Palmitoylation of GNAQ/11 is critical for tumor cell proliferation and survival in GNAQ/11-mutant uveal melanoma

Abstract

More than 85% of patients with uveal melanoma (UM) carry a GNAQ or GNA11 mutation at a hotspot codon (Q209) that encodes G protein α subunit q/11 polypeptides (Gα_q/11). GNAQ/11 relies on palmitoylation for membrane association and signal transduction. Despite the palmitoylation of GNAQ/11 was discovered long before, its implication in UM remains unclear. Here, results of palmitoylation-targeted mutagenesis and chemical interference approaches revealed that the loss of GNAQ/11 palmitoylation substantially affected tumor cell proliferation and survival in UM cells. Palmitoylation inhibition through the mutation of palmitoylation sites suppressed GNAQ/11^Q209L-induced malignant transformation in NIH3T3 cells. Importantly, the palmitoylation-deficient oncogenic GNAQ/11 failed to rescue the cell death initiated by the knock down of endogenous GNAQ/11 oncogenes in UM cells, which are much more dependent on Gα_q/11 signaling for cell survival and proliferation than other melanoma cells without GNAQ/11 mutations. Furthermore, the palmitoylation inhibitor, 2-bromopalmitate, also specifically disrupted Gα_q/11 downstream signaling by interfering with the MAPK pathway and BCL2 survival pathway in GNAQ/11-mutant UM cells and showed a notable synergistic effect when applied in combination with the BCL2 inhibitor, ABT-199, in vitro. The findings validate that GNAQ/11 palmitoylation plays a critical role in UM and may serve as a promising therapeutic target for GNAQ/11-driven UM.

Keywords

uveal melanoma / mutant GNAQ/11 / palmitoylation / BCL2 / combination target therapy

1 Introduction

Activating GTPase proteins, including guanine nucleotide binding proteins (G proteins) and RAS superfamily members, plays a pivotal role in the tumorigenesis of various human cancers by transducing aberrant signaling cascades from membrane receptors to downstream effectors [1,2]. Recurrent oncogenic mutations in genes encoding the Gα subunits, GNAQ and GNA11, are highly prominent in uveal melanoma (UM) [3]. UM is the most common primary intraocular malignancy in adults and has a strong tendency for lethal hepatic metastasis with a median survival of less than 6 months and a 5-year survival rate of ~15% [4,5]. Missense mutations in GNAQ or GNA11 at codon glutamine Q209, like Q209L or Q209P, are the initiating genetic alterations that prevail in most patients with UM (~85%) [4]. Currently, radiotherapy and surgical excision are the primary treatment options for localized disease, but no effective targeted therapy is available for UM, especially at advanced stages [6].

Similar to RAS oncoproteins, mutational G proteins are difficult to target by simply reversing the activated GTP-bound state to the inactive GDP-bound state owing to the intrinsic “switch-off” activity of GTPase and the vast intracellular molecular pool of GTP [7]. In this scenario, major efforts to develop targeted therapy for UM have focused on inhibiting these downstream effectors to block Gα_q/11 signaling, including protein kinase C (PKC) [8], mitogen-activated protein kinase (MAPK) [9], focal adhesion kinase (FAK) [10], and yes-associated protein (YAP) pathways [11–13]. However, little progress has been made in developing these monotherapeutic strategies [14]. For instance, patients with metastatic UM failed to respond to the inhibition of extracellular signal-regulated kinase (ERK) [15–18], which prompted different combination therapies, such as co-inhibitions of the MEK and PI3K pathways [19], the PKC and MEK pathways [20], and the FAK and MEK pathways [21], to improve the clinical outcome for patients with UM. However, these targeted therapies are supposed to inhibit early and late mutational events for maximal efficacy [22]. For GNAQ/11 orchestrating most downstream signaling in UM, targeting GNAQ/11 directly is still an ideal candidate to inhibit oncogenic signaling networks. Gα_q/11 inhibitors YM-254890 [23,24] and FR900359 [25,26] mediated the inhibition of Gα subunits to prevent canonical nucleotide exchange, which inactivated downstream signaling, but no available solution for clinical use was found. Thus, novel strategies that target Gα_q/11 are worth developing for UM treatment.

