Src-dependent DBL family members drive resistance to vemurafenib in human melanoma

Abstract

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...

Functional Genomic Screening Independently Identifies CUL3 as a Mediator of Vemurafenib Resistance via Src-Rac1 Signaling Axis

“…The partial loss of RAC1 in the A375 shNT cells did not affect their sensitivity to vemurafenib in a 72h dose response assay ( Figures 7A,B and Supplementary Figure 5D). However, RAC1 knockdown increased sensitivity of these cells to vemurafenib (3 µM) exposure in a 5 day growth assay, consistent with previously reported results (19) (Figure 7C). In contrast, expression of the shRAC1#1 and shRAC1#2 constructs resulted in a moderate increase in vemurafenib sensitivity in A375 CUL3 KD cells with both assays ( Figures 7A-C and Supplementary Figures 5E,F).…”

“…Indeed, constitutively active mutant of RAC1 (RAC1 P29S ) is found in 10% of MAPKi progression melanoma tumors and overexpression of PAK may promote MAPKi resistance in some settings (9,10,(16)(17)(18). Based on our previous work showing that RAC-driven mechanisms of resistance can be ablated with the addition of the Src family inhibitor (Srci) saracatinib (19), we evaluated the effect of vemurafenib in combination with saracatinib. The drug combination decreased the level of pMEK S298 in both A375 NT and CUL3 KD cells compared to vemurafenib alone (Figures 4A,B) whereas it had no effect on pMEK S217 level (Figures 4A,C).…”

Ibrutinib blocks YAP1 activation and reverses BRAFi resistance in melanoma cells

“…Our data show that this is not due to on-target BTK inhibition, but is due to off-target inhibition, presumably of at least one SFK. Other studies have also identified a role for SFKs in BRAFi resistance (69)(70)(71), further supporting the idea that off-target anti-SFK activity of potential melanoma therapeutics may be mechanistically important. One study in particular characterized a novel dual RAF/SRC inhibitor which retains activity against melanoma tumors which had previously developed resistance to dabrafenib/trametinib therapy (72).…”

A simplified transposon mutagenesis method to perform phenotypic forward genetic screens in cultured cells

“…Our first application of this mutagenesis approach was to derive human melanoma cells resistant to the BRAF inhibitor vemurafenib (Fig.2) (Feddersen et al 2019). This screen identified nine candidate genes as drivers of vemurafenib resistance in A375 cells ( Supplemental Table 1).…”

“…Of the nine genes identified by gCIS2, eight were predicted to drive resistance through over-expression and one was predicted to drive resistance via gene disruption ( Supplemental Table 1). Importantly, we have recently described experiments that functionally validated the role of four of these candidates (VAV1, MCF2, BRAF, RAF1) in driving vemurafenib resistance in human melanoma cell lines (Feddersen et al 2019). We next evaluated the eight genes predicted to drive vemurafenib resistance via over-expression, as determined by gCIS2.…”