Abstract 394: Saturation mutagenesis of KRAS reveals the functional landscape of missense variants

Abstract

Abstract KRAS is the most commonly mutated oncogenes and is a major driver of tumor initiation and progression. Understanding the functional consequences of cancer-associated KRAS variants may have important clinical implications. For example, KRAS mutation status defines those that are likely to respond to EGFR-directed therapy in KRAS-mutant metastatic colorectal cancer. A compendium of all possible oncogenic KRAS alleles would serve as a roadmap for future therapeutic strategies directed at KRAS itself or downstream signaling effectors. Comprehensive mutagenesis of KRAS may also elucidate structure-function relationships that reveal novel biochemical properties that may be exploited for therapeutic gain. We performed saturation mutagenesis of both a wild-type (WT) and a G12D mutant form of KRAS cDNA and generated lentiviral expression libraries of 3,553 and 3,534 single amino acid substitution mutants of each backbone. We utilized these WT and G12D mutagenesis libraries for functional genetic screening to identify gain- and loss-of-function missense variants that alter critical oncogenic properties of KRAS. First, we sought to comprehensively identify all possible oncogenic missense mutations in KRAS that mediate oncogenic transformation. We stably transduced the WT library into immortalized human epithelial cells and evaluated growth in low attachment (GILA), an assay that is highly correlated with in vivo tumor formation. We identified all previously known hotspot oncogenic alleles of KRAS as well as many functionally relevant alleles that are also discovered at lower frequency in human tumors. Moreover, we also discovered a group of transforming KRAS variants that have not been well described in human tumors, thus revealing potentially novel activating mechanisms for oncogenic KRAS. In parallel, we utilized the G12D mutagenesis library to perform second-site suppressor screening to identify loss-of-function single amino acid changes that abrogate the transforming ability of oncogenic KRAS. We performed positive-selection screening in primary cell lines for variants that enable bypass of oncogene-induced senescence. Additionally, we conducted a negative-selection screen with the G12D library in a KRAS-dependent cancer cell line with inducible suppression of endogenous KRAS, thus identifying all possible second-site mutations that abolish KRAS-driven signaling necessary for maintenance of cellular proliferation and viability. Structure-function analysis of these data may reveal novel patterns of amino-acid changes that result in inactivation of oncogenic KRAS. In summary, this comprehensive dictionary of gain- and loss-of-function KRAS mutants will facilitate understanding of clinically important mutations and also yield novel insights into structure-function relationships that may improve our understanding of the KRAS oncogene. Citation Format: Eejung Kim, Seav Huong Ly, Nicole S. Persky, Belinda Wang, Xiaoping Yang, Federica Piccioni, Katherine Labella, Mihir Doshi, Robert E. Lintner, Cong Zhu, Scott Steelman, David E. Root, Cory M. Johannessen, Alex B. Burgin, Laura E. MacConaill, William C. Hahn, Andrew J. Aguirre. Saturation mutagenesis of KRAS reveals the functional landscape of missense variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 394. doi:10.1158/1538-7445.AM2017-394