MET Oncogene Research Articles

The MET Oncogene: Thirty Years of Insights into Molecular Mechanisms Driving Malignancy.

The discovery and subsequent research on the MET oncogene's role in cancer onset and progression have illuminated crucial insights into the molecular mechanisms driving malignancy. The identification of MET as the hepatocyte growth factor (HGF) receptor has paved the path for characterizing the MET tyrosine kinase activation mechanism and its downstream signaling cascade. Over the past thirty years, research has established the importance of HGF/MET signaling in normal cellular processes, such as cell dissociation, migration, proliferation, and cell survival. Notably, genetic alterations that lead to the continuous activation of MET, known as constitutive activation, have been identified as oncogenic drivers in various cancers. The genetic lesions affecting MET, such as exon skipping, gene amplification, and gene rearrangements, provide valuable targets for therapeutic intervention. Moreover, the implications of MET as a resistance mechanism to targeted therapies emphasize the need for combination treatments that include MET inhibitors. The intriguing "flare effect" phenomenon, wherein MET inhibition can lead to post-treatment increases in cancer cell proliferation, underscores the dynamic nature of cancer therapeutics. In human tumors, increased protein expression often occurs without gene amplification. Various mechanisms may cause an overexpression: transcriptional upregulation induced by other oncogenes; environmental factors (such as hypoxia or radiation); or substances produced by the reactive stroma, such as inflammatory cytokines, pro-angiogenic factors, and even HGF itself. In conclusion, the journey to understanding MET's involvement in cancer onset and progression over the past three decades has not only deepened our knowledge, but has also paved the way for innovative therapeutic strategies. Selective pharmacological inactivation of MET stands as a promising avenue for achieving cancer remission, particularly in cases where MET alterations are the primary drivers of malignancy.

Current status of molecular diagnostics for lung cancer.

The management of lung cancer (LC) requires the analysis of a diverse spectrum of molecular targets, including kinase activating mutations in EGFR, ERBB2 (HER2), BRAF and MET oncogenes, KRAS G12C substitutions, and ALK, ROS1, RET and NTRK1-3 gene fusions. Administration of immune checkpoint inhibitors (ICIs) is based on the immunohistochemical (IHC) analysis of PD-L1 expression and determination of tumor mutation burden (TMB). Clinical characteristics of the patients, particularly age, gender and smoking history, significantly influence the probability of finding the above targets: for example, LC in young patients is characterized by high frequency of kinase gene rearrangements, while heavy smokers often have KRAS G12C mutations and/or high TMB. Proper selection of first-line therapy influences overall treatment outcomes, therefore, the majority of these tests need to be completed within no more than 10 working days. Activating events in MAPK signaling pathway are mutually exclusive, hence, fast single-gene testing remains an option for some laboratories. RNA next-generation sequencing (NGS) is capable of detecting the entire repertoire of druggable gene alterations, therefore it is gradually becoming a dominating technology in LC molecular diagnosis.

NGS and Sequence Analysis with Biopython for Prospective Brain Cancer Therapeutic Studies

Abstract: Malignant primary brain tumour in adults and in analysis driven by variousgenomic alterations. Next generation sequencing (NGS) provides about the geneticlandscape of tumours and might detect targetable mutations. Genomic DNA wasextracted from blood samples or tumor tissues number of biological pathways incancer involve the products of genes that are not mutated, but epigenetically whenwe found regulated, for example by altered transcription factor availability,repressor activity or gene methylation receptor-type protein tyrosine phosphatases(RPTPs) comprises PTPRZ and PTPRG,primary human glioblastomas suggested aclose association between PTPRZ1 (human PTPRZ) expression and cancerstemness. The functional roles of PTPRZ activity in glioma that sphere-forming cellsin human U251 glioblastoma cell lines showed high expression levels of PTPRZ-B.Potential PTPRZ1-MET fusion, that is caused when PTPRZ1 and the MET oncogene are fused in 8% to 16% of gliomas and is linked to a less favorable outcome nevertheless is also a promising biomarker for kinase inhibitor therapy. In accordance to the studies that are currently available, receptor-type tyrosine-protein phosphatase zeta (PTPRZ1) is found in various tumour tissues and plays a role in migration, cell proliferation, the EMT and adhesions, as well as treatment resistance by binding with a variety of substances.

