Combined Inhibition of Gα_q and MEK Enhances Therapeutic Efficacy in Uveal Melanoma.

Abstract

Purpose

All uveal melanoma and a fraction of other melanoma subtypes are driven by activation of the Gα_q pathway. Targeting these melanomas has proven difficult despite advances in the molecular understanding of key driver signaling pathways in the disease pathogenesis. Inhibitors of Gα_q have shown promising preclinical results, but their therapeutic activity in distinct Gα_q mutational contexts and in vivo have remained elusive.

Experimental Design

We used an isogenic melanocytic cellular system to systematically examine hotspot mutations in GNAQ (e.g., G48V, R183Q, Q209L) and CYSLTR2 (L129Q) found in human uveal melanoma. This cellular system and human uveal melanoma cell lines were used in vitro and in vivo xenograft studies to assess the efficacy of Gα_q inhibition as a single agent and in combination with MEK inhibition.

Results

We demonstrate that the Gα_q inhibitor YM-254890 inhibited downstream signaling and in vitro growth in all mutants. In vivo, YM-254890 slowed tumor growth but did not cause regression in human uveal melanoma xenografts. Through comprehensive transcriptome analysis, we observed that YM-254890 caused inhibition of the MAPK signaling with evidence of rebound by 24 hours and combination treatment of YM-254890 and a MEK inhibitor led to sustained MAPK inhibition. We further demonstrate that the combination caused synergistic growth inhibition in vitro and tumor shrinkage in vivo.

Conclusions

These data suggest that the combination of Gα_q and MEK inhibition provides a promising therapeutic strategy and improved therapeutic window of broadly targeting Gα_q in uveal melanoma.

Introduction

Uveal Melanoma (UVM) is the most common intraocular malignancy with approximately 3,000 new cases per year in the U.S. (1,2). Metastatic UVM has a median survival of less than six months and a five-year survival rate of ~15% (3). Unlike cutaneous melanoma, where there has been significant progress in targeted therapy and immunotherapy, there remains no effective systemic therapeutic option for advanced UVM.

UVM is characterized by aberrant activation of the heterotrimeric G-protein alpha-q (Gα_q) pathway, which canonically activates phospholipase Cβ (PLCB) and downstream effectors, including inositol 1,4,5-triphosphate (IP₃), diacylglycerol (DAG), and protein kinase C (PKC) (4). Approximately 90% of UVMs harbor activating mutations in two homologous α subunits of Gα_q GNAQ and GNA11; the remaining cases harbor activating mutations in the upstream G protein-coupled receptor (GPCR) cysteinyl leukotriene receptor 2 (CYSLTR2) (5–7) and downstream target PLCB4 (8). These mutations are not exclusive to UVM, as they have been identified in a majority of blue nevi, leptomeningeal melanocytic neoplasms (LMNs), hepatic small vessel neoplasms, Sturge-Weber syndrome, and in a small subset of cutaneous and mucosal melanomas (7,9–15). The identification of similar genetic aberrations among these diseases demonstrates the significance of this pathway and the need for effective therapeutic options against it.

The MAPK pathway is an important downstream signaling output of Gα_q; prior studies highlighted the high levels of MAPK activity in the absence of canonical drivers found in cutaneous melanoma (6,7,16). Despite this, single agent MEK inhibitors (MEKi) have failed to provide clinical benefit (1,17,18). Recent mechanistic studies identified the RAS guanine exchange factor, RasGRP3, as an essential intermediary between Gα_q and RAS/MAPK signaling (19,20). Inhibition of RasGRP3 reduced ERK signaling and cell growth further implicating the dependence of MAPK signaling in UVM (19). Promising preclinical studies have pointed to the combination of a pan-PKC inhibitor with MEKi as a potential therapeutic approach. However, clinical applications of these compounds have been hampered by toxicity from pan-PKC inhibition and therefore the lack of therapeutic window (1,17,18,21,22). Direct inhibition of mutant Gα_q signaling could potentially circumvent such toxicity and provide an effective therapeutic window and conceivably durable response. The recent development of allele-specific RAS inhibitors revealed mutations in KRAS, another small GTPase can continue to cycle between the GTP-GDP states, albeit at reduced rates compared to wild-type (23). In fact, KRAS^G12C-specific inhibitors, similar to YM, target the GDP-bound state and are efficacious in inhibiting KRAS^G12C-driven cancers (24–26). These studies have shown that direct targeting of a mutant GTPase is not only possible, but highly efficacious in KRAS^G12C-driven cancers.

YM-254890 (YM) is a naturally-occurring cyclic depsipeptide that inhibits platelet aggregation by perturbing Gα_q-mediated Ca²⁺ mobilization (27,28). YM is an allosteric inhibitor that binds to the hydrophobic cleft between two inter-domain linkers of Gα_q, stabilizing the inactive GDP-bound form by hindering the flexibility of the linkers (29). YM inhibition of Gα_q prevents canonical nucleotide exchange; without binding GTP, the protein is unable to activate downstream signaling partners. Studies have shown YM can act as a therapeutic agent for inhibition of platelet aggregation and hypocalcemia (30–32). However, initial studies using overexpression systems indicated YM was unable to inhibit mutant Gα_q^Q209L (27). The lack of activity was hypothesized to be due to the reduced GTPase activity of the Gα_q^Q209L mutant and thereby locking the mutant protein in the GTP-bound state similar to the paralogous Gα_s^Q227L mutant (33). Three recent studies have highlighted the use of FR900359 (FR), an analog of YM, to directly target Q209-mutant Gα_q. These studies demonstrated FR’s ability to inhibit signaling and growth in Q209 mutant UVM cell lines, and efficacy against one human xenograft model (34–36). These reports illustrate direct inhibition of Q209 mutant Gα_q as a potential therapeutic avenue for UVM. However, both YM and FR are potent inhibitors of physiologically active wild-type Gα_q signaling (27–29,37), which raises concerns of therapeutic window of YM and FR in targeting mutant Gα_q in UVM. Thus, discovery of novel synergistic combinations that can potentiate therapeutic efficacy and broaden the therapeutic window of YM or FR is imperative. Furthermore, there remains a need for a comprehensive understanding of direct Gα_q inhibition across the mutational landscape of UVM.

Here, we identified a novel GNAQ mutational hotspot, G48, and showed exogenous expression of GNAQ^G48V, similar to the canonical GNAQ mutants, transformed melanocytes. We demonstrated YM effectively inhibited cellular growth and downstream Gα_q signaling in four UVM mutants, GNAQ (G48V, R183Q, Q209L) and CYSLTR2 (L129Q). We further confirmed the efficacy of YM in a series of in vivo experiments with human UVM cell lines harboring Gα_q^Q209 mutations. Furthermore, transcriptomic and synergistic analysis revealed that together, YM and MEKi led to enhanced and sustained inhibition of MAPK signaling to significantly decrease tumor growth. These results suggest direct Gα_q inhibition provides an effective therapeutic strategy in UVM and synergizes with MEKi to further increase therapeutic potential.

