Abstract IA31: Genetic approaches for testing candidate drug targets for Ras-induced tumorigenesis in vivo

Despite remarkable progress over the past few years, developing chemical inhibitors that directly and selectively target mutant Ras proteins remains a fundamental challenge. Given this, inhibitors of proteins that function upstream or downstream of oncogenic Ras are a promising alternative therapeutic strategy for RAS mutant tumors. However, the complexity of Ras signaling networks and adaptive mechanisms that result in pathway “rewiring” complicates identifying genes and proteins that are essential for the growth and survival of these cancers. Genetically engineered mice that express mutant Ras alleles from the endogenous locus are a robust system for uncovering pathway dependencies and potential therapeutic vulnerabilities. We have generated novel strains of mice to address two specific questions. First, we collaborated with Kevin Haigis to generate a “second site” C181S substitution within a conditional NrasLSL-G12D mutant allele (Nat Genet 2008;40:600) to ask if palmitoylation is essential for myeloid transformation in vivo. We intercrossed mice carrying this mutation with the Mx1-Cre strain and induced Cre recombinase expression in the hematopoietic compartment by injecting compound mutant pups with polyI:polyC. As previously reported, homozygous NrasLSL-G12D mice uniformly developed an aggressive myeloproliferative disorder (MPD) characterized by markedly elevated blood leukocyte counts, splenomegaly, and death by age 9 months. By contrast, homozygous NrasLSL-G12D,C181S mice remained healthy with no evidence of hematologic disease despite constitutively elevated levels of RasGTP in bone marrow cells that were similar to those observed in NrasLSL-G12D mice. Whereas normal NRas and N-RasG12D proteins are predominantly in the plasma membrane, N-RasG12D, C181S was largely mis-localized in the cytosol. We also generated Mx1-Cre, NrasG12D,C181S/G12D compound heterozygous mutant mice to investigate potential interactions between oncogenic NrasG12D alleles with and without the C181S substitution. Over half of these mice developed aggressive hematologic malignancies, albeit with prolonged latency compared to homozygous NrasG12D mice. Importantly, genetic analysis of diseased tissues revealed reduced frequency or complete absence of the NrasG12D,C181S allele due to secondary somatic genetic events, which indicates strong selective pressure to overcome the growth-suppressive properties of unpalmitoylated N-Ras. Together, these studies validate the palmitoylation/depalmitoylation cycle as a therapeutic target in NRAS mutant hematologic malignancies and establish a novel model system for addressing this question in other cancers. In another ongoing project, we collaborated with Pedro Perez-Macera and David Tuveson to modify a LSL-KrasG12D knock-in allele to introduce a second-site amino acid Y64G substitution that severely impairs binding to PI3 kinase. Surprisingly, mice expressing KrasG12D,Y64G in the germline are viable and phenotypically normal, although they are born at a lower than expected Mendelian frequency. Whereas Mx1-Cre, KrasG12D/+ mice uniformly develop aggressive MPD and die by 4 months of age, KrasG12D,Y64G animals have normal blood counts at 12-18 months of age. However, 100% of KrasG12D,Y64G mice develop lung lesions by 1 year of age, and ultimately succumb from lung tumors with a median survival of 496 days. The majority of these lesions are classified pathologically as atypical lymphoid proliferation or papillary adenomas, with a few mice developing adenocarcinomas. These studies demonstrating that expressing a Y64G amino acid substitution in the context of oncogenic KrasG12D rescues embryonic lethality, abrogates myeloid disease, and attenuates lung tumorigenesis are generally consistent with and also extend elegant studies of Pi3kca mutant mice from the Downward lab (Cell 2007;129:957; Cancer Cell 2013;24:617). Beyond the bone marrow and lung, KrasG12D,Y64G mice are a potent genetic tool for dissecting the role of aberrant PI3 kinase signaling in lung, pancreatic, colon, and other tissues characterized by tumors driven by somatic KRAS mutations, and these data have implications for treating human cancers with KRAS mutations. Citation Format: Kevin M. Shannon. Genetic approaches for testing candidate drug targets for Ras-induced tumorigenesis in vivo [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA31.