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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.

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 than can be targeted therapeutically in cancer.

S1. Doxycycline-inducible shRNA knockdown of BAP1 results in robust decrease of GLO1 expression in uveal melanoma cell lines. S2. Phenotypic rescue and toxicity analysis of three HDAC inhibitors in Xenopus embryos injected with control morpholino CtrlMO or bap1-targeting morpholino BAP1MO. S3. Quisinostat and trametinib combination exhibits mostly additive cytotoxic effect in uveal melanoma in vitro.

Table S2. Results of epigenetic library screen.

Drug screens leading to successful targeted therapies in cancer have been mainly based on cell viability assays identifying inhibitors of dominantly acting oncogenes. In contrast, there has been little success in discovering targeted therapies that reverse the effects of inactivating mutations in tumor-suppressor genes. BAP1 is one such tumor suppressor that is frequently inactivated in a variety of cancers, including uveal melanoma, renal cell carcinoma, and mesothelioma. Because BAP1 is an epigenetic transcriptional regulator of developmental genes, we designed a two-phase drug screen involving a cell-based rescue screen of transcriptional repression caused by BAP1 loss, followed by an in vivo screen of lead compounds for rescue of a BAP1-deficient phenotype with minimal toxicity in Xenopus embryos. The first screen identified 9 compounds, 8 of which were HDAC inhibitors. The second screen eliminated all except one compound due to inefficacy or toxicity. The resulting lead compound, quisinostat, has a distinctive activity spectrum, including high potency against HDAC4, which was recently shown to be a key target of BAP1. Quisinostat was further validated in a mouse model and found to prevent the growth of BAP1-mutant uveal melanomas. This innovative strategy demonstrates the potential for identifying therapeutic compounds that target tumor-suppressor mutations in cancer.Implications:Few drugs have been identified that target mutations in tumor suppressors. Using a novel 2-step screening approach, strategy, we identified quisinostat as a candidate for therapy in BAP1-mutant uveal melanoma. HDAC4 is implicated as a key target in uveal melanoma and perhaps other BAP1-mutant cancers.

Table S1. Results of differential expression analysis

TCGA_Methyl_DMP: All significantly methylated probes (FDR < 0.05) in Class 2 uveal melanomas compared to Class 1 uveal melanomas from the 80 TCGA cases. JWH_Methyl_DMP: All significantly methylated probes (p < 0.05) in Class 2 uveal melanomas compared to Class 1 uveal melanomas from 12 cases of the senior author (JWH). TCGA_D12_PC1_PC2_Methyl: The 67,261 probes shared between the top 20% most variable methylated probes from the TCGA and JWH datasets used in the principal component analyses. The percentage contribution to PC1 and PC2 are displayed for all probes. TCGA_D12_Probes_Top5000PC1: The 204 probes shared between the TCGA and JWH datasets out of the Top 5000 shared most variable methylated probes contributing to PC1 in the principal component analyses.

GViz integrated chromosomal visualization plots.

Purpose:The strong association between BAP1 mutations and metastasizing Class 2 uveal melanoma (UM) suggests that epigenetic alterations may play a significant role in tumor progression. Thus, we characterized the impact of BAP1 loss on the DNA methylome in UM.Experimental Design: Global DNA methylation was analyzed in 47 Class 1 and 45 Class 2 primary UMs and in UM cells engineered to inducibly deplete BAP1. RNA-Seq was analyzed in 80 UM samples and engineered UM cells.Results:Hypermethylation on chromosome 3 correlated with downregulated gene expression at several loci, including 3p21, where BAP1 is located. Gene set analysis of hypermethylated and downregulated genes identified axon guidance and melanogenesis as deregulated pathways, with several of these genes located on chromosome 3. A novel hypermethylated site within the BAP1 locus was found in all Class 2 tumors, suggesting that BAP1 itself is epigenetically regulated. Highly differentially methylated probes were orthogonally validated using bisulfite sequencing, and they successfully distinguished Class 1 and Class 2 tumors in 100% of cases. In functional validation experiments, BAP1 knockdown in UM cells induced methylomic repatterning similar to UM tumors, enriched for genes involved in axon guidance, melanogenesis, and development.Conclusions:This study, coupled with previous work, suggests that the initial event in the divergence of Class 2 UM from Class 1 UM is loss of one copy of chromosome 3, followed by mutation of BAP1 on the remaining copy of chromosome 3, leading to the methylomic repatterning profile characteristic of Class 2 UMs.