Methods

Study approval

MSK-IMPACT testing for UVM patients was ordered by the treating physician who signed informed consent to research protocol (ClinicalTrials.gov, NCT01775072). All animal studies were performed in accordance to MSKCC IACUC 11–12-029.

Drugs and Chemicals

YM-254890 was purchased from Wako Pure Chemical Industries (CAT# 257–00631). MEK162 (binimetinib) was purchased from Array BioPharma Inc. Trametinib was purchased from Selleck Chemicals (Catalog No. S2673).

Mutational Analysis

For the MSKCC uveal melanoma cohort, all patients provided informed consent for tissue procurement and mutational testing, and the study was approved by the institutional review board (ClinicalTrials.gov, NCT01775072). Patients had a confirmed diagnosis of uveal melanoma. A total of 124 specimens from 116 patients were analyzed including 47 specimens from primary UVM and 77 from metastasis (56 liver, 6 lymph node, 5 lung, 10 other). In the 7 patients with multiple samples, the Gα_q mutational status were identical. Tumor samples were sequenced using MSK-IMPACTv3, v5, or v6 that captures 341, 410, and 468 genes respectively. The MSK-IMPACT panel includes GNAQ and GNA11 in all versions and CYSLTR2 was added to v6.

For integrative analysis of published cohorts, Level 2 whole-exome mutational data were downloaded from the NIH TCGA server. Processed whole-exome sequencing data from the UNI-UDE (n = 22) and MDACC/MEEI (n = 52) uveal melanoma cohorts and processed whole-genome sequencing data from the CRUK (n = 12) and QIMR (n = 28) cohorts were extracted from the supplementary tables of relevant publications (20–24). For downstream analysis, we merged the data for 3 duplicate samples present in both the CRUK and TCGA databases and removed 3 samples from the QIMR database that lacked any somatic mutations, leaving 188 samples. The OncoPrint was generated using MSKCC cBioPortal.

Exogenous Gene Expression

For transient transfection experiments in HEK-293T, we used a synthetic gene for codon optimized CYSLTR2 (5) and WT cDNA for GNAQ (cDNA.org). We performed site-directed mutagenesis to generate mutants Q209L, R183Q and G48V using QuickChange (Agilent Technologies) site-directed mutagenesis. HEK-293T cells were transiently transfected with the plasmids encoding for GNAQ WT and mutants subcloned into pcDNA3.1(+) using Lipofectamine according to the manufacturer’s instructions. Briefly, 7,000 HEK-293T cells were transfected in low-volume 384-well plates with 11 ng of total DNA/well for 24 hours.

For stable expression in melan-a cells, we employed human cDNAs for CYSLTR2 and GNAQ obtained from Origene, mutagenized using QuickChange and cloned into the retroviral vector MSCV-Puro (Addgene plasmid #68469) (38). NRAS^Q61K (Addgene plasmid #49404) and MEKDD (Addgene plasmid #15268) (39) constructs for rescue experiments were purchased from addgene. Retroviral production was performed through transfection of retroviral expression vector with the pCl-Ampho packaging vector with X-tremeGENE 9 (Sigma-Aldrich). QuikChange primers were used to introduce the various mutant constructs. All constructs were confirmed by sequencing. Site directed mutagenesis primers are listed in Supplementary Table S1.

Cell Culture

Mutation dependent melan-a cells were generated by transducing cells with cDNAs in MSCV-PURO (retrovirus) and then selecting cells with puromycin (1μg/ml) for two days. TPA (Sigma-Aldrich) was then withdrawn from cultures. Cells with activating mutations (CysLT₂R^L129Q, GNAQ^{G48V/R183Q/Q209L}, KRAS^G12V, BRAF^V600E, and NRAS^Q61K) continued to proliferate over several passages (>2 weeks). Vector and WT controls were unable to maintain proliferation or pigmentation after a few passages without TPA. All cells were cultured in media containing 10% FBS, L-glutamine (2nM), penicillin (100U/ml), streptomycin (100 μg/ml). Melan-a cells provided by D. Bennett, St. George’s Hospital, University of London were cultured in RPMI supplemented with 200nM TPA unless otherwise noted (40). HEK-293T (from ATCC) and A375 cells were cultured in DMEM and UVM cells were cultured in RPMI. All cells tested negative for mycoplasma. Human UVM cell lines Mel202, OMM1.3, and OMM1 were described previously (19) and MP41 was purchased from ATCC (CRL-3297).

Cell Growth Assays and Dose Response Curves

Cell growth and dose response curves for YM were assayed using CellTiter-Glo 2.0 (Promega) and readout on a Glomax Luminometer (Promega). For growth assays, melan-a cells were seeded in the absence of TPA, 1,000 cells/well in a 96-well plate and then counted on days 1, 3, and 6. Growth assays were baselined to day 1 readings. For dose response curves melan-a and human UVM cells were seeded 1,000–3,500 cells/well in a 96-well plate and treated 24 hours later with YM for 5 days. Data was baselined to vehicle. Growth assays and dose response curves were analyzed and IC₅₀ values calculated using GraphPad Prism 7.0 software. Data shown is representative of at least three independent experiments with at least three technical replicates.

Crystal Violet Growth Assay

Cells were seeded in 12- or 6-well plates, media and drug were replenished every two days. Cells were washed twice with ice cold 1X PBS, fixed with ice cold 100% methanol, and stained with 0.5% crystal violet (sigma) solution in 25% ethanol. Plates were imaged with a GELCOUNT (Oxford Optronix).

Drug Synergy Analysis

Synergy assays were performed in 96 well plates (1,000–3,000 cells/well) for 5 days with Trametinib and YM, readout by CellTiter-Glo 2.0. Viability was baselined to vehicle (100%), input into Combenefit software to obtain HSA synergy and viability plots (41). Data shown is representative of at least three independent experiments with three technical replicates.

Mouse experiments

For melan-a allograft studies, 2.0 × 10⁶ cells were resuspended in 100μL of 1:1 mix of RPMI media and Matrigel (BD Biosciences) and subcutaneously injected into 6–8-week-old C57BL/6J mice (Jackson Laboratory). For Mel202 xenograft studies, 3.0 × 10⁶ cells were injected into 6–8-week-old CB17-SCID mice (Taconic). Tumor sizes were measured twice a week with calipers starting from three weeks post graft and were calculated using the following formula: tumor volume = (D2 × d2× h2)/6, whereby D, d and h refers to long diameter, short diameter and height of the tumor, respectively. Treatment began at a tumor size of ~100mm³. Mice were treated with YM-254890 (daily intraperitoneal injection), binimetinib (twice daily oral gavage), a combination of both drugs, or a matched vehicle (0.5% DMSO in 1X PBS and 1% Carboxymethyl cellulose + 0.5% Tween 80 in ddH₂O, or both, respectively). Mice were treated for five days and then given two days for recovery for the length of the experiment. Mice were euthanized in response but not limited to the following: tumor ulceration, tumors located too close to the trunk of the mice to impede movement and blood flow, and tumor burden. For all experiments, mice were grafted double flank, except Fig. 6A (single flank).

