment of many effective targeted therapies, with more to come. PMID:25302162

  16. 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.

  17. ALK-rearranged adenocarcinoma with extensive mucin production can mimic mucinous adenocarcinoma: clinicopathological analysis and comprehensive histological comparison with KRAS-mutated mucinous adenocarcinoma.

    PubMed

    Cha, Yoon Jin; Han, Joungho; Hwang, Soo Hyun; Lee, Tae Bum; Kim, Hojoong; Zo, Jea Ill

    2016-06-01

    We aimed to investigate clinicopathological features and histology of ALK-rearranged adenocarcinomas with extensive mucin production (AEM) that mimic mucinous adenocarcinoma (MA). Retrospectively, 12 cases of AEM and 25 cases of MA harbouring KRAS mutation were retrieved. The clinicopathological profile and detailed histological features were analysed and compared based on the ALK and KRAS status. AEMs occurred in younger patients (p = 0.044) and were characterised by floating tubulopapillary pattern (p < 0.001), prominent nucleolus (p < 0.001), and apical cytoplasmic snouts (p < 0.001). In contrast, KRAS-mutated MAs lacked ALK-specific histological patterns (p < 0.05). Instead, tumour-infiltrating leukocytes (p = 0.018) and smooth cytoplasmic borders (p < 0.001) with vesicular nuclei (p = 0.004) were prominent in KRAS-mutated MAs. AEMs demonstrated characteristic tubulopapillary pattern and apical snouts, which were distinguishing features from MAs with KRAS mutation. Apical snouts can be a useful histological surrogate for ALK rearrangement in the pulmonary adenocarcinomas showing extensive mucin that mimic MA. PMID:27114375

  18. 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.

  19. Inflammatory myofibroblastic tumor with RANBP2 and ALK gene rearrangement with bland cytological features mimicking desmoid-type fibromatosis: A case report and review of the literature

    PubMed Central

    HUANG, YU-HUA; TIAN, YU-FENG; LI, CHIEN-FENG

    2016-01-01

    Here, we present an uncommon case of inflammatory myofibroblastic tumor (IMT) involving the mesentery. The tumor was composed of loosely arranged round-to-spindle-shaped tumor cells with amphophilic cytoplasm in an inflammatory and myxoid background. The mitotic activity was low (1 per 50 high-power fields) and the tumor cells lacked cellular atypism. Immunohistochemically, the tumor cells demonstrated strong nuclear membranous staining with anaplastic lymphoma kinase (ALK). In situ hybridization for ALK gene rearrangement revealed a splitting apart of the two signals within the tumor cells. Reverse transcription-polymerase chain reaction revealed that the tumor harbored a ran-binding protein 2 (RANBP2)-ALK rearrangement. IMTs are usually characterized by epithelioid-to-round cells featuring increased mitotic activity, occasionally demonstrating unusual tumor cells and more aggressive clinical behavior. To date, 23 IMTs have been reported with RANBP2 and ALK gene rearrangements. However, the present case demonstrated indolent cytological features, leading to a difficulty in differentiating it from desmoid-type fibromatosis. PMID:26893756

  20. Constitutively Activated ALK2 and Increased SMAD1/5 Cooperatively Induce Bone Morphogenetic Protein Signaling in Fibrodysplasia Ossificans Progressiva*S⃞

    PubMed Central

    Fukuda, Toru; Kohda, Masakazu; Kanomata, Kazuhiro; Nojima, Junya; Nakamura, Atsushi; Kamizono, Jyunji; Noguchi, Yasuo; Iwakiri, Kiyofumi; Kondo, Takeo; Kurose, Junichi; Endo, Ken-ichi; Awakura, Takeshi; Fukushi, Junichi; Nakashima, Yasuharu; Chiyonobu, Tomohiro; Kawara, Akira; Nishida, Yoshihiro; Wada, Ikuo; Akita, Masumi; Komori, Tetsuo; Nakayama, Konosuke; Nanba, Akira; Maruki, Yuichi; Yoda, Tetsuya; Tomoda, Hiroshi; Yu, Paul B.; Shore, Eileen M.; Kaplan, Frederick S.; Miyazono, Kohei; Matsuoka, Masaru; Ikebuchi, Kenji; Ohtake, Akira; Oda, Hiromi; Jimi, Eijiro; Owan, Ichiro; Okazaki, Yasushi; Katagiri, Takenobu

    2009-01-01

    Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by congenital malformation of the great toes and by progressive heterotopic bone formation in muscle tissue. Recently, a mutation involving a single amino acid substitution in a bone morphogenetic protein (BMP) type I receptor, ALK2, was identified in patients with FOP. We report here that the identical mutation, R206H, was observed in 19 Japanese patients with sporadic FOP. This mutant receptor, ALK2(R206H), activates BMP signaling without ligand binding. Moreover, expression of Smad1 and Smad5 was up-regulated in response to muscular injury. ALK2(R206H) with Smad1 or Smad5 induced osteoblastic differentiation that could be inhibited by Smad7 or dorsomorphin. Taken together, these findings suggest that the heterotopic bone formation in FOP may be induced by a constitutively activated BMP receptor signaling through Smad1 or Smad5. Gene transfer of Smad7 or inhibition of type I receptors with dorsomorphin may represent strategies for blocking the activity induced by ALK2(R206H) in FOP. PMID:18684712

  1. Spectral classifications of Gaia16alk (AT2016brw) as type II SN and AT2016brv as type Ia SN

    NASA Astrophysics Data System (ADS)

    Piascik, A. S.; Steele, I. A.

    2016-04-01

    We conducted a spectroscopic observation of transient Gaia16alk (AT2016brw) at 2016-04-24T01:58:41 UT. This transient was observed by the Gaia Photometric Science survey on 2016-04-18T09:16:07 UT at position RA = 17:08:26.60, DEC = 25:30:51.00.

  2. Solitary pulmonary metastasis from lung cancer harboring EML4-ALK after a 15-year disease-free interval.

    PubMed

    Tomizawa, Kenji; Ito, Simon; Suda, Kenichi; Fukui, Takayuki; Usami, Noriyasu; Hatooka, Shunzo; Kuwano, Hiroyuki; Yatabe, Yasushi; Mitsudomi, Tetsuya

    2013-04-01

    It is often difficult to differentiate metachronous primary lung cancers from local pulmonary recurrences when the histopathological findings are similar. A 43-year-old man underwent right upper lobectomy with lymph node dissection for primary lung adenocarcinoma (p-T2aN0M0, stage IB). Fifteen years later, he developed a lung nodule in his right middle lobe. The tumor was preoperatively thought to be a metachronous second primary lung adenocarcinoma, and was surgically resected. Histopathological findings for both tumors were of poorly differentiated adenocarcinoma with mucus production. Both tumors also harbored the EML4 (echinoderm microtubule-associated protein-like 4)-ALK (anaplastic lymphoma kinase) fusion gene (variant 3a+b). Based on this molecular finding, the pulmonary nodule was considered to be a recurrence after very long latent period. PMID:23279872

  3. ALK-Positive Inflammatory Myofibroblastic Tumor of the Nipple During Pregnancy-An Unusual Presentation of a Rare Disease.

    PubMed

    Kovács, Anikó; Máthé, Gyöngyvér; Mattsson, Jan; Stenman, Göran; Kindblom, Lars-Gunnar

    2015-01-01

    Inflammatory myofibroblastic tumors (IMT) is a benign to low-grade malignant neoplasm most commonly occurring in the viscera and soft tissues of children and young adults. Involvement of the breast is very rare. This report presents the first case of IMT of the nipple and highlights the histologic features and differential diagnosis at this unusual anatomical site. The patient was a 31-years-old pregnant woman with a palpable mass at the upper half of the left nipple. The lesion appeared after breastfeeding of her first child and increased in size during her second pregnancy. A conservative, incomplete surgical excision was performed in the 24th week of the second pregnancy. The residual tumor subsequently underwent spontaneous regression. There was no evidence of disease 5 years after surgery. FISH and immunohistochemical analyses revealed rearrangement and overexpression of the ALK gene, a typical feature of both pulmonary and extrapulmonary IMT. PMID:25772857

  4. 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

  5. Structure-activity relationships of anthraquinone derivatives derived from bromaminic acid as inhibitors of ectonucleoside triphosphate diphosphohydrolases (E-NTPDases)

    PubMed Central

    Baqi, Younis; Weyler, Stefanie; Iqbal, Jamshed; Zimmermann, Herbert

    2008-01-01

    Reactive blue 2 (RB-2) had been characterized as a relatively potent ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) inhibitor with some selectivity for NTPDase3. In search for the pharmacophore and to analyze structure-activity relationships we synthesized a series of truncated derivatives and analogs of RB-2, including 1-amino-2-sulfo-4-ar(alk)ylaminoanthraquinones, 1-amino-2-methyl-4-arylaminoanthraquinones, 1-amino-4-bromoanthraquinone 2-sulfonic acid esters and sulfonamides, and bis-(1-amino-4-bromoanthraquinone) sulfonamides, and investigated them in preparations of rat NTPDase1, 2, and 3 using a capillary electrophoresis assay. Several 1-amino-2-sulfo-4-ar(alk)ylaminoanthraquinone derivatives inhibited E-NTPDases in a concentration-dependent manner. The 2-sulfonate group was found to be required for inhibitory activity, since 2-methyl-substituted derivatives were inactive. 1-Amino-2-sulfo-4-p-chloroanilinoanthraquinone (18) was identified as a nonselective competitive blocker of NTPDases1, 2, and 3 (Ki 16–18 μM), while 1-amino-2-sulfo-4-(2-naphthylamino)anthraquinone (21) was a potent inhibitor with preference for NTPDase1 (Ki 0.328 μM) and NTPDase3 (Ki 2.22 μM). Its isomer, 1-amino-2-sulfo-4-(1-naphthylamino)anthraquinone (20), was a potent and selective inhibitor of rat NTPDase3 (Ki 1.5 μM). PMID:18528783

  6. [Proteasome inhibitor].