Gene ontology analysis of genes differentially expressed between Class1PRAME+ tumors and Class1PRAME- tumors.

Gene Set Enrichment Analysis (GSEA) of genes differentially expressed between Class1PRAME+ tumors and Class1PRAME- tumors with respect to molecular function.

Comparison of features between uveal melanoma subgroups.

Molecular analyses and the order in which they were conducted in this study.

Significance Analysis of Microarrays (SAM) for differentially expressed genes between Class1met+ tumors and Class1met- tumors.

Clinicopathologic and genomic features in the 12 patients with Class1met+ uveal melanomas.

Purpose: Uveal melanoma (UM) can be classified by gene expression profiling (GEP) into Class 1 (low metastatic risk) and Class 2 (high metastatic risk), the latter being strongly associated with mutational inactivation of the tumor suppressor BAP1. Nevertheless, a small percentage of Class 1 tumors give rise to metastatic disease. The purpose of this study was to identify biomarkers of metastasis in Class 1 tumors.Experimental Design: A total of 389 consecutive patients with UM were assigned to Class 1 or Class 2 using a prospectively validated 12-gene prognostic classifier. Selected tumors were further analyzed using global GEP and single nucleotide polymorphism microarrays. PRAME (preferentially expressed antigen in melanoma) mRNA expression was analyzed in 64 Class 1 tumors by qPCR.Results: Among Class 1 UMs, the most significant predictor of metastasis was PRAME mRNA expression (P = 0.0006). The 5-year actuarial rate of metastasis was 0% for Class1PRAME−, 38% for Class1PRAME+, and 71% for Class 2 tumors. Median metastasis-free survival for Class1PRAME+ patients was 88 months, compared to 32 months for Class 2 patients. Findings were validated using three independent datasets, including one using disomy 3 to identify low-risk UM. Chromosome copy number changes associated with Class1PRAME+ tumors included gain of 1q, 6p, 8q, and 9q and loss of 6q and 11q. PRAME expression was associated with larger tumor diameter (P = 0.05) and SF3B1 mutations (P = 0.003).Conclusions: PRAME is an independent prognostic biomarker in UM, which identifies increased metastatic risk in patients with Class 1 or disomy 3 tumors. This finding may further enhance the accuracy of prognostic testing and precision medicine for UM. Clin Cancer Res; 22(5); 1234–42. ©2016 AACR.

BAP1 knockdown alters the epigenomic profile in uveal melanoma (UM) cells. (A) Western blot of tetracycline-inducible 92.1 and Mel202 shBAP1 UM cells without (-) and with (+) induction by doxycycline. (B) Multidimensional scaling (MDS) plot of the 1000 most variably methylated probes in the BAP1KD cell experiment, comparing the tetracycline-induced BAP1KD cells (BAP1KD, red) to the un-induced BAP1 control cells (BAP1WT, green). (C) Hierarchical clustering dendrogram, using all methylation probes, plotting the induced (BAP1KD) and uninduced (BAP1WT) 92.1 and Mel202 biological replicates.

Statistical, molecular, and survival analyses.

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.

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, 16 whole transcriptomes, 5 single-cell transcriptomes, and 4 whole genomes from primary Rb tumors to identify novel 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 novel dependency of Rb cells on ESRRG, and they implicate ESRRG as a potential therapeutic vulnerability in Rb.

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.

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 up-regulated 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 up-regulated 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 co-culture with BAP1-/- UMCs, and this phosphorylation was blocked by depletion of PROS1 in the UMCs. These findings were corroborated by multi-color immunohistochemistry, where class 2/BAP1-mutant UMs demonstrated increased PROS1 expression in tumor cells and increased MERTK phosphorylation in CD163+ macrophages compared to class 1/BAP1-wildtype UMs. Taken together, these findings provide a mechanistic link between BAP1 loss and 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.

