Activity and specificity of KRAS variants-directed Revolution Medicines Company (RMC) compounds for pleural effusion-derived non-small cell lung cancer cell lines.

Background: The clinical therapy for KRAS G12C mutated lung cancers has been revolutionized by the approval of the covalent inhibitors Sotorasib and Adagrasib. However, the restriction to KRAS G12C, the response rates of approximately 45% and the duration of response (DOR) of approximately 1 year leaves room for improvements. A completely new approach uses intracellular glues to block KRAS variants by steric hindrance through binding of cyclophilin A (CYPA) to the switch regions of KRAS. A series of Revolution Medicines Company (RMC) compounds were developed, targeting all KRAS variants and KRAS wildtype, namely (RMC-6236, daraxonrasib), KRAS G12C (RMC-6291, elironrasib), KRAS G12D (RMC-9805, zoldonrasib) and KRAS G12V (RMC-5217) with more agents under development. This study aimed to reveal the specificity of RMC compounds and to discover underlying resistance mechanisms in real-world tumor cell lines. Methods: Four RMC compounds were tested against a panel of 22 tumor cell lines, including 15 non-small cell lung cancer (NSCLC) lines carrying a range of KRAS mutations that were recently established from pleural effusions of lung cancer patients. Compounds were used in cytotoxicity tests and Western blot arrays comprising oncology-related proteins as well as for KRAS Western blots. Results: The RMC compounds were highly active against the wild-type KRAS cell lines but showed limited activity against the KRAS variant cell lines with approximately 50% of the NSCLC lines surviving even at the highest concentration of 2 µM with. The distinct RMC compounds targeting KRAS G12C, G12D and G12V exhibited poor selectivity for the respective NSCLC cell lines, although phosphoproteome analysis showed a uniform downregulation of the phosphorylation of ERK1/2. Cells exposed to RMC-6236 exhibited increased resistance to this drug and Western blots of KRAS revealed either increased protein expression or a shift of KRAS likely attributed to monoubiquitylation that was reported to increase KRAS activity. Conclusions: The RMC compounds directed at KRAS G12C, D and V mutations exhibit cytotoxic activity against the correspondingly mutated newly established NSCLC cell lines und suppress the MAPK pathway. However, a significant fraction of the cancer cells survives at high drug concentrations and in most cases, the compounds show a poor selectivity for the targeted variant cell lines although they partially contain specific warheads. Increased resistance to RMC-6236 has been associated with elevated expression or likely monoubiquitylation of KRAS. The limited activity of the RMC KRAS(ON) compounds may be supplemented by conventional KRAS(OFF) compounds or other glues or degraders.