MET Amplification In Lung Cancer Research Articles

PAI-1 mediates acquired resistance to MET-targeted therapy in non-small cell lung cancer.

Mechanisms underlying primary and acquired resistance to MET tyrosine kinase inhibitors (TKIs) in managing non-small cell lung cancer remain unclear. In this study, we investigated the possible mechanisms acquired for crizotinib in MET-amplified lung carcinoma cell lines. Two MET-amplified lung cancer cell lines, EBC-1 and H1993, were established for acquired resistance to MET-TKI crizotinib and were functionally elucidated. Genomic and transcriptomic data were used to assess the factors contributing to the resistance mechanism, and the alterations hypothesized to confer resistance were validated. Multiple mechanisms underlie acquired resistance to crizotinib in MET-amplified lung cancer cell lines. In EBC-1-derived resistant cells, the overexpression of SERPINE1, the gene encoding plasminogen activator inhibitor-1 (PAI-1), mediated the drug resistance mechanism. Crizotinib resistance was addressed by combination therapy with a PAI-1 inhibitor and PAI-1 knockdown. Another mechanism of resistance in different subline cells of EBC-1 was evaluated as epithelial-to-mesenchymal transition with the upregulation of antiapoptotic proteins. In H1993-derived resistant cells, MEK inhibitors could be a potential therapeutic strategy for overcoming resistance with downstream mitogen-activated protein kinase pathway activation. In this study, we revealed the different mechanisms of acquired resistance to the MET inhibitor crizotinib with potential therapeutic application in patients with MET-amplified lung carcinoma.

Abstract 5101: Comparative genomic analysis reveals distinctive immune profiles in MET exon 14 mutated and MET amplified lung cancer

Abstract Background: In non-small cell lung cancer (NSCLC), the MET tyrosine kinase receptor can be mutated or amplified resulting in dysregulation of receptor function leading to tumor proliferation. MET exon 14 (METEx14) mutated and MET amplified NSCLC tumors have shown varied response to immunotherapy. Therefore, we sought to compare the genomic and immune landscape of MET-altered NSCLC. Methods: The genomic profiles of 3,821 NSCLC tumors sequenced using the Strata Select assay on the Strata Oncology Platform were analyzed. Tumors were sorted based on METex14 mutations, MET amplification (METamp) defined as copy number gain (CNG) ≥6 and MET wild-type (METwt). The RNA expression of 19 immune regulatory genes, tumor mutation burden (TMB), Strata Immunotherapy Response Signature (IRS) score and the frequency of gene co-alterations were compared across groups. Statistical comparisons of medians were done with Kruskal-Wallis testing and categorical comparisons with Chi square using R Studio version 4.3.2. Results: 122 (3.2%) METex14, 70 (1.8%) METamp, and 3,629 (95.0%) METwt were identified. Patients with METex14 were older and more frequently female. MET gene expression was found to be higher in METamp (median 14.4) compared to METex14 (median 12.3) (P<0.01). TMB was lowest in METex14 (median 2.7) group compared to METamp (median 7.1) and METwt (median 5.1) (P<0.001). High PD-L1 expression occurred in 71.4% of METamp, 68.0% of METex14 and 41.3% of METwt (P<0.001). Overall, METamp tumors had a greater proportion of high IRS scores (52.9%) compared to METex14 (35.2%) and METwt (45.4%) (P<0.01). mRNA expression of immune checkpoints CTLA4, PD-1, LAG3, IDO1, TIGIT and HAVCR2 were all higher in METex14 compared to METamp and METwt (P<0.001 for all). METex14 tumors also exhibited higher gene expression of CD4, CD8A and FOXP3 compared to METamp and METwt (p<0.001). METex14 had higher prevalence of MDM2 amplification (34% vs 3% vs 4%) and lower prevalence of alterations in EGFR (0.8% vs 22% vs 21%), KRAS (0.8% vs 10% vs 26%), CDKN2A (17% vs 29% v 28%) and TP53 (34% vs 87% vs 64%) compared with METamp and METwt tumors, respectively (p<0.05). Conclusion: METex14 and METamp tumors harbor distinct immune-gene expression profiles and co-occurring gene alterations that distinguish themselves from each other and METwt tumors. Further analysis must be conducted to determine how these variations impact immunotherapy in MET-altered NSCLC. Citation Format: Saahil Javeri, Rachel Minne, Shrey Ramesh, Andrew Baschnagel, Randy Kimple, Laura Lamb, Scott Tomlins, Bryan Johnson, Nickolay Khazanov. Comparative genomic analysis reveals distinctive immune profiles in MET exon 14 mutated and MET amplified lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5101.