Uveal melanoma (UM) is a highly metastatic cancer that, in contrast to cutaneous melanoma, is largely unresponsive to checkpoint immunotherapy. Here, we interrogate the tumor microenvironment at single-cell resolution using scRNA-seq of 59,915 tumor and non-neoplastic cells from 8 primary and 3 metastatic samples. Tumor cells reveal novel subclonal genomic complexity and transcriptional states. Tumor-infiltrating immune cells comprise a previously unrecognized diversity of cell types, including CD8+ T cells predominantly expressing the checkpoint marker LAG3, rather than PD1 or CTLA4. V(D)J analysis shows clonally expanded T cells, indicating that they are capable of mounting an immune response. An indolent liver metastasis from a class 1B UM is infiltrated with clonally expanded plasma cells, indicative of antibody-mediated immunity. This complex ecosystem of tumor and immune cells provides new insights into UM biology, and LAG3 is identified as a potential candidate for immune checkpoint blockade in patients with high risk UM., Uveal melanoma is highly metastatic and unresponsive to checkpoint immunotherapy. Here, the authors present single-cell transcriptomics of 59,915 cells in 8 primary and 3 metastatic samples, highlighting the diversity of the tumour microenvironment.

Here, we present an O2-independent/HIF-1α (Hypoxia inducible factor-1α)-dependent mechanism that regulates developmental and regenerative growth of mammalian cardiomyoblasts. An autocrine feedback mechanism of GH/IGF1/SST (Growth hormone/Insulin-like growth factor 1/Somatostatin) signaling, mediated by GHRH/GHRH-R (Growth hormone-releasing hormone/GHRH-Receptor), is established specifically in NKX2-5 (NK2 Homeobox 5) expressing myocardial cells which affects HIF-1α stability through cAMP (cyclic adenosine monophosphate) or cGMP (cyclic guanosine monophosphate) activity. cAMP-mediated HIF-1α stabilization fuels Warburg metabolism and enhances NKX2-5 expression, limiting the developmental and regenerative growth of cardiomyoblasts. In contrast, cGMP-mediated HIF-1α inhibition (or knock-out of HIF-1α) redirects glycolytically derived citrate toward long-chain saturated fatty acid biosynthesis, leading to enhanced developmental and regenerative growth of cardiomyoblasts. These findings suggest that HIF1α-mediated glycolysis serves as a rate-limiting, O2-independent sensor of cardiomyogenesis and that targeting GHRH/GHRH-R signaling could be a therapeutic strategy for regenerating the mammalian heart post-injury.

This paper presents a general framework for the motion design of cam mechanisms using Non-Uniform Rational B-Spline (NURBS). The follower motion is described by the NURBS function. To establish motion curves, the system of linear equations is set up by arbitrary boundary conditions of the follower motion on displacements, velocities, accelerations, and jerks. Moreover, the computation of the NURBS parameters (the knot vector and the weight factor) is formulated in order to reduce peak values of the acceleration and jerk. The example demonstrates that the motion curves used by NURBS obtain advantageous characteristics and the maximum values of the acceleration and jerk on the motion curves decrease considerably compared with the polynomial and B-spline functions. Therefore, inertial forces and the tendency of vibration can be reduced in cam dynamics of high-speed cam systems. The results show that using NURBS for synthesizing motion curves is robust and effective because this can be applied for arbitrary motion curves of cam mechanisms.

Uveal Melanoma (UM) is the most common primary ocular cancer in adults. Virtually all UM have activating mutations in the Gq signaling pathway. Metastatic risk in patients correlates with additional secondary mutations, where the lowest metastatic risk (class 1) is associated with EIF1AX and SF3B1, and the highest metastatic risk (class 2) is associated with BAP1 mutations. The interplay between the initiating Gq pathway mutations, and the secondary mutations found in all UM has not been well understood yet. Here, we utilize a multi-omics approach, to describe gene expression changes, the proteome, and phosphatome, following GNAQ mutation and BAP1 knockout in primary human uveal melanocytes. We generated human uveal melanocyte cell lines with inducible and specific mutations and analyzed via RNAseq, ATACseq, and mass spectrometry. Results reveal significant changes in pathways related to p53, chromatin regulation/modification, gene expression, and RNA processing with noted dysregulation of genes JUN, HDAC4, YAP, and SOX10, which cause senescence in melanocytes following GNAQ mutation, and are relieved through additional mutation in BAP1. Citation Format: Dawn Alexandra Owens, Stefan Kurtenbach, Jeffim N. Kuznetsoff, Daniel A. Rodriguez, Anthony Cruz, J. Harbour. Analysis of canonical uveal melanoma mutations reveals novel signaling effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 855.

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 in order 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 one color leads to repetitive and unintuitive representations. Here, we present Pie Party, 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 individual genes. 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.