Interfering with protein post-translational modifications (PTMs) is a practical therapeutic approach that can affect oncoproteins in an alternative way aside from targeting catalytic functions [27]. Palmitoylation is one such unique PTM that is present in G proteins, as well as RAS superfamily proteins [28]. Palmitoylation confers substantial membrane-binding abilities to these signal transductors and regulates their subcellular localization and physiologic function [29,30]. Our previous studies demonstrated that palmitoylation is essential for NRAS-induced leukemogenesis by regulating its plasma membrane translocation and signal transduction in vitro and in vivo [31,32]. More recently, we uncovered that the palmitoylation status of GNA13 regulates its protein stability and downstream signaling in human lymphomas [33]. GNAQ and GNA11 can be palmitoylated on two adjacent cysteine residues (Cys 9/10) at their N-terminals, which is sufficient for membrane attachment [28,34,35]. In hypoxic pulmonary arteries, Gα_q palmitoylation on both cysteines is required for Gα_q membrane localization and Ca²⁺ mobilization, which determines its association with the thromboxane receptor [36]. Palmitoylation and interactions with Gβγ subunits are required for the plasma membrane localization of the Gα subunit [37]. Interestingly, Fukata et al. identified DHHC3 and DHHC7 as the major palmitoyl transferases for Gα subunits in HEK293T cells and showed that their knockdown causes GNAQ/11 mislocalization in hippocampal neurons and reduces the efficiency of Gα_q-mediated signaling in HeLa cells [38]. All of these studies demonstrated that Gα_q palmitoylation plays an important role in different physical and pathological processes by regulating membrane attachment and signaling cascades. However, the role of GNAQ/11 palmitoylation in UM has never been fully realized, and whether the inhibition of GNAQ/11 palmitoylation could be applied in UM treatment is unclear.

In this paper, through GNAQ/11 site-directed mutagenesis and pharmacological intervention for palmitoylation, we investigated the therapeutic effects of the inhibition of GNAQ/11 palmitoylation in UM. We found that palmitoylation considerably impaired the capability of driver oncogenes in tumor cell proliferation and survival, especially in UM cells with GNAQ or GNA11 mutations. Moreover, we also discovered that BCL2 inhibitors could cooperate with palmitoylation inhibition in treating UM cells. This finding implies that targeting palmitoylation and BCL2 may serve as an encouraging therapeutic combination for future clinical application.

2 Materials and methods

2.1 Cell lines

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; basal media, China) containing 10% fetal bovine serum (FBS; Moregate, Batch No. 827106). Mutant UM cells (OMM1, OMM2.3, and 92.1) and wild-type human melanoma cells (Mel285, Mel290, and Mum2B; herein called hM) were cultured in RPMI-1640 supplemented with 10% FBS, except for Mel285 (20% FBS). NIH3T3 cells were cultured in DMEM supplemented with 10% bovine serum (BS). All cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂. Mycoplasma contamination was excluded using the Plasmocin (Invivogen, San Diego, CA, USA) and periodically examined using MycoFluor Mycoplasma Detection Kit (Invitrogen, #M7006).

2.2 Compounds

2-Bromopalmitate (2-BP) was purchased from Sigma-Aldrich, Inc. (MO, USA). BCL2 inhibitor venetoclax (ABT-199) was purchased from CSNpharm (Arlington Heights, IL, USA). The BCL2 inhibitor navitoclax (ABT-263) was purchased from Selleckchem (Houston, TX, USA). For the in vitro study, all compounds were dissolved in dimethyl sulfoxide (DMSO) to 10 mmol/L (BCL2 inhibitors) or 100 mmol/L (2-BP) and stored at –20 °C.

2.3 Immunoblot assay

Western blot assays were performed as described previously [39]. HRP-conjugated secondary antibodies were applied and the images were taken with Amersham Imager 600 system (GE America).

