PRAME induces genomic instability in uveal melanoma.

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.

BAP1 Loss Promotes Suppressive Tumor Immune Microenvironment via Upregulation of PROS1 in Class 2 Uveal Melanomas.

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.

RB1 loss triggers dependence on ESRRG in retinoblastoma.

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.

Multiple Genetically Distinct Uveal Melanomas Arise in the Same Eye of Two Patients with Melanosis Oculi.

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.

PieParty: visualizing cells from scRNA-seq data as pie charts.

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.

Mechanism Sharing Between Genetic and Gestational Hypoxia-Induced Cardiac Anomalies.

Background: Cardiac development is a dynamic process both temporally and spatially. These complex processes are often disturbed and lead to congenital cardiac anomalies that affect approximately 1% of live births. Disease-causing variants in several genetic loci lead to cardiac anomalies, with variants in transcription factor NKX2-5 gene being one of the largest variants known. Gestational hypoxia, such as seen in high-altitude pregnancy, has been known to affect cardiac development, yet the incidence and underlying mechanisms are largely unknown. Methods and Results: Normal wild-type female mice mated with heterozygous Nkx2-5 mutant males were housed under moderate hypoxia (14% O2) or normoxia (20.9% O2) conditions from 10.5 days of gestation. Wild-type mice exposed to hypoxia demonstrate excessive trabeculation, ventricular septal defects, irregular morphology of interventricular septum as well as atrial septal abnormalities, which overlap with those seen in heterozygous Nkx2-5 mutant mice. Genome-wide transcriptome done by RNA-seq of a 2-day hypoxic exposure on wild-type embryos revealed abnormal transcriptomes, in which approximately 60% share those from Nkx2-5 mutants without hypoxia. Gestational hypoxia reduced the expression of Nkx2-5 proteins in more than one-half along with a reduction in phosphorylation, suggesting that abnormal Nkx2-5 function is a common mechanism shared between genetic and gestational hypoxia-induced cardiac anomalies, at least at a specific developing stage. Conclusion: The results of our study provide insights into a common molecular mechanism underlying non-genetic and genetic cardiac anomalies.

Single Genome Sequencing of Expressed and Proviral HIV-1 Envelope Glycoprotein 120 (gp120) and nef Genes.

The current study provides detailed protocols utilized to amplify the complete HIV-1 gp120 and nef genes from single copies of expressed or integrated HIV present in fresh-frozen autopsy tissues of patients who died while on combined antiretroviral therapy (cART) with no detectable plasma viral load (pVL) at death ( Lamers et al., 2016a and 2016b; Rose et al., 2016 ). This method optimizes protocols from previous publications ( Palmer et al., 2005 ; Norström et al., 2012 ; Lamers et al., 2015 ; 2016a and 2016b; Rife et al., 2016 ) to produce single distinct PCR products that can be directly sequenced and includes several cost-saving and time-efficient modifications.

Identifying Transmission Clusters with Cluster Picker and HIV-TRACE.

We compared the behavior of two approaches (Cluster Picker and HIV-TRACE) at varying genetic distances to identify transmission clusters. We used three HIV gp41 sequence datasets originating from the Rakai Community Cohort Study: (1) next-generation sequence (NGS) data from nine linked couples; (2) NGS data from longitudinal sampling of 14 individuals; and (3) Sanger consensus sequences from a cross-sectional dataset (n = 1,022) containing 91 epidemiologically linked heterosexual couples. We calculated the optimal genetic distance threshold to separate linked versus unlinked NGS datasets using a receiver operating curve analysis. We evaluated the number, size, and composition of clusters detected by Cluster Picker and HIV-TRACE at six genetic distance thresholds (1%-5.3%) on all three datasets. We further tested the effect of using all NGS, versus only a single variant for each patient/time point, for datasets (1) and (2). The optimal gp41 genetic distance threshold to distinguish linked and unlinked couples and individuals was 5.3% and 4%, respectively. HIV-TRACE tended to detect larger and fewer clusters, whereas Cluster Picker detected more clusters containing only two sequences. For NGS datasets (1) and (2), HIV-TRACE and Cluster Picker detected all linked pairs at 3% and 4% genetic distances, respectively. However, at 5.3% genetic distance, 20% of couples in dataset (3) did not cluster using either program, and for >1/3 of couples cluster assignment were discordant. We suggest caution in choosing thresholds for clustering analyses in a generalized epidemic.

HIV Maintains an Evolving and Dispersed Population in Multiple Tissues during Suppressive Combined Antiretroviral Therapy in Individuals with Cancer.

UNLABELLED: While combined antiretroviral therapy (cART) can result in undetectable plasma viral loads, it does not eradicate HIV infection. Furthermore, HIV-infected individuals while on cART remain at an increased risk of developing serious comorbidities, such as cancer, neurological disease, and atherosclerosis, suggesting that during cART, tissue-based HIV may contribute to such pathologies. We obtained DNA and RNA env, nef, and pol sequences using single-genome sequencing from postmortem tissues of three HIV(+) cART-treated (cART(+)) individuals with undetectable viral load and metastatic cancer at death and performed time-scaled Bayesian evolutionary analyses. We used a sensitive in situ hybridization technique to visualize HIV gag-pol mRNA transcripts in cerebellum and lymph node tissues from one patient. Tissue-associated virus evolved at similar rates in cART(+) and cART-naive (cART(-)) patients. Phylogenetic trees were characterized by two distinct features: (i) branching patterns consistent with constant viral evolution and dispersal among tissues and (ii) very recently derived clades containing both DNA and RNA sequences from multiple tissues. Rapid expansion of virus near death corresponded to wide-spread metastasis. HIV RNA(+) cells clustered in cerebellum tissue but were dispersed in lymph node tissue, mirroring the evolutionary patterns observed for that patient. Activated, infiltrating macrophages were associated with HIV RNA. Our data provide evidence that tissues serve as a sanctuary for wild-type HIV during cART and suggest the importance of macrophages as an alternative reservoir and mechanism of virus spread. IMPORTANCE: Combined antiretroviral therapy (cART) reduces plasma HIV to undetectable levels; however, removal of cART results in plasma HIV rebound, thus highlighting its inability to entirely rid the body of infection. Additionally, HIV-infected individuals on cART remain at high risk of serious diseases, which suggests a contribution from residual HIV. In this study, we isolated and sequenced HIV from postmortem tissues from three HIV(+) cART(+) individuals who died with metastatic cancer and had no detectable plasma viral load. Using high-resolution evolutionary analyses, we found that tissue-based HIV continues to replicate, evolve, and migrate among tissues during cART. Furthermore, cancer onset and metastasis coincided with increased HIV expansion, suggesting a linked mechanism. HIV-expressing cells were associated with tissue macrophages, a target of HIV infection. Our results suggest the importance of tissues, and macrophages in particular, as a target for novel anti-HIV therapies.