High-throughput chemogenetic drug screening reveals PKC-RhoA/PKN as a targetable signaling vulnerability in GNAQ-driven uveal melanoma

Abstract

Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.

Keywords: FAK; GNAQ; PKC; PKN/PRK; chemogenetic drug screening; combination therapy; melanoma; precision medicine; synthetic lethality.

Graphical abstract

Figure 1

Mechanistic and target-level dependencies enriched in GNAQ-mutant UM (A) Schematic of screening pipeline in GNAQ-mutant and BRAF-mutant cell lines. Created with Biorender. (B) Heatmap depicting Z-transformed area under the curve (AUC) scores for all cell lines versus MIPE 5.0 compound library. Compounds (rows) were sorted based on the difference in average Z-AUC (ΔZ-AUC) between UM and SKCM cell contexts. Unsupervised hierarchical clustering was performed on cell lines (columns). Context-selective drugs (rows) are marked on the right. (C) Average Z-AUCs for UM plotted against SKCM. Hits identified in the UM context are highlighted in blue and SKCM hits are highlighted in red. (D) Enrichment plot for PKC-targeting drugs in UM cell lines. (E) Top 20 selective drugs ranked by Z-AUC score (UM selective above, SKCM selective below). PKC-targeting drugs are shaded in darker blue, and BRAF-targeting drugs are shaded in darker red. (F) IC₅₀ values for all tested PKCi across UM cell lines. (G) Cell viability dose response of darovasertib in all cell lines screened. (H) Two-class comparison of PRKCE CRISPR gene effect plotted against –log10 p value for UM versus SKCM cell lines from DepMap 21Q1 Public Dataset. Genes in red have a q value < 0.05. (I) Gene effect for PRKCE in UM and SKCM cell lines from DepMap 22Q2 Public+Score, Chronos Dataset. See also Tables S1, S2, and S3.

Figure 2

Multi-targeted activity of darovasertib underlies its potency in UM (A) Dose-dependent effects on phosphorylated FAK and ERK in 92.1 UM cells in response to treatment with VS-4718, darovasertib, or Go6983 for 2 h. (B) Impact of siRNA-mediated knockdown of PKCδ+ε on phosphorylated FAK, ERK, and MEK in 92.1 UM cells. (C) Kinome profiling of darovasertib. Node size and color indicate degree of kinase inhibition in response to 1 μM darovasertib, with reduction in kinase activity as red, and increase in kinase activity as blue. The figure was generated using Coral. (D) Percent kinase activity remaining after treatment with 1 μM darovasertib for the top 15 kinases with highest inhibition. (E) IC₅₀ and 95% CI of darovasertib on recombinant enzymes for a sub-panel of AGC kinases. (F) Phosphorylation of PKNs and ERK in response to treatment with a panel of PKCi: 1 μM darovasertib, 1 μM sotrastaurin, or 1 μM Go6983 for 1 h in 92.1 UM cells.

Figure 3

PKN converges with ROCK to control FAK downstream of the Gαq-RhoA signaling axis (A) Phosphorylation of FAK and PKNs in HEK293 cells transfected with empty vector, Gαq-QL, or RhoA-QL active mutants. (B) Schematic of affinity purification mass spectrometry pipeline used to identify RhoA and its associated protein binding partners after doxycycline-inducible FLAG-tagged RhoA was expressed in HEK293 cells with stable overexpression of Gαq. Created with Biorender. (C) Label-free quantification (LFQ) intensity of RhoA binding partners after RhoA expression was induced by 1 μM doxycycline treatment for 42 h. (D) Phosphorylation of FAK and PKNs in response to expression of Gαq-QL alone or in combination with RhoA blockade using 2 μg/mL C3 toxin for 16 h in HEK293 cells (left) or UM cells (right). (E) Phosphorylation of FAK after expression of Gαq-QL alone or in combination with siRNA-mediated knockdown of PKNs in HEK293 cells. (F) Phosphorylation of FAK in UM cells in response to siRNA-mediated knockdown of PKNs. (G) Quantification of pFAK signal normalized to total FAK levels from (F) (mean ± SEM, n = 3). (H) Phosphorylation of FAK in UM cells in response to siRNA-mediated knockdown of PKNs, alone or in combination with ROCK inhibition using 10 μM ROCKi (Y-27632) for 1 h. (I) Phosphorylation of FAK and ERK in UM cells in response to a panel of inhibitors, all used at 1 μM for 1 h. (J) FAK phosphorylation in response to overexpression of PKN2 in HEK293 cells.