Figure 6.

Combination of YM-254890 and binimetinib in vivo inhibits tumor growth and signaling. (A) Percent tumor volume of individual tumors (left) and averaged (right) of GNAQ^Q209L melan-a allografts treated with vehicle, YM, binimetinib, or Combo for 20 days (mean ± SEM). Origin (Y=100) represents no change in tumor volume. (B) Percent tumor volume of CysLT₂R^L129Q melan-a allografts treated with vehicle, YM, binimetinib, or Combo for 22 days (mean ± SEM, n = 10). (C) Percent photon flux of OMM1.3 xenografts treated with vehicle, YM, binimetinib, or Combo daily for 21 days (mean ± SEM, n = 4–10). Cells were transduced with pBMN for luciferase expression. (D) Western blot of OMM1.3 tumors treated for 4 hours with vehicle, YM, binimetinib, and Combo. (E) Percent tumor volume of Mel202 xenografts treated with vehicle, YM, binimetinib, or Combo for 20 days (mean ± SEM, n = 4–6). (F) Western blot of Mel202 tumors treated for 1 week with vehicle, YM, binimetinib, and Combo. (G) Immunohistochemistry of Mel202 tumors from (Figure 6F) for Ki67, p-ERK, and p-S6R. Scale bar, 50μm for insert (left) and 2mm for whole tumor (right). For all cases P > .05; ns, P < 0.005; **, P < 0.0005; ***, P < 0.0001, ****.

Luciferase Imaging

OMM1.3 cells were transduced with pBMN (CMV-copGFP-Luc2-Puro) for luciferase expression (Addgene plasmid # 80389) (42). OMM1.3 xenografts were setup the same as Mel202 grafts described above. Treatment was started 3 days after grafting, following the methods described above. Mice were injected with 15mg/ml D-luciferin potassium salt (GoldBio) in 1X PBS and imaged 15 minutes later using Xenogen IVIS Spectrum. The raw photon flux was calculated using Living Image 4.4 Software and baselined to day 1 readings.

Western Blotting Analysis

Cell lysates were harvested after indicated treatments as previously described (5,19). For western blots of OMM1.3 tumors, mice were treated once (q.d.) with vehicle, YM, binimetinib, or combo and then scarified four hours later. For western blots of Mel202 tumors, mice were treated with vehicle, YM (q.d.), binimetinib (b.i.d), or combo for a week and then scarified four hours after the last treatment. Tumor lysates were generated as previously described (19). Primary antibodies were incubated at a 1:1,000 dilution, unless noted otherwise. Western blot antibodies are listed in Supplementary Table S2 and raw blots are shown in Supplementary Fig. S7.

Immunohistochemistry

All tissues were fixed at 4°C overnight in 4% paraformaldehyde. Tissue processing, embedding, sectioning, and H&E staining were performed by Histoserv. Staining was done as previously described (19). IHC antibodies and dilutions are listed in Supplementary Table S3.

RNA Isolation and qRT-PCR

RNA was extracted from cells using Trizol (Invitrogen) following the manufacturers standard Trizol extraction protocol. 2 μg of RNA from each sample was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Power SYBR Green PCR Master Mix (Applied Biosystems) was then used for PCR on a QuantStudio 6 Flex System (Applied Biosystems). Expression was normalized to ribosomal protein RPL27. Relative expression of mRNA was plotted as 2^-ΔΔCt and each experiment was performed in triplicate and repeated in at least three independent experiments. qRT-PCR primers are listed in Supplementary Table S4.

Bioinformatics Analysis

TCGA uveal melanoma (UVM) and skin cutaneous melanoma (SKCM) mutational and RNA-seq data were download from cBioPortal (www.cbioportal.org) using the PanCancer Atlas Version. The six SKCM samples with GNAQ or GNA11 Q209L mutations are TCGA-RP-A6K9–06, TCGA-RP-A690–06, TCGA-ER-A3ET-06, TCGA-ER-A3ES-06, TCGA-ER-A2NF-06, TCGA-ER-A2NF-01. All RNA-seq was performed by MSKCC genomics core facility using poly-A capture. The libraries were sequenced on an Illumina HiSeq-2500 platform with 50 bp paired-end to obtain a minimum yield of 40 million reads per sample. The sequence data for all melan-a cells were mapped to the mouse reference genome (GRCm38), whereas, Mel202 and OMM1.3 were mapped to the human reference genome (GRCh38) using STAR v2.330 (43). Gene counts were quantified using STAR to Ensembl gene annotations GRCm38.91 and GRCh38.90 for mouse and human samples, respectively. Counts were r-log transformed using DESeq2 (44). Hierarchical clustering and heatmaps of wild-type melan-a cells grown in TPA and GNAQ^Q209L and KRAS^G12V transduced melan-a cells grown without TPA was performed on r-log transformed genes with STDEV>1.5 using Partek Genomics Suite. K-means clustering and heatmaps of drug treated melan-a and human uveal melanoma cells were performed on r-log transformed, Z-scored (mean = 0, stdev = 1), and then partition clustered (k-means) were performed using Partek Genomics Suite. Sum Z-scores were calculated by adding transformed gene expression data from the specified gene signatures. Gene sets are listed in Supplementary Table S5. Cluster and gene set (Hallmark) enrichment analysis were obtained from https://www.gsea-msigdb.org/gsea/index.jsp and are shown in Supplementary Table S6–S11. Raw and processed data are deposited in Gene Expression Omnibus (GEO) Accession #: GSE152705 and GSE160112.