2.4 Plasmid construction and transfection

GNAQ/11 cDNA was subcloned into MigR1-YFP to generate plasmids MSCV-HA-GNAQ^WT-YFP (herein called GNAQ^WT) and MSCV-HA-GNA11^WT-YFP (herein called GNA11^WT). All primers are listed in Table S1. Then, the two plasmids were used as templates to generate plasmids MSCV-HA-GNAQ-Q209L-YFP (herein called GNAQ^Q209L) and MSCV-HA-GNA11-Q209L-YFP (herein called GNA11^Q209L), respectively. GNAQ^Q209L was used as the template to generate plasmid MSCV-HA-GNAQ-Q209P-YFP (GNAQ^Q209P). Then, all five plasmids were used as templates to generate the palmitoylation-deficient plasmids MSCV-HA-GNAQ^WT-C9/10S-YFP (herein called GNAQ^WT-CS), MSCV-HA-GNA11^WT-C9/10S-YFP (herein called GNA11^WT-CS), MSCV-HA-GNAQ-Q209L-C9/10S-YFP (herein called GNAQ^Q209L-CS), MSCV-HA-GNA11-Q209L-C9/10S-YFP (herein called GNA11^Q209L-CS), and MSCV-HA-GNAQ-Q209P-C9/10S-YFP (herein called GNAQ^Q209P-CS). Point mutations were generated using the QuikChange site-directed mutagenesis kit by following the manufacturer’s instructions (Agilent Genomics, CA). ShGNAQ/11-resistant plasmids were generated using the same kit. The plasmids were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

2.5 Cell viability assay

Cell growth assays were performed as described previously [39,40]. Cells were cultured in 96-well tissue culture plates (3000–5000 cells/well) and treated with drugs at various concentrations for 72 h. Cell lysis was achieved using the CellTiter-Glo Luminescent Cell Viability Reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The resulting luminescence after 30 min of incubation at room temperature was measured using an Envision plate reader (PerkinElmer, Akron, OH, USA) to complete the assay.

2.6 Short hairpin RNAs

Two distinct shRNA oligonucleotides were designed for knocking down GNAQ/11. Paired oligonucleotides were annealed and inserted into lentiviral expression vectors (pLKO.1-GFP). The GNAQ/11-targeted or non-specific control (NC) shRNA plasmid was co-transfected with the lentiviral packaging vectors, psPAX2 and pMD2G, using Lipofectamine 3000 in HEK293T cells to produce shGNAQ, shGNA11, or NC lentiviruses.

2.7 Synergistic analysis of drug combinations

Cells were plated in triplicate into 96-well plates at 3000–5000 cells per well depending on doubling times. Mixtures of inhibitors were added to the cells according to the planned dose matrices. Cell viability was analyzed 72 h later using CellTiter Glo (Promega, Madison, WI, USA) according to the manufacturer’s instruction. The plates were read in an Envision plate reader (PerkinElmer, Akron, OH, USA). Three technical replicates and three independent biological replicates were performed for each treatment and each cell model. The CompuSyn software (ComboSyn, Paramus, NJ, USA) was used to calculate the combination index (CI) to indicate additive or synergistic effects. CI < 1 was interpreted as synergistic.

2.8 Crystal violet growth assays

Cells were plated in 12-well plates at 10 000–45 000 cells per well. On the second day, drugs were added as single agent or in combinations. Two 2-BP concentrations and three ABT-199 concentrations were prepared, which resulted in six different combinations of 2-BP and ABT-199. The cells were grown before being stained with 0.04% crystal violet/2% ethanol in PBS for 30 min. Photographs of the stained cells were captured.

2.9 In vitro synergy assays

Human melanoma cell lines were transfected with non-targeting or GNAQ/11-targeting shRNA, seeded in triplicates in 96-well plates, and simultaneously treated with serial 1:3 dilutions of ABT-199 (0–10 µmol/L). The effects on cell viability were studied after 72 h of treatment with ABT-199 using the CellTiter Glo Cell Viability Reagent.

2.10 Soft agar colony formation assay

A 2 mL of 0.6% soft agar (incubated at 42 °C) was plated into each well of a 6-well tissue culture plate as the bottom agar. FACS-purified NIH3T3 cell lines expressing GNAQ^WT/GNA11^WT, oncogenic GNAQ/11, or palmitoylation-deficient oncogenic GNAQ/11 were diluted to 1×10³ cells per 3 mL of medium. Triplicate 3 mL cells suspended in 0.3% soft agar (cell dilution + 0.6% soft agar (w/v) at a 1:1 ratio) were added on top of the jellified bottom agar for each type of transduced cell and incubated in a CO₂ incubator at 37 °C. Colonies were counted under a light microscope on day 14 to day 21 after plating.

2.11 Colony formation assay

Human melanoma (mutant UM and wild-type melanoma) cells transfected with various viruses were seeded onto 6-well plates. The cells were subsequently placed in an incubator at 37 °C with 5% CO₂. Cell colonies were grown and then stained with 0.04% crystal violet/2% ethanol in PBS for 30 min. 2-BP was administrated at serial concentrations on the second day after mutant or wild-type human melanoma cells were seeded onto 6-well plates to test the cell proliferation upon inhibition with a palmitoylation inhibitor. Photographs of the stained colonies were captured, and the colony formation assay was performed in duplicate.

