KRAS GTPases are activated in one-third of cancers and KRAS G12C is the most common activating alteration in lung adenocarcinoma 1,2 . KRAS G12C inhibitors 3,4 are in Phase-I clinical trials and early data show partial responses in ~50% of lung cancer patients. How cancer cells bypass inhibition, to prevent maximal response to therapy, is not understood. Because KRAS G12C cycles between an active and inactive conformation [4][5][6] , and the inhibitors only bind to the latter, we tested if isogenic cell populations respond non-uniformly by studying the effect of treatment at Reprints and permissions information is available at www.nature.com/reprints.

KRAS GTPases are activated in one-third of cancers and KRAS G12C is the most common activating alteration in lung adenocarcinoma. KRAS G12C-specific inhibitors (G12Ci) are in Phase-I clinical trials and early data show only partial responses in lung cancer patients. How cancer cells bypass inhibition, to prevent maximal responses to therapy, is not understood. Because KRAS G12C cycles between an active and inactive conformation, and the covalent G12Ci only bind to the latter, we tested whether isogenic cell populations respond non-uniformly by studying the effect of treatment at a single-cell resolution. Using single-cell RNA sequencing and a fluorescent quiescence biosensor, we show that shortly after treatment, most cancer cells are sequestered in a quiescent state with low KRAS activity, while a small population reactivates KRAS to resume proliferation. This rapid divergent response is due to synthesis of new, drug-free KRAS protein, resulting from increased KRAS transcription in response to suppressed MAPK signaling. Combining cell fate-specific gene expressions and results from a CRISPR-Cas9 screen, we identified that adaptive signals such as epidermal growth-factor receptor and aurora kinase A signaling modulate the heterogeneous response to treatment with G12Ci. These upstream signals help to maintain new KRAS G12C protein in its active, drug-insensitive state, which restores KRAS signaling and transcriptional output in a subset of cells to allow escape from G12Ci-induced quiescence. Cells without these adaptive changes (or cells where they are pharmacologically inhibited) remain sensitive to G12Ci treatment, because new KRAS G12C is either not available, or it exists in its inactive, drug-sensitive state. Combined inhibition of these adaptive signals along with KRAS G12C produced more potent antitumor effects in xenograft models. The direct targeting of KRAS oncoproteins has been a longstanding objective in precision oncology. Our study uncovers a flexible non-uniform fitness mechanism that enables groups of cells within a population to rapidly bypass the effect of treatment. This adaptive process must be overcome to maximize the therapeutic potential of conformation-specific KRAS G12C inhibitors in the clinic. Citation Format: Jenny Y. Xue, Yulei Zhao, Jordan Aronowitz, Trang T. Mai, Alberto Vides, Besnik Qeriqi, Dongsung Kim, Chuanchuan Li, Elisa de Stanchina, Linas Mazutis, Davide Risso, Piro Lito. Rapid non-uniform adaptation to conformation-specific KRAS G12Cinhibition [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 622.

KRAS GTPases are activated in one-third of cancers and KRAS G12C is the most common activating alteration in lung adenocarcinoma. KRAS G12C inhibitors are in Phase-I clinical trials and early data show only partial responses in lung cancer patients. How cancer cells bypass inhibition, to prevent maximal responses to therapy, is not understood. Because KRAS G12C cycles between an active and inactive conformation, and the inhibitors only bind to the latter, we hypothesized that isogenic cell populations respond non-uniformly. Here we studied the effect of treatment at the single cell level and showed that shortly after treatment, some cancer cells were sequestered in a quiescent state with low KRAS activity, while others reactivated KRAS to resume proliferation. By combining cell fate-specific gene expressions and results from a CRISPR-Cas9 screen, we identified that this rapid divergent response is due to new KRAS G12C produced in response to suppressed MAPK output. Upstream-acting adaptive signals, such as epidermal growth-factor receptor and aurora kinase signaling, maintain new KRAS G12C protein in its active/drug-insensitive state to restore KRAS output. Cells without these adaptive changes (or cells where they are pharmacologically inhibited) remain sensitive to drug treatment, because new KRAS G12C is either not available, or it exists in its inactive/drug-sensitive state. Combined inhibition of these adaptive signals along with KRAS G12C produced more potent antitumor effects in vivo. Our study uncovers a flexible non-uniform fitness mechanism that enables groups of cells within a population to rapidly bypass the effect of treatment. This adaptive process must be overcome to maximize the therapeutic potential of conformation-specific KRAS G12C inhibitors in the clinic. Citation Format: Jenny Xue, Yulei Zhao, Jordan Aronowitz, Trang T Mai, Alberto Vides, Besnik Qeriqi, Dongsung Kim, Chuanchuan Li, Elisa de Stanchina, Linas Mazutis, Davide Risso, Piro Lito. Rapid non-uniform adaptation to conformation-specific KRAS G12C inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A04. doi:10.1158/1535-7163.TARG-19-LB-A04