ALK and IGF-1R as independent targets in crizotinib resistant lung cancer.
ABSTRACT: ALK positive non-small cell lung cancer is highly responsive to ALK inhibitors such as crizotinib, but drug resistance typically develops within a year of treatment. In this study we investigated whether IGF-1R is an independent druggable target in ALK-positive lung cancer cells. We confirmed that combination ALK and IGF-1R inhibitor treatment is synergistically cytotoxic to ALK-positive lung cancer cells and that this remains the case for at least 12 days after initial exposure to crizotinib. ALK-positive cells with acquired resistance to crizotinib did not acquire cross-resistance to IGF-1R inhibition, though combination treatment in the resistant cells gave additive rather than synergistic cytotoxicity. We concluded that IGF-1R is an independent druggable target in ALK-positive lung cancer and support the trial of combination treatment.
Project description:Despite the impressive efficacy of crizotinib for the treatment of ALK-positive non-small cell lung cancer, patients invariably develop therapeutic resistance. Suppression of the IGF-1R signaling pathway may abrogate this acquired mechanism of drug resistance to crizotinib. Metformin, a widely used antidiabetic agent, may reverse crizotinib resistance through inhibition of IGF-1R signaling.The present study revealed that metformin effectively increased the sensitivity of both crizotinib-sensitive and -resistant non-small cell lung cancer cells to crizotinib, as evidenced by decreased proliferation and invasion and enhanced apoptosis. Metformin reduced IGF-1R signaling activation in crizotinib-resistant cells. Furthermore, the addition of IGF-1 to crizotinib-sensitive H2228 cells induced crizotinib resistance, which was overcome by metformin.The effects of metformin to reverse crizotinib resistance were examined in vitro by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), invasion assay, ki67 incorporation assay, flow cytometry analysis, Western blot analysis, and colony-forming assay.Metformin may be used in combination with crizotinib in ALK+ NSCLC patients to overcome crizotinib resistance and prolong survival.
Project description:Crizotinib, a selective tyrosine kinase inhibitor (TKI), shows marked activity in patients whose lung cancers harbor fusions in the gene encoding anaplastic lymphoma receptor tyrosine kinase (ALK), but its efficacy is limited by variable primary responses and acquired resistance. In work arising from the clinical observation of a patient with ALK fusion-positive lung cancer who had an exceptional response to an insulin-like growth factor 1 receptor (IGF-1R)-specific antibody, we define a therapeutic synergism between ALK and IGF-1R inhibitors. Similar to IGF-1R, ALK fusion proteins bind to the adaptor insulin receptor substrate 1 (IRS-1), and IRS-1 knockdown enhances the antitumor effects of ALK inhibitors. In models of ALK TKI resistance, the IGF-1R pathway is activated, and combined ALK and IGF-1R inhibition improves therapeutic efficacy. Consistent with this finding, the levels of IGF-1R and IRS-1 are increased in biopsy samples from patients progressing on crizotinib monotherapy. Collectively these data support a role for the IGF-1R-IRS-1 pathway in both ALK TKI-sensitive and ALK TKI-resistant states and provide a biological rationale for further clinical development of dual ALK and IGF-1R inhibitors.
Project description:Anaplastic lymphoma kinase (ALK) rearrangement, a key oncogenic driver in a small subset of non-small cell lung cancers, confers sensitivity to ALK tyrosine kinase inhibitors (TKIs). Crizotinib, a first generation ALK-TKI, has superiority to standard chemotherapy with longer progression-free survival and higher objective response rate. However, clinical benefit is limited by development of resistance, typically within a year of therapy. In this study the combined effect of crizotinib and the MEK inhibitor selumetinib was investigated in both crizotinib naïve (H3122) and crizotinib resistant (CR-H3122) ALK-positive lung cancer cells. Results showed that combination treatment potently inhibited the growth of both H3122 and CR-H3122 cells, resulting from increased apoptosis and decreased cell proliferation as a consequence of suppressed downstream RAS/MAPK signalling. The drug combination also elicited a greater than 3-fold increase in Bim, a mediator of apoptosis, and p27, a cyclin dependent kinase inhibitor compared to crizotinib alone. The results support the hypothesis that combining MEK inhibitors with ALK inhibitor can overcome ALK inhibitor resistance, and identifies Bim, PARP and CDK1 as druggable targets for possible triple drug therapy.
