Abstract B08: Identification and characterization of Rho family GTPases as drivers of drug resistance in BRAFV600 mutant melanoma

Abstract

Abstract The serine/threonine protein kinase BRAF is mutated in approximately 50% of cutaneous melanomas, leading to hyperactivation of the MAPK/ERK pathway. The most common mutations, BRAFV600, can be targeted by selective kinase inhibitors, such as vemurafenib. Although initial clinical response to BRAF inhibition (BRAFi) is encouraging, 90% of patients develop drug resistance within a few months. Drug resistance can be delayed, but not prevented, by combining BRAFi with an MEK inhibitor (MEKi), such as cobimetinib. While some resistance mechanisms are known, disease progression on drug cannot be explained in all patients. We performed a gain-of-function mutagenesis screen utilizing the Sleeping Beauty transposon system to identify novel drivers of resistance in BRAFV600E mutant melanoma cells sensitive to current therapies. We chose four of the top candidates from our screen and validated the ability to drive resistance to both vemurafenib and vemurafenib-cobimetinib combination treatment in multiple melanoma cell lines. In an effort to determine the broader role of candidate vemurafenib-resistance drivers, we conducted an additional in vivo mutagenesis screen, of which genetic analysis is ongoing. Our initial cell-based screen identified two members of the Dbl family of guanine nucleotide exchange factors (GEFs), VAV1 and MCF2, as candidate drivers of vemurafenib resistance. A375 melanoma cells overexpressing VAV1 or MCF2 maintain significant growth under vemurafenib treatment, while control cells do not. Functional tests of VAV1 and MCF2 identified that the active form of two Rho family members, RAC1 and CDC42, increases following treatment with vemurafenib, suggesting a PAK-mediated pathway of resistance. In addition, all candidates that were tested elevated ERK signaling in the presence of vemurafenib. Many of the extracellular signaling pathways known to drive increased vemurafenib resistance activate Rho signaling. Our results suggest that Dbl family members may play an important role in this process. Understanding how Rho activation occurs and its consequences for drug resistance in melanoma will provide critical insights into the design and validation of future targeted therapies. Citation Format: Jacob L. Schillo, Charlotte R. Feddersen, Afshin Varzavand, Hayley R. Vaughn, Lexy S. Wadsworth, Andrew P. Voigt, Eliot Y. Zhu, Jesse D. Riordan, Christopher S. Stipp, Adam J. Dupuy. Identification and characterization of Rho family GTPases as drivers of drug resistance in BRAFV600 mutant melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B08.