    PubMed

    Yagi, Hideo

    2014-06-01

    The ubiquitin-proteasome system plays an essential role in degradation of eukaryotic intracellular protein, including cell cycle regulation, cell growth and proliferation, and survival. Cancer cells generally have higher level of proteasome activity compared with normal cells, suggesting proteasome inhibition could be therapeutic target in oncology. Bortezomib, the first proteasome inhibitor introduced into the clinic, is approved for the treatment of patients with multiple myeloma (MM). Although it was approved as single agent in the relapsed setting, bortezomib is now predominantly used in combination with conventional and novel targeted agents because bortezomib has demonstrated additive and synergistic activity in preclinical studies. Recently, several second-generation proteasome inhibitors, such as carfilzomib and MLN9708, have been developed and entered into clinical trials. These agents were investigated in frontline MM in combination with lenalidomide and low-dose dexamethasone. These studies demonstrated positive efficacy and safety, and it is expected that they will be approved in near future. PMID:25016815

  7. 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

  8. 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

  9. Specific Activin Receptor–Like Kinase 3 Inhibitors Enhance Liver Regeneration

    PubMed Central

    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.

    2014-01-01

    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

  10. The Atomic Resolution Structure of Human AlkB Homolog 7 (ALKBH7), a Key Protein for Programmed Necrosis and Fat Metabolism*

    PubMed Central

    Wang, Guoqiang; He, Qingzhong; Feng, Chong; Liu, Yang; Deng, Zengqin; Qi, Xiaoxuan; Wu, Wei; Mei, Pinchao; Chen, Zhongzhou

    2014-01-01

    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. PMID:25122757

  11. Autophagy inhibitors.

    PubMed

    Pasquier, Benoit

    2016-03-01

    Autophagy is a lysosome-dependent mechanism of intracellular degradation. The cellular and molecular mechanisms underlying this process are highly complex and involve multiple proteins, including the kinases ULK1 and Vps34. The main function of autophagy is the maintenance of cell survival when modifications occur in the cellular environment. During the past decade, extensive studies have greatly improved our knowledge and autophagy has exploded as a research field. This process is now widely implicated in pathophysiological processes such as cancer, metabolic, and neurodegenerative disorders, making it an attractive target for drug discovery. In this review, we will summarize the different types of inhibitors that affect the autophagy machinery and provide some potential therapeutic perspectives. PMID:26658914

  12. Biochemical Analysis of Recombinant AlkJ from Pseudomonas putida Reveals a Membrane-Associated, Flavin Adenine Dinucleotide-Dependent Dehydrogenase Suitable for the Biosynthetic Production of Aliphatic Aldehydes

    PubMed Central

    Kirmair, Ludwig

    2014-01-01

    The noncanonical alcohol dehydrogenase AlkJ is encoded on the alkane-metabolizing alk operon of the mesophilic bacterium Pseudomonas putida GPo1. To gain insight into the enzymology of AlkJ, we have produced the recombinant protein in Escherichia coli and purified it to homogeneity using His6 tag affinity and size exclusion chromatography (SEC). Despite synthesis in the cytoplasm, AlkJ was associated with the bacterial cell membrane, and solubilization with n-dodecyl-β-d-maltoside was necessary to liberate the enzyme. SEC and spectrophotometric analysis revealed a dimeric quaternary structure with stoichiometrically bound reduced flavin adenine dinucleotide (FADH2). The holoenzyme showed thermal denaturation at moderate temperatures around 35°C, according to both activity assay and temperature-dependent circular dichroism spectroscopy. The tightly bound coenzyme was released only upon denaturation with SDS or treatment with urea-KBr and, after air oxidation, exhibited the characteristic absorption spectrum of FAD. The enzymatic activity of purified AlkJ for 1-butanol, 1-hexanol, and 1-octanol as well as the n-alkanol derivative ω-hydroxy lauric acid methyl ester (HLAMe) was quantified in the presence of the artificial electron acceptors phenazine methosulfate (PMS) and 2,6-dichlorophenolindophenol (DCPIP), indicating broad substrate specificity with the lowest activity on the shortest alcohol, 1-butanol. Furthermore, AlkJ was able to accept as cosubstrates/oxidants the ubiquinone derivatives Q0 and Q1, also in conjunction with cytochrome c, which suggests coupling to the bacterial respiratory chain of this membrane-associated enzyme in its physiological environment. Using gas chromatographic analysis, we demonstrated specific biocatalytic conversion by AlkJ of the substrate HLAMe to the industrially relevant aldehyde, thus enabling the biotechnological production of 12-amino lauric acid methyl ester via subsequent enzymatic transamination. PMID:24509930

  13. Novel roles of Akt and mTOR in suppressing TGF-β/ALK5-mediated Smad3 activation

    PubMed Central

    Song, Kyung; Wang, Hui; Krebs, Tracy L; Danielpour, David

    2006-01-01

    Insulin-like growth factor-I inhibits transforming growth factor-β (TGF-β) signaling by blocking activation of Smad3 (S3), via a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent pathway. Here we provide the first report that the kinase activity of Akt is necessary for its ability to suppress many TGF-β responses, including S3 activation and induction of apoptosis. Wild-type and myristoylated Akts (AktWT and AktMyr) suppress TGF-β-induced phospho-activation of S3 but not Smad2 (S2), whereas kinase-dead Akt1 (Akt1K179M) or dominant-negative PI3K enhances TGF-β-induced phospho-activation of both S2 and S3. Using siRNA, rapamycin (Rap), and adenoviral expression for FKBP12-resistant and constitutively active TGF-β type I receptor (ALK5), we demonstrate that mammalian target of Rap (mTOR) mediates Akt1 suppression of phospho-activation of S3. These and further data on Akt1-S3 binding do not support a recently proposed model that Akt blocks S3 activation through physical interaction and sequestration of S3 from TGF-β receptors. We propose a novel model whereby Akt suppresses activation of S3 in an Akt kinase-dependent manner through mTOR, a likely route for loss of tumor suppression by TGF-β in cancers. PMID:16362038

  14. 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

  15. Inflammatory myofibroblastic tumor with RANBP2 and ALK gene rearrangement: a report of two cases and literature review

    PubMed Central

    2013-01-01

    Abstract Inflammatory myofibroblastic tumors (IMTs) are categorized as intermediate biologic neoplasms, whereas IMTs with genetic features of ran-binding protein 2 (RANBP2) and anaplastic lymphoma kinase (ALK) rearrangement (IMT-RAs) are possibly related to a more aggressive clinical course. However, fewer than 10 cases of IMT-RA have been reported to date. Herein, we present 2 new cases of IMT-RA in which both tumors recurred quickly after primary surgery; one patient died 3 months later from the disease, and the other patient has been living with the disease for 12 months. IMT-RAs are characterized by noncohesive epithelioid and rounded tumoral cell morphology, commonly derived from pelvic and peritoneal cavities, and frequently show larger tumor sizes. The relation between the clinicopathologic features and poor prognosis of IMT-RA is discussed. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3314123381007714 PMID:24034896

  16. Combined point mutation in KRAS or EGFR genes and EML4-ALK translocation in lung cancer patients.

    PubMed

    Jürgens, Jessica; Engel-Riedel, Walburga; Prickartz, Alexander; Ludwig, Corinna; Schildgen, Oliver; Tillmann, Ramona-Liza; Stoelben, Erich; Brockmann, Michael; Schildgen, Verena

    2014-03-01

    A total of three cases with novel constellations regarding mutation patterns in non-small-cell lung cancer (NSCLC) are reported. The mutation patterns that are observed are novel and unexpected. First, a combined simultaneous KRAS mutation and EML4-ALK translocation, both in the main tumor and a bone metastasis, were observed, these mutations are assumed to mutually exclude each other. A further two cases include a father and a daughter, both of whom are suffering from NSCLC with different EGFR mutation patterns. A common cause was assumed; however, could not be deduced to mutations in the KRAS, BRAF and EGFR genes. The aforementioned cases are important, as it must be taken into account that mutations previously assumed to be exclusive can occur in combination, may influence the clinical outcome and may require different therapy compared with single mutated tumors. It has to be discussed whether diagnostic algorithms need to be adapted. The cases of father and daughter show that further unknown factors can influence development of NSCLC. PMID:24754584

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. Alterations in genes other than EGFR/ALK/ROS1 in non-small cell lung cancer: trials and treatment options.

    PubMed

    Desai, Arpita; Menon, Smitha P; Dy, Grace K

    2016-03-01

    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

  3. 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 Central

    Smoktunowicz, Natalia; Alexander, Robert E.; Franklin, Linda; Williams, Andrew E.; Holman, Beverley; Mercer, Paul F.; Jarai, Gabor; Scotton, Chris J.; Chambers, Rachel C.

    2015-01-01

    ABSTRACT 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. PMID:26138704

  4. 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. PMID:24350757

  5. The dichloromethane extract of the ethnomedicinal plant Neurolaena lobata inhibits NPM/ALK expression which is causal for anaplastic large cell lymphomagenesis.

    PubMed

    Unger, Christine; Popescu, Ruxandra; Giessrigl, Benedikt; Laimer, Daniela; Heider, Susanne; Seelinger, Mareike; Diaz, Rene; Wallnöfer, Bruno; Egger, Gerda; Hassler, Melanie; Knöfler, Martin; Saleh, Leila; Sahin, Emine; Grusch, Michael; Fritzer-Szekeres, Monika; Dolznig, Helmut; Frisch, Richard; Kenner, Lukas; Kopp, Brigitte; Krupitza, Georg

    2013-01-01

    The present study investigates extracts of Neuolaena lobata, an anti-protozoan ethnomedicinal plant of the Maya, regarding its anti-neoplastic properties. Firstly, extracts of increasing polarity were tested in HL-60 cells analyzing inhibition of cell proliferation and apoptosis induction. Secondly, the most active extract was further tested in anaplastic large cell lymphoma (ALCL) cell lines of human and mouse origin. The dichloromethane extract inhibited proliferation of HL-60, human and mouse ALCL cells with an IC50 of ~2.5, 3.7 and 2.4 µg/ml, respectively and arrested cells in the G2/M phase. The extract induced the checkpoint kinases Chk1 and Chk2 and perturbed the orchestrated expression of the Cdc25 family of cell cycle phosphatases which was paralleled by the activation of p53, p21 and downregulation of c-Myc. Importantly, the expression of NPM/ALK and its effector JunB were drastically decreased, which correlated with the activation of caspase 3. Subsequently also platelet derived growth factor receptor β was downregulated, which was recently shown to be transcriptionally controlled by JunB synergizing with ALK in ALCL development. We show that a traditional healing plant extract downregulates various oncogenes, induces tumor suppressors, inhibits cell proliferation and triggers apoptosis of malignant cells. The discovery of the 'Active Principle(s)' is warranted. PMID:23135783

  6. DNA Damage: Quantum Mechanics/Molecular Mechanics Study on the Oxygen Binding and Substrate Hydroxylation Step in AlkB Repair Enzymes

    PubMed Central

    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 N1-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. PMID:24339041

  7. Proton pump inhibitors

    MedlinePlus

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by glands in ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This is a ...

