Dataset Information

Selective CRAF Inhibition Elicits Transactivation.

ABSTRACT: Discovering molecules that regulate closely related protein isoforms is challenging, and in many cases the consequences of isoform-specific pharmacological regulation remains unknown. RAF isoforms are commonly mutated oncogenes that serve as effector kinases in MAP kinase signaling. BRAF/CRAF heterodimers are believed to be the primary RAF signaling species, and many RAF inhibitors lead to a "paradoxical activation" of RAF kinase activity through transactivation of the CRAF protomer; this leads to resistance mechanisms and secondary tumors. It has been hypothesized that CRAF-selective inhibition might bypass paradoxical activation, but no CRAF-selective inhibitor has been reported and the consequences of pharmacologically inhibiting CRAF have remained unknown. Here, we use bio-orthogonal ligand tethering (BOLT) to selectively target inhibitors to CRAF. Our results suggest that selective CRAF inhibition promotes paradoxical activation and exemplify how BOLT may be used to triage potential targets for drug discovery before any target-selective small molecules are known.

SUBMITTER: Morgan CW

PROVIDER: S-EPMC8041278 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Selective CRAF Inhibition Elicits Transactivation.

Morgan Charles W CW Dale Ian L IL Thomas Andrew P AP Hunt James J Chin Jason W JW Chin Jason W JW

Journal of the American Chemical Society 20210322 12

Discovering molecules that regulate closely related protein isoforms is challenging, and in many cases the consequences of isoform-specific pharmacological regulation remains unknown. RAF isoforms are commonly mutated oncogenes that serve as effector kinases in MAP kinase signaling. BRAF/CRAF heterodimers are believed to be the primary RAF signaling species, and many RAF inhibitors lead to a "paradoxical activation" of RAF kinase activity through transactivation of the CRAF protomer; this leads ...[more]

PMID: 33750116

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Project description: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. SIGNIFICANCE: c-MET inhibition causes profound metabolic reprogramming that can be targeted by drug combination therapies.

Dataset Information

Selective CRAF Inhibition Elicits Transactivation.

Publications

Selective CRAF Inhibition Elicits Transactivation.

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