Abstract 3277: PAI-1 mediates resistance to MET-targeted therapy in non-small cell lung cancer

Abstract [Purpose] The mesenchymal epithelial transition factor receptor (MET) is a potential therapeutic target in various cancers, including non-small cell lung cancer (NSCLC). In NSCLC, the activation of MET pathway is thought to occur through diverse mechanisms that influence properties affecting cancer cell survival, growth, and invasiveness. These tumors show oncogene addiction to MET and exhibit remarkable responses to MET-Tyrosine Kinase Inhibitors (TKIs). However, the long-term effectiveness of MET-TKIs is usually limited due to acquired drug resistance. The mechanisms underlying primary and acquired resistance to MET-TKIs in the management of NSCLC remain unclear. In this study, we investigated the mechanisms acquired for crizotinib in MET-amplified lung carcinoma cell lines. [Experimental Designs] Two MET-amplified lung cancer cell lines, EBC-1 and H1993, were established for acquired resistance to MET-TKI crizotinib and were functionally elucidated. The factors contributing to the resistance mechanism were assessed by genomic and transcriptomic data, and the alterations hypothesized to confer resistance were validated. [Results] Multiple mechanisms underlay acquired resistance to crizotinib in MET-amplified lung cancer cell lines. In EBC-1-derived resistant cells, the drug resistance mechanism was mediated by the overexpression of SERPINE1, the gene encoding plasminogen activator inhibitor-1 (PAI-1). Crizotinib resistance was overcome by combination therapy with PAI-1 inhibitor and PAI-1 knockdown. Another mechanism of resistance in different subline cells of EBC-1 was evaluated as epithelial to mesenchymal transition (EMT) with the upregulation of antiapoptotic proteins. In the H1993-derived resistant cells, the phosphorylated forms of MEK and ERK were observed to be upregulated and MEK inhibitors could be a potential therapeutic strategy for overcoming the resistance. [Conclusions] In this study, we revealed the different mechanisms of drug resistance to MET-TKI treatment and presented some possible strategies to overcome the resistance. Citation Format: Tomoaki Higashihara, Yin Min Thu, Ken Suzawa, Keiichi Date, Atsushi Matsuoka, Daisuke Mizuno, Ryo Yoshichika, Naohiro Hayashi, Fumiaki Mukohara, Mao Yoshikawa, Kazuhiko Shien, Hiromasa Yamamoto, Shinichi Toyooka. PAI-1 mediates resistance to MET-targeted therapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3277.

Single targeting of MET in EGFR-mutated and MET-amplified non-small cell lung cancer.

In EGFR-mutant and MET-amplified lung cancer resistant to EGFR inhibitors, double blockade of EGFR and MET is considered as a reasonable strategy despite increasing toxicity. This study evaluated the single MET inhibition in these specific tumours. We investigated the efficacy of a single MET inhibitor in EGFR-mutant, MET-amplified lung cancer cells (HCC827GR) and the matched clinical cases and patient-derived cells. Acquired resistance mechanisms to single MET inhibitor were further explored. Single MET inhibitor sufficiently inhibited the EGFR downstream signalling and proliferation in the HCC827GR cells. The MET-inhibitor-sensitive clones had similar EGFR mutation allele frequency as the MET-inhibitor-resistant clones. The patients with EGFR-mutant, MET-amplified lung cancer resistant to EGFR inhibitors showed definite response to single MET inhibitor but the response duration was not durable. The MET gene copy number in their plasma circulating tumour DNA was significantly decreased during the treatment and was not re-increased after progression. In the cells resistant to single MET inhibitor, the EGFR pathway was reactivated, and gefitinib alone successfully suppressed their growth. Single MET inhibition produced a short-lived response in EGFR-mutant and MET-amplified lung cancer. A further study of a novel combination therapy schedule is needed to achieve long-lasting efficacy and less toxicity.

