Search

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults, which preferentially metastasizes to the liver in approximately half of all cases. Metastatic UM is notoriously resistant to therapy and is almost uniformly fatal. UM metastasis is most strongly associated with mutational inactivation of the BAP1 tumor suppressor gene. Given the role of BAP1 in epigenetic regulation as the ubiquitin hydrolase subunit of the polycomb repressive deubiquitinase (PR-DUB) complex, we conducted high-throughput drug screening using a well-characterized epigenetic compound library to identify new therapeutic vulnerabilities. We identified several promising new lead compounds, in particular the extra-terminal domain protein (BET) inhibitor mivebresib (ABBV-075). Mivebresib significantly improved survival rates in a metastatic uveal melanoma xenograft mouse model and entirely prevented detectable metastases to the bones, spinal cord, and brain. RNA sequencing revealed a notable overlap between the genes and pathways affected by HDAC and BET inhibition, including the reversal of gene signatures linked to high metastatic risk and upregulation of genes associated with a neuronal phenotype. Together, we found that UM cells are particularly vulnerable to class I HDAC and BET inhibition, and highlight the BET inhibitor mivebresib as a promising candidate for further clinical evaluation., Competing Interests: The authors declare no conflicts of interest.

PURPOSEValidated and accurate prognostic testing is critical for precision medicine in uveal melanoma (UM). Our aims were to (1) prospectively validate an integrated prognostic classifier combining a 15-gene expression profile (15-GEP) and PRAME RNA expression and (2) identify clinical variables that enhance the prognostic accuracy of the 15-GEP/ PRAME classifier.MATERIALS AND METHODSThis study included 1,577 patients with UM of the choroid and/or ciliary body who were enrolled in the Collaborative Ocular Oncology Group Study Number 2 (COOG2) and prospectively monitored across 26 North American centers. Test results for 15-GEP (class 1 or class 2) and PRAME expression status (negative or positive) were available for all patients. The primary end point was metastasis-free survival (MFS).RESULTS15-GEP was class 1 in 1,082 (68.6%) and class 2 in 495 (31.4%) patients. PRAME status was negative in 1,106 (70.1%) and positive in 471 (29.9%) patients. Five-year MFS was 95.6% (95% CI, 93.9 to 97.4) for class 1/ PRAME (-), 80.6% (95% CI, 73.9 to 87.9) for class 1/ PRAME (+), 58.3% (95% CI, 51.1 to 66.4) for class 2/ PRAME (-), and 44.8% (95% CI, 37.9 to 52.8) for class 2/ PRAME (+). By multivariable Cox proportional hazards analysis, 15-GEP was the most important independent predictor of MFS (hazard ratio [HR], 5.95 [95% CI, 4.43 to 7.99]; P < .001), followed by PRAME status (HR, 1.82 [95% CI, 1.42 to 2.33]; P < .001). The only clinical variable demonstrating additional prognostic value was tumor diameter.CONCLUSIONIn the largest prospective multicenter prognostic biomarker study performed to date in UM to our knowledge, the COOG2 study validated the superior prognostic accuracy of the integrated 15-GEP/ PRAME classifier over 15-GEP alone and clinical prognostic variables. Tumor diameter was found to be the only clinical variable to provide additional prognostic information. This prognostic classifier provides an advanced resource for risk-adjusted metastatic surveillance and adjuvant trial stratification in patients with UM.

Epigenetic regulatory mechanisms are underappreciated, yet are critical for enteric nervous system (ENS) development and maintenance. We discovered that fetal loss of the epigenetic regulator Bap1 in the ENS lineage caused severe postnatal bowel dysfunction and early death in Tyrosinase-Cre Bap1fl/fl mice. Bap1-depleted ENS appeared normal in neonates; however, by P15, Bap1-deficient enteric neurons were largely absent from the small and large intestine of Tyrosinase-Cre Bap1fl/fl mice. Bowel motility became markedly abnormal with disproportionate loss of cholinergic neurons. Single-cell RNA sequencing at P5 showed that fetal Bap1 loss in Tyrosinase-Cre Bap1fl/fl mice markedly altered the composition and relative proportions of enteric neuron subtypes. In contrast, postnatal deletion of Bap1 did not cause enteric neuron loss or impaired bowel motility. These findings suggest that BAP1 is critical for postnatal enteric neuron differentiation and for early enteric neuron survival, a finding that may be relevant to the recently described human BAP1-associated neurodevelopmental disorder.

PRAME is a CUL2 ubiquitin ligase subunit that is normally expressed in the testis but becomes aberrantly overexpressed in many cancer types in association with aneuploidy and metastasis. Here, we show that PRAME is expressed predominantly in spermatogonia around the time of meiotic crossing-over in coordination with genes mediating DNA double strand break repair. Expression of PRAME in somatic cells upregulates pathways involved in meiosis, chromosome segregation and DNA repair, and it leads to increased DNA double strand breaks, telomere dysfunction and aneuploidy in neoplastic and non-neoplastic cells. This effect is mediated at least in part by ubiquitination of SMC1A and altered cohesin function. PRAME expression renders cells susceptible to inhibition of PARP1/2, suggesting increased dependence on alternative base excision repair pathways. These findings reveal a distinct oncogenic function of PRAME that can be targeted therapeutically in cancer., (© 2023. The Author(s).)

