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

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.

Engaging community members in cancer research: an assessment of an NCI-designated cancer center.

PURPOSE: Despite the importance of engaging community members in research, multiple barriers exist. We conducted a mixed-methods evaluation to understand the opportunities and challenges of engaging community members in basic, clinical, translational, and population science research. METHODS: We designed a survey and an interview guide based on the constructs of the Consolidated Framework for Implementation Research. Surveys were distributed electronically to all cancer center investigators and interviews were conducted virtually with a select group of basic, clinical, and population science investigators. Survey data (n = 77) were analyzed across all respondents using frequency counts and mean scores; bivariate analyses examined differences in responses by research program affiliation, gender, race, and faculty rank. Interviews (n = 16) were audio recorded, transcribed verbatim, and analyzed using a reflective thematic approach. RESULTS: There was strong agreement among investigators that "Community engagement in research will help the SKCC address cancer disparities in the catchment area" (M 4.2, SD 0.9) and less agreement with items such as "I know how to find and connect with community members who I can engage in my research" (M 2.5, SD 1.3). Investigators mentioned challenges in communicating complex science to a lay audience but were open to training and workshops to acquire skills needed to integrate community members into their research. CONCLUSION: Cancer centers should develop and promote training and collaborative opportunities for investigators and community members. Overcoming challenges will lead to more patient- and community-centered cancer research in the future.

Targeting BRD/BET proteins inhibits adaptive kinome upregulation and enhances the effects of BRAF/MEK inhibitors in melanoma.

BACKGROUND: BRAF-mutant melanoma patients respond to BRAF inhibitors and MEK inhibitors (BRAFi/MEKi), but drug-tolerant cells persist, which may seed disease progression. Adaptive activation of receptor tyrosine kinases (RTKs) has been associated with melanoma cell drug tolerance following targeted therapy. While co-targeting individual RTKs can enhance the efficacy of BRAFi/MEKi effects, it remains unclear how to broadly target multiple RTKs to achieve more durable tumour growth inhibition. METHODS: The blockage of adaptive RTK responses by the new BET inhibitor (BETi), PLX51107, was measured by RPPA and Western blot. Melanoma growth was evaluated in vitro by colony assay and EdU staining, as well as in skin reconstructs, xenografts and PDX models following BRAFi, MEKi and/or PLX51107 treatment. RESULTS: Treatment with PLX51107 limited BRAFi/MEKi upregulation of ErbB3 and PDGFR-ß expression levels. Similar effects were observed following BRD2/4 depletion. In stage III melanoma patients, expression of BRD2/4 was strongly correlated with ErbB3. PLX51107 enhanced the effects of BRAFi/MEKi on inhibiting melanoma growth in vitro, in human skin reconstructs and in xenografts in vivo. Continuous triple drug combination treatment resulted in significant weight loss in mice, but intermittent BETi combined with continuous BRAFi/MEKi treatment was tolerable and improved durable tumour inhibition outcomes. CONCLUSIONS: Together, our data suggest that intermittent inhibition of BET proteins may improve the duration of responses following BRAFi/MEKi treatment in BRAF-mutant melanoma.

Murine Cytomegalovirus Infection of Melanoma Lesions Delays Tumor Growth by Recruiting and Repolarizing Monocytic Phagocytes in the Tumor.