Recent advances in single cell sequencing technologies allow for greater resolution in assessing tumor clonality using chromosome copy number variations (CNVs), which can be inferred from single cell RNA-seq (scRNA-seq) data using applications such as inferCNV. Inferences regarding tumor clonality are frequently visualized using phylogenetic plots, which previously required time-consuming and tedious manual analysis. Here, we present UPhyloplot2, a python script that generates phylogenetic plots directly from inferCNV output files. The tool is publicly available at https://github.com/harbourlab/UPhyloplot2/.

Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We generated a Vav1 promoter-driven Flag-Asxl1Y588X transgenic mouse model, Asxl1Y588X Tg, to express a truncated FLAG-ASXL1aa1-587 protein in the hematopoietic system. The Asxl1Y588X Tg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patients with ASXL1 mutations. ATAC- and RNA-sequencing analyses revealed that the ASXL1aa1-587 truncating protein expression results in more open chromatin in cKit+ cells compared with wild-type cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation experiments showed that ASXL1aa1-587 acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hypersensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors. This study demonstrates that ASXL1aa1-587 plays a gain-of-function role in promoting myeloid malignancies. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies.

Cancer is thought to arise through the accumulation of genomic aberrations evolving under Darwinian selection. However, it remains unclear when the aberrations associated with metastasis emerge during tumor evolution. Uveal melanoma (UM) is the most common primary eye cancer and frequently leads to metastatic death, which is strongly linked to BAP1 mutations. Accordingly, UM is ideally suited for studying the clonal evolution of metastatic competence. Here we analyze sequencing data from 151 primary UM samples using a customized bioinformatic pipeline, to improve detection of BAP1 mutations and infer the clonal relationships among genomic aberrations. Strikingly, we find BAP1 mutations and other canonical genomic aberrations usually arise in an early punctuated burst, followed by neutral evolution extending to the time of clinical detection. This implies that the metastatic proclivity of UM is “set in stone” early in tumor evolution and may explain why advances in primary treatment have not improved survival., Uveal melanoma (UM), the most common primary eye cancer, is strongly linked to mutations in the tumor suppressor BAP1. Here, the authors analyze 151 primary UM samples to find that BAP1 and other canonical genomic aberrations arise in an early punctuated burst followed by neutral tumor evolution.

Retinoblastoma, which is usually initiated by the biallelic mutational inactivation of RB1, is the most common primary eye cancer in children and is fatal if left untreated. While retinoblastoma is initially sensitive to chemotherapy, it demonstrates a strong propensity for therapeutic escape, which is a leading cause of ocular morbidity, eye loss, and reduced survival. This problem is exacerbated by a poor understanding of how retinoblastoma progresses after the initial loss of RB1. Genomic analysis of primary retinoblastoma tumors has revealed the corepressor BCOR as the second most common target of mutations in retinoblastoma. Here, we explored the role of BCOR in normal retinal development to provide insights into its role in retinoblastoma. Mass spectrometry following immunoprecipitation of wildtype BCOR in primary retinoblastoma cells revealed novel BCOR interacting proteins in the Polycomb and MiDAC epigenetic complexes. Subsequent studies revealed potential functional consequences of BCOR mutation that may promote retinoblastoma progression. This work reveals new insights the role of epigenetic deregulation in retinoblastoma progression, and they nominate new candidates for therapeutic intervention. Citation Format: Michelle Garying Zhang, Dawn Owens, Stefan Kurtenbach, Jeffim Kuznetsov, Matthew Fields, Daniel Pelaez, James W. Harbour. Role of BCOR in retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3027.

Uveal melanoma (UM) is the most common primary eye cancer and has a high rate of metastasis with liver tropism. UM can be stratified according to metastatic risk into low risk Class 1 and high risk Class 2 tumor. Biallelic mutational inactivation of the tumor suppressor BAP1 is the quintessential molecular feature of Class 2 UM, but the mechanism by which loss of BAP1 leads to metastasis is poorly understood. We recently showed using single-cell RNA sequencing that the tumor microenvironment of primary and metastatic Class 2 UM is characterized by large numbers of inhibitory T cells and macrophages. Taken together, these findings led us to hypothesize that loss of BAP1 triggers changes in tumor-immune cell interactions that lead to an immunosuppressive microenvironment. To test this hypothesis, we performed RNA-seq and proteomic mass spectrometry in uveal melanocytes and UM cells engineered to lack BAP1 expression. These experiments identified a small cassette of immune modulatory genes that are up-regulated at the mRNA and protein levels following BAP1 loss. These proteins are known to drive monocytes towards type 2 macrophage polarization, which secrete cytokines that can inhibit T cell activation. Our findings suggest that altered interaction between UM cells and monocytes may be key to the emergence of metastatic competence in UM, and we are exploring this possibility with function experiments that may open the door to targeted therapy to subvert this process. Citation Format: Christopher J. Kaler, James J. Dollar, Stefan Kurtenbach, Jeffim N. Kuznetsov, Michael A. Durante, J. William Harbour. Mechanisms of genomic-microenvironmental interactions in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2764.