  8. Abundance and diversity of n-alkane-degrading bacteria in a forest soil co-contaminated with hydrocarbons and metals: a molecular study on alkB homologous genes.

    PubMed

    Pérez-de-Mora, Alfredo; Engel, Marion; Schloter, Michael

    2011-11-01

    Unraveling functional genes related to biodegradation of organic compounds has profoundly improved our understanding of biological remediation processes, yet the ecology of such genes is only poorly understood. We used a culture-independent approach to assess the abundance and diversity of bacteria catalyzing the degradation of n-alkanes with a chain length between C(5) and C(16) at a forest site co-contaminated with mineral oil hydrocarbons and metals for nearly 60 years. The alkB gene coding for a rubredoxin-dependent alkane monooxygenase enzyme involved in the initial activation step of aerobic aliphatic hydrocarbon metabolism was used as biomarker. Within the area of study, four different zones were evaluated: one highly contaminated, two intermediately contaminated, and a noncontaminated zone. Contaminant concentrations, hydrocarbon profiles, and soil microbial respiration and biomass were studied. Abundance of n-alkane-degrading bacteria was quantified via real-time PCR of alkB, whereas genetic diversity was examined using molecular fingerprints (T-RFLP) and clone libraries. Along the contamination plume, hydrocarbon profiles and increased respiration rates suggested on-going natural attenuation at the site. Gene copy numbers of alkB were similar in contaminated and control areas. However, T-RFLP-based fingerprints suggested lower diversity and evenness of the n-alkane-degrading bacterial community in the highly contaminated zone compared to the other areas; both diversity and evenness were negatively correlated with metal and hydrocarbon concentrations. Phylogenetic analysis of alkB denoted a shift of the hydrocarbon-degrading bacterial community from Gram-positive bacteria in the control zone (most similar to Mycobacterium and Nocardia types) to Gram-negative genotypes in the contaminated zones (Acinetobacter and alkB sequences with little similarity to those of known bacteria). Our results underscore a qualitative rather than a quantitative response of

  9. 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.

  10. Safety of cranial radiotherapy concurrent with tyrosine kinase inhibitors in non-small cell lung cancer patients: A systematic review.

    PubMed

    Hendriks, Lizza E L; Schoenmaekers, Janna; Zindler, Jaap D; Eekers, Danielle B P; Hoeben, Ann; De Ruysscher, Dirk K M; Dingemans, Anne-Marie C

    2015-07-01

    Recently, non-small cell lung cancer (NSCLC) has been partly subclassified into molecularly-defined oncogene "addicted" tumors for which targeted agents are available. Tyrosine kinase inhibitors (TKI) are currently approved for patients with an activating epidermal growth factor receptor (EGFR) mutation or anaplastic lymphoma kinase (ALK) rearrangement. In these patients, brain metastases are often the first site of progression while on TKI treatment. The TKI may however still be active on extra-cranial sites and clinicians are thus faced with the question if the TKI may be continued during cranial radiotherapy. Advantages of combining TKI with cranial radiotherapy would be a possible synergistic effect on the brain metastases and the prevention of a systemic disease flare-up. A disadvantage is the possibly increased risk of (neuro)toxicity. The present systematic review addresses the toxicity of combining TKI with cranial radiotherapy in NSCLC patients. PMID:25990950

  11. 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

  12. 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

  13. 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

  14. [Inhibitors of xanthine oxidoreductase].

    PubMed

    Okamoto, Ken

    2008-04-01

    Inhibitors of xanthine oxidoreductase decrease production of uric acid, thus they act as hypouricemic drugs. Allopurinol, a prototypical xanthine oxidoreductase inhibitor, has been widely prescribed for treatment of gout and hyperuricemia. However, severe side effects of allopurinol may occur in patients with renal insufficiency. Recently, novel nonpurine selective inhibitors of xanthine oxidoreductase have been developed as potential alternatives to allopurinol. They have different inhibition mechanisms, utilizing the enzyme structure and the reaction mechanism. Such variation of the inhibition mechanism affects/in vivo/hypouricemic effects of the inhibitors. PMID:18409526

  15. Targeted therapies for treatment of non-small cell lung cancer-Recent advances and future perspectives.

    PubMed

    Minguet, Joan; Smith, Katherine H; Bramlage, Peter

    2016-06-01

    Non-small cell lung cancer (NSCLC) is one of the most deadly cancers worldwide, with poor prognosis once the disease has progressed past the point at which surgery is a viable option. Whilst chemotherapy has improved survival over recent decades, there is still great need for improvements in treatments for patients with advanced disease. Over the last decade, a variety of such drugs have received market approval for treating NSCLC, with a variety of others in the pipeline. Here, we review the development of targeted therapies for the treatment of advanced or metastatic NSCLC, including those already in clinical practice and those in early trials. The epidermal growth factor receptor (EGFR) inhibitors, gefitinib, erlotinib and afatinib; the anaplastic lymphoma kinase (ALK) inhibitor, crizotinib; and the anti-vascular endothelial growth factor receptor monoclonal antibody, bevacizumab, are already providing improved survival for patients with NSCLC. Moreover, the discovery of EGFR mutations and ALK rearrangements has enabled the identification of patients who are more likely to benefit from a specific drug. The recent approval of the immune checkpoint inhibitor nivolumab, along with the designation of alectinib and MPDL3280A as breakthrough therapies by the FDA, demonstrates how rapidly this area of research is expanding. Over the last decade there has been significant progress made in the treatment of advanced NSCLC, and the large and varied selection of drugs currently undergoing trials provide great promise for improving the prognosis of this highly prevalent and deadly form of cancer. PMID:26537995

  16. 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.

  17. Screening of telomerase inhibitors.

    PubMed

    Kleideiter, Elke; Piotrowska, Kamilla; Klotz, Ulrich

    2007-01-01

    Shortening of telomeres prevents cells from uncontrolled proliferation. Progressive telomere shortening occurs at each cell division until a critical telomeric length is reached. Telomerase expression is switched off after embryonic differentiation in most normal cells, but it is expressed in a very high percentage of tumors of different origin. Thus, telomerase is regarded as the best tumor marker and a promising novel molecular target for cancer treatment. Therefore, different strategies to inhibit telomerase have been developed. However, systematic screening of telomerase inhibitors has not been performed to compare their therapeutic potential. We propose a suitable strategy for estimation of the therapeutic potential of telomerase inhibitors, which is based on a systematic screening of different inhibitors in the same cell system. From the long list of compounds discussed in the literature, we have selected four telomerase inhibitors of different structure and mode of action: BRACO19 (G-quadruplex-interactive compound), BIBR1532 (non-nucleosidic reverse transcriptase inhibitor), 2'-O-methyl RNA, and peptide nucleic acids (PNAs; hTR antisense oligonucleotides). To determine minimal effective concentrations for telomerase inhibition, telomerase activity was measured using the cell-free telomerase repeat amplification protocol (TRAP) assay. We also tested inhibitors in long-term cell-culture experiments by exposing A-549 cells to non-cytotoxic concentrations of inhibitors for a period of 99 days. Subsequently, telomerase activity of A-549 cells was investigated using the TRAP assay, and telomere length of samples was assessed by telomere restriction fragment (TRF) Southern blot analysis. PMID:18369824

  18. 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.

  19. 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

  20. 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

  1. Aromatase and its inhibitors.

    PubMed

    Brodie, A; Lu, Q; Long, B

    1999-01-01

    Inhibitors of aromatase (estrogen synthetase) have been developed as treatment for postmenopausal breast cancer. Both steroidal substrate analogs, type I inhibitors, which inactivate the enzyme and non-steroidal competitive reversible, type II inhibitors, are now available. 4-hydroxyandrostenedione (4-OHA), the first selective aromatase inhibitor, has been shown to reduce serum estrogen concentrations and cause complete and partial responses in approximately 25% of patients with hormone responsive disease who have relapsed from previous endocrine treatment. Letrozole (CGS 20, 269) and anastrozole (ZN 1033) have been recently approved for treatment. Both suppress serum estrogen levels to the limit of assay detection. Letrozole has been shown to be significantly superior to megace in overall response rates and time to treatment failure, whereas anastrozole was found to improve survival in comparison to megace. Both were better tolerated than the latter. The potential of aromatase within the breast as a significant source of estrogen mediating tumor proliferation and which might determine the outcome of inhibitor treatment was explored. Using immunocytochemistry and in situ hybridization, aromatase and mRNAarom was detected mainly in the epithelial cells of the terminal ductal lobular units (TDLU) of the normal breast and also in breast tumor epithelial cells as well as some stromal cells. Increase in proliferation, measured by increased thymidine incorporation into DNA and by PCNA immunostaining in response to testosterone was observed in histocultures of breast cancer samples. This effect could be inhibited by 4-OHA and implies that intratumoral aromatase has functional significance. An intratumoral aromatase model in the ovariectomized nude mouse was developed which simulated the hormone responsive postmenopausal breast cancer patient. This model also allows evaluation of the efficacy of aromatase inhibitors and antiestrogens in tumors of estrogen receptor positive

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. Protein protease inhibitors in insects and comparison with mammalian inhibitors.