Preventing or effectively treating metastatic uveal melanoma (UM) is critical because it occurs in about half of patients and confers a very poor prognosis. There is emerging evidence that hepatocyte growth factor (HGF) and insulin-like growth factor 1 (IGF-1) promote metastasis and contribute to the striking metastatic hepatotropism observed in UM metastasis. However, the molecular mechanisms by which HGF and IGF-1 promote UM liver metastasis have not been elucidated. ASAP1, which acts as an effector for the small GTPase ARF6, is highly expressed in the subset of uveal melanomas most likely to metastasize. Here, we found that HGF and IGF-1 hyperactivate ARF6, leading to its interaction with ASAP1, which then acts as an effector to induce nuclear localization and transcriptional activity of NFAT1. Inhibition of any component of this pathway impairs cellular invasiveness. Additionally, knocking down ASAP1 or inhibiting NFAT signaling reduces metastasis in a xenograft mouse model of UM. The discovery of this signaling pathway represents not only an advancement in our understanding of the biology of uveal melanoma metastasis but also identifies a novel pathway that could be targeted to treat or prevent metastatic uveal melanoma., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Uveal melanoma (UM) displays a high frequency of metastasis; however, effective therapies for metastatic UM are limited. Identifying unique metabolic features of UM may provide a potential targeting strategy. A lipid metabolism protein expression signature was induced in a normal choroidal melanocyte (NCM) line transduced with GNAQ (Q209L), a driver in UM growth and development. Consistently, UM cells expressed elevated levels of fatty acid synthase (FASN) compared to NCMs. FASN upregulation was associated with increased mammalian target of rapamycin (mTOR) activation and sterol regulatory element-binding protein 1 (SREBP1) levels. FASN and mTOR inhibitors alone significantly reduced UM cell growth. Concurrent inhibition of FASN and mTOR further reduced UM cell growth by promoting cell cycle arrest and inhibiting glucose utilization, TCA cycle metabolism, and de novo fatty acid biosynthesis. Our findings indicate that FASN is important for UM cell growth and co-inhibition of FASN and mTOR signaling may be considered for treatment of UM.

Purpose: To report the clinical findings, treatments, and outcomes in a series of patients with vitreous metastasis from cutaneous melanoma., Methods: This single-center, retrospective, interventional case series included patients with biopsy-confirmed vitreous metastasis from cutaneous melanoma diagnosed between 1997 and 2020. Standard 23- or 25-gauge pars plana vitrectomy was performed for diagnostic sampling. Sclerotomies were treated with double or triple freeze-thaw cryotherapy. Perioperative intravitreal injections of melphalan (32 µg/0.075 mL) were administered, when indicated. Visual acuity, intraocular pressure, and systemic and ocular treatment responses were reported., Results: Five eyes of five patients with unilateral vitreous metastasis from cutaneous melanoma were identified. The median age at diagnosis was 84 (range, 37-88) years. The median follow-up after ophthalmic diagnosis was 28 (8.5-36) months; one patient did not have a follow-up. The initial visual acuity ranged from 20/30 to hand motions. Baseline clinical findings included pigmented or non-pigmented cellular infiltration of the vitreous (5/5), anterior segment (4/5), and retina (3/5). Four patients had secondary glaucoma. Systemic therapy included checkpoint inhibitor immunotherapy (n=3, all with partial/complete response), systemic chemotherapy (n=2), surgical resection (n=3), and radiation (n=2). The median time from primary diagnosis to vitreous metastasis was 2 (2-15) years. One patient had an active systemic disease at the time of vitreous metastasis. The final visual acuity ranged from 20/40 to no light perception. Ophthalmic treatment included vitrectomy in all five patients, intravitreal administration of melphalan in three, and intravitreal administration of methotrexate in one. One patient required enucleation, and histopathology revealed extensive invasion by melanoma cells., Conclusions: Vitreous metastasis from cutaneous melanoma can present as a diffuse infiltration of pigmented or non-pigmented cells into the vitreous and may be misdiagnosed as uveitis. Diagnostic pars plana vitrectomy and periodic intravitreal chemotherapy may be indicated.

Purpose: To describe a patient with an unusual presentation of iris metastasis from breast cancer and her response to systemic therapy., Methods: Retrospective chart review of one patient., Results: A 57-year-old woman presented with a superonasal translucent vascularized iris stromal mass with fish egg-like structures budding from the surface. High-frequency anterior segment ultrasonography demonstrated a solid iris stromal mass measuring 6.0 mm × 3.3 mm × 1.9 mm. On optical coherence tomography, the egglike structures appeared as hyperreflective spheres, some of which were detached from the main iris stromal tumor. Oncologic evaluation revealed metastatic breast cancer involving the brain and lung. She was treated with oral abemaciclib and letrozole, as well as external beam radiotherapy to the brain. The iris mass had completely regressed within 4 months and remained undetectable through the 8-month follow-up. The other metastatic lesions responded well to therapy., Conclusion: A case of iris metastasis was reported as the presenting sign of cancer dissemination that was successfully treated with targeted systemic therapy without ocular radiotherapy.

Melanomas arising in the uveal tract of the eye are a rare form of the disease with a biology and clinical phenotype distinct from their more common cutaneous counterparts. Treatment of primary uveal melanoma with radiotherapy, enucleation or other modalities achieves local control in more than 90% of patients, although 40% or more ultimately develop distant metastases, most commonly in the liver. Until January 2022, no systemic therapy had received regulatory approval for patients with metastatic uveal melanoma, and these patients have historically had a dismal prognosis owing to the limited efficacy of the available treatments. A series of seminal studies over the past two decades have identified highly prevalent early, tumour-initiating oncogenic genomic aberrations, later recurring prognostic alterations and immunological features that characterize uveal melanoma. These advances have driven the development of a number of novel emerging treatments, including tebentafusp, the first systemic therapy to achieve regulatory approval for this disease. In this Review, our multidisciplinary and international group of authors summarize the biology of uveal melanoma, management of primary disease and surveillance strategies to detect recurrent disease, and then focus on the current standard and emerging regional and systemic treatment approaches for metastatic uveal melanoma., (© 2023. Springer Nature Limited.)