Why loss of Y? A pan-cancer genome analysis of tumors with loss of Y chromosome

Loss of the Y chromosome (LoY) is frequently observed in somatic cells of elderly men. However, LoY is highly increased in tumor tissue and correlates with an overall worse prognosis. The underlying causes and downstream effects of LoY are widely unknown. Therefore, we analyzed genomic and transcriptomic data of 13 cancer types (2375 patients) and classified tumors of male patients according to loss or retain of the Y chromosome (LoY or RoY, average LoY fraction: 0.46). The frequencies of LoY ranged from almost absence (glioblastoma, glioma, thyroid carcinoma) to 77% (kidney renal papillary cell carcinoma). Genomic instability, aneuploidy, and mutation burden were enriched in LoY tumors. In addition, we found more frequently in LoY tumors the gate keeping tumor suppressor gene TP53 mutated in three cancer types (colon adenocarcinoma, head and neck squamous carcinoma, lung adenocarcinoma) and oncogenes MET, CDK6, KRAS, and EGFR amplified in multiple cancer types. On the transcriptomic level, we observed MMP13, known to be involved in invasion, to be up-regulated in LoY of three adenocarcinomas and down-regulation of the tumor suppressor gene GPC5 in LoY of three cancer types. Furthermore, we found enrichment of a smoking-related mutation signature in LoY tumors of head and neck and lung cancer. Strikingly, we observed a correlation between cancer type-specific sex bias in incidence rates and frequencies of LoY, in line with the hypothesis that LoY increases cancer risk in males. Overall, LoY is a frequent phenomenon in cancer that is enriched in genomically unstable tumors. It correlates with genomic features beyond the Y chromosome and might contribute to higher incidence rates in males.

Abstract 1104: Loss of SUV420H2 drives EGFR inhibitor resistance in EGFR mutant NSCLC cell lines

Abstract Although important advancements have been achieved in the treatment of cancer, lung cancer is still the leading cause of cancer-related deaths in both men and women, with an estimated 1.6 million deaths each year. Non-small cell lung cancer (NSCLC), which makes up ~85% of lung cancer cases, is mainly treated with radiotherapy, chemotherapies, and targeted agents. Epidermal growth factor receptor (EGFR) is commonly mutated in NSCLC patients and leads to increased proliferation and cell survival. The standard-of-care treatment for patients harboring activating mutations in EGFR is treatment with tyrosine kinase inhibitors (TKI), such as erlotinib. While tumors initially respond to TKIs, after 1-2 years most of the patients develop resistance. In ~60% of patients, TKI resistance is the results of a secondary mutation in EGFR, leading to sustained receptor activation. Whereas, in the remaining 20%, tumors turn on bypass tracks-signals to overcome inhibition of the EGFR pathway. However, in 15-20% of the cases the mechanisms underlying resistance are unknown. Hence, there is a need to identify additional mechanisms involved in TKIs resistance. Our laboratory performed a genome-wide CRISPR Cas9 knock-out screen to identify genes that when knocked-out would drive erlotinib resistance. Fold enrichment analysis of sgRNAs, identified the protein coding gene SUV420H2 as a top candidate. SUV420H2 is a histone methyltransferase that trimethylates H4K20 (H4K20me3), enabling the establishment of constitutive and facultative heterochromatin. The process by which SUV420H2 is reduced in tumors is unknown, yet data from human samples suggests that SUV420H2 is globally downregulated in NSCLC. Additionally, loss of H4K20me3, the modification made by SUV420H2, in preneoplasia influences prognosis of NSCLC, indicating that loss of SUV420H2 function is a crucial mechanism in carcinogenesis. SUV420H2 expression has historically been associated with generating constitutive heterochromatin. However, more recently SUV420H2 was found to silence euchromatin regions. Our recent data suggests that SUV420H2 leads to reduced transcription of the oncogene MET by silencing the oncogenic long non-coding RNA LINC01510 upstream of MET. These findings led us to hypothesize that loss of SUV420H2 leads to erlotinib resistance through to upregulation of oncogenes including MET. Moreover, we aim to identify genes that are dynamically regulated by SUV420H2 using chromatin immunoprecipitation in NSCLC cell lines to further understand SUV420H2-mediated gene regulation and its role in TKIs resistance. Citation Format: Alejandra Agredo, Arpita Pal, Nadia Lanman, Jihye Son, Andrea L. Kasinski. Loss of SUV420H2 drives EGFR inhibitor resistance in EGFR mutant NSCLC cell lines [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 1104.

Genomic and clinical characteristics of MET alterations in solid tumors among the 10,475 Chinese patients.

3115 Background: Somatic alterations of the MET oncogene are emerging as an attractive target in human cancers, thus, understanding the molecular epidemiology of MET alterations is essential. While the occurrence of MET exon 14 skipping mutations (MET ex14) in US lung cancer patients is well defined, it is not widely published for Chinese patients. In addition, reports on the occurrence of METex14 outside lung cancer and other clinically relevant MET alterations across all cancers are limited. Methods: The MET ex14 alterations and amplification data of 10475 Chinese cancer patients from 16 types of cancer were obtained in Acronmed database, including non-small cell lung cancer (NSCLC, n = 5719), Hepatocellular carcinoma (HCC, n = 511), colorectal cancer (CRC, n = 1779), renal cell carcinoma (RCC, n = 1169), Gastric carcinoma (GC, n = 679), etc. Genomic profiling of DNA was performed through a next-generation sequencing. Results: Of all pan-cancer patients, 141 cases (1.3%) with MET alterations were identified, including MET Amp (0.9%) and MET ex14 (0.7%). Compared to Western population (̃3%), the frequency of MET alterations is much lower in Chinese cancer patients (1.3%). MET Amp were most commonly found in HCC (1.7%), GC (1.3%), NSCLC (0.7%), RCC (0.7%), CRC (0.2%). MET ex14 occurred most in NSCLC (0.5%), HCC (0.3%), CRC (0.2%). HCC were significantly to have MET Amp than NSCLC (P = 0.01), while MET ex14 mutations were more likely to be observed in NSCLC (p = 0.02). We further analyzed the MET mutation characteristics of NSCLC. The most frequently co-mutated genes in MET ex14 cohort were TP53 (48%,15/31), KMT2B (23%,7/31) and EGFR (16%, 5/31). Except for TP53 (65%,22/34) and EGFR (56%,19/34) mutations. Interestingly,other gene mutations were rare in the patients with MET Amp. In addition, MET ex14 patients showed significant lower EGFR mutation comparing to the MET amp patients (P = 0.002). Conclusions: Our study demonstrated a landscape of MET alterations among the Chinese population. MET mutations occurs in a variety of solid tumors, indicating that these patients may benefit from MET inhibitors.

