Purpose: The mesenchymal-epithelial transition factor (c-Met) receptor, also known as hepatocyte growth factor receptor (HGFR), controls morphogenesis, a process that is physiologically required for embryonic development and tissue repair. Aberrant c-Met activation is associated with a variety of human malignancies including cancers of the lung, kidney, stomach, liver, and brain. In this study, we investigated the properties of two novel compounds developed to selectively inhibit the c-Met receptor in antitumor therapeutic interventions.Experimental Design: The pharmacologic properties, c-Met inhibitory activity, and antitumor effects of EMD 1214063 and EMD 1204831 were investigated in vitro and in vivo, using human cancer cell lines and mouse xenograft models.Results: EMD 1214063 and EMD 1204831 selectively suppressed the c-Met receptor tyrosine kinase activity. Their inhibitory activity was potent [inhibitory 50% concentration (IC 50 ), 3 nmol/L and 9 nmol/L, respectively] and highly selective, when compared with their effect on a panel of 242 human kinases. Both EMD 1214063 and EMD 1204831 inhibited c-Met phosphorylation and downstream signaling in a dose-dependent fashion, but differed in the duration of their inhibitory activity. In murine xenograft models, both compounds induced regression of human tumors, regardless of whether c-Met activation was HGF dependent or independent. Both drugs were well tolerated and induced no substantial weight loss after more than 3 weeks of treatment.Conclusions: Our results indicate selective c-Met inhibition by EMD 1214063 and EMD 1204831 and strongly support clinical testing of these compounds in the context of molecularly targeted anticancer strategies.

Summary Tepotinib is an oral MET inhibitor approved for metastatic non-small cell lung cancer (NSCLC) harboring MET exon 14 (METex14) skipping mutations. Examining treatment-naive or tepotinib-resistant cells with MET amplification or METex14 skipping mutations identifies other receptor tyrosine kinases (RTKs) that co-exist in cells prior to tepotinib exposure and become more prominent upon tepotinib resistance. In a small cohort of patients with lung cancer with MET genetic alterations treated with tepotinib, gene copy number gains of other RTKs were found at baseline and affected treatment outcome. An Src homology 2 domain-containing phosphatase 2 (SHP2) inhibitor delayed the emergence of tepotinib resistance and synergized with tepotinib in treatment-naive and tepotinib-resistant cells as well as in xenograft models. Alternative signaling pathways potentially diminish the effect of tepotinib monotherapy, and the combination of tepotinib with an SHP2 inhibitor enables the control of tumor growth in cells with MET genetic alterations.

Background Advanced non-small cell lung cancer (NSCLC) patients with MET exon 14 skipping mutations (METex14) or MET amplification (METamp) derive benefit from MET tyrosine kinase inhibitors (TKIs). Tepotinib is an investigational selective MET TKI. Co-occurring MAPK pathway alterations in MET dependent tumors are frequent and may induce primary or acquired resistance to MET TKIs. Methods METamp (EBC1, NCI-H1993 and MNK-45) and METex14 (Hs746T) cells were obtained from commercial vendors. Tepotinib-resistant (TR) cells (EBC1-TR1, -TR2 and Hs746T-TR) were generated upon continuous exposure of EBC1 and Hs746T cells to tepotinib. The phosphorylation of 49 receptor tyrosine kinases (RTK) and 43 non-RTKs was assessed using Human Phospho-RTK kinase Array Kits (R&D Systems, Minneapolis, MN). Cell viability was evaluated by Resazurin Cell Viability Assay (R&D Systems, Minneapolis, MN) and level of proteins by western blot. Results First, we assessed the short- and long-term effect of tepotinib on MET downstream signaling. Exposure of EBC1 cells to 0.01 µM of tepotinib for 1h abrogated MET, ERK and AKT phosphorylation, an effect that was sustained even 20h after tepotinib withdrawal. Next, we evaluated the phosphorylation status of other than MET RTKs and non-RTKs on the parental EBC1 and EBC1-TR1 cells. We observed activation of several RTKs, including ERBB (EGFR, ErbB2 and ErbB3), FGFR3, AXL, RET, DDR1, and M-CSFR in both cell lines. EBC1-TR1 cells displayed elevated levels of phosphorylated AXL and EGFR compared to EBC1 parental cells. We also observed increased phosphorylation of ERK1/2, AKT, c-Jun and YES on EBC1-TR1 compared to parental EBC1 cells. Similar results were achieved with EBC1-TR2 and Hs746T-TR cells. RTK signaling is mediated by the MAPK pathway which is related among others, with the Src-homology 2 domain-containing protein tyrosine phosphatase (SHP2). Therefore, we tested tepotinib in combination with a MEK inhibitor, pimasertib, or a SHP2 inhibitor, RMC-4550, initially in a panel of treatment-naïve MET dependent cells. A moderate or strong synergism was found when tepotinib was combined with either of the two compounds. A stronger decrease of MET phosphorylation was observed upon treatment with a SHP2 inhibitor combined with tepotinib compared to tepotinib alone. Further molecular characterization of the TR cells as well as combinations in vitro and in vivo of tepotinib with drugs that may prolong its effect and overcome resistance, are ongoing. Conclusions Our results indicate that co-occurring RAS-MAPK alterations may mediate innate or acquired resistance to tepotinib, despite its prolonged on-target activity. The development of therapeutic strategies co-targeting the MAPK pathway may enhance the initial magnitude and duration of response to tepotinib, delay or overcome acquired resistance and ultimately improve clinical outcomes. Citation Format: Linda Pudelko, Frank Jaehrling, Christopher Stroh, Nina Linde, Michael Sanderson, Doreen Musch, Marina Keil, Christina Esdar, Andree Blaukat, Niki Karachaliou. Unraveling mechanisms of resistance to tepotinib and future treatment options [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 3774.

