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 signaling. 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.

Figure 1

CYSLTR2 mutation encoding p.Leu129Gln is a hotspot mutation and is mutually exclusive with known drivers in uveal melanoma. (a) OncoPrint of a selection of frequently mutated genes in uveal melanomas from four data sets: TCGA (n = 80), UNI-UDE (n = 22), CRUK (n = 9), and QIMR (n = 25). (b) CoMEt plots of two distinct, mutually exclusive modules in uveal melanoma. Each node is a mutated gene, and the number inside each node is the number of samples with a mutation in that gene. The number on each edge between nodes is edge weight (δ), which represents the fraction of permutations where mutations in the two genes are significantly mutually exclusive. The P value, also known as Φ, represents the mid P value that the genes in the module are mutated in a mutually exclusive manner. Coverage is the percentage of samples with at least one mutation in the genes in the module. (c) A schematic of the pathway activated in uveal melanoma. GPCR (CYSLTR2) activation of G_αq/11 (GNAQ or GNA11) promotes the exchange of GDP for GTP and binding of G_αq/11 to phospholipase C β (PLCβ; PLCB4) to activate cleavage of phosphatidylinositol 4,5-bisphosphate (PIP₂) to produce diacylglycerol (DAG) and inositol triphosphate (IP₃), resulting in subsequent calcium release.

Figure 2

Leu129Gln CysLT₂R exhibits high basal coupling to G_αq. (a) Structural homology model of CysLT₂R based on the structure for PAR1. Transmembrane (TM) segments are labeled. Leu129 (3.43) is shown in orange space fill. Boxed segments are expanded to show residues that interact with position 3.43. The bottom panels show the model rotated by 90° relative to the top panels; the intracellular surface faces outward. (b) Representative 100-s time course of calcium flux with 10 nM LTD₄ added at 20 s (arrow) in HEK293T cells transfected with empty vector or expressing wild-type (WT) or Leu129Gln CysLT₂R. Data are presented as relative fluorescence units (RFU). Error bars, s.e.m. from three biological replicates, each with three technical replicates. (c) LTD₄ concentration-dependent calcium flux in HEK293T cells transfected with empty vector or expressing wild-type or Leu129Gln CysLT₂R. The EC₅₀ (half-maximal effective concentration) for wild-type CysLT₂R is 3.07 nM. Expression of Leu129Gln CysLT₂R causes sustained elevated calcium flux that is unaffected by LTD₄. Data are presented as the percentage of relative fluorescence units at LTD₄ concentrations and correspond to the means ± s.e.m. of seven independent experiments, each carried out in triplicate. (d) Dose-dependent G_αs activation by LTD₄ in HEK293T cells cotransfected with vector encoding RLuc3-EPAC-GFP10 together with empty vector or vector encoding wild-type or Leu129Gln CysLT₂R. An overlay shows activity for a control receptor (calcitonin receptor–like receptor (CLR); cotransfected with the RAMP2 accessory protein) in cells treated with adrenomedullin to cause dose-dependent production of cAMP. Data are expressed as the percentage of cAMP accumulation relative to that observed with forskolin induction and correspond to the means ± s.e.m. from two independent experiments, each carried out in triplicate. (e) Analysis of dose-dependent G_αi activation performed as in d with forskolin pretreatment. An overlay shows activity for a control receptor (C-C chemokine receptor 5 (CCR5)) in cells treated with RANTES to cause dose-dependent inhibition of forskolin-induced cAMP production. Data are expressed as the percentage of cAMP accumulation relative to that observed with forskolin induction and correspond to the means ± s.e.m. from four independent experiments, each carried out in triplicate.

