Activation Of Bypass Signaling Pathways Research Articles

Mechanism of Resistance to the WDR5 Inhibitor in MLL-Rearranged Leukemia

Drug resistance is a major problem often limiting the long-term effectiveness of targeted cancer therapeutics. Resistance can be acquired through mutations or amplification of the primary drug targets or activation of bypass signaling pathways. Considering the multifaceted function of WDR5 in human malignancies, WDR5 has emerged as an attractive drug target for the discovery of small-molecule inhibitors. In this study, we investigated if cancer cells might develop resistance to a highly potent WDR5 inhibitor. We established a drug-adapted cancer cell line and discovered that WDR5P173L mutation occurs in the resistant cells, which confers resistance by preventing target engagement of the inhibitor. This work elucidated the WDR5 inhibitor's potential resistance mechanism in a preclinical study as a reference for future study in the clinical stage.

The Landscape of ALK-Rearranged Non-Small Cell Lung Cancer: A Comprehensive Review of Clinicopathologic, Genomic Characteristics, and Therapeutic Perspectives.

Simple SummaryIn recent years, prognosis of non-small cell lung cancer (NSCLC) patients significantly improved thanks to the introduction of tyrosine kinase inhibitors (TKIs) in clinical practice. ALK-rearranged NSCLC patients benefit from treatment with ALK inhibitors (ALK-i), which have shown a greater efficacy and a better intracranial activity than chemotherapy. Comparative studies between next-generation ALK-i are still lacking and clinicians are looking for reliable tools to determine which drug suits best for each patient. The aim of this review is to deepen the role of clinical and pathological characteristics influencing patients’ prognosis during treatment with ALK-i and to provide an overview of molecular mechanisms of ALK-i resistance. In this setting, liquid biopsy may play an important role in predicting tumor response and monitoring resistance mutations. We will summarize ongoing trials developing new ALK-i or combinations between ALK-i and other agents, which may represent future scenarios in the field of NSCLC research.During the last decade, the identification of oncogenic driver mutations and the introduction of tyrosine kinase inhibitors (TKIs) in daily clinical practice have substantially revamped the therapeutic approach of oncogene-addicted, non-small cell lung cancer (NSCLC). Rearrangements in the anaplastic lymphoma kinase (ALK) gene are detected in around 3–5% of all NSCLC patients. Following the promising results of Crizotinib, a first-generation ALK inhibitor (ALK-i), other second-generation and more recently third-generation TKIs have been developed and are currently a landmark in NSCLC treatment, leading to a significant improvement in patients prognosis. As clinical trials have already demonstrated high efficacy of each ALK-i, both in terms of systemic and intracranial disease control, comparative studies between second and third generation ALK-i are still lacking, and primary or secondary ALK-i resistance inevitably limit their efficacy. Resistance to ALK-i can be due to ALK-dependent or ALK-independent mechanisms, including the activation of bypass signaling pathways and histological transformation: these findings may play an important role in the future to select patients’ subsequent therapy. This review aims to provide an overview of underlying molecular alterations of ALK-i resistance and point out promising role of liquid biopsy in predicting tumor response and monitoring resistance mutations. The purpose of this review is also to summarize current approval for ALK-rearranged NSCLC patients, to help clinicians in making decisions on therapeutic sequence, and to deepen the role of clinicopathological and genomic characteristics influencing patients’ prognosis during treatment with ALK-i.

A pharmacological exploration of targeted drug therapy in non-small cell lung cancer

Lung cancer is the prime cause of cancer-related deaths globally, with a contribution of 85% from non-small cell lung cancer. Before a few decades back, conventional chemotherapy was the most chosen treatment option for NSCLC but with side effects. Now, the treatment approaches have shifted to a new trend, targeted therapy, and a better treatment strategy with minimal side effects compared to chemotherapy. Advances in technologies and understanding the pathways lead to the discovery of new targets and through which it is possible to improve treatment outcomes and patient compliance. Unlike chemotherapy, targeted therapy focuses on the tumor cells and does not produce toxicity to healthy cells. The last two decades were very crucial in the development of many small molecules with the capability to target-specific proteins or genes in the disease progression pathway. Although the targeted therapy approach was a gemstone with many successful drugs for the treatment of NSCLC, various resistance mechanisms and activation of bypass signaling pathways put many of these drugs in the trash. In this review, we will discuss the major targeted proteins involved in NSCLC as well as the inhibitor drugs developed to target them for now and along with the future directions.