Abstract 417: Targeting aurora kinase b overcomes acquired resistance to met-tyrosine kinase inhibitor in met-amplified lung cancer

Abstract Mesenchymal-epithelial transition factor receptor (MET) mediated signaling emerged as an important clinical target for the treatment of many cancer types including non-small cell lung cancer. MET amplification is prevalent in 2%~4% of primary lung cancer and 15%~50% of EGFR-mutated lung cancer resistant to EGFR inhibitors. Currently, MET-targeting drugs have been actively investigated in these MET-amplified lung cancers. However, the acquired resistance mechanism for MET-targeted therapy remains unknown. Our study aims to provide effective treatment for overcoming MET-TKI-acquired resistance in MET-amplified non-small cell lung cancer. To discover the resistance mechanism, we established the resistant cell line (PR-S2) by chronic exposure of MET-amplified lung cancer cells (H1993) to MET kinase inhibitor, PHA665752. Through high-throughput screening of a custom library of 276 compounds, we found that Aurora kinase B (AURKB) inhibitor, barasertib was more sensitive to the PR-S2 cells than H1993 cells. AURKB inhibition by barasertib induced apoptotic cell death with G2/M arrest and polyploid cell formation in the PR-S2 cells. The expression of p-AURKB was increased with no change in the expression of p-histone H3, a major product of AURKB in the PR-S2 cells. AURKB knockdown using multiple siRNA sequences increases the sensitivity to PHA665752 in the PR-S2 cells. In mRNA sequencing data analysis, we found the expression of signal transducer and activator of transcription 3 (STAT3) was elevated in the PR-S2 cells compared to the H1993 cells. We confirmed increased STAT3 expression in both protein and mRNA levels in the PR-S2 cells. p-STAT3 expression was decreased after barasertib treatment in the PR-S2 cells. However, in the immunoprecipitation assay, there was no direct interaction between AURKB and STAT3. To know the target of STAT3, we checked the mRNA expression of downstream genes of STAT3. The expression of BCL-2, one of the downstream targets of STAT3 and the central apoptosis regulator, was highly upregulated in the resistant cells. The siRNA-mediated knockdown of STAT3 expression decreased BCL2 expression in the PR-S2 cells, whereas not in the H1993 cells. Furthermore, we investigated apoptosis-related gene expressions and found that the BH3-only proapoptotic member of the BCL-2 protein family, BIM was significantly decreased in the PR-S2 cells. AURKB inhibition induced BIMEL accumulation and reduced p-BIM expression. Collectively, our study suggests that AURKB activation induces resistance to MET-TKI through anti-apoptosis mechanism with upregulation of STAT3/BCL2 axis in MET-amplified lung cancer cells. Citation Format: Yura Choi, Mihwa Hwang, Sunshin Kim, Jae J. Song, Youngjoo Lee. Targeting aurora kinase b overcomes acquired resistance to met-tyrosine kinase inhibitor in met-amplified lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 417.

Abstract 5235: The role of transient single MET inhibition in MET-amplified and EGFR-mutant nonsmall cell lung cancer resistant to EGFR inhibitors

Abstract Background: In recent clinical trials for EGFR-mutant lung cancer acquiring MET amplification as resistance to EGFR inhibitor, two-drug combination treatment showed promising anti-tumor activity. However, these combination treatments also resulted in increased toxicity with intolerability. Thus, this study evaluated the possible role of single MET inhibition in these distinct tumors. Methods: Using gefitinib-resistant MET-amplified lung cancer cells (HCC827GR), we tested the sensitivity to single MET inhibitor with various conditions. Supporting preclinical data with cell lines, the matched case reports and drug-sensitivity tests using patient-derived cells were presented. Furthermore, we chronically treated HCC827GR with single MET inhibitor to establish MET inhibitor-resistant cell line (HCC827GR_PR). And then, we explored putative resistance mechanisms of the HCC827GR_PR cells. Results: The HCC827GR cells were partially inhibited by single MET inhibitor regardless of gefitinib-free duration or drug exposure time. Three patients with stage IV MET-amplified and EGFR-mutant lung cancer resistant to EGFR inhibitor showed initially definite response to single crizotinib treatment but their disease progressed within two or three months. The copy number of MET gene in plasma circulating tumor DNA was decreased significantly during crizotinib treatment and not increased even after progression to crizotinib. In their post-crizotinib tumor, MET amplification was disappeared in fluorescence in situ hybridization test. In vitro drug test using patient-derived cells revealed the sensitivity to EGFR inhibitor was recovered after single crizotinib treatment. We established HCC827GR_PR resistant to single MET inhibitor. In the major clones of HCC827GR_PR cells, EGFR signaling pathway was reactivated and gefitinib successfully suppressed their survival. Furthermore, increased MET gene copy number or bypass activation of FGFR1 were observed as resistance mechanism in the minor clones of HCC827GR_PR cells. Conclusions: Single MET inhibitor could be effective but its efficacy could not durable in patients with MET-amplified and EGFR-mutant lung cancer resistant to EGFR inhibitors because these resistant cells might be subclones. A novel combination strategy is needed to get both long-term efficacy and less toxicity in these lung cancer patients. Citation Format: Youngjoo Lee, Yu-Ra Choi, Eun Hye Kang, Sunshin Kim, SeogYun Park, Ji-Youn Han. The role of transient single MET inhibition in MET-amplified and EGFR-mutant nonsmall cell lung cancer resistant to EGFR inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5235.