We previously demonstrated that pan-HDAC inhibitors could limit escape from MEK inhibitor (MEKi) therapy in uveal melanoma (UM) through suppression of AKT and YAP/TAZ signaling. Here, we focused on the role of specific HDACs in therapy adaptation. Class 2 UM displayed higher expression of HDACs 1, 2, and 3 than Class 1, whereas HDACs 6, 8, and 11 were uniformly expressed. Treatment of UM cells with MEKi led to modulation of multiple HDACs, with the strongest increases observed in HDAC11. RNA-seq analysis showed MEKi to decrease the expression of multiple HDAC11 target genes. Silencing of HDAC11 significantly reduced protein deacetylation, enhanced the apoptotic response to MEKi and reduced growth in long-term colony formation assays across multiple UM cell lines. Knockdown of HDAC11 led to decreased expression of TAZ in some UM cell lines, accompanied by decreased YAP/TAZ transcriptional activity and reduced expression of multiple YAP/TAZ target genes. Further studies showed this decrease in TAZ expression to be associated with increased LKB1 activation and modulation of glycolysis. In an in vivo model of uveal melanoma, silencing of HDAC11 limited the escape to MEKi therapy, an effect associated with reduced levels of Ki67 staining and increased cleaved caspase-3. We have demonstrated a novel role for adaptive HDAC11 activity in UM cells, that in some cases modulates YAP/TAZ signaling leading to MEKi escape., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Introduction: Iodine-125 brachytherapy is an effective eye-sparing treatment for uveal melanoma. Previous work has shown that uveal melanomas cluster into distinct molecular classes based on gene expression profiles - discriminating low-grade from high-grade tumors. Our objective was to identify clinical and molecular predictors of local recurrence (LR) and progression-free survival (PFS)., Methods: We constructed a retrospective database of uveal melanoma patients from the University of Miami's electronic medical records that were treated between January 8, 2012, and January 5, 2019, with either COMS-style or Eye Physics plaque. Data on tumor characteristics, pretreatment retinal complications, post-plaque treatments, LR, and PFS were collected. Univariate and multivariate Cox models for cumulative incidence of LR and PFS were conducted using SAS version 9.4., Results: We identified 262 patients, with a median follow-up time of 33.5 months. Nineteen patients (7.3%) had LR, and 56 patients (21.4%) were classified as PFS. We found that ocular melanocytosis (hazard ratio = 5.55, p < 0.001) had the greatest impact on PFS. Genetic expression profile did not predict LR outcomes (hazard ratio = 0.51, p = 0.297)., Conclusion: These findings help physicians identify predictors for short-term brachytherapy outcomes, allowing better shared decision making with patients preoperatively when deciding between brachytherapy versus enucleation. Patients stratified to higher risk groups based on preoperative characteristics such as ocular melanocytosis should be monitored more closely. Future studies must validate these findings using a prospective cohort study., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2022 by S. Karger AG, Basel.)

Purpose: I 125 Eye Plaque brachytherapy is the standard treatment for medium-sized uveal melanomas (UM). Patients develop radiation toxicities (RTT), including radiation maculopathy (RM), radiation neovascular glaucoma/iris neovascularization (RNGI) and radiation optic neuropathy (RON). We aim to investigate demographics, pretreatment tumor characteristics and posttreatment complications as predictors of RTT., Methods and Materials: An IRB-approved single-institution retrospective chart review was performed from 2011 to 2019 for patients with posterior UM treated with brachytherapy. We collected demographics, pretreatment tumor characteristics and posttreatment complications. Univariate analysis (UVA) and multivariate analysis (MVA) were performed using logistic regression model. Hazard ratios (HR) and corresponding p-values were reported. All tests were two-sided; statistical significance was considered when p<0.05., Results: Two hundred and fifty eight patients were evaluated. Median follow-up was 33.50 months (range 3.02-97.31). 178 patients (69.0%) had RTT. 131 patients (50.8%) developed RM. Fifty-six patients (21.7%) developed RON. Nineteen patients (7.4%) developed RNGI. UVA found shorter distance to fovea (DF) (p = 0.04), posttreatment exudative retinal detachment (PERD) (p = 0.001) and posttreatment vitreous hemorrhage (PVH) (p = 0.001) are associated with RTT. MVA found shorter DF (HR=1.03, p = 0.04), PERD (HR=2.52, p = 0.01) and PVH (HR=3.34, p = 0.006) are associated with RTT. MVA found female sex (HR=1.731, p = 0.031) and tumor height (HR=1.13, p = 0.013) are associated with RM and pretreatment retinal detachment (HR=3.41, p<0.001) is associated with RON., Conclusions: Shorter DF, PERD and PVH are associated with RTT; female sex and tumor height are associated with RM and tumor height is associated with RON. These findings serve as prognostic tools to counsel patients and promote early intervention in management of RTT., (Copyright © 2022 American Brachytherapy Society. Published by Elsevier Inc. All rights reserved.)

Retinoblastoma is the most common eye cancer in children and is fatal if left untreated. Over the past three decades, chemotherapy has become the mainstay of eye-sparing treatment. Nevertheless, chemoresistance continues to represent a major challenge leading to ocular and systemic toxicity, vision loss, and treatment failure. Unfortunately, the mechanisms leading to chemoresistance remain incompletely understood. Here, we engineered low-passage human retinoblastoma cells to study the early molecular mechanisms leading to resistance to carboplatin, one of the most widely used agents for treating retinoblastoma. Using single-cell next-generation RNA sequencing (scRNA-seq) and single-cell barcoding technologies, we found that carboplatin induced rapid transcriptomic reprogramming associated with the upregulation of PI3K-AKT pathway targets, including ABC transporters and metabolic regulators. Several of these targets are amenable to pharmacologic inhibition, which may reduce the emergence of chemoresistance. We provide evidence to support this hypothesis using a third-generation inhibitor of the ABCB1 transporter., Competing Interests: The authors declare no conflict of interest.