    PubMed

    Eguchi, M

    1993-01-01

    1. Studies on insect protein protease inhibitors are summarized. Biochemical, genetic and physiological investigations of the silkworm are performed. 2. In addition, the properties and characteristics of fungal protease inhibitors from the silkworm (Bombyx mori) are described and their importance as defensive functions is emphasized. 3. This review also concerns comparative and evolutionary studies of protease inhibitors from various sources. 4. The biological significance of inhibitors is discussed in view of the extensive experimental results. PMID:8365101

  10. Sunflower trypsin inhibitor-1.

    PubMed

    Korsinczky, Michael L J; Schirra, Horst Joachim; Craik, David J

    2004-10-01

    SFTI-1 is a bicyclic 14 amino acid peptide that was originally isolated from the seeds of the sunflower Helianthus annuus. It is a potent inhibitor of trypsin, with a sub-nanomolar K(i) value and is homologous to the active site region of the well-known family of serine protease inhibitors known as the Bowman-Birk trypsin inhibitors. It has a cyclic backbone that is cross-braced by a single disulfide bridge and a network of hydrogen bonds that result in a well-defined structure. SFTI-1 is amenable to chemical synthesis, allowing for the creation of synthetic variants. Alterations to the structure such as linearising the backbone or removing the disulfide bridge do not reduce the potency of SFTI-1 significantly, and minimising the peptide to as few as nine residues results in only a small decrease in reactivity. The creation of linear variants of SFTI-1 also provides a tool for investigating putative linear precursor peptides. The mechanism of biosynthesis of SFTI-1 is not yet known but it seems likely that it is a gene-coded product that has arisen from a precursor protein that may be evolutionarily related to classic Bowman-Birk inhibitors. PMID:15544530

  11. Inhibitors of rhomboid proteases.

    PubMed

    Wolf, Eliane V; Verhelst, Steven H L

    2016-03-01

    Rhomboid proteases form one of the most widespread families of intramembrane proteases. They utilize a catalytic serine-histidine dyad located several Å below the surface of the membrane for substrate hydrolysis. Multiple studies have implicated rhomboid proteases in biologically and medically relevant processes. Several assays have been developed that are able to monitor rhomboid activity. With the aid of these assays, different types of inhibitors have been found, all based on electrophiles that covalently react with the active site machinery. Although the currently available inhibitors have limited selectivity and moderate potency, they can function as research tools and as starting point for the development of activity-based probes, which are reagents that can specifically detect active rhomboid species. Structural studies on complexes of inhibitors with the Escherichia coli rhomboid GlpG have provided insight into how substrate recognition may occur. Future synthetic efforts, aided by high-throughput screening or structure-based design, may lead to more potent and selective inhibitors for this interesting family of proteases. PMID:26166068

  12. Conversion of fatty aldehydes into alk (a/e)nes by in vitro reconstituted cyanobacterial aldehyde-deformylating oxygenase with the cognate electron transfer system

    PubMed Central

    2013-01-01

    Background Biosynthesis of fatty alk(a/e)ne in cyanobacteria has been considered as a potential basis for the sunlight-driven and carbon-neutral bioprocess producing advanced solar biofuels. Aldehyde-deformylating oxygenase (ADO) is a key enzyme involved in that pathway. The heterologous or chemical reducing systems were generally used in in vitro ADO activity assay. The cognate electron transfer system from cyanobacteria to support ADO activity is still unknown. Results We identified the potential endogenous reducing system including ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) to support ADO activity in Synechococcus elongatus PCC7942. ADO (Synpcc7942_1593), FNR (SynPcc7942_0978), and Fd (SynPcc7942_1499) from PCC7942 were cloned, overexpressed, purified, and characterized. ADO activity was successfully supported with the endogenous electron transfer system, which worked more effectively than the heterologous and chemical ones. The results of the hybrid Fd/FNR reducing systems demonstrated that ADO was selective against Fd. And it was observed that the cognate reducing system produced less H2O2 than the heterologous one by 33% during ADO-catalyzed reactions. Importantly, kcat value of ADO 1593 using the homologous Fd/FNR electron transfer system is 3.7-fold higher than the chemical one. Conclusions The cognate electron transfer system from cyanobacteria to support ADO activity was identified and characterized. For the first time, ADO was functionally in vitro reconstituted with the endogenous reducing system from cyanobacteria, which supported greater activity than the surrogate and chemical ones, and produced less H2O2 than the heterologous one. The identified Fd/FNR electron transfer system will be potentially useful for improving ADO activity and further enhancing the biosynthetic efficiency of hydrocarbon biofuels in cyanobacteria. PMID:23759169

  13. Recent advances for FLAP inhibitors.

    PubMed

    Pettersen, Daniel; Davidsson, Öjvind; Whatling, Carl

    2015-07-01

    A number of FLAP inhibitors have been progressed to clinical trials for respiratory and other inflammatory indications but so far no drug has reached the market. With this Digest we assess the opportunity to develop FLAP inhibitors for indications beyond respiratory disease, and in particular for atherosclerotic cardiovascular disease. We also show how recently disclosed FLAP inhibitors have structurally evolved from the first generation FLAP inhibitors paving the way for new compound classes. PMID:26004579

  14. Alpha glucosidase inhibitors.

    PubMed

    Kalra, Sanjay

    2014-04-01

    Alpha glucosidase inhibitors (AGIs) are a unique class of anti-diabetic drugs. Derived from bacteria, these oral drugs are enzyme inhibitors which do not have a pancreato -centred mechanism of action. Working to delay carbohydrate absorption in the gastrointestinal tract, they control postprandial hyperglycaemia and provide unquestioned cardiovascular benefit. Specially suited for a traditional Pakistani carbohydrate-rich diet, AGIs have been termed the 'untapped diamonds' of diabetology. The use of these oral antidiabetic drugs (OADs) that target pathophysiology in the early stages of type 2 diabetes, notably to reduce postprandial hyperglycaemia and hyperinsulinaemia will inevitably increase with time. This review describes the history of their development, mechanism of action, basic and clinical pharmacology, and suggests practical, evidence-based guidance for their optimal use. PMID:24864650

  15. [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

  16. PARP inhibitors and more.

    PubMed

    Bose, Chinmoy K; Basu, Nirban

    2015-01-01

    Polyadenosine diphosphate (ADP) ribose polymerase (PARP) lends a panoramic view to the inner mystery of protection of integrity of deoxyribonucleic acid (DNA) in a cell genome. They are a balancing part of an even more dynamic equilibrium of normalcy against daily assaults. PARP finds its companion candidates in other tumor suppressors, with the most prominent and glaring one being breast cancer (BRCA) 1 and 2. The strength of both is split by PARP inhibitors, inculcating the synthetic lethality of tumor cell, which is now in the market for ovarian cancer treatment. There are many reasons for the resistance of such inhibitors, which are now becoming clinically important. These are seen along with other damage repair approaches. PMID:26097394

  17. PARP inhibitors and more

    PubMed Central

    Bose, Chinmoy K.; Basu, Nirban

    2015-01-01

    Polyadenosine diphosphate (ADP) ribose polymerase (PARP) lends a panoramic view to the inner mystery of protection of integrity of deoxyribonucleic acid (DNA) in a cell genome. They are a balancing part of an even more dynamic equilibrium of normalcy against daily assaults. PARP finds its companion candidates in other tumor suppressors, with the most prominent and glaring one being breast cancer (BRCA) 1 and 2. The strength of both is split by PARP inhibitors, inculcating the synthetic lethality of tumor cell, which is now in the market for ovarian cancer treatment. There are many reasons for the resistance of such inhibitors, which are now becoming clinically important. These are seen along with other damage repair approaches. PMID:26097394

  18. Benzoylurea Chitin Synthesis Inhibitors.

    PubMed

    Sun, Ranfeng; Liu, Chunjuan; Zhang, Hao; Wang, Qingmin

    2015-08-12

    Benzoylurea chitin synthesis inhibitors are widely used in integrated pest management (IPM) and insecticide resistance management (IRM) programs due to their low toxicity to mammals and predatory insects. In the past decades, a large number of benzoylurea derivatives have been synthesized, and 15 benzoylurea chitin synthesis inhibitors have been commercialized. This review focuses on the history of commercial benzolyphenylureas (BPUs), synthetic methods, structure-activity relationships (SAR), action mechanism research, environmental behaviors, and ecotoxicology. Furthermore, their disadvantages of high risk to aquatic invertebrates and crustaceans are pointed out. Finally, we propose that the para-substituents at anilide of benzoylphenylureas should be the functional groups, and bipartite model BPU analogues are discussed in an attempt to provide new insight for future development of BPUs. PMID:26168369

  19. Mineralization by Inhibitor Exclusion

    PubMed Central

    Price, Paul A.; Toroian, Damon; Lim, Joo Eun

    2009-01-01

    One of our goals is to understand the mechanisms that deposit mineral within collagen fibrils, and as a first step we recently determined the size exclusion characteristics of the fibril. This study revealed that apatite crystals up to 12 unit cells in size can access the water within the fibril, whereas molecules larger than a 40-kDa protein are excluded. Based on these observations, we proposed a novel mechanism for fibril mineralization: that macromolecular inhibitors of apatite growth favor fibril mineralization by selectively inhibiting crystal growth in the solution outside of the fibril. To test this mechanism, we developed a system in which crystal formation is driven by homogeneous nucleation at high calcium phosphate concentration and the only macromolecule in solution is fetuin, a 48-kDa inhibitor of apatite growth. Our experiments with this system demonstrated that fetuin determines the location of mineral growth; in the presence of fetuin mineral grows exclusively within the fibril, whereas in its absence mineral grows in solution outside the fibril. Additional experiments showed that fetuin is also able to localize calcification to the interior of synthetic matrices that have size exclusion characteristics similar to those of collagen and that it does so by selectively inhibiting mineral growth outside of these matrices. We termed this new calcification mechanism “mineralization by inhibitor exclusion,” the selective mineralization of a matrix using a macromolecular inhibitor of mineral growth that is excluded from that matrix. Future studies will be needed to evaluate the possible role of this mechanism in bone mineralization. PMID:19414589

  20. 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

  1. Development of scale inhibitors

    SciTech Connect

    Gill, J.S.