Review of Therapeutic Strategies for Anaplastic Lymphoma Kinase-Rearranged Non-Small Cell Lung Cancer.

Simple SummaryAnaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) was first reported in 2007. Following the development of crizotinib as a tyrosine kinase inhibitor (TKI) targeting ALK, the treatment of advanced NSCLC with ALK-rearrangements has made remarkable progress. Currently, there are five ALK-TKIs approved by the FDA, and the development of new agents, including fourth-generation TKI, is ongoing. Clinical trials with angiogenesis inhibitors and immune checkpoint inhibitors are also underway, and further progress in the treatment of ALK-rearranged advanced NSCLC is expected. The purpose of this manuscript is to provide information on the recent clinical trials of ALK-TKIs, angiogenesis inhibitors, immune checkpoint inhibitors, and chemotherapy, to describe tissue and liquid biopsy as a method to investigate the mechanisms of resistance against ALK-TKIs and suggest a proposed treatment algorithm.Non-small cell lung cancer (NSCLC) with anaplastic lymphoma kinase rearrangement (ALK) was first reported in 2007. ALK-rearranged NSCLC accounts for about 3–8% of NSCLC. The first-line therapy for ALK-rearranged advanced NSCLC is tyrosine kinase inhibitors (TKI) targeting ALK. Following the development of crizotinib, the first ALK-TKI, patient prognosis has been greatly improved. Currently, five TKIs are approved by the FDA. In addition, clinical trials of the novel TKI, ensartinib, and fourth-generation ALK-TKI for compound ALK mutation are ongoing. Treatment with angiogenesis inhibitors and immune checkpoint inhibitors is also being studied. However, as the disease progresses, cancers tend to develop resistance mechanisms. In addition to ALK mutations, other mechanisms, including the activation of bypass signaling pathways and histological transformation, cause resistance, and the identification of these mechanisms is important in selecting subsequent therapy. Studies on tissue and liquid biopsy have been reported and are expected to be useful tools for identifying resistance mechanisms. The purpose of this manuscript is to provide information on the recent clinical trials of ALK-TKIs, angiogenesis inhibitors, immune checkpoint inhibitors, and chemotherapy to describe tissue and liquid biopsy as a method to investigate the mechanisms of resistance against ALK-TKIs and suggest a proposed treatment algorithm.

Circulating tumor cell copy-number heterogeneity in ALK-rearranged non-small-cell lung cancer resistant to ALK inhibitors

Gatekeeper mutations are identified in only 50% of the cases at resistance to Anaplastic Lymphoma Kinase (ALK)-tyrosine kinase inhibitors (TKIs). Circulating tumor cells (CTCs) are relevant tools to identify additional resistance mechanisms and can be sequenced at the single-cell level. Here, we provide in-depth investigation of copy number alteration (CNA) heterogeneity in phenotypically characterized CTCs at resistance to ALK-TKIs in ALK-positive non-small cell lung cancer. Single CTC isolation and phenotyping were performed by DEPArray or fluorescence-activated cell sorting following enrichment and immunofluorescence staining (ALK/cytokeratins/CD45/Hoechst). CNA heterogeneity was evaluated in six ALK-rearranged patients harboring ≥ 10 CTCs/20 mL blood at resistance to 1st and 3rd ALK-TKIs and one presented gatekeeper mutations. Out of 82 CTCs isolated by FACS, 30 (37%) were ALK+/cytokeratins-, 46 (56%) ALK-/cytokeratins+ and 4 (5%) ALK+/cytokeratins+. Sequencing of 43 CTCs showed highly altered CNA profiles and high levels of chromosomal instability (CIN). Half of CTCs displayed a ploidy >2n and 32% experienced whole-genome doubling. Hierarchical clustering showed significant intra-patient and wide inter-patient CTC diversity. Classification of 121 oncogenic drivers revealed the predominant activation of cell cycle and DNA repair pathways and of RTK/RAS and PI3K to a lower frequency. CTCs showed wide CNA heterogeneity and elevated CIN at resistance to ALK-TKIs. The emergence of epithelial ALK-negative CTCs may drive resistance through activation of bypass signaling pathways, while ALK-rearranged CTCs showed epithelial-to-mesenchymal transition characteristics potentially contributing to ALK-TKI resistance. Comprehensive analysis of CTCs could be of great help to clinicians for precision medicine and resistance to ALK-targeted therapies.