Advanced nccRCC: what therapeutic options in 2022?

Non-clear-cell renal cell carcinomas (nccRCC) represent around 25% of all renal cancers and are a very heterogeneous group of tumours in terms of both biological features and prognosis. Papillary renal cell carcinomas (pRCC) are the most frequent subtype with 15% to 20% of all kidney cancers. Improved biological knowledge of these tumours has led to better identification of each subtype. Among pRCC, some exhibit mutations of the MET oncogene and others mutations of the gene coding for fumarate hydratase. The management of nccRCC, in particular the pRCC subtype, has evolved considerably in recent times, spearheaded by the advent of targeted therapies including anti-angiogenics but also new immunotherapy agents. Several studies have in the last few years prompted a new standard of care for these nccRCC. We propose to present throughout this article the latest available efficacy data on different compounds assessed in the treatment of the most frequent nccRCC, including the pRCC, chromophobe carcinoma, collecting duct carcinoma, MiT family translocation renal cell carcinoma and renal medullary carcinoma subtypes.

HOA-DN30: a highly effective humanized single-arm MET antibody inducing remission of ‘MET-addicted’ cancers

BackgroundThe tyrosine kinase receptor encoded by the MET oncogene is a major player in cancer. When MET is responsible for the onset and progression of the transformed phenotype (MET-addicted cancers), an efficient block of its oncogenic activation results in potent tumor growth inhibition.MethodsHere we describe a molecular engineered MET antibody (hOA-DN30) and validate its pharmacological activity in MET-addicted cancer models in vitro and in vivo. Pharmacokinetics and safety profile in non-human primates have also been assessed.ResultshOA-DN30 efficiently impaired MET activation and the intracellular signalling cascade by dose and time dependent removal of the receptor from the cell surface (shedding). In vitro, the antibody suppressed cell growth by blocking cell proliferation and by concomitantly inducing cell death in multiple MET-addicted human tumor cell lines. In mice xenografts, hOA-DN30 induced an impressive reduction of tumor masses, with a wide therapeutic window. Moreover, the antibody showed high therapeutic efficacy against patient-derived xenografts generated from MET-addicted gastric tumors, leading to complete tumor regression and long-lasting effects after treatment discontinuation. Finally, hOA-DN30 showed a highly favorable pharmacokinetic profile and substantial tolerability in Cynomolgus monkeys.ConclusionshOA-DN30 unique ability to simultaneously erase cell surface MET and release the ‘decoy’ receptor extracellular region results in a paramount MET blocking action. Its remarkable efficacy in a large number of pre-clinical models, as well as its pharmacological features and safety profile in non-human primates, strongly envisage a successful clinical application of this novel single-arm MET therapeutic antibody for the therapy of MET-addicted cancers.

EPCO-21. CORE REGULATORY CIRCUIT TRANSCRIPTION FACTORS DRIVE EXPRESSION FROM HIGH LEVEL AMPLICONS IN PEDIATRIC HIGH-GRADE GLIOMAS

Abstract BACKGROUND Pediatric High-Grade Gliomas (pHGGs) show recurrent high-level amplifications around the oncogenes MET, MYCN and EGFR. However what drives expression of the oncogenes from these amplicons remains unclear. We aim to discover enhancers on these amplicons that are responsible for oncogene expressions and the core regulatory transcription factors (TFs) they bind. METHOD Using RNA-seq from 12 pHGG cell lines, we identified groups of high and low-expressing pHGG lines for MET, MYCN and EGFR. We then compared the H3K27Ac ChIP-seq between the two groups using diffbind. This allowed us to identify statistically significant peaks that are differentially activated in the oncogene-high v.s. oncogene-low expressing groups. Additionally, we overlapped the positions of these candidate oncogene enhancers with the regions that are recurrently incorporated into high-level amplicons based on published whole genome sequencing data. Using a previously defined set of core regulatory TFs we determined which TF binds the amplified oncogene enhancers and could be driving oncogenic expressions of MET, MYCN and EGFR in pHGGs. RESULTS We identify 3 cell lines for both the high- and low-expressing groups for each oncogene. Cell lines with high expression of the oncogene showed distinct enhancers with significant enrichment in H3K27Ac compared to the cell lines with low expression for each oncogene. Of all enhancers with enrichment high oncogene expression groups those with binding sites for known pHGG core regulatory circuit TF were preferentially incorporated into the high-level amplicons of the oncogene. We also identified core TFs that bind enhancers for MYCN, EGFR and MET as well as core TFs that are unique to a single oncogene. CONCLUSION We identified candidate core transcription factor that drives expression of multiple oncogenes in pHGG. These could serve as a potential novel therapeutic target for pHGGs with addiction to MYCN or RTK signaling.