Objective: To investigate the impact of global and local genetic ancestry and neighborhood socioeconomic status (nSES), on breast cancer (BC) subtype, and gene expression., Background: Higher rates of aggressive BC subtypes [triple negative breast cancer (TNBC)] and worse overall BC survival are seen in black women [Hispanic Black (HB) and non-Hispanic Black (NHB)] and women from low nSES. However, the complex relationship between genetic ancestry, nSES, and BC subtype etiology remains unknown., Methods: Genomic analysis was performed on the peripheral blood from a cohort of 308 stage I to IV non-Hispanic White (NHW), Hispanic White (HW), HB, and NHB women with BC. Patient and tumor characteristics were collected. Global and local ancestral estimates were calculated. Multinomial logistic regression was performed to determine associations between age, stage, genetic ancestry, and nSES on rates of TNBC compared to estrogen receptor (ER+)/epidermal growth factor receptor 2 (HER2-), ER+/HER2+, and ER-/HER2+ disease., Results: Among 308 women, we identified a significant association between increasing West African (WA) ancestry and odds of TNBC [odds ratio (OR): 1.06, 95% confidence interval (95% CI): 1.001-1.126, P =0.046] as well as an inverse relationship between higher nSES and TNBC (OR: 0.343, 95% CI: 0.151-0.781, P =0.011). WA ancestry remained significantly associated with TNBC when adjusting for patient age and tumor stage, but not when adjusting for nSES (OR: 1.049, 95% CI: -0.987-1.116, P =0.120). Local ancestry analysis, however, still revealed nSES-independent enriched WA ancestral segment centered at χ 2 =42004914 ( p =3.70×10 -5 ) in patients with TNBC., Conclusions: In this translational epidemiologic study of genetic ancestry and nSES on BC subtype, we discovered associations between increasing WA ancestry, low nSES, and higher rates of TNBC compared to other BC subtypes. Moreover, on admixture mapping, specific chromosomal segments were associated with WA ancestry and TNBC, independent of nSES. However, on multinomial logistic regression adjusting for WA ancestry, women from low nSES were more likely to have TNBC, independent of genetic ancestry. These findings highlight the complex nature of TNBC and the importance of studying potential gene-environment interactions as drivers of TNBC., Competing Interests: J.W.H. is an inventor of intellectual property related to gene expression profiling in uveal melanoma. He is a paid consultant for Castle Biosciences, licensee of this intellectual property, and he receives royalties from its commercialization. The remaining authors report no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: Approximately 40% of patients with uveal melanoma (UM) will develop metastatic disease. Tumors measuring at least 12mm in basal diameter with a class 2 signature, as defined by a widely used gene expression-profiling test, are associated with significantly higher risk of metastasis, with a median time to recurrence of 32 months. No therapy has been shown to reduce this risk., Materials and Methods: This was a single-arm, multicenter study in patients with high-risk UM who received definitive treatment of primary disease and had no evidence of metastasis. Patients were consecutively enrolled to receive 12 four-week cycles of adjuvant crizotinib at a starting dose of 250mg twice daily and were subsequently monitored for 36 months. The primary outcome of this study was to assess recurrence-free survival (RFS) of patients with high-risk UM who received adjuvant crizotinib., Results: 34 patients enrolled and received at least one dose of crizotinib. Two patients were unevaluable due to early withdrawal and loss to follow-up, leaving 32 patients evaluable for efficacy. Eight patients (25%) did not complete the planned 48-week course of treatment due to disease recurrence (n=5) or toxicity (n=3). All patients experienced at least one adverse event (AE), with 11/34 (32%) experiencing a Common Terminology Criteria for Adverse Events (CTCAE) grade 3 or 4 AE. After a median duration of follow up of 47.1 months, 21 patients developed distant recurrent disease. The median RFS was 34.9 months (95% CI (Confidence Interval), 23-55 months), with a 32-month recurrence rate of 50% (95% CI, 33-67%). Analysis of protein contents from peripheral blood extracellular vesicles in a subset of patient samples from baseline, on-treatment, and off-treatment, revealed a change in protein content associated with crizotinib exposure, however without a clear association with disease outcome., Conclusions: The use of adjuvant crizotinib in patients with high-risk UM did not result in improved RFS when compared to historical controls. Analysis of blood extracellular vesicles revealed changes in protein content associated with treatment, raising the possibility of future use as a biomarker. Further investigation of adjuvant treatment options are necessary for this challenging disease., Competing Interests: SK reports honoraria from Castle Biosciences. JL reports advisory/consulting fees from Regeneron, Sapience Therapeutics, Agenus, and research funding from Bristol Myers Squibb, Novartis, Agenus, Vyriad, Regeneron, Immunocore, Foghorn, Replimmune, InstilBio, Iovance, InflaRx, and Trisalus. ANS reports advisory/personal fees from Bristol Myers Squibb, Immunocore, Novartis, and research funding from Pfizer, Bristol Myers Squibb, Immunocore, Novartis, Targovax, Polaris, Checkmate Pharmaceuticals, Foghorn Therapeutics, Linneaus Therapeutics, and Prelude Therapeutics. GKS reports stock or other ownership interests in GenCirq, Bionaut Labs, and January Therapeutics; advisory/consulting/personal fees from Bionaut Labs, Ellipses Pharma,GenCirq, Epizyme, Array BioPharma, Apexigen, Oncogenuity, OnCusp Therapeutics, Concarlo, Shanghai Pharma, Astex Pharmaceuticals, January Therapeutics, Sellas Life Sciences, PureTech Health, and Killys Therapeutics; research funding from Astex Pharmaceuticals, Incyte, Calithera Biosciences, Lilly, Daiichi Sankyo, Fortress Biotech, Karyopharm Therapeutics, Oxford Biotherapeutics, TopAlliance Biosciences, Adaptimmune, Springworks Therapeutics, and TRACON Pharma. RDC reports consulting fees from Alkermes, Bristol Myers Squibb, Castle Biosciences, Delcath Systems, Eisai, Jiangsu Hengrui Pharmaceuticals, Ideaya Biosciences, Immunocore, InxMed, Iovance, Merck, Novartis, OncoSec, Pierre Fabre, PureTech Health, Regeneron, Sanofi Genzyme, Sorrento Therapeutics, Trisalus and advisory board fees from Aura Biosciences, Chimeron, and Rgenix. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2022 Khan, Lutzky, Shoushtari, Jeter, Marr, Olencki, Cebulla, Abdel-Rahman, Harbour, Sender, Nesson, Singh-Kandah, Hernandez, King, Katari, Dimapanat, Izard, Ambrosini, Surriga, Rai, Chiuzan, Schwartz and Carvajal.)