Cytomegalovirus (CMV) is a ubiquitous betaherpesvirus that infects many different cell types. Human CMV (HCMV) has been found in several solid tumors, and it has been hypothesized that it may promote cellular transformation or exacerbate tumor growth. Paradoxically, in some experimental situations, murine CMV (MCMV) infection delays tumor growth. We previously showed that wild-type MCMV delayed the growth of poorly immunogenic B16 melanomas via an undefined mechanism. Here, we show that MCMV delayed the growth of these immunologically "cold" tumors by recruiting and modulating tumor-associated macrophages. Depletion of monocytic phagocytes with clodronate completely prevented MCMV from delaying tumor growth. Mechanistically, our data suggest that MCMV recruits new macrophages to the tumor via the virus-encoded chemokine MCK2, and viruses lacking this chemokine were unable to delay tumor growth. Moreover, MCMV infection of macrophages drove them toward a proinflammatory (M1)-like state. Importantly, adaptive immune responses were also necessary for MCMV to delay tumor growth as the effect was substantially blunted in Rag-deficient animals. However, viral spread was not needed and a spread-defective MCMV strain was equally effective. In most mice, the antitumor effect of MCMV was transient. Although the recruited macrophages persisted, tumor regrowth correlated with a loss of viral activity in the tumor. However, an additional round of MCMV infection further delayed tumor growth, suggesting that tumor growth delay was dependent on active viral infection. Together, our results suggest that MCMV infection delayed the growth of an immunologically cold tumor by recruiting and modulating macrophages in order to promote anti-tumor immune responses.IMPORTANCE Cytomegalovirus (CMV) is an exciting new platform for vaccines and cancer therapy. Although CMV may delay tumor growth in some settings, there is also evidence that CMV may promote cancer development and progression. Thus, defining the impact of CMV on tumors is critical. Using a mouse model of melanoma, we previously found that murine CMV (MCMV) delayed tumor growth and activated tumor-specific immunity although the mechanism was unclear. We now show that MCMV delayed tumor growth through a mechanism that required monocytic phagocytes and a viral chemokine that recruited macrophages to the tumor. Furthermore, MCMV infection altered the functional state of macrophages. Although the effects of MCMV on tumor growth were transient, we found that repeated MCMV injections sustained the antitumor effect, suggesting that active viral infection was needed. Thus, MCMV altered tumor growth by actively recruiting macrophages to the tumor, where they were modulated to promote antitumor immunity.

Development and optimization of orthotopic liver metastasis xenograft mouse models in uveal melanoma.

BACKGROUND: Patients with metastatic uveal melanoma (MUM) in the liver usually die within 1 year. The development of new treatments for MUM has been limited by the lack of diverse MUM cell lines and appropriate animal models. We previously reported that orthotopic xenograft mouse models established by direct injection of MUM cells into the liver were useful for the analysis associated with tumor microenvironment in the liver. However, considering that patients with UM metastasize to the liver hematogenously, direct liver injection model might not be suitable for investigation on various mechanisms of liver metastasis. Here, we aim to establish new orthotopic xenograft models via hematogenous dissemination of tumor cells to the liver, and to compare their characteristics with the hepatic injection model. We also determine if hepatic tumors could be effectively monitored with non-invasive live imaging. METHODS: tdtTomate-labeled, patient-derived MUM cells were injected into the liver, spleen or tail vein of immunodeficient NSG mice. Tumor growth was serially assessed with In Vivo Imaging System (IVIS) images once every week. Established hepatic tumors were evaluated with CT scan and then analyzed histologically. RESULTS: We found that splenic injection could consistently establish hepatic tumors. Non-invasive imaging showed that the splenic injection model had more consistent and stronger fluorescent intensity compared to the hepatic injection model. There were no significant differences in tumor growth between splenic injection with splenectomy and without splenectomy. The splenic injection established hepatic tumors diffusely throughout the liver, while the hepatic injection of tumor cells established a single localized tumor. Long-term monitoring of tumor development showed that tumor growth, tumor distribution in the liver, and overall survival depended on the number of tumor cells injected to the spleen. CONCLUSION: We established a new orthotopic hepatic metastatic xenograft mouse model by splenic injection of MUM cells. The growth of orthotopic hepatic tumors could be monitored with non-invasive IVIS imaging. Moreover, we evaluated the therapeutic effect of a MEK inhibitor by using this model. Our findings suggest that our new orthotopic liver metastatic mouse model may be useful for preclinical drug screening experiments and for the analysis of liver metastasis mechanisms.

Novel therapeutic strategies and targets in advanced uveal melanoma.

