Mutant Uveal Melanoma Research Articles

Abstract 4025: New candidate therapy for BAP1-mutant cancer identified using novel screen

Abstract Inactivation of the oncogene BAP1 is associated with poor prognosis in many cancers, including uveal melanoma (UM). Therapies for mediating the detrimental effects of BAP1 mutations have yet to be developed. Previously, we found that BAP1 depletion in Xenopus causes a striking developmental phenotype and a global dysregulation of differentiation markers across the genetic landscape. This was caused by impaired promoter assembly at commitment genes, marked by decreased H3K27ac levels. Inhibition of HDAC4 protein rescues the affected BAP1-depleted phenotype and genetic regulations, further emphasizing the role of H3K27ac in BAP1 function. Pairing these findings with a lab-devised multitiered screening process, we identified an epigenetic compound that successfully reverses the detrimental effects of the BAP1 deficient phenotype back to phenotypically normal conditions. This multitiered screening process tests prospective compounds for the ability to mediate transcriptional aberrations caused by BAP1 loss in UM cells (phase 1), rescue the BAP1 loss phenotype in Xenopus (phase 2), and provide significant inhibition of UM tumor growth in a mouse model (phase 3). These findings reveal a new application for a promising epigenetic compound as a therapeutic and reversing agent for BAP1 mutant UM. Furthermore, our streamlined process for the testing of promising therapeutic agents can accelerate the testing of such agents and getting them to clinical trials in a timely manner. Citation Format: DAWN A. OWENS, Jeffim N. Kuznetsov, Andy Lopez, Stefan Kurtenbach, Daniel Bilbao, Evan R. Roberts, Claude-Henry Volmar, Claes R. Wahlestedt, Shaun P. Brothers, J. William Harbour. New candidate therapy for BAP1-mutant cancer identified using novel screen [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4025.

Metabolic Adaptations to MEK and CDK4/6 Cotargeting in Uveal Melanoma.

Frequent GNAQ and GNA11 mutations in uveal melanoma hyperactivate the MEK-ERK signaling pathway, leading to aberrant regulation of cyclin-dependent kinases (CDK) and cell-cycle progression. MEK inhibitors (MEKi) alone show poor efficacy in uveal melanoma, raising the question of whether downstream targets can be vertically inhibited to provide long-term benefit. CDK4/6 selective inhibitors are FDA-approved in patients with estrogen receptor (ER)-positive breast cancer in combination with ER antagonists/aromatase inhibitors. We determined the effects of MEKi plus CDK4/6 inhibitors (CDK4/6i) in uveal melanoma. In vitro, palbociclib, a CDK4/6i, enhanced the effects of MEKi via downregulation of cell-cycle proteins. In contrast, in vivo CDK4/6 inhibition alone led to cytostasis and was as effective as MEKi plus CDK4/6i treatment at delaying tumor growth. RNA sequencing revealed upregulation of the oxidative phosphorylation (OxPhos) pathway in both MEKi-resistant tumors and CDK4/6i-tolerant tumors. Furthermore, oxygen consumption rate was increased following MEKi + CDK4/6i treatment. IACS-010759, an OxPhos inhibitor, decreased uveal melanoma cell survival in combination with MEKi + CDK4/6i. These data highlight adaptive upregulation of OxPhos in response to MEKi + CDK4/6i treatment in uveal melanoma and suggest that suppression of this metabolic state may improve the efficacy of MEKi plus CDK4/6i combinations.

Detection of GNAQ/ 11 Mutations in Uveal Melanoma Patients' FFPE DNA with Droplet Digital PCR

