Single-cell analysis reveals new evolutionary complexity in uveal melanoma.

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.

BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to 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.

BAP1 Loss Is Associated with DNA Methylomic Repatterning in Highly Aggressive Class 2 Uveal Melanomas.

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.

Physiological and hypoxic oxygen concentration differentially regulates human c-Kit+ cardiac stem cell proliferation and migration.

Cardiac stem cells (CSCs) are being evaluated for their efficacy in the treatment of heart failure. However, numerous factors impair the exogenously delivered cells' regenerative capabilities. Hypoxia is one stress that contributes to inadequate tissue repair. Here, we tested the hypothesis that hypoxia impairs cell proliferation, survival, and migration of human CSCs relative to physiological and room air oxygen concentrations. Human endomyocardial biopsy-derived CSCs were isolated, selected for c-Kit expression, and expanded in vitro at room air (21% O2). To assess the effect on proliferation, survival, and migration, CSCs were transferred to physiological (5%) or hypoxic (0.5%) O2 concentrations. Physiological O2 levels increased proliferation (P < 0.05) but did not affect survival of CSCs. Although similar growth rates were observed in room air and hypoxia, a significant reduction of ß-galactosidase activity (-4,203 fluorescent units, P < 0.05), p16 protein expression (0.58-fold, P < 0.001), and mitochondrial content (0.18-fold, P < 0.001) in hypoxia suggests that transition from high (21%) to low (0.5%) O2 reduces senescence and promotes quiescence. Furthermore, physiological O2 levels increased migration (P < 0.05) compared with room air and hypoxia, and treatment with mesenchymal stem cell-conditioned media rescued CSC migration under hypoxia to levels comparable to physiological O2 migration (2-fold, P < 0.05 relative to CSC media control). Our finding that physiological O2 concentration is optimal for in vitro parameters of CSC biology suggests that standard room air may diminish cell regenerative potential. This study provides novel insights into the modulatory effects of O2 concentration on CSC biology and has important implications for refining stem cell therapies.

Epigenetic reprogramming and aberrant expression of PRAME are associated with increased metastatic risk in Class 1 and Class 2 uveal melanomas.

BACKGROUND: We previously identified PRAME as a biomarker for metastatic risk in Class 1 uveal melanomas. In this study, we sought to define a threshold value for positive PRAME expression (PRAME+) in a large dataset, identify factors associated with PRAME expression, evaluate the prognostic value of PRAME in Class 2 uveal melanomas, and determine whether PRAME expression is associated with aberrant hypomethylation of the PRAME promoter. RESULTS: Among 678 samples analyzed by qPCR, 498 (73.5%) were PRAME- and 180 (26.5%) were PRAME+. Class 1 tumors were more likely to be PRAME-, whereas Class 2 tumors were more likely to be PRAME+ (P < 0.0001). PRAME expression was associated with shorter time to metastasis and melanoma specific mortality in Class 2 tumors (P = 0.01 and P = 0.02, respectively). In Class 1 tumors, PRAME expression was directly associated with SF3B1 mutations (P < 0.0001) and inversely associated with EIF1AX mutations (P = 0.004). PRAME expression was strongly associated with hypomethylation at 12 CpG sites near the PRAME promoter. MATERIALS AND METHODS: Analyses included PRAME mRNA expression, Class 1 versus Class 2 status, chromosomal copy number, mutation status of BAP1, EIF1AX, GNA11, GNAQ and SF3B1, and genomic DNA methylation status. Analyses were performed on 555 de-identified samples from Castle Biosciences, 123 samples from our center, and 80 samples from the TCGA. CONCLUSIONS: PRAME is aberrantly hypomethylated and activated in Class 1 and Class 2 uveal melanomas and is associated with increased metastatic risk in both classes. Since PRAME has been successfully targeted for immunotherapy, it may prove to be a companion prognostic biomarker.

Metformin induces apoptosis through AMPK-dependent inhibition of UPR signaling in ALL lymphoblasts.

The outcome of patients with resistant phenotypes of acute lymphoblastic leukemia (ALL) or those who relapse remains poor. We investigated the mechanism of cell death induced by metformin in Bp- and T-ALL cell models and primary cells, and show that metformin effectively induces apoptosis in ALL cells. Metformin activated AMPK, down-regulated the unfolded protein response (UPR) demonstrated by significant decrease in the main UPR regulator GRP78, and led to UPR-mediated cell death via up-regulation of the ER stress/UPR cell death mediators IRE1α and CHOP. Using shRNA, we demonstrate that metformin-induced apoptosis is AMPK-dependent since AMPK knock-down rescued ALL cells, which correlated with down-regulation of IRE1α and CHOP and restoration of the UPR/GRP78 function. Additionally rapamycin, a known inhibitor of mTOR-dependent protein synthesis, rescued cells from metformin-induced apoptosis and down-regulated CHOP expression. Finally, metformin induced PIM-2 kinase activity and co-treatment of ALL cells with a PIM-1/2 kinase inhibitor plus metformin synergistically increased cell death, suggesting a buffering role for PIM-2 in metformin's cytotoxicity. Similar synergism was seen with agents targeting Akt in combination with metformin, supporting our original postulate that AMPK and Akt exert opposite regulatory roles on UPR activity in ALL. Taken together, our data indicate that metformin induces ALL cell death by triggering ER and proteotoxic stress and simultaneously down-regulating the physiologic UPR response responsible for effectively buffering proteotoxic stress. Our findings provide evidence for a role of metformin in ALL therapy and support strategies targeting synthetic lethal interactions with Akt and PIM kinases as suitable for future consideration for clinical translation in ALL.

Inhibition of Akt potentiates 2-DG-induced apoptosis via downregulation of UPR in acute lymphoblastic leukemia.

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-D-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with D-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG-treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DG's cytotoxicity in ALL. 2-DG-treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival.

AMPK and Akt determine apoptotic cell death following perturbations of one-carbon metabolism by regulating ER stress in acute lymphoblastic leukemia.

AICAr is a cell-permeable nucleotide that has been used in vivo and in vitro to activate AMPK. Our previous findings have shown that AICAr as a single agent induces dose- and time-dependent growth inhibition in acute lymphoblastic leukemia (ALL) cell lines. In addition, the combination of AICAr with antifolates [methotrexate (MTX) or pemetrexed] has been shown to further potentiate AMPK activation and to lead to greater cytotoxicity and growth inhibition in leukemia and other malignant cell types. Our data presented herein show that sustained endoplasmic reticulum (ER) stress is the predominant mechanism behind the synergistic induction of cell death by the combination of AICAr plus the inhibitor of one-carbon metabolism, MTX, in Bp- and T-ALL, as evidenced by induction of several unfolded protein response markers leading to apoptosis. We also show for the first time that AICAr in combination with MTX significantly induces Akt phosphorylation in ALL. Under these conditions, the concomitant inhibition of Akt, a cellular antagonist of AMPK, leads to further upregulation of AMPK activity and alleviates AICAr plus MTX-induced ER stress and apoptosis. Therefore, we also show that the concomitant activation of AMPK actually rescues the cells from AICAr plus MTX-induced ER stress and apoptosis. Our data suggest that the effects of AMPK activation on cell death or survival differ contextually depending on its signaling alterations with related oncogenic pathways and provide insight into the reported paradoxical proapoptotic versus prosurvival effects of AMPK activation.