Retinoblastoma (Rb) is a deadly childhood eye cancer that is classically initiated by inactivation of the RB1 tumor suppressor. Clinical management continues to rely on nonspecific chemotherapeutic agents that are associated with treatment resistance and toxicity. Here, we analyzed 103 whole exomes, 20 whole transcriptomes, 5 single-cell transcriptomes, and 4 whole genomes from primary Rb tumors to identify previously unknown Rb dependencies. Several recurrent genomic aberrations implicate estrogen-related receptor gamma (ESRRG) in Rb pathogenesis. RB1 directly interacts with and inhibits ESRRG, and RB1 loss uncouples ESRRG from negative regulation. ESRRG regulates genes involved in retinogenesis and oxygen metabolism in Rb cells. ESRRG is preferentially expressed in hypoxic Rb cells in vivo. Depletion or inhibition of ESRRG causes marked Rb cell death, which is exacerbated in hypoxia. These findings reveal a previously unidentified dependency of Rb cells on ESRRG, and they implicate ESRRG as a potential therapeutic vulnerability in Rb.

BRCA1-associated protein 1 (BAP1) is a tumor suppressor gene that is mutated in cancer, including uveal melanoma. Loss-of-function BAP1 mutations are associated with uveal melanoma metastasis and poor prognosis, but the mechanisms underlying these effects remain unclear. Upregulation of cell-cell adhesion proteins is involved with collective migration and metastatic seeding of cancer cells. Here, we show that BAP1 loss in uveal melanoma patient samples is associated with upregulated gene expression of multiple cell adhesion molecules (CAM), including E-cadherin (CDH1), cell adhesion molecule 1 (CADM1), and syndecan-2 (SDC2). Similar findings were observed in uveal melanoma cell lines and single-cell RNA-sequencing data from uveal melanoma patient samples. BAP1 reexpression in uveal melanoma cells reduced E-cadherin and CADM1 levels. Functionally, knockdown of E-cadherin decreased spheroid cluster formation and knockdown of CADM1 decreased growth of BAP1-mutant uveal melanoma cells. Together, our findings demonstrate that BAP1 regulates the expression of CAMs which may regulate metastatic traits., Implications: BAP1 mutations and increased metastasis may be due to upregulation of CAMs., (©2022 American Association for Cancer Research.)

Uveal melanoma (UM) is the most common primary cancer of the eye and is associated with a high rate of metastatic death. UM can be stratified into two main classes based on metastatic risk, with class 1 UM having a low metastatic risk and class 2 UM having a high metastatic risk. Class 2 UM have a distinctive genomic, transcriptomic, histopathologic, and clinical phenotype characterized by biallelic inactivation of the BAP1 tumor-suppressor gene, an immune-suppressive microenvironment enriched for M2-polarized macrophages, and poor response to checkpoint-inhibitor immunotherapy. To identify potential mechanistic links between BAP1 loss and immune suppression in class 2 UM, we performed an integrated analysis of UM samples, as well as genetically engineered UM cell lines and uveal melanocytes (UMC). Using RNA sequencing (RNA-seq), we found that the most highly upregulated gene associated with BAP1 loss across these datasets was PROS1 , which encodes a ligand that triggers phosphorylation and activation of the immunosuppressive macrophage receptor MERTK. The inverse association between BAP1 and PROS1 in class 2 UM was confirmed by single-cell RNA-seq, which also revealed that MERTK was upregulated in CD163+ macrophages in class 2 UM. Using ChIP-seq, BAP1 knockdown in UM cells resulted in an accumulation of H3K27ac at the PROS1 locus, suggesting epigenetic regulation of PROS1 by BAP1. Phosphorylation of MERTK in RAW 264.7 monocyte-macrophage cells was increased upon coculture with BAP1 -/- UMCs, and this phosphorylation was blocked by depletion of PROS1 in the UMCs. These findings were corroborated by multicolor immunohistochemistry, where class 2/BAP1-mutant UMs demonstrated increased PROS1 expression in tumor cells and increased MERTK phosphorylation in CD163+ macrophages compared with class 1/BAP1-wildtype UMs. Taken together, these findings provide a mechanistic link between BAP1 loss and the suppression of the tumor immune microenvironment in class 2 UMs, and they implicate the PROS1-MERTK pathway as a potential target for immunotherapy in UM.

