Abstract 3410: Adaptive resistance mechanisms to KRASG12C specific inhibitors
Authors: Solanki Hitendra Singh , Fang Bin , Welsh Eric A. , Tan Aik C. , Kinose Fumi , Rix Uwe , Haura Eric B. 
Published: September 2020

Covalent inhibitors of KRASG12C mutants allow specific targeting of KRAS driven signaling yet early studies suggest bypass signaling as a mechanism of resistance. Understanding adaptive signaling mechanisms enabling cells to survive KRASG12C inhibition could identify combination therapies and associated predictive biomarkers. We employed KRASG12C mutant lung cancer cell lines and evaluated their sensitivity in 2D cell culture to mimic the resistant state. We combined small molecule targeted agents (N=292 compounds) screens alongside KRASG12C inhibition as well as global mass spectrometry based phosphoproteomics (pY/S/T) to examine adaptive signaling changes. KRASG12C mutant lung cancer cell lines have differential sensitivity to KRASG12C inhibition with ARS-1620. Signaling analysis indicates inhibition of p-ERK and to some degree pAKT after 6 hours with evidence of rebound by 24 hr. To expand our understanding of cell type specific response and pathway reactivation upon KRASG12C inhibition, TMT-based quantitative phosphoproteomics was performed after phosphotyrosine (pY) enrichment in 3 cell lines (H358, Calu1 and H1792) after ARS-1620 treatment (6h and 24h). We identified 2275 human phosphosites in total, corresponding to 1153 proteins in 987 protein groups, out of which 433 phosphosites were differentially expressed (1.5-fold change and p-value &lt; 0.05) between any treatment time point and control. Preliminary analysis revealed some common and cell line specific response to ARS-1620. MAPK1, MAPK3, MET, CTNND1, and VTI1B exhibit the strongest down-regulation, across all time points in all cell lines. We observed 84 phosphosites corresponding to 77 proteins that were more abundant in ARS-1620 treated cells indicating a potential for adaptive resistance signaling. Results from the small molecule screens indicate compounds targeting ERBB/HER family of kinases and PI3K pathway showed better synergistic effects with ARS-1620. Signaling analysis in H358, H23 and H2122 cells revealed more abundant tyrosine phosphorylated and total HER2 and HER3 upon ARS-1620 treatment. Further suppression of pERK and pAKT was observed when afatinib was added to ARS-1620 resulting in more combined effects on cell viability. When compared to more specific EGFR inhibition (erlotinib), afatinib enhanced inhibition of pERK and pAKT by ARS-1620 and had more effects on cell viability. However, in H1792 cells, phosphoproteomics identified increased tyrosine phosphorylation of FGFR1 following ARS-1620 and this correlated with combination effects on cell viability of ARS-1620 with the FGFR inhibitor AZD-4547. Lastly, our results suggest that cells sensitive to combination with either HER TKI or FGFR TKI with ARS-1620 are also demonstrating combination effects with SHP2 inhibition (SHP099). Our combined chemical biology and phosphoproteomics results suggest widespread adaptive signaling changes, some of which are contributing to adaptive resistance. Results suggest a more important role of pan-HER inhibition compared with EGFR inhibition. These data may guide biomarker development strategies, especially those that reflect adaptive signaling responses, to predict clinically effective combination strategies in diverse group of KRASG12C mutant lung cancer.

Citation Format: Hitendra Singh Solanki, Bin Fang, Eric A. Welsh, Aik C. Tan, Fumi Kinose, Uwe Rix, Eric B. Haura. Adaptive resistance mechanisms to KRASG12C specific inhibitors [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 3410.