To quantitatively detect GNAQ/11 mutations in uveal melanoma (UM) by droplet digital PCR (ddPCR). Formaldehyde-fixed paraffin-embedded (FFPE) tumor samples were taken from 78 UM patients with enucleation in West China Hospital between 2009 and 2015. None of the patients received radiotherapy or chemotherapy before enucleation. A retrospective study was conducted to detect GNAQ/11 mutation in UM by ddPCR. To compare the consistency of the results of the two detection methods, DNA sequencing was performed on the target gene by Sanger sequencing. 78 patients with UM were studied retrospectively. GNAQ/11 mutations in uveal melanoma was detected by ddPCR. The consistency of the results of the two detection methods was analyzed. GNAQ/11 mutations frequency was 91.9%. The consistency test between Sanger sequencing and ddPCR of GNAQ/11 mutations in 74 patients with UM was conducted. Kappa coefficient=0.436, P=0.001. The error rate of Sanger sequencing results was significantly higher in the heterogeneous group than in the homogeneous group (12/37 vs. 3/16, P=0.53), but the difference was not statistically significant. The results of ddPCR and Sanger sequencing showed good consistency, and the mutation ratio of GNAQ/11 in UM was significantly different. GNAQ/11 mutation frequency in UM patients detected by ddPCR was close to the reported frequency. It is more recommended to use ddPCR with high sensitivity to detect gene mutations in samples of tumor tissue DNA derived from FFPE. Sanger sequencing is prone to false negative results.

Circulating tumor DNA (ctDNA) in patients (pts) with metastatic uveal melanoma (UM) treated with protein kinase C inhibitor (PKCi).

e22054 Background: UM is the most common primary intraocular malignancy. About 50% of pts with UM will develop metastatic disease and currently there is no effective treatment in metastatic UM. Nearly 95% of UM harbour mutually exclusive activating mutations in GNAQ, GNA11, CYSTLR2 and PLCb4. We sought to evaluate ctDNA in metastatic UM pts receiving experimental early phase clinical trial of LXS196, a PKCi, using digital droplet PCR (ddPCR) and targeted ion torrent next generation sequencing (NGS). Methods: 17 pts with metastatic UM were identified from a single institution. Pt characteristics including mutation status, disease volume using sum of product of disease (SPOD), best response and clinical benefit defined as partial response (PR) or stable disease (SD) > 6 months were assessed. Plasma samples at baseline and early on treatment (EOT) (14 – 30 days on treatment) were analysed for ctDNA using mutation specific ddPCR. A custom made NGS panel covering 95% of UM mutations was used on plasma ctDNA samples at baseline and on treatment. The allele frequency (AF) of activating mutations identified using ion torrent NGS analysis were then validated using ddPCR. Results: Using ddPCR, 16/17 pts had a +ve ctDNA at baseline with baseline ctDNA copies correlating with LDH (n = 17, p = < 0.001, Spearman’s rank r = 0.8015) and SPOD (n = 17, p = 0.005, r = 0.6642). 16/17 pts had paired samples at baseline and EOT and only 4/16 pts were undetectable at EOT. These 4 pts were GNA11 Q209L positive, had below median SPOD (median 5986, range 200 – 16782), and low numbers of liver metastases (median 9, range 1 – 49). Of these 4 pts, one had PR and three had SD (two with SD > 6 months) as best response. A further 8 pts had +ve ctDNA at baseline and showed a reduction in EOT (reduction range 46 – 99%). Of these 8 pts, one had PR, four had SD (two with SD > 6 months) and three had progressive disease (PD) as best response. The remaining 4 pts showed increasing ctDNA from baseline to EOT and all had SD/PD. Using ROC analysis, EOT ctDNA predicted clinical benefit to PKCi (AUC 0.84, [95% confidence interval, 0.65-1.0, p = 0.026]). AF of ion torrent NGS correlated significantly with ddPCR AF (n = 30, p = < 0.001, r = 0.968). Ion torrent NGS was able to detect additional mutations implicated in UM prognosis including SF3B1 mutations in 4 pts. Conclusions: Baseline ctDNA correlates with baseline LDH level and disease volume. EOT ctDNA predicted clinical benefit to PKCi. The ctDNA AF derived from ddPCR and NGS was comparable and targeted ion torrent NGS was useful in detecting driver as well as additional mutations in UM.

Presence and prevalence of UV related genetic mutations in uveal melanoma: similarities with cutaneous melanoma.