2.12 Statistical analysis

All data analysis was performed using GraphPad Prism version 7.03 for Windows (GraphPad Software, CA, USA). The data were analyzed by t-test (*P < 0.05, ** P < 0.01, *** P < 0.001).

3 Results

3.1 Genetic perturbation of palmitoylation sites in GNAQ/11 suppresses tumor transformation in NIH3T3 cells

To investigate the effect of GNAQ/11 palmitoylation on tumorigenesis, we first evaluated tumor transformation activity in NIH3T3 cells by performing focus-forming assays and soft agar colony formation assays. As shown in Fig.1, GNAQ^Q209L initiated spindle-shaped growth and anchorage-independent growth in NIH3T3 cells, which were not observed in NIH3T3 cells that expressed GNAQ^WT, whereas the palmitoylation-deficient GNAQ^Q209L-CS abolished cell malignant transformation (Fig.1). In Fig.1, the colonies generated by NIH3T3 cells expressing GNAQ^Q209L-CS were fewer than those generated by GNAQ^Q209L-expressing cell lines and were almost equal number to those generated by GNAQ^WT-expressing cell lines in soft agar colony formation assays. Moreover, NIH3T3 cells transfected with GNAQ^Q209L were resistant to BS withdrawal after 48 h. However, NIH3T3 cells expressing palmitoylation-deficient GNAQ^Q209L-CS appeared to be apoptotic and died (Fig.1). The RAS/MEK/ERK pathway is activated by oncogenic GNAQ [9]. Cells were serum-starved for 30 h before lysis to minimize the RAS signaling background activated by serum growth factors. As expected, GNAQ^Q209L-CS conferred a reduction in MEK1/2 and ERK1/2 phosphorylation in NIH3T3 cells, whereas these genes exhibited increased phosphorylation in NIH3T3 cells infected with GNAQ^Q209L (Fig.1). In addition, the apoptosis of NIH3T3 cells expressing GNAQ^WT and GNAQ^Q209L-CS in response to BS withdrawal was indicated by obvious PARP (poly ADP-ribose polymerase) cleavage and the reduced amount of the antiapoptotic protein, BCL2 (Fig.1). Similar results were obtained in GNA11 (Fig.1–1D). Overall, these data indicated that palmitoylation plays an important role in the oncogenic GNAQ/11 initiation of cell transformation and signal transduction in NIH3T3 cells.

3.2 GNAQ/11 mutations determine the effect of Gα_q/11 signaling in UM cells

To confirm the function of GNAQ/11 in melanoma tumor, we applied three commonly used GNAQ/11-mutant UM cell lines (OMM2.3 (GNAQ^Q209P), OMM1 (GNA11^Q209L), and 92.1 (GNAQ^Q209L)) and three GNAQ/11 wild-type human melanoma cell lines (Mum2B, Mel290, and Mel285; used as controls) in this study. Notably, Mel285 and Mel290 are widely-used wild-type GNAQ/11-expressing UM cell lines in many ocular cancer research works [8,12,41–43], although they have been genetically shifted to some extent and differ from their primary tumors in chromosome status and several melanocyte markers [44], whereas Mum2B cells are derived from cutaneous melanomas [45]. All these cell lines were infected with shGNAQ/11 lentivirus directed against GNAQ/11 mRNA or infected with control shRNA (NC). As shown in Fig.2, S1A, and S2A, shGNAQ and shGNA11 remarkably knocked down the protein levels of GNAQ and GNA11. MAPK pathway activation is a contributing factor to GNAQ/11-mediated oncogenesis and has an important role in generating cell-proliferation signals [8,46]. GNAQ/11 knockdown considerably reduced the expression levels of p-ERK1/2 in GNAQ/11-mutant melanoma cell lines (Fig.2 and S1A).

