Differential pathway engagement and the biological consequences of KRAS variants in cancer

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Project Summary KRAS is the most frequently mutated proto-oncogene in human cancer and encodes a small GTPase that regulates multiple cellular processes such as cell proliferation, metabolism, migration, and survival. Point mutations in amino acids G12, G13 and Q61 prevent KRAS inactivation by regulatory GTPase-activating proteins and facilitate tumorigenesis. Although the frequency of specific KRAS mutant variants differ by cancer type, the mechanistic basis for this observation is unknown. It has been postulated that specific mutants induce a “sweet spot” of signaling alterations to induce a cell state optimized for tumor development and maintenance in specific tissues. In support of this hypothesis, KRAS mutants exhibit different biochemical properties in GTP hydrolysis rates and binding affinity to downstream effectors, supporting divergence in their activation of signaling networks. Furthermore, preclinical and clinical data revealed allele-specific differences in tumor initiation capacity and patient prognosis in pancreatic ductal adenocarcinoma (PDAC), suggesting that divergent signaling output could lead to altered phenotypic properties. A systematic and comprehensive evaluation of allele-specific signaling networks would be valuable to better understand KRAS diversity and reveal variant-specific dependencies. The overarching objective of this proposal is to understand KRAS mutant-specific differences in signaling and how these alter cellular fitness. Our preliminary data revealed differential engagement of global signaling networks and canonical amongst KRAS mutants. Therefore, our central hypothesis is that the biochemical differences between KRAS variants result in differential signaling pathway engagement, affecting cellular behavior, tumorigenic properties, and response to therapy. To test this hypothesis, I will re-express a large panel of KRAS mutants observed in human cancer in our recently generated isogenic KRAS deficient PDAC cell lines to dissect KRAS variant-specific differences in signaling networks using data-independent acquisition mass spectroscopy (DIA-MS). Experiments proposed in Aim 1 will investigate differences in signaling pathway activation and dependency of KRAS mutants and validate them in genetically engineered mouse models, PDX models, and human tumor biospecimens. Aim 2 will explore the cellular fitness of these mutants combining in vitro and in vivo competitive assays. Collectively, these data will provide fundamental insights into the biology of KRAS mutants that could potentially explain differences in clinical prevalence and response to therapy. More broadly, this work could inform new allele-specific therapeutic strategies for PDAC and other KRAS mutant cancers. Finally, this highly interdisciplinary and collaborative effort will train me in the application of cutting-edge molecular, biochemical, and computational techniques to facilitate my career goal of becoming an independent scientist in cancer biology.