Supplementary Figures and Tables from Molecular Landscape of Acquired Resistance to Targeted Therapy Combinations in BRAF-Mutant Colorectal Cancer

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posted on 2023-03-31, 00:29 authored by Daniele Oddo, Erin M. Sennott, Ludovic Barault, Emanuele Valtorta, Sabrina Arena, Andrea Cassingena, Genny Filiciotto, Giulia Marzolla, Elena Elez, Robin M.J.M. van Geel, Alice Bartolini, Giovanni Crisafulli, Valentina Boscaro, Jason T. Godfrey, Michela Buscarino, Carlotta Cancelliere, Michael Linnebacher, Giorgio Corti, Mauro Truini, Giulia Siravegna, Julieta Grasselli, Margherita Gallicchio, René Bernards, Jan H.M. Schellens, Josep Tabernero, Jeffrey A. Engelman, Andrea Sartore-Bianchi, Alberto Bardelli, Salvatore Siena, Ryan B. Corcoran, Federica Di Nicolantonio

Supplementary Tables S1-S3 - List of drug concentrations to generate resistant cell lines (S1); Drug concentrations applied in the screening depicted in Figure 6 (S2); Primers for gene amplification and sequencing (S3). Supplementary Figure S1. Synergistic activity of targeted therapy combinations in BRAF mutant colorectal cancer cells. Supplementary Figure S2. Amplification of mutant BRAF V600E confers resistance to combined EGFR and MEK targeting in colorectal cancer cells. Supplemental Figure S3. Cytotoxicity induced by ERK inhibition in VACO432 resistant to BRAF combination therapies.

Funding

AIRC

Fondo per la Ricerca Locale

MIUR

Ministero della Salute

Fondazione Oncologia Niguarda Onlu

NIH

NCI

Seventh Framework Programme

European Union's Horizon

IMI

International Translational Cancer Research

Entertainment Industry Foundation

Dutch Cancer Society

Fondazione Umberto Veronesi

History

ARTICLE ABSTRACT

Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1. These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504–15. ©2016 AACR.

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Keywords

Drug Mechanisms Cellular responses to anticancer drugs Drug Resistance Novel mechanisms Drug Targets Cell surface receptor drug targets Protein kinase & phosphatase drug targets Gastrointestinal Cancers Colorectal cancer Small Molecule Agents

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