BAP1 deficiency causes loss of melanocytic cell identity in uveal melanoma

BACKGROUND: Uveal melanoma is a highly aggressive cancer with a strong propensity for metastasis, yet little is known about the biological mechanisms underlying this metastatic potential. We recently showed that most metastasizing uveal melanomas, which exhibit a class 2 gene expression profile, contain inactivating mutations in the tumor suppressor BAP1. The aim of this study was to investigate the role of BAP1 in uveal melanoma progression. METHODS: Uveal melanoma cells were studied following RNAi-mediated depletion of BAP1 using proliferation, BrdU incorporation, flow cytometry, migration, invasion, differentiation and clonogenic assays, as well as in vivo tumorigenicity experiments in NOD-SCID-Gamma mice. RESULTS: Depletion of BAP1 in uveal melanoma cells resulted in a loss of differentiation and gain of stem-like properties, including expression of stem cell markers, increased capacity for self-replication, and enhanced ability to grow in stem cell conditions. BAP1 depletion did not result in increased proliferation, migration, invasion or tumorigenicity. CONCLUSIONS: BAP1 appears to function in the uveal melanocyte lineage primarily as a regulator of differentiation, with cells deficient for BAP1 exhibiting stem-like qualities. It will be important to elucidate how this effect of BAP1 loss promotes metastasis and how to reverse this effect therapeutically

Chronic Infection Depletes Hematopoietic Stem Cells through Stress-Induced Terminal Differentiation

Chronic infections affect a third of the world’s population and can cause bone marrow suppression, a severe condition that increases mortality from infection. To uncover the basis for infection-associated bone marrow suppression, we conducted repeated infection of WT mice with Mycobacterium avium. After 4–6 months, mice became pancytopenic. Their hematopoietic stem and progenitor cells (HSPCs) were severely depleted and displayed interferon gamma (IFN-γ) signaling-dependent defects in self-renewal. There was no evidence of increased HSPC mobilization or apoptosis. However, consistent with known effects of IFN-γ, transcriptome analysis pointed toward increased myeloid differentiation of HSPCs and revealed the transcription factor Batf2 as a potential mediator of IFN-γ-induced HSPC differentiation. Gain- and loss-of-function studies uncovered a role for Batf2 in myeloid differentiation in both murine and human systems. We thus demonstrate that chronic infection can deplete HSPCs and identify BATF2 as a mediator of infection-induced HSPC terminal differentiation

The Role of Melanocyte Differentiation in Uveal Melanoma Progression

Uveal melanoma is a rare, but extremely deadly disease. Gaining a better understanding of disease progression has huge implications for the treatment of metastatic disease. The relatively non-aggressive class 1 tumors, which rarely metastasize, closely resemble a normal, differentiated melanocyte, whereas the aggressive class 2 tumors, which almost always metastasize, are much less differentiated and more stem-like in nature. This clear distinction led us to further investigate the role that differentiation is playing in uveal melanoma disease progression. The retinoblastoma protein (Rb) is functionally inactivated in almost all uveal melanomas and upon investigation of Rb\u27s potential role in the differentiation of melanocytes, we found that Rb physically interacts with the master regulator of melanocyte differentiation, MITF, and that it is able to cooperate with MITF to induce expression of melanocyte differentiation genes. Our evidence suggests that this role of Rb is dependent on it being in its active, hypophosphorylated form, which implies that the inactivated form of Rb that is found in uveal melanoma maybe unable to cooperate with MITF to induce differentiation. In addition to this potential role for Rb in uveal melanoma progression, we also identified a metastasis suppressor gene, BAP1 that is mutated in the overwhelming majority of the aggressive class 2 tumors. We found that loss of BAP1 within the melanocytic lineage does not affect traditional measures of tumorigenicity, but rather