    1996-12-01

    During the last fifty years, scale inhibition has gone from an art to a science. Scale inhibition has changed from simple pH adjustment to the use of optimized dose of designer polymers from multiple monomers. The water-treatment industry faces many challenges due to the need to conserve water, availability of only low quality water, increasing environmental regulations of the water discharge, and concern for human safety when using acid. Natural materials such as starch, lignin, tannin, etc., have been replaced with hydrolytically stable organic phosphates and synthetic polymers. Most progress in scale inhibition has come from the use of synergistic mixtures and copolymerizing different functionalities to achieve specific goals. Development of scale inhibitors requires an understanding of the mechanism of crystal growth and its inhibition. This paper discusses the historic perspective of scale inhibition and the development of new inhibitors based on the understanding of the mechanism of crystal growth and the use of powerful tools like molecular modeling to visualize crystal-inhibitor interactions.

  2. Antineoplastic Treatment and Renal Injury: An Update on Renal Pathology Due to Cytotoxic and Targeted Therapies.

    PubMed

    Troxell, Megan L; Higgins, John P; Kambham, Neeraja

    2016-09-01

    Cancer patients experience kidney injury from multiple sources, including the tumor itself, diagnostic procedures, hypovolemia, infection, and drug exposure, superimposed upon baseline chronic damage. This review will focus on cytotoxic or targeted chemotherapy-associated renal injury. In this setting, tubulointerstitial injury and thrombotic microangiopathy (vascular injury) are more common than other forms of kidney injury including glomerular. Cisplatin, pemetrexed, and ifosfamide are well-known causes of acute tubular injury/necrosis. Acute interstitial nephritis seems underrecognized in this clinical setting. Interstitial nephritis is emerging as an "immune-related adverse effect" (irAE's) with immune checkpoint inhibitors in small numbers of patients. Acute kidney injury is rarely reported with targeted therapies such as BRAF inhibitors (vemurafinib, dabrafenib), ALK inhibitors (crizotinib), and mTOR inhibitors (everolimus, temsirolimus), but additional biopsy data are needed. Tyrosine kinase inhibitors and monoclonal antibodies that block the vascular endothelial growth factor pathway are most commonly associated with thrombotic microangiopathy. Other causes of thrombotic microangiopathy in the cancer patients include cytotoxic chemotherapies such as gemcitabine and mitomycin C, hematopoietic stem cell transplant, and cancer itself (usually high-stage adenocarcinoma with marrow and vascular invasion). Cancer patients are historically underbiopsied, but biopsy can reveal type, acuity, and chronicity of renal injury, and facilitate decisions concerning continuation of chemotherapy and/or initiation of renoprotective therapy. Biopsy may also reveal unrelated and unanticipated findings in need of treatment. PMID:27403615

  3. Impact of different green manures on the content of S-alk(en)yl-L-cysteine sulfoxides and L-ascorbic acid in leek (Allium porrum).

    PubMed

    Lundegårdh, B; Botek, P; Schulzov, V; Hajslov, J; Strömberg, A; Andersson, H C

    2008-03-26

    This field study investigated the impact of various fertilization strategies with red clover ( Trifolium pratense L.) green manure on the levels of S-alk(en)yl- l-cysteine sulfoxides (ACSO) and l-ascorbic acid in leek. Two of the 12 treatments were controls, one without fertilizers and the other with a commercial mineral fertilizer. The remaining 10 treatments were different forms and quantities of green manure prepared from red clover. One treatment consisted of direct incorporation into soil of the preceding red clover crop. The other 9 treatments comprised three types of red clover green manure [anaerobically digested red clover biomass (biodigestate), composted red clover, fresh red clover as mulch] applied at three different doses. Yield was increased only at the highest dose of compost and the highest dose of mulch. High doses of green manure decreased dry matter content in leek. The fertilizer treatments increased the nitrogen uptake and the nitrogen content of leek. Sulfur uptake and sulfur levels were increased only by the mineral fertilizer and by the compost. Nonfertilized leek contained 20.4 +/- 5.8 g/kg of dry weight (dw) ACSOs as determined by LC-MS/MS and 1.57 +/- 0.01 g/kg of dw ascorbic acid as determined by HPLC. The ACSOs were to 92-96% isoalliin, the rest being methiin. Alliin was identified in only 1 of 72 samples. The ACSO level was increased by 37% by the mineral fertilizer. Whereas direct incorporation of red clover, mulch, and red clover biodigestate had no influence on the ACSO level, the highest dose of compost increased the ACSO level by 55%. Ascorbic acid levels were not influenced by the mineral treatment. Green manures increased ascorbic acid levels only on a dry weight basis. A high correlation between the content of sulfur and ACSO indicated that delivering capacity of sulfur from the manure to the plant strongly affected the ASCO content of the leek. In conclusion, the composted green manure was the most useful organic fertilizer

  4. Entrectinib is a potent inhibitor of Trk-driven neuroblastomas in a xenograft mouse model.

    PubMed

    Iyer, Radhika; Wehrmann, Lea; Golden, Rebecca L; Naraparaju, Koumudi; Croucher, Jamie L; MacFarland, Suzanne P; Guan, Peng; Kolla, Venkatadri; Wei, Ge; Cam, Nicholas; Li, Gang; Hornby, Zachary; Brodeur, Garrett M

    2016-03-28

    Neuroblastoma (NB) is one of the most common and deadly childhood solid tumors. These tumors are characterized by clinical heterogeneity, from spontaneous regression to relentless progression, and the Trk family of neurotrophin receptors plays an important role in this heterogeneous behavior. We wanted to determine if entrectinib (RXDX-101, Ignyta, Inc.), an oral Pan-Trk, Alk and Ros1 inhibitor, was effective in our NB model. In vitro effects of entrectinib, either as a single agent or in combination with the chemotherapeutic agents Irinotecan (Irino) and Temozolomide (TMZ), were studied on an SH-SY5Y cell line stably transfected with TrkB. In vivo growth inhibition activity was studied in NB xenografts, again as a single agent or in combination with Irino-TMZ. Entrectinib significantly inhibited the growth of TrkB-expressing NB cells in vitro, and it significantly enhanced the growth inhibition of Irino-TMZ when used in combination. Single agent therapy resulted in significant tumor growth inhibition in animals treated with entrectinib compared to control animals [p < 0.0001 for event-free survival (EFS)]. Addition of entrectinib to Irino-TMZ also significantly improved the EFS of animals compared to vehicle or Irino-TMZ treated animals [p < 0.0001 for combination vs. control, p = 0.0012 for combination vs. Irino-TMZ]. We show that entrectinib inhibits growth of TrkB expressing NB cells in vitro and in vivo, and that it enhances the efficacy of conventional chemotherapy in in vivo models. Our data suggest that entrectinib is a potent Trk inhibitor and should be tested in clinical trials for NBs and other Trk-expressing tumors. PMID:26797418

  5. Thymidylate synthase inhibitors.

    PubMed

    Danenberg, P V; Malli, H; Swenson, S

    1999-12-01

    Thymidylate synthase (TS) is a critical enzyme for DNA replication and cell growth because it is the only de novo source of thymine nucleotide precursors for DNA synthesis. TS is the primary target of 5-fluorouracil (5-FU), which has been used for cancer treatment for more than 40 years. However, dissatisfaction with the overall activity of 5-FU against the major cancers, and the recognition that TS still remains an attractive target for anticancer drugs because of its central position in the pathway of DNA synthesis, led to a search for new inhibitors of TS structurally analogous to 5,10-methylenetetrahydrofolate, the second substrate of TS. TS inhibitory antifolates developed to date that are in various stages of clinical evaluation are ZD 1694 and ZD9331 (Astra-Zeneca, London, UK), (Eli Lilly, Indianapolis, IN), LY231514 (BW1843U89 (Glaxo-Wellcome, Research Triangle Park, NC), and AG337 and AG331 (Agouron, La Jolla, CA). Although each of these compounds has TS as its major intracellular site of action, they differ in propensity for polyglutamylation and for transport by the reduced folate carrier. LY231514 also has secondary target enzymes. As a result, each compound is likely to have a different spectrum of antitumor activity and toxicity. This review will summarize the development and properties of this new class of TS inhibitors. PMID:10606255

  6. 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.

  7. [Kinase inhibitors and their resistance].

    PubMed

    Togashi, Yosuke; Nishio, Kazuto

    2015-08-01

    Kinase cascades are involved in all stages of tumorigenesis through modulation of transformation and differentiation, cell-cycle progression, and motility. Advances in molecular targeted drug development allow the design and synthesis of inhibitors targeting cancer-associated signal transduction pathways. Potent selective inhibitors with low toxicity can benefit patients especially with several malignancies harboring an oncogenic driver addictive signal. This article evaluates information on solid tumor-related kinase signals and inhibitors, including receptor tyrosine kinase or serine/threonine kinase signals that lead to successful application in clinical settings. In addition, the resistant mechanisms to the inhibitors is summarized. PMID:26281685

  8. Repositioning of DHFR Inhibitors.

    PubMed

    Lele, Arundhati Chandrashekhar; Mishra, Deepak Amarnath; Kamil, Tengku Karmila; Bhakta, Sanjib; Degani, Mariam Sohel

    2016-01-01

    Development of new drugs is a time-consuming, hugely expensive and an uncertain endeavor. The pharmaceutical industry is looking for cost-effective alternatives with reduced risks of drug failure. Validated target machinery along with established inhibitors indicates usefulness in drug design, discovery and further development. Folate metabolism, found in both prokaryotes and eukaryotes, represents an essential druggable target for chemotherapy. Numerous enzymes in the cell replication cycle use folate either as a cofactor or as a substrate. DHFR, an enzyme of the folate biosynthesis pathway is an established chemotherapeutic target, initially explored for anti-cancer drug discovery. Diaminopteridines e.g. methotrexate and aminopterin, primarily used as anti-cancer agents, are folic acid analogues, first reported in late 1940's, used to produce temporary remission of acute leukaemia in children. However, due to the toxicity of these drugs, they could not be used for other therapeutic implications such as in the treatment of infectious diseases. Development of newer diaminopteridine derivatives has helped in repositioning their therapeutic usefulness. These analogues have now been proven as anti-parasitic, immuno-suppressants, anti-bacterial agents, to enlist a few therapeutic applications. Likewise, diaminopyrimidine, diaminoquinazoline and diaminodihydrotriazines are being explored for structural modifications by which they can be repurposed from their originally developed medicinal applicability and exploited for various other infectious disease conditions. In this review, we encompass the study of DHFR inhibitors potentially to be repurposed for different infectious disease case scenario and also highlight the novel anti-infective drug discovery benefits therein. PMID:26881719

  9. 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

  10. 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 ...