The relative viability of the mutant (OMM2.3 and OMM1) and wild-type cells (Mum2B and Mel290) infected with shGNAQ/11 was measured via short-term CellTiter Glo assays (over 5 days) to observe the oncolytic effects of the treatments. Greater antitumor effects are defined as reduced growth rates and increased cell death. As opposed to shGNAQ-infected wild-type melanoma cells (Mum2B and Mel290), the survival rate of the GNAQ-mutated OMM2.3 and GNA11-mutated OMM1 cells decreased sharply after shGNAQ/11 treatment (Fig.2). In long-term colony formation assays, the mutant (OMM2.3 and OMM1) and wild-type cells (Mum2B and Mel290) infected with the indicated shRNA or not were plated and stained with crystal violet after 7–13 days. As shown in Fig.2, shGNAQ/11-mediated GNAQ/11 knockdown notably decreased the number and size of OMM2.3 and OMM1 cell colonies but had little or no effect on the number or size of Mum2B and Mel290 cell coloniess (Fig.2). Consistently, GNAQ/11-mutant melanoma cell lines exhibited downregulated GNAQ/11 and BCL2 levels and increased PARP cleavage (Fig.2 and S1A), but these apoptotic proteins were unaltered in wild-type melanoma cell lines (Fig.2, S1A, and S2A). Interestingly, GNA11 knockdown also slightly inhibited the cell proliferation and colony formation ability of the wild-type cells, which indicates that a subtle difference in sustaining the viability of melanoma cells exists between GNA11 and GNAQ (Fig. S2B and S2C). The results suggest that GNAQ/11 mutation is important for GNAQ/11-mediated cell proliferation and survival, which implies that GNAQ/11 is a suitable node for the targeted therapy of UM.

3.3 Loss of GNAQ palmitoylation inhibits GNAQ-dependent UM cell proliferation

To further assess the effect of defective palmitoylation on the oncogenic potential of GNAQ/11 in UM , we constructed OMM2.3-shGNAQ and OMM1-shGNA11 cell lines using shGNAQ/11 lentivirus to infect OMM2.3 and OMM1 cell lines, respectively. Then the OMM2.3-shGNAQ cell line was overexpressed with palmitoylation-intact GNAQ^Q209P or palmitoylation-deficient GNAQ^Q209P-CS, as well as with palmitoylation-active GNA11^Q209L or palmitoylation-deficient GNA11^Q209L-CS cells, overexpressed in OMM1-shGNA11 cell lines. As previously described, GNAQ/11 downregulation by shGNAQ/11 decreased the levels of p-ERK1/2 and BCL2, increased PARP cleavage (Fig.3 and 3B), and impaired cell viability of mutant UM cell lines. However, as shown by Western blot analysis, GNAQ^Q209P in OMM2.3-shGNAQ cells can also remarkably rescue the expression levels of p-ERK1/2 and BCL2 rather than palmitoylation-deficient GNAQ^Q209P-CS (Fig.3). GNA11^Q209L can substantially rescue the decreased expression levels of p-ERK1/2 and BCL2 mediated by shGNA11 in OMM1 cells rather than the palmitoylation-deficient GNA11^Q209L-CS protein (Fig.3). Additionally, PARP cleavage was absent in the mutant OMM2.3-shGNAQ/OMM1-shGNA11 cells when GNAQ^Q209P/GNA11^Q209L was overexpressed but not absent when GNAQ^Q209P-CS/GNA11^Q209L-CS was overexpressed. Correspondingly, GNAQ^Q209P/GNA11^Q209L overexpression in OMM2.3-NC or OMM1-NC cells increased the BCL2 and p-ERK1/2 levels. By contrast, palmitoylation-deficient GNAQ^Q209P-CS/GNA11^Q209L-CS had little effect on p-ERK1/2 and BCL2 levels (Fig.3 and 3B). However, GNAQ^WT or palmitoylation-deficient GNAQ^WT-CS did not affect these signaling in wild-type cells (Mum2B and Mel290, Fig.3 and 3D). In line with the signaling, as shown in the cell viability (Fig.3) and colony formation assays (Fig. S3A), GNAQ^Q209P in OMM2.3-shGNAQ cells and GNA11^Q209L in OMM1-shGNA11 cells rather than palmitoylation-deficient GNAQ^Q209P-CS or GNA11^Q209L-CS can partially restore cell viability impaired by GNAQ/11 knockdown. However, GNAQ^WT or palmitoylation-deficient GNAQ^WT-CS also had no effect on the growth kinetics of wild-type cells transfected with shGNAQ (Fig.3 and S3A). The results implicate that palmitoylation plays an important role in the tumorigenesis of UM cells carrying oncogenic GNAQ/11.

