Research ArticleDRUG DEVELOPMENT

Targeting nucleotide exchange to inhibit constitutively active G protein α subunits in cancer cells

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Science Signaling  04 Sep 2018:
Vol. 11, Issue 546, eaao6852
DOI: 10.1126/scisignal.aao6852

Targeting mutant Gα

Activating mutations in G protein α subunits cause various diseases, including some forms of uveal melanoma (UM), an aggressive eye cancer. Onken et al. found that the plant-derived compound FR900359 blocked the growth of Gα-mutant UM cells in culture. FR900359 allosterically inhibited the guanine nucleotide exchange activity of constitutively active Gαq, thereby trapping it in inactive heterotrimers. The loss of Gαq signaling in UM cells induced redifferentiation and cell death. Targeting this compound, if safe, or synthetic derivatives to the uveal tumor tissue may be an effective treatment for patients.

Abstract

Constitutively active G protein α subunits cause cancer, cholera, Sturge-Weber syndrome, and other disorders. Therapeutic intervention by targeted inhibition of constitutively active Gα subunits in these disorders has yet to be achieved. We found that constitutively active Gαq in uveal melanoma (UM) cells was inhibited by the cyclic depsipeptide FR900359 (FR). FR allosterically inhibited guanosine diphosphate–for–guanosine triphosphate (GDP/GTP) exchange to trap constitutively active Gαq in inactive, GDP-bound Gαβγ heterotrimers. Allosteric inhibition of other Gα subunits was achieved by the introduction of an FR-binding site. In UM cells driven by constitutively active Gαq, FR inhibited second messenger signaling, arrested cell proliferation, reinstated melanocytic differentiation, and stimulated apoptosis. In contrast, FR had no effect on BRAF-driven UM cells. FR promoted UM cell differentiation by reactivating polycomb repressive complex 2 (PRC2)–mediated gene silencing, a heretofore unrecognized effector system of constitutively active Gαq in UM. Constitutively active Gαq and PRC2 therefore provide therapeutic targets for UM. The development of FR analogs specific for other Gα subunit subtypes may provide novel therapeutic approaches for diseases driven by constitutively active Gα subunits or multiple G protein–coupled receptors (GPCRs) where targeting a single receptor is ineffective.

INTRODUCTION

Heterotrimeric G proteins transduce signals from hundreds of cell surface G protein–coupled receptors (GPCRs) to intracellular signaling networks that regulate diverse biological processes. By undergoing GPCR-stimulated guanosine diphosphate–for–guanosine triphosphate (GDP/GTP) exchange followed by GTP hydrolysis, G protein α subunits cycle between inactive GDP-bound and active GTP-bound states to determine the duration, magnitude, and specificity of biological responses (1). In cholera, certain cancers (2), Sturge-Weber syndrome (3), and other disorders, this cycle is disrupted by mutant or covalently modified Gα subunits that, by failing to hydrolyze GTP, are constitutively active.

Constitutively active mutant forms of Gαq or its close relative Gα11 are the oncogenic drivers in nearly 90% of uveal melanoma (UM) patients (46). UM is the most common cancer of the eye, and the eye is the second most common site of melanoma. Regardless of primary tumor treatment, nearly half of UM patients develop metastatic disease (7) with a mean survival of less than 1 year (8). Therapies to treat primary tumors and treat or prevent metastatic disease are needed. Inhibitors of individual signaling pathways downstream of Gαq/11 are being studied in UM clinical trials, but all have failed thus far (9). Thus, therapeutic approaches that directly target constitutively active Gαq/11 may be required to inhibit all necessary downstream oncogenic signaling networks.

Constitutively active Gα subunits have yet to be targeted pharmacologically in disease due to challenges analogous to those of inhibiting oncogenic Ras (1012). GTP hydrolysis defects would be extremely difficult to correct pharmacologically, and the high affinity of Gα subunits for GTP or GDP precludes the generation of effective competitive inhibitors of guanine nucleotide binding.

However, other evidence led us to consider that constitutively active Gαq can be targeted in UM by pharmacologically inhibiting GDP/GTP exchange. Although nucleotide exchange by soluble Gαq is very slow in vitro (13), it is enhanced markedly by lipid membranes (14, 15) and Ric-8a (16, 17), a nonreceptor guanine nucleotide exchange factor (GEF) and folding chaperone. Nucleotide exchange, therefore, may occur at appreciable rates in cells; however, constitutively active Gαq still would exist predominantly in the active GTP-bound state because average GTP/GDP ratios in human cells are ~8:1 (18), and GTP dissociates ~10-fold slower than GDP (13). Nevertheless, we reasoned that this equilibrium might be driven toward the GDP-bound state by inhibiting GDP dissociation, which would cause constitutively active Gαq to assemble into inactive GDP-bound Gαβγ heterotrimers and thereby attenuate all downstream oncogenic signaling networks.

Here, we found that constitutively active Gαq can be targeted pharmacologically in UM cells by FR900359 (FR), a naturally occurring, cyclic depsipeptide that has been shown previously to inhibit wild-type Gαq by interfering allosterically with GDP dissociation (1921). We show that FR can trap constitutively active Gαq in the GDP-bound state and inhibit downstream signaling in UM cells. We also show that constitutively active Gαq drives oncogenesis by a previously unknown mechanism that antagonizes epigenetic silencing. Our results suggest that targeting nucleotide exchange is a novel, general strategy for inhibiting Gα subunits in cancer and other diseases.

RESULTS

FR traps mutant, constitutively active Gαq in the GDP-bound state

To determine whether constitutively active Gαq undergoes appreciable GDP/GTP exchange in cells, we investigated whether Gαq(Q209L), a common oncogenic guanosine triphosphatase–defective mutant in UM, could be trapped in the GDP-bound state by FR. The GDP- and GTP-bound states of Gαq(Q209L) were assessed by detecting interaction with Gβγ subunits, which bind preferentially to GDP-loaded Gα subunits (1), or with regulator of G protein signaling 2 (RGS2), which binds GTP-loaded but not GDP-loaded Gαq (22). To detect protein-protein interactions, we used split luciferase complementation assays (23) using click beetle green (CBG) luciferase to study activation state–dependent interaction between Gα subunits and cognate binding partners in living cells (24) or copurification with tandem affinity-tagged [FLAG and StrepII (FS)] wild-type or constitutively active Gαq.

