Abstract B03: Biologic and biochemical interactions of NF1 GAP on KRAS G13x mutations

Abstract

KRAS mutations are common among 30% of cancers and promote tumor growth by constitutively activating the MAPK pathway independent of mitogenic stimuli. KRAS mutations at codons 12, 13, or 61 stabilize the KRAS-GTP complex by preventing GAP protein-stimulated GTP hydrolysis. There are two major RAS-GAP proteins, NF1 and p120, and genetic deletions or inactivating mutations of NF1 GAP also constitutively activate RAS proteins and the MAPK pathway by increasing cellular RAS-GTP levels, but KRAS and NF1 mutations are thought to be functionally redundant and rarely occur in the same tumor. Surprisingly, results from clinical trials in mCRC patients with KRAS G13D mutations suggest that inhibition of upstream receptor tyrosine kinases may be effective in a subset of patients, but the underlying mechanism has never been explained. Here, we show that approximately 50% of cell lines with KRAS codon 13 mutations also carry NF1 mutations, but NF1 mutations are rarely observed in cell lines with KRAS codon 12 or 61 mutations. We hypothesize that presence or lack of a functional NF1 determines the response to EGFR inhibitors. Here we used SW48 isogenic KRAS-mutated cell lines to show that presence of NF1 sensitizes KRAS wild-type and KRAS G13D mutated cells to EGFR inhibition, and NF1 knockdown rescues proliferation in KRAS G13D but not KRAS G12D cells. Furthermore, introducing NF1 into G13D mutated cancer cells impairs KRAS signal transduction, suggesting that NF1 may be active in KRAS G13x mutated cells. To test this directly, we used biochemical assays to study G13D KRAS mutants with NF1. The intrinsic GTPase activity of KRAS is very low, and NF1 or p120 GAP accelerates GTP hydrolysis of KRAS wild-type significantly. NF1 and p120 GAP are inactive on KRAS G12D. However, NF1, but not p120 GAP, notably stimulates GTP hydrolysis of KRAS G13D. These results demonstrate that KRAS G13D is partially responsive to NF1-GAP stimulated hydrolysis and suggest that KRAS G13x cancers that have a functional NF1 may respond to EGFR inhibition. Citation Format: Dana Rabara, Robert Stephens, Matthew Holderfield. Biologic and biochemical interactions of NF1 GAP on KRAS G13x mutations [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 B03.