Figure 4

Impact of darovasertib on the FAK/YAP signaling axis in UM (A) YAP and FAK phosphorylation in response to 1 μM VS-4718 or 1 μM darovasertib over a time course in OMM1.3 UM cells. (B) YAP/TAZ luciferase reporter assay after 1 μM VS-4718 or 1 μM darovasertib treatment for 2 or 24 h in UM cells (mean ± SEM, n = 3) in 92.1 UM cells. (C) Monitoring of endogenous YAP subcellular localization by immunofluorescent staining (green), and DAPI staining for nuclear DNA (blue) in UM cells after 1 μM VS-4718 or 1 μM darovasertib treatment for 24 h, vehicle treatment was used as a control in OMM1.3 UM cells. Scale bar, 50 μm. (D) Quantification of (C) showing fraction of cells with nuclear YAP localization in gray, and cytoplasmic fraction in color (vehicle as black, VS-4718 as gold, darovasertib as blue) (mean ± SEM, n = 3). (E) mRNA expression of YAP target genes (CTGF, CYR61, AMOTL2) in response to 1 μM VS-4718, 1 μM darovasertib, and 1 μM Go6983 for 24 h (mean ± SEM, n = 3) in 92.1 UM cells. (F) Schematic depicting the non-canonical signaling pathway regulating FAK activation by Gαq. Signaling downstream of RhoA is co-regulated by ROCK and PKN, converging on FAK activity. Created with Biorender.

Figure 5

High-throughput and targeted combinatorial screens reveal that FAKi and darovasertib are synergistic in UM in vitro (A) Assessment of synergy in UM cells treated with a combination of VS-4718 and darovasertib. Cell viability was measured using CellTiter Glo assay 48 h after treatment (left). Combination index (CI) was determined using the ΔBliss method (CI < 1 synergism, CI = 1 additivity, CI > 1 antagonism) (middle). Apoptosis was measured by CaspaseGlo assay, 18 h after treatment (right). (B) Distribution of CI in a diverse panel of UM and mUM cells with distinct BAP1 status. CI was determined using the HSA method (CI > 10 synergism, 0 < CI < 10 additivity, CI < 0 antagonism). (C) CI in a panel of UM cells combining darovasertib or Go6983 with various FAKi in OMM1.3, OMM1.5, and Mel202 cells determined using the HSA method. CI of PKCi combined with BRAFi (dabrafenib, vemurafenib) used as a comparison. (D) Apoptosis of UM cells measured by CaspaseGlo-3/7 assay, in response to vehicle, 1 μM VS-4718, 1 μM darovasertib, or 1 μM VS-4718 + 1 μM darovasertib for 24 h (mean ± SEM, n = 3). (E) Immunoblot showing cleaved-PARP, pFAK, and pERK in response to treatment with 1 μM VS-4718, 1 μM darovasertib, or 1 μM VS-4718 + 1 μM darovasertib for 24 h in UM cells.

Figure 6

Impact of darovasertib and VS-4718 on UM tumor growth in preclinical models (A) UM 92.1 tumor xenograft growth kinetics in SCID/NOD mice treated with vehicle (control), VS-4718 50 mg/kg BID PO, darovasertib 50 mg/kg BID PO, or combination of VS-4718 50 mg/kg BID PO + darovasertib 50 mg/kg BID PO. Data are mean ± SEM (>5 mice/group). (B) H&E staining of representative xenograft tumor sections from (A) after 25 days of treatment.Scale bar, 2mm. (C) Waterfall plot depicting tumor objective response rate from (A). (D) Representative IHC staining tumor sections for Ki67, pERK, and cleaved caspase-3 (cl-Casp3). Scale bar, 100 μm. YAP was detected by immunofluorescence. Scale bar, 50 μm. (E) Quantification of stained tumor sections in (D). Control is in gray, VS-4718 is in gold, darovasertib is in blue, and VS-4718 + darovasertib combination is in magenta (mean ± SEM, n = 3). (F) Schematic of UM metastatic model. Splenic injection of 92.1 GFP-luc cells is followed by a short period of hematogenous dissemination, splenectomy, and subsequent monitoring of hepatic metastasis by IVIS. Created with Biorender. (G) Hepatic tumor burden measured by IVIS imaging after injection of 92.1 GFP-luc UM cells in SCID/NOD mice. Mice were treated with vehicle (control) VS-4718 50 mg/kg BID PO, darovasertib 50 mg/kg BID PO, or combination of VS-4718 50 mg/kg BID PO + darovasertib 50 mg/kg BID PO. Data are mean ± SEM (>5 mice/group). (H) IVIS imaging of representative mice from (G).

Figure 7

Parallel and converging signaling mechanisms driven by Gαq (A) Lateral inhibition of Gαq-regulated signaling mechanisms represents a promising signal-transduction-based precision therapy against UM. The multi-targeted kinase activity of darovasertib primes its activity on specific Gαq-regulated growth-promoting signaling networks and, in combination with FAKi, target the core survival mechanisms in UM. Created with Biorender.