Lung cancer is the number one cause of cancer-related deaths worldwide. The most prevalent type of lung cancer is Non-Small Cell Lung Cancer (NSCLC). A significant number of patients with NSCLC carry oncogenic KRAS mutations. However, the efforts to target KRAS directly have thus far proven unsuccessful and tumors harboring mutations in this gene remain the most difficult to treat, highlighting the need for alternative approaches. One promising strategy is to target KRAS-dependent cancers through synthetic lethality. However, KRAS activates multiple effector pathways, suggesting that targeting one gene may not be sufficient to fully inhibit KRAS oncogenesis. Therefore, we propose that targeting combinations of genes that together are synthetic lethal with KRAS may constitute a better therapeutic strategy. Furthermore, we hypothesize that a targeted approach focused on the protein-protein interaction network proximal to KRAS may be more effective than the current emphasis on genome-wide screens. To discover novel, combinatorial KRAS synthetic lethal genes, we used affinity purification/mass spectrometry (AP/MS), to systematically identify KRAS interacting proteins and 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 KRAS-interacting genes. Using this library we simultaneously knocked-out pairs of 119 genes in two KRAS-driven non-small cell lung cancer (NSCLC) cell lines (A549 and H23). Knock-out of many gene pairs synergistically impaired growth of these cells, while the knock-out of each of the genes alone had no or little effect. We chose 20 most promising targets for further screening in vitro and in vivo in a panel of 9 KRAS-mutant and KRAS wild type Cas9-expressing NSCLC cell lines. We also selected six gene pairs that had the most synergistic effect on growth in A549 and H23 cells for individual validation in Cas9-expressing NSCLC cell lines and normal human bronchial epithelial cells (HBECs). We found that the simultaneous knock-out of one pair of genes, Rap1GDS1 and RhoA, significantly decreased growth of KRAS-dependent NSCLC cells, while having a limited effect on KRAS-independent cells or HBECs. Moreover the knock-out of either of these genes alone had no effect on growth in any of the cell lines, suggesting that only the combination of these two genes is synthetically lethal with KRAS. We are currently performing further validation in organoid cultures and in vivo. Additional validation and human relevance will be determined using patient-derived xenografts (PDX).

Citation Format: Kaja Kostyrko, Marcus R. Kelly, Kyuho Han, Edwin E. Jeng, David W. Morgens, Michael C. Bassik, Peter K. Jackson, Alejandro Sweet-Cordero. Identification of novel combinatorial synthetic lethal vulnerabilities in KRAS-driven lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4362.