Ultraviolet (UV) radiation is an accepted etiological factor in cutaneous melanoma (CM), however its role in uveal melanoma (UM) is controversial. Partly as a consequence, CM and UM are often considered to be separate conditions, and advances in the treatment of CM have not led to joint clinical trials or parallel improvements in survival of UM. This study hypothesized that a subset of UM tumors displays evidence of genetic changes consistent with UV-related damage similar to that shown in CM. Analysis of the Broad Institute's Firebrowse depository of 80 UM samples and 343 CM samples, together with the Sanger Institute's Catalogue of Somatic Mutations in Cancer depository of 995 UM and 12,447 CM samples was undertaken to identify the most frequently mutated genes, mutation types, and specific nucleotide variants (SNVs) in each condition. Somatic mutation data were cross-correlated and shared mutations assessed against known effects of UV radiation. The proportion of samples with C>T substitutions (a classic genetic marker of UV-related damage) was higher in UM than CM on both DNA strands (17.0% vs 13.1%, p=0.038). The most frequently encountered cross-correlated mutated genes between UM and CM were, in order, BRAF, NRAS, TP53, CDKN2A, TERT, PTEN, ARID2, and KMT2C, with multiple common BRAF point mutations. Each cross-correlated mutation, and each common point mutation in BRAF, was associated with UV-related mechanistic changes. These findings support the hypothesis that the etiology of a substantial minority of UMs may be more UV dependent than previously recognized.

Synthetic Lethal Screen to Identify Molecular Mechanisms that Drive Uveal Melanoma

Melanoma that strikes the uveal cells of the eye represents about 5% of all melanoma cases. Uveal melanoma is a highly aggressive form of cancer that exhibits a propensity to metastasize to the liver. At this time there are no effective treatments for metastatic uveal melanoma and new therapeutic strategies are urgently needed. The majority of uveal melanomas are derived from early activating mutations in the G-proteins GNA11 or GNAQ. In order to further explore and identify molecular mechanisms that uveal melanoma cell lines rely upon for proliferation and survival, we will perform CRISPR/Cas9 mediated synthetic lethality screening. To accomplish this, we will generate a uveal melanoma cell line 92.1 that expresses the endonuclease Cas9 and introduce a guide RNA library into it that targets the functional domains of epigenetic regulator proteins. Thus, we will be able to determine which epigenetic regulator genes are required for the survival and proliferation of these cells by measuring which guide RNAs "drop out" over time. The guide RNAs that drop out likely disrupt essential genes that the cell relies on for growth. Cells carrying these gRNAs are consequently depleted from the culture. This will indicate that Cas9 mediated disruption of the gene targeted by that guide RNA causes cell death and will therefore identify the key dependencies of the uveal melanoma cells. We expect that the results of these experiments will reveal novel genetic mechanisms characteristic of GNAQ mutant uveal melanoma cells which could be therapeutically targeted in the future.

Mutations of GNAQ, GNA11, SF3B1, EIF1AX, PLCB4 and CYSLTR in Uveal Melanoma in Chinese Patients

Background: The purpose of this study is to determine the mutation frequencies of key driver genes in uveal melanoma (UM) in Chinese patients and to detect associations between metastasis and the mutation of these genes. Method: A total of 85 patients with UM were enrolled in this study, including 18 patients with metastasis and 67 without metastasis. Sanger sequencing covering the mutational hotspot regions of the G protein subunit alpha Q (GNAQ), GNA11, splicing factor 3B subunit 1 (SF3B1), X-linked eukaryotic translation initiation factor 1A (EIF1AX), phospholipase C beta 4 (PLCB4) and cysteinyl leukotriene receptor 2 (CYSLTR2) genes was used to analyse the mutations in Chinese patients. Results: The frequencies of GNAQ and GNA11 mutations in UM were 45% (38/85) and 35% (30/85) respectively. The frequencies of SF3B1 and EIF1AX mutations were 37% (31/85) and 9% (8/85) respectively. Only 2 mutations were detected in exon 4 of GNAQ, and no mutations were detected in exon 4 of GNA11. A novel mutation, c.627G>T (Q209H) in GNA11 was found. The detected mutations affecting SF3B1 were c.1873C>T (R625C), c.1874G>A (R625H) and c.1874G>T (R625L). The association between the mutations in SF3B1 and low risk of metastasis was statistically significant (OR 0.17, 95% CI 0.035–0.819). The mutations affecting EIF1AX were –23G>A (5′-UTR), c.5C>G (P2R), c.23G>A (G8Q), c.25G>C (G9A) and c.38_39GC>CT (R13P). No mutations were found in the PLCB4 and CYSLTR2 genes. Unfortunately, information on BRCA1-associated protein 1 could not be obtained. Conclusions: These data indicate that mutations in the PLCB4 and CYSLTR2 genes are rare in Chinese UM patients. The mutations in GNAQ, GNA11 and EIF1AX were not associated with metastasis, whereas SF3B1 mutations were correlated with low risk of metastasis and demonstrated a protective effect in UM patients in China.