PURPOSE OF REVIEW: Currently, there are no U.S. Food and Drug Administration-approved or effective treatment options for advanced-stage uveal melanoma. In this article, we focus on therapeutic targets in pathways/mechanisms associated with common mutations in uveal melanoma. We review the challenges associated with targeting of these pathways and novel treatment strategies. RECENT FINDINGS: Common mutations that promote uveal melanoma initiation and progression include alterations in G protein subunit alpha q/11 (GNAQ/GNA11) and breast cancer gene 1-associated protein 1 (BAP1). Mutant GNAQ/GNA11 induces constitutive activation of tumorigenic pathways such as extracellular signal-regulated kinase (ERK)1/2 and yes-associated protein. Inhibition of mitogen-activated protein kinase kinase (MEK) downstream of ERK1/2, however, was shown in trials to have limited clinical benefit. Recent reports suggested that combination therapies of MEK inhibition and modulators of mechanisms of drug resistance may improve tumor responses to MEK inhibitors. BAP1 has been shown to be involved in modulating chromatin dynamics and deubiquitination of proteins. Hence, epigenetic inhibitors are being investigated in BAP1 mutant uveal melanoma. However, other functions of BAP1, such as in DNA damage repair and cell cycle regulation, indicate additional targets for treatment of BAP1 mutant uveal melanoma. In addition, the frequent delayed development of uveal melanoma macrometastases is likely due to cellular dormancy mechanisms. Nuclear receptor subfamily 2, group F, member 1 and transforming growth factor beta 2 were among factors that have been shown in other cancers to induce dormant phenotypes. SUMMARY: Findings from studies in uveal melanoma and in other cancers provide evidence for potential strategies that may be tested preclinically and clinically in advanced-stage uveal melanoma to improve treatment outcome and overall survival of patients.

Establishment of an orthotopic patient-derived xenograft mouse model using uveal melanoma hepatic metastasis.

BACKGROUND: Metastatic uveal melanoma is a highly fatal disease; most patients die from their hepatic metastasis within 1 year. A major drawback in the development of new treatments for metastatic uveal melanoma is the difficulty in obtaining appropriate cell lines and the lack of appropriate animal models. Patient-derived xenograft (PDX) tumor models, bearing ectopically implanted tumors at a subcutaneous site, have been developed. However, these ectopically implanted PDX models have obstacles to translational research, including a low engraftment rate, slow tumor growth, and biological changes after multiple passages due to the different microenvironment. To overcome these limitations, we developed a new method to directly transplant biopsy specimens to the liver of immunocompromised mice. RESULTS: By using two metastatic uveal melanoma cell lines, we demonstrated that the liver provides a more suitable microenvironment for tumor growth compared to subcutaneous sites and that surgical orthotopic implantation (SOI) of tumor pieces allows the creation of a liver tumor in immunocompromised mice. Subsequently, 10 of 12 hepatic metastasis specimens from patients were successfully xenografted into the immunocompromised mice (83.3% success rate) using SOI, including 8 of 10 needle biopsy specimens (80%). Additionally, four cryopreserved PDX tumors were re-implanted to new mice and re-establishment of PDX tumors was confirmed in all four mice. The serially passaged xenograft tumors as well as the re-implanted tumors after cryopreservation were similar to the original patient tumors in histologic, genomic, and proteomic expression profiles. CT imaging was effective for detecting and monitoring PDX tumors in the liver of living mice. The expression of Ki67 in original patient tumors was a predictive factor for implanted tumor growth and the success of serial passages in PDX mice. CONCLUSIONS: Surgical orthotopic implantation of hepatic metastasis from uveal melanoma is highly successful in the establishment of orthotopic PDX models, enhancing their practical utility for research applications. By using CT scan, tumor growth can be monitored, which is beneficial to evaluate treatment effects in interventional studies.

Establishment and Characterization of Orthotopic Mouse Models for Human Uveal Melanoma Hepatic Colonization.