Project description:Despite the advances in new targeted therapies in ALK positive population, most patients progress under ALK inhibitors within first 2 years; being the brain the most frequent site of relapse. ALK mutations, in ~30% of patients, are the main known mechanism of resistance. Classically, second-generation ALK inhibitors have been the standard of care in the crizotinib-resistant population; however, each ALK inhibitor has a different spectrum of sensitivity to ALK mutations, complicating the optimal treatment strategy for the resistant population. Brigatinib (AP26113) is a novel highly selective and potent inhibitor of ALK and ROS1 with a high degree of selectivity. In vitro, brigatinib not only inhibited ALK with 12-fold higher potency compared to crizotinib, but also inhibited IGF-1R, FLT3 and EGFR mutants, with some activity against the EGFRT790M resistance mutation. In xenograft models, brigatinib overcomes resistance to ALK inhibitors, including the ALK G1202R mutation, which is resistant to first- and second-generation inhibitors. The efficacy of brigatinib in crizotinib-resistant, ALK-positive patients has been demonstrated in two early studies, which led to its approval in this setting, and it is currently being investigated as the first-line therapy versus crizotinib in tyrosine kinase inhibitor-naïve patients. Brigatinib demonstrates not only promising whole-body activity, but also an impressive improvement of intracranial outcomes in terms of both objective response rate and progression-free survival in the crizotinib-resistant population, with optimal efficacy at 180 mg (following a 90 mg run-in for 7 days) and good tolerance. These data confirm brigatinib as an excellent therapeutic strategy after crizotinib failure, particularly in the setting of central nervous system involvement. In this review, we summarize the two main clinical studies reported to date with brigatinib in ALK-positive advanced NSCLC patients, in particular, in the crizotinib-resistant population. We also address the mechanism of action for development of resistance and the challenging issues of optimal implementation for sequences of administration for ALK inhibitors.
Project description: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.
Project description: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.
Project description:Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance.
Project description:BACKGROUND:Na+/H+ exchanger regulatory factor 1 (NHERF1) is an important scaffold protein participates in the modulation of a variety of intracellular signal pathways. NHERF1 was able to enhance the effects of chemo-drugs in breast and cervical cancer cells. Anaplastic lymphoma kinase (ALK) fusion mutations are validated molecules targeted therapy in lung cancers, where crizotinib can be used as the specific inhibitor to suppress tumor progression. However, due to the less frequent occurrence of ALK mutations and the complexity for factors to determine drug responses, the genes that could alter crizotinib sensitivity are unclear. METHODS:Both ALK-translocated and ALK-negative lung adenocarcinoma specimens in tissue sections were collected for immunohistochemistry. The possible mechanisms of NHERF1 and its role in the cell sensitivity to crizotinib were investigated using an ALK-positive and crizotinib-sensitive lung adenocarcinoma cell line H3122. Either a NHERF1 overexpression vector or agents for NHERF1 knockdown was used for crizotinib sensitivity measures, in association with cell viability and apoptosis assays. RESULTS:The expression level of NHERF1 in ALK-translocated NSCLC was significantly higher than that in other lung cancer tissues. NHERF1 expression in ALK positive lung cancer cells was regulated by ALK activities, and was in return able to alter the sensitivity to crizotinib. The function of NHERF1 to influence crizotinib sensitivity was depending on its subcellular distribution in cytosol instead of its nucleus localized form. CONCLUSION:Ectopically overexpressed NHERF1 could be a functional protein for consideration to suppress lung cancers. The determination of NHERF1 levels in ALK positive NSCLC tissues might be useful to predict crizotinib resistance, especially by distinguishing cytosolic or nuclear localized NHERF1 for the overexpressed molecules.
Project description:Anaplastic large cell lymphoma (ALCL) is a rare aggresive non-Hodgkin lymphoma, of which over 50% of cases have an aberrant nucleophosmin (NPM)?anaplastic lymphoma kinase (ALK) fusion protein. Both mechanistic target of rapamycin (mTOR) inhibitor everolimus and ALK inhibitor crizotinib have shown promising antitumor activity in ALK-positive cancer cell lines. However, their combined effect has not yet been investigated.We evaluated the anti-proliferative effects of everolimus and/or crizotinib in ALK-positive ALCL cell lines, Karpas 299 and SU-DHL-1, and lung adenocarcinoma cell line, NCI-H2228.We found that individually, both everolimus and crizotinib potently inhibited cell growth in a dose-dependent manner in both Karpas 299 and SU-DHL-1 cells. A combination of these agents synergistically inhibited proliferation in the two cell lines. Crizotinib down-regulated aberrant AKT and ERK phosphorylation induced by everolimus. Combination treatment also significantly increased G0/G1 cell-cycle arrest, DNA damage, and apoptosis compared with everolimus or crizotinib alone in ALK-positive ALCL cells. In the Karpas 299 xenograft model, the combination treatment exerted a stronger antitumor effect than monotherapies, without significant change in body weight. The synergistic effect of everolimus and crizotinib was also reproduced in the ALK-positive lung adenocarcinoma cell line NCI-H2228. The combination treatment abrogated phosphoinositide 3-kinase/AKT and mTOR signaling pathways with little effect on the Ras/ERK pathway in NCI-H2228 cells.Crizotinib combinedwith everolimus synergistically inhibits proliferation of ALK-positive ALCL cells. Our results suggest that this novel combination is worthy of further clinical development in patients with ALK-positive ALCL.
Project description: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 being one of the newest and most appealing. Presence of ALK fusions is associated with some particular clinical and pathological features, including a preferential seeding into the central nervous system (CNS). In addition, ALK rearrangements are recognized as the strongest predictor for benefit of anti-ALK therapy. Crizotinib, the first ALK inhibitor (ALK-I) licensed in clinical practice, is the standard of care for newly diagnosed patients. Unfortunately, within the first year of treatment the majority of patients become insensitive to crizotinib, with approximately one third of them developing brain metastases (BMs). Optimal management of BMs is one of the major challenges in treating ALK positive NSCLC. Several novel and highly CNS penetrant ALK-Is are currently under investigation and available data clearly indicated their ability in controlling intracranial disease.