SRSF2 Mutations in Uveal Melanoma: A Preference for In-Frame Deletions?

Background: Uveal melanoma (UM) is the most common primary ocular malignancy in adults in the Western world. UM with a mutation in SF3B1, a spliceosome gene, is characterized by three or more structural changes of chromosome 1, 6, 8, 9, or 11. Also UM without a mutation in SF3B1 harbors similar chromosomal aberrations. Since, in addition to SF3B1, mutations in U2AF1 and SRSF2 have also been observed in hematological malignancies, UM without a SF3B1 mutation—but with the characteristic chromosomal pattern—might harbor mutations in one of these genes. Methods: 42 UMs were selected based on their chromosomal profile and wildtype SF3B1 status. Sanger sequencing covering the U2AF1 (exon 2 and 7) hotspots and SRSF2 (exon 1 and 2) was performed on DNA extracted from tumor tissue. Data of three UM with an SRSF2 mutation was extracted from the The Cancer Genome Atlas (TCGA). Results: Heterozygous in-frame SRSF2 deletions affecting amino acids 92–100 were detected in two UMs (5%) of 42 selected tumors and in three TGCA UM specimens. Both the UM with an SRSF2 mutation from our cohort and the UM samples from the TCGA showed more than four structural chromosomal aberrations including (partial) gain of chromosome 6 and 8, although in two TCGA UMs monosomy 3 was observed. Conclusions: Whereas in myelodysplastic syndrome predominantly missense SRSF2 mutations are described, the observed SRSF2 mutations in UM are all in-frame deletions of 8–9 amino acids. This suggests that the R625 missense SF3B1 mutations and SRSF2 mutations in UM are different compared to the spliceosome gene mutations in hematological cancers, and probably target a different, as yet unknown, set of genes involved in uveal melanoma etiology.

Tris DBA palladium is an orally available inhibitor of GNAQ mutant uveal melanoma in vivo.

Uveal melanoma is a rare but often lethal malignancy and is the leading cause of death due to an ophthalmic condition. Uveal melanoma is often diagnosed at a late stage and has a strong propensity to hepatic metastasis. Recently, the most common driver mutations in uveal melanoma have been identified, predominantly in the G-proteins GNAQ. This pattern differs from that of cutaneous melanoma in which Braf and Nras predominate. There are no current clinically used agents that target GNAQ mutations, unlike the use of Braf inhibitors in cutaneous melanoma. We tested the novel agent Tris DBA palladium and found that it was markedly more effective against GNAQ mutant melanomas than wild type uveal melanomas. Given that ARF6 has recently been discovered as a node in GNAQ mutations, we evaluated the efficacy of Tris DBA palladium on ARF6 signaling and found that it was effective in inhibiting ARF6 activation. Finally, Tris DBA palladium was orally effective against GNAQ mutant melanoma in vivo. Tris DBA Palladium deserves further evaluation as a systemic agent for uveal melanoma.

Abstract 1764: Targeting nucleotide exchange to inhibit Gq/11 driver mutations in uveal melanoma

Abstract Constitutively activating mutations in the G-protein alpha subunits GNAQ(Gαq) and GNA11(Gα11) act as oncogenic drivers in over 90% of uveal (eye) melanoma (UM) tumors. We show that constitutively active Gαq and Gα11 can be targeted in UM cells by the cyclic depsipeptide FR900359 (FR). FR inhibits GDP/GTP guanine nucleotide exchange allosterically to trap constitutively active Gαq/11 in inactive GDP-bound Gαβγ heterotrimers. FR inhibits second messenger signaling, arrests proliferation and reinstates melanocytic differentiation in UM cells driven by constitutively active Gαq or Gα11. At higher doses, FR also induces apoptosis. The re-differentiation and anti-proliferative effects of FR are not seen in UM cells that lack mutations in Gαq or Gα11. FR promotes UM cell differentiation by reactivating polycomb repressive complex 2 (PRC2)-mediated gene silencing, and this re-differentiation can be blocked with an EZH2 inhibitor. The effector system regulating PRC2 downstream of constitutively active Gαq/11 in UM is currently under investigation. Preliminary data from human primary tumor samples suggest that targeting constitutively active Gαq/11 with FR could provide an important therapeutic approach for UM. Citation Format: Michael D. Onken, Carol M. Makepeace, Kevin M. Kaltenbronn, Stanley M. Kanai, Tyson D. Todd, Shiqi Wang, Thomas J. Broekelmann, Prabakar Kumar Rao, John A. Cooper, Kendall J. Blumer. Targeting nucleotide exchange to inhibit Gq/11 driver mutations in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1764.

