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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.

HyperMethvsDecGE_ChrLoc: Chromosomal loci enriched (FDR < 0.05) for hypermethylated/downregulated genes in Class 2 uveal melanomas compared to Class 1 uveal melanomas HypoMethvsIncGE_ChrLoc: Chromosomal loci enriched (FDR < 0.05) for hypomethylated/upregulated genes in Class 2 uveal melanomas compared to Class 1 uveal melanomas HyperMethvsDecGE_KEGG_REACTOME: Functional (KEGG and REACTOME) pathways enriched (FDR < 0.05) for hypermethylated/downregulated genes in Class 2 uveal melanomas compared to Class 1 uveal melanomas HypoMethvsIncGE_KEGG_REACTOME: Functional (KEGG and REACTOME) pathways enriched (FDR < 0.05) for hypomethylated/upregulated genes in Class 2 uveal melanomas compared to Class 1 uveal melanomas HyperMethvsDecGE_GO: Gene ontology molecular functions enriched (FDR < 0.05) for hypermethylated/downregulated genes in Class 2 uveal melanomas compared to Class 1 uveal melanomas HypoMethvsIncGE_GO: Gene ontology molecular functions enriched (FDR < 0.05) for hypomethylated/upregulated genes in Class 2 uveal melanomas compared to Class 1 uveal melanomas HyperMethvsDecGE_TF: Promoter region transcription factor binding sites enriched (FDR < 0.05) for hypermethylated/downregulated genes in Class 2 uveal melanomas compared to Class 1 uveal melanomas HypoMethvsIncGE_TF: Promoter region transcription factor binding sites enriched (FDR < 0.05) for hypomethylated/upregulated genes in Class 2 uveal melanomas compared to Class 1 uveal melanomas

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.

BAP1KDvsWT_HyperMethDecGE: All significantly hypermethylated probes from hypermethylated/downregulated genes (p < 0.05) with an absolute value (log2(RNA fold change)) > 1 and absolute value (methylation Delta Beta value) > 0.01 in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KDvsWT_HypoMethIncGE: All significantly hypomethylated probes from hypomethylated/upregulated genes (p < 0.05) with an absolute value (log2(RNA fold change)) > 1 and absolute value (methylation Delta Beta value) > 0.01 in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HyperMethDecGE_ChrLoc: Chromosomal loci enriched (FDR < 0.05) for hypermethylated/downregulated genes in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HypoMethIncGE_ChrLoc: Chromosomal loci enriched (FDR < 0.05) for hypomethylated/upregulated genes in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HyperMethDecGE_KEGG: Functional (KEGG and REACTOME) pathways enriched (FDR < 0.05) for hypermethylated/downregulated genes in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HypoMethIncGE_KEGG: Functional (KEGG and REACTOME) pathways enriched (FDR < 0.05) for hypomethylated/upregulated genes in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_TCGA_HyperMethDecGE_Gene: All significantly hypermethylated probes (FDR < 0.05) from hypermethylated/downregulated genes shared in common between the shBAP1KD (p < 0.05) and TCGA (FDR < 0.05) datasets. BAP1KD_TCGA_HypoMethIncGE_Gene: All significantly hypomethylated probes (FDR < 0.05) from hypomethylated/upregulated genes shared in common between the shBAP1KD (p < 0.05) and TCGA (FDR < 0.05) datasets.

Comparison of features between uveal melanoma subgroups.

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

HyperMethvsDecGE_Cutoffs: All significantly hypermethylated probes from hypermethylated/downregulated genes (FDR < 0.05) with an absolute value (log2(RNA fold change)) > 1 and absolute value (methylation Delta Beta value) > 0.05 in Class 2 uveal melanomas compared to Class 1 uveal melanomas. HypoMethvsIncGE_Cutoffs: All significantly hypomethylated probes from hypomethylated/upregulated genes (FDR < 0.05) with an absolute value (log2(RNA fold change)) > 1 and absolute value (methylation Delta Beta value) > 0.05 in Class 2 uveal melanomas compared to Class 1 uveal melanomas. HyperMethvsDecGE: All significantly hypermethylated probes from hypermethylated/downregulated genes (FDR < 0.05) in Class 2 uveal melanomas compared to Class 1 uveal melanomas. HypoMethvsIncGE: All significantly hypomethylated probes significant hypomethylated/upregulated genes (FDR < 0.05) in Class 2 uveal melanomas compared to Class 1 uveal melanomas.