3.4 2-BP selectively inhibits UM cells that harbor GNAQ mutations

To study the essential role of palmitoylation in tumorigenesis, particularly GNAQ/11 palmitoylation in UM, we tested the antitumor effect of palmitoylation inhibition on melanoma cells using the pan-palmitoylation inhibitor, 2-BP. Mutant UM cells and wild-type melanoma cells were treated with varying amounts of 2-BP for 72 h and subjected to CellTiter Glo assay. As shown in Fig.4, 2-BP exhibited greater antiproliferative effects on UM cell lines carrying mutant GNAQ/11 than on melanoma cells with wild-type GNAQ or GNA11. The IC50 ranged from 2 μmol/L to 35 μmol/L for the mutant cell lines, but the cell lines without GNAQ or GNA11 mutations were insensitive at doses up to 80 μmol/L and higher. Two mutant and two wild-type cells were plated at appropriate densities that can confer these cell lines to grow sustainedly and then treated with different doses of 2-BP on the second day to confirm the selective effect of 2-BP in long-term proliferation assays. After 7–13 days, the cells were stained with crystal violet. The result showed that 2-BP also exerts greater antiproliferative activity on UM cells that harbor GNAQ/11 mutations than on wild-type cells (Fig. S4A). The selective effect of 2-BP on cells with GNAQ or GNA11 mutations was also reflected in the growth curves, which showed that GNAQ/11-mutant cell lines showed less proliferation than wild-type cells after the triple administration of the indicated 2-BP dose for over 6 days (Fig.4 and S5A). These findings suggest that the inhibition of GNAQ/11 palmitoylation by 2-BP can exert an antiproliferative action on UM cells with GNAQ/11 mutations.

Next, to determine the role of GNAQ/11 mutations in signaling repression using 2-BP, we treated the melanoma cell lines with 2-BP at the indicated concentrations for 6 h up to 48 h to test whether 2-BP can suppress MAP-kinase signaling. As expected, ERK1/2 phosphorylation was remarkably suppressed by 2-BP in all three GNAQ/11-mutant cell lines at 3 h to 6 h after administration (Fig.4 and S5B). By contrast, ERK1/2 phosphorylation was not affected by 2-BP in the GNAQ/11 wild-type line (Fig.4 and S5B). In addition, 2-BP decreased the BCL2 level in GNAQ/11-mutant UM cells at 6 h (Fig.4 and S5B), but this trend is more notable at 24 h in OMM2.3 and OMM1 cells or at 48 h in 92.1 cells with an increased level of PARP cleavage (Fig.4, S5C, and S5D). The apoptosis-associated marker Bcl-XL mirrored the change of BCL2 (Fig.4, 4D, and S5B–S5D). However, 2-BP had little effect on apoptosis-associated markers in wild-type cells (Fig.4, 4D, and S5B–S5D). These findings suggest that 2-BP can decrease the MAPK pathway downstream of mutant GNAQ/11 and the BCL2 level in UM cells, which suggests that inhibiting GNAQ/11 palmitoylation can confer antiproliferative activity.

3.5 Elevated BCL2 expression in GNAQ/11-mutant UM cells confers sensitivity to BCL2 inhibitors

According to our data in Fig.3–3D, we noticed that the shGNAQ/11-mediated knockdown of mutant GNAQ/11 reduced the BCL2 levels, whereas the overexpression of mutant GNAQ/11 was associated with elevated BCL2 levels. This finding implicates the potential association between the expression level of BCL2 protein and GNAQ/11 activity. The RNA sequencing data of 80 UM cases from The Cancer Genome Atlas (TCGA) were analyzed to confirm the correlation between BCL2 and oncogenic GNAQ/11 in UM. We found that BCL2 expression and GNAQ/11 expression were positively correlated (Fig.5 and 5B). The expression level of BCL2 in human UM tissues far exceeds the expression levels of other cancer tissues according to the TCGA data (Fig.5). This result indicates the specific role of this protein in UM. Interestingly, 6 out of 448 samples of cutaneous melanoma that harbor GNAQ or GNA11 hotspot mutations also had elevated BCL2 mRNA levels that were comparable with those present in UM cells (Fig.5). Consistently, the level of BCL2 protein in mutant UM cells was remarkably higher than that in wild-type cells (Fig.5). A similar finding was reported in other melanomas with or without GNAQ/11 mutation [47]. These data suggest that elevated BCL2 expression is specifically associated with the presence of oncogenic GNAQ/11. Furthermore, the BCL2 inhibitor ABT-199 or the BCL2/Bcl-XL/W inhibitor ABT-263 exerted greater antiproliferative activity to UM cells that harbor GNAQ/11 mutations (Fig.5 and 5G), which indicates that BCL2 could be an alternative therapeutic target for UM treatment.