A receptor-antibody hybrid hampering MET-driven metastatic spread

BackgroundThe receptor encoded by the MET oncogene and its ligand Hepatocyte Growth Factor (HGF) are at the core of the invasive-metastatic behavior. In a number of instances genetic alterations result in ligand-independent onset of malignancy (MET addiction). More frequently, ligand stimulation of wild-type MET contributes to progression toward metastasis (MET expedience). Thus, while MET inhibitors alone are effective in the first case, combination therapy with ligand inhibitors is required in the second condition.MethodsIn this paper, we generated hybrid molecules gathering HGF and MET inhibitory properties. This has been achieved by ‘head-to-tail’ or ‘tail-to-head’ fusion of a single chain Fab derived from the DN30 MET antibody with a recombinant ‘ad-hoc’ engineered MET extracellular domain (decoyMET), encompassing the HGF binding site but lacking the DN30 epitope.ResultsThe hybrid molecules correctly bind MET and HGF, inhibit HGF-induced MET downstream signaling, and quench HGF-driven biological responses, such as growth, motility and invasion, in cancer cells of different origin. Two metastatic models were generated in mice knocked-in by the human HGF gene: (i) orthotopic transplantation of pancreatic cancer cells; (ii) subcutaneous injection of primary cells derived from a cancer of unknown primary. Treatment with hybrid molecules strongly affects time of onset, number, and size of metastatic lesions.ConclusionThese results provide a strategy to treat metastatic dissemination driven by the HGF/MET axis.

Abstract 3407: Anti-tumor activity of tepotinib in orthotopic models of lung cancer patient-derived brain metastases with MET amplification

Abstract Background Patients with advanced non-small cell lung cancer (NSCLC) harboring oncogenic MET alterations, such as MET exon 14 skipping or MET amplification (MET amp), benefit from MET tyrosine kinase inhibitor (TKI) treatment. Brain metastases are common in patients with advanced NSCLC. In preclinical studies in rats, tepotinib had relatively high binding to brain tissue (fu br=0.4%, fu pl=4%) and free tepotinib concentrations in brain were 25% of the concentrations found in plasma (Kp u,u=0.25). In this study we investigated the efficacy of the MET TKIs tepotinib, capmatinib, savolitinib and crizotinib in mice implanted orthotopically with MET-dependent tumor explants derived from human NSCLC brain metastases. Methods The antitumor activity of tepotinib (30 mg/kg qd) was investigated in a screen of 21 subcutaneous lung cancer patient-derived xenograft (PDX) models of brain metastases grown in NOD-SCID mice (n=1/model). 20 of these tumors were retrospectively analyzed for cancer-specific mutations, gene copy number (GCN) and gene expression by Nanostring. 2 responding models, both found to harbor MET amp, were tested again subcutaneously in NOD-SCID mice treated with vehicle or tepotinib (125 mg/kg qd; n=5). Both models were orthotopically implanted into brains of NOD-SCID mice and tumor growth was monitored by MRI. Established tumors were treated with MET TKIs (n=10) tepotinib (125 mg/kg qd), capmatinib (30 mg/kg bid), savolitinib (60 mg/kg qd) or crizotinib (50 mg/kg qd). Results 2 of 21 subcutaneous PDX tumors (LU5406 and LU5349) regressed in response to tepotinib treatment. When implanted subcutaneously again into mice, both models regressed completely upon treatment with tepotinib. Molecular profiles revealed that these were the only tumors of the 20 models investigated by Nanostring that had MET amp (high-level increase in MET GCN of >10). Both models grew orthotopically when implanted into brains of mice. Contrast-weighted MRI indicated regions with intact and disrupted blood–brain barrier (BBB) in all implanted tumors. Tumor regression of orthotopic LU5406 tumors was observed with all MET TKIs (% median tumor volume changes [%TV]: tepotinib, –63%; capmatinib, –24%; savolitinib, –38%; crizotinib, –27%). In the orthotopic LU5349 tumors, treatment with crizotinib or savolitinib led to growth inhibition (%TV: +88% and –13%, respectively), whereas tepotinib and capmatinib induced tumor regression (%TV: –84% and –63%, respectively). Conclusions The heterogenous pattern of regions with intact and disrupted BBB in the orthotopic brain metastases models is thought to mirror the clinical situation. Tepotinib was efficacious in the two MET-driven orthotopic brain metastases PDX tumors characterized by pronounced tumor regression and may be relevant to patients with brain metastases. Relationships between efficacy and BBB leakiness will be discussed. Citation Format: Manja Friese-Hamim, Anderson Clark, Lindsey Crowley, Christof Reusch, Olga Bogatyrova, Claudia Wilm, Hong Zhang, Timothy Crandall, Jing Lin, Jianguo Ma, David Bachner, Jürgen Schmidt, Martin Schaefer, Christopher Stroh. Anti-tumor activity of tepotinib in orthotopic models of lung cancer patient-derived brain metastases with MET amplification [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 3407.