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.

Methylation validation probe targets with forward and reverse primers and the optimized annealing temperature

BAP1KDvsWT_HyperMethyl: All significantly hypermethylated probes (FDR < 0.05) with an absolute value (log2(RNA fold change)) > 1 and absolute value (methylation Delta Beta value) > 0.01 in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KDvsWT_HypoMethyl: All significantly hypomethylated probes (FDR < 0.05) with an absolute value (log2(RNA fold change)) > 1 and absolute value (methylation Delta Beta value) > 0.01 in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HyperMeth_ChrLoc: Chromosomal loci enriched (FDR < 0.05) for top 1000 hypermethylated genes in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HypoMeth_ChrLoc: Chromosomal loci enriched (FDR < 0.05) for top 1000 hypomethylated genes in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HyperMeth_KEGG_REACTOME: Functional (KEGG and REACTOME) pathways enriched (FDR < 0.05) for top 1000 hypermethylated genes in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HypoMeth_KEGG_REACTOME: Functional (KEGG and REACTOME) pathways enriched (FDR < 0.05) for for top 1000 hypomethylated genes in BAP1KDvsWT 92.1 and Mel202 cells.

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.

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.

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.

The presence of active neurogenic niches in adult humans is controversial. We focused attention to the human olfactory neuroepithelium, an extracranial site supplying input to the olfactory bulbs of the brain. Using single-cell RNA sequencing analyzing 28,726 cells, we identified neural stem cell and neural progenitor cell pools and neurons. Additionally, we detailed the expression of 140 olfactory receptors. These data from the olfactory neuroepithelium niche provide evidence that neuron production may continue for decades in humans.

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. TRANSLATIONAL RELEVANCE: We provide evidence that bi-allelic loss of BAP1 leads to extensive methylomic repatterning that results in the highly aggressive Class 2 phenotype, thereby providing a more complete picture of UM genomic evolution and potentially explaining the loss of melanocytic differentiation and gain of neural crest-like migratory behavior in Class 2 UM. Highly differentially methylated sites were identified that could form the basis for liquid biopsy biomarkers. These findings suggest a potential role for DNA methylation modulators in the treatment of UM.

Purpose Sebaceous carcinoma (SC) is a malignant eyelid tumor of the ocular adnexa that is primarily treated via surgical excision. Few therapies exist in advanced cases, and medical therapy is limited because of our incomplete understanding of SC biology. Herein, we describe a technique to culture human ocular adnexal SC for use as an in vitro model. Methods Human ocular adnexal SC tumor cells were isolated from a patient undergoing orbital exenteration surgery and named Bascom Palmer 50 (BP50). They were cultured in Dulbecco's modified Eagle medium/nutrient mixture F-12 supplemented with 10% fetal bovine serum and antibiotics and were maintained at 37°C in humidified 5% CO2. The cells were characterized by immunohistochemistry, exome sequencing, and short tandem repeats analysis. In vitro drug screening against mitomycin-C (MMC) was performed using a cell viability assay. Results BP50 grew past 40 passages with a doubling time of 52.3 hours. Immunocytochemical staining revealed expression of SC-associated markers adipophilin, epithelial membrane antigen, p53, and androgen receptor. Whole exome sequencing showed a significant carryover in somatic mutations between the tumor tissue and corresponding cell line, revealing genetic markers consistent with SC. MMC affected cell viability in a dose-dependent manner. Conclusions BP50 displays characteristics of ocular adnexal SC and therefore may facilitate improved understanding of SC biology and the high throughput assessment of novel therapeutic compounds and new drug combinatorial approaches targeted for this disease. Translational relevance Drug screening with MMC against these cells shows in vitro evidence to support its continued clinical use in SC.

