Activation of KRAS Mediates Resistance to Targeted Therapy in MET Exon 14-mutant Non-small Cell Lung Cancer

Abstract

Purpose: MET exon 14 splice site alterations that cause exon skipping at the mRNA level (METex14) are actionable oncogenic drivers amenable to therapy with MET tyrosine kinase inhibitors (TKI); however, secondary resistance eventually arises in most cases while other tumors display primary resistance. Beyond relatively uncommon on-target MET kinase domain mutations, mechanisms underlying primary and acquired resistance remain unclear.

Experimental design: We examined clinical and genomic data from 113 patients with lung cancer with METex14. MET TKI resistance due to KRAS mutation was functionally evaluated using in vivo and in vitro models.

Results: Five of 113 patients (4.4%) with METex14 had concurrent KRAS G12 mutations, a rate of KRAS cooccurrence significantly higher than in other major driver-defined lung cancer subsets. In one patient, the KRAS mutation was acquired post-crizotinib, while the remaining 4 METex14 patients harbored the KRAS mutation prior to MET TKI therapy. Gene set enrichment analysis of transcriptomic data from lung cancers with METex14 revealed preferential activation of the KRAS pathway. Moreover, expression of oncogenic KRAS enhanced MET expression. Using isogenic and patient-derived models, we show that KRAS mutation results in constitutive activation of RAS/ERK signaling and resistance to MET inhibition. Dual inhibition of MET or EGFR/ERBB2 and MEK reduced growth of cell line and xenograft models.

Conclusions: KRAS mutation is a recurrent mechanism of primary and secondary resistance to MET TKIs in METex14 lung cancers. Dual inhibition of MET or EGFR/ERBB2 and MEK may represent a potential therapeutic approach in this molecular cohort.

Figure 1:. A patient with lung adenocarcinoma harboring MET exon14 alteration and acquired KRAS G12S

A. The clinical course and treatment history of PT-5 with a MET exon 14 alteration (KRAS wild type) at the time of progression on cytotoxic chemotherapy. The next biopsy took place after progression on MET bivalent antibody and showed a newly acquired KRAS G12S mutation. B. Computed tomography scans of the patient before and after progression during crizotinib treatment. C. MSK-IMPACT sequence analysis of paired samples before and after crizotinib resistance. D. The copy number plots for paired samples before and after crizotinib resistance. E. The top and bottom 20 genes differentially expressed in METex14 (n=3) vs MET wildtype amplified (n=5) lung adenocarcinomas from BCCA. F. The same analysis but for TCGA tumors with 7 METex14 and 7 MET wildtype amplified tumors. G. The top GSEA Oncogenic Signatures upregulated in METex14 vs MET wildtype amplified tumors. Gene sets related to KRAS signaling are indicated in bold. H. Representative GSEA enrichment plots for the top KRAS-related signatures in BCCA (top) and TCGA (bottom).

Figure 2:. KRAS mutations mediates resistance to MET directed therapies in METex14 skipping cells.

A. Isogenic stably transfected 3T3-METex14-KRAS cell lines were treated with crizotinib for 96 hours. Cell viability was determined using AlamarBlue. Each condition was assayed in six-replicate determinations and data are representative of three independent experiments (mean ± SE). B. Isogenic stable 3T3-METex14-KRAS lines were serum starved for 6 hours, and subsequently treated with increasing concentrations of crizotinib for 4 hours and lysates were subjected to immunoblotting. C. Caspase 3/7 activity was analyzed in stable 3T3-METex14-KRAS lines that were treated with crizotinib for 48 hours. Each condition was assayed in triplicate determinations and data are representative of two independent experiments (mean ± SE). *p<0.05, compared to either 3T3-METex14-empty or -KRAS WT group. D. Direct sequencing confirms co-occurrence of METex14 skipping and KRAS G12S mutation in the LUAD12C cell line. E. LUAD12C cells were treated with crizotinib or cabozantinib for 96 hours. Cell viability was determined using AlamarBlue viability dye. Each condition was assayed in six-replicate determinations and data are representative of three independent experiments (mean ± SE). F. LUAD12C cells were treated with increasing concentrations of crizotinib for 4 hours and lysates were subjected to immunoblotting. G. LUAD12C cells were infected with lentivirus expressing shRNAs targeting KRAS or non-targeting shRNA as a control, followed by selection with puromycin for 2 days. Lysates were subjected to immunoblotting. H. A total 2.5 × 10⁴ of KRAS knockdown LUAD12C cells were plated in 6-well plates in HGF supplemented medium, and growth rate determined. Each condition was assayed in duplicate determinations and data are representative of three independent experiments (mean ± SE). *p<0.05, compared to non-targeting shRNA cells. I. KRAS knockdown LUAD12C cells were treated with crizotinib in HGF-supplemented medium for 120 hours. Cell viability was determined using AlamarBlue. Each experiment was assayed in six-replicate determinations and data are representative of three independent experiments (mean ± SE).

Figure 3:. Trametinib synergizes with MET inhibitors in concurrent METex14 alteration and KRAS mutant cells.