Selected Articles from This Issue

Genomic alterations in cell-cycle components and the PI3K pathway are often observed in pediatric brain tumors, suggesting that combined CDK and mTOR inhibition may provide clinical benefit to these children. DeWire and colleagues performed a phase I trial to identify the maximum tolerated dose (MTD)/recommended phase II dose (RP2D) of ribociclib and everolimus in children with recurrent or refractory brain tumors. This combination was well tolerated, and an RP2D was identified for both agents. Ribociclib concentrations in plasma, tumor, and CSF samples were assessed, and ribociclib was determined to enter the tumor and CSF at therapeutic concentrations. This work serves as a basis for further trials to assess the efficacy of this combination in children with brain tumors.Although checkpoint inhibition has benefited patients with microsatellite instable (MSI-H) CRC, patients with microsatellite stable (MSS) CRC are resistant to this treatment regimen. Preclinical studies have demonstrated that focal radiation can stimulate antitumor immunity. Monjazeb and colleagues conducted a randomized phase II study evaluating the combination of durvalumab, a PD-L1 inhibitor, and tremelimumab, a CTLA-4 inhibitor with either hypofractionated radiation (HFRT), or low-dose fractionated radiation (LDFRT) in patients with advanced MSS CRC. This combination led to stable disease in one patient with out-of-field tumor shrinkage. HFRT treatment increased CD8+ T-cell tumor infiltration and decreased circulating immune cells.Immunotherapies have enhanced cancer therapeutics, but only for a minority of patients. Ways to reliably enhance immunogenicity, T-cell infiltration into tumors, and predict responsiveness are critically needed. Telli and colleagues demonstrate that plasmid IL-12 injected intratumorally followed by electroporation (Tavo) induces a CXCR3 gene signature (CXCR3-GS) with enhanced antigen presentation, expansion, and licensing of T cells systemically in both mice and humans. A previously unresponsive patient with melanoma demonstrated significant clinical response to nivolumab following Tavo therapy. This safe, effective intratumoral therapy and induced CXCR3-GS represents a potential tool for treatment of nonimmunogenic tumors.KRASG12C inhibitors, including AMG-510, have demonstrated efficacy in KRASG12C mutant lung cancer. However, resistance occurs in many patients due to activation of bypass signaling pathways. Although multiple agents are being tested in combination with KRASG12C inhibitors in clinical trials, it is unclear how best to identify the appropriate combination for individual patients. Solanki and colleagues conducted a proteomics analysis of KRASG12C cell lines after short term treatment with ARS-1620 to identify molecular responses to KRASG12C inhibition. ERBB2/3 signaling led to resistance to KRASG12C inhibitors in epithelial-like lung cancer cells, while FGFR1 or AXL signaling led to resistance in mesenchymal-like cells. These results support clinical trials assessing these combinations in patients with KRASG12C mutant lung cancer, and this approach may identify similar resistance mechanisms in other cancer settings.

Abstract 3003: MET mediates entrectinib resistance in ROS1 gene fusion positiveNSCLC

Abstract ROS1 gene fusions are predominantly found in non-small cell lung cancer (NSCLC), and ROS1 gene fusion-positive (ROS1+) tumors comprise 1-2% of all diagnosed lung adenocarcinomas. Currently, there are two ROS1 tyrosine kinase inhibitors (TKIs) which are US FDA approved, crizotinib and entrectinib, both of which generate tumor response rates in excess of 70% and demonstrate prolonged disease control. However, acquired resistance to TKIs is inevitable, and has been reported in ROS1+ NSCLC tumors or cancer cell models by our group and others and include ROS1 kinase domain mutations (e.g., L1951R, S1986Y/F, F2004V, L2026M, and G2032R), activation of bypass signaling pathways (e.g., EGFR, RAS, or KIT), or phenotypic transformation (such as epithelial to mesenchymal transition). In order to better understand mechanisms of acquired TKI resistance in ROS1 NSCLC, our lab utilized primary, patient-derived cancer cell models harboring these rare oncogenic kinase rearrangements. Herein, using a patient-derived NSCLC cancer cell line (CUTO28) harboring a TPM3-ROS1 fusion, we derived an entrectinib-resistant cell line (CUTO28-ER) through in vitro culture under drug selection. In these CUTO28-ER cells, we identified: (1) MET-mediated bypass signaling as an acquired resistance mechanism to entrectinib, (2) upregulation of MET signaling is accomplished via MET gene amplification, and (3) resistance could be overcome by the dual ROS1/MET inhibitor crizotinib. To our knowledge, this is the first reported case of MET-mediated acquired resistance in a ROS1+ cancer. This cell line model finding was supported by another primary, patient-derived cell line (CUTO38) harboring a CD74-ROS1 fusion; this cell line was derived from a NSCLC patient following disease progression on entrectenib in the clinic. CUTO38 is resistant to entrectinib in vitro and displays elevated MET expression, MET activation with increased phosphorylation, and dependence on MET for cell proliferation and survival. Notably, MET activation in CUTO38 was not generated by gene amplification, MET exon 14 splicing, MET gene fusion, or autocrine HGF expression, suggesting alternate means of MET activation. These findings suggest that interrogation of MET gene amplification or other MET biomarkers should be performed in ROS1+ NSCLC patients who experience disease progression on entrectinib or other ROS1 TKIs that do not inhibit MET, since there are clinically available MET inhibitors, some of which are dual ROS1/MET inhibitors, that may overcome drug resistance in these patients. Citation Format: Logan Tyler, Anh Le, Hala Nijmeh, Robert Doebele. MET mediates entrectinib resistance in ROS1 gene fusion positiveNSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3003.