<p>PDF file - 240K, Dose-dependent inhibition of c-Met auto-phosphorylation in vivo. Panel A: Mice bearing established tumors, derived from s.c. injection of Hs746T gastric cancer cells, received a single dose of 3 mg/kg , 10 mg/kg , 30 mg/kg , and 100 mg/kg EMD 1214063. The pharmacokinetic profile of EMD 1214063 was defined by measuring the compound levels in plasma and tumor tissue. The lower limits of detection for EMD 1214063 were 0.007 �M (4 ng/mL) in the plasma and 0.037 �M (20 ng/mL) in tumor samples. The levels of c-Met auto-phosphorylation (Y1234/Y1235) were assessed by Western blot analysis and used as a pharmacodynamic marker of activity. Each data point represents the mean � SD of the values obtained from four mice per experimental group. A data point was not shown if any of the values within the experimental group was below the detection limit. Panel B: A similar experimental approach to that already described for EMD 1214063 was used to define the pharmacokinetic profile and pharmacodynamic effects of EMD 1204831. In brief, mice bearing established Hs746T subcutaneous tumors received a single dose of 3 mg/kg, 10 mg/kg, 30 mg/kg, or 100 mg/kg EMD 1204831. In the pharmacokinetic studies, the lower limits of detection for EMD 1204831 were 0.014 �M (8 ng/mL) in the plasma and tumor samples. The pharmacodynamic effects were determined by Western blot analyses of c-Met auto-phosphorylation (Y1234/Y1235). Each data point represents the mean � SD of the values obtained from four mice per experimental group. A data point was not shown if any of the values within the experimental group was below the detection limit.</p>

<p>PDF file - 45K, ATP competition studies with EMD 1214063. EMD 1214063 inhibited c-Met phosphorylation in an ATP-competitive fashion. In brief, binding competition of EMD 1214063 and ATP to c-Met was analyzed by a kinase reaction followed by Western blotting. ATP in the indicated concentrations was mixed with 1 nM EMD 1214063 and added to 100 ng recombinant human c-Met kinase (Carna Biosciences 08-151). The reaction mixtures were incubated at room temperature for 30 min. The reaction was stopped by addition of 4x LDS Sample Buffer (Invitrogen NP-0007) containing 25 mM DTT and heating at 80{degree sign}C for 20 min. The reaction mixtures were analyzed by Western blotting using 1 ng c-Met kinase per lane in gel electrophoresis. After blotting on nitrocellulose, detection was performed with an anti-phosphotyrosine antibody (clone PY99, Santa Cruz sc-7020) and an anti-GST-tag antibody (clone B-14, Santa Cruz sc-138) as a loading control. Chemiluminescence read out was measured with a VersDoc analyzer (BioRad) and band densities were quantified with the inherent software (ImageLab).</p>