PieParty visualizes every cell in scRNA-seq experiments as pie charts, where every slice of a pie chart corresponds to the expression of a single gene., 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.

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.

Chromatin immunoprecipitation (ChIP) has ushered in a new era of scientific discovery by allowing new insights into DNA-protein interactions. ChIP is used to quantify enriched genomic regions using qPCR, and more recently is combined with next generation sequencing (ChIP-seq) to obtain a genome wide profile of protein binding sites. Nevertheless, ChIP-qPCR remains an integral component of this technology for quality control purposes, before the library preparation and sequencing steps. In addition, ChIP-qPCR remains more time- and cost-effective for many focused projects in which the DNA regions of interest are already known. However, the DNA oligonucleotide primers needed for ChIP-qPCR are more challenging to design than for other qPCR projects. Here, we present the first public repository for ChIP oligonucleotides that have been verified to perform well in ChIP-qPCR experiments. ChIPprimersDB was developed by manual screening of publications to ensure primer quality and provide additional specific information on the ChIP experiments where the primers have been used. In addition to the primer sequences, the database includes information about the antibody, cells and tissues used in the experiment, information on the experimental design, and a direct link to the original publication. The database is linked at https://umiamihealth.org/bascom-palmer-eye-institute/research/laboratory-research/ocular-oncology-laboratory/chip-primers and hosted at https://www.chipprimers.com/.

Pannexins (Panx) are integral membrane proteins, with Panx1 being the best-characterized member of the protein family. Panx1 is implicated in sensory processing, and knockout (KO) animal models have become the primary tool to investigate the role(s) of Panx1 in sensory systems. Extending previous work from our group on primary olfaction, the expression patterns of Panxs in the vomeronasal organ (VNO), an auxiliary olfactory sense organ with a role in reproduction and social behavior, were compared. Using qRT-PCR and Immunohistochemistry (IHC), we confirmed the loss of Panx1, found similar Panx2 expression levels in both models, and a significant upregulation of Panx3 in mice with a global ablation of Panx1. Specifically, Panx3 showed upregulated expression in nerve fibers of the non-sensory epithelial layer in juvenile and adult KO mice and in the sensory layer of adults, which overlaps with Panx1 expression areas in WT populations. Since both social behavior and evoked ATP release in the VNO was not compromised in KO animals, we hypothesized that Panx3 could compensate for the loss of Panx1. This led us to compare Panx1 and Panx3 channels in vitro, demonstrating similar dye uptake and ATP release properties. Outcomes of this study strongly suggest that Panx3 may functionally compensate for the loss of Panx1 in the VNO of the olfactory system, ensuring sustained chemosensory processing. This finding extends previous reports on the upregulation of Panx3 in arterial walls and the skin of Panx1 KO mice, suggesting that roles of Panx1 warrant uncharacterized safeguarding mechanisms involving Panx3.

Usher syndrome is a genetically and clinically heterogeneous disease in humans, characterized by sensorineural hearing loss, retinitis pigmentosa and vestibular dysfunction. This disease is caused by mutations in genes encoding proteins that form complex networks in different cellular compartments. Currently, it remains unclear whether the Usher proteins also form networks within the olfactory epithelium (OE). Here, we describe Usher gene expression at the mRNA and protein level in the OE of mice and showed interactions between these proteins and olfactory signaling proteins. Additionally, we analyzed the odor sensitivity of different Usher syndrome mouse models using electro-olfactogram recordings and monitored significant changes in the odor detection capabilities in mice expressing mutant Usher proteins. Furthermore, we observed changes in the expression of signaling proteins that might compensate for the Usher protein deficiency. In summary, this study provides novel insights into the presence and purpose of the Usher proteins in olfactory signal transduction.