Molecular characterization and prognostic analysis of non-small cell lung cancer: A single centric real-world study.

e21646 Background: Identifying targetable genetic variants may help maximize clinical benefit of non-small cell lung cancer (NSCLC). Here, we retrospectively analyzed the benefit of next-generation sequencing (NGS) for NSCLC in routine clinical practice. Methods: A total of 59 NSCLC patients were enrolled in the study from Jun. 2018 to Dec. 2019. Formalin-fixed paraffin-embedded tissues samples were collected and sequenced by NGS, which detects single-nucleotide variants, small insertions/deletions, rearrangements, and copy-number alterations of either 59 or 1021 tumor related genes. The tumor response was evaluated using RECIST v1.1. Results: The detection rate of targetable variants was 59.32% (35/59), including EGFR mutation 49.15% (29/59), ALK rearrangement 5.08% (3/59), ROS1 rearrangement 1.69% (1/59), BRAF V600E mutation 1.69% (1/59), and ERBB2 mutation 1.69% (1/59). Of 29 patients with EGFR mutations, 5 patients (17.24%) had multiple EGFR mutations, including 2 patients (6.89%) with uncommon mutations. 25patients (86.21%) harbored concurrent mutations that may limit the efficacy of EGFR-TKIs, including EGFR amplification, activation of bypass signaling pathways, TP53 exon8 mutation, BCL2L11 deletion polymorphism, and PI3K-AKT-mTOR and cell cycle gene alterations. Among 25 patients received EGFR-TKIs, TKI effectiveness was evaluated in 18 patients. The disease control rate was 100% and overall response rate was 27.78%. The proportion of patients with TMB-H and/or PD-L1≥1, and targetable variants negative was 33.89% (20/59). Only one patient of this group received immunotherapy with pembrolizumab and achieved SD at the 2nd month. The proportion of patients with targetable variants negative, TMB-L, and PD-L1 < 1 or unknow patients was 6.78% (4/59), two patients of them left hospital without additional treatment, two patients received chemotherapy and the KRAS mutation present in one of them suggested that he might be resistant to EGFR-TKIs. Conclusions: More than half of NSCLC patients have targetable genetic variants and more than 30% patients have a molecular signature that can be treated with immunotherapy. Compared with immunotherapy, targeted therapy has higher acceptability in real-world. NGS-based genetic testing may have clinical value to predict the effectiveness of targeted therapy and immunotherapy. It can be widely implemented and standardized into clinical use.

Clinical outcomes of various resistance mechanisms of osimertinib in Chinese advanced non-small cell lung cancer patients.

