Abstract A23: A second site KrasG12D mutation that impairs PI3K binding rescues embryonic lethality, abrogates myeloproliferative disease, and delays lung tumorigenesis
Authors: Wong Jasmine C. , Perez-Mancera Pedro A. , Kim Jangkyung , Jen Kuang-Yu , Kogan Scott C. , Firestone Ari J. , Collisson Eric A. , Tuveson David A. , Shannon Kevin 
Published: June 2021

Abstract
Phosphatidylinositol 3-kinase (PI3K) signaling is essential for RAS-driven transformation. To directly investigate the role of oncogenic K-Ras binding to PI3K in development and tumorigenesis, we generated KrasG12D/+,Y64G/+ mice that express, from the endogenous locus, a K-Ras oncoprotein that also contains a “second site” amino acid substitution at tyrosine 64 (Y64G) that disrupts the interaction between oncogenic K-Ras and PI3K. Surprisingly, KrasG12D,Y64G mice are viable, fertile, and phenotypically unremarkable, although they are born at a lower than expected Mendelian frequency (26% versus the expected 50% on a C57BL/6 strain background). As opposed to the enhanced proliferative rate observed in mouse embryonic fibroblasts (MEFs) from K-RasG12D mice, KrasG12D,Y64G MEFs exhibit wild-type rates of proliferation. Detailed analysis of the hematopoietic compartment in KrasG12D,Y64G mice reveals a reduced proportion of long-term hematopoietic stem cells, but no evidence of the aggressive myeloproliferative neoplasm observed when a conditional mutant KrasG12D is activated in hematopoietic cells. Consistent with these observations, bone marrow cells isolated from K-RasG12D,Y64G mice exhibit a normal pattern of myeloid progenitor colony growth in response to cytokine stimulation. In contrast to the lack of observed hematologic malignancy, 100% of KrasG12D,Y64G mice we examined at 1 year of age showed lung lesions, ultimately succumbing to lung tumors with a median survival of 496 days. Despite the ubiquitous KrasG12D,Y64G expression, these mice survive longer than models with mosaic, adenoviral-Cre recombinase-controlled KrasG12D (median survival of 185 days). The majority of the lung lesions that arise in KrasG12D, Y64G mice are low grade, classified pathologically as atypical lymphoid proliferation or papillary adenomas; a few adenocarcinomas are also observed. These studies reinforce the importance of oncogenic KRas-mediated activation of PI3K for transformation and demonstrate that expressing a Y64G amino acid substitution in the context of oncogenic KrasG12D normalizes cell proliferation, rescues embryonic lethality, abrogates myeloid disease, and attenuates lung tumorigenesis. Beyond the bone marrow and lung, this mutant strain is a potent genetic tool for dissecting the role of aberrant PI3K signaling in pancreatic, colon, and other tissues characterized by tumors driven by somatic KRAS mutations, and also have implications for treating human cancers with KRAS mutations.
Citation Format: Jasmine C. Wong, Pedro A. Perez-Mancera, Jangkyung Kim, Kuang-Yu Jen, Scott C. Kogan, Ari J. Firestone, Eric A. Collisson, David A. Tuveson, Kevin Shannon. A second site KrasG12D mutation that impairs PI3K binding rescues embryonic lethality, abrogates myeloproliferative disease, and delays lung tumorigenesis [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 A23.