Abstract B136: Highly accurate DNA-based detection of MET exon 14 skipping mutations in non-small cell lung cancer and clinical response upon targeted treatment

Abstract Background: Approximately 1.6%-5.7% of non-small cell lung cancers (NSCLC) are described to be driven by oncogenic MET exon 14 skipping mutations (METex14del). Although currently no therapy is registered specifically against METex14del, Phase I/II clinical trials and named patient based programs with cMET inhibitors show promising results. RNA-based analysis seems most optimal for METex14del detection, because theoretically, it would capture all MET exon 13-exon 15 fusions, regardless of the underlying DNA changes. However, acquiring sufficient RNA is often problematic. An alternative is DNA-based analysis, but commercially available DNA-based panels only detect up to 63% of known METex14del alterations. The goal of this study is to describe an optimized DNA-based diagnostic test for METex14del, and to report the histologic and clinical features of a real world METex14del non-squamous NSCLC patient cohort, including follow-up of patients treated with cMET-targeted therapy and consequent resistance mechanisms to cMET inhibition. Methods: Routine diagnostic pathology NSCLC FFPE specimens were investigated by a custom-made DNA-based targeted amplicon-based next generation sequencing (NGS) panel which includes 4 amplicons for METex14del detection. Retrospectively, histopathological characteristics and clinical follow up were investigated for advanced NSCLC with METex14del. We only present data of patients receiving crizotinib in a named patient based program (Pfizer Oncology). Results: In silico analysis showed that our custom-made NGS panel can detect 96% of reported METex14 alterations. From Jan 2016 - May 2018, METex14del was found in 2% of patients with NSCLC tested for therapeutic purposes (31/1496). Outside above mentioned timeframe and tested for other purposes, an additional 15 patients were detected, making a total of 46 NSCLC patients with METex14del, since introduction of our panel in May 2015. Adenocarcinoma was the most common histological type (31/46, 68.9%), followed by sarcomatoid carcinoma (8/46, 17.8%). Thirty-six patients had advanced NSCLC, they were predominantly male (69% vs 31%) and mostly (ex) smokers (64% vs 31%), with a median age of 76.5 years at disease onset [range 53-90]. Eleven patients were treated with targeted therapy, either with crizotinib in named patient based program or in clinical trials. With crizotinib, disease control was achieved for 4 out of 5 patients (3 PR [PFS 4 -12 mo], 1 SD [14 mo]). Biopsy at progression revealed a resistance mechanism (MET c.3682G>A; p.D1228N) to crizotinib in two patients, respectively, 6 and 12 months after start of crizotinib treatment. Conclusions: In this study, we show that we can detect 96% of the known MET exon 14 skipping mutations using our custom-made DNA based NGS approach for DNA isolated from FFPE tissue. In this large consecutive series, METex14del was found in 2% of NSCLC, mostly elderly patients, in a good condition and with a smoking history. Although a small cohort, patients responded well to targeted treatment, underlining the need for routine testing of METex14del in advanced non-squamous NSCLC to guarantee optimal personalized treatment. Citation Format: Melinda Pruis, WRR Geurts, JH von der Thüsen, IC Meijssen, WNM Dinjens, JGJV Aerts, MP Lolkema, HJ Dubbink, MS Paats. Highly accurate DNA-based detection of MET exon 14 skipping mutations in non-small cell lung cancer and clinical response upon targeted treatment [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B136. doi:10.1158/1535-7163.TARG-19-B136

Icotinib-resistant HCC827 cells produce exosomes with mRNA MET oncogenes and mediate the migration and invasion of NSCLC

BackgroundIcotinib has been widely used in patients with non-small cell lung cancer (NSCLC), and have significantly enhanced the overall survival rate of NSCLC patients. However, acquired drug resistance limits its clinical efficacy. Tumor cell-derived exosomes have been reported to participate in various biological processes, including tumor invasion, metastasis and drug resistance.Materials and methodsIn the present study, drug resistance was measured by MTT assay. Exosomes were extracted from the cell supernatant using ultracentrifugation and identified by exosomal marker. HCC827 cells were treated with exosomes derived from icotinib-resistant (IR) HCC827 to observe the invasion and migration of parent cells. The expression of exo-mRNA was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-PCR). In addition, 10 exo-mRNAs detecting from the plasma and bronchoalveolar lavage fluid (BALF) of NSCLC patients with icotinib treatment were used to establish a new drug resistant-warning formula.ResultsThe oncogene MET into exosomes was identified from icotinib-resistant lung cancer cells, and this was also presented in exosomes in NSCLC patients diagnosed with cancer metastasis after icotinib treatment. The knockdown of MET in exosomes significantly decreased the ability of invasion and migration in HCC827 cells.ConclusionIt was suggested that MET might be specifically package and transferred by exosomes to modify the invasion and migration ability of the surrounding icotinib-sensitive cells.