A. LUAD12C, 3T3-METex14-KRAS WT, and -KRAS G12D cells were treated with crizotinib (1 μM), trametinib (25 nM), or a combination of trametinib (25 nM) and crizotinib (1μM) for 4 hours. Lysates were then subjected to immunoblotting. B. LUAD12C, 3T3-METex14-KRAS WT, and -KRAS G12D cells were treated with a combination of trametinib and either crizotinib or cabozantinib for 96 hours. Cell viability was determined by AlamarBlue. Data represented the mean value of growth inhibition ratio at each concentration of the drugs in four independent experiments. C. Dot plot indicates the combination index and fraction affected (inhibition ratio) of various drug concentration. D. 3T3-METex14-empty, -KRAS WT, and -KRAS G12D cells were implanted into a subcutaneous flank of athymic nude mice. When tumors reached approximately 100 mm³, mice were treated with vehicle or 25 mg/kg crizotinib, 1 mg/kg trametinib, or a combination of 25 mg/kg crizotinib and 1 mg/kg trametinib daily for 10 days. The relative change in volume from baseline of Individual tumors are shown in the waterfall plots. E. Tumors volume in animals with crizotinib treatment group are shown stratified by cell lines. *p<0.05, compared to the respective empty group. F. Tumors volume of 3T3-METex14-KRAS G12D xenografts are shown stratified by treatment group. ^#p<0.05, compared to vehicle-treated group.

Figure 4:. Trametinib treatment induces feedback activation of AKT and ERBB family and combination of MEK and ERBB inhibition induces synergetic suppressive effect on LUAD12C cells.

A. LUAD12C cells were treated with crizotinib (1 μM), trametinib (25 nM), or a combination of trametinib (25 nM) and crizotinib (1μM) for 48 hours. Lysates were then applied to phospho-RTK arrays. B. LUAD12C cells were treated with trametinib (25 nM) for indicated time. Lysates were subjected to immunoblotting. C. LUAD12C cells were treated with trametinib (25 nM) for 48 hours and mRNA expression level of ERBB families were determined by RT-qPCR. Experiments were conducted in triplicate and error bars represent mean ± SD. D. LUAD12C cells were treated with trametinib (25 nM) for 48 hours and mRNA expression level of ligands for ERBB families were determined by RT-qPCR. Experiments were conducted in triplicate and error bars represent mean ± SD. E. LUAD12C cells were infected with lentivirus expressing shRNAs targeting genes or non-targeting shRNA as a control, followed by selection with puromycin. Subsequently, a total 1.5 × 10⁵ of knockdown LUAD12C cells were plated in 6-well plates, and treated with DMSO or trametinib (5nM) for 10 days. Relative cell numbers were shown. Each condition was assayed in duplicate determinations in 2 independent experiments and error bar represent mean ± SE. F. Lysates from knockdown LUAD12C cells after selection were subjected to immunoblotting.

Figure 5:. MEK inhibition suppresses MET through transcription and protein level.

A. Lysate from stably transfected 3T3-METex14-KRAS WT, G12D, or G12S cells were collected at the indicated time points after addition of cycloheximide (CHX, 100 μg/mL) and subjected to immunoblotting. B. The amount of MET protein was quantified and is shown relative to the amount of MET expressed in absence of CHX. Data are representative of three independent experiments (mean ± SE).*p<0.05, compared to 3T3-METex14-KRAS WT cells. C. MET mRNA expression level of was determined by RT-qPCR in KRAS knockdown LUAD12C cells after selection. Each condition was assayed in triplicate determinations (mean ± SD). D. LUAD12C cells were treated with trametinib (25 nM) for the indicated time and mRNA expression levels of MET were analyzed by RT-qPCR. Experiments were conducted in triplicate and error bars represent mean ± SD. E. LUAD12C cells were treated with trametinib (25 nM) for the indicated time. Lysates were subjected to immunoblotting.

Figure 6:. Trametinib synergizes with afatinib in concurrent METexon14 alteration and KRAS mutant cells.

A. LUAD12C cells were treated with crizotinib (1 μM), trametinib (25 nM), erlotinib (1 μM), afatinib (1 μM), osimertinib (1 μM), or a combination of them for 48 hours. Lysates were then subjected to immunoblotting. B. A total 3 × 10⁴ of LUAD12C cells were plated in 6-well plates, and treated with DMSO, crizotinib (200nM), trametinib (5nM), afatinib (100 nM), or a combination of them for 11 days. Relative cell numbers are shown. Each condition was assayed in duplicate determinations in 3 independent experiments and error bar represent mean ± SE. C. LUAD12C cells were treated with a combination of trametinib and arfatinib for 96 hours. Cell viability was determined by AlamarBlue. Data represent the mean value of growth inhibition ratio at each concentration of the drugs in three independent experiments. Dot plot indicates the combination index and fraction affected (inhibition ratio) of various drug concentration. D. LUAD12C cells were implanted into a subcutaneous flank of NSG mice. When tumors reached approximately 100 mm³, mice were treated with vehicle or 25 mg/kg crizotinib, 1 mg/kg trametinib, 12.5 mg/kg afatinib, or a combination of them 4 days a week. Tumor volume was determined on the indicated days after the onset of treatment. Data represent mean ± SE (n = 5). *p<0.05, compared to trametinib-treated group. E. A strategy for the treatment of METex14 and KRAS-mutant lung cancer.