IP₁ Accumulation Assay

IP₁ concentrations in transfected HEK-293T cells, stably transduced melan-a cells, and uveal melanoma cells were measured using a competitive homogenous time resolved fluorescence (HTRF) assay (CisBio). Briefly, 7,000 transiently transfected HEK-293T cells, 5,000 melan-a cells and 5,000 uveal melanoma cells were seeded in low-volume 384-well plates in 7 μL media for 24 hours prior to YM treatment. Cells were then treated with various concentrations of YM at 37°C for 3 or 24 hours. 1 hour and 45 minutes prior lysis, cells were supplemented with 1X Stimulation Buffer provided by the manufacturer (HEPES 10 mM, CaCl2 1 mM, MgCl2 0.5 mM, KCl 4.2 mM, NaCl 146 mM, glucose 5.5 mM, LiCl 50 mM, pH 7.4) with 0.2% BSA and 50 mM of LiCl (to prevent IP₁ degradation). Following incubation, cells were lysed by addition of 3 μL/well of d2-labeled IP₁ analogue as the fluorescence acceptor and the Terbium cryptate-labeled anti-IP1 mAb as the fluorescence donor, diluted in the kit-supplied lysis buffer. The plates were incubated overnight at RT and time-resolved fluorescence signals were read using the BioTek Synergy NEO plate reader (BioTek Instruments, Winooski, VT) at 620 nm and 665 nm. Results were calculated as a 665nm/620nm signal ratio, and IP₁ concentrations were interpolated from a standard curve prepared using the supplied IP₁ calibrator. Results are shown as IP₁ (nM). Dose response curves and bar graphs were analyzed using GraphPad Prism 7.0 software.

Statistical analysis

All statistical comparisons were done with Graphpad Prism 7.0 software and used a two-tailed Student’s t-test for comparison between groups. Data is shown as the mean ± SEM (unless otherwise noted) from triplicate samples from at least three independent experiments. P less than 0.05 was used to designate significance. Significant differences between groups are indicated by P > .05; ns, P < 0.05; *, P < 0.005; **, P < 0.0005; ***, P < 0.0001, ****.

Results

Gα_q mutations occur at three hotspot residues that are known to perturb GTP hydrolysis

We examined the Gα_q pathway mutations from a series of 116 consecutive patients with UVM seen at Memorial Sloan Kettering Cancer Center (MSKCC) and who had undergone clinical sequencing on the MSK-IMPACT platform (45). Similar to previously reported cohorts, 107 of 116 patients harbored mutually exclusive activating mutations in either GNAQ or GNA11 and two patients had a CYSLTR2^L129Q mutation (Fig. 1A) (5). For GNAQ, in addition to previously known recurrent mutations at amino acids Q209 and R183, one sample harbored a novel G48L mutation. To further evaluate the GNAQ^G48 mutation, we next integrated 188 uveal melanoma patients from 5 published whole-exome or whole-genome uveal melanoma cohorts (8,46–49). We identified two patients with GNAQ^G48L and one patient with a GNAQ^G48V mutation, indicating G48 as a third mutational hotspot in GNAQ (Fig. 1B). The codon G48 resides in the phosphate binding loop (P-loop) of Gα_q and is paralogous to codon G12 in RAS GTPases, which is frequently mutated in various cancers (23). Furthermore, the paralogous G47V mutation in GNAS exhibits constitutive activity (33). Structural studies of active Gα_q illustrate G48 of the P-loop, R183 of switch I, and Q209 of switch II are in spatial proximity adjacent to the nucleotide-binding pocket (Fig. 1C) (50), indicating mutations in G48, like Q209 and R183 would hinder GTPase activity.

Figure 1.

Gα_q mutations occur at three hotspot residues that are known to affect the guanine-nucleotide binding pocket. (A) Oncoprint of Gα_q pathway mutations in 116 UVM patients who have undergone MSK-IMPACT testing. (B) Oncoprint of Gα_q pathway mutations in 188 UVM patients from five published cohorts including TCGA (46), Cancer Research UK (CRUK) (47), QIMR Berghofer Medical Research Institute (8), University of Duisburg-Essen (UNI-UDE) (48) and MD Anderson Cancer Center/Massachusetts Eye and Ear Infirmary (MDACC/MEEI) (49). (C) 3D cartoon structure of GNAQ (left) highlighting Gly48 (magenta) of the P-loop (yellow), Arg183 (green) of Switch 1 (red), and Gln209 (blue) of Switch 2 (red). GDP (orange) and AlF₄^- (grey) are both shown as sticks and the three residues are shown as mesh spheres. Close up view of GTP binding pocket (right). Structure from Protein Data Bank Entry 5DO9 (50). (D) Growth assay for melan-a cells in the absence of TPA for six days. Growth was assayed by Celltiter-Glo 2.0 at D1, D3, and D6. Fold increase in luminescence is shown relative to D1 cell number. (E) Phase contrast images of engineered melan-a cells after TPA withdrawal for 2 weeks (scale bar 100μm). (F) Basal level of IP₁ accumulation in melan-a cells. Vector and WT controls are cultured with TPA whereas the remaining samples were cultured without TPA. (G) Western blot of melanocyte lineage markers (MITF, TRP2/DCT, c-KIT, and RASGRP3) upon TPA withdrawal for 2 weeks. For all cases (mean ± SEM, n = 3), P < 0.0005; ***, vector compared against each condition.

Generation of isogenic melanocytes dependent on mutations found in UVM

To determine the role of distinct UVM driver mutations in a genetically defined context, we stably expressed human cDNAs encoding CYSLTR2^L129Q, GNAQ^G48V, GNAQ^R183Q, GNAQ^Q209L, WT controls, as well as BRAF^V600E, KRAS^G12V, and NRAS^Q61K in melan-a cells (Supplementary Fig. S1A and S1B) (40). BRAF^V600E, KRAS^G12V, and NRAS^Q61K hyperactivate the RAS-RAF-MEK-ERK kinase (MAPK) signaling pathway, distinct from the GPCR-Gα_q-PLCβ signaling axis. Melan-a cells are immortalized mouse melanocytes that require media supplemented with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a DAG analog, for continued proliferation and are characterized by pigmentation and melanocytic morphology (5,40). After TPA withdrawal, melan-a cells expressing empty vector, CYSLTR2^WT or GNAQ^WT lost pigmentation and eventually growth arrested (Fig. 1D and 1E; Supplementary Fig. S1C). However, cells expressing activating mutations of GNAQ and CYSLTR2 exhibited TPA-independent growth and enhanced melanocytic features (e.g. dark pigmentation, enlarged melanosomes). Interestingly, expression of BRAF^V600E, KRAS^G12V, and NRAS^Q61K that hyperactivate MAPK signaling also conveyed TPA-independence but failed to maintain pigmentation (Fig. 1D and 1E; Supplementary Fig. S1C and S1D).

