Abstract 619: Comprehensive cell line profiling of monomer- and dimer-selective small molecule RAF inhibitors uncovers determinants of cellular responses
Authors: Kooijman Jeffrey J. , Koerdi Awan Al , van der Leeuw Sjoerd B. , Kluitmans Daphne J. , van den Bossche Esmee , Dylus Jelle , de Roos Jeroen A. , Melis Janneke J. , Willemsen-Seegers Nicole , Zaman Guido J. 
Published: March 2024

Dysregulation of the Mitogen-Activated Protein Kinase (MAPK) pathway is a frequent event in many cancers. The RAF family of kinases, which includes ARAF, BRAF, and CRAF (RAF1), plays a key role in MAPK signaling. Of the three isoforms, only BRAF is currently targeted by approved RAF inhibitors. These inhibitors were designed to target BRAF-mutant variants that act as oncogenic monomers. However, BRAF-monomer inhibitors can induce activation of wild-type RAF by the formation of dimers, resulting in hyperproliferative lesions of BRAF wild-type cells. Additionally, BRAF-monomer inhibitors do not target RAF-dimers, although this is a potential vulnerability of many MAPK-driven cancers. Novel inhibitors are in development to circumvent these limitations. Further insights into the precise targeting and mechanism of action of RAF inhibitors can help in determining the most promising avenues for drug development. We performed extensive cellular profiling experiments with three approved and five clinical stage inhibitors of RAF to gain more insight into the differences and cellular targeting of these agents. The inhibitors were tested in cell viability assays on a panel of 130 human cancer cell lines from diverse tissue origins. Cell lines were exposed to dose ranges of each inhibitor spanning four logarithmic orders, and viability was determined by measurement of intracellular ATP. Effects of compound on cell viability were related to genomic, transcriptomic, and proteomic status using bioinformatics, with the aim to identify predictive biomarkers for drug response, and to determine selective targeting of MAPK-driven cell lines. Additionally, the primary mechanism of cellular inhibitor activity was determined by relating cell line responses to gene dependency data from large CRISPR knockout screens.

Our results showed striking similarity in the overall inhibitory profile of BRAF-monomer inhibitors and the vemurafenib-derivative and paradox breaker plixorafenib, which all selectively targeted BRAF-mutant cell lines. However, whereas vemurafenib increased cell viability in RAS-mutant cell lines at high drug concentrations, this effect was greatly reduced for plixorafenib. Western blot analysis of phosphorylated MEK and ERK levels in RAS-mutant cell lines treated with BRAF-monomer inhibitors confirmed MAPK pathway activation as cause of the increased cell viability. In addition, RAF-dimer inhibitors potently targeted BRAF-mutant cell lines, as well as several KRAS- and NRAS-mutant cell lines. Lastly, correlation analyses of cell line profiling data with CRISPR knockout data revealed the preferential targeting of RAF1-dependent cell lines by the dual RAF/MEK inhibitor avutometinib. The results of our analyses shed light on the intricacies of cellular targeting by RAF inhibitors and provide insights to guide the development of new RAF inhibitors.

Citation Format: Jeffrey J. Kooijman, Awan Al Koerdi, Sjoerd B. van der Leeuw, Daphne J. Kluitmans, Esmee van den Bossche, Jelle Dylus, Jeroen A. de Roos, Janneke J. Melis, Nicole Willemsen-Seegers, Guido J. Zaman. Comprehensive cell line profiling of monomer- and dimer-selective small molecule RAF inhibitors uncovers determinants of cellular responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 619.