Abstract LB-A34: Modeling convergent therapeutic strategies to overcome the heterogeneity of acquired resistance in BRAF-mutant colorectal cancer
Authors: Hazar-Rethinam Mehlika , Kleyman Marianna , Han G. Celine , Liu David , Ahronian Leanne G. , Shahzade Heather A. , Chen Lifeng , Parikh Aparna R. , Allen Jill N. , Clark Jeffrey W. , Kwak Eunice L. , Faris Jason E. , Murphy Janet E. , Hong Theodore S. , Seventer Emily E. Van , Nadres Brandon , Hong Catriona B. , Gurski Joseph M. , Jessop Nicholas A. , Dias-Santagata Dora , Iafrate A. John , Van Allen Eli M. , Corcoran Ryan B. 
Published: August 2018

Abstract
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.