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. Targeting cancer with kinase inhibitors

    PubMed Central

    Gross, Stefan; Rahal, Rami; Stransky, Nicolas; Lengauer, Christoph; Hoeflich, Klaus P.

    2015-01-01

    Kinase inhibitors have played an increasingly prominent role in the treatment of cancer and other diseases. Currently, more than 25 oncology drugs that target kinases have been approved, and numerous additional therapeutics are in various stages of clinical evaluation. In this Review, we provide an in-depth analysis of activation mechanisms for kinases in cancer, highlight recent successes in drug discovery, and demonstrate the clinical impact of selective kinase inhibitors. We also describe the substantial progress that has been made in designing next-generation inhibitors to circumvent on-target resistance mechanisms, as well as ongoing strategies for combining kinase inhibitors in the clinic. Last, there are numerous prospects for the discovery of novel kinase targets, and we explore cancer immunotherapy as a new and promising research area for studying kinase biology. PMID:25932675

  17. Aromatase inhibitors for male infertility.

    PubMed

    Schlegel, Peter N

    2012-12-01

    Some men with severely defective sperm production commonly have excess aromatase activity, reflected by low serum testosterone and relatively elevated estradiol levels. Aromatase inhibitors can increase endogenous testosterone production and serum testosterone levels. Treatment of infertile males with the aromatase inhibitors testolactone, anastrazole, and letrozole has been associated with increased sperm production and return of sperm to the ejaculate in men with non-obstructive azoospermia. Use of the aromatase inhibitors anastrazole (1 mg/day) and letrozole (2.5 mg/day) represent off-label use of these agents for impaired spermatogenesis in men with excess aromatase activity (abnormal testosterone/estradiol [T/E] ratios). Side effects have rarely been reported. Randomized controlled trials are needed to define the magnitude of benefit of aromatase inhibitor treatment for infertile men. PMID:23103016

  18. 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

  19. [Cancer therapy by PARP inhibitors].

    PubMed

    Seimiya, Hiroyuki

    2015-08-01

    Poly(ADP-ribose) polymerases(PARP) synthesize the ADP-ribose polymers onto proteins and play a role in DNA repair. PARP inhibitors block the repair of single-strand breaks, which in turn gives rise to double-strand breaks during DNA replication. Thus, PARP inhibitors elicit synthetic lethality in cancer with BRCA1/2 loss-of-function mutations that hamper homologous recombination repair of double-strand breaks. Olaparib, the first-in-class PARP inhibitor, was approved for treatment of BRCA-mutated ovarian cancer in Europe and the United States in 2014. Other PARP inhibitors under clinical trials include rucaparib, niraparib, veliparib, and the "PARP-trapping" BMN-673. BRCA1/2 sequencing is an FDA-approved companion diagnostics, which predicts the cancer vulnerability to PARP inhibition. Together, synthetic lethal PARP inhibition is a novel promising strategy for cancer intervention even in cases without prominent driver oncogenes. PMID:26281686

  20. [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

  1. Anaplastic lymphoma kinase status in rhabdomyosarcomas.

    PubMed

    Yoshida, Akihiko; Shibata, Tatsuhiro; Wakai, Susumu; Ushiku, Tetsuo; Tsuta, Koji; Fukayama, Masashi; Makimoto, Atsushi; Furuta, Koh; Tsuda, Hitoshi

    2013-06-01

    Rhabdomyosarcoma is a rare soft tissue sarcoma that typically affects children, adolescents, and young adults. Despite treatment via a multidisciplinary approach, the prognosis of advance-stage rhabdomyosarcomas remains poor, and a new treatment strategy is needed. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is a potential target for specific inhibitors. In this study, we investigated 116 rhabdomyosarcomas using a polymer-based ALK immunostaining method and correlated the results with clinicopathological parameters. In addition, we examined ALK status using dual-color fluorescence in situ hybridization, PCR, and sequencing. In immunohistochemical analysis, ALK was detected in 2 (6%) of 33 embryonal rhabdomyosarcomas, 42 (69%) of 61 alveolar rhabdomyosarcomas, and 0 (0%) of 22 other subtypes, including pleomorphic, adult-spindle-cell/sclerosing, and epithelioid variants. Compared with ALK-negative alveolar rhabdomyosarcomas, ALK-positive ones are presented with metastatic spread more frequently and showed a greater extent of myogenin reactivity. Overall survival was not associated with ALK expression. FOXO1 rearrangement was significantly associated with ALK immunoreactivity. The median ALK copy number was greater in ALK-positive tumors than in ALK-negative tumors. Most (93%) cases tested showed no selective increase in the ALK gene dosage. ALK selective amplification and low-level selective gain were noted in one and three cases, respectively. Further, a high-polysomy pattern (≥4 ALK copies in ≥40% of cells) was observed in seven cases. A significant increase in the ALK copy number was exclusive to the ALK-immunopositive cohort, but it was uncommon, accounting for only 30% of the 37 ALK-positive rhabdomyosarcomas. ALK gene rearrangement was not observed in either cohort, while an ALK somatic mutation (I1277T) was found in one ALK-negative embryonal case. Although it remains controversial whether ALK expression without gene rearrangement

  2. Corrosion inhibitor selection for wet pipelines

    SciTech Connect

    Buck, E.

    1995-12-31

    Selection of corrosion inhibitors for wet pipelines is based on laboratory testing and field confirmation. Both the use and selection of corrosion inhibitors are driven by economics. Economics of alternative corrosion protection methods is not treated in this paper, but the economics of proper inhibitor selection are. The key to successful inhibitor selection is careful analysis of pipeline flow conditions and experimental emulation of its corrosive environment. Transportation of inhibitor to the corroding interface must be explicitly considered in the emulation. Standard corrosion rate measurement methods are used to evaluate inhibitors. Inhibitor properties tabulated during evaluation form a core database for continuing quality control.

  3. [The synthesis of specific enzyme inhibitors].

    PubMed

    Iakovleva, G M

    1987-04-01

    The review deals with directed synthesis of specific enzyme inhibitors. They are classified within the framework of the mechanistic approach, namely, stable analogues of substrates, which form enzyme complexes mimicking the Michaelis complex or those which influence the chemical stages of enzyme catalysis; conformational inhibitors; substrate analogues participating in enzyme reactions and producing modified products; suicide inhibitors; stage inhibitors (inhibitors influencing certain stages of enzyme reaction); transition state analogues; multisubstrate analogues and collected substrates. Types of chemical modification used in synthesis of the specific inhibitors are discussed. Some possibilities of the quantity structure-activity relationship methods, computer modelling and molecular graphics in designing the optimal structure of inhibitors are mentioned. PMID:3300658

  4. Pharmacology of phosphodiesterase-5 inhibitors.

    PubMed

    Corbin, J D; Francis, S H

    2002-01-01

    The clinical properties (efficacy and safety profile) of a medicine are related not only to its mode of action, but also to its selectivity for its target (usually a receptor or enzyme) and are also influenced by its pharmacokinetic properties (absorption, distribution, metabolism and elimination). The growing number of phosphodiesterase inhibitors that are selective for phosphodiesterase-5 (PDE5) represent a promising new class of compounds that are useful for the treatment of erectile dysfunction and perhaps other disorders. Some of the basic pharmacodynamic and pharmacokinetic parameters that describe drug action are discussed with regard to the new PDE5 inhibitors. Central topics reviewed are the concentration that produces a given in vitro response, or potency (IC50), maximum plasma concentration (Cmax), time to Cmax (Tmax), half-life (t 1/2), area under the curve (AUC), bioavailability, onset and duration of action, and the balance to achieve optimum safety and efficacy. To illustrate these concepts, a group of inhibitors with varying selectivities and potencies for PDE5 (theophylline, IBMX, zaprinast, sildenafil, tadalafil and vardenafil) are discussed. Each drug has its own set of unique pharmacological characteristics based on its specific molecular structure, enzyme inhibition profile and pharmacokinetic properties. Each PDE5 inhibitor has a distinct selectivity that contributes to its safety profile. As with all new drugs, and especially those in a new class, careful evaluation will be necessary to ensure the optimal use of the PDE5 inhibitors. PMID:12166544

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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%. PMID:22561212

  11. 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.

  12. Monoglyceride lipase: Structure and inhibitors.

    PubMed

    Scalvini, Laura; Piomelli, Daniele; Mor, Marco

    2016-05-01

    Monoglyceride lipase (MGL), the main enzyme responsible for the hydrolytic deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG), is an intracellular serine hydrolase that plays critical roles in many physiological and pathological processes, such as pain, inflammation, neuroprotection and cancer. The crystal structures of MGL that are currently available provide valuable information about how this enzyme might function and interact with site-directed small-molecule inhibitors. On the other hand, its conformational equilibria and the contribution of regulatory cysteine residues present within the substrate-binding pocket or on protein surface remain open issues. Several classes of MGL inhibitors have been developed, from early reversible ones, such as URB602 and pristimerin, to carbamoylating agents that react with the catalytic serine, such as JZL184 and more recent O-hexafluoroisopropyl carbamates. Other inhibitors that modulate MGL activity by interacting with conserved regulatory cysteines act through mechanisms that deserve to be more thoroughly investigated. PMID:26216043

  13. STAT inhibitors for cancer therapy

    PubMed Central

    2013-01-01

    Signal Transducer and Activator of Transcription (STAT) proteins are a family of cytoplasmic transcription factors consisting of 7 members, STAT1 to STAT6, including STAT5a and STAT5b. STAT proteins are thought to be ideal targets for anti-cancer therapy since cancer cells are more dependent on the STAT activity than their normal counterparts. Inhibitors targeting STAT3 and STAT5 have been developed. These included peptidomimetics, small molecule inhibitors and oligonucleotides. This review summarized advances in preclinical and clinical development of these compounds. PMID:24308725

  14. SGLT2 inhibitors: new reports.

    PubMed

    2015-10-12

    A significant decrease in cardiovascular mortality has been reported with use of the SGLT2 inhibitor empagliflozin (Jardiance) to treat patients with type 2 diabetes who have established cardiovascular disease. The mechanism of this reduction is unclear, and these results may not apply to patients with type 2 diabetes and less advanced cardiovascular disease. Whether the increase in fractures reported with canagliflozin (Invokana) could also occur with empagliflozin remains to be established. All SGLT2 inhibitors are only modestly effective for treatment of diabetes. PMID:26445203

  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. 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 Central

    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.

