Figure S1. Crizotinib resistant cells reveal a reprogrammed tumor metabolism. Figure S2. Crizotinib modulates fatty acid metabolism and mitochondrial parameters. Figure S3. Tracing analysis reveals distinct modulation of metabolism by MET inhibition. Figure S4. Acute and chronic MET inhibition drives oxygen consumption rate and ECAR. Figure S5. Specific silencing of MET enhances oxidative metabolism in patient-derived xenograft cells. Figure S6. Inhibition of complex I and complex V of the electron transport chain and MET synergistically reduce viability of glioblastoma cells. Figure S7. Gamitrinib and etomoxir synergize with crizotinib to reduce cellular viability in glioblastoma model systems. Figure S8. Combined Met inhibition and Gamitrinib treatment elicits cleavage of caspases and modulate the expression of Bcl2 family members. Figure S9. Crizotinib and gamitrinib elicit cell death with features of apoptosis. Figure S10. Gamitrinib and crizotinib is superior over kinase inhibitor combination treatments to elicit apoptosis. Figure S11. The combination treatment of etomoxir and crizotinib elicits a reduction in tumor proliferation without induction of organ toxicity. Supplementary Table 1. Primer sequences for real time PCR and chromatin immunoprecipitation qPCR
ARTICLE ABSTRACT
The receptor kinase c-MET has emerged as a target for glioblastoma therapy. However, treatment resistance emerges inevitably. Here, we performed global metabolite screening with metabolite set enrichment coupled with transcriptome and gene set enrichment analysis and proteomic screening, and identified substantial reprogramming of tumor metabolism involving oxidative phosphorylation and fatty acid oxidation (FAO) with substantial accumulation of acyl-carnitines accompanied by an increase of PGC1α in response to genetic (shRNA and CRISPR/Cas9) and pharmacologic (crizotinib) inhibition of c-MET. Extracellular flux and carbon tracing analyses (U-13C-glucose, U-13C-glutamine, and U-13C-palmitic acid) demonstrated enhanced oxidative metabolism, which was driven by FAO and supported by increased anaplerosis of glucose carbons. These findings were observed in concert with increased number and fusion of mitochondria and production of reactive oxygen species. Genetic interference with PGC1α rescued this oxidative phenotype driven by c-MET inhibition. Silencing and chromatin immunoprecipitation experiments demonstrated that cAMP response elements binding protein regulates the expression of PGC1α in the context of c-MET inhibition. Interference with both oxidative phosphorylation (metformin, oligomycin) and β-oxidation of fatty acids (etomoxir) enhanced the antitumor efficacy of c-MET inhibition. Synergistic cell death was observed with c-MET inhibition and gamitrinib treatment. In patient-derived xenograft models, combination treatments of crizotinib and etomoxir, and crizotinib and gamitrinib were significantly more efficacious than single treatments and did not induce toxicity. Collectively, we have unraveled the mechanistic underpinnings of c-MET inhibition and identified novel combination therapies that may enhance its therapeutic efficacy.
c-MET inhibition causes profound metabolic reprogramming that can be targeted by drug combination therapies.