Figure 3

Leu129Gln CysLT₂R promotes TPA-independent growth in vitro and enforces a melanocyte-lineage-specific signature. (a) Cell growth of melan-a cells expressing empty-vector control, wild-type CysLT₂R, or Leu129Gln CysLT₂R and assayed by CellTiter-Glo in the presence or absence of 200 nM TPA for 3 d. The fold increase in growth relative to cell numbers at 0 d is shown and corresponds to means ± s.e.m. from six technical replicates. *P < 0.005. (b) Representative phase-contrast microscopy images of melan-a cells expressing empty-vector control, wild-type CysLT₂R, or Leu129Gln CysLT₂R and grown in the presence or absence of 200 nM TPA for 3 d. Scale bar, 50 μm. (c) Cellular pellets of melan-a cells expressing empty-vector control, wild-type CysLT₂R, or Leu129Gln CysLT₂R and grown in the absence of TPA for 18 d. (d) Relative mRNA levels of melanocyte-lineage-specific genes (Kit, Dct, and Mitf) in melan-a cells for each group in the presence or absence of 200 nM TPA for 18 d as assessed by RT–qPCR. Error bars, s.d. from three technical replicates. *P < 0.05. (e) Cropped immunoblots of CysLT₂R and melanocyte-lineage markers in melan-a cells expressing empty- vector control, wild-type CysLT₂R, or Leu129Gln CysLT₂R and grown in the presence (left) or absence (right) of TPA for 18 d. Full-length immunoblots are presented in Supplementary Figure 5. (f) Relative mRNA expression of melanocyte-lineage-specific genes (DCT, TYRP1, and TYR) in MEL290 cells (a human melanoma cell line) expressing empty-vector control, wild-type CysLT₂R, or Leu129Gln CysLT₂R and grown for 14 d as assessed by RT–qPCR. Error bars, s.d. from three technical replicates. *P < 0.05.

Figure 4

Leu129Gln CysLT₂R promotes tumorigenesis in vivo and enforces a melanocyte-lineage-specific signature. (a) Tumor volume over time of SCID mice subcutaneously injected with melan-a cells expressing empty-control vector, wild-type CysLT₂R, or Leu129Gln CysLT₂R (n = 8 mice per group). Error bars, s.e.m.*P < 0.001. (b) Photograph of six representative melan-a xenograft tumors per group explanted at 32 d after implantation. Scale bar, 1.51 cm. (c) Representative hematoxylin and eosin (H&E) images of melan-a xenograft tumors expressing empty-vector control, wild-type CysLT₂R, or Leu129Gln CysLT₂R at 32 d after implantation. Scale bar, 50 μm. (d) Relative mRNA levels of melanocyte-lineage-specific genes (Mitf, Kit, Dct, Tyrp1, and Tyr) assessed by RT–qPCR in three explanted xenograft tumors for each group. Each data point represents the mRNA level of a single tumor normalized to Rpl27. Statistical significance of P < 0.05 was observed for differences between tumors expressing Leu129Gln CysLT₂R and tumors expressing empty-vector control or wild-type CysLT₂R. (e) Cropped immunoblots of CysLT₂R and melanocyte markers in three explanted melan-a xenograft tumors for each group. Full-length immunoblots are presented in Supplementary Figure 5.

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. (a) Growth curves of melan-a cells expressing wild-type or Leu129Gln CysLT₂R transfected with either scrambled siRNA (siSCR) or siRNA targeting CYSLTR2 (siCYSLTR2) and assayed by CellTiter-Glo. Melan-a cells expressing wild-type CysLT₂R were grown in the presence of 200 nM TPA, whereas cells expressing Leu129Gln CysLT₂R were grown in the presence or absence of 200 nM TPA. All cells were grown for 3 d. The fold increase in growth is shown relative to cell numbers at 1 d and corresponds to the means ± s.e.m. from six technical replicates. *P < 0.05. (b) Representative phase-contrast microscopy images of melan-a cells expressing wild-type or Leu129Gln CysLT₂R transfected with either scrambled siRNA or siRNA targeting CYSLTR2 at day 5 after transfection. Scale bar, 50 μm. (c) Relative mRNA levels of melanocyte-lineage-specific genes (Mitf, Tyrp1, and Tyr) in melan-a cells expressing Leu129Gln CysLT₂R with scrambled siRNA or siRNA targeting CYSLTR2 grown in the presence of TPA. Error bars, s.d. from three technical replicates. *P < 0.05.