The data indicate that FR was able to trap constitutively active Gαq in the GDP-bound state. Split luciferase complementation between Gβ1/Gγ2 and wild-type or constitutively active Gαq was increased by FR [half-maximal effective concentration (EC50), 3 and 9 nM, respectively; Fig. 1, A and B]. FR also increased the association between Gβγ and affinity-tagged wild-type or constitutively active Gαq (19- and 9-fold, respectively), as indicated by copurification (Fig. 1C). Conversely, FR drove constitutively active Gαq out of the active GTP-bound state, as indicated by inhibition of split luciferase complementation between constitutively active Gαq and RGS2 [half-maximal inhibitory concentration (IC50), 0.4 nM; Fig. 1D]. FR was selective for Gαq, as revealed by its lack of effect on the interaction between wild-type or constitutively active Gα13(Q226L) and Gβ1γ2 (Fig. 1A) or between constitutively active Gα13 and the RGS domain of leukemia-associated Rho GEF (LARG) (Fig. 1D). Thus, FR drives constitutively active Gαq from its active GTP-bound state into inactive GDP-bound Gαβγ complexes.

Fig. 1 FR traps mutant constitutively active Gαq in the inactive GDP-bound state.

(A) Split luciferase complementation assays in human embryonic kidney (HEK) 293 cells, measured as reconstitution of CBG luciferase activity normalized to cotransfected, constitutively expressed Renilla luciferase to assess the effect of FR on the activity state of Gα subunits as determined by interaction of Gβ1γ2 with the indicated wild-type (WT) or mutant constitutively active forms of Gαq (q) or Gα13 (13) (Q209L and Q226L, respectively). Data are means ± SEM of three experiments. (B) Split luciferase complementation assays in HEK293 cells, as described in (A), to assess the potency of FR as a driver of interaction between Gβ1γ2 and wild-type or mutant constitutively active Gαq. Data are means ± SEM from three experiments. CBGC, C-terminal portion of CBG luciferase; CBGN, N-terminal portion of CBG luciferase. (C) Effect of FR treatment of HEK293 cells on copurification of endogenous Gβγ with overexpressed, affinity-tagged wild-type or constitutively active Gαq(Q209L). Affinity-tagged Gαq and Gβγ were detected by immunoblotting (IB) with FLAG and Gβ antibodies, respectively. Data shown are representative of three independent experiments. (D) Split luciferase complementation assays in HEK293 cells, as described in (A), to assess the potency of FR as an inhibitor of interaction between RGS2 and mutant constitutively active Gαq(Q209L) or between the RGS domain of LARG [LARG(RGS)] and mutant constitutively active Gα13(Q226L). Data are means ± SEM from three experiments. (E) Potency of FR as inhibitor of signal transduction by constitutively active Gαq or Gα13 detected with an SRE(L) promoter-driven transcriptional reporter in HEK293 cells. Data are means ± SEM from three experiments. Constitutively active Gαq(Q209L), constitutively active Gαq bearing the indicated FR-binding site mutations, and constitutively Gα13(Q226L) were studied. Concentration-response curves were fit by nonlinear regression to derive EC50 or IC50 values, which were compared by t test. *P < 0.05, **P < 0.01 by t test; significance was confirmed using q < 0.05 or q < 0.01 by the false discovery rate (FDR) method of Benjamini and Hochberg. n.s., not significant.

As a further test of the ability of FR to inhibit constitutively active Gαq, we measured downstream signaling. FR inhibited the induction of a transcriptional reporter driven by constitutively active Gαq (IC50, 1 nM; Fig. 1E) but had no effect on the expression of the reporter when driven by constitutively active Gα13 (Fig. 1E). Crystallographic and mutagenesis studies of wild-type Gαq identified amino acid residues (Arg60, Val184, and Ile190) that are important for inhibition by YM-254890 (19), an inhibitor nearly identical to FR. We found that single amino acid substitutions at any of these sites (R60K, V184S, and I190N) in constitutively active Gαq were sufficient to blunt the inhibitory potency of FR (IC50, 30 to 70 nM; Fig. 1E), demonstrating that FR targets constitutively active and wild-type Gαq by using the same binding site.

FR inhibits Gαi1 bearing an engineered FR-binding site

FR inhibits receptor-evoked signaling by wild-type Gαq and its close relatives Gα11 and Gα14 but not by other Gα subunits (20). We therefore determined whether an FR-insensitive Gα subunit could be converted into an FR-sensitive form by introducing an FR-binding site. This might be possible because (i) all Gα subunits release GDP by a common allosteric mechanism (25), (ii) structural elements of the allosteric relay include the FR-binding site (19, 25), and (iii) FR-insensitive Gα subunits contain a similar but diverged form of the FR-binding site.

To test this hypothesis, we engineered an FR-binding site into Gαi1 by changing eight diverged amino acids to match their counterparts in the FR-binding site of Gαq, producing “Gi/q” α subunits (illustrated in Fig. 2A). We chose Gαi1 because it is insensitive to FR (20) and because its function is studied readily in biochemical and cell-based assays. As a control, we also made a Gαi/q(R54K) mutant, corresponding to a Gαq mutant that is less sensitive to FR (19). We found that FR inhibited the rate of nucleotide exchange by Gαi/q in vitro (IC50, 4.6 μM), as indicated by the binding kinetics of a fluorescent, nonhydrolyzable GTP analog (BODIPY-GTPγS; Fig. 2B and fig. S1A), which is rate-limited by GDP release. As expected, FR was >10-fold less potent toward Gαi/q(R54K) (IC50, 76 μM; Fig. 2B and fig. S1A). Similarly, we found that FR inhibited Gαi/q signaling in cells. FR attenuated the ability of Gαi/q activated by cannabinoid type 1 receptors to inhibit forskolin-induced cyclic adenosine 3′,5′-monophosphate (cAMP) accumulation (IC50, 25 nM; Fig. 2C and fig. S2, C and D). FR was ~30-fold less potent toward Gαi/q(R54K) (IC50, ~800 nM; Fig. 2C and fig. S2, C and D). Thus, FR targets Gαi/q and Gαq by using the same binding site. This finding suggests that FR-like molecules could be created to target the analogous, but distinct, binding sites of other Gα subtypes, providing a general approach to discover novel chemical probes of Gα function and potential therapeutics for various diseases.

Fig. 2 Inhibition of Gαi1 bearing an engineered FR-binding site.

