Abstract PR17: Sleeping Beauty mutagenesis reveals a Src-dependent DBL GEF family signaling mechanism driving MAPK inhibitor resistance in BRAF mutant melanoma
Authors: Feddersen Charlotte R. , Schillo Jacob , Varzavand Afshin , Vaughn Hayley , Voight Andrew , Zhu Eliot , Riordan Jesse D , Stipp Christopher S. , Dupuy Adam J. 
Published: June 2022

Abstract
BRAF/MEK inhibition remains an important treatment option for patients with BRAF V600 mutant melanoma who show disease progression on immunotherapy; however, the majority of patients treated with BRAF/MEK (MAPKi) therapy develop MAPKi-resistant disease within two years of initiating treatment. Genomic analysis of drug-resistant melanomas has identified somatic mutations associated with resistance, including BRAF amplification or truncation and RAS mutation. In addition, other forms of acquired resistance involving adaptation and reprogramming have been described, with different studies identifying different drivers in different melanoma cell lines and patient-derived xenografts. The heterogeneity of acquired resistance mechanisms presents a major clinical challenge for identifying novel drug combinations with potential activity in significant subsets of patients. To enable the rapid identification of context-dependent drivers of MAPKi resistance, we adapted our Sleeping Beauty (SB) transposon mutagenesis for use in cell culture. SB mutagenesis in this setting identifies predominantly gain-of-function mechanisms and thus has the ability to identify both genetic mutations (such as expression of truncated proteins) and genes within alternative acquired resistance pathways whose upregulated expression drives MAPKi resistance. Validating our approach, our screen identified N-terminal truncation of BRAF—a known mechanism of vemurafenib resistance. In addition, we identified DBL family guanine exchange factors (GEFs), MCF2 and VAV1, as drivers of MAPKi resistance that we have functionally validated in multiple BRAF V600E mutant melanoma cell lines. Crucially, many DBL family GEFs are well known to be regulated by Src family kinases (SFKs), and our data indicate that GEF-driven MAPKi resistance can be blocked by combination treatment with vemurafenib and saracatinib, a selective SFK inhibitor. Expansion of resistant cells in the presence of vemurafenib or vemurafenib with cobimetinib can be reversed by switching to vemurafenib plus saracatinib. Consistently, we find that adding saracatinib converts MAPKi-mediated cytostasis into cell killing and blocks spontaneous MAPKi resistance in responsive melanoma cell lines. The GEFs signal downstream to a RAC1-PAK kinase module that is required for GEF-driven resistance. Importantly, the potential utility of both SFK inhibitors and PAK inhibitors for overcoming MAPKi resistance has been proposed, but the connection between SFK-dependent and PAK-dependent resistance mechanisms had not been obvious. We show that combined BRAF/SFK inhibition is effective in cell lines in which the GEF-driven mechanism operates, but not in cell lines in which the GEFs do not drive resistance. Our SB mutagenesis system has the ability to rapidly survey the context-dependent landscape of MAPKi resistance in any transfectable cell system, with the potential to reveal connections between seemingly disparate resistance mechanisms.
This abstract is also being presented as Poster B04.
Citation Format: Charlotte R. Feddersen, Jacob Schillo, Afshin Varzavand, Hayley Vaughn, Andrew Voight, Eliot Zhu, Jesse D Riordan, Christopher S. Stipp, Adam J. Dupuy. Sleeping Beauty mutagenesis reveals a Src-dependent DBL GEF family signaling mechanism driving MAPK inhibitor resistance in BRAF 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 PR17.