P59.02 Profile of Next-Generation Sequencing (NGS) on MET exon 14 Skipping Mutation and MET Amplification in Lung Cancer: A Calibration Project in China

MET is an oncogenic driver and becomes a significant therapeutic target in lung cancer. Detection of MET alterations is essential for clinical treatment decision-making. With the advancement of diagnostic technology, next-generation sequencing (NGS) becomes an option. However, it has limited probe design and coverage of specific regions of introns, and the quality of testing varies widely among different laboratories. Moreover, clinically defined cut-off values for MET amplification vary considerably and lack uniform criteria.

MET inhibition downregulates DR4 expression in MET-amplified lung cancer cells with acquired resistance to EGFR inhibitors through suppressing AP-1-mediated transcription

Death receptor 4 (DR4) is a cell surface protein that is generally thought to mediate apoptosis upon binding to its ligand named TRAIL. However, its contribution to apoptosis resistance has also been reported. MET (or c-MET) gene amplification represents an important mechanism for acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) against EGFR mutant non-small cell lung cancer (NSCLC). This study focuses on demonstrating the impact of MET inhibition on DR4 modulation in MET-amplified EGFR mutant NSCLC cell lines and the underlying mechanisms. Several MET inhibitors decreased DR4 levels in MET-amplified HCC827 cell lines resistant to EGFR-TKIs with no or limited effects on modulating DR5 levels, while increasing DR4 levels in HCC827 parental cells and other NSCLC cell lines. MET inhibitors did not affect DR4 stability, but decreased DR4 mRNA levels with suppression of AP-1-dependent DR4 promoter transactivation. Moreover, these inhibitors suppressed ERK and c-Jun phosphorylation accompanied with decreasing c-Jun levels. Hence, it is likely that MET inhibition downregulates DR4 expression in MET-amplified EGFR mutant NSCLC cells through suppressing AP-1-mediated DR4 transcription. Osimertinib combined with MET inhibition synergistically induces apoptosis in the MET-amplified EGFR mutant NSCLC cells accompanied with augmented DR4 reduction both in vitro and in vivo. Furthermore, MET inhibition combined with TRAIL enhanced killing of MET-amplified EGFR mutant HCC827/AR cells, but not HCC827 parental cells. These data collectively suggest that DR4 may possess an unrecognized anti-apoptotic function, contributing to apoptosis resistance under given conditions.

Characteristics and response to crizotinib in lung cancer patients with MET amplification detected by next-generation sequencing

ObjectivesMesenchymal-epithelial transition (MET) amplification is a rare gene alteration in lung cancer. The aim of this study was to investigate the clinical characteristics of MET amplification in lung cancer and the response to crizotinib by subsets of patients with MET amplification detected by next-generation sequencing (NGS). Patients and methodsWe collected NGS sequencing data for patients with MET amplification in our institution from January 2018 to April 2019. The efficacy of crizotinib in MET amplification was retrospectively analyzed. ResultsA total of 2694 patients received NGS tests, 3.27 % (82/2507) of patients had primary MET amplification, and acquired MET amplification accounted for 16.04 % (30/187) of re-biopsy patients. Only 19 patients received monotherapy with crizotinib. In survival analysis, ten patients with copy number greater than 4 (CN > 4) had longer median PFS (mPFS) (4.76 months; 95 %CI: 1.67–7.85 months) compared with other nine patients (CN ≤ 4) (2.10 months; 95 %CI: 1.53–2.68 months; P = 0.063), but failed to get a statistical significance. No significant differences were observed between median PFS (mPFS) of the patients with primary and acquired MET amplification (4.04 months vs 2.76 months; P = 0.310). ConclusionsPrimary and acquired MET amplification were detected in 3.27 % and 16.04 % of lung cancer patients, respectively. Patients with CN > 4 seemed to have longer PFS after crizotinib treatment. No significant differences in PFS were observed between patients with primary and acquired MET amplification.

