Mutations in the serine/threonine kinase BRAF are found in more than 60% of melanomas. The most prevalent melanoma mutation is BRAF(V600E), which constitutively activates downstream MAPK signalling. Vemurafenib is a potent RAF kinase inhibitor with remarkable clinical activity in BRAF(V600E)-positive melanoma tumours. However, patients rapidly develop resistance to vemurafenib treatment. One resistance mechanism is the emergence of BRAF alternative splicing isoforms leading to elimination of the RAS-binding domain. Here we identify interference with pre-mRNA splicing as a mechanism to combat vemurafenib resistance. We find that small-molecule pre-mRNA splicing modulators reduce BRAF3-9 production and limit in-vitro cell growth of vemurafenib-resistant cells. In xenograft models, interference with pre-mRNA splicing prevents tumour formation and slows growth of vemurafenib-resistant tumours. Our results identify an intronic mutation as the molecular basis for a RNA splicing-mediated RAF inhibitor resistance mechanism and we identify pre-mRNA splicing interference as a potential therapeutic strategy for drug resistance in BRAF melanoma.
Bibliographical noteFunding Information:
We thank Paola Scaffidi and Travis Dittmer for advice and technical help, Antoni Ribas for the M397/M397AR cell lines, Meenhard Herlyn for 451Lu/451Lu BR and WM938B/WM983B BR cell lines, Minoru Yoshida for the kind gift of SSA, Plexxikon Inc for PLX4720, Kazunori Koide for MAMB and Liang Cao for the use of the Sector Imager. This work was in part performed at the NCI High-Throughput Imaging Facility. This research was supported by the Intramural Research Program of the National Institutes of Health, NCI, Center for Cancer Research and was funded in part with federal funds from the Frederick National Laboratory for Cancer Research, National Institutes of Health, under Contract HHSN261200800001E to W.K.K.
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