Uveal melanoma (UM) is a rare type of melanoma, although it is the most common primary ocular malignant tumor in adults. Nearly one-half the patients with primary UM subsequently develop systemic metastasis, preferentially to the liver. Currently, no treatment is effective for UM hepatic metastasis, and the prognosis is universally poor. The main challenge in designing a treatment strategy for UM hepatic metastasis is the lack of suitable animal models. We developed two orthotopic mouse models for human UM hepatic metastases: direct hepatic implantation model (intrahepatic dissemination model) and splenic-implantation model (hematogenous dissemination model) and investigated the tumorgenesis in the liver. A human UM cell line, established from a hepatic metastasis and nonobese diabetic severe combined immunodeficient γ mice, were used for development of in vivo tumor models. In the direct hepatic implantation model, a localized tumor developed in the liver in all cases and intrahepatic dissemination was subsequently seen in about one-half of cases. However, in the splenic implantation model, multiple hepatic metastases were observed after splenic implantation. Hepatic tumors subsequently seeded intra-abdominal metastasis; however, lung metastases were not seen. These findings are consistent with those observed in human UM hepatic metastases. These orthotopic mouse models offer useful tools to investigate the biological behavior of human UM cells in the liver.

Fibroblast-derived neuregulin 1 promotes compensatory ErbB3 receptor signaling in mutant BRAF melanoma.

Rapidly accelerated fibrosarcoma (RAF) inhibitors are first-line treatments for patients harboring V600E/K mutant BRAF melanoma. Although RAF inhibitors produce high response rates, the degree of tumor regression is heterogeneous. Compensatory/adaptive responses to targeted inhibitors are frequently initiated by the activation of growth factor receptor tyrosine kinases, including ErbB3, and factors from the tumor microenvironment may play an important role. We have shown previously that mutant v-raf murine sarcoma viral oncogene homolog B1 (BRAF) melanoma cells have enhanced activation of ErbB3 following RAF inhibition. However, the source of neuregulin 1 (NRG1), the ligand for ErbB3, is unknown. In this study, we demonstrate that NRG1 is highly expressed by dermal fibroblasts and cancer-associated fibroblasts (CAFs) isolated from mutant BRAF melanomas. Conditioned medium from fibroblasts and CAFs enhanced ErbB3 pathway activation and limited RAF inhibitor cytotoxicity in V600 mutant BRAF-harboring melanomas. Targeting the ErbB3/ErbB2 pathway partially reversed the protective effects of fibroblast/CAF-derived NRG1 on cell growth properties of RAF inhibitor-treated melanoma cells. These findings support the idea that NRG1, acting in a paracrine manner, promotes resistance to RAF inhibitors and emphasize that targeting the ErbB3/ErbB2 pathway will likely improve the efficacy of RAF inhibitors for mutant BRAF melanoma patients.

PAX3 and FOXD3 Promote CXCR4 Expression in Melanoma.

Metastatic melanoma is an aggressive and deadly disease. The chemokine receptor CXCR4 is active in melanoma metastasis, although the mechanism for the promotion and maintenance of CXCR4 expression in these cells is mostly unknown. Here, we find melanoma cells express two CXCR4 isoforms, the common version and a variant that is normally restricted to cells during development or to mature blood cells. CXCR4 expression is driven through a highly conserved intronic enhancer element by the transcription factors PAX3 and FOXD3. Inhibition of these transcription factors slows melanoma cell growth, migration, and motility, as well as reduces CXCR4 expression. Overexpression of these transcription factors drives the production of increased CXCR4 levels. Loss of PAX3 and FOXD3 transcription factor activity results in a reduction in cell motility, migration, and chemotaxis, all of which are rescued by CXCR4 overexpression. Here, we discover a molecular pathway wherein PAX3 and FOXD3 promote CXCR4 gene expression in melanoma.

Jak2-Stat5a/b Signaling Induces Epithelial-to-Mesenchymal Transition and Stem-Like Cell Properties in Prostate Cancer.

