Characterization of the PKM2-RAS Interaction as a Novel Metabolic Vulnerability of RAS-driven Tumors

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Project Summary/Abstract RAS is the most prevalent oncogene in human cancer and is disproportionately present in aggressive cancers, such as lung, pancreas, and colon adenocarcinomas, that are leading causes of cancer-related deaths in the US. Unfortunately, attempts to target RAS have largely failed such that cytotoxic chemotherapy remains the standard of care for RAS-driven tumors. Hence, there exists a need to identify vulnerabilities of RAS-driven tumors that can be targeted by novel directed therapies. RAS-mutant tumors rewire glycolysis in order to divert glucose-carbons away from mitochondrial oxidative phosphorylation and into glycolytic shunt pathways for the biosynthesis of nucleotides, amino acids, and reducing equivalents needed to sustain proliferation. One mechanism by which tumors achieve shunting of glucose-carbons into biosynthetic processes is through expression of the M2 isoform of pyruvate kinase (PKM2), as its dynamic enzymatic activity allows malignant cells to regulate glycolytic flux. Strikingly, our lab recently identified PKM2 as a potential metabolic effector of RAS proteins. Preliminary data suggests that RAS directly binds PKM2 in a GTP-dependent manner that diminishes the stability of PKM2 multimers and inhibits its enzymatic function. The proposed work in this fellowship will determine the precise molecular mechanisms employed by oncogenic RAS to undermine PKM2 tetramer stability and test whether inhibition of PKM2 by RAS alters central carbon metabolism in RAS-mutant tumor cells. To understand the molecular mechanism of inhibition, PKM2 domains and critical residues that mediate the interaction with RAS will be identified by mutational analysis and binding assays. Given the essential role of multimerization for PKM2 function, the effects of oncogenic RAS proteins on PKM2 multimer stability and formation will be measured utilizing sucrose velocity gradients. The effects of RAS on PKM2 multimerization will be validated in vivo by analysis of PKM2 multimer profiles across a panel of oncogenic and wildtype RAS-bearing colorectal adenocarcinoma cell lines. We hypothesize that inhibition of PKM2 by oncogenic RAS rewires glycolysis to divert glucose-carbons towards the biosynthesis of macromolecules. To test this hypothesis, the central carbon metabolism of cell lines engineered to express wildtype PKM2 or PKM2 mutants with diminished affinity to RAS will be measured by coupling stable isotope tracing with liquid-chromatography and mass spectrometry. We hypothesize that RAS-driven tumor cells rely on inhibition of PKM2 to sustain the biosynthetic requirements of sustained malignant proliferation and, therefore, represents a metabolic vulnerability that can be targeted therapeutically. To test this, cellular viability and proliferation of a panel of oncogenic and wildtype RAS-bearing colorectal adenocarcinoma cell lines will be measured in response to pharmacological stimulation of PKM2. Collectively, this work will determine the molecular mechanisms of PKM2 inhibition by RAS, elucidate the metabolic consequences of the interaction, and evaluate a novel directed therapy for RAS-driven tumors.