3.6 2-BP synergizes with the BCL2 inhibitor ABT-199 in treating GNAQ-dependent UM cells

Next, we further tested the combination treatment of 2-BP and ABT-199 in mutant UM and wild-type melanoma cells. As shown in Fig.6 and S6A, the combination of 2-BP and BCL2 inhibitors remarkably reduced the cell viability of mutant UM cells when compared with 2-BP or BCL2 inhibitors alone. The representative CI values show that the inhibition of GNAQ/11 palmitoylation by 2-BP has a strong synergistic effect with ABT-199 at most of the doses tested in GNAQ/11-mutant UM cells (Fig.6 and S6B). In comparison, the wild-type melanoma cell lines treated with 2-BP showed no sensitivity to ABT-199 without synergistic effects (Fig.6 and S6A). The drug sensitivity of hM-shGNAQ/11 cells to the BCL2 inhibitor compared with control cells was tested to rule out the toxicity or non-specific effect evoked by the pan-palmitoylation inhibitor, 2-BP. As expected, the mutant UM cell lines with knocked down GNAQ/11 were more susceptible to ABT-199 compared with the mutant hM-NC cells (Fig. S7A), but the wild-type hM-shGNAQ cell lines did not show sensitivity (Fig. S7B). The synergistic effect was further confirmed in long-term proliferation assays. These melanoma cell lines showed variable sensitivity to 2-BP or ABT-199, and the combination of the two drugs led to fewer colonies than the application of single agents to mutant UM cells (Fig. S8A). However, wild-type melanoma cells showed no sensitivity to single agents, and the combination of 2-BP and ABT-199 also did not lead to a decrease in wild-type melanoma cell colonies at the limited tested doses (Fig. S8B). Western blot was performed on two human mutant UM cell lines treated with a mock treatment, 2-BP, ABT-199, or a combination of 2-BP and ABT-199 for 16 or 24 h to better understand the synergistic effect of the drug combination. Undoubtedly, 2-BP suppressed the BCL2 level (Fig.6). ABT-199 was unable to suppress BCL2 and p-ERK1/2 and even increased the level of p-ERK1/2 in OMM1 cells. The combination of 2-BP and ABT-199 led to greater suppression of BCL2 and p-ERK1/2 than incubation with ABT-199 alone. Moreover, the synergistic apoptotic effect was indicated by the increased levels of cleaved PARP in OMM2.3 and OMM1 cells. Together, the results suggest the synergistic efficacy conferred by the combined inhibition of BCL2 and GNAQ/11 palmitoylationin vitro.

4 Discussion

Aberrantly activated Gα proteins or RAS superfamily members often contribute to tumor cell proliferation, tumorigenesis induction, and cancer metastasis [48,49]. For most GTPases, lipid modifications, such as farnesylation and palmitoylation, are essential in facilitating membrane attachment and signal transduction [50,51]. Previously, we described palmitoylation as a post-translational modification for targeting NRAS- and GNA13-driven hematological malignancies [31,33]. Encouraged by these findings, we attempted to expand our research scope to solid tumors, such as UM, which is another devastating cancer driven by the constitutive activation of Gα_q/11-mediated signaling. In this study, we found that GNAQ/11 palmitoylation determines the malignant transformation potential of mutant GNAQ/11 and regulates the downstream signaling in NIH3T3 cells. Importantly, we also demonstrated that Gα_q/11 palmitoylation in human UM cell lines is critical for UM cell proliferation and survival, which rely on oncogenic GNAQ/11^Q209 mutations. Furthermore, we uncovered that the inhibitory effect on tumorigenesis imposed by genetic deficiency or pharmacological interference on palmitoylation can be synergistically enhanced by the BCL2 inhibitor, ABT-199, which suggests that coupling palmitoylation inhibition and BCL2 inhibition is a promising combined therapeutic strategy to treat UM with GNAQ/11 mutation.

