P-038 MET activation via exon 14 skipping mutations (METex14del): gastrointestinal prevalence and sensitivity to MET inhibitor AMG337

Abstract

Introduction: Activation of the MET oncogene can result from amplification or activating mutation. MET mutations affecting the 14 5' splice acceptor site or the 3' splice donor site cause in-frame skipping of exon 14. The resulting protein lacks a portion of the juxtamembrane domain, which eliminates binding of Cbl, an E3 ubiquitin ligase involved in Met down-regulation. Met protein lacking exon 14 results in a relative over-expression of Met protein, MET activation and oncogenesis. Case reports are the only clinical evidence of response to MET inhibitors in patients harboring METex14del. To date, partial responses have been reported in 13 cases of lung cancer cases treated with MET inhibitors; the duration of response ranged from 5 weeks to >13 months. In these reports, treatment with MET inhibitors decreased the size of tumors throughout the body, including a metastatic lesion in the brain. The ongoing crizotinib trial that led to approval of crizotinib for ALK- and ROS1-rearranged non-small cell lung cancer is now enrolling patients with METex14del. METex14del has been identified in gastric cancer cell lines and in RNA extracted from patients with gastric cancer (3/42;7%) and colon cancer (4/43;9%). Here we report that the small-molecule MET inhibitor AMG 337 demonstrates nanomolar inhibition of the Met kinase activity in gastric cell lines harboring METex14del. Methods: The effect of the MET inhibitor AMG 337 on HGF-induced proliferation was evaluated by MTT assay in two gastric cancer cell lines harboring either wild-type Met or METex14del (Hs746T). IC50 values were obtained. To assess tyrosine kinase activity, the Meso Scale Discovery assay measured phosphotyrosine levels of the activated wild-type Met and MetEx14 proteins. Clonogenic assay determined the effectiveness of AMG 337. Further evidence of AMG337 antagonism of METex14del signaling was derived from soft agar colony formation assays. Results: Basal phospho-MET (pMet) content was low in both gastric cancer cell lines and was significantly increased by recombinant full-length HGF. In both cell lines, treatment with AMG337 reversed HGF-driven increases in pMet as well as HGF-enhanced gastric cancer cell invasion, proliferation and anchorage-independent growth. Growth rate and Met activation state were 10-fold lower in METex14del cells stimulated with purified HGF and treated with AMG337 as compared with untreated cells. Soft agar colony formation by Hs746T cells was suppressed by ∼50% in cultures grown in the presence of 50 nM AMG337 relative to untreated cultures, and completely abolished in the presence of 100 nM AMG337. Conclusion: Our results provide the first evidence that AMG 337 can block migration, invasion and anchorage-independent growth in gastric cell lines harboring METex14del. METex14del is a potential actionable driver mutation in gastrointestinal malignancies. This finding provides opportunities for clinical trials of AMG337 in gastrointestinal cancer to include not only MET-amplified, but also METex14del-positive patients. Identification of an additional 7%-9% of gastrointestinal cancer patients with MET alterations would increase the population eligible for MET inhibitor therapy.