e20530 Background: Increasing efforts have been invested in elucidating the resistance mechanisms to osimertinib. Major resistance mechanisms include but not limited to acquired EGFR mutations, predominantly C797, mutations in bypass pathways and small cell lung cancer (SCLC) transformation. However, no study has comprehensively investigated clinical outcomes of various mechanisms of resistance. Methods: 103 T790M positive advanced Chinese non-small cell lung cancer (NSCLC) patients who progressed on 1st generation EGFR-TKI were enrolled. Targeted sequencing, using a panel consisting of 168 lung cancer related genes, was performed on paired plasma samples collected prior to osimertinib and after the development of disease progression (PD) to profile mutation spectrum. 7 patients with no mutation detected at PD were excluded from analyses. Results: Major acquired mutations included 25% EGFR mutations, predominantly C797 and L792, 16% MET amplification, 8% TP53 mutations, 4% KRAS mutations, 4% RET fusions, 4% ERBB2 amplification and 6.25% RB1 mutations. Acquired RB1 mutation may indicate the possibility of SCLC transformation. Approximately, 30% of patients with no known resistance mechanisms at PD. In this cohort, we had 61 patients with 19 deletion and 35 patients with EGFR L858R prior to the initiation of osimertinib. We revealed patients with 19del acquired more mutations ( p= 0.014) and were more likely to acquire mutations in MAP/PI3Kpathway ( p= 0.04) and TP53 at PD ( p= 0.021). On the other hand, acquired ERBB2 amplifications were only detected in L858R-mutant patients ( p= 0.047). Furthermore, 36 patients preserved T790M and 60 patients lost T790M at PD. Our data revealed patients retaining T790M, often associated with activation of bypass signaling pathways or continued EGFR activation through tertiary mutations, had a longer progression-free survival (PFS) ( p= 0.047) and overall survival (OS) ( p= 0.04) comparing to patients with T790M loss, often with diverse and EGFR-independent mechanisms. We also show that patients with acquired C797S had significantly longer PFS ( p= 0.031), while patients with acquired MET amplifications had significantly shorter PFS ( p= 0.033). Conclusions: Collectively, we revealed differential clinical outcomes associated with various resistance mechanisms, representing an important step in advancing the understanding of resistance mechanisms of osimertinib.

Abstract 1007: SHP2 inhibition restores sensitivity to ALK inhibition in resistant ALK-rearranged non-small cell lung cancer (NSCLC)

Abstract Despite development of highly potent and selective inhibitors (e.g., ceritinib, alectinib, lorlatinib) targeting anaplastic lymphoma kinase (ALK), resistance invariably develops and limits the efficacy of these inhibitors in the clinic. The major classes of resistance are on-target genetic alterations (e.g., secondary ALK kinase domain mutations) and activation of alternative or bypass signaling pathways. While most patients are responsive to sequential treatment with two or more ALK inhibitors, ALK-independent resistance eventually emerges and leads to failure of further ALK-directed monotherapy. We used a synthetic lethal pooled shRNA screen to discover loss-of-function events that could sensitize resistant patient-derived cell lines to ALK inhibition. In addition to identifying known bypass targets such as FGFR, EGFR and SRC, we also identified PTPN11 (which encodes SHP2, a non-receptor protein tyrosine phosphatase that modulates signaling downstream of growth factor receptors) as a common hit shared by cell lines exhibiting different mechanisms of bypass activation. In parallel with the shRNA screen, we also performed a high throughput combination compound screen in the same patient-derived models, and identified activation of the same bypass signaling pathways. We showed that the highly potent and selective small-molecule SHP2 inhibitor SHP099 could sensitize resistant cell lines to ALK inhibition. In biochemical studies, co-targeting of ALK and SHP2 overcame resistance mediated by ALK-independent bypass mechanisms by decreasing RAS-GTP loading potential of cells and inhibiting phospho-ERK rebound. These results suggest that dual ALK and SHP2 inhibition may represent a new therapeutic strategy for ALK-positive patients, whose lung cancers have evolved ALK-independent mechanisms of resistance, including activation of bypass signaling pathways. Citation Format: Leila Dardaei, Hui Qin Wang, Paul Fordjour, Manrose Singh, Grainne Kerr, Satoshi Yoda, Jinsheng Liang, Yichen Cao, Yan Chen, Justin F. Gainor, Luc Friboulet, Ibiayi Dagogo-Jack, David T. Myers, Emma Labrot, David Ruddy, Melissa Parks, Dana Lee, Richard H. DiCecca, Susan Moody, Huaixiang Hao, Morvarid Mohseni, Matthew LaMarche, Juliet Williams, Keith Hoffmaster, Giordano Caponigro, Cyril H. Benes, Alice T. Shaw, Aaron N. Hata, Fang Li, Jeffrey A. Engelman. SHP2 inhibition restores sensitivity to ALK inhibition in resistant ALK-rearranged non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1007. doi:10.1158/1538-7445.AM2017-1007

Marsdenia tenacissima extract overcomes Axl- and Met-mediated erlotinib and gefitinib cross-resistance in non-small cell lung cancer cells.