Growth Hormone Upregulates Melanocyte-Inducing Transcription Factor Expression and Activity via JAK2-STAT5 and SRC Signaling in GH Receptor-Positive Human Melanoma.

Growth hormone (GH) facilitates therapy resistance in the cancers of breast, colon, endometrium, and melanoma. The GH-stimulated pathways responsible for this resistance were identified as suppression of apoptosis, induction of epithelial-to-mesenchymal transition (EMT), and upregulated drug efflux by increased expression of ATP-binding cassette containing multidrug efflux pumps (ABC-transporters). In extremely drug-resistant melanoma, ABC-transporters have also been reported to mediate drug sequestration in intracellular melanosomes, thereby reducing drug efficacy. Melanocyte-inducing transcription factor (MITF) is the master regulator of melanocyte and melanoma cell fate as well as the melanosomal machinery. MITF targets such as the oncogene MET, as well as MITF-mediated processes such as resistance to radiation therapy, are both known to be upregulated by GH. Therefore, we chose to query the direct effects of GH on MITF expression and activity towards conferring chemoresistance in melanoma. Here, we demonstrate that GH significantly upregulates MITF as well as the MITF target genes following treatment with multiple anticancer drug treatments such as chemotherapy, BRAF-inhibitors, as well as tyrosine-kinase inhibitors. GH action also upregulated MITF-regulated processes such as melanogenesis and tyrosinase activity. Significant elevation in MITF and MITF target gene expression was also observed in mouse B16F10 melanoma cells and xenografts in bovine GH transgenic (bGH) mice compared to wild-type littermates. Through pathway inhibitor analysis we identified that both the JAK2-STAT5 and SRC activities were critical for the observed effects. Additionally, a retrospective analysis of gene expression data from GTEx, NCI60, CCLE, and TCGA databases corroborated our observed correlation of MITF function and GH action. Therefore, we present in vitro, in vivo, and in silico evidence which strongly implicates the GH–GHR axis in inducing chemoresistance in human melanoma by driving MITF-regulated and ABC-transporter-mediated drug clearance pathways.

A phase I open prospective cohort trial of curcumin plus tyrosine kinase inhibitors for EGFR-mutant advanced non-small cell lung cancer.

e20611 Background: An increasing number of patients with lung cancer use natural products concurrently with conventional treatments, one of which is curcumin, or diferuloylmethane, the main ingredient in the popular spice known as curry. Curcumin has been shown to have anti-carcinogenic effects on human lung cancer cell lines and in vivo models, as well as the potential to modulate all three known pathways accounting for resistance to EGFR-TKIs. More specifically, curcumin dose-dependently suppresses EGFR expression, reduces transactivation of the MET oncogene, and reduces inflammation in the tumor microenvironment by selectively inhibiting TGF-β, IL-6, C-reactive protein (CRP) as well as TNF-α. Despite these promising findings, no studies have yet assessed the safety of introducing curcumin alongside EGFR-TKIs in a lung cancer population. Methods: We performed a phase 1 open prospective cohort study to assess the safety and feasibility of using an enhanced bioavailable formulation of curcumin (CURCUViva TM at 80 mg/ 1 capsule per day) approved and licensed by Health Canada (NPN 80027414) in conjunction with an EGFR-TKI in patients with advanced NSCLC (ClinicalTrials.gov: NCT02321293). The primary objective was to determine safety and feasibility. Exploratory objectives were monitoring changes in health-related quality of life using the standardized FACT-L questionnaire and evaluating anti-inflammatory properties of curcumin by measuring CRP. Results: 55 patients were approached, of whom 75% were interested in using curcumin. Completion rate (80%) and adherence (82%) levels were very high; most patients perceived the product as easy to take and were willing to take it again if it were available. Nine patients refused to participate solely due to frequent clinic visits. A total of 37 adverse events were reported, all of which were mild (Grade 1 or 2). The vast majority were pre-existing and related to TKI treatment. No biochemical or hematological evidence of toxicity was observed. Overall, there was improvement in quality of life, and improvement in the LCS of the FACT-L (p = 0.06). There was no significant change in CRP levels. Conclusions: This study provides preliminary evidence that short-term use of curcumin in EGFR-positive patients treated with TKIs is feasible and safe. We report high treatment adherence and improved quality of life with curcumin. These findings, as well as efficacy data and the effect of curcumin on other inflammation-associated biomarkers, warrant investigation in a larger phase 2 study. Clinical trial information: NCT02321293.