Retinoblastoma (RB) is the most common intraocular pediatric malignancy. Advancements in intra-arterial chemotherapy (IAC) for treatment of RB have resulted in dramatic improvement in eye salvage rates. Data regarding IAC outcomes and associated hematologic toxicities are limited. The objective of this retrospective study was to analyze baseline characteristics, efficacy, and hematologic complications associated with IAC treatment in children with RB at a single international referral institution. Ninety-five sessions of IAC were performed in 28 patients. Mean age at RB diagnosis was 12.5 months (SD, 9.2 mo). Fourteen patients had bilateral RB. IAC agents included melphalan, carboplatin, and topotecan. The most common regimens were triple-agent IAC and single-agent melphalan (66.3% and 15.8%, respectively). Median number of IAC sessions was 3 (mean: 3.39, range: 1 to 9). Eye salvage rate was 83.7% with an overall survival rate of 100% at a median follow-up of 29 months (mean: 29.8 mo, range: 1 to 63 mo). A total of 26 patients (92.9%) experienced at least 1 hematologic toxicity during their treatment course Prevalence of neutropenia, anemia, and thrombocytopenia were 89.3%, 85.7%, and 25%, respectively. While IAC is effective in salvaging most eyes with advanced intraocular RB, over half of patients experienced clinically significant neutropenia and anemia. Clinicians should be vigilant in monitoring patients for IAC-related complications., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: To determine whether unilateral multifocal uveal melanomas (UM) in the setting of ocular melanosis (melanosis oculi) represent genetically independent tumors., Design: Clinical case series., Methods: Two patients with unilateral multifocal UM in the setting of melanosis oculi were included. Tumors were evaluated for gene expression profile (GEP) and next generation sequencing (NGS) for uveal melanoma-associated mutations. Histopathologic analysis of enucleated specimens was also performed when available., Results: Patients were both female, ages 73 and 83 years. In Patient #1, the tumors both exhibited Class 2 GEP but each harbored a unique BAP1 mutation. In Patient #2, one tumor was Class 1 and harbored an SF3B1 mutation, whereas the other tumor was Class 2 and harbored a BAP1 mutation., Conclusions: Unilateral multifocal UM in the setting of melanosis oculi can arise due to the development of genetically independent primary tumors, which is detectable based on the mutation profile of each tumor. This is the first report of genetically-confirmed independent primary tumors in the setting of unilateral multifocal UM., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Effective therapeutic options are still lacking for uveal melanoma (UM) patients who develop metastasis. Metastatic traits of UM are linked to BRCA1-associated protein 1 (BAP1) mutations. Cell metabolism is re-programmed in UM with BAP1 mutant UM, but the underlying mechanisms and opportunities for therapeutic intervention remain unclear. BAP1 mutant UM tumors have an elevated glycolytic gene expression signature, with increased expression of pyruvate dehydrogenase (PDH) complex and PDH kinase (PDHK1). Furthermore, BAP1 mutant UM cells showed higher levels of phosphorylated PDHK1 and PDH that was associated with an upregulated glycolytic profile compared to BAP1 wild-type UM cells. Suppressing PDHK1-PDH phosphorylation decreased glycolytic capacity and cell growth, and induced cell cycle arrest of BAP1 mutant UM cells. Our results suggest that PDHK1-PDH phosphorylation is a causative factor of glycolytic phenotypes found in BAP1 mutant UM and propose a therapeutic opportunity for BAP1 mutant UM patients., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Mutations in the titin (TTN) gene are among the most common genomic aberrations in ocular surface squamous neoplasia (OSSN), the most common cancer of the external eye. Further, TTN mutations are associated with resistance to standard therapy with topical interferon alpha-2b (IFN-α2b). However, it remains unclear how TTN mutations drive OSSN pathogenesis and treatment resistance. TTN encodes the largest protein in the human body and its best understood function is as a myofibril scaffold in striated muscle. However, recent evidence indicates that TTN has additional functions in non-muscle cells and in cancer. Here, we performed a disorder-based bioinformatics analysis which revealed that intrinsically disordered protein regions are abundant in TTN and provide mechanistic insights into its function as a nuclear protein in epithelial cells. Specific mutations found in OSSN are predicted to affect its intrinsically disordered protein regions (IDPRs), promoting chromosomal instability, oncogenesis, and altered response to IFN-α2b treatment., (Copyright © 2021. Published by Elsevier B.V.)