Tyrosine kinase inhibitors (TKIs) are an effective treatment strategy for non-small cell lung cancer (NSCLC) patients harboring mutations that result in constitutive activation of the epidermal growth factor receptor (EGFR). However, most patients eventually develop resistance to TKIs. This occurs due to additional EGFR mutations or the activation of bypass signaling pathways. In our previous work, we demonstrated that Marsdenia tenacissima extract (MTE) restored gefitinib sensitivity in resistant NSCLC cells with EGFR T790M or K-ras mutations. However, the potential efficacy of MTE in NSCLC cells with resistance mediated by Axl and c-Met, and the related molecular mechanisms need to be elucidated. In this study we evaluated the ability of MTE to restore erlotinib/gefitinib sensitivity in TKI resistant HCC827/ER cells and xenograft mice models. Our results demonstrate that MTE overcomes erlotinib and gefitinib resistance driven by Axl and c-Met in vitro and in vivo. Combination therapy significantly suppressed EGFR downstream molecules and the c-Met and Axl activated bypass signaling pathways. Moreover, we observed that MTE is more efficient at restoring resistance to erlotinib than gefitinib. As the Axl and c-Met mediated bypass pathways share the same downstream signaling cascade as EGFR, simultaneous targeting of these pathways is a promising strategy to overcome acquired resistance of TKIs. Our results demonstrate that MTE treatment attenuates Axl phosphorylation and the associated epithelial-mesenchymal transition, suggesting MTE treatment may be a potential therapeutic strategy for overcoming erlotinib and gefitinib cross-resistance in NSCLC, especially for erlotinib resistance.

Abstract IA31: Mechanisms of resistance to EGFR inhibitors in lung cancer

Abstract Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs), including geftinib, erlotinib and afatinib are the standard of care for patients with advanced EGFR mutant non-small cell lung cancer. Treatment with an EGFR TKI leads to a response rate of ~60% and a median progression free survival ranging from 9 to 11 months. Acquired drug resistance inevitably develops in most of not all EGFR mutant lung cancer patients treated with EGFR inhibitors. The most common mechanism of acquired resistance is the development of a secondary EGFR T790M mutation. Other mechanism of resistance include activation of bypass signaling pathways (such as MET or HER2) and histological transformation. Over the last few years a series of new structurally distinct EGFR inhibitors have been identified and entered the clinic that are capable of inhibiting EGFR T790M. This class of mutant selective EGFR TKIs are effective both against the activating EGFR mutation and the drug resistant T790M but spare inhibition of wild type (WT) EGFR. This has enormous potential therapeutic implications as the side effects of all approved EGFR inhibitors is derived from inhibition of WT EGFR. Several of these agents, including AZD9291 and rociletinib (CO-1686) are in advanced stage clinical development. Both agents have been granted FDA’s breakthrough designation. In EGFR inhibitor resistant patients who have developed resistance to current clinical EGFR inhibitors through T790M, the response rates to either AZD9291 or rociletinib is over 60% with median progression free survivals of ~ 10 months. Current research efforts and clinical studies are focusing on a.) understanding mechanisms of drug resistance to this new class of EGFR inhibitors, b.) the development of rationale combination therapies and c.) use of these agents as initial treatment (instead of currently approved EGFR inhibitors) for patients with advanced EGFR mutant NSCLC. Citation Format: Pasi Jänne. Mechanisms of resistance to EGFR inhibitors in lung cancer. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA31.