    2016-01-01

    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 bed-side 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 2 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

  17. 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.

  18. 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

  19. Less-toxic corrosion inhibitors

    NASA Technical Reports Server (NTRS)

    Humphries, T. S.

    1981-01-01

    Combinations of borates, nitrates, phosphates, silicates, and sodium MBT protect aluminum from corrosion in fresh water. Most effective combinations contained sodium phosphate and were alkaline. These inhibitors replace toxic chromates which are subject to governmental restrictions, but must be used in larger quantities. Experimental exposure times varied from 1 to 14 months depending upon nature of submersion solution.

  20. CREATE: Cross-tumoral Phase 2 With Crizotinib

    ClinicalTrials.gov

    2016-07-06

    Locally Advanced and/or Metastatic Anaplastic Large Cell Lymphoma; Locally Advanced and/or Metastatic Inflammatory Myofibroblastic Tumor; Locally Advanced and/or Metastatic Papillary Renal Cell Carcinoma Type 1; Locally Advanced and/or Metastatic Alveolar Soft Part Sarcoma; Locally Advanced and/or Metastatic Clear Cell Sarcoma; Locally Advanced and/or Metastatic Alveolar Rhabdomyosarcoma

  1. 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 ...

  2. 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

  3. 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

  4. 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

  5. Mutation-Independent Activation of the Anaplastic Lymphoma Kinase in Neuroblastoma.

    PubMed

    Regairaz, Marie; Munier, Fabienne; Sartelet, Hervé; Castaing, Marine; Marty, Virginie; Renauleaud, Céline; Doux, Camille; Delbé, Jean; Courty, José; Fabre, Monique; Ohta, Shigeru; Viehl, Philippe; Michiels, Stefan; Valteau-Couanet, Dominique; Vassal, Gilles

    2016-02-01

    Activating mutations of anaplastic lymphoma kinase (ALK) have been identified as important players in neuroblastoma development. Our goal was to evaluate the significance of overall ALK activation in neuroblastoma. Expression of phosphorylated ALK, ALK, and its putative ligands, pleiotrophin and midkine, was screened in 289 neuroblastomas and 56 paired normal tissues. ALK was expressed in 99% of tumors and phosphorylated in 48% of cases. Pleiotrophin and midkine were expressed in 58% and 79% of tumors, respectively. ALK activation was significantly higher in tumors than in paired normal tissues, together with ALK and midkine expression. ALK activation was largely independent of mutations and correlated with midkine expression in tumors. ALK activation in tumors was associated with favorable features, including a younger age at diagnosis, hyperdiploidy, and detection by mass screening. Antitumor activity of the ALK inhibitor TAE684 was evaluated in wild-type or mutated ALK neuroblastoma cell lines and xenografts. TAE684 was cytotoxic in vitro in all cell lines, especially those harboring an ALK mutation. TAE684 efficiently inhibited ALK phosphorylation in vivo in both F1174I and R1275Q xenografts but demonstrated antitumor activity only against the R1275Q xenograft. In conclusion, ALK activation occurs frequently during neuroblastoma oncogenesis, mainly through mutation-independent mechanisms. However, ALK activation is not associated with a poor outcome and is not always a driver of cell proliferation and/or survival in neuroblastoma. PMID:26687816

  6. 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

  7. 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.

  8. [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

  9. 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

  10. 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

  11. Bromodomains and their pharmacological inhibitors.

    PubMed

    Gallenkamp, Daniel; Gelato, Kathy A; Haendler, Bernard; Weinmann, Hilmar

    2014-03-01

    Over 60 bromodomains belonging to proteins with very different functions have been identified in humans. Several of them interact with acetylated lysine residues, leading to the recruitment and stabilization of protein complexes. The bromodomain and extra-terminal domain (BET) proteins contain tandem bromodomains which bind to acetylated histones and are thereby implicated in a number of DNA-centered processes, including the regulation of gene expression. The recent identification of inhibitors of BET and non-BET bromodomains is one of the few examples in which effective blockade of a protein-protein interaction can be achieved with a small molecule. This has led to major strides in the understanding of the function of bromodomain-containing proteins and their involvement in diseases such as cancer and inflammation. Indeed, BET bromodomain inhibitors are now being clinically evaluated for the treatment of hematological tumors and have also been tested in clinical trials for the relatively rare BRD-NUT midline carcinoma. This review gives an overview of the newest developments in the field, with a focus on the biology of selected bromodomain proteins on the one hand, and on reported pharmacological inhibitors on the other, including recent examples from the patent literature. PMID:24497428

  12. Enhancing CHK1 inhibitor lethality in glioblastoma.

    PubMed

    Tang, Yong; Dai, Yun; Grant, Steven; Dent, Paul

    2012-04-01

    The present studies were initiated to determine whether inhibitors of MEK1/2 or SRC signaling, respectively, enhance CHK1 inhibitor lethality in primary human glioblastoma cells. Multiple MEK1/2 inhibitors (CI-1040 (PD184352); AZD6244 (ARRY-142886)) interacted with multiple CHK1 inhibitors (UCN-01, AZD7762) to kill multiple primary human glioma cell isolates that have a diverse set of genetic alterations typically found in the disease. Inhibition of SRC family proteins also enhanced CHK1 inhibitor lethality. Combined treatment of glioma cells with (MEK1/2 + CHK1) inhibitors enhanced radiosensitivity. Combined (MEK1/2 + CHK1) inhibitor treatment led to dephosphorylation of ERK1/2 and S6 ribosomal protein, whereas the phosphorylation of JNK and p38 was increased. MEK1/2 + CHK1 inhibitor-stimulated cell death was associated with the cleavage of pro-caspases 3 and 7 as well as the caspase substrate (PARP). We also observed activation of pro-apoptotic BCL-2 effector proteins BAK and BAX and reduced levels of pro-survival BCL-2 family protein BCL-XL. Overexpression of BCL-XL alleviated but did not completely abolish MEK1/2 + CHK1 inhibitor cytotoxicity in GBM cells. These findings argue that multiple inhibitors of the SRC-MEK pathway have the potential to interact with multiple CHK1 inhibitors to kill glioma cells. PMID:22313687

  13. 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

  14. Xylanase inhibitors bind to nonstarch polysaccharides.

    PubMed

    Fierens, Ellen; Gebruers, Kurt; Courtin, Christophe M; Delcour, Jan A

    2008-01-23

    This study is an in-depth investigation of the interaction between polysaccharides and the proteinaceous xylanase inhibitors, Triticum aestivum xylanase inhibitor (TAXI), xylanase inhibitor protein (XIP), and thaumatin-like xylanase inhibitor (TLXI). The binding affinities of all three known types of xylanase inhibitors from wheat are studied by measuring the residual xylanase inhibition activity after incubation of the inhibitors in the presence of different polysaccharides, such as beta-glucans and (arabino)xylans. The binding affinities of all three xylanase inhibitors for (arabino)xylans increased with a decreasing arabinose/xylose ratio (A/X ratio). This phenomenon was observed both with water-extractable and water-unextractable (arabino)xylans. The inhibitors also interacted with different soluble and insoluble beta-glucans. None of the inhibitors tested had the ability to hydrolyze the polysaccharides investigated. The present findings contribute to the unraveling of the function of xylanase inhibitors in nature and to the prediction of the effect of added xylanases in cereal-based biotechnological processes, such as bread making and gluten-starch separation. PMID:18092758

  15. Cytological Diagnosis of Bilateral Breast Implant-Associated Lymphoma of the ALK-Negative Anaplastic Large-Cell Type. Clinical Implications of Peri-Implant Breast Seroma Cytological Reporting.