1967P - Triple MET/SRC/PIM inhibition in MET addicted tumors

BackgroundMET addicted tumors are known to show very short response to single MET inhibition. In MET addicted cells, MET inhibition leads to activation of proviral integration site for Moloney murine leukemia virus-1 (PIM1). PIM1 and SRC can regulate the expression of receptor tyrosine kinases (RTKs), potentially being responsible of resistance to MET inhibition. We previously showed that the dual inhibition of MET and PIM1 with class I MET inhibitors and the pan-PIM inhibitor AZD1208, as well as MET and SRC (with dasatinib), is synergistic in MET addicted cell lines. MethodsWe evaluated the activity of class I and class II MET inhibitors, MET/SRC/CSF1R inhibitor TPX-02226 and pan-PIM inhibitors AZD1208 and PIM447 in four MET addicted cell lines: 2 MET amplified lung cancer cell lines (EBC1 and H1993), 1 MET exon 14 mutant cell line (Hs746T) and 1 MET exon 7-8 splicing variant cell line (E98). We assessed the effect of the dual inhibition of MET and PIM, and the triple inhibition of MET, SRC and PIM in cell viability by combining the TPX-02226 with the pan-PIM inhibitors. ResultsAll cell lines were sensitive to class I-II MET inhibitors (IC50s in nM range) except for cabozantinib in H1993. Only the MET amplified cell lines were sensitive to TPX-02226. All cell lines were resistant to PIM inhibition. The combination of class I MET inhibitors with PIM447 showed stronger synergism in the MET amplified cell lines, compared to the combination with AZD1208. Class I-II MET inhibitors and PIM inhibitor combination showed only additive effect in exon 7-8 cell line E98, while the combination of TPX-02226 and PIM447 was strongly synergistic. ConclusionsWe identified potential role of PIM inhibition in MET amplified tumors. Even though single agent TPX-02226, AZD1208 or PIM447 showed no activity and dual MET/PIM inhibition showed only additive effect, the triple inhibition of MET, SRC and PIM was strongly effective in MET exon 7-8 splicing variant, suggesting a crucial role of SRC-PIM interaction in this still not well recognized MET addicted tumor. Further investigation on this triple inhibition is ongoing in MET addicted cell lines and role of co-presence of MET and PIM1 and/or SRC alterations in tumor samples. Legal entity responsible for the studyIGTP, Germans Trias i Pujol Research Institute, Badalona, Barcelona, Spain. FundingFundació Obra Social “La Caixa”. DisclosureJ.J. Cui: Leadership role, Officer / Board of Directors: TP Therapeutics. N. Karachaliou: Officer / Board of Directors: Merck KgaA. All other authors have declared no conflicts of interest.

Abstract 1732: Switch to MET oncogene dependence as a novel mechanism of drug resistance in EGFR-mutant lung cancer