We next examined the ability of Gα_q to stimulate PLCβ and generate IP₃, by measuring accumulation of the IP₃ degradation product IP₁. Melan-a cells expressing CYSLTR2^L129Q and all three GNAQ mutations exhibited enhanced IP₁ accumulation, whereas the CYSLTR2^WT-, GNAQ^WT-, BRAF^V600E-, KRAS^G12V-, and NRAS^Q61K-expressing cells did not, indicating that this cellular system faithfully recapitulates the distinct signaling pathways driven by CysLT₂R and Gα_q oncoproteins (Fig. 1F; Supplementary Fig. S1E). Western blot analysis of melan-a cells following TPA-withdrawal showed that UVM associated CysLT₂R and Gα_q oncoproteins maintained expression of melanocyte markers MITF, c-KIT, TRP2/DCT, and RASGRP3 (UVM specific), whereas wild-type CYSLTR2^WT, GNAQ^WT, KRAS^G12V, and NRAS^Q61K did not (Fig. 1G; Supplementary Fig. S1F). In TCGA datasets, we found these genes (MITF, KIT, DCT, and RASGRP3) were expressed higher in uveal melanoma (UVM) and Gα_q -mutated skin cutaneous melanoma (SKCM) compared to RAS/RAF mutated SKCM (Supplementary Fig. S1G), suggesting that the engineered melan-a cells capture important oncogene-specific biology of human melanomas (5,19).

To determine the transcriptome response to expression of activated Gα_q and RAS/RAF pathway in melan-a cells, we performed RNA-seq in wild-type melan-a cells grown in TPA, melan-a expressing GNAQ^Q209L and melan-a expressing KRAS^G12V cells after TPA-withdrawal. Unsupervised hierarchical clustering showed wild-type cells in TPA were more similar with GNAQ^Q209L cells than KRAS^G12V cells, consistent with direct Gα_q signaling to phospholipase C-beta to generate DAG and IP3 (Supplementary Fig. S1H). To determine pathways activated in these melan-a lines, we performed enrichment analysis of gene ontology (GO), Hallmark, and KEGG gene sets as well as custom gene sets defined by genes differentially expressed between human Gα_q vs BRAF/NRAS/KRAS mutated melanoma from TCGA uveal and cutaneous melanoma datasets (46,51) and by genes differentially expressed in genetically engineered mouse melanoma in Bap1^KO;Gna11^Q209L vs Bap1^KO;BRAF^V600E mice (19). We found that GNAQ^Q209L melan-a cells had increased expression of genes in human Gα_q mutated melanomas and mouse Bap1^KO;Gna11^Q209L tumors while KRAS^G12V melan-a cells had increased expression of genes in human RAS/RAF mutated tumors and mouse Bap1^KO;Braf^V600E tumors (Supplementary Fig. S1I). In addition gene sets including calcium mediated signaling and melanogenesis were strongly enriched in GNAQ^Q209L cells, whereas MAPK signaling was enriched in KRAS^G12V cells (Supplementary Fig. S1I). (19). These data indicate Gα_q activation generates a distinct oncogenic phenotype that maintains melanocyte lineage specification, and is consistent with previous observations in genetically engineered murine models of UVM (19,52). This system of engineered mutant oncoprotein-transformed melanocytes allows for context-relevant systematic evaluation of Gα_q-pathway activating mutations that are not available in human cancer cell lines or patient-derived xenograft (PDX) UVM models.

YM-254890 inhibits mutation dependent melan-a cells in vitro and in vivo

We next determined the ability of YM to inhibit the activity of distinct UVM mutations. Since YM is thought to stabilize the GDP-bound state of Gα_q^WT, we hypothesized CysLT₂R^L129Q would be particularly sensitive to YM. First, we tested the ability of YM to inhibit Gα_q signaling in a panel of HEK-293T cells transfected with oncoprotein cDNA constructs and assayed the dose response of IP₁ accumulation 24-hours following treatment of YM. In this system, CysLT₂R^L129Q and Gα_q^G48V appeared to be most sensitive to YM with subnanomolar potency (Supplementary Fig. S2A). Gα_q^R183Q was ~20-fold less sensitive to YM compared to CysLT₂R^L129Q and Gα_q^G48V, whereas Gα_q^Q209L was the least sensitive to YM and its signaling was incompletely inhibited within 24-hour YM treatment, consistent with previous reports (Supplementary Fig. S2A) (27).

Transient transfection experiments in HEK-293T cells result in non-physiologic overexpression of oncoproteins and can lead to misleading observations. In the more physiologic context of Gα_q pathway mutant-transformed melan-a cells, when we assayed IP₁ after 3 hours of treatment, rapid reduction of IP₁ was observed with UVM oncoproteins except Gα_q^Q209L, which achieved only half-maximal inhibition (Fig. 2A). Interestingly, 24-hour treatment with YM completely inhibited IP₁ accumulation across all UVM activating mutants, including Gα_q^Q209L. CysLT₂R^L129Q and Gα_q^G48V were most sensitive to YM, whereas Gα_q^R183Q and Gα_q^Q209L mutants were approximately 10-fold less sensitive (Fig. 2A). These data suggest activating Gα_q mutants, while prominently GTP-bound, undergo GTP hydrolysis, albeit presumably at decreased levels compared to wild-type, allowing for YM to bind in the Gα_q GDP-state, reminiscent of covalent KRAS^G12C inhibitors (24–26,29).

Figure 2.

YM-254890 inhibits mutation dependent melan-a cell tumor growth and signaling. (A) IP₁ accumulation assays in mutation dependent melan-a cells. Cells were treated with increasing concentrations of YM for 3 hours (left) and 24 hours (right). Data are expressed as IP₁ concentration (nM) (mean ± SEM, n = 3). (B) Western blot analysis of MAPK signaling in melan-a cells treated with 500nM YM at 0, 2, 4, 8, and 24 hours; the last sample for each cell line was also treated with TPA for 24 hours showing rescue of the pathway. (C) Dose response of melan-a cells treated with YM for 5 days at increasing dose and readout by CellTiter Glo 2.0. Data are expressed as the percentage RLU relative to that observed with vehicle. (mean ± SEM, n = 3). (D) Western blot of lysates from CysLT₂R^L129Q melan-a tumors harvested at the indicated time after a single dose YM (7.5mg/kg). (E) Percent tumor volume of CysLT₂R^L129Q and (F) GNAQ^Q209L melan-a allografts treated with vehicle or YM daily. Pictures of representative tumors are vehicle (top) and YM treated (bottom) at the end of treatment (mean ± SEM, n = 6–10). For all cases P < 0.005; **, P < 0.0005; ***, P < 0.0001; ****.