Heterogeneity in Mitogen-Activated Protein Kinase (MAPK) Pathway Activation in Uveal Melanoma With Somatic GNAQ and GNA11 Mutations.

PurposeThe activation of the mitogen-activated protein kinase (MAPK) pathway has been suggested as the major downstream target when GNAQ and GNA11 (GNAQ/11) are mutated in uveal melanoma (UM). However, clinical trials with single agent MEK inhibitor showed no clinical significance in altering the overall outcome of the disease in UM; therefore, we investigated the correlation between naturally occurring mutations in GNAQ/11 and activation of MAPK pathway in vivo in primary UM.MethodsScreening for activating mutations in codons 183 and 209 of GNAQ/11 was carried out by sequencing and restriction fragment length polymorphism (RFLP) in a cohort of 42 primary UM. Activation of the MAPK pathway and other potential downstream signals was assessed by immunohistochemistry and/or Western blot analysis. Potential downstream signaling of mutant and wild type GNAQ/11 was studied by transient transfection assay in nonmutant cell lines.ResultsSomatic mutations in GNAQ/11 were observed in 35/42 (83.3%) of primary UM. Tumors with GNAQ/11 mutations showed variations in the activation of ERK1/2 with significant tumor heterogeneity. Weak and undetectable ERK1/2 activation was observed in 4/35 (11.4%) and 8/35 (22.9%) of the GNAQ/11 mutant UM, respectively. Tumor heterogeneity of GNAQ/11 mutations was also observed in a subset of tumors.ConclusionsOur results indicate that there is marked variation in MAPK activation in UM with GNAQ/11 mutations. Thus, GNAQ/11 mutational status is not a sufficient biomarker to adequately predict UM patient responses to single-agent selective MEK inhibitor therapy.

Potential Mutations in Uveal Melanoma Identified Using Targeted Next-Generation Sequencing.

Background/objective: Uveal melanoma (UM) is the most common intraocular malignancy and has a high tendency to metastasize to the liver. Although primary tumours can be successfully treated, there is currently no effective treatment for metastatic UM. To gain insight into the genetics of UM, we performed the targeted next-generation sequencing (NGS) of UM samples from a non-Caucasian population.Methods: This study included tumour samples and blood samples from 107 UM patients at Peking University Cancer Hospital & Institute. Clinical data were collected. DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) specimens. Using the HaloPlex Target Enrichment System (Agilent Technologies), NGS was performed to investigate mutations in a 35-gene panel composed of cancer-related genes.Results: Recurrent coding mutations were found in the known UM drivers GNAQ and GNA11. FOXO1, PIK3R1 and HIF1A were also found to harbour somatic mutations in more than 20% of patients, a result that may indicate previously undescribed associations between these genes and UM pathogenesis. Patients with HIF1A and FOXO1 mutations exhibited worse overall survival (OS). In multivariate analysis, FOXO1 mutation was an independent prognostic factor for OS (P<0.05) that was associated with an increase in the risk ratio by a factor of 1.35. Notably, we found that HIF1A and FOXO1 mutations were associated with metastatic transformation of UM (P<0.05 and P<0.001, respectively).Conclusion: Our findings from analyses of targeted NGS data shed new light on the molecular genetics of UM and facilitate the exploration of mutations associated with metastatic potential.

