Co-occurring Alterations in the RAS-MAPK Pathway Limit Response to MET Inhibitor Treatment in MET Exon 14 Skipping Mutation-Positive Lung Cancer

Abstract

Purpose: Although patients with advanced-stage non-small cell lung cancers (NSCLC) harboring MET exon 14 skipping mutations (METex14) often benefit from MET tyrosine kinase inhibitor (TKI) treatment, clinical benefit is limited by primary and acquired drug resistance. The molecular basis for this resistance remains incompletely understood.

Experimental design: Targeted sequencing analysis was performed on cell-free circulating tumor DNA obtained from 289 patients with advanced-stage METex14-mutated NSCLC.

Results: Prominent co-occurring RAS-MAPK pathway gene alterations (e.g., in KRAS, NF1) were detected in NSCLCs with METex14 skipping alterations as compared with EGFR-mutated NSCLCs. There was an association between decreased MET TKI treatment response and RAS-MAPK pathway co-occurring alterations. In a preclinical model expressing a canonical METex14 mutation, KRAS overexpression or NF1 downregulation hyperactivated MAPK signaling to promote MET TKI resistance. This resistance was overcome by cotreatment with crizotinib and the MEK inhibitor trametinib.

Conclusions: Our study provides a genomic landscape of co-occurring alterations in advanced-stage METex14-mutated NSCLC and suggests a potential combination therapy strategy targeting MAPK pathway signaling to enhance clinical outcomes.

Figure 1.. Co-occurring genomic alterations are common in non-small cell lung cancer (NSCLC) with a MET exon 14 mutation.

A. Distribution of co-occurring genomic alterations in a targeted list of cancer-associated genes (Supplemental Table 2) as detected by cfDNA in 332 samples from 289 patients with advanced-stage METex14-mutated NSCLC. Results are filtered to exclude synonymous variants, variants predicted to result in an unknown or neutral function impact (via COSMIC, GENIE, ClinVar, and mutation assessor prediction algorithms) and mutations previously reported as associated with clonal hematopoiesis. Only genomic alterations occurring at a 2% or greater frequency are displayed. CNG, copy number gain.

Figure 2.. RAS-MAPK pathway alterations are common in METex14-mutated NSCLC

A. Comparative frequency of genomic alterations as measured by cfDNA in a cohort of advanced-stage METex14 NSCLC patients (n=289) compared to an independent cohort of patients (n=1489) with a known canonical EGFR activating mutation (EGFR exon 19 deletions, EGFR L858R). A previously published independent cohort (11) of cfDNA from patients without either a canonical EGFR-activating mutation or METexon14 mutation (METex14 neg/EGFR wildtype, n = 918) is shown for comparison. Gene alterations with a statistically significant difference between METex14-mutated and EGFR-mutated NSCLC are displayed. q-values were calculated using Benjamini-Hochberg correction for false discovery rate < 0.2, with significant differences marked by an asterisk (*). B. Spectrum of gene alterations detectable by cfDNA in the subset of METex14-mutated NSCLC patients (n=61) without prior MET TKI treatment. Genomic alterations with a frequency of greater than 2% displayed. C. Genomic alterations detectable by cfDNA in the subset of patients with METex14-mutated NSCLC without prior MET TKI treatment (n=61) as compared to the subset of patients with EGFR-mutated NSCLC without prior EGFR TKI treatment (n = 58). q-values >0.95 not shown. CNG, copy number gain.

Figure 3.. MET second-site and bypass pathway alterations are newly detectable after MET TKI treatment.

Oncoprints show detectable genomic alterations by in paired samples from 12 patients, obtained either (A) before known MET TKI exposure or (B) after known MET TKI exposure. All samples reflect the results of cfDNA analysis (Guardant360), with the exception of three patients for whom only tissue NGS was available pre-MET TKI. Genes not common to the cfDNA panel were excluded from analysis. Additional details included in Table 1. C. Spectrum of newly detectable genomic alterations following MET TKI exposure by functional category.

Figure 4.. KRAS overexpression or NF1 downregulation promotes MET TKI resistance that is overcome by combined crizotinib and trametinib polytherapy in METex14-mutated preclinical models.

A. Cell viability curves demonstrating a shift in the half maximal inhibitory concentration (IC50) to crizotinib with overexpression of wild type KRAS in MET exon 14-mutant expressing Ba/F3 cells. The cells were grown in culture with HGF supplementation (50 ng/mL). B. Cell viability curve for crizotinib treatment in Ba/F3 cells with both METex14 expression and wild-type KRAS overexpression (KRAS OE), in the setting of treatment with trametinib at 0.01 μM and supplementation with HGF 50 ng/mL. The KRAS OE, trametinib negative curves in panels A and B reflect the same experimental data, displayed on two graphs for clarity. C Ba/F3 cells with stable expression of wild type MET, MET with an exon 14 skipping mutation (METex14), or mCherry ORF control were treated with 50 ng/mL HGF with or without 24 hours treatment with crizotinib at 0.1 μM and/or trametinib (0.01μM). Treatment with crizotinib inhibits MET phosphorylation and inhibits downstream Erk phosphorylation, with associated increase in apoptosis as measured by cleaved PARP (cPARP). KRAS overexpression (KRAS OE) restored downstream Erk phosphorylation and reduced cleaved PARP, despite crizotinib treatment. Addition of trametinib inhibited Erk phosphorylation and increased cleaved PARP, consistent with induction of apoptosis, despite the presence of KRAS overexpression. D. Cell viability curves for crizotinib-treated MET exon 14-mutant expressing Ba/F3 cells with either NF1 knockdown (shNF1) or a negative control scrambled shRNA (shScr). E. Cell viability curves for crizotinib-treated MET exon-14 mutant expressing Ba/F3 cells with NF1 knockdown, with and without addition of 0.01 μM trametinib. The shNF1, trametinib negative curves in panels C and D reflect the same data displayed on two graphs for clarity. F. NF1 knockdown (NF1 KD) restores downstream Erk phosphorylation and decreases cleaved PARP in crizotinib-treated tumor cells. The addition of trametinib reduced Erk phosphorylation and restored PARP cleavage consistent with induction of apoptosis despite the presence of NF1. G. Summary graph of IC50 to crizotinib with trametinib with and without KRAS overexpression. H. IC50s of crizotinib with trametinib with or without NF1 downregulation. *** p-value < 0.001 by student’s t-test.