(A) Amino acid residues in Gαi1 identical to or diverged from the FR-binding site in Gαq are indicated in blue and red, respectively. An FR-binding site was engineered in Gαi1 by introducing the eight indicated amino acid substitutions so as to match the corresponding residues of Gαq, thereby producing a chimeric Gα subunit termed Gi/q. (B) Effect of FR on guanine nucleotide exchange by Gαi/q in vitro. Nucleotide exchange was assayed by measuring increase in BODIPY-GTPγS fluorescence upon binding to the indicated purified His-tagged Gα subunits in the absence or presence of the indicated concentrations of FR. Gi/q(R54K) corresponds to a Gαq mutant that is less sensitive to inhibition by FR. Nucleotide exchange rates (kobs) of Gαi/q and Gαi/q(R54K) in the absence of FR were 0.24 and 0.12 min−1, respectively. Data are means ± SEM from three independent experiments. (C) Effect of FR on agonist-evoked signaling mediated by Gi/q. Inhibition of forskolin-induced cAMP Förster by Gi-coupled cannabinoid receptors was measured in Neuro2A cells transfected with a cAMP fluorescence resonance energy transfer (FRET) reporter and pertussis toxin (PTX)–resistant and EE epitope–tagged forms of the indicated Gα subunits and treated with PTX to inactivate endogenous Gi. Inhibition of forskolin-induced cAMP formation by a cannabinoid receptor agonist (WIN 55,212-2; WIN) was measured by FRET. Attenuation of this inhibitory effect by FR was quantified relative to vehicle controls. Data are means ± SEM from three experiments. IC50 values of FR toward cells expressing Gi/q and Gi/q(R54K) were 4.6 and 76 μM, respectively. (D) Failure of FR to correct the GTP hydrolysis defect of Gi/q(Q204L) in vitro. Hydrolysis of γ32P-GTP by the indicated Gα subunits present at 10-fold molar excess over GTP was measured over time. FR was added at the indicated time point (arrow). Data are means ± SEM from three independent experiments. (E) Effect of FR on guanine nucleotide exchange by constitutively active Gi/q(Q204L) in vitro, as determined by methods described in (B). The rate of nucleotide exchange (kobs) by Gi/q(Q204L) in the absence of FR was 0.14 min−1. Concentration-response curves were fit by nonlinear regression to derive IC50 values, which were compared by t test. *P < 0.05, **P < 0.01 by t test; significance was confirmed using q < 0.05 or q < 0.01 by the FDR method of Benjamini and Hochberg.

FR targets constitutively active Gα subunits by inhibiting nucleotide exchange

In principle, FR could target constitutively active Gα subunits by inhibiting nucleotide exchange or restoring GTP hydrolysis. We tested both hypotheses by using Gαi/q; Gαq was unsuitable due to its unusual nucleotide binding properties that confound measuring GTP hydrolysis in vitro. We found that constitutively active Gαi/q(Q204L) [equivalent to Gαq(Q209L)] exhibited a severe defect in the catalytic rate of GTP hydrolysis that was not corrected by FR (Fig. 2D), whereas wild-type Gαi/q hydrolyzed GTP effectively (Fig. 2D). In contrast, FR effectively inhibited nucleotide exchange by constitutively active Gαi/q (IC50, 2.7 μM; Fig. 2E). Thus, FR targets constitutively active Gα subunits by inhibiting nucleotide exchange rather than restoring GTP hydrolysis.

FR inhibits signaling by constitutively active Gαq in UM cells

To determine whether FR inhibits signal transduction by constitutively active Gαq in UM cells, we analyzed two UM cell lines (Mel202 and 92.1) driven by constitutively active Gαq(Q209L). A third UM cell line (OCM-1A) driven by constitutively active BRAF(V600E) served as a negative control. Signaling by Gαq-stimulated phospholipase Cβ was quantified on the basis of the abundance of inositol monophosphate (IP1), a metabolically stable product of inositol 1,4,5-trisphosphate produced by cleavage of phosphatidylinositol 4,5-bisphosphate. In the absence of FR, IP1 was >50-fold more abundant in Gαq(Q209L)-driven Mel202 and 92.1 cells relative to BRAF(V600E)-driven OCM-1A cells (Fig. 3A). FR reduced IP1 abundance in Mel202 and 92.1 cells >50-fold (Fig. 3B) but had only modest effect (~2-fold) on OCM-1A cells (Fig. 3B). Thus, FR markedly inhibited second messenger production driven by constitutively active Gαq in UM cells.

Fig. 3 FR inhibits signaling by constitutively active Gαq in UM cells.

(A) Inhibition of signaling by constitutively active Gαq in UM cell lines was quantified by measuring intracellular IP1, a metabolically stable product of inositol 1,4,5-trisphosphate produced by Gαq-stimulated phospholipase Cβ. Basal IP1 values in UM cell lines driven by constitutively active Gαq(Q209L) (92.1 and Mel202 cells) and BRAF(V600E) (OCM-1A cells). Data are means ± SEM from three experiments performed in triplicate. (B) Effect of FR on IP1 abundance in 92.1, Mel202, and OCM-1A cells. Data are means ± SEM of four experiments performed in triplicate. *P < 0.01 by t test; significance was confirmed using q < 0.01 by the FDR method of Benjamini and Hochberg.

FR inhibits UM tumor cell proliferation and survival

During the preceding experiments, we also observed that FR treatment decreased the viability of Gαq(Q209L)-driven UM tumor cells. Quantification confirmed that FR inhibited the proliferation of Gαq(Q209L)-driven Mel202 and 92.1 UM cells (EC50, 6 and 2 nM, respectively; Fig. 4, A and B), with no effect on proliferation of BRAF(V600E)-driven OCM-1A cells even when applied at a high concentration (10 μM). Flow cytometry revealed that FR induced cell cycle inhibition (decreased fraction of S or G2-M phase cells) and apoptosis (increased fraction of sub-G1 phase cells) in Mel202 and 92.1 cells but had no effect on OCM-1A cells (Fig. 4, C and D). FR therefore inhibited the proliferation and survival of UM cells in which constitutively active Gαq is the oncogenic driver.

Fig. 4 FR-sensitive growth and viability of UM cells driven by constitutively active Gαq.

(A) Changes in viability of UM cells treated with FR. Cell viability was quantified using a water-soluble tetrazolium salt assay. The fold change in cell viability over time is shown for Gαq(Q209L)-driven 92.1 and Mel202 cells and for BRAF(V600E)-driven OCM-1A cells in response to increasing concentrations of FR. Data are means ± SEM from four experiments performed in triplicate. (B) Potency of FR as an inhibitor of UM cell viability measured in 92.1, Mel202, and OCM-1A cell lines as in (A). Data are means ± SEM from four experiments performed in triplicate. The indicated UM cell lines were treated for 3 days with the indicated concentrations of FR and analyzed for DNA content. (C) Representative histograms from flow cytometry of the Gαq(Q209L)-driven cell lines (92.1 and Mel202) and BRAF(V600E)-driven OCM-1A cells. PI, propidium iodide. (D) Potency of FR as inducer of apoptosis (sub-G1 cells) and inhibitor of cell proliferation (S and G2-M phase cells) in 92.1, Mel202, and OCM-1A cell lines; data are means ± SEM from four experiments. *P < 0.01 by t test; significance was corrected for multiple comparisons using the Holm-Sidak method.

