Kinome profiling identifies MARK3 and STK10 as potential therapeutic targets in uveal melanoma

Abstract

Most uveal melanoma cases harbor activating mutations in either GNAQ or GNA11. Despite activation of the mitogen-activated protein kinase (MAPK) signaling pathway downstream of Gαq/11, there are no effective targeted kinase therapies for metastatic uveal melanoma. The human genome encodes numerous understudied kinases, also called the "dark kinome". Identifying additional kinases regulated by Gαq/11 may uncover novel therapeutic targets for uveal melanoma. In this study, we treated GNAQ-mutant uveal melanoma cell lines with a Gαq/11 inhibitor, YM-254890, and conducted a kinase signaling proteomic screen using multiplexed-kinase inhibitors followed by mass spectrometry. We observed downregulated expression and/or activity of 22 kinases. A custom siRNA screen targeting these kinases demonstrated that knockdown of microtubule affinity regulating kinase 3 (MARK3) and serine/threonine kinase 10 (STK10) significantly reduced uveal melanoma cell growth and decreased expression of cell cycle proteins. Additionally, knockdown of MARK3 but not STK10 decreased ERK1/2 phosphorylation. Analysis of RNA-sequencing and proteomic data showed that Gαq signaling regulates STK10 expression and MARK3 activity. Our findings suggest an involvement of STK10 and MARK3 in the Gαq/11 oncogenic pathway and prompt further investigation into the specific roles and targeting potential of these kinases in uveal melanoma.

Keywords: cancer; g protein; guanine nucleotide-binding protein G(q) subunit alpha; melanoma; microtubule affinity-regulating kinase 3; mitogen-activated protein kinase (MAPK); proteomics; serine/threonine-protein kinase 10.

Figure 1

Profiling the uveal melanoma kinome using quantitative MIB-MS after YM-254890 treatment shows downregulation of cell cycle, signal transduction, and cell division kinases.A, GNAQ-mutant uveal melanoma cell lines MP38, MP46, and OMM1.3 were treated for 24 h with 0.5 μM or 1.0 μM YM-254890. Cell lysates were analyzed by Western blotting for phospho-ERK1/2 and total ERK1/2. β-actin was used as loading control. B, schematic representation of kinome profiling using MIB-MS. Lysates from GNAQ-mutant uveal melanoma cells treated with either DMSO (vehicle control) or 1 μM YM-254890 were run through MIB columns followed by MS (n = 3). C, heatmap of altered kinases after 24 h of 1 μM YM-254890 treatment in uveal melanoma cell lines. Statistical significance was determined by the Student’s t test p-value < 0.05.

Figure 2

Loss-of-function screen identifies MARK3 and STK10 as potential therapeutic targets. Histogram of cell viability measured by CellTiter-Glo post-siRNA knockdown of 22 candidate kinases in MP38 (left panel) and OMM1.3 (right panel) cells. Targets are ordered based on average cell viability in all three cell lines.

Figure 3

MARK3 and STK10 knockdown effects on target proteins and cell growth.A, representative Western blots of MARK3, STK10, and respective downstream targets in knockdown cells. B, effects of siRNA-mediated knockdown of MARK3 or STK10 on MP38 and OMM1.3 cell growth were analyzed using the IncuCyte Live Cell Analysis Imaging System by measuring % plate coverage (n = 3). ∗p < 0.05 as determined by t test, and error bars are ± SEM. C, annexin-V staining (%) following MARK3 or STK10 knockdown in MP38 or OMM1.3 (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as determined by t test, and error bars are ± SEM. D, analysis of TCGA data for uveal melanoma patient disease-specific survival according to MARK3 and STK10 expression, above or below median RNA expression. Log-rank test was used to determine the significance of disease-specific survival.

Figure 4

MARK3 and STK10 knockdown effects on cell cycle proteins.A, heatmap of median-centered, log₂-transformed group average expression RPPA data for differentially expressed proteins (p < 0.05, log₂fc > 0.58496) when comparing siRNA-mediated knockdown of MARK3 or STK10 cells to untreated or siCTL. Each lysate was collected in quadruplets (n = 4). B, representative Western blots validating the effects of MARK3 or STK10 knockdown on cell cycle and cell growth proteins.

Figure 5

Gαq/11 and MAPK pathway regulates STK10 mRNA expression.A, publicly available RNA-seq data was analyzed for MARK3 and STK10 expression following 24-h YM-254890 treatment in Mel202 and OMM1.3 cells. Expression data is shown as a log₂ fold change of normalized counts compared to DMSO. B, RNA-seq of PDX4 cells treated with MEK (5 μM binimetinib or 50 nM trametinib) or ERK1/2 (2 μM BVD523 or 500 nM SCH772984) inhibitors for 24 h. Data are shown as fold change relative to DMSO (n = 3). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 6

Gαq/11 regulates STK10 expression and MARK3 activity.A, representative Western blots of MARK3 and STK10 after OMM1.3 cells were treated with 1 μM of YM-254890 over a 24-h time course (n = 3). B, representative Western blots showing the effect of siGNAQ on MARK3 and STK10 expression. C, Western blot of lysates from OMM1.3 cells treated with YM-254890 for 0 to 24 h were analyzed for phosphorylation of the endogenous MARK3 substrate, CDC25C; WCL, whole cell lysate.