Analysis of Alternative mRNA Splicing in Vemurafenib-Resistant Melanoma Cells

SIMPLE SUMMARY: Alternative splicing (AS) is one of the hallmarks of human cancer. One of the most common mechanisms of vemurafenib resistance in malignant melanoma is AS of BRAF, occurring in 15–30% of patients. The aim of our study was to investigate the transcriptome and AS D04landscape in the isogenic BRAF V600E cell line pair SK-MEL-239, where the vemurafenib-resistant derivative expresses a truncated BRAF transcript that lacks the RAS-binding domain. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. AS analysis, by four different tools, DEXSeq, rMATS, ASpli, and LeafCutter, has identified genes enriched for cell motility and melanin synthesis in vemurafenib-resistant cells. Overlapping predictions for all four tools have been experimentally validated. Our study expands the understanding of melanoma drug resistance from a new perspective and supports the need to investigate in detail the aberrant AS landscape in patients with malignant melanoma. ABSTRACT: Alternative mRNA splicing is common in cancers. In BRAF V600E-mutated malignant melanoma, a frequent mechanism of acquired resistance to BRAF inhibitors involves alternative splicing (AS) of BRAF. The resulting shortened BRAF protein constitutively dimerizes and conveys drug resistance. Here, we have analysed AS in SK-MEL-239 melanoma cells and a BRAF inhibitor (vemurafenib)-resistant derivative that expresses an AS, shortened BRAF V600E transcript. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. As there is no consensus approach to analysing AS events, we used and compared four common AS softwares based on different principles, DEXSeq, rMATS, ASpli, and LeafCutter. Two of them correctly identified the BRAF V600E AS in the vemurafenib-resistant cells. Only 12 AS events were identified by all four softwares. Testing the AS predictions experimentally showed that these overlapping predictions are highly accurate. Interestingly, they identified AS caused alterations in the expression of melanin synthesis and cell migration genes in the vemurafenib-resistant cells. This analysis shows that combining different AS analysis approaches produces reliable results and meaningful, biologically testable hypotheses.