Clinical significance of intronic variants in BRAF inhibitor resistant melanomas with altered BRAF transcript splicing
- Gulietta M. Pupo1, 3,
- Suzanah C. Boyd2, 3,
- Carina Fung2, 3,
- Matteo S. Carlino1, 3, 4,
- Alexander M. Menzies3, 5,
- Bernadette Pedersen1, 3,
- Peter Johansson7,
- Nicholas K. Hayward7,
- Richard F. Kefford1, 2, 3, 5,
- Richard A. Scolyer3, 6, 8,
- Georgina V. Long3, 5 and
- Helen Rizos2, 3Email authorView ORCID ID profile
© The Author(s). 2017
Received: 14 March 2017
Accepted: 2 May 2017
Published: 11 May 2017
Alternate BRAF splicing is the most common mechanism of acquired resistance to BRAF inhibitor treatment in melanoma. Recently, alternate BRAF exon 4–8 splicing was shown to involve an intronic mutation, located 51 nucleotides upstream of BRAF exon 9 within a predicted splicing branch point. This intronic mutation was identified in a single cell line but has not been examined in vivo. Herein we demonstrate that in three melanomas biopsied from patients with acquired resistance to BRAF inhibitors, alternate BRAF exon 4–8 splicing is not associated with this intronic branch point mutation. We also confirm that melanoma cells expressing BRAF splicing variants retain exquisite sensitivity to existing FDA-approved MEK inhibitors.
The serine/threonine kinase BRAF is mutated and constitutively activated in 40–60% of cutaneous melanomas. Selective inhibitors of mutant BRAF, including dabrafenib and vemurafenib, improve the progression-free and overall survival of BRAF-mutant melanoma patients [1, 2]. Despite this activity, acquired resistance develops in most melanoma patients and multiple mechanisms of resistance have been described. Most of these resistance effectors, including alterations in MEK1, BRAF and N-RAS, promote the reactivation of the mitogen activated protein kinase pathway . Alternate splicing of oncogenic BRAF is the most common driver of acquired resistance to BRAF inhibitors and is evident in approximately 30% of resistant melanomas [4–6]. Splice variants of BRAF encode an active kinase, but lack an intact RAS binding domain. These truncated proteins are prone to dimerization and have reduced affinity for the class I BRAF inhibitors, vemurafenib and dabrafenib . Predictably, melanomas expressing BRAF splice variants display MAPK re-activation in the presence of BRAF inhibitors, and retain sensitivity to the inhibition of the downstream kinases MEK and ERK [5, 7].
One mechanism of altered BRAF splicing was recently shown to involve a C-to-G mutation in intron 8 within a predicted splicing branch point, 51 nucleotides upstream of BRAF exon 9 (i.e C > G − 51). This intron 8, variant was sufficient to promote the expression of a BRAF transcript lacking exons 4–8 (BRAF exon 4–8∆; also referred to as BRAF3–9) in a single vemurafenib resistant melanoma cell line . The significance of this intronic mutation was not clinically validated, although we are aware of five patients with acquired BRAF inhibitor resistance driven by the BRAF exon 4–8∆ alternate transcript [4–6].
Predicted effects of −51 mutation in intron 8
Significantly, the expression of BRAF splice variants is insufficient to confer MEK inhibitor resistance in melanoma (Additional file 2: Figure S2). Consequently, BRAFV600-mutant metastatic melanoma patients treated with combination BRAF and MEK inhibitors, rarely develop resistance due to alternate BRAF splicing . In this clinical setting, therefore, the therapeutic potential of splicing modulators, such as spliceostatin A, which inhibit the formation of the BRAF exon 4–8∆ via inhibition of the SF3B1 splicing factor , is diminished.
BRAF inhibitor resistance mediated by alternate BRAF splicing is likely a result of cis-acting alterations that disrupt the use of allele-specific constitutive splice sites. Trans-acting mutations can also affect basal and alternate splicing machinery, but these mutations would be predicted to alter the processing of both wild type and mutant BRAF pre-mRNA. Several studies have now confirmed that the wild type BRAF allele is transcribed and processed correctly in mutant BRAF melanoma, and the shorter, variant BRAF transcripts are derived only from the mutant BRAF allele [4, 5]. Our data show that alternate BRAF exon 4–8∆ splicing in melanomas derived from patients with resistance to BRAF inhibitor monotherapy is not associated with a mutation in the −51 position in intron 8. Regardless of the precise mechanisms altering the splicing of BRAF, downstream MEK and ERK inhibition effectively inhibit the proliferation of BRAF inhibitor resistant melanoma cells expressing BRAF splice variants [5, 7].
All patients included in this study had BRAFV600 mutant metastatic melanoma, and were treated with either dabrafenib or vemurafenib . All patients had a pre-treatment melanoma tissue sample obtained before commencing BRAF inhibitor and a matched progressing melanoma metastasis (Prog). Informed consent was obtained for each patient under approved Human Research Ethics Committee protocols.
BRAF sequence analysis
Sequences were amplified using Taq polymerase (Fisher Biotech) using primers shown in Additional file 3: Table S1. PCR products were purified using QIAquick PCR purification kit (Qiagen, Limburg, Netherlands) followed by Sanger sequencing on the 3730xl DNA Analyser (AGRF, Westmead, NSW, Australia). The Human Splicing Finder system  was used to identify and analyse BRAF splice sites and branch point sites and auxiliary splicing enhancer sequences.
Melanoma cell lines
The short-term patient-derived dabrafenib-resistant melanoma cells, WMD009 and SMU027, as well as SKMel28 parental and the dabrafenib-resistant BR4 cell line  were grown in in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS and glutamine (Gibco-BRL).
Clinicians and Staff at Melanoma Institute Australia, Royal Prince Alfred Hospital Department of Pathology and Crown Princess Mary Cancer Centre, Westmead Hospital.
RAS, HR, GVL and NKH are supported by NHMRC Fellowship programs. This work is supported by Program Grant 1093017 and project grants of the National Health and Medical Research Council of Australia (NHMRC).
Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.
GMP and HR designed the study. CF, SCB, BP, GMP, NKH and PJ performed the experiments. GVL, AMM, MSC, NKH, PJ, RAS, RFK and HR analysed the data. GVL, AMM, MSC, RAS and RFK recruited the patients and provided the reagents/tissues. All authors contributed to the final version of the paper. All authors read and approved the final manuscript.
A.M.M – Travel Support, GlaxoSmithKline; Honoraria GlaxoSmithKline; M.S.C - Honoraria GlaxoSmithKline and Novartis; J.F.T - Travel support, GlaxoSmithKline, Provectus; Consultant Advisor – Roche, GlaxoSmithKline, Provectus; Honoraria – GlaxoSmithKline, Provectus; R.F.K - Consultant Advisor Roche, GlaxoSmithKline and Novartis; R.A.S - Consultant Advisor Roche, GlaxoSmithKline and Novartis; G.V.L - Consultant Advisor Roche, GlaxoSmithKline and Novartis; Travel Support Roche; Honoraria Roche, GlaxoSmithKline.
Consent for publication
Ethics approval and consent to participate
Written consent was obtained from all patients under approved Human Research ethics committee protocols from Royal Prince Alfred Hospital (Protocol X15-0454 & HREC/11/RPAH/444).
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