Atypical activation of the G protein Gαq by the oncogenic mutation Q209P

The causative role of G protein-coupled receptor (GPCR) pathway mutations in uveal melanoma (UM) has been well-established. Nearly all UMs bear an activating mutation in a GPCR pathway mediated by G proteins of the Gq/11 family, driving tumor initiation and possibly metastatic progression. Thus, targeting this pathway holds therapeutic promise for managing UM. However, direct targeting of oncogenic Gαq/11 mutants, present in ∼90% of UMs, is complicated by the belief that these mutants structurally resemble active Gαq/11 WT. This notion is solidly founded on previous studies characterizing Gα mutants in which a conserved catalytic glutamine (Gln-209 in Gαq) is replaced by leucine, which leads to GTPase function deficiency and constitutive activation. Whereas Q209L accounts for approximately half of GNAQ mutations in UM, Q209P is as frequent as Q209L and also promotes oncogenesis, but has not been characterized at the molecular level. Here, we characterized the biochemical and signaling properties of Gαq Q209P and found that it is also GTPase-deficient and activates downstream signaling as efficiently as Gαq Q209L. However, Gαq Q209P had distinct molecular and functional features, including in the switch II region of Gαq Q209P, which adopted a conformation different from that of Gαq Q209L or active WT Gαq, resulting in altered binding to effectors, Gβγ, and regulators of G-protein signaling (RGS) proteins. Our findings reveal that the molecular properties of Gαq Q209P are fundamentally different from those in other active Gαq proteins and could be leveraged as a specific vulnerability for the ∼20% of UMs bearing this mutation.

Comparison of Germline versus Somatic BAP1 Mutations for Risk of Metastasis in Uveal Melanoma

BackgroundGermline mutations in BAP1 have been associated with BAP1-Tumor Predisposition Syndrome (BAP1-TPDS), a predisposition to multiple tumors within a family that includes uveal melanoma (UM), cutaneous melanoma, malignant mesothelioma and renal cell carcinoma. Alternatively, somatic mutations in BAP1 in UM have been associated with high risk for metastasis. In this study, we compare the risk of metastasis in UM that carry germline versus somatic BAP1 mutations and mutation-negative tumors.MethodsDNA extracted from 142 UM and matched blood samples was sequenced using Sanger or next generation sequencing to identify BAP1 gene mutations.ResultsEleven of 142 UM (8%) carried germline BAP1 mutations, 43 (30%) had somatic mutations, and 88 (62%) were mutation-negative. All BAP1 mutations identified in blood samples were also present in the matched UM. There were 52 unique mutations in 54 tumors. All were pathogenic or likely pathogenic.A comparison of tumors carrying somatic vs. germline mutations, or no mutations, showed a higher frequency of metastasis in tumors carrying somatic mutations: 74% vs. 36%, P=0.03 and 74% vs. 26% P<0.001, respectively. Tumors with a somatic mutation compared to mutation-negative had an older age of diagnosis of (61.8 vs. 52.2 years, P=0.002), and shorter time to metastasis (16 vs. 26 months, P=0.04). Kaplan-Meier analysis further showed that tumors with somatic (vs. germline) mutations demonstrated a greater metastatic risk (P=0.03). Cox multivariate analysis showed in addition to chromosome-3 monosomy and larger tumor diameter, the presence of BAP1 somatic, but not germline mutations, was significantly associated with risk of metastasis(P=0.02).Personal or family history of BAP1-TPDS was available for 79 of the cases. All eight cases with germline mutations reported a history of BAP1-TPDS, which was significantly greater than what was observed in cases with somatic mutations (10 of 23, P=0.009) or mutation-negative cases (11 of 48, P<0.001).ConclusionsDefining germline vs. somatic nature of BAP1 mutations in UM can inform the individual about both the risk of metastasis, and the time to metastasis, which are critically important outcomes for the individual. This information can also change the cascade screening and surveillance of family members.

An insight into the molecular genetics of a uveal melanoma patient cohort

Uveal melanoma (UM) is a highly aggressive malignancy and presents a clinically significant unmet need in cancer therapeutics. The aim of this study was to identify previously unreported mutations in UM among an Irish cohort of patients which may have potential clinical relevance. DNA was extracted from 36 intraocular melanoma patient samples and 4 metastatic melanoma samples among the patient cohort by microdissection from formalin-fixed paraffin embedded tissue blocks and underwent genotyping to test for known single nucleotide polymorphisms in 42 cancer associated genes. These mutations were analysed using a custom-designed sequenom panel. Using high-throughput genotyping, mutually exclusive GNAQ and GNA11 mutations were detected in 31 of 34 UM patients together with a number of non-synonymous changes in established cancer driver genes, PHLPP2, MET, PIK3R1 and IDH-1, variants which have not been previously associated with UM. Given the lack of knowledge regarding the clinical relevance of the variants identified in this UM cohort and their likely pathogenic nature in other cancers, further studies of the functional impact of these variant mutations are warranted to establish possible previously, undescribed roles in UM pathogenesis, which may provide additional targets for future therapies.

