Abstract 6425: Sensitivity of oncogenic KRAS-expressing cells to CDK9 inhibition identified by a phenotypic compound screen
Authors: Lai Lick P. , Brel Viviane , Sharma Kanika , Frappier Julia , Le-Henanf Nadia , Vivet Bertrand , Muzet Nicolas , Schell Emilie , Morales Renaud , Rooney Eamonn , Holderfield Matthew , Lacroix Frederic , Englaro Walter , Marcireau Christophe , Debussche Laurent , Nissley Dwight V. , Mccormick Frank 
Published: September 2020

The normal RAS signaling pathway provides a primary switch whereby cell proliferation is controlled through activation of RAS proteins and downstream signaling. Oncogenic mutants of RAS genes often drive hyper-activation of cell proliferation and signaling resulting in tumorigenesis. Oncogenic forms of KRAS protein are known to be drivers of pancreatic, colorectal and lung cancers. These cancers are particularly lethal and are associated with poor prognosis. The goal of this study is to identify chemical leads that inhibit oncogenic KRAS signaling with phenotypic screen. We first developed an isogenic panel of mouse embryonic fibroblast (MEF) cell lines that carry wild-type and oncogenic RAS. We validated these cell lines by screening against a tool compound library of 1402 annotated inhibitors in the CellTiter-Glo® cell viability assay. We identified previously known specific RAS allele vulnerabilities, confirming the biological properties of these cell lines and their utility in future screens. Subsequently, MEFs from this panel expressing KRASG12D, KRASG12V, KRASG13D, KRASQ61R, HRASWT, and NRASWT, were used to conduct a high throughput screen with a proprietary compound library of about 931,000 compounds in the CellTiter-Glo® cell viability assay. 126 compounds (29 chemical clusters and 10 singletons) showed specific activity against mutant KRAS (IC50: RASWT/mutant KRAS ≥ 5), and were selected for secondary assays including a series of cell-based, biophysical and biochemical assays, in order to prioritize and eliminate compounds with undesired properties. Finally, five chemical clusters were chosen. They have specific activity against SW620 (KRASG12V) and LS513 (KRASG12D) over Colo320 (RASWT) colon cancer cell lines. In addition, they have no effects on BRAFV600E, MEK1, ERK2, PI3K alpha, AKT1, or mTOR as tested in in vitro enzymatic activity assays. Biophysical assays, SPR and DSF, demonstrated that these compounds do not bind directly to KRAS. We further identified the mechanism of action of these five chemical clusters, with three of them having CDK9 inhibitory activity. In conclusion, we have developed and validated an isogenic MEF panel that can be used successfully to identify specific RAS oncogenic or wild-type allele vulnerabilities. Furthermore, we identified sensitivity of oncogenic KRAS-expressing cell to CDK9 inhibitors, which warrants future studies of potential use of CDK9 inhibitors in treating KRAS-driven cancers.

Citation Format: Lick P. Lai, Viviane Brel, Kanika Sharma, Julia Frappier, Nadia Le-Henanf, Bertrand Vivet, Nicolas Muzet, Emilie Schell, Renaud Morales, Eamonn Rooney, Matthew Holderfield, Frederic Lacroix, Walter Englaro, Christophe Marcireau, Laurent Debussche, Dwight V. Nissley, Frank Mccormick. Sensitivity of oncogenic KRAS-expressing cells to CDK9 inhibition identified by a phenotypic compound screen [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6425.