SPOT-007 Identifying novel combinatorial synthetic lethal vulnerabilities in KRAS-driven lung cancer
Kaja Kostyrko, Kyuho Han, Marcus R. Kelly, Edwin E. Jeng, David W. Morgens, Michael Cory Bassik, Peter K. Jackson, Alejandro Sweet-Cordero · ESMO Open · 2018
Introduction KRAS is one of the most frequently mutated genes in human cancer, but the efforts to target KRAS directly have thus far been unsuccessful, highlighting the need for alternative approaches. One promising strategy is to target KRAS through synthetic lethality. However, KRAS activates multiple effector pathways, suggesting that targeting one gene may not be sufficient to fully inhibit KRAS-driven oncogenesis. Therefore, targeting combinations of genes that together are synthetic lethal with KRAS may constitute a better therapeutic approach. Material and methods 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 centred on KRAS. Based on this network we designed a CRISPR 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 expressing Cas9. Knock-out of several 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 5 KRAS mutant and 4 wild type NSCLC cell lines. We also selected six gene pairs that had the most synergistic effect on growth for individual validation in Cas9-expressing NSCLC cells and normal human bronchial epithelial cells (HBECs). The cells were cultured in 3D, which was shown to more faithfully recapitulate important aspects of cancer biology than cells grown as monolayers. Results and discussions Out of the six gene combinations, the simultaneous knock-out of one pair of genes, Rap1GDS1 and RhoA, selectively impaired sphere growth of KRAS-dependent lung cancer cells but had little effect on the growth of KRAS-independent cells or HBECs. Moreover the individual knock-out of these genes had no effect on 3D growth in any of the cell lines, suggesting that only the combination of these two genes is synthetically lethal with KRAS. We are now performing further validation in vivo . Finally, human relevance will be determined using patient-derived xenograft (PDX) models. Conclusion Combinatorial inhibition of Rap1GDS1 and RhoA appears to be synthetically lethal with mutated KRAS and therefore these genes may constitute attractive drug targets for the treatment of KRAS-dependent NSCLC and other KRAS-driven cancers.