Metastatic uveal melanoma (UM) responds poorly to targeted therapies and immune checkpoint inhibitors. Loss of BRCA1-associated protein 1 (BAP1) via inactivating mutations in the BAP1 gene is associated with UM progression. Thus, molecular alterations caused by BAP1 dysfunction may be novel therapeutic targets for metastatic UM. Here, we found that phosphorylation of AMP-dependent kinase (AMPK) was elevated in BAP1-altered (or mutant) compared to BAP1-unaltered (or wild-type [WT]) UM tumors. As a readout of AMPK pathway activation, phosphorylation of an AMPK downstream effector, acetyl-CoA-carboxylase (ACC), was also elevated. BAP1 re-expression in BAP1-null UM cell lines decreased phospho-AMPK (pAMPK) and phospho-ACC (pACC) levels. AMPK phosphorylation is mediated by calcium/calmodulin dependent protein kinase kinase 2 (CaMKK2) and potentially liver kinase B1 (LKB1) in BAP1 mutant UM cells. Knockdown of AMPKα1/2 reduced the viability of BAP1 mutant UM cells, indicating a survival function of AMPK in BAP1 mutant UM. Our data suggest that the AMPK pathway is an important mechanism mediating the survival of BAP1 mutant UM. Targeting the AMPK pathway may be a novel therapeutic strategy for metastatic UM., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: Gene expression profiling (GEP) is widely used for prognostication in patients with uveal melanoma (UM). Because biopsy tissue is limited, it is critical to obtain as much genomic information as possible from each sample. Combined application of both GEP and next-generation sequencing (NGS) allows for analysis of RNA and DNA from a single biopsy sample, offers additional prognostic information, and can potentially inform therapy selection. This study evaluated the analytical performance of a targeted custom NGS panel for mutational profiling of 7 genes commonly mutated in UM., Methods: One hundred five primary UM tumors were analyzed, including 37 formalin-fixed paraffin-embedded (FFPE) and 68 fine-needle aspiration biopsy specimens. Sequencing was performed on the Ion GeneStudio S5 platform to an average read depth of >500X per region of interest., Results: The 7-gene panel achieved a positive percent agreement of 100% for detection of both single-nucleotide variants and insertions/deletions, with a technical positive predictive value of 98.8% and 100%, respectively. Intra-assay and inter-assay concordance studies confirmed the assay's reproducibility and repeatability., Discussion/conclusion: The 7-gene panel is a robust, highly accurate NGS test that can be successfully performed, along with GEP, from a single small-gauge needle biopsy sample or FFPE specimen., Competing Interests: K.M.A., L.M.S., K.R.C., S.M.A., J.K.W., J.F.S., S.B., K.M.O., F.A.M., and R.W.C. were employees and stockholders of Castle Biosciences, Inc. at the time of this study. J.W.H. is the inventor of intellectual property related to prognostic testing for uveal melanoma. He is a paid consultant for Castle Biosciences, Inc., Friendswood, TX, USA licensee of this intellectual property, and he receives royalties from its commercialization., (Copyright © 2021 by S. Karger AG, Basel.)

Purpose: Tilting of the posterior plaque margin during eye plaque brachytherapy can lead to tumor underdosing and increased risk of local recurrence. We performed a quantitative analysis of the dosimetric effects of plaque tilt as a function of tumor position, basal dimension, height and plaque type using 3D treatment planning software., Materials and Methods: Posterior and anterior tumors with largest basal dimensions of 6, 12 and 18 mm and heights of 4, 7 and 10 mm were modeled. Both Eye Physics and COMS plaques were simulated and uniformly loaded. Plans were normalized to 85 Gy at the tumor apex. Posterior plaque tilts of 1, 2, 3 and 4 mm were simulated., Results: Volumetric coverage is more sensitive to tilt than the area coverage. Wide, flat tumors are more susceptible to tilt. Apical dose changed significantly as a function of tumor height and diameter. No other parameter exhibited significant differences. Posterior tumors are slightly more susceptible to tilt due to the use of notched plaques. Plaque type does not significantly alter the effect of plaque tilt., Conclusions: Wide, flat tumors are the most susceptible to plaque tilt. Tumor location or plaque type does not have a significant effect on dosimetry changes from plaque tilt. Robust clinical procedures such as the use of mattress sutures, pre- and post-implant ultrasound and post-implant dosimetry can all mitigate the risk associated with plaque tilt., (Copyright © 2021 American Brachytherapy Society. Published by Elsevier Inc. All rights reserved.)

In recent years, there has been tremendous enthusiasm with respect to detailing the genetic basis of many neoplasms, including conjunctival melanoma (CM). We aim to analyze five proteins associated with CM, namely BRAF, NRAS, c-KIT, NF1, and PTEN. We evaluated each protein for its intrinsically disordered protein regions (IDPRs) and its protein-protein interactions (PPI) with the Predictor of Natural Disordered Protein Regions (PONDR ® ) and the Search Tool for the Retrieval of Interacting Genes (STRING ® ). Our PONDR ® analysis found high levels of IDPRs in all five proteins with mutations linked to CM. The highest levels of IDPRs were in BRAF (45.95%), followed by PTEN (31.76%), NF1 (22.19%), c-KIT (21.82%), and NRAS (14.81%). Our STRING analysis found that each of these five proteins had more predicted interactions then expected ( p -value < 1.0 × 10 -16 ). Our analysis demonstrates that the mutations linked to CM likely affected IDPRs and possibly altered their highly complex PPIs. Quantifying IDPRs in BRAF, NRAS, c-KIT, NF1, and PTEN and understanding these protein regions are important processes as IDPRs can be possible drug targets for novel targeted therapies for treating CM.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults and leads to deadly metastases for which there is no approved treatment. Genetic events driving early tumor development are well-described, but those occurring later during metastatic progression remain poorly understood. We performed multiregional genomic sequencing on 22 tumors collected from two patients with widely metastatic UM who underwent rapid autopsy. We observed multiple seeding events from the primary tumors, metastasis-to-metastasis seeding, polyclonal seeding, and late driver variants in ATM, KRAS, and other genes previously unreported in UM. These findings reveal previously unrecognized temporal and anatomic complexity in the genetic evolution of metastatic uveal melanoma, and they highlight the distinction between early and late phases of UM genetic evolution with implications for novel therapeutic approaches., (© 2021. The Author(s).)

