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The three RAS genes are the most commonly mutated oncogenes in cancer and have long been considered an “undruggable” target. Alterations in KRAS occur at the highest frequency, with G12D, G12V, and G12C mutations being the most common. The recent development of novel covalent inhibitors selective for the KRASG12C mutation offers the unprecedented opportunity to target KRAS directly. However, prior efforts to target the RAS-MAPK pathway in have been hampered by adaptive feedback, which drives pathway reactivation and resistance. We find evidence of rapid adaptive RAS-MAPK pathway feedback reactivation following KRASG12C inhibition in the majority of KRASG12C models, including lung, colon, and pancreatic lines. Reactivation of the RAS-MAPK pathway correlates with activation of multiple upstream RTKs and subsequent increase of wild-type RAS activity. We find that co-inhibition of SHP2, a common node downstream of RTKs, or co-inhibition of individual RTKs such as EGFR or FGFR enhances the efficacy of KRASG12C inhibition both in vitro and in vivo by overcoming adaptive reactivation of the RAS-MAPK pathway. Notably, only the combination of KRASG12C and SHP2 inhibitor treatment displays tumor regressions in vivo in multiple models when compared to either inhibitor alone. We also see evidence that combining KRASG12C inhibitors with MEK or ERK inhibitors as an additional potential vertical inhibition strategy in KRASG12C models. We propose a vertical inhibition strategy anchored with KRASG12C inhibitors with either RTK or SHP2 inhibitors as a potential strategy for the treatment of KRASG12C mutant tumors. Citation Format: Meagan B Ryan, Ferran Fece de la Cruz, Sarah Phat, David T Myers, Heather A Shahzade, Catriona B Hong, Ryan B Corcoran. Vertical inhibition overcomes adaptive resistance to KRASG12C 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 A129. doi:10.1158/1535-7163.TARG-19-A129

Clonal heterogeneity associated with the emergence of acquired resistance to therapy presents a critical challenge for therapeutic strategies to overcome resistance. We investigated the molecular landscape of acquired resistance in BRAF-mutant colorectal patients treated with BRAF inhibitor combinations. Through whole-exome sequencing of paired pre-treatment and post-progression tumor biopsies and targeted sequencing of pre-treatment and post-progression plasma circulating tumor DNA (ctDNA), we identified 14 unique alterations in MAPK pathway components driving acquired resistance, affecting KRAS, NRAS, BRAF, MEK1 and MEK2. Analysis of ctDNA at the time of disease progression revealed profound tumor heterogeneity associated with acquired resistance, with multiple concurrent resistance alterations detectable in ctDNA in individual patients, with one patient harboring as many as 8 co-existing resistance alterations. These findings necessitate development of a strategy capable of simultaneously overcoming multiple heterogeneous resistance mechanisms. To evaluate potential strategies for convergent targeting of multiple concurrent resistance alterations, we generated individual resistant models harboring the full spectrum of clinically observed mutations driving acquired resistance. Individually, these alterations drove resistance to BRAF inhibitor combinations currently in clinical trials by maintaining MAPK signaling. However, since acquired resistance is thought to arise from pre-existing clones that emerge during treatment, we developed a novel pooled clone model system to study clonal outgrowth under the selective pressure of therapy. In this system, each resistant clone was pooled at an abundance of 1% in a background of the sensitive parental cells and exposed to an array of potential therapies, both in vitro, and in vivo as xenografts. The change in clonal abundance from baseline to the completion of therapy was assessed for each clone by digital PCR to measure the degree of clonal outgrowth. Moreover, in vitro, we were able to monitor clonal dynamics in real-time by isolating cell-free DNA (cfDNA) from the cell culture media every 3-4 days during therapy. We observed rapid outgrowth of resistant clones during BRAF-EGFR and BRAF-MEK therapy, and delayed, but robust outgrowth during BRAF-MEK-EGFR therapy, all of which are therapies that have been evaluated in recent clinical trials. However, ERK inhibitor alone, and to a greater degree BRAF-ERK and BRAF-ERK-EGFR combinations markedly abrogated the clonal outgrowth of resistant clones. Moreover, in xenograft tumors derived from clonal pools, BRAF-ERK-EGFR triple combination resulted in profound tumor regressions and completely prevented the outgrowth of all resistant clones. In conclusion, we observed the potential for profound heterogeneity of acquired resistance mechanisms in BRAF-mutant colorectal cancer patients, with multiple alterations observed in ctDNA from individual patients. Our data suggest that convergent, upfront therapy with RAF-ERK or RAF-ERK-EGFR inhibitor combinations may suppress outgrowth of clones harboring clinically observed resistant alterations, offering the potential for improved clinical outcome. Citation Format: Mehlika Hazar-Rethinam, Marianna Kleyman, G. Celine Han, David Liu, Leanne G. Ahronian, Heather A. Shahzade, Lifeng Chen, Aparna R. Parikh, Jill N. Allen, Jeffrey W. Clark, Eunice L. Kwak, Jason E. Faris, Janet E. Murphy, Theodore S. Hong, Emily E. Van Seventer, Brandon Nadres, Catriona B. Hong, Joseph M. Gurski Jr., Nicholas A. Jessop, Dora Dias-Santagata, A. John Iafrate, Eli M. Van Allen, Ryan B. Corcoran. Modeling convergent therapeutic strategies to overcome the heterogeneity of acquired resistance in BRAF-mutant colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A34.