The use of selective BRAF inhibitors (BRAFi) has produced remarkable outcomes for patients with advanced cutaneous melanoma harboring a BRAF V600E mutation. Unfortunately, the majority of patients eventually develop drug-resistant disease. We employed a genetic screening approach to identify gain-of-function mechanisms of BRAFi resistance in two independent melanoma cell lines. Our screens identified both known and unappreciated drivers of BRAFi resistance, including multiple members of the DBL family. Mechanistic studies identified a DBL/RAC1/PAK signaling axis capable of driving resistance to both current and next-generation BRAFis. However, we show that the SRC inhibitor, saracatinib, can block the DBL-driven resistance. Our work highlights the utility of our straightforward genetic screening method in identifying new drug combinations to combat acquired BRAFi resistance.Significance: A simple, rapid, and flexible genetic screening approach identifies genes that drive resistance to MAPK inhibitors when overexpressed in human melanoma cells.

The use of selective BRAF inhibitors (BRAFi) has produced remarkable outcomes for patients with advanced cutaneous melanoma harboring a BRAF V600E mutation. Unfortunately, the majority of patients eventually develop drug-resistant disease. We employed a genetic screening approach to identify gain-of-function mechanisms of BRAFi resistance in two independent melanoma cell lines. Our screens identified both known and unappreciated drivers of BRAFi resistance, including multiple members of the DBL family. Mechanistic studies identified a DBL/Rac1/Pak signaling axis capable of driving resistance to both current and next-generation BRAF inhibitors. However, we show that the Src inhibitor, saracatinib, can block the DBLdriven resistance. Our work highlights the utility of our straightforward genetic screening method in identifying new drug combinations to combat acquired BRAFi resistance. KEYWORDSBRAF inhibitor resistance, cutaneous melanoma, VAV1, MCF2, RAC1, vemurafenib, saracatinib Melanoma is the deadliest form of skin cancer, with around 90,000 diagnoses of invasive disease and ~10,000 deaths per year(1). Patients had few treatment options until the development of vemurafenib, a highly selective kinase inhibitor that specifically targets the BRAF V600E mutant protein present in ~50% of all melanoma cases(2). Initially, vemurafenib provided complete or partial response in over 50% of patients and increased progression-free survival (3). Unfortunately, most patients relapse once tumors acquire resistance to vemurafenib.Genetic analysis of progression samples has identified resistance mechanisms, including amplification of BRAF V600E , expression of truncated BRAF V600E , and RAS mutation (4-6). However, these mechanisms explain only ~60% of cases of BRAF inhibitor (BRAFi) resistance (5,7,8). Drug resistance can be delayed by combining vemurafenib with cobimetinib, a MEK inhibitor (MEKi), but most patients eventually develop progressive disease via resistance mechanisms that have not been well characterized (7). Thus, unexplained cases of resistance to MAPK inhibition (MAPKi) in human melanoma represent an important unmet clinical need.Mechanisms of vemurafenib resistance have been studied in BRAF V600E mutant human melanoma cell lines using genome-wide shRNA and CRISPR loss-of-function screens (9-11). Overall, these studies showed little overlap in candidate mechanisms. Two screens have been reported that attempted to identify drivers of vemurafenib resistance by high throughput overexpression of genes via lentiviral libraries (12,13). Importantly, these screens failed to identify known mechanism of vemurafenib resistance (e.g. BRAF V600E amplification or N-terminal truncation)(4, 6). These observations led us to develop a simple insertional mutagenesis screening approach using the Sleeping Beauty (SB) transposon system to identify novel drivers of vemurafenib resistance in an unbiased forward genetic screen.The SB system is a well-established tool for developing mouse models of spontaneous cancer in which transposo...

<p>Supplemental figures showing more detailed analysis of the genetic screen results (S1-S3), additional experiments showing biological validation of major candidate genes (S4-S7), and characterization of spontaneous Vemurafenib resistance in A375 cells (S8).</p>

<div>Abstract<p>The use of selective BRAF inhibitors (BRAFi) has produced remarkable outcomes for patients with advanced cutaneous melanoma harboring a <i>BRAF^V600E</i> mutation. Unfortunately, the majority of patients eventually develop drug-resistant disease. We employed a genetic screening approach to identify gain-of-function mechanisms of BRAFi resistance in two independent melanoma cell lines. Our screens identified both known and unappreciated drivers of BRAFi resistance, including multiple members of the DBL family. Mechanistic studies identified a DBL/RAC1/PAK signaling axis capable of driving resistance to both current and next-generation BRAFis. However, we show that the SRC inhibitor, saracatinib, can block the DBL-driven resistance. Our work highlights the utility of our straightforward genetic screening method in identifying new drug combinations to combat acquired BRAFi resistance.</p>Significance:<p>A simple, rapid, and flexible genetic screening approach identifies genes that drive resistance to MAPK inhibitors when overexpressed in human melanoma cells.</p></div>

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.