FR promotes melanocytic redifferentiation of UM cell lines

We observed that FR treatment caused Gαq(Q209L)-driven Mel202 and 92.1 cells to undergo morphological changes indicative of redifferentiation. FR-treated Mel202 and 92.1 cells lost spindle morphology, became flatter, and produced multiple projections when compared with vehicle-treated cells or FR-treated OCM-1A cells (Fig. 5A). Furthermore, FR increased melanocytic pigmentation in Mel202 and 92.1 cells as compared to vehicle-treated cells or FR-treated OCM-1A cells (Fig. 5B). Similarly, FR increased the expression of two pigmentation enzymes [tyrosinase (TYR) and dopachrome tautomerase (DCT)] and premelanosome protein (PMEL) in Mel202 and 92.1 cells but not in OCM-1A cells (Fig. 5C). FR increased the proportion of DCT-positive 92.1 and Mel202 cells (3.5- and 1.6-fold, respectively), TYR-positive Mel202 cells (1.8-fold), and PMEL-positive 92.1 and Mel202 cells (6.8- and 3.9-fold, respectively). Thus, FR antagonized the dedifferentiation process driven by constitutively active Gαq in UM cells.

Fig. 5 FR induces redifferentiation of UM cells driven by constitutively active Gαq.

(A) Morphological changes elicited by FR in UM cell lines driven by constitutively active Gαq (92.1 and Mel202) or BRAF-V600E (OCM-1A) treated for 3 days with FR and imaged by phase-contrast microscopy. Representative fields from one of three experiments are shown. (B) Melanocytic differentiation of FR-treated UM cells indicated by pigmentation. 92.1, Mel202, and OCM-1A UM cell lines were treated for 3 days with FR. Cells were pelleted and examined macroscopically; representative images from one of three experiments performed in triplicate. (C) Induction of melanocytic markers by FR as indicated by immunofluorescence staining of tyrosinase (TYR), dopachrome tautomerase (DCT), and premelanosome protein (PMEL) of Gαq-mutant cell lines (92.1 and Mel202) but not BRAF-driven UM cells (OCM-1A). Images are representative fields from one of three experiments. Scale bars, 50 μm.

FR alters expression of genes regulated by constitutively active Gαq in UM cells

To explore how FR regulates phenotypes of UM cells driven by constitutively active Gαq, we analyzed global gene expression by RNA sequencing (RNA-seq). Although FR caused an apoptotic response in UM cells, it did not markedly increase the expression of proapoptotic genes or decrease the expression of survival genes. Instead, FR modestly decreased the expression of two proapoptotic BCL2 family members (BBC3/PUMA by 3.5-fold and PMAIP1/NOXA by 2.5-fold; fig. S2). Other BCL2 family members showed insignificant changes in expression, and broader examination of apoptosis-related genes showed only small effects (fig. S2). These results are consistent with evidence that intrinsic and extrinsic apoptotic pathways function independently of gene transcription (26, 27).

Cell cycle genes were also relatively unaffected by FR, as indicated by gene set enrichment analysis (GSEA) and direct comparison of the RNA-seq data (fig. S2). The cyclin-dependent kinase inhibitor p21CIP1 (CDKN1A, down threefold) showed reduced expression, and several cell cycle genes showed upward trends lacking statistical significance (fig. S2). Targets of E2F, a transcription factor positively regulated by cyclin-dependent kinases, were positively enriched (data file S1). These results suggest that the antiproliferative effects of FR are not mediated primarily by short-term changes in cell expression of cycle genes.

FR restores UM cell differentiation by promoting polycomb-mediated gene repression

In contrast to the results obtained for apoptosis and cell cycle genes, large changes in expression were observed for differentiation and developmental genes in FR-treated Gαq(Q209L)-driven 92.1 cells (data files S1 and S2). Results of a multidimensional gene expression analysis comparing relative patterns of expression of all genes across all samples showed that a large number of gene expression changes were associated specifically with FR treatment (Fig. 6A). Most strikingly, a distinct gene cluster showed marked reduction in expression [4- to ~160-fold; Fig. 6B (circled) and data file S3]. Among genes in this cluster, 38% are associated with cell differentiation and development, as revealed by gene ontology (GO) analysis (Fig. 6C and data file S4). A total of 42% of genes in this cluster are known targets of the polycomb repressive complex 2 (PRC2) during differentiation from embryonic stem cells, as indicated by GSEA (Fig. 6D and data file S5). These results were confirmed by quantitative real-time polymerase chain reaction (PCR) analysis of FR-treated 92.1 cells (fig. S3). In contrast, expression of PRC2-regulated genes in BRAF(V600E)-driven OCM-1A cells was unaffected by FR (fig. S3), demonstrating specificity of FR for developmental and differentiation genes targeted by constitutively active Gαq in UM cells.

Fig. 6 FR represses expression of differentiation genes by restoring function of the PRC2.

(A) Gαq-mutant 92.1 UM cells were treated with FR or vehicle, and RNA was collected 1 and 3 days (1d and 3d, respectively) after treatment for RNA-seq analysis. Results of a multidimensional gene expression analysis that compares the relative patterns of expression of all genes across all samples and groups genes with similar patterns. The graph shows samples positioned by their relative gene expression values within each pattern. Dimension 1 (x axis; the most represented pattern) shows separation based on vehicle treatment (red balls) versus FR treatment (blue balls), whereas dimension 2 (y axis; the second most represented pattern) shows separation based on time in culture (indicated by 1d or 3d on balls). (B) MA plot (M, log ratio; A, mean average) comparing gene expression between FR- and vehicle-treated 92.1 samples identifies a group of significantly reduced genes (circled; fold change, >2; FDR, q < 0.01) associated with FR treatment. (C) GO analysis of the FR-repressed gene set [circled in (B) with arrow]. (D) FR-repressed genes [circled in (B) with arrow] identified as targets of the polycomb repressive complex 2 (PRC2) by GSEA. EGF, epidermal growth factor; BMP2, bone morphogenetic protein; hESC, human embryonic stem cells. (E) Effect of the EZH1/2 inhibitor GSK503 on morphological differentiation elicited by FR. Representative fields are shown from one of three experiments of 92.1 UM cells treated for 7 days with GSK503 and for 3 days with FR and then imaged by phase-contrast microscopy. Scale bar, 100 μm. (F) Effect of GSK503 on pigmentation of FR-treated cells, visualized by macroscopic inspection. 92.1 cells were treated for 7 days with GSK503 and for 3 days with FR and pelleted; representative images from one of three experiments. (G) PRC2 inhibition by GSK503. Immunoblots of 92.1 cells treated for 7 days with GSK503 show reduced histone H3K27 trimethylation. Plot shows relative fraction of trimethyl-histone H3K27 compared to dimethyl sulfoxide (DMSO) control and normalized to total histone H3 from densitometry data from three independent experiments. *P < 0.01 by t test; significance was confirmed using q < 0.01 by the FDR method of Benjamini and Hochberg.