A novel BRET biosensor for Gαq‐GTP reveals unique properties of cancer‐associated GNAQ mutants

Heterotrimeric G proteins are critical signal transducers and dysregulation of their activity is emerging a critical driver of cancer. In fact, the mutational landscape of heterotrimeric G protein signaling networks in cancer has expanded dramatically in the recent years through large scale sequencing campaigns. However, the rate at which novel mutations are identified clearly outpaces the ability to analyze their functional consequences in a systematic and reliable manner, making evident the need for developing novel tools for their investigation. Optical reporters that directly monitor G protein activity in intact cells are ideally suited for this purpose as they allow the rapid characterization of signaling mechanisms with high sensitivity and temporal resolution. Despite that nucleotide exchange in the α-subunit is the event that defines G protein activation, all reporters described to date rely on measuring the dissociation and/or rearrangement of Gα-Gβγ complexes as an indirect readout of G protein activation. Here we describe a Bioluminescence Resonance Energy Transfer (BRET)-based biosensor that specifically detects GTP-bound Gαq in cells with sub-second resolution and its implementation to characterize cancer-associated Gαq mutants. Using this novel biosensor, we show that Gαq activation by GPCR stimulation leads to a robust increase in BRET that is rapidly reserved by receptor antagonists. This sensor can also detect modulation of the amplitude and kinetics of Gαq responses by GTPase Activating Proteins (GAPs). By combining this Gαq-GTP BRET sensor with other approaches, we dissected the mechanisms of action for several cancer-associated Gαq mutants to make novel and, in some cases, unexpected observations. For example, we have identified mutants that display normal basal activity and GPCR-mediated activation, but deactivate slowly due to GAP-insensitivity, thereby suggesting a novel cancer-promoting mechanism. Strikingly, we found that Gαq Q209L and Q209P, the two most frequent mutations in uveal melanoma, lead to signaling hyperactivation via different molecular mechanisms. Our data suggests that this difference arises from unique structural features of the Q209P mutant, which might represent a novel vulnerability of the 20–25% uveal melanomas that bear this mutation. In summary, we have validated a first-in-class biosensor for the direct measurement of the bona fide active species of Gα and demonstrated its implementation to gain further insights into the properties of G protein mutants in cancer. Support or Funding Information NIH grants R01GM112631 and R01GM108733, American Cancer Society grants RSG-13-362-01-TBE and IRG-72-001-36, and the Karin Grunebaum Cancer Research Foundation. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel therapeutic strategies and targets in advanced uveal melanoma.

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. 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. 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.

Prognostic impact of chromosomal aberrations and GNAQ, GNA11 and BAP1 mutations in uveal melanoma.

To evaluate clinico-pathological and molecular prognostic factors in a well-defined series of posterior uveal melanoma (UM) with focus on chromosomal aberrations and mutations in the GNAQ, GNA11 and BRCA1-associated protein 1 (BAP1) genes. Formalin-fixed paraffin-embedded (FFPE) tissue samples were obtained from 50 consecutive eyes enucleated for UM between 1993 and 2005. The material was tested for loss of chromosome 3 and gain of chromosome 8q gene signatures by selective molecular gene markers using multiplex ligation-dependent probe amplification (MLPA), and for DNA mutations in the GNAQ, GNA11 and BAP1 genes. After a mean follow-up of 83months (range, 8-205months), 21 patients had died of metastatic UM and 16 patients of other causes. Tumour diameter, ciliary body involvement, mixed/epithelioid cell types, mitotic index, Ki-67 proliferation index, loss of chromosome 3 and gain of chromosome 8q showed statistically significant associations with metastatic disease. There were no significant differences in the prevalence of GNAQ and GNA11 mutations between patients with or without metastatic disease. Mutational analysis of the BAP1 gene was performed in 32 primary UM and in five UM liver metastases. Nine different BAP1 missense mutations were identified. BAP1 mutations were not more common in metastasizing than in nonmetastasizing UM. The molecular gene markers showing loss of chromosome 3 and gain of 8q gene signatures were associated with an increased risk of metastatic disease. BRCA1-associated protein 1 (BAP1) gene mutation status had no prognostic significance. The frequency and spectrum of BAP1 mutations in UM may be more dependent on ethnicity and demographic variables than hitherto considered.