Reviving oncogenic addiction to MET bypassed by BRAF (G469A) mutation

Cancer clonal evolution is based on accrual of driving genetic alterations that are expected to cooperate and progressively increase malignancy. Little is known on whether any genetic alteration can hinder the oncogenic function of a coexisting alteration, so that therapeutic targeting of the one can, paradoxically, revive the function of the other. We report the case of a driver oncogene (MET) that is not only bypassed, but also disabled by the mutation of a downstream transducer (BRAF), and reignited by inhibition of the latter. In a metastasis originated from a cancer of unknown primary (CUP), the MET oncogene was amplified eightfold, but unexpectedly, the kinase was dephosphorylated and inactive. As result, specific drugs targeting MET (JNJ-38877605) failed to inhibit growth of xenografts derived from the patient. In addition to MET amplification, the patient harbored, as sole proliferative driver, a mutation hyperactivating BRAF (G469A). Surprisingly, specific blockade of the BRAF pathway was equally ineffective, and it was accompanied by rephosphorylation of the amplified MET oncoprotein and by revived addiction to MET. Mechanistically, MET inactivation in the context of the BRAF-activating mutation is driven through a negative feedback loop involving inactivation of PP2A phosphatase, which in turn leads to phosphorylation on MET inhibitory Ser985. Disruption of this feedback loop allows PP2A reactivation, removing the inhibitory phosphorylation from Ser985 and thereby unleashing MET kinase activity. Evidence is provided for a mechanism of therapeutic resistance to single-oncoprotein targeting, based on reactivation of a genetic alteration functionally dormant in targeted cancer cells.

Abstract 828: Targeting the MET oncogene by concomitant inhibition of receptor and ligand by an antibody-‘decoy' strategy

Abstract Background: MET, a master gene sustaining ‘invasive growth', is a relevant target for cancer precision therapy. In a limited number of cases, a MET genetic lesion drives the malignant phenotype in ligand-independent manner (oncogene ‘addiction'). In the vast majority of tumors, however, wild-type MET behaves as a ‘stress-response' gene and relies on the ligand (HGF) to sustain cell ‘scattering', invasive growth and apoptosis protection (oncogene ‘expedience'). In this context -i.e. ligand-dependent MET activation- concomitant targeting of MET and HGF could be crucial to reach effective inhibition. Methods: To test this hypothesis we combined an anti-MET antibody (MvDN30), characterized by the property of inducing ‘shedding' (i.e. removal of MET from the cell surface), with a ‘decoy' (i.e. the soluble extracellular domain of the MET receptor) endowed with HGF-sequestering ability. To avoid antibody/decoy interaction -and subsequent neutralization- we identified by site directed mutagenesis a single aminoacid in the extracellular domain of MET -lysine 842- that is critical for MvDN30 binding, and engineered the corresponding recombinant decoyMET (K842E). The effect of MvDN30 and decoyMETK842E in combination on the inhibition of MET phosphorylation and on the impairment of different MET driven biological responses (motility, invasion, cell proliferation, apoptosis and anchorage independent cell growth) was evaluated on a panel of cancer cells (lines and patient derived-primary cultures) expressing wild-type MET sensitive to HGF stimulation. Therapeutic efficacy was assessed in an orthotopic model of pancreatic cancer generated in SCID mice engineered to express human HGF. Results: The decoyMETK842E retains the ability to bind HGF with high affinity and inhibits HGF-induced MET phosphorylation. In HGF-dependent cellular models, MvDN30 antibody and decoyMETK842E used in combination cooperate in restraining invasive growth, and synergize in blocking cancer cell ‘scattering'. The antibody and the decoy unbridle apoptosis of colon cancer stem cells grown in vitro as spheroids. In the preclinical model of MET ‘expedience' concomitant treatment with antibody and decoy significantly reduces metastatic spread. Conclusions: The data reported indicate that vertical targeting of the MET/HGF axis results in powerful inhibition of ligand-dependent MET activation, providing proof of concept in favor of combined targeted therapy of MET ‘expedience'. Citation Format: Cristina Basilico, Chiara Modica, Federica Maione, Elisa Vigna, Paolo M. Comoglio. Targeting the MET oncogene by concomitant inhibition of receptor and ligand by an antibody-‘decoy' strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 828.

Randomized phase 2 study of tivantinib plus erlotinib versus single-agent chemotherapy in previously treated KRAS mutant advanced non-small cell lung cancer

BackgroundKRAS mutations are identified in approximately 25% of non-small cell lung cancer (NSCLC) cases and are associated with resistance to currently available targeted therapies. The MET oncogene may be implicated in malignant progression of KRAS-mutant tumors. In a pre-specified subset analysis of KRAS mutant cancers in an earlier phase 2 study of erlotinib plus the oral MET inhibitor tivantinib, combination therapy was associated with substantial clinical benefit compared to erlotinib alone (progression-free survival [PFS] HR 0.18; P < 0.01). The current study was conducted to evaluate this combination further in KRAS mutant non-small cell lung cancer (NSCLC). Materials and methodsPreviously treated patients with advanced KRAS mutant NSCLC were randomized to receive either oral tivantinib (360 mg twice daily) plus erlotinib (150 mg daily) (ET) or single-agent chemotherapy (investigator’s choice of pemetrexed, docetaxel, or gemcitabine) (C). The primary endpoint was PFS. At progression, crossover from C to ET was permitted. ResultsNinety-six patients were randomly assigned to ET (n = 51) or to C (n = 45). Median PFS was 1.7 months (mos) for ET and 4.3 mos for C (HR 1.19; 95% CI, 0.71-1.97; P = 0.50). There was no difference in overall survival (HR 1.20; 95% CI, 0.76-1.88; P = 0.44). There were 4 partial responses in the C arm, and none in the ET arm. Overall, adverse events occurred more frequently in the C arm, with more cytopenias, nausea, fatigue, and alopecia. Dermatologic toxicities were more common in the ET arm. ConclusionIn previously treated patients with advanced KRAS mutant NSCLC, the combination of the MET inhibitor tivantinib and erlotinib is not superior to conventional single-agent chemotherapy.