The RNA-seq analyses suggested a novel mechanism for Gαq-induced oncogenesis in UM in which constitutively active Gαq antagonizes PRC2-mediated gene repression, thereby reactivating genes associated with stemness and driving dedifferentiation of UM cells into a more stem-like phenotype. FR treatment inhibits constitutively active Gαq, relieves blockade of PRC2-mediated repression, resilences these genes, and returns UM cells to a melanocytic state. Consistent with this hypothesis, we found that inhibiting the catalytic subunits of PRC2 complexes (EZH1/2) with GSK503 maintained 92.1 cells in an undifferentiated state and blocked the ability of FR to redifferentiate these cells, as indicated by morphology (Fig. 6E) and pigmentation (Fig. 6F). The effectiveness of GSK503 at inhibiting histone H3 Lys27 (H3K27) methylation was confirmed by immunoblotting histones isolated from control and FR-treated 92.1 cells (Fig. 6G).

DISCUSSION

GDP/GTP exchange can be exploited as an Achilles heel of constitutively active Gα subunits

The most important discovery provided by our studies is that GDP/GTP exchange is an underappreciated vulnerability of constitutively active Gα subunits and one that can be exploited pharmacologically in UM and other diseases. Although Gα subunits undergo GDP/GTP exchange slowly in vitro, we discovered that nucleotide exchange occurs in cells at rates sufficient for constitutively active Gαq to be trapped in the inactive GDP-bound state by treatment with FR, an allosteric inhibitor of GDP release. When trapped by FR, GDP-bound constitutively active Gαq assembles into Gαβγ heterotrimers, further suppressing GDP release and stabilizing the inactive state. Because FR-bound Gq heterotrimers are refractory to activation by GPCRs (20), signaling networks downstream of constitutively active Gαq are attenuated.

Although our study involved constitutively active Gαq in UM, we anticipate that constitutively active forms of Gα subunit subtypes that drive other types of cancer may also be vulnerable to allosteric inhibitors of GDP release. Constitutively active Gα11 in UM (12) and Gα14 in vascular tumors (28) should be susceptible because wild-type forms of these Gα subunits are sensitive to FR (20). Although other subtypes of Gα subunits are insensitive to FR, all Gα subunits have a diverged but related form of the allosteric regulatory site in Gαq that binds FR. This site includes conserved features of linker 1, which stabilizes the GDP-bound state by interacting with helix 1, helix A, and helix F as part of the universal mechanism that regulates GDP release. As predicted by this hypothesis, we found that engineering an FR-binding site into an FR-insensitive Gα subunit was sufficient to confer FR sensitivity. Thus, we speculate that a collection of FR-like inhibitors, each of which selectively targets the diverged allosteric regulatory site of certain Gα subunits, may provide a novel approach toward therapeutic development in cancers associated with other mutant constitutively active Gα subunits (2), cholera, and Sturge-Weber syndrome. In addition, this approach may be efficacious for diseases that are driven by multiple GPCRs in which blocking a single receptor is ineffective.

UM cells are addicted to constitutively active Gαq

Another important discovery emerging from our study is that constitutively active Gαq has unexpectedly diverse functional roles as an oncogenic driver in UM. Instead of affecting a single-cell biological process, FR inhibits proliferation, triggers apoptosis, and drives melanocytic redifferentiation of UM cells. Our findings help to explain why previous studies using mitogen-activated protein kinase kinase, Akt, and protein kinase C inhibitors to target individual signaling pathways downstream of Gαq/11 failed in clinical trials of UM (9). By inhibiting constitutively active Gαq and consequently attenuating all downstream signaling networks, FR or related inhibitors may impair the growth and survival of cancer cells in UM primary tumors, and they may also decrease the probability or extent of metastasis because melanocytic differentiation correlates inversely with metastatic potential in UM (29, 30). For primary tumors, FR may slow conversion from the indolent state to aggressive class 2 tumors (31), or it may impair the growth or spread of metastatic lesions, including those that are clinically undetectable. Tumor-targeted or focal delivery of FR may be required because systemic administration might cause unacceptable side effects by inhibiting Gαq/11 in normal tissues. Nevertheless, the marked FR-sensitivity of UM tumor cells driven by constitutively active Gαq suggests that clinical investigation of this or related inhibitors could be considered.

Constitutively active Gαq antagonizes gene silencing by the PRC2

We found a marked and unanticipated consequence of inhibiting constitutively active Gαq in UM—the repression of gene sets that control differentiation and development. Many of these repressed genes are involved in embryonic stem cell lineage specification and differentiation and are targets of epigenetic silencing by the PRC2, which acts through H3K27 trimethylation (3234). These repressed genes include ADRA2A2A-adrenergic receptor) and HAND2 (heart and neural crest derivatives expressed-2). The ADRA2A gene promoter was identified in independent screens for PRC2 subunit binding and for H3K27 trimethylation (3234), and ADRA2A has been linked to cancer progression and severity (35). The HAND2 gene promoter is also targeted by PRC2 binding and H3K27 trimethylation (3234), especially in migrating cranial neural crest cells, where HAND2 expression distinguishes neural crest cell lineages during facial development (36).

Together, our findings reveal a novel mechanism in which signaling by constitutively active Gαq in UM cells antagonizes PRC2-mediated gene silencing, thereby maintaining UM cells in a less differentiated state similar to premelanocytic cranial neural crest cells (36). This finding, coupled with previous studies of BAP1 (BRCA1-associated protein-1) (30, 37), indicates that a temporal hierarchy of epigenetic regulation drives tumorigenesis and progression in UM. Early in tumorigenesis, mutations that constitutively activate Gαq are acquired, which inhibits PRC2-mediated repression. Subsequent loss of BAP1, a histone H2A(Lys119) deubiquitinase that antagonizes repression by PRC1, then leads to metastasis.