Palmitoylation is a versatile protein post-translational lipid modification that helps regulate the subcellular localization, trafficking, and protein activity of substrates [51]. In theory, the palmitoylation of G proteins, such as Gα and RAS, may contribute to their association with cellular membranes where physiological signaling is passed on to downstream molecules. Consequently, one of the most striking phenotypic changes for palmitoylation mutant NRAS is its retention within the cytosol and restriction from the plasma membrane [31]. By contrast, Gαq signaling appears to occur in the endomembrane system in the cytoplasm, as the knockdown or chemical inhibition of ARF6 induced the relocalization of GNAQ from the cytoplasm to the plasma membrane with a concomitant decrease in the signal transduction of many GNAQ-mediated pathways [52]. These data challenge the conventional model and raise a new hypothesis that most of the signaling from active GNAQ in UM cells emanates from endocytic vesicular membranes rather than the plasma membrane. In addition, in terms of the enzymatic activity of mutant GNAQ/11, we demonstrated that palmitoylation is indispensable for mutant GNAQ/11 to maintain full functionality in UM cells as shown in Fig.1 and Fig.3. Nevertheless, these inconsistent yet unique findings in GNAQ/11 palmitoylation are worth further studying to elucidate the role of palmitoylation and GNAQ/11 in UM.

In UM, activating mutations in GNAQ/11 promotes the activation of the ERK1/2, p38, JNK, FAK and YAP signaling pathways [10,11,47,53]. Here, we confirmed that GNAQ/11 palmitoylation determines UM cell proliferation through the regulation of downstream Gα_q/11 signaling. The data presented herein clearly showed that the loss of palmitoylation and the subsequent inactivation of ERK1/2 signaling are among the most important signaling events in UM cells. Moreover, we found that GNAQ and GNA11 with hotspot mutations can elevate the expression of BCL2 in UM cells, which eventually conferred drug sensitivity to the BCL2 inhibitor, ABT-199. Interestingly, this result coincides with our previous finding that the loss-of-function mutant GNA13 positively regulated BCL2 accumulation in a palmitoylation-dependent manner in diffuse large B cell lymphoma, where venetoclax (ABT-199) exhibited outstanding efficacy [33]. Additionally, the GNAQ inhibitor, FR900359, moderately decreased the expression of two proapoptotic BCL2 family members, BBC3 and PMAIP1, in GNAQ-mutant cells [54]. The PKC inhibitor, enzastaurin, also reduced the expression of antiapoptotic BCL2 and survivin in GNAQ-mutant cells rather than in wild-type cells [47]. All of these pieces of evidence imply that patients with UM with GNAQ/11 mutations might be suitable for treatment with the BCL2 inhibitor, ABT-199, which is now being tested to extend its application against UM.

Approaches that individually inhibit each arm of an activated oncogenic pathway had been adopted in the past but were proven to be inefficient. This outcome spurred interest in combinatorial trials as an alternative approach. Our work highlights that the combination of palmitoylation inhibitor 2-BP and BCL2 inhibitor ABT-199 dramatically inhibited the proliferation and growth of GNAQ/11-mutant UM cells. Consistently, the mutant UM cells with GNAQ/11 knockdown were more sensitive to treatment with BCL2 inhibitors. The results demonstrate that the combined inhibition of GNAQ/11 palmitoylation and BCL2 had a synergistic effect on the repression of oncogenic proliferation and exhibited superior efficacy compared with singular treatment using either approach alone. For instance, ABT-199 alone cannot decrease p-ERK1/2 and BCL2 protein levels but can decrease these proteins when used in combination with GNAQ/11 palmitoylation inhibition. This finding justifies the synergistic antiproliferation effect demonstrated by increased PARP cleavage.

Several preliminary studies have shown that 2-BP can be applied in some preclinical animal treatments and exert notable synergistic effect with other drugs. For instance, blocking PD-L1 palmitoylation by 2-BP to activate T cell cytotoxicity suppressed the growth of MC38 tumorsin vivo [55]. In another study, Chen and other coworkers found that 2-BP presents synergistic differentiation induction with all-trans retinoic acid (ATRA) in acute promyelocytic leukemic (APL) cells and murine model by targeting RARα, which implies that 2-BP would be a promising candidate for APL therapy, especially for overcoming the ATRA resistance of patients with relapsed APL [56]. Likewise, the application of BCL2 inhibitors induces antitumor activity in xenografts derived from patients with UM [57]. Thus, thein vivo synergistic efficacy of the combination of GNAQ/11 palmitoylation inhibition and BCL2 inhibition in UM is worth testing in the near future.

Together, the findings suggest that the combination of GNAQ/11 palmitoylation inhibition and BCL2 inhibition might be a rational therapeutic strategy for patients with UM with GNAQ or GNA11 mutations.

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