Expression of the c-MET, HGF and VEGF biomarkers in intestinal and diffuse gastric cancer in the Brazilian population: a pilot study for the standardization of the quantitative PCR technique

BackgroundGastric carcinoma (GC) is the third leading cause of death among malignant tumors worldwide, causing approximately 900,000 deaths/year. Changes in oncogenes that encode tyrosine kinase receptors play an important role in the pathogenesis of GC. MET gene is a proto-oncogene that encodes a tyrosine kinase receptor c-MET and it is required for embryonic development and tissue repair. The hepatocyte growth factor (HGF) is the only known ligand for c-Met receptor. The MET oncogene activation suppresses apoptosis and promotes the survival, proliferation, migration, differentiation and angiogenesis of cells. Among the angiogenic factors, VEGF is the main regulator. Its biological function includes the promotion of endothelial cells mitosis to stimulate cells proliferation. These biomarkers expression in GC is relatively recent and population-based studies are required to define the expression pattern. The aim of this study was to determine qPCR technical standardization to evaluate quantitatively, in paraffin tissue samples, the presence of gene 23 expression of the MET, HGF and VEGF in diffuse and intestinal GC types.MethodsTwenty GC patients were studied, 10 patients were intestinal-type GC (average age 72.1 years) and 10 diffuse-type (average age 50.1 years). In all patients, tissue samples were analyzed from the tumor and distant areas of the tumor tissue. The relative expressions of the tumor markers c-Met, HGF and VEGF were performed by qPCR technique by comparing tumor and non-tumoral samples and they were normalized with the GAPDH constitutive gene. Statistical analysis was performed through T-test.ResultsFor c-Met, 18/20 (90%) patients expressed the marker and 9/20 (45%) overexpressed this gene, in which three were intestinal-type GC and six were diffuse-type GC. For HGF, only 7/20 (35%) patients expressed this gene and it was overexpressed in 4/20 (20%), in which two were intestinal-type GC and two were diffuse-type GC. For VEGF, 20/20 (100%) patients expressed this marker and in 12/20 (60%) were observed overexpression, in which eight patients had diffuse-type GC and four had intestinal-type GC.ConclusionsqPCR technique was standardized and suitable for expression analysis of the three biomarkers using paraffin embedded tissue samples. Further studies should be carried out to characterize the expression pattern of these biomarkers in GC in the Brazilian population.

In bone metastasis miR-34a-5p absence inversely correlates with Met expression, while Met oncogene is unaffected by miR-34a-5p in non-metastatic and metastatic breast carcinomas.

The highlight of the molecular basis and therapeutic targets of the bone-metastatic process requires the identification of biomarkers of metastasis colonization. Here, we studied miR-34a-5p expression, and Met-receptor expression and localization in bone metastases from ductal breast carcinomas, and in ductal carcinomas without history of metastasis (20 cases). miR-34a-5p was elevated in non-metastatic breast carcinoma, intermediate in the adjacent tissue and practically absent in bone metastases, opposite to pair-matched carcinoma. Met-receptor biomarker was highly expressed and inversely correlated with miR-34a-5p using the same set of bone-metastasis tissues. The miR-34a-5p silencing might depend on aberrant-epigenetic mechanisms of plastic-bone metastases, since in 1833 cells under methyltransferase blockade miR-34a-5p augmented. In fact, 1833 cells showed very low endogenous miR-34a-5p, in respect to parental MDA-MB231 breast carcinoma cells, and the restoration of miR-34a-5p with the mimic reduced Met and invasiveness. Notably, hepatocyte growth factor (HGF)-dependent Met stabilization was observed in bone-metastatic 1833 cells, consistent with Met co-distribution with the ligand HGF at plasma membrane and at nuclear levels in bone metastases. Met-protein level was higher in non-metastatic (low grade) than in metastatic (high grade) breast carcinomas, notwithstanding miR-34a-5p-elevated expression in both the specimens. Thus, mostly in non-metastatic carcinomas the elevated miR-34a-5p unaffected Met, important for invasive/mesenchymal phenotype, while possibly targeting some stemness biomarkers related to metastatic phenotype. In personalized therapies against bone metastasis, we suggest miR-34a-5p as a suitable target of epigenetic reprogramming leading to the accumulation of miR-34a-5p and the down-regulation of Met-tyrosine kinase, a key player of the bone-metastatic process.