Uveal Melanomas with SF3B1 Mutations: A Distinct Subclass Associated with Late-Onset Metastases

To investigate the prevalence and prognostic value of SF3B1 and EIF1AX mutations in uveal melanoma (UM) patients. Case series. Cohort of 151 patients diagnosed with and treated for UM. SF3B1 and EIF1AX mutations in primary tumors were investigated using whole-exome sequencing (n= 25) and Sanger sequencing (n= 151). For the detection of BAP1 mutations, a previously reported cohort of 90 patients was extended using BAP1 sequencing or immunohistochemistry. The status of SF3B1, EIF1AX, and BAP1 in tumors of patients were correlated to clinical, histopathologic, and genetic parameters. Survival analyses were performed for patients whose tumors had SF3B1, EIF1AX, and BAP1 mutations. Patients with tumors harboring EIF1AX mutations rarely demonstrated metastases (2 of 28 patients) and overall had a longer disease-free survival (DFS; 190.1 vs. 100.2 months; P < 0.001). Within the patient group with disomy 3, UM patients with an SF3B1 mutation had an increased metastatic risk compared with those without an SF3B1 mutation (DFS, 132.8 vs. 174.4 months; P= 0.008). Patients with such a mutation were more prone to demonstrate late metastases (median, 8.2 years; range, 23-145 months). Patients with UM and loss of BAP1 expression had a significantly decreased survival (DFS, 69.0 vs. 147.9 months; P < 0.001). According to our data, patients with UM can be classified into 3 groups, of which EIF1AX-mutated tumors and tumors without BAP1, SF3B1, or EIF1AX mutations are associated with prolonged survival and low metastatic risk, SF3B1-mutated tumors are associated with late metastasis, and tumors with an aberrant BAP1 are associated with an early metastatic risk and rapid decline in patient DFS.

Clinical Characteristics of Uveal Melanoma in Patients With GermlineBAP1Mutations

Somatic mutations in BAP1 (BRCA1-associated protein 1 gene) are frequently identified in uveal melanoma. To date, the role of germline BAP1 mutations in uveal melanoma has not been characterized. To characterize the clinical phenotype of uveal melanoma in patients with germline BAP1 mutations. Retrospective cohort study at an academic ophthalmology referral center among 507 patients with uveal melanoma who consented for collection of blood samples. The study dates were June 22, 1992, to December 14, 2010. Clinical characteristics of uveal melanoma and the development of metastases. BAP1 gene sequencing from blood samples of patients with uveal melanoma was correlated with clinical characteristics. Of 507 blood samples analyzed, 25 patients (4.9%) exhibited 18 BAP1 polymorphisms, of which 9 were novel. Computational analyses predicted that 8 BAP1 mutations in 8 patients (1.6%) were likely to result in damaged BAP1 protein. Five of these 8 mutations were novel. These 8 patients were compared with 482 patients in whom no BAP1 polymorphisms were identified. In univariate analyses, patients with germline BAP1 mutations exhibited larger tumor diameters (mean, 15.9 vs 12.3 mm; P = .004) and higher rates of ciliary body involvement (75.0% vs 21.6%, P = .002) and metastases (71.4% vs 18.0%, P = .003) compared with control subjects. Patients with germline BAP1 mutations exhibited increased frequency of family history of cancer (100% vs 65.9%, P = .06), particularly cutaneous melanoma (62.5% vs 9.9%, P < .001) and ocular melanoma (25.0% vs 1.9%, P = .01). No differences were identified in age at diagnosis, sex, history of other malignant neoplasm, presenting visual acuity, distance of the tumor from the optic nerve or fovea, iris involvement, extrascleral extension, or tumor pigmentation. Germline BAP1 mutations increased risk of metastasis independent of ciliary body involvement (P = .02). Germline BAP1 mutation approached significance as an independent risk factor for metastasis (P = .09). These data suggest that germline BAP1 mutations occur infrequently in uveal melanoma and are associated with larger tumors and higher rates of ciliary body involvement, 2 known risk factors for metastasis.