This paper proposes a vertical planar 2 P RP-2 P PR parallel manipulator along with a serial planar RRR passive orthosis (exoskeleton/supporting system) for performing sitting/lying type (stationary trainer) lower limb rehabilitation therapies in the sagittal plane. Proposed system's kinematic arrangement along with forward and inverse solutions is presented. System dynamics is derived to understand the behavior of the over-constrained manipulator-orthosis system. Further, a robust motion control scheme is proposed and the motion control scheme is based on non-singular fast terminal sliding mode control along with a nonlinear disturbance observer. The effectiveness and usefulness of the proposed manipulator is shown with the implementation of the motion controller through computer based numerical simulations using a clinical gait motion pattern. The proposed motion control scheme is also validated on an in-house fabricated prototype through motion control experiments. The controller parameter sensitivity and controller robustness are further analyzed at different working conditions. In comparison to the conventional controllers, the proposed control scheme possess few advantages namely better robustness, less chattering, high precision and fast finite time convergence, and can work in the presence of parameter uncertainties. From the demonstration, the proposed manipulator has certain advantages over existing stationary lower limb rehabilitation trainers namely, simple design, larger workspace, higher stiffness, modular design and low cost.

Pannexins are membrane channel proteins that play a role in a number of critical biological processes (Panchin et al., 2000; Shestopalov, Panchin, 2008). Among other cellular functions, pannexin hemichannels serve as purine nucleoside conduits providing ATP efflux into the extracellular space (Dahl, 2015), where it is rapidly degraded to adenosine. Pannexin1 (Panx1) is abundantly expressed in the brain and has been shown to contribute to adenosine signaling in nervous system tissues (Prochnow et al., 2012). We hypothesized that pannexin1 may contribute to sleep-wake cycle regulation through extracellular adenosine, a well-established paracrine factor in slow wave sleep. To investigate this link, EEG and movement activity throughout the light/dark cycle were compared in Panx1-/- and Panx1+/+ mice. We found a significant increase in waking and a correspondent decrease in slow wave sleep percentages in the Panx1-/- animals. These changes were especially pronounced during the dark period. Furthermore, we found a significant increase in movement activity of Panx1-/- mice. These findings are consistent with the hypothesis that extracellular adenosine is relatively depleted in Panx1-/- animals due to the absence of the ATP-permeable hemichannels. At the same time, sleep rebound after a 6-h sleep deprivation remained unchanged in Panx1-/- mice as compared to the control animals. Behavioral tests revealed that Panx1-/- mice were significantly faster during their descent along the vertical pole but more sluggish during their run through the horizontal pole as compared to the control mice.

BAP1 regulates developmental switch in lineages commonly affected by BAP1-mutant cancers., The BAP1 tumor suppressor is mutated in many human cancers such as uveal melanoma, leading to poor patient outcome. It remains unclear how BAP1 functions in normal biology or how its loss promotes cancer progression. Here, we show that Bap1 is critical for commitment to ectoderm, mesoderm, and neural crest lineages during Xenopus laevis development. Bap1 loss causes transcriptional silencing and failure of H3K27ac to accumulate at promoters of key genes regulating pluripotency-to-commitment transition, similar to findings in uveal melanoma. The Bap1-deficient phenotype can be rescued with human BAP1, by pharmacologic inhibition of histone deacetylase (HDAC) activity or by specific knockdown of Hdac4. Similarly, BAP1-deficient uveal melanoma cells are preferentially vulnerable to HDAC4 depletion. These findings show that Bap1 regulates lineage commitment through H3K27ac-mediated transcriptional activation, at least in part, by modulation of Hdac4, and they provide insights into how BAP1 loss promotes cancer progression.