We next assayed the effect of YM on downstream Gα_q signaling, including RASGRP3 phosphorylation by PKC, MAPK signaling by CRAF, MEK, and ERK phosphorylation and cyclin D1 expression that integrates signaling to promote cell cycle progression. YM inhibited these downstream signaling targets, with slower kinetics in Gα_q^R183Q and Gα_q^Q209L expressing cells compared to CysLT₂R^L129Q and Gα_q^G48V expressing cells (Fig. 2B), consistent with the IP₁ biochemical assay. Since TPA is a DAG analog and acts downstream of Gα_q, supplementing it in the media restored downstream signaling targets 24 hours after YM treatment (Fig. 2B). YM was ineffective in inhibiting MAPK signaling in BRAF^V600E, KRAS^G12V, and NRAS^Q61K-expressing cells (Supplementary Fig. S2B), confirming the specificity of YM to Gα_q inhibition. Expressing KRAS^G12V in CysLT₂R^L129Q and Gα_q^Q209L melan-a cells rescued YM-mediated MAPK inhibition (Supplementary Fig. S2E). We next examined if YM could effectively inhibit TPA-independent cell growth in the engineered melan-a cells. Consistent with IP₁ accumulation, all UVM activating mutants were highly sensitive to YM. CysLT₂R^L129Q and Gα_q^G48V cells exhibited low-nanomolar sensitivity to YM, and the Gα_q^R183Q- and Gα_q^Q209L-expressing cells were modestly less sensitive, whereas the BRAF^V600E, KRAS^G12V, and NRAS^Q61K -expressing cells were insensitive (Fig. 2C; Supplementary Fig. S2C). TPA supplementation or KRAS^G12V expression rescued YM-mediated growth inhibition in CysLT₂R^L129Q-expressing cells (Supplementary Fig. S2D).

We performed pilot pharmacokinetic studies suggesting YM has a short serum half-life in mice (data not shown). However, given its potency, YM may engage and inhibit Gα_q more durably. We thus performed a pharmacodynamic study examining downstream signaling in CysLT₂R^L129Q cells allografted into C57BL/6J mice. After a single dose of YM given at 7.5 mg kg⁻¹ intraperitoneally, we observed inhibition of downstream signaling in CysLT₂R^L129Q tumors as early as 30 minutes (Fig. 2D). By 24 hours, a sustained decreased in ERK phosphorylation and cyclin D was observed despite rebound of CRAF phosphorylation. We observed a paradoxical increase in RASGRP3 phosphorylation levels which may indicate feedback activation in this model.

Given these data, we proceeded to dose YM daily and assessed efficacy. We observed YM significantly inhibited tumor growth of CysLT₂R^L129Q or Gα_q^Q209L allografts (Fig. 2E and 2F). Importantly, YM treatment did not cause adverse effects such as weight loss (Supplementary Fig. S2F and S2G). These data suggest YM is a highly selective inhibitor against Gα_q signaling and can effectively inhibit proliferation both in vitro and in vivo.

YM-254890 inhibits growth of human UVM cells and xenograft models

Given the sensitivity of all three activating hotspot mutations to YM, including the most common GNAQ^Q209L mutation, in the melan-a system, we proceeded to study the effect of YM on a panel of human UVM cell lines that harbor GNAQ^Q209L/P or GNA11^Q209L mutations and A375 (BRAF^V600E) cutaneous melanoma cell line as control. UVM cells exhibited high basal activity of IP₁ accumulation, which was inhibited upon YM treatment at 24 hours (Fig. 3A; Supplementary Fig. S3A). Analysis of downstream signaling showed RASGRP3, MAPK output, and cyclin D1 proteins were inhibited in UVM cells, but not in A375 cells, upon treatment with YM (Fig. 3B; Supplementary Fig. S3B). Expressing NRAS^Q61K or MEK^DD in UVM cells completely rescued YM-mediated MAPK inhibition (Supplementary Fig. S3G). In OMM1.3 and Mel270 cells, treatment with YM rapidly inhibited the MAPK pathway while suppression of the intermediary target, p-RASGRP3, required prolonged drug exposure. In contrast, YM treatment in Mel202 cells showed potent inhibition of p-RASGRP3 at early time points, but the MAPK pathway was only mildly inhibited; p-ERK1/2 rebounded by 24 hours. These results indicate UVM cells may have different wiring downstream of Gα_q. Regardless, YM potently and effectively inhibited cell viability in Gα_q mutant UVM cell lines but not A375 cells (Fig. 3C; Supplementary Fig. S3C and S3D). TPA supplementation, NRAS^Q61K, or MEK^DD expression rescued YM-mediated growth inhibition in UVM cells (Supplementary Fig. S3C, S3E, and S3F).

Figure 3.

YM-254890 inhibits human UVM cell signaling and tumor growth. (A) IP₁ accumulation assay in UVM cells and A375 cells (cutaneous BRAF^V600E). Cells were treated with increasing concentrations of YM for 3 hours (left) and 24 hours (right). Data are expressed as IP₁ concentration (nM) (mean ± SEM, n = 3) (B) Western blot analysis of indicated proteins in UVM cells and A375 cells treated with 500nM YM at 0, 2, 4, 8, and 24 hours. (C) Dose response of UVM and A375 cells treated with YM for 5 days at increasing dose and readout by CellTiter Glo 2.0. Data are expressed as the percentage RLU relative to that observed with vehicle. (mean ± SEM, n = 3). (D) Percent photon flux of OMM1.3 xenografts treated with vehicle or YM (2.5mg/kg) for 21 days (mean ± SEM, n = 10). Cells were transduced with pBMN for luciferase expression. Arrow indicates start of treatment at day 3. P < 0.05; *, P < 0.0005; ***, P < 0.0001, ****.

We next used a UVM xenograft model to test the efficacy of YM in vivo. In mice with xenografts of OMM1.3 cells, YM inhibited tumor formation compared to vehicle and no adverse weight loss was observed (Fig. 3D; Supplementary Fig. S3H). Taken together, these data indicate that Gα_q inhibition is effective against human UVM xenografts in vivo, in agreement with previously reported data using FR (53).

Gα_q inhibition and MEKi synergistically sustain MAPK inhibition and suppress growth in UVM models

To gain further mechanistic insight, we compared the transcriptional response to YM with that of trametinib treatment for 4- or 24-hours in the CysLT₂R^L129Q melan-a cells. Perturbed genes were partitioned into six clusters by k-means clustering algorithm (Fig. 4A). Genes downregulated by either YM or trametinib behaved similarly to one another (Clusters 1, 2, 3), whereas upregulated genes were distinct (Clusters 4, 5, 6). We then computed the top five GSEA HALLMARK gene set overlaps with each cluster to better understand the impact of treatment on these clusters (Supplementary Fig. S4A). YM, similar to trametinib, effectively inhibited the cell cycle progression (E2F targets) of CysLT₂R^L129Q cells at 24-hours (cluster 1; Fig. 4B). However, trametinib more potently suppressed KRAS and inflammatory signaling than YM (cluster 2; Fig. 4B; Supplementary Fig. S4A). To further understand the suppression of KRAS genes, we used a 52-gene set comprised of genes rapidly downregulated by MEK inhibition in melanoma cells (54), PRATILAS_MAPK signature. This gene signature was downregulated by both YM and trametinib at 4 hours, but by 24 hours, genes rebounded with YM treatment while trametinib further suppressed this gene signature (Fig. 4C). Specifically, this rebound pattern was seen in MAPK signaling output genes DUSP6 and SPRY2/4 (Fig. 4D). This suggests that YM does not durably suppress MAPK signaling at 24 hours, potentially limiting therapeutic effect.