MATERIALS AND METHODS

FR900359

FR was purified from Ardisia crenata according to published methods (20). The structure of purified FR relative to a commercially available equivalent (UBO-QIC; University of Bonn, Germany) was established by nuclear magnetic resonance.

Biochemical assays

Split luciferase assays were performed as described previously (24). The N-terminal portion of CBG luciferase (CBGN) was inserted into the αB-αC loop within the helical domain of wild-type and constitutively active (c.626A>T; Q209L) mutant forms of GNAQ (Gαq) and GNA13 (Gα13) (Q226L). Insertion of foreign proteins at this site preserves Gα subunit function (38). The C-terminal region of CBG luciferase (CBGC) was fused to the N termini of GNB1 (Gβ1), which was cotransfected with untagged GNG2 (Gγ2); RGS2; and the RGS domain of ARHGEF12 (LARG), which interacts with Gα13 only in the active GTP-bound state (39). HEK293 cells transiently transfected with various combinations of fusion constructs generating tagged proteins were treated for 18 hours with vehicle (DMSO) or FR, and luciferase assays were performed to measure reconstituted luciferase activity. Assays of transcriptional reporters driven by Gα subunits were performed as described previously (40). Gα-driven firefly luciferase activity was normalized to cotransfected Renilla luciferase expressed from a constitutive promoter.

Experiments used to measure agonist-evoked inhibition of forskolin-induced cAMP formation in Neuro2A cells were performed in a 96-well plate format, as described in (24) with slight modifications. Cells were transfected with a cAMP FRET reporter and PTX-resistant forms of Gαi1, Gαi/q, or Gαi/q(R54K). After cells were treated for 16 hours with PTX (100 ng/ml) to inactivate endogenously expressed Gi, FRET was used to measure inhibition of forskolin (FSK)–induced cAMP formation by a CB1 cannabinoid receptor agonist (WIN 55,212-2; WIN). A Synergy H4 hybrid plate reader (BioTek) was used to measure FRET every 57 s by exciting the FRET donor (420/20-nm bandpass filter) and simultaneously detecting donor and acceptor emissions (480/20- and 540/20-nm bandpass filters, respectively). FRET measurements of changes in intracellular cAMP were expressed ratiometrically asΔRR0=FRET ratiobaseline FRET ratiobaseline FRET ratiowhere FRET ratio is (480-nm emission/540-nm emission) at a given time point, and the baseline FRET ratio is the average of (480-nm emission/540-nm emission) before FSK stimulation. Independent experiments were performed in triplicate. Effects of FR on agonist-evoked adenylyl cyclase inhibition were quantified as the percentage of ΔR/Ro relative to vehicle control. FR concentration-response curves were generated by a three-parameter fit.

Accumulation of IP1 in UM cells was measured using the IP-One Kit (catalog number 62IPAPEB, Cisbio Inc.), according to the supplier’s instructions. A total of 10,000 Mel 202 cells, 20,000 92.1 cells, and 20,000 OCM-1A cells were seeded into white-bottom tissue culture grade 384-well plates. After an overnight incubation, cells were treated with FR or DMSO and returned to the incubator. The next day, stimulation buffer was added for 1 hour after which IP1-d2 and Ab-Cryp were added, and the cells were incubated at room temperature for 60 min. Plates were read in a Synergy H4 hybrid plate reader. Standard curves were generated using reagents supplied with the kit.

Copurification of tandem affinity-tagged Gαq and endogenous Gβγ was assayed as follows. A gBlock gene fragment (Integrated DNA Technologies) encoding a glycine/serine linker (GGGSSGGG) followed by a FLAG-StrepII-StrepII (FS) tag and another glycine/serine linker (GGGSSGGG) was inserted between sequences encoding amino acid residues 124 and 125 of mouse Gαq (wild-type and Q209L mutant), cloned into pcDNA3.1+, and verified by DNA sequencing. HEK293 cells were plated in 10-cm dishes and transfected the next day with the indicated plasmids. After 24 hours, transfection medium was removed and replaced with fresh media containing one FR (1 μM) or vehicle (DMSO). Twenty-four hours after treatment, media were removed, and cells were washed with Dulbecco’s phosphate-buffered saline (PBS) and processed for tandem affinity purification (TAP). TAP was performed as described previously (24) with the following modifications: All subsequent steps were performed on ice or at 4°C. HEK293 cells were lysed in [50 mM tris (pH 8.0), 5 mM EDTA, 100 mM NaCl, 0.5% (v/v) IGEPAL CA-630, 1 mM MgCl2, and complete protease inhibitor mix (catalog number 11697498001, Roche), with or without 1 μM FR] by sonication on ice for 2 min (30 s on, 30 s off, 60% A), rotated end-over-end for 30 min, and cleared by ultracentrifugation at 100,000g for 15 min. Cleared lysates were incubated with Strep-Tactin resin (catalog number 2-1206-010, lot number 1206-0350, IBA) overnight with end-over-end rotation and washed three times in batch with 10 column volumes of wash buffer [50 mM tris (pH 8.0), 5 mM EDTA, 100 mM NaCl, 0.5% (v/v) IGEPAL CA-630, 1 mM MgCl2, complete protease inhibitor mix, and ±1 μM FR]. Protein complexes were eluted two consecutive times by incubating with a fivefold column volume of elution buffer [desthiobiotin buffer E (catalog number 2-1000-025, lot number 1000-5170, IBA), 0.1% (v/v) IGEPAL CA-630, 1 mM magnesium chloride, complete protease inhibitor mixture, and ±1 μM FR] for 30 min in batch. Strep-Tactin elution fractions were combined and incubated with anti-FLAG M2-Agarose (catalog number A2220, lot number SLBW1929, Sigma-Aldrich) in batch for 2 hours and washed three times in batch with a 10-fold column volume of wash buffer. Protein complexes were eluted from FLAG agarose by incubating with a fourfold column volume of FLAG elution buffer [3xFLAG peptide (200 μg/ml); catalog number F4799, lot number SLBM1190V in wash buffer, Sigma-Aldrich) for 30 min in batch. Lysates and FLAG eluates were resolved on 12% SDS–polyacrylamide gel electrophoresis (PAGE) gels and transferred to Immobilon(PSQ) polyvinylidene difluoride (PVDF) membranes (catalog number ISEQ00010, Millipore). Membranes were blocked with 5% (w/v) milk in TBST [25 mM tris (pH 7.2), 150 mM NaCl , 2.7 mM KCl, and 0.1% (v/v) Tween 20] and incubated with primary antibodies overnight at 4°C. Lysate primary antibody mixture was ANTI-FLAG M2 (1:50,000; catalog number F1804, lot number SLBN5629V, Sigma-Aldrich) and Gβ H-1 (1:500; catalog number sc-166123, lot number G2414, Santa Cruz Biotechnology). TAP eluates were probed with a primary antibody mixture consisting of anti-FLAG M2 (1:50,000; catalog number F1804, lot number SLBN5629V, Sigma-Aldrich) and anti-Gβ1 H-1 (1:500; catalog number sc-166123, lot number G2414, Santa Cruz Biotechnology). Membranes were washed with TBST at least three times and incubated with goat anti-mouse immunoglobulin G coupled to IRDye 800CW (catalog number 926-32210, lot number C70712-15, LI-COR Biosciences). After incubation, membranes were washed at least three times with TBST, and signals were detected using an Odyssey model 9120 imaging system (LI-COR Biosciences).