BEACH domain proteins are involved in membrane protein traffic and human diseases, but their molecular mechanisms are not understood. The BEACH protein LRBA has been implicated in immune response and cell proliferation, and human LRBA mutations cause severe immune deficiency. Here, we report a first functional and molecular phenotype outside the immune system of LRBA-knockout mice: compromised olfaction, manifesting in reduced electro-olfactogram response amplitude, impaired food-finding efficiency, and smaller olfactory bulbs. LRBA is prominently expressed in olfactory and vomeronasal chemosensory neurons of wild-type mice. Olfactory impairment in the LRBA-KO is explained by markedly reduced concentrations (20–40% of wild-type levels) of all three subunits αolf, β1 and γ13 of the olfactory heterotrimeric G-protein, Golf, in the sensory cilia of olfactory neurons. In contrast, cilia morphology and the concentrations of many other proteins of olfactory cilia are not or only slightly affected. LRBA is also highly expressed in photoreceptor cells, another cell type with a specialized sensory cilium and heterotrimeric G-protein-based signalling; however, visual function appeared unimpaired by the LRBA-KO. To our knowledge, this is the first observation that a BEACH protein is required for the efficient subcellular localization of a lipid-anchored protein, and of a ciliary protein.

Within the further development of a special mechanical representation of the human circulatory system the CVELoop at the Department of Cardiovascular Engineering at RWTH Aachen University, a contractive MockHeart was developed. This MockHeart simulates a healthy heart in the CVELoop. In opposition to all previous cardiovascular system simulators, the left ventricle is externally driven by several cam mechanisms. These cam mechanisms provide the necessary power to create the pressure-volume-work for the fluid circulation but also the important time dependent radial contraction, which is the most influential degree of Freedom in the human heart. The aim is to produce a MockHeart providing valid boundary conditions for the connected VAD (Ventricular Assist Device). The systematic approach of the development of the mechanism is performed based on an accurate measurement of the kinematic properties of a healthy human heart at RWTH Aachen University with the speckle tracking echocardiography-procedure.

A Non Uniform Rational B-Spline (NURBS) is used for synthesizing the motion curve of cam mechanisms because it is flexible and satisfies arbitrary boundary conditions from the working requirement of machinery systems. For using NURBS curve as motion curves of cam mechanisms, selecting the knot vector is very important. This work presents the effect of the knot vector on the displacement, velocity, acceleration, and jerk curves. The linear system of equations for solving the cam motion is also presented. A general computation of the knot vector of NURBS for synthesizing the motion curve is presented. Several examples illustrate this research.

In cam design process, synthesizing the motion curves is very important because of the effect of motion curves to cam size, force, and vibration. Thus, improving the kinematics of cam curves is significant. This paper presents a general synthesis of motion curves of cam mechanisms. A Non Uniform Rational B-Spline (NURBS) is used to improve the kinematics. The linear system of equations is established to determine motion curves for arbitrary boundary conditions of displacement, velocity, acceleration, and jerk. By controlling the weight parameters of NURBS, the peak values of acceleration and jerk can decrease. Several examples are presented to demonstrate this research. The results are also compared with traditional motion curves.

BEACH domain proteins are involved in membrane protein traffic and human diseases, but their molecular mechanisms are not understood. The BEACH protein LRBA has been implicated in immune response and cell proliferation, and human LRBA mutations cause severe immune deficiency. Here, we report a first functional and molecular phenotype outside the immune system of LRBA-knockout mice: compromised olfaction, manifesting in reduced electro-olfactogram response amplitude, impaired food-finding efficiency, and smaller olfactory bulbs. LRBA is prominently expressed in olfactory and vomeronasal chemosensory neurons of wild-type mice. Olfactory impairment in the LRBA-KO is explained by markedly reduced concentrations (20–40% of wild-type levels) of all three subunits αolf, β1 and γ13 of the olfactory heterotrimeric G-protein, Golf, in the sensory cilia of olfactory neurons. In contrast, cilia morphology and the concentrations of many other proteins of olfactory cilia are not or only slightly affected. LRBA is also highly expressed in photoreceptor cells, another cell type with a specialized sensory cilium and heterotrimeric G-protein-based signalling; however, visual function appeared unimpaired by the LRBA-KO. To our knowledge, this is the first observation that a BEACH protein is required for the efficient subcellular localization of a lipid-anchored protein, and of a ciliary protein.