Abstract Introduction: Non-small cell lung cancers harboring sensitizing gain-of-function mutations in the epidermal growth factor receptor (EGFR) are treated with small molecule EGFR kinase inhibitors including erlotinib and osimertinib. Unfortunately, the clinical efficacy of these inhibitors is limited by progression to drug resistance. Genomic amplification of the hepatocyte growth factor receptor (MET) is a prevalent mechanism of resistance to clinical EGFR inhibitors. EGFR mutant lung cancers with MET amplification are co-dependent on signaling through EGFR and MET kinases, whereby either oncogene can phosphorylate the EGFR family receptor ERBB3, activating downstream signaling and promoting survival. These EGFR/MET co-dependent cancers are characteristically resistant to treatment with individual drugs, but retain sensitivity to combination treatment with EGFR and MET inhibitors. Here, we describe three patient-derived EGFR mutant, MET amplified lung cancer models that exhibit a switch away from EGFR, to MET oncogene dependence. Methods: We established and characterized patient-derived lung cancer cell lines and xenograft models harboring concurrent EGFR activating mutations (EGFR L858R or EGFR Del19) and genomic MET copy number gain. Three patient-derived models—DFCI81, DFCI161, and DFCI307—were developed from pleural effusion and core biopsy specimens of patients whose tumors progressed on erlotinib. Clinical samples were immediately sorted and stably cultured in vitro or engrafted into immunodeficient mice. Results: DFCI81, DFCI161, and DFCI307 cell lines and xenograft models retained mutant EGFR expression, but exhibited resistance to clinical EGFR inhibitors and sensitivity to single-agent c-MET inhibitors, including crizotinib and savolitinib. Comparing our EGFR-mutant and MET-dependent cell lines to EGFR/MET-codependent models, we observed that EGFR expression was significantly reduced in our models compared to controls. Ectopic overexpression of EGFR Del19 and EGFR L858R in DFCI81 and DFCI161 cell lines, respectively, was sufficient to confer crizotinib resistance and induce EGFR/MET co-dependency. We demonstrated that MET-mediated ERBB3 phosphorylation drives downstream PI3K activation to promote cell proliferation and survival in DFCI81 and DFCI161 cells. In these contexts, ERBB3 reactivation by recombinant ligand treatment was sufficient to induce EGFR-mediated ERBB3 activation, conferring resistance to single-agent crizotinib treatment. Conclusions: We have identified and characterized three patient-derived models of treatment refractory EGFR mutant lung cancer that exhibit a switch to MET oncogene dependency. Clinically, we predict a subset of EGFR mutant, MET-dependent tumors exists, and can be identified de novo by a reduced EGFR to MET expression ratio: a potential biomarker predictive of sensitivity to single-agent MET inhibition. Citation Format: Pinar Ö. Eser, Raymond M. Paranal, Michael J. Poitras, Man Xu, Stephen Wang, Atsuko Ogino, Jihyun Choi, Pavlos Missios, Heidi M. Haikala, Jieun Son, Mika Lin, Masahiko Yanagita, Prafulla C. Gokhale, George Q. Daley, Pasi A. Jänne. Switch to MET oncogene dependence as a novel mechanism of drug resistance in EGFR-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1732.

Cyclosporine A sensitizes lung cancer cells to crizotinib through inhibition of the Ca2+/calcineurin/Erk pathway.

BackgroundCrizotinib has potent anti-tumor activity in patients with advanced MET-amplified non-small cell lung cancer (NSCLC). However, the therapeutic effect is still not satisfying. Thus, developing approaches that improve the efficacy of crizotinib remains a significant challenge.MethodsMET-amplified NSCLC cell lines were treated with crizotinib and cyclosporine A (CsA). Cell viability was determined by MTS assay. The changes of apoptosis, cell cycle and calcineurin-Erk pathways were assessed by western blot. Xenograft mouse model, primary human NSCLC cells and hollow fiber assays were utilized to confirm the effects of CsA.FindingsWe demonstrated that CsA significantly increased the anti-tumor effect of crizotinib on multiple MET-amplified NSCLC cells in vitro and in vivo. Mechanistically, crizotinib treatment led to the activation of Ca2+-calcineurin (CaN)-Kinase suppressor of Ras 2 (KSR2) signaling, resulting in Erk1/2 activation and enhanced survival of cancer cells. CsA effectively blocked CaN-KSR2-Erk1/2 signaling, promoting crizotinib-induced apoptosis and G2/M arrest. Similarly, pharmacologic or genetic inhibition of Erk1/2 also enhanced crizotinib-induced growth inhibition in vitro. Xenograft studies further confirmed that CsA or Erk1/2 inhibitor PD98059 enhanced the anti-cancer activity of crizotinib through inhibition of CaN-Erk1/2 axis. The results were also validated by primary human NSCLC cells in vitro and hollow fiber assays in vivo.InterpretationThis study provides preclinical evidences that combination therapy of CsA and crizotinib is a promising approach for targeted treatment of MET-amplified lung cancer patients.FundThis work was supported by the National Natural Science Foundation of China, the Key Projects of Natural Foundation of Zhejiang Province, the Ten thousand plan youth talent support program of Zhejiang Province, the Zhejiang Natural Sciences Foundation Grant, and the Zhejiang medical innovative discipline construction project-2016.

Correlation of MET gene amplification and TP53 mutation with PD-L1 expression in non-small cell lung cancer.