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

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

O10.1 - Next generation ALK inhibitors and mechanisms of resistance to therapy

ABSTRACT Anaplastic lymphoma kinase (ALK) gene rearrangements are important therapeutic targets in non-small cell lung cancer (NSCLC) that impart sensitivity to treatment with ALK tyrosine kinase inhibitors (TKIs), such as crizotinib. Indeed, crizotinib is now considered a standard of care for ALK-positive NSCLC patients based upon randomized trials in which crizotinib produced significant improvements in objective response rate (ORR) and progression-free survival (PFS) compared to first- and second-line cytotoxic chemotherapy. Nonetheless, patients almost invariably relapse on crizotinib—typically within 1-2 years. Molecular mechanisms of resistance to crizotinib include: 1) acquired secondary mutations in the ALK kinase domain (e.g., L1196M, G1269A), 2) ALK fusion gene amplification, and 3) activation of bypass signaling pathways, such as EGFR and c-KIT. To date, therapeutic strategies to overcome crizotinib resistance have focused on the use of more potent and/or structurally distinct, next-generation ALK inhibitors. Ceritinib (LDK378) is one such next-generation ALK inhibitor that recently gained accelerated regulatory approval in the United States. In a recent phase I study, ceritinib was associated with an ORR of 58% among 114 patients receiving at least 400 mg per day. Importantly, among patients previously treated with crizotinib, the response rate to ceritinib was 56%. Similarly, the next-generation ALK inhibitor alectinib has shown impressive activity in ALK-positive NSCLC. In a phase I/II study conducted in Japan, alectinib was associated with an ORR of 93.5% in TKI-naive patients. More recently, data on alectinib in the crizotinib-resistant setting has also emerged. Among 44 patients previously treated with crizotinib, the ORR on alectinib was 55%. Of note, both alectinib and ceritinib have also shown activity against central nervous system metastases—an important sanctuary site in ALK-positive NSCLC. Finally, at least 6 other ALK inhibitors are currently in clinical development. Determining the mechanisms of resistance to these agents as well as the optimal sequencing of ALK inhibitors in the clinic are important emerging questions.

Oncogene addiction: pathways of therapeutic response, resistance, and road maps toward a cure.

A key goal of cancer therapeutics is to selectively target the genetic lesions that initiate and maintain cancer cell proliferation and survival. While most cancers harbor multiple oncogenic mutations, a wealth of preclinical and clinical data supports that many cancers are sensitive to inhibition of single oncogenes, a concept referred to as 'oncogene addiction'. Herein, we describe the clinical evidence supporting oncogene addiction and discuss common mechanistic themes emerging from the response and acquired resistance to oncogene-targeted therapies. Finally, we suggest several opportunities toward exploiting oncogene addiction to achieve curative cancer therapies.

Therapeutic strategies utilized in the setting of acquired resistance to EGFR tyrosine kinase inhibitors.

Patients with EGFR-mutant lung cancer derive significant therapeutic benefit from treatment with EGFR tyrosine kinase inhibitors (TKI). Unfortunately, acquired resistance is an inevitable consequence of this treatment strategy, with a broad variety of resistance mechanisms including acquired EGFR mutations (e.g., T790M) and activation of bypass signaling pathways, such as MET and HER2. Several therapeutic strategies hypothesized to delay or overcome resistance have been tested in clinical trials, including "next-generation" EGFR TKIs and rational combinations of targeted agents. However, to date, there are no FDA-approved therapies for patients with acquired resistance to first-line EGFR TKI therapy. There remains a critical need for more effective and better tailored treatments in this setting to match treatments to the individual patient and specific resistance mechanism at hand. In this review, we discuss known mechanisms of resistance to first-line EGFR TKI therapy and describe previous and ongoing strategies to overcome resistance.

ROR1 as a novel therapeutic target for EGFR-mutant non-small-cell lung cancer patients with the EGFR T790M mutation.

Activation of bypass signaling pathways, impairment of apoptosis and mutation of epidermal growth factor receptor (EGFR) to a drug-resistant state are well known mechanisms of resistance to single-agent erlotinib therapy in non-small-cell lung cancer (NSCLC) driven by EGFR mutations. Orphan receptor 1 (ROR1) knockdown inhibited the growth of NCI-H1975 cells (harboring EGFR L858R and T790M mutations). A pro-survival function for ROR1/MEK/ERK signaling in cooperation with AKT has been demonstrated. We have assessed ROR1 expression in 45 patients from the EURTAC trial (clinicaltrials.gov NCT00446225), 27 of whom harbored pretreatment concomitant EGFR T790M mutations, and correlated results with outcome. Progression-free survival (PFS) was 11.8 months for erlotinib-treated patients with low/intermediate and 5.8 months for those with high ROR1 levels. PFS for chemotherapy-treated patients was 5.6 and 9 months, respectively (P=0.0165). A total of 15 erlotinib-treated patients harbored concomitant T790M mutations; for these patients, PFS was 10.8 months for those with low/intermediate compared to 2.7 months for those with high ROR1 levels. In contrast, among 12 chemotherapy-treated patients with concomitant T790M mutations, PFS was 5.8 months for those with low/intermediate, compared to 14.2 months for those with high ROR1 levels (P=0.0138). ROR1 expression has a differential effect on outcome to erlotinib and chemotherapy in EGFR-mutant NSCLC patients. High ROR1 expression significantly limits PFS in erlotinib-treated patients with T790M mutations and ROR1-directed therapies can enhance the efficacy of treatment. In contrast, high ROR1 expression confers longer PFS to chemotherapy in the same group of patients. The role of chemotherapy and erlotinib in EGFR-mutant NSCLC patients with high ROR1 expression warrants further investigation.

