Abstract PR07: Combinatorial knockout of Rap1GDS1 and RhoA leads to lethality in KRAS-driven non-small cell lung cancer

Synthetic lethal-based approaches to targeting KRAS driven cancers have recently been receiving increasing attention. However, as KRAS activates multiple effector pathways, targeting single genes may not be sufficient to fully inhibit KRAS-driven oncogenesis. In addition, genome-wide screens can be limited by high noise and low signal. Furthermore, it is increasingly clear that standard screens based on 2D proliferation can miss critical phenotypes relevant to cancer biology. Therefore, we developed a multistage screening strategy that includes a) identification of KRAS-proximal protein-protein interaction (PPI) networks, b) targeted CRISPR-based screening for pairs of genes that together are synthetic lethal with KRAS, and c) validation in a 3D sphere system using both human and mouse NSCLC cells. We used affinity purification/mass spectrometry (AP/MS) to construct a detailed map of protein-protein interactions centered on KRAS. Based on this network, we designed a CRISPR/Cas9 library targeting pairwise combinations of 119 KRAS-interacting genes. We screened two KRAS-driven non-small cell lung cancer (NSCLC) cell lines (A549 and H23). This screen yielded a large number of gene pairs that synergistically impaired cell growth. We selected the 20 most promising targets for further screening in a panel of 5 KRAS-mutant and 4 KRAS wild-type NSCLC cell lines. From these screens we identified a KRAS-specific synergistic interaction between two genes—Rap1GDS1 and RhoA. Validation in 3D cultures in a panel of cell lines showed that combinatorial Rap1GDS1 and RhoA knockout selectively impairs sphere growth of KRAS-mutant NSCLC cells but has minimal effects on KRAS wild-type NSCLC cell lines or nontransformed lung cells. We demonstrate that this effect is primarily due to the induction of apoptosis. Rap1GDS1 has previously been reported to encode two different isoforms with distinct cellular functions. We observe that only one of the isoforms is involved in the mediation of the KRAS-dependent lethal effect. Current efforts are directed at elucidating the mechanistic basis for the isoform-specific combinatorial lethality between Rap1GDS1 and RhoA. Furthermore, we are actively exploring how to exploit this knowledge to define potential novel therapies for KRAS-driven cancer. Overall, we demonstrate that an integrated strategy combining proteomics, paired CRISPR screens and innovative 3D validation systems can identify novel synthetic lethal combinations. This approach may help identify novel synthetic lethal drug combinations for KRAS and other to-date undruggable oncogenes. This abstract is also being presented as Poster A40. Citation Format: Kaja Kostyrko, Marcus Kelly, Kyuho Han, Edwin Jeng, David Morgens, Michael Bassik, Peter Jackson, Alejandro Sweet-Cordero. Combinatorial knockout of Rap1GDS1 and RhoA leads to lethality in KRAS-driven non-small cell lung cancer [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 PR07.