BackgroundThe role of MET amplification in lung cancer, particularly in relation to checkpoint inhibition and EGFR WT, has not been fully explored. In this study, we correlated PD-L1 expression with MET amplification and EGFR, KRAS, or TP53 mutation in primary lung cancer.MethodsIn this retrospective study, tissue collected from 471 various tumors, including 397 lung cancers, was tested for MET amplification by FISH with a MET/centromere probe. PD-L1 expression was evaluated using clone SP142 and standard immunohistochemistry, and TP53, KRAS, and EGFR mutations were tested using next generation sequencing.ResultsOur results revealed that PD-L1 expression in non-small cell lung cancer is inversely correlated with EGFR mutation (P=0.0003), and positively correlated with TP53 mutation (P=0.0001) and MET amplification (P=0.004). Patients with TP53 mutations had significantly higher MET amplification (P=0.007), and were more likely (P=0.0002) to be EGFR wild type. There was no correlation between KRAS mutation and overall PD-L1 expression, but significant positive correlation between PD-L1 expression and KRAS with TP53 co-mutation (P=0.0002). A cut-off for the ratio of MET: centromere signal was determined as 1.5%, and 4% of lung cancer patients were identified as MET amplified.ConclusionsThis data suggests that in lung cancer both MET and TP53 play direct roles in regulating PD-L1 opposing EGFR. Moreover, KRAS and TP53 co-mutation may cooperate to drive PD-L1 expression in lung cancer. Adding MET or TP53 inhibitors to checkpoint inhibitors may be an attractive combination therapy in patients with lung cancer and MET amplification.

414 - Acquired resistance mechanisms to INC280, a MET inhibitor in MET-amplified lung cancer cells

414 - Acquired resistance mechanisms to INC280, a MET inhibitor in MET-amplified lung cancer cells

Abstract 873: A novel MET-EGFR bispecific antibody LY3164530 shows advantage over combining MET and EGFR antibodies in tumor inhibition and overcome resistance

Abstract The epidermal growth factor receptor (EGFR) and the mesenchymal-epithelial transition factor (MET) are receptor tyrosine kinases that each plays a key role in cancer signaling. Co-expression and activation of MET and EGFR are found in a number of tumor types, including non-small cell lung, colorectal, gastric, and head and neck cancers (Nanjo et al. 2013). Blocking one receptor tends to up-regulate the other, leading to resistance to single-agent treatment (Engelman et al. 2007). Amplification of MET and/or high levels of HGF expression has been observed in non-small cell lung cancer (NSCLC) patients with intrinsic or acquired resistance to tyrosine kinase inhibitors of EGFR (Engelman et al. 2007; Yano et al. 2011). Conversely, MET-amplified lung cancer cells exposed to MET-inhibiting agents for a prolonged period develop resistance via the EGFR pathway (McDermott et al. 2010). The crosstalk between the MET and EGFR pathways suggests that dual inhibition of these targets may lead to improved outcomes for patients with MET- and EGFR-positive cancers, and that simultaneous inhibition may overcome or delay resistance compared to the blockade of just a single pathway. LY3164530 is an engineered mAb-scFv bispecific antibody that consists of an immunoglobulin G4 (IgG4) antibody to MET and a single-chain variable fragment (scFv) to EGFR fused to the N-terminus of each heavy chain (HC). LY3164530 binds to extracellular domains of MET and EGFR with high affinity and inhibits signaling via both MET and EGFR receptors by blocking ligand binding and internalizing and degrading both receptors. In tumor cells, it binds and co-immunoprecipitates both receptors. LY3164530 has increased avidity binding to MET in cells expressing higher level of EGFR. This increased avidity binding leads to better neutralization of HGF compared to parental MET antibody in these cells. Surprisingly, LY3164530 has superior activity in internalizing/degrading EGFR (wild type and mutant forms) in vitro and in vivo relative to the combination of LY2875358 (i.e., emibetuzumab) and cetuximab in cells expressing high MET and EGFR. In addition, LY3164530 has superior activity in overcoming HGF-mediated resistance to erlotinib, gefitinib, lapatinib, or vemurafenib as compared to the combination of individual monoclonal antibodies targeting these receptors in cell-based assays. In vivo, administration of LY3164530 results in dose-dependent antitumor activity in multiple cell line-derived NSCLC and gastric xenografts. The antitumor activity of LY3164530 is equivalent, and in some cases superior to the combination of emibetuzumab and cetuximab in NSCLC and gastric tumor models. The Phase 1 study with LY3164530 is on-going (NCT02221882). Citation Format: Ling Liu, Wei Zeng, Marcio Chedid, Yi Zeng, Sheng-hung Tschang, Yu Tian, Ying Tang, Jirong Lu. A novel MET-EGFR bispecific antibody LY3164530 shows advantage over combining MET and EGFR antibodies in tumor inhibition and overcome resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 873.