    PubMed

    Granados, Rosario; Lumbreras, Eva M; Delgado, Manuel; Aramburu, José A; Tardío, Juan C

    2016-07-01

    The cytological examination of peri-prosthetic breast effusions allowed the diagnosis of bilateral breast-implant ALK-negative anaplastic large cell lymphoma (BI-ALCL) in the case reported. Ten years after reconstructive surgery with bilateral breast implants, a large unilateral seroma developed and was cytologically analyzed. The presence of CD30 and CD4-positive large-sized atypical lymphoid cells exhibiting horseshoe-shaped nuclei and a brisk mitotic activity rendered the diagnosis of BI-ALCL. Similar cells were seen in the peri-prosthetic fluid intraoperatively collected from the contralateral breast. Although initial histological analysis of the capsulectomy specimens showed unilateral tumor, the cytological findings prompted a more thorough tissue sampling, resulting in the diagnosis of bilateral disease. BI-ALCL usually follows an indolent clinical course; however, there are reported cases with an aggressive behavior. While the presence of bilateral disease is a putative risk factor for a bad prognosis, the small number of cases reported precludes a definitive assessment of this risk. Since most BI-ALCL present with late seromas, cytologic analysis of these effusions in women with breast implants should be mandatory. Cytology is a safe tool for diagnosis and follow-up of patients with breast implant-related late seromas, sometimes proven more sensitive than histological analysis. Complete bilateral capsulectomy and a detailed histological analysis should follow a cytological diagnosis of BI-ALCL in a breast effusion in order to avoid false negative diagnoses. Our case constitutes the first published report of a bilateral BI-ALCL diagnosed by cytology. Diagn. Cytopathol. 2016;44:623-627. © 2016 Wiley Periodicals, Inc. PMID:27079579

  16. Biomarkers associated with checkpoint inhibitors.

    PubMed

    Manson, G; Norwood, J; Marabelle, A; Kohrt, H; Houot, R

    2016-07-01

    Checkpoint inhibitors (CPI), namely anti-CTLA4 and anti-PD1/PD-L1 antibodies, demonstrated efficacy across multiple types of cancer. However, only subgroups of patients respond to these therapies. Additionally, CPI can induce severe immune-related adverse events (irAE). Biomarkers that predict efficacy and toxicity may help define the patients who may benefit the most from these costly and potentially toxic therapies. In this study, we review the main biomarkers that have been associated with the efficacy (pharmacodynamics and clinical benefit) and the toxicity (irAE) of CPIs in patients. PMID:27122549

  17. Monoamine Oxidase Inhibitors: Clinical Review

    PubMed Central

    Remick, Ronald A.; Froese, Colleen

    1990-01-01

    Monoamine oxidase inhibitors (MAOIs) are effective antidepressant agents. They are increasingly and effectively used in a number of other psychiatric and non-psychiatric medical syndromes. Their potential for serious toxicity (i.e., hypertensive reaction) is far less than original reports suggest, and newer reversible substrate-specific MAOIs may offer even less toxicity. The author reviews the pharmacology, mechanism of action, clinical indications, and dosing strategies of MAOIs. The common MAOI side-effects (hypotension, weight gain, sexual dysfunction, insomnia, daytime sedation, myoclonus, and hypertensive episodes) are described and management techniques suggested. Recent clinical developments involving MAOIs are outlined. PMID:21233984

  18. Reduced SMAD7 leads to overactivation of TGF-beta signaling in MDS that can be reversed by a specific inhibitor of TGF-beta receptor I kinase.

    PubMed

    Zhou, Li; McMahon, Christine; Bhagat, Tushar; Alencar, Cristina; Yu, Yiting; Fazzari, Melissa; Sohal, Davendra; Heuck, Christoph; Gundabolu, Krishna; Ng, Chun; Mo, Yongkai; Shen, Wa; Wickrema, Amittha; Kong, Guanghui; Friedman, Ellen; Sokol, Lubomir; Mantzaris, Ioannis; Mantzaris, Giannis; Pellagatti, Andrea; Boultwood, Jacqueline; Platanias, Leonidas C; Steidl, Ulrich; Yan, Lei; Yingling, Jonathan M; Lahn, Michael M; List, Alan; Bitzer, Markus; Verma, Amit

    2011-02-01

    Even though myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, the molecular alterations that lead to marrow failure have not been well elucidated. We have previously shown that the myelosuppressive TGF-β pathway is constitutively activated in MDS progenitors. Because there is conflicting data about upregulation of extracellular TGF-β levels in MDS, we wanted to determine the molecular basis of TGF-β pathway overactivation and consequent hematopoietic suppression in this disease. We observed that SMAD7, a negative regulator of TGF-β receptor I (TBRI) kinase, is markedly decreased in a large meta-analysis of gene expression studies from MDS marrow-derived CD34(+) cells. SMAD7 protein was also found to be significantly decreased in MDS marrow progenitors when examined immunohistochemically in a bone marrow tissue microarray. Reduced expression of SMAD7 in hematopoietic cells led to increased TGF-β-mediated gene transcription and enhanced sensitivity to TGF-β-mediated suppressive effects. The increased TGF-β signaling due to SMAD7 reduction could be effectively inhibited by a novel clinically relevant TBRI (ALK5 kinase) inhibitor, LY-2157299. LY-2157299 could inhibit TGF-β-mediated SMAD2 activation and hematopoietic suppression in primary hematopoietic stem cells. Furthermore, in vivo administration of LY-2157299 ameliorated anemia in a TGF-β overexpressing transgenic mouse model of bone marrow failure. Most importantly, treatment with LY-2157199 stimulated hematopoiesis from primary MDS bone marrow specimens. These studies demonstrate that reduction in SMAD7 is a novel molecular alteration in MDS that leads to ineffective hematopoiesis by activating of TGF-β signaling in hematopoietic cells. These studies also illustrate the therapeutic potential of TBRI inhibitors in MDS. PMID:21189329

  19. Reduced SMAD7 leads to overactivation of TGF-beta signaling in MDS that can be reversed by a specific inhibitor of TGF-beta receptor I kinase

    PubMed Central

    Zhou, L; McMahon, C; Bhagat, T; Alencar, C; Yu, Y; Fazzari, M; Sohal, D; Heuck, C; Gundabolu, K; Ng, C; Mo, Y; Shen, W; Wickrema, A; Kong, G; Friedman, E; Sokol, L; Mantzaris, G; Pellagatti, A; Boultwood, J; Platanias, LC.; Steidl, U; Yan, L; Yingling, JM; Lahn, MM; List, A; Bitzer, M; Verma, A

    2011-01-01

    Even though myelodysplastic syndromes are characterized by ineffective hematopoiesis, the molecular alterations that lead to marrow failure have not been well elucidated. We have previously shown that the myelosuppressive TGF-β pathway is constitutively activated in MDS progenitors. Since there is conflicting data about upregulation of extracellular TGF-b levels in MDS, we wanted to determine the molecular basis of TGF-β pathway overactivation and consequent hematopoietic suppression in this disease. We observed that SMAD7, a negative regulator of TGF-β receptor I (TBRI) kinase is markedly decreased in a large meta-analysis of gene expression studies from MDS marrow derived CD34+ cells. SMAD7 protein was also found to be significantly decreased in MDS marrow progenitors when examined immunohistochemically in a bone marrow tissue microarray. Reduced expression of SMAD7 in hematopoietic cells led to increased TGF-β mediated gene transcription and enhanced sensitivity to TGF-β mediated suppressive effects. The increased TGF-β signaling due to SMAD7 reduction could be effectively inhibited by a novel clinically relevant TBRI (ALK5 kinase) inhibitor, LY-2157299. LY-2157299 could inhibit TGF-β mediated SMAD2 activation and hematopoietic suppression in primary hematopoietic stem cells. Furthermore, in vivo administration of LY-2157299 ameliorated anemia in a TGF-β overexpressing transgenic mouse model of bone marrow failure. Most importantly, treatment with LY-2157199 stimulated hematopoiesis from primary MDS bone marrow specimens. These studies demonstrate that reduction in SMAD7 is a novel molecular alteration in MDS that leads to ineffective hematopoiesis by activating of TGF-β signaling in hematopoietic cells. These studies also illustrate the therapeutic potential of TBRI inhibitors in MDS. PMID:21189329

  20. Mouse Model for ROS1-Rearranged Lung Cancer

    PubMed Central

    Takahashi, Hiroyuki; Nakamura, Hiromi; Hama, Natsuko; Kohno, Takashi; Tsuta, Koji; Yoshida, Akihiko; Asamura, Hisao; Mutoh, Michihiro; Hosoda, Fumie; Tsuda, Hitoshi; Shibata, Tatsuhiro

    2013-01-01

    Genetic rearrangement of the ROS1 receptor tyrosine kinase was recently identified as a distinct molecular signature for human non-small cell lung cancer (NSCLC). However, direct evidence of lung carcinogenesis induced by ROS1 fusion genes remains to be verified. The present study shows that EZR-ROS1 plays an essential role in the oncogenesis of NSCLC harboring the fusion gene. EZR-ROS1 was identified in four female patients of lung adenocarcinoma. Three of them were never smokers. Interstitial deletion of 6q22–q25 resulted in gene fusion. Expression of the fusion kinase in NIH3T3 cells induced anchorage-independent growth in vitro, and subcutaneous tumors in nude mice. This transforming ability was attributable to its kinase activity. The ALK/MET/ROS1 kinase inhibitor, crizotinib, suppressed fusion-induced anchorage-independent growth of NIH3T3 cells. Most importantly, established transgenic mouse lines specifically expressing EZR-ROS1 in lung alveolar epithelial cells developed multiple adenocarcinoma nodules in both lungs at an early age. These data suggest that the EZR-ROS1 is a pivotal oncogene in human NSCLC, and that this animal model could be valuable for exploring therapeutic agents against ROS1-rearranged lung cancer. PMID:23418494

  1. Targeting the MET gene for the treatment of non-small-cell lung cancer.

    PubMed

    Gelsomino, F; Facchinetti, F; Haspinger, E R; Garassino, M C; Trusolino, L; De Braud, F; Tiseo, M

    2014-02-01

    Recently, a better understanding of the specific mechanisms of oncogene addiction has led to the development of antitumor strategies aimed at blocking these abnormalities in different malignancies, including lung cancer. These abnormalities trigger constitutive activation of tyrosine kinase receptors (RTKs) involved in fundamental cell mechanisms such as proliferation, survival, differentiation and migration, and consequently the aberrant signaling of RTKs leads to cancer growth and survival. The inhibition of aberrant RTKs and downstream signaling pathways has opened the door to the targeted therapy era. In non-small-cell lung cancer (NSCLC), molecular research has allowed the discrimination of different aberrant RTKs in lung cancer tumorigenesis and progression, and thus the identification of several targetable oncogenic drivers. Following the development of small molecules (gefitinib/erlotinib and crizotinib) able to reversibly inhibit the epidermal growth factor receptor (EGFR) and signaling pathways mediated by anaplastic lymphoma kinase (ALK), respectively, the MET signaling pathway has also been recognized as a potential target. Moreover, according to current knowledge, MET could be considered both as a secondary oncogenic mechanism and as a prognostic factor. Several therapeutic strategies for inhibiting activated hepatocyte growth factor receptor (HGFR) and the subsequent downstream signaling transduction have been improved in order to block tumor growth. This review will focus on the MET pathway and its role in resistance to EGFR TK (tyrosine kinase) inhibitors, the different strategies of it