BAP1 is an ubiquitin hydrolase whose deubiquitinase activity is mediated by polycomb group-like protein ASXL2. Cancer-related BAP1 mutations/deletions lead to loss-of-function by targeting the catalytic ubiquitin C-terminal hydrolase (UCH) or UCH37-like domain (ULD) domains of BAP1, and the latter disrupts binding to ASXL2, an obligate partner for BAP1 enzymatic activity. However, the biochemical and biophysical properties of domains involved in forming the enzymatically active complex are unknown. Here, we report the molecular dynamics, kinetics, and stoichiometry of these interactions. We demonstrate that interactions between BAP1 and ASXL2 are direct, specific, and stable to biochemical and biophysical manipulations as detected by isothermal titration calorimetry (ITC), GST association, and optical biosensor assays. Association of the ASXL2-AB box greatly stimulates BAP1 activity. A stable ternary complex is formed, comprised of the BAP1-UCH, BAP1-ULD, and ASXL2-AB domains. Stoichiometric analysis revealed that one molecule of the ULD domain directly interacts with one molecule of the AB box. Real-time kinetic analysis of the ULD/AB protein complex to the BAP1-UCH domain, based on surface plasmon resonance, indicated that formation of the ULD/AB complex with the UCH domain is a single-step event with fast association and slow dissociation rates. In vitro experiments validated in cells that the ASXL-AB box directly regulates BAP1 activity. IMPLICATIONS: Collectively, these data elucidate molecular interactions between specific protein domains regulating BAP1 deubiquitinase activity, thus establishing a foundation for small-molecule approaches to reactivate latent wild-type BAP1 catalytic activity in BAP1 -mutant cancers., (©2021 American Association for Cancer Research.)

Background: Recent advances in single cell sequencing technologies allow for greater resolution in assessing tumor clonality using chromosome copy number variations (CNVs). While single cell DNA sequencing technologies are ideal to identify tumor sub-clones, they remain expensive and in contrast to single cell RNA-seq (scRNA-seq) methods are more limited in the data they generate. However, CNV data can be inferred from scRNA-seq and bulk RNA-seq, for which several tools have been developed, including inferCNV, CaSpER, and HoneyBADGER. Inferences regarding tumor clonality from CNV data (and other sources) are frequently visualized using phylogenetic plots, which previously required time-consuming and error-prone, manual analysis., Results: Here, we present Uphyloplot2, a python script that generates phylogenetic plots directly from inferred RNA-seq data, or any Newick formatted dendrogram file. The tool is publicly available at https://github.com/harbourlab/UPhyloplot2/ ., Conclusions: Uphyloplot2 is an easy-to-use tool to generate phylogenetic plots to depict tumor clonality from scRNA-seq data and other sources.

Through a synthetic lethal screen, ERK activation was found to mediate resistance to FAK inhibition in GNAQ-mutant uveal melanoma. With PLCB-PKC-ERK and Trio-FAK-Yap representing compensatory effectors of mutant Gαq signaling, combined inhibition of both pathways may be a promising therapeutic strategy in metastatic uveal melanoma. See related article by Paradis et al., p. 3190 ., (©2021 American Association for Cancer Research.)

Purpose: Exophytic juxtapapillary retinal capillary hemangioblastoma (JRCH) can be difficult to diagnose. We explore the value of multimodal imaging to aid in the diagnosis., Design: Retrospective case series., Methods: Medical records and multimodal imaging studies were reviewed on all patients diagnosed with RCH at Bascom Palmer Eye Institute, Miami, Florida, between January 2013 and December 2019. Patients with exophytic lesions within 2 mm of the disc were included. One patient from the Baylor College of Medicine, Houston, Texas was included. Patient demographics, referring diagnosis, history of von Hippel-Lindau syndrome, initial and last visual acuity, and treatments were recorded. Fundus photography, fundus autofluorescence, fluorescein angiography, indocyanine green angiography, optical coherence tomography (OCT), OCT angiography, and B-scan images were reviewed., Results: Twelve patients were identified with exophytic JRCH. The mean age was 54 years (range 38-73 years). Five patients had von Hippel-Lindau syndrome. The most common referral diagnoses were choroidal neovascularization and neuroretinitis. Imaging features included nodular outer retinal thickening with shadowing and intra-/subretinal fluid on OCT, hypoautofluorescence on fundus autofluorescence, middle to outer retinal hypervascularity on OCT angiography, early hyperfluorescence with late leakage on fluorescein angiography, and lack of choroidal vascular lesion on indocyanine green angiography. Treatments included photodynamic therapy (6 patients), intravitreal anti-vascular endothelial growth factor therapy (6 patients), argon laser photocoagulation (2 patients), intravitreal or sub-Tenon's triamcinolone (3 patients), and observation (4 patients)., Conclusions: A key to the accurate diagnosis of exophytic JRCH is its intraretinal location, typically involving the outer retinal layers, which results in a clinical appearance that is distinct from the more common and easily recognizable endophytic RCH. Multimodal imaging can aid in ruling out choroidal neovascularization and disc edema by demonstrating an absence of involvement of those structures., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Single-cell RNA sequencing (scRNA-seq) has been a transformative technology in many research fields. Dimensional reduction techniques such as UMAP and tSNE are used to visualize scRNA-seq data in two or three dimensions for cells to be clustered in biologically meaningful ways. Subsequently, gene expression is frequently mapped onto these plots to show the distribution of gene expression across the plots, for instance to distinguish cell types. However, plotting each cell with only a single color leads to repetitive and unintuitive representations. Here, we present PieParty, which allows scRNA-seq data to be plotted such that every cell is represented as a pie chart, and every slice in the pie charts corresponds to the gene expression of a single gene. This allows for the simultaneous visualization of the expression of multiple genes and gene networks. The resulting figures are information dense, space efficient, and highly intuitive. PieParty is publicly available on GitHub at https://github.com/harbourlab/PieParty., (© 2021 Stefan et al.)