Active Stat5a/b predicts early recurrence and disease-specific death in prostate cancer (PC), which both typically are caused by development of metastatic disease. Herein, we demonstrate that Stat5a/b induces epithelial-to-mesenchymal transition (EMT) of PC cells, as shown by Stat5a/b regulation of EMT marker expression (Twist1, E-cadherin, N-cadherin, vimentin, and fibronectin) in PC cell lines, xenograft tumors in vivo, and patient-derived PCs ex vivo using organ explant cultures. Jak2-Stat5a/b signaling induced functional end points of EMT as well, indicated by disruption of epithelial cell monolayers and increased migration and adhesion of PC cells to fibronectin. Knockdown of Twist1 suppressed Jak2-Stat5a/b-induced EMT properties of PC cells, which were rescued by re-introduction of Twist1, indicating that Twist1 mediates Stat5a/b-induced EMT in PC cells. While promoting EMT, Jak2-Stat5a/b signaling induced stem-like properties in PC cells, such as sphere formation and expression of cancer stem cell markers, including BMI1. Mechanistically, both Twist1 and BMI1 were critical for Stat5a/b induction of stem-like features, because genetic knockdown of Twist1 suppressed Stat5a/b-induced BMI1 expression and sphere formation in stem cell culture conditions, which were rescued by re-introduction of BMI1. By using human prolactin knock-in mice, we demonstrate that prolactin-Stat5a/b signaling promoted metastases formation of PC cells in vivo. In conclusion, our data support the concept that Jak2-Stat5a/b signaling promotes metastatic progression of PC by inducing EMT and stem cell properties in PC cells.

Targeting mutant NRAS signaling pathways in melanoma.

Cutaneous melanoma is a devastating form of skin cancer and its incidence is increasing faster than any other preventable cancer in the United States. The mutant NRAS subset of melanoma is more aggressive and associated with poorer outcomes compared to non-NRAS mutant melanoma. The aggressive nature and complex molecular signaling conferred by this transformation has evaded clinically effective treatment options. This review examines the major downstream effectors of NRAS relevant in melanoma and the associated advances made in targeted therapies that focus on these effector pathways. We outline the history of MEK inhibition in mutant NRAS melanoma and recent advances with newer MEK inhibitors. Since MEK inhibitors will likely be optimized when combined with other targeted therapies, we focus on recently identified targets that can be used in combination with MEK inhibitors.

SOX10 Loss Sensitizes Melanoma Cells to Cytokine-Mediated Inflammatory Cell Death.

The transcription factor, SOX10, plays an important role in the differentiation of neural crest precursors to the melanocytic lineage. Malignant transformation of melanocytes leads to the development of melanoma, and SOX10 promotes melanoma cell proliferation and tumor formation. SOX10 expression in melanomas is heterogeneous, and loss of SOX10 causes a phenotypic switch toward an invasive, mesenchymal-like cell state and therapy resistance; hence, strategies to target SOX10-deficient cells are an active area of investigation. The impact of cell state and SOX10 expression on antitumor immunity is not well understood but will likely have important implications for immunotherapeutic interventions. To this end, we tested whether SOX10 status affects the response to CD8+ T cell-mediated killing and T cell-secreted cytokines, TNFα and IFNγ, which are critical effectors in the cytotoxic killing of cancer cells. We observed that genetic ablation of SOX10 rendered melanoma cells more sensitive to CD8+ T cell-mediated killing and cell death induction by either TNFα or IFNγ. Cytokine-mediated cell death in SOX10-deficient cells was associated with features of caspase-dependent pyroptosis, an inflammatory form of cell death that has the potential to increase immune responses. IMPLICATIONS: These data support a role for SOX10 expression altering the response to T cell-mediated cell death and contribute to a broader understanding of the interaction between immune cells and melanoma cells.

