Protein Kinase Signaling Networks Driven by Oncogenic Gq/11 in Uveal Melanoma Identified by Phosphoproteomic and Bioinformatic Analyses

Onken, Michael D.; Erdmann-Gilmore, Petra; Zhang, Qiang; Thapa, Kisan; King, Emily; Kaltenbronn, Kevin M.; Noda, Sarah E.; Makepeace, Carol M.; Goldfarb, Dennis; Babur, Özgün; Townsend, R. Reid; Blumer, Kendall J.

doi:10.1016/j.mcpro.2023.100649

Cited by 5 publications

(1 citation statement)

References 62 publications

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“…Synthetic lethality approaches revealed non-canonical pathways as the main drivers of oncogenic GNAQ signaling [8]. Recent phosphoproteomic and genome-wide synthetic lethality strategies and chemogenetic drug screening have revealed novel targetable signaling vulnerabilities in GNAQ-driven uveal melanoma [6,7,9]. To expand the possibilities to understand the molecular intricacies of unconventional pathways activated by oncogenic GNAQ, we focused our current studies on the hypothetical role of PDZ-RhoGEF/RhoJ as a direct signaling axis activated by Gαq Q209L, an oncogenic mutant, and used a rational data mining strategy, focusing on their relational signaling partners in cancer patients, to highlight those that, according to synthetic lethality datasets of cancer cells, represent vulnerabilities.…”

Section: Introductionmentioning

confidence: 99%

Oncogenic Gαq activates RhoJ through PDZ-RhoGEF

Cervantes-Villagrana,

Color-Aparicio,

Castillo-Kauil

et al. 2023

IJMS

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Oncogenic Gαq causes uveal melanoma via non-canonical signaling pathways. This constitutively active mutant GTPase is also found in cutaneous melanoma, lung adenocarcinoma, and seminoma, as well as in benign vascular tumors, such as congenital hemangiomas. We recently described that PDZ-RhoGEF (also known as ARHGEF11), a canonical Gα12/13 effector, is enabled by Gαs Q227L to activate CdcIn addition, and we demonstrated that constitutively active Gαq interacts with the PDZ-RhoGEF DH-PH catalytic module, but does not affect its binding to RhoA or Cdc. This suggests that it guides this RhoGEF to gain affinity for other GTPases. Since RhoJ, a small GTPase of the Cdc42 subfamily, has been involved in tumor-induced angiogenesis and the metastatic dissemination of cancer cells, we hypothesized that it might be a target of oncogenic Gαq signaling via PDZ-RhoGEF. Consistent with this possibility, we found that Gαq Q209L drives full-length PDZ-RhoGEF and a DH-PH construct to interact with nucleotide-free RhoJ-G33A, a mutant with affinity for active RhoJ-GEFs. Gαq Q209L binding to PDZ-RhoGEF was mapped to the PH domain, which, as an isolated construct, attenuated the interaction of this mutant GTPase with PDZ-RhoGEF’s catalytic module (DH-PH domains). Expression of these catalytic domains caused contraction of endothelial cells and generated fine cell sprouts that were inhibited by co-expression of dominant negative RhoJ. Using relational data mining of uveal melanoma patient TCGA datasets, we got an insight into the signaling landscape that accompanies the Gαq/PDZ-RhoGEF/RhoJ axis. We identified three transcriptional signatures statistically linked with shorter patient survival, including GPCRs and signaling effectors that are recognized as vulnerabilities in cancer cell synthetic lethality datasets. In conclusion, we demonstrated that an oncogenic Gαq mutant enables the PDZ-RhoGEF DH-PH module to recognize RhoJ, suggesting an allosteric mechanism by which this constitutively active GTPase stimulates RhoJ via PDZ-RhoGEF. These findings highlight PDZ-RhoGEF and RhoJ as potential targets in tumors driven by mutant Gαq.

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Section: Introductionmentioning

confidence: 99%

Oncogenic Gαq activates RhoJ through PDZ-RhoGEF

Cervantes-Villagrana,

Color-Aparicio,

Castillo-Kauil

et al. 2023

IJMS

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Examination of INPP5A in uveal melanoma uncovers novel calcium transients

Onken,

Kaltenbronn,

Makepeace

et al. 2024

Preprint

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Uveal melanoma is a highly aggressive intraocular cancer that metastasizes in about half of patients whereupon it is inexorably fatal. Uveal melanomas (UM) are distinct from other melanomas because they are driven by constitutively activating mutation in the heterotrimeric G protein alpha subunits Gq (GNAQ) and G11 (GNA11). This results in constitutive production of inositol trisphosphate (IP3) by phospholipase C-beta downstream of Gq/11. In normal cells, increased IP3 causes calcium release from the endoplasmic reticulum, which would be cytotoxic if maintained chronically, but UM cells are able to survive constitutive IP3 production. INPP5A, which dephosphorylates and thus inactivates IP3, is highly upregulated in UM cells compared to other melanomas and another study has shown that INPP5A is necessary for UM cell survival. To understand the mechanism of calcium regulation in response to IP3, we collected single-cell calcium measurements and found that UM cells driven by constitutively active Gq/11 produce spontaneous calcium transients. These calcium oscillations are not seen in any other melanoma cell lines unless induced by an agonist, but they are present in patient UM tumor samples. Moreover, these calcium oscillations are lost in UM cells treated with the Gq/11 inhibitor FR900359, demonstrating their dependence on constitutive Gq/11 activity. We found that the INPP5A inhibitor YU144369 causes significant changes in calcium oscillations in UM cells, demonstrating a role for INPP5A in this system. INPP5A is tethered to membranes by C-terminal prenylation and palmitoylation, suggesting that localization may play a role in INPP5A regulation of IP3 levels. GFP-tagged INPP5A was localized to plasma membrane, nuclear envelop, endoplasmic reticulum, and lysosomes. Mutation of the palmitoylation site significantly reduced localization to the plasma membrane, while mutation of the prenylation site resulted in purely nucleoplasmic localization of INPP5A. These results demonstrate a role of palmitoylation in the regulation of INPP5A localization and mobilization in UM cells.

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Meeting Report From the 2023 Cure Ocular Melanoma (CURE OM) Global Science Meeting, Philadelphia, PA, November 2023

Seedor,

Aplin,

Bertolotto

et al. 2024

Pigment Cell Melanoma Res

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The 2023 Cure Ocular Melanoma (CURE OM) Global Science Meeting was held in Philadelphia on November 6, 2023. There is increased awareness and dedicated research in uveal melanoma (UM), but unmet needs remain in the prevention, detection, and treatment of UM. The purpose of this meeting was to provide an international forum for the exchange of research ideas, to allow for discussion of basic science as well as clinical research on UM, and to gather input about advocacy and patient needs.

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Protein Kinase Signaling Networks Driven by Oncogenic Gq/11 in Uveal Melanoma Identified by Phosphoproteomic and Bioinformatic Analyses

Cited by 5 publications

References 62 publications

Oncogenic Gαq activates RhoJ through PDZ-RhoGEF

Oncogenic Gαq activates RhoJ through PDZ-RhoGEF

Examination of INPP5A in uveal melanoma uncovers novel calcium transients

Meeting Report From the 2023 Cure Ocular Melanoma (CURE OM) Global Science Meeting, Philadelphia, PA, November 2023

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