Abstract 4999: Signaling-associated complexes to define targetable vulnerabilities in lung cancer

Abstract Lung cancer is the leading cause of cancer related death in the U.S. Despite successes targeting tyrosine kinase drivers such as EGFR and EML4-ALK, identification of patients who will benefit from emerging targeted therapy regimens remains a challenge. This is exemplified by the disappointing results in the recent Phase III evaluation of ornatuzumab in combination with erlotinib in advanced non-small cell lung cancer (NCT01456324), targeting both MET and EGFR in biomarker-defined patient populations. We have previously shown that proximity ligation assay (PLA) technology can be harnessed to evaluate EGFR pathway activation by annotating the presence of EGFR in complex with its major signaling adaptor GRB2. Presence of these “signaling-associated complexes” correlates with EGFR activity, reveals erlotinib pharmacodynamics and associates with improved outcomes to EGFR-directed therapies. Here, we show that PLA technology can also be harnessed to assess MET signaling activity in lung cancer. We have found that MET is constitutively associated with GRB2 in MET-amplified lung cancer cell lines and this interaction is abrogated by the MET-specific tyrosine kinase inhibitor PHA665,752 (MET TKI) as measured by biochemical approaches and PLA. In parallel experiments, siRNA-mediated knockdown of GRB2 and 100nM MET TKI both lead to dramatic reductions in cell viability, indicating that this interaction is critical in MET-driven cells. Conversely, GRB2 knockdown has no effect in MET-amplified H1648 cells which are also completely resistant to MET TKI. In MET¬-amplified patient-derived xenograft models of lung cancer we observe MET:GRB2 complexes in regions that also stain strongly using pMET(Y1234/5) immunohistochemistry (IHC). However, only a subset of regions exhibiting strong staining for total MET by IHC had MET:GRB2 complexes or pMET. Surprisingly, we have observed a very low frequency (2%) of lung adenocarcinoma patients (N = 169) with MET:GRB2 signaling complexes, even among high MET-expressing tumors, defined by MET IHC. These data suggest that MET:GRB2 is critical in the amplified setting, but may be less important in stromal-induced MET signaling. Consistent with this, GRB2 knockdown in non-amplified A549 and H292 cells fails to impair HGF-induced increases in pAKT and pERK. We are currently conducting mass spectrometry experiments to decipher differences between MET complexes in the amplified vs. ligand-stimulating setting and updated results will be presented. We suggest that only a subset of high MET-expressing cells has evidence of MET in complex with GRB2, potentially explaining the relatively low response rates observed with MET-directed therapies in lung cancer. We believe our approach of combining biochemical, PLA and mass spectrometry-based techniques will also be applicable to the development of improved biomarker strategies for newly emerging targets such as FGFR1. Citation Format: Matthew Smith, Thomas Licata, Yun Bai, Guolin Zhang, Vincent Vuaroqueaux, Heinz-Herbert Fiebig, Eric B. Haura. Signaling-associated complexes to define targetable vulnerabilities in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4999. doi:10.1158/1538-7445.AM2015-4999

MET amplification status in therapy-naïve adeno- and squamous cell carcinomas of the lung.

MET is a potential therapeutic target in lung cancer and both MET tyrosine kinase inhibitors and monoclonal antibodies have entered clinical trials. MET signaling can be activated by various mechanisms, including gene amplification. In this study, we aimed to investigate MET amplification status in adeno- and squamous cell carcinomas of the lung. We propose clearly defined amplification scores and provide epidemiologic data on MET amplification in lung cancer. We evaluated the prevalence of increased MET gene copy numbers in 693 treatment-naïve cancers by FISH, defined clear cutoff criteria, and correlated FISH results to MET IHC. Two thirds (67%) of lung cancers do not have gains in MET gene copy numbers, whereas 3% show a clear-cut high-level amplification (MET/centromer7 ratio ≥2.0 or average gene copy number per nucleus ≥6.0 or ≥10% of tumor cells containing ≥15 MET copies). The remaining cases can be subdivided into intermediate- (6%) and low-level gains (24%). Importantly, MET amplifications occur at equal frequencies in squamous and adenocarcinomas without or with EGFR or KRAS mutations. MET amplification is not a mutually exclusive genetic event in therapy-naïve non-small cell lung cancer. Our data suggest that it might be useful to determine MET amplification (i) before EGFR inhibitor treatment to identify possible primary resistance to anti-EGFR treatment, and (ii) to select cases that harbor KRAS mutations additionally to MET amplification and, thus, may not benefit from MET inhibition. Furthermore, our study provides comprehensive epidemiologic data for upcoming trials with various MET inhibitors.