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Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma

Abstract

Uveal melanomas are molecularly distinct from cutaneous melanomas and lack mutations in BRAF, NRAS, KIT, and NF1. Instead, they are characterized by activating mutations in GNAQ and GNA11, two highly homologous α subunits of Gαq/11 heterotrimeric G proteins, and in PLCB4 (phospholipase C β4), the downstream effector of Gαq signaling1,2,3. We analyzed genomics data from 136 uveal melanoma samples and found a recurrent mutation in CYSLTR2 (cysteinyl leukotriene receptor 2) encoding a p.Leu129Gln substitution in 4 of 9 samples that lacked mutations in GNAQ, GNA11, and PLCB4 but in 0 of 127 samples that harbored mutations in these genes. The Leu129Gln CysLT2R mutant protein constitutively activates endogenous Gαq and is unresponsive to stimulation by leukotriene. Expression of Leu129Gln CysLT2R in melanocytes enforces expression of a melanocyte-lineage signature, drives phorbol ester–independent growth in vitro, and promotes tumorigenesis in vivo. Our findings implicate CYSLTR2 as a uveal melanoma oncogene and highlight the critical role of Gαq signaling in uveal melanoma pathogenesis.

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Figure 1: CYSLTR2 mutation encoding p.Leu129Gln is a hotspot mutation and is mutually exclusive with known drivers in uveal melanoma.
Figure 2: Leu129Gln CysLT2R exhibits high basal coupling to Gαq.
Figure 3: Leu129Gln CysLT2R promotes TPA-independent growth in vitro and enforces a melanocyte-lineage-specific signature.
Figure 4: Leu129Gln CysLT2R promotes tumorigenesis in vivo and enforces a melanocyte-lineage-specific signature.
Figure 5: CYSLTR2 is required for the growth and maintenance of the melanocyte-lineage-specific signature in melan-a cells transformed to express Leu129Gln CysLT2R.

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Acknowledgements

We gratefully acknowledge the members of the Molecular Diagnostics Service in the Department of Pathology and the Marie-Josée and Henry R. Kravis Center for Molecular Oncology. We thank T. Wiesner (MSKCC and Medical University of Graz) for the melan-a and MEL290 cell lines and intellectual input, D. Abramson for intellectual input, C. Kandoth and the TCGA for data sharing, M. Berger and N. Bouvier for obtaining patient DNA, Y.A. Berchiche for the flow cytometry analysis and scientific input, and M. Bouvier (Université de Montréal) for the kind gift of the EPAC construct. This work was supported in part by grants from the NIH to P.C. (P50CA140146, CDA; DP2CA174499; K08CA151660), the NIH to Y.C. (K08CA140946), the Sidney Kimmel Foundation to P.C. (Kimmel Scholar Award), the Cycle for Survival Fund to P.C. and Y.C., the Geoffrey Beene Award to P.C. and Y.C., the Prostate Cancer Foundation to Y.C., the STARR Cancer Consortium to P.C. and Y.C., and the François Wallace ç Monahan Fellowship to E.C.

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Authors and Affiliations

Authors

Contributions

Project planning and experimental design: P.C., Y.C., A.R.M., T.P.S., M.A.K., and E.C. Bioinformatic analysis of exome sequencing data: Y.C., A.R.M., M.T.C., and B.S.T. Clinical specimen acquisition, annotation, and analysis: A.N.S. Immunoblots, growth curves, qPCR, and all cellular assays: A.R.M., E.C., and Y.G. Xenograft assays: A.R.M., J.J.S., and E.G.W. Generation of the expression vectors: J.Q.Z., E.C., and A.R.M. Molecular modeling: T.H. Manuscript writing: A.R.M., P.C., Y.C., E.C., and T.P.S. All authors reviewed the final manuscript.

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Correspondence to Yu Chen.

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Integrated supplementary information

Supplementary Figure 1 Raw sequencing reads of CYSLTR2 mutations.

(a) IGV view and wild-type and mutant read count (right) of sequencing reads around the CYSLTR2 p.Leu129Gln (c.386T>A) mutation of germline DNA, tumor DNA, and tumor RNA (when available) of four mutant samples from next-generation sequencing. The wild-type T allele is in red, and the mutant A allele is in green. Note that the mutant reads are found in tumor DNA and RNA but not in germline DNA. (b) Sanger sequencing trace of tumor and germline DNA from an MSKCC patient with uveal melanoma and is wild-type for GNAQ and GNA11.

Supplementary Figure 2 CYSLTR2 mutations in TCGA data sets.

(a) Prevalence of CYSLTR2 mutations in TCGA cohorts. (b) Location of all CYSLTR2 mutations in TCGA cohorts. Two amino acids, Leu129 and Arg136, are mutated in more than one sample. There are three p.Leu129Gln mutations, all in uveal melanoma (red arrow), two p.Arg136His mutations (one colorectal and one adrenal cortical), and one p.Arg136Cys mutation (colorectal) (blue arrow). (c) Gene expression of CYSLTR2 in wild-type and mutant samples in selected TCGA cohorts. Data show that, other than p.Leu129Gln in uveal melanoma, CYSLTR2 is not highly expressed in mutated samples in other cancer types. A blue arrow indicates samples with Arg136 mutation. A red arrow indicates samples with p.Leu129Gln mutation. (d) Total mutational count of tumor samples in selected TCGA cohorts in CYSLTR2 wild-type and mutated samples. This highlights the low mutational burden of uveal melanoma. A blue arrow indicates samples with Arg136 mutation. A red arrow indicates samples with Leu129Gln mutation.

Supplementary Figure 3 HEK293T expression of wild-type and Leu129Gln CysLT2R.

(a) Immunoblotting for the N-terminal FLAG tag, C-terminal 1D4 tag, and -tubulin in HEK293T cells transfected with empty-vector, wild-type, or Leu129Gln CysLT2R. (b) Representative image of immunofluorescence for 1D4 (green) and FLAG (red) in HEK293T cells transfected with wild-type CysLT2R, or Leu129Gln CysLT2R. Hoechst (blue) was used to counterstain the nucleus. Scale bar, 10 m. (c) Flow cytometry analysis of live transfected HEK293T cells using FLAG antibody against the extracellular FLAG tag showing the respective cell surface expression of the receptor.

Supplementary Figure 4 Effect of Leu129Gln CysLT2R on tumorigenesis and TPA independent of cell growth.

(a) Tumor volume of mice implanted with NIH3T3 cells expressing empty-vector, wild-type CysLT2R, Leu129Gln CysLT2R, or Arg136His CysLT2R (n = 10). Error bars, ±s.e.m. *P < 0.005. (b) Photographs of cellular pellets of melan-a cells expressing emptyvector, wild-type CysLT2R or Leu129Gln CysLT2R grown in the absence of TPA for 4 and 10 d.

Supplementary Figure 5 Uncropped immunoblots.

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Supplementary Figures 1–5 and Supplementary Table 1. (PDF 1325 kb)

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Moore, A., Ceraudo, E., Sher, J. et al. Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma. Nat Genet 48, 675–680 (2016). https://doi.org/10.1038/ng.3549

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