Mcl-1 mediates intrinsic resistance to RAF inhibitors in mutant BRAF papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most frequent form of thyroid cancer. PTC commonly presents with mutations of the serine/threonine kinase BRAF (BRAFV600E), which drive ERK1/2 pathway activation to support growth and suppress apoptosis. PTC patients often undergo surgical resection; however, since the average age of PTC patients is under 50, adverse effects associated with prolonged maintenance therapy following total thyroidectomy are a concern. The development of mutant-selective BRAF inhibitors (BRAFi), like vemurafenib, has been efficacious in patients with metastatic melanoma, but the response rate is low for mutant BRAF PTC patients. Here, we assay the therapeutic response of BRAFi in a panel of human PTC cell lines and freshly biopsied patient samples. We observed heterogeneous responses to BRAFi, and multi-omic comparisons between susceptible and resistant mutant BRAF PTC revealed overrepresented stress response pathways and the absence of compensatory RTK activation - features that may underpin innate resistance. Importantly, resistant cell lines and patient samples had increased hallmarks of failed apoptosis; a cellular state defined by sublethal caspase activation and DNA damage. Further analysis suggests that the failed apoptotic phenotypes may have features of "minority mitochondrial outer membrane permeabilization (MOMP)" - a stress-related response characterized by fragmented and porous mitochondria known to contribute to cancer aggressiveness. We found that cells presenting with minority MOMP-like phenotypes are dependent on the apoptotic regulator, Mcl-1, as treatment with the Mcl-1 inhibitor, AZD5991, potently induced cell death in resistant cells. Furthermore, PI3K/AKT inhibitors sensitized resistant cells to BRAFi; an effect that was at least in part associated with reduced Mcl-1 levels. Together, these data implicate minority MOMP as a mechanism associated with intrinsic drug resistance and underscore the benefits of targeting Mcl-1 in mutant BRAF PTC.

Lineage commitment pathways epigenetically oppose oncogenic Gαq/11-YAP signaling in dormant disseminated uveal melanoma.

The mechanisms driving late relapse in uveal melanoma (UM) patients remains a medical mystery and major challenge. Clinically it is inferred that UM disseminated cancer cells (DCCs) persist asymptomatic for years-to-decades mainly in the liver before they manifest as symptomatic metastasis. Here we reveal using Gαq/11 mut /BAP wt human uveal melanoma models and human UM metastatic samples, that the neural crest lineage commitment nuclear receptor NR2F1 is a key regulator of spontaneous UM DCC dormancy in the liver. Using a quiescence reporter, RNA-seq and multiplex imaging we revealed that rare dormant UM DCCs upregulate NR2F1 expression and genes related to neural crest programs while repressing gene related to cell cycle progression. Gain and loss of function assays showed that NR2F1 silences YAP1/TEAD1 transcription downstream of Gαq/11 signaling and that NR2F1 expression can also be repressed by YAP1. YAP1 expression is repressed by NR2F1 binding to its promoter and changing the histone H3 tail activation marks to repress YAP1 transcription. In vivo CRISPR KO of NR2F1 led dormant UM DCCs to awaken and initiate relentless liver metastatic growth. Cut&Run and bulk RNA sequencing further confirmed that NR2F1 epigenetically stimulates neuron axon guidance and neural lineage programs, and it globally represses gene expression linked to G-protein signaling to drive dormancy. Pharmacological inhibition of Gαq/11 mut signaling resulted in NR2F1 upregulation and robust UM growth arrest, which was also achieved using a novel NR2F1 agonist. Our work sheds light on the molecular underpinnings of UM dormancy revealing that transcriptional programs driven by NR2F1 epigenetically short-circuit Gαq/11 signaling to its downstream target YAP1. Highlights: Quiescent solitary uveal melanoma (UM) DCCs in the liver up- and down-regulate neural crest and cell cycle progression programs, respectively.NR2F1 drives solitary UM DCC dormancy by antagonizing the Gαq/11-YAP1 pathway; small molecule Gαq/11 inhibition restores NR2F1 expression and quiescence. NR2F1 short-circuits oncogenic YAP1 and G-protein signaling via a chromatin remodeling program. Loss of function of NR2F1 in dormant UM DCCs leads to aggressive liver metastasis.