ALK-positive non-small cell lung cancer: mechanisms of resistance and emerging treatment options.

Targeted therapy has emerged as an effective treatment option for certain molecular subsets of advanced stage non-small cell lung cancer (NSCLC). The discovery of the echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) translocation as an oncogenic driver has led to the development of novel therapies with activity in vitro and in the clinic. The first-in-class tyrosine kinase inhibitor crizotinib is effective against ALK-positive NSCLC and is currently used as first-line or salvage therapy in the setting of advanced disease. However, resistance inevitably develops through a variety of mechanisms, including point mutations affecting the fusion protein, activation of bypass signaling pathways, copy number gain of ALK, and other means. Increased understanding of these pathways is essential for tailoring treatment choices to improve outcomes and minimize toxicities. Potent second-generation ALK inhibitors currently in trials are producing encouraging results in ALK-positive NSCLC, even in patients with acquired resistance to crizotinib. The success in identifying the ALK translocations and rapidly developing targeted drugs to exploit it paves the way for a better understanding of NSCLC biology and the quest to provide effective, personalized treatment for lung cancer patients.

Abstract IA36: Treatment strategies to overcome EGFR TKI resistance.

Abstract IA36: Treatment strategies to overcome EGFR TKI resistance.

Abstract C90: Dependence of afatinib and cetuximab resistant lung adenocarcinomas on mTOR signaling.

Abstract Lung adenocarcinomas harboring mutations in the tyrosine kinase domain of the EGFR are sensitive to tyrosine kinase inhibitors (TKIs) erlotinib or gefitinib. Despite a ∼70% response rate to these agents, patients almost inevitably develop resistance on average within a year of starting drug treatment. In 50% of cases, acquired resistance to EGFR TKIs is due to the emergence of a secondary mutation in the tyrosine kinase domain of the receptor (the T790M mutation). Additional less common mechanisms of resistance include activation of bypass signaling pathways via amplification of other receptor tyrosine kinases (RTKs) like MET and HER2 or mutations in genes encoding downstream signaling components such as PIK3CA or BRAF. In rare instances, tumors biopsied at progression show phenotypic transformations such as epithelial-to-mesenchymal transition (EMT) or neuroendocrine differentiation. We previously generated transgenic mice that develop EGFRL858R+T790M-induced lung adenocarcinomas and showed that T790M-mediated resistance could be overcome using a combination of a second generation TKI BIBW-2992 (afatinib) with the anti-EGFR antibody, cetuximab. This preclinical study prompted a Phase IB/II clinical trial testing this drug combination in patients with progressive disease after erlotinib or gefitinib treatment that is showing a promising 30% response rate. Unfortunately patients eventually develop disease progression on this drug combination and the mechanisms of resistance to afatinib+cetuximab are currently unknown. Here, we used an intermittent dosing strategy, previously used to generate erlotinib-resistant tumors in mice, to generate afatinib+cetuximab resistant tumors in xenograft and transgenic mouse models of EGFR mutant lung cancer. In these models, afatinib+cetuximab resistant tumors lacked detectable mutations in the EGFR transgene, the ERBB2 kinase domain or KRAS. Molecular analysis of resistant tumors revealed high levels of activation of the mTOR signaling pathway. Consistent with these findings, two patients with afatinib+cetuximab-resistant tumors exhibited alterations in genes (TSC1 and NF2) that activate the mTOR pathway. In cell culture and in mouse models, such resistance can be overcome by addition of the mTOR pathway inhibitor, rapamycin. These studies are the first to demonstrate mechanisms of acquired resistance to dual inhibition of EGFR in EGFR mutant lung cancer and provide new insight into the biology of this subset of lung cancers with immediate therapeutic implications for patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C90. Citation Format: Valentina Pirazzoli, Caroline Nebhan, Xiaoling Song, Zenta Walter, Guoping Cai, Anna Wurtz, Zhongming Zhao, Elisa Elisa de Stanchina, Leora Horn, David Carbone, Philip J. Stevens, Vincent Miller, Scott Gettinger, William Pao, Katerina Politi. Dependence of afatinib and cetuximab resistant lung adenocarcinomas on mTOR signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C90.