Figure 4.

Transcriptomics implicates synergistic action between Gα_q inhibition and MEK inhibition in melan-a cells. (A) Heatmap of RNA-seq gene expression from CysLT₂R^L129Q melan-a cells treated with vehicle, YM (100nM), or trametinib (100nM) for 4 or 24 hours (duplicates shown). Data was r-log transformed, Z-scored, and then k-means clustered. Clusters are labeled 1–6 and color coordinated (left). (B) Sum Z-scores of E2F_Targets, KRAS_SIGNALING_UP, and (C) Pratilas_MAPK are shown (top) with heatmaps (bottom) showing expression of individual signature genes (mean ± SEM, n = 2). (D) RNA-seq based expression (log₂ count) of Dusp6, Spry2, and Spry4 (mean ± SEM, n = 2). (E) Viability curves for melan-a cell lines treated with increasing doses of YM in combination with four doses of trametinib (left) and corresponding HSA synergy diagrams (right) (mean ± SEM, n = 3). (F) Growth assays for parental melan-a cells (+TPA) and CysLT₂R^L129Q melan-a cells (-TPA) in the presence of vehicle, YM, trametinib, or Combo for 10 or 26 days, respectively. (G) Western blot analysis of indicated proteins in CysLT₂R^L129Q melan-a cells. Cells were treated with vehicle, YM, trametinib, or Combo for 4, 24, 48, and 72 hours.

This prompted us to ask whether YM could be used in combination with MEKi to prevent potential MAPK rebound and induction. In our melan-a cellular system, we found significant synergy in an HSA assay between YM and trametinib in cells dependent on CYSLTR2 or GNAQ mutants but not in melan-a cells still dependent on TPA (Fig. 4E; Supplementary Fig. S4B). Long term treatment of YM plus trametinib greatly reduced proliferation of CysLT₂R^L129Q cells at 26 days compared to single agent treatments (Fig. 4F). In melan-a cells with TPA, only trametinib showed long term effects on proliferation. Combination treatment in CysLT₂R^L129Q melan-a cells led to rapid MAPK signaling inhibition that was sustained for 72 hours, whereas p-ERK had returned to basal levels in single treatment groups (Fig. 4G). To further investigate these findings, we used UVM cell lines to assess the effect of YM and MEKi on viability, transcriptomics, and signaling. We performed HSA synergy assays on four UVM cell lines and found significant synergy between YM and trametinib (Fig. 5A; Supplementary Fig. S5A). Growth assays in these UVM cell lines showed variable sensitivity to either YM or MEKi, but combination of the two drugs lead to fewer colonies than single agents in all UVM cells (Fig. 5B). However, BRAF^V600E mutant A375 cells did not show synergy and only showed sensitivity to MEKi and combination with YM did not lead to a decrease in colonies (Supplementary Fig. S5B and S5C).

Figure 5.

YM-254890 and MEKi lead to sustained MAPK inhibition in UVM cells. (A) Viability curves for four UVM cell lines treated with increasing doses of YM in combination with four doses of trametinib (left) and corresponding HSA synergy diagrams (right). (mean ± SEM, n = 3). (B) Growth assays of Mel202, OMM1.3, MP41, and OMM1 cells treated with vehicle, YM, trametinib, or Combo. (C) Mel202 and OMM1.3 sum Z scores of Pratilas_MAPK are shown (top) with heatmaps (bottom) showing expression of individual signature genes (mean ± SEM, n = 2). (D) Expression counts (log₂) of DUSP6, SPRY2, and SPRY4 from RNA-seq data for Mel202 and OMM1.3 cells (mean ± SEM, n = 2). (E) Western blot analysis of indicated proteins in Mel202, (F) OMM1.3, and (G) MP41 cells. Cells were treated with vehicle, YM, trametinib, or Combo for 4, 24, 48, and 72 hours.

To better understand the synergistic effect of the combination, we performed RNA-seq in two human UVM cell lines treated with vehicle, single agent YM or trametinib, or a combination of both for 24 hours (Supplementary Fig. S5D and S5E). Similar to the melan-a cells, we observed trametinib suppressed the PRATALIS_MAPK gene signature with a greater effect than YM (Fig. 5C). Combination resulted in more dramatic gene expression changes when compared to single agent or vehicle and also led to greater inhibition of MAPK, cell cycle, and MYC gene sets, with the exception of cell cycle in Mel202 cells (Fig. 5C; Supplementary Fig. S5D and S5E). Specifically, combination led to greater suppression of MAPK signaling output genes DUSP6 and SPRY2/4 (Fig. 5D). This transcriptomic data indicates that YM and trametinib have very similar effects on gene expression, however combination of the drugs further enhances the effects on gene expression.

We next determined the effects of YM, trametinib and combination treatment on signaling. In Mel202 cells, YM treatment alone inhibited RASGRP3, CRAF, and MEK phosphorylation at all time points but downstream ERK and cyclin D inhibition was modest (Fig. 5E). Trametinib treatment alone inhibited ERK phosphorylation at 24 hours with rebound by 48 hours, and combination treatment sustained robust inhibition at all timepoints. Similarly, in OMM1.3 and MP41 cells, YM single agent was very effective at inhibiting RASGRP3, CRAF, and MEK phosphorylation but ERK phosphorylation rebounded by 72 hours (Fig. 5F and 5G). Combination treatment led to sustained inhibition of ERK phosphorylation and cyclin D. Expression of KRAS^G12V in OMM1.3 cells was sufficient to rescue YM-mediated p-ERK inhibition, but not trametinib- or combination-mediated p-ERK inhibition (Supplementary Fig. S5F). These data suggest that Gα_q and MEK inhibition are more effective against UVM cell growth and signaling than single agents, indicating that combination therapy may have an increased clinical benefit.

In vivo combination of YM-254890 and binimetinib inhibits tumor growth and potently suppresses the MAPK pathway

We tested the combination of YM with an FDA-approved MEK inhibitor binimetinib in vivo. Using the Gα_q^Q209L melan-a allograft model, we tested the combination on immunocompetent C57BL/6J mice treated over the course of 20 days. Mice treated with YM or binimetinib alone had a significant inhibition in tumor growth, with about a two-fold increase compared to day one size (Fig. 6A). However, combination treatment led to significant tumor reduction compared to vehicle or single agents (Fig. 6A). Similarly, we found that the combination greatly reduced tumor growth in the CysLT₂R^L129 allograft model. (Fig. 6B). No treatment group resulted in weight loss (Supplementary Fig. S6A and S6B). These experiments show that together, YM and MEKi further reduced tumor burden in allograft models driven by two different activating mutations found in patients.