Nucleotide exchange by purified His-tagged Gα subunits was assayed as follows. Plasmid pet14B-6xHIS-Gαi1 was a gift from M. Linder (Cornell University, New York). A pet14B-6xHIS-Gαi/q plasmid was generated by cloning a custom synthesized gBlock gene fragment (Integrated DNA Technologies) containing mutations encoding eight amino acid substitutions (V50I, K54R, Y69F, V72L, K180P, V185I, T187Y, and H188P) in 6xHIS-Gαi1. Site-directed mutagenesis of this plasmid was used to generate pet14B-6xHIS-Gαi/q(R54K). Recombinant His-tagged Gα subunits were expressed and purified from Escherichia coli according to published methods (41). To detect nucleotide exchange, fluorescence of the GTP analog BODIPY-GTPγS (catalog number G22183, Thermo Fisher Scientific) was recorded with a Synergy H4 hybrid plate reader in the absence or presence of recombinant Gα subunits (42). The indicated Gα subunits (1 μM) were incubated in a black-wall clear-bottom 96-well plate (catalog number 3603, Costar) with vehicle (DMSO) or FR at the indicated concentrations for 20 min at 25°C in reaction buffer [50 mM tris-HCl (pH 8.0), 1 mM EDTA, and 10 mM MgCl2]. BODIPY-GTPγS then was added to the reaction mixture at a final concentration of 25 nM to initiate nucleotide exchange. The intensity of fluorescence emission was recorded at 30°C every 10 s for 30 min with excitation and emission wavelengths of 485 and 528 nm, respectively, and the monochromator was set to a bandwidth of 9 nm. Specific fluorescence was normalized to background fluorescence as followsΔFF0=(fluorescence from BODIPY-GTPγS–bound Gα)(fluorescence from BODIPY-GTPγS alone)fluorescence from BODIPY-GTPγS aloneGraphPad Prism was used to fit fluorescence (ΔF/F0) curves to a pseudo first-order rate equation and obtain kobs values relative to vehicle controls. FR concentration-response curves were generated by a three-parameter fit of normalized kobs values.

Assays of GTP hydrolysis by His-tagged Gαi/q subunits were performed as follows. Reactions contained wild-type Gαi/q (1.5 μM) or constitutively active Gαi/q(Q204L) (3 μM) in reaction buffer [50 mM tris-HCl, 5 mM MgCl2, 1 mM EDTA, and 0.5 mM dithiothreitol (pH 8.0)]. Reactions performed in triplicate were started by adding reaction buffer containing γ32P-GTP (final concentration, 100 nM; specific activity, 100 Ci/mmol). Gα subunits were present at >10-fold molar excess over GTP to assess the catalytic rather than steady-state rate of GTP hydrolysis. As indicated, vehicle (DMSO) or FR (final concentration, 25 μM) was added. Aliquots were removed at intervals, quenched by addition of 1 N formic acid, and spotted on polyethyleneimine-cellulose thin-layer plates (catalog number Z122882, Sigma-Aldrich). Plates were developed in 0.5 M LiCl2 and 0.5 M formic acid, dried, wrapped in plastic, and exposed 1 hour to a phosphor storage screen (GE Healthcare Life Sciences) that was visualized on a Typhoon FLA 9500 scanner (GE Healthcare Life Sciences) at a resolution of 100 μm and photomultiplier voltage of 750 V. Scans were analyzed using Image Studio Lite to quantify the amount of labeled GTP and orthophosphate in each lane. GTP hydrolysis was calculated by dividing the magnitude of the orthophosphate signal by the sum of the orthophosphate and GTP signals. Data were fit using GraphPad Prism to a pseudo first-order rate equation.

UM cell culture assays

Cells were cultured at 37°C in 5% CO2. Human UM cell lines 92.1, Mel202, and OCM-1A were derived by M. Jager (Laboratory of Ophthalmology, Leiden University), B. Ksander (Schepens Eye Institute, Massachusetts Eye and Ear Infirmary), and J. Kan-Mitchell (Biological Sciences, University of Texas at El Paso). UM cell lines were grown in RPMI 1640 medium (Life Technologies), supplemented with 10% fetal bovine serum and antibiotics. Cell viability was measured using a water-soluble tetrazolium salt, WTS-8 (Bimake), following the manufacturer’s protocol. Flow cytometry for analysis of cell proliferation and apoptosis was performed at the Siteman Cancer Center Flow Cytometry Core on a FACScan analyzer (BD Biosciences) using a standard propidium iodide staining protocol, as described previously (43).

Immunofluorescence staining of UM cell lines was carried out by adding an equal volume of 2× fixative (PBS with 4% paraformaldehyde and 0.4% glutaraldehyde) to UM cells in RPMI 1640 growth medium. After 15 min at 37°C, cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min, washed with PBS, and blocked with 2% fish gelatin (Sigma-Aldrich) in PBS. Primary and secondary antibodies were diluted in 2% fish gelatin in PBS. Primary antibodies included mouse monoclonal anti-premelanosomal protein (One World Lab), rabbit polyclonal anti-TYR (One World Lab), rabbit polyclonal anti-DCT (One World Lab), rabbit polyclonal anti-S100 (DakoCytomation), and mouse monoclonal anti-BrdU (Life Technologies). Secondary antibodies were Alexa Fluor conjugates (Life Technologies), and the mounting agent was ProLong Gold (Life Technologies). Cell morphology was assessed by phase-contrast imaging with an inverted microscope (Olympus IX72) using a 10× objective. Images were analyzed to compare the percentage of cell populations that were positive for a given marker in representative fields from three independent samples. Significant difference relative to vehicle control was defined as P < 0.01 by Fisher’s exact test for all comparisons.