Slow proliferation of BAP1-deficient uveal melanoma cells is associated with reduced S6 signaling and resistance to nutrient stress.

Uveal melanoma (UM) is the deadliest form of eye cancer in adults. Inactivating mutations and/or loss of expression of the gene encoding BRCA1-associated protein 1 (BAP1) in UM tumors are associated with an increased risk of metastasis. To investigate the mechanisms underlying this risk, we explored the functional consequences of BAP1 deficiency. UM cell lines expressing mutant BAP1 grew more slowly than those expressing wild-type BAP1 in culture and in vivo. The ability of BAP1 reconstitution to restore cell proliferation in BAP1-deficient cells required its deubiquitylase activity. Proteomic analysis showed that BAP1-deficient cells had decreased phosphorylation of ribosomal S6 and its upstream regulator, p70S6K1, compared with both wild-type and BAP1 reconstituted cells. In turn, expression of p70S6K1 increased S6 phosphorylation and proliferation of BAP1-deficient UM cells. Consistent with these findings, BAP1 mutant primary UM tumors expressed lower amounts of p70S6K1 target genes, and S6 phosphorylation was decreased in BAP1 mutant patient-derived xenografts (PDXs), which grew more slowly than wild-type PDXs in the liver (the main metastatic site of UM) in mice. BAP1-deficient UM cells were also more resistant to amino acid starvation, which was associated with diminished phosphorylation of S6. These studies demonstrate that BAP1 deficiency slows the proliferation of UM cells through regulation of S6 phosphorylation. These characteristics may be associated with metastasis by ensuring survival during amino acid starvation.

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

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.

SHOC2 and CRAF mediate ERK1/2 reactivation in mutant NRAS-mediated resistance to RAF inhibitor.

ERK1/2 signaling is frequently dysregulated in tumors through BRAF mutation. Targeting mutant BRAF with vemurafenib frequently elicits therapeutic responses; however, durable effects are often limited by ERK1/2 pathway reactivation via poorly defined mechanisms. We generated mutant BRAF(V600E) melanoma cells that exhibit resistance to PLX4720, the tool compound for vemurafenib, that co-expressed mutant (Q61K) NRAS. In these BRAF(V600E)/NRAS(Q61K) co-expressing cells, re-activation of the ERK1/2 pathway during PLX4720 treatment was dependent on NRAS. Expression of mutant NRAS in parental BRAF(V600) cells was sufficient to by-pass PLX4720 effects on ERK1/2 signaling, entry into S phase and susceptibility to apoptosis in a manner dependent on the RAF binding site in NRAS. ERK1/2 activation in BRAF(V600E)/NRAS(Q61K) cells required CRAF only in the presence of PLX4720, indicating a switch in RAF isoform requirement. Both ERK1/2 activation and resistance to apoptosis of BRAF(V600E)/NRAS(Q61K) cells in the presence of PLX4720 was modulated by SHOC-2/Sur-8 expression, a RAS-RAF scaffold protein. These data show that NRAS mutations confer resistance to RAF inhibitors in mutant BRAF cells and alter RAF isoform and scaffold molecule requirements to re-activate the ERK1/2 pathway.

Targeting TEAD-ious resistance.

Recent advances in targeting mutant KRAS are limited by resistance. A recent study in Nature Cancer by Hagenbeek et al. utilizes a novel inhibitor that targets the TEAD transcription factor, GNE-7883, to overcome resistance to KRAS inhibitors. Thus, TEAD inhibitors may maximize the durability of KRAS inhibitors in patients with cancer.

Site matters in metastatic melanoma.

Metastasis and cross-therapy resistance to mitogen-activated protein kinase (MAPK) inhibition and immune checkpoint blockade (ICB) are significant clinical issues in melanoma. A new study in NatureMedicine by Liu et al. utilizes metastatic melanoma (MM) tumors from a rapid autopsy cohort to dissect genomic and transcriptomic features of therapy resistance, organ-specific gene signatures, and crosstalk between MM and organ sites.