In the OMM1.3 luciferase xenograft model, single agent treatments did inhibit growth, but combination treatment decreased tumor luminescence by 1000-fold, and we were unable to identify the grafts at the end of treatment (Fig. 6C; Supplementary Fig. S6C). OMM1.3 tumors from mice treated for 4 hours showed that combination potently inhibited both PKC and MAPK signaling (Fig. 6D). Single agent binimetinib inhibited p-ERK but increased p-MEK, typical of MAPK feedback (55), whereas YM treatment potently inhibited RASGRP3 and MAPK signaling despite minimal changes in ERK phosphorylation (Fig. 6D). Immunohistochemistry of these OMM1.3 tumors showed no change in Ki67 staining, most likely due to the short treatment time, but did show a dramatic decrease in p-S6R staining (Supplementary Fig. S6E). We also tested this combination in Mel202 xenografts, which exhibit much slower in vivo growth. YM and binimetinib both showed a tumor reduction of about 30% whereas combination led to a greater than 75% average tumor reduction (Fig. 6E; Supplementary Fig. S6D). Tumors from Mel202 xenografted mice, treated for one-week, displayed low basal levels of activated RASGRP3, CRAF, and MEK but did have measurable p-ERK levels (Fig. 6F). YM treatment decreased p-RASGRP3 and downstream ERK signaling, while p-ERK remained unchanged, whereas binimetinib potently inhibited p-ERK and downstream targets but caused rebound in p-RASGRP3, p-CRAF, and p-MEK. Combination treatment suppressed the reactivation of p-MEK and p-CRAF compared to single agent binimetinib and lead to more potent inhibition of p-ERK and p-P90RSK. Immunohistochemistry of these Mel202 tumors show residual Ki67 and p-S6R staining and no significant p-ERK staining in combination treated mice (Fig. 6G), which corroborates the western blot analysis. These in vivo data indicate that combination treatment in mice is feasible, highly active, and more efficacious than single agent YM or MEKi.

Discussion

Advances in targeted therapy have shown that direct inhibition of mutated oncoproteins, such as EGFR, KIT, KRAS and BRAF can be highly efficacious, due to the high selectivity against the oncoprotein over wild-type. Inhibition of downstream signaling is more challenging and often limited by on-target toxicity and narrow therapeutic window. UVM is molecularly defined by mutational activation of the Gα_q pathway and harbor low mutational burden suggesting effective direct Gα_q inhibition could have high efficacy. Compared to kinases, rational design of drugs against mutant GTPases, such as Gα_q and RAS, have been challenging for a number of reasons: activating mutations in GTPases are enzymatically impaired whereas those in kinases are enzymatically hyperactive; GTPases activate downstream effectors through protein-protein interactions whereas kinases activate effectors through enzymatic modification; the abundance of intracellular GTP also makes it difficult to design high-affinity inhibitors that can directly access and bind to the GTP-binding pocket (56). Therefore, GTPases cannot be targeted through inhibition of enzymatic activity but require allosteric drugs that affect conformation or effector binding. Nature has evolved strategies that target human GTPases, including brefeldin A that inhibits ARF, pertussis toxin inhibits Gα_i, YM from bacteria and FR from the plant Ardisia crenata inhibit Gα_q. In each case, the inhibitor functions through stabilization of the GDP-bound inactive or transitional states, suggesting that active cycling is critical for inhibitor function.

The varying susceptibility to YM across our panel of Gα_q mutants underscores the biochemical differences of these mutants. These novel findings have strong parallels to mutant RAS proteins. Structurally, RAS and Gα_q share homologous mutational “hotspots”, Q61/Q209 and G12/G48, respectively. Interestingly, codon Q61 mutations in KRAS are thought to be more active than those at codon G12, with higher rates of intrinsic nucleotide exchange and a greater reduction of intrinsic hydrolysis (23). This is consistent with our observations of increased IP₁ output for Gα_q^Q209L and could also explain the differences in YM sensitivity we see between Gα_q mutants. The GDP-bound state of Gα_q^Q209L may be short-lived compared to Gα_q^G48V or wild-type Gα_q in CysLT₂R^L129Q cells, making it less susceptible to inhibition by YM. While YM effectively inhibits the panel of mutants both in vitro and in vivo, these biochemical differences require foresight clinically.

As observed in RAS-mutant tumors, allele-specific inhibitors show limited efficacy as monotherapies and will require combinations with other inhibitors (23). As oncogenic mutations in UVM activate RAS proteins through RasGRP3, we hypothesize resistance mechanisms to direct inhibitors of Gα_q could occur through reactivation of the MAPK pathway. In fact, compared to trametinib, we observed YM fail to fully suppress the MAPK gene signature. Therefore, the combination strategy of YM plus MEKi could prevent reactivation and durably suppress MAPK signaling. This strategy may also circumvent some of the disadvantages of targeting wild-type Gα_q, which allows for lower doses of YM and MEKi and broadens the therapeutic window for maximal clinical benefit.

Our work and recent studies highlight the possibility of directly targeting oncogenic Gα_q signaling characteristic of UVM and the importance of pursuing this strategy further (34–36). One important limitation of this study and others is the lack of testing Gα_q inhibition in liver metastasis models of UVM. Although these models are technically difficult and typically require intrasplenic injection of cells it is important to understand Gα_q inhibition efficacy in the liver tumor microenvironment. Recently, complete synthesis of YM as well as FR, and novel analogs of each have been reported (37,57–60) and a large toolbox of Gα_q inhibitors will likely soon be available.

In summary, we showed cells harboring activating mutations at one of three residues in GNAQ, as well as wild-type Gα_q driven by CYSLTR2^L129Q were exquisitely sensitive to YM treatment. Transcriptomic and synergy analysis revealed combination of YM with MEKi provides an efficacious and durable response. Our work demonstrates that combination of YM and MEKi leads to enhanced reduction in tumor growth and signaling in UVM, making it an ideal treatment strategy to pursue clinically.

Translational Relevance.

All uveal melanoma and a fraction of other melanoma subtypes are driven by activation of the Gα_q pathway. There are no clinically efficacious systemic therapies for these melanomas. Recent work has shown promising preclinical activity of natural compounds that directly inhibit Gα_q, but their clinical use is potentially limited by narrow therapeutic window from inhibition of normal Gα_q. Here, we describe synergistic efficacy of the Gα_q inhibitor YM-254890 and clinically used MEK inhibitors in engineered models driven by distinct mutations in Gα_q and in CYSLTR2 as well as in multiple human uveal melanoma cell lines. Our work suggests that the combination leads to sustained inhibition of the MAPK pathway and synergistic growth inhibition. Combination treatment in vivo led to increased MAPK pathway inhibition and tumor shrinkage. Together, this work nominates combination of Gα_q and MEK inhibition as a strategy for targeting Gα_q driven melanoma clinically.