Immunoblotting of UM cell lines was performed by lysing cells in a radioimmunoprecipitation assay buffer [150 mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 50 mM tris (pH 8.0)] with 1× complete protease inhibitor mix (catalog number 11697498001, Roche). Cleared lysates were analyzed by immunoblotting, as described above for HEK293 cells.

Histones were isolated from UM cell lines by using the Active Motif Histone Purification Mini Kit (catalog number 40026, Active Motif). Lysates were resolved on 15% SDS-PAGE gels and transferred to an Immobilon(P) PVDF membrane (catalog number IPVH00010, Millipore). Membranes were blocked with 5% (w/v) milk in TBST [25 mM tris (pH 7.2), 150 mM NaCl, 2.7 mM KCl, and 0.1% (v/v) Tween 20] and incubated with primary antibodies. Membranes were washed with TBST at least three times and incubated with IRDye 680–coupled goat anti-rabbit and IRDye 800 or goat anti-mouse antibodies (LI-COR Biosciences). After incubation, membranes were washed at least three times with TBST, and signals were detected using Odyssey model 9120 imaging system (LI-COR Biosciences). Other primary antibodies used for immunoblotting were anti-EE (catalog number MMS-115P, lot number E12BF00285, Covance), anti-actin C4 (catalog number MAB1501, Millipore), anti-histone H3 (clone A3S; catalog number 05-928, MIllipore), and anti-histone H3-trimethyl-K27 (catalog number 6002, Abcam).

Statistical analyses

All statistical analyses were performed with GraphPad Prism. For split luciferase complementation assays, IP1 assays, and viability assays, cells were plated in triplicate wells and treated in parallel (technical replicates), and each experiment was performed three times on different days (biological replicates). Mean values and SEMs were calculated from at least nine replicate values for each condition, and t tests were performed comparing each condition to controls to determine statistical significance of FR treatment. For guanine nucleotide exchange and GTP hydrolysis assays, experiments were performed in triplicate, and each experiment was performed at least three times on different days with different protein preparations. Mean values and SEMs were calculated from at least nine replicate values for each condition, and t tests were performed comparing each condition to controls to determine statistical significance of FR treatment. For FRET reporter assays, FRET fluorescence signals were quantified relative to vehicle controls from three independent experiments, and mean values and SEM were calculated from the combined data of three experiments. IC50 values and confidence intervals were calculated by the least-square nonlinear curve-fitting method for normalized response to FR. For histone methylation assays, densitometry was performed on immunoblots for trimethyl-histone H3K27 and compared to DMSO control and normalized to total histone H3 from three independent experiments. The statistical significance of response to FR treatment was determined with t tests.

Gene expression analysis

92.1 UM cells were treated with 100 nM FR or vehicle (DMSO) in RPMI growth medium and collected after 1 and 3 days of treatment. RNA was isolated using the RNeasy Mini Kit (QIAGEN) following the manufacturer’s protocol and including the optional DNase I treatment step. RNA quality was assessed on a Bioanalyzer 2100 (Agilent Technologies). mRNA was extracted from total RNA using a Dynal mRNA Direct kit, fragmented, and reverse-transcribed to double-stranded complementary DNA with random primers before addition of adapters for library preparation. Library preparation and HiSeq 2500 sequencing were performed by the Washington University Genome Technology Access Center (gtac.wustl.edu). FastQ files were aligned to the transcriptome and the whole genome with STAR (Spliced Transcripts Alignment to a Reference). Biologic replicates were simultaneously analyzed by edgeR and Sailfish analyses of gene-level/exon-level features. Unexpressed genes and exons were removed from the analyses. Unsupervised principal component analysis and volcano plots were generated in Bioconductor using edgeR. Significance Analysis of Microarrays version 4.0 was used to generate a ranked gene list, and a threshold of q < 10% and a fold change of >2.0 were then used to select the most highly statistically significant genes that showed reduced expression in FR-treated versus vehicle control cells. This list was used as signature gene sets for GO analysis and GSEA (44). GO groups were assembled by merging the lists of genes from related GO terms that were significantly enriched (P < 0.01 using the Kolmogorov-Smirnov statistic) in the signature gene set. Significant gene sets (P < 0.01 using the Kolmogorov-Smirnov statistic) from GSEA analyses were combined such that genes associated with multiple related signatures were only counted once and each gene was assigned only to a single combined group based on the signature with the highest enrichment score for that gene. Gene expression changes were validated in all three UM cell lines by quantitative PCR using fast SYBR Green Master Mix (Thermo Fisher Scientific), following the manufacturers’ protocol. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an endogenous control. Primer sets used for the assay are listed in data file S6.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/11/546/eaao6852/DC1

Fig. S1. FR inhibition of Gαi1 bearing an engineered FR-binding site.

Fig. S2. Heatmaps of cell cycle and apoptosis gene expression in response to FR.

Fig. S3. Validation of selected FR target genes.

Data file S1. Gene sets with positive correlation to FR treatment in GSEA.

Data file S2. Gene sets with negative correlation to FR treatment in GSEA.

Data file S3. Genes within the FR-responsive, reduced-expression cluster.

Data file S4. GO analysis results for the FR-responsive gene cluster.

Data file S5. GSEA results for the FR-responsive gene cluster.

Data file S6. Primers used for quantitative PCR gene expression analysis.

REFERENCES AND NOTES

Acknowledgments: We are grateful to J. Y. Niederkorn (University of Texas Southwestern Medical Center) for providing UM cell lines and R. P. Mecham (Washington University in St. Louis) for supporting this project. Funding: This study was supported by a multi-investigator grant from the Siteman Cancer Center and Pedal the Cause (to K.J.B. and M.D.O.) and grants from the NIH (GM044592 and GM124093 to K.J.B. and GM118171 to J.A.C.). Author contributions: M.D.O. and K.J.B. planned experiments. M.D.O., C.M.M., S.W., K.M.K., S.M.K., T.D.T., and K.J.B. performed experiments. T.J.B. and K.J.B. purified FR. M.D.O., T.J.B., P.K.R., J.A.C., and K.J.B. analyzed data and provided comments. M.D.O., C.M.M., K.M.K., S.M.K., T.D.T., P.K.R., J.A.C., and K.J.B. wrote the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The RNA-seq data used in this manuscript are deposited at National Center for Biotechnology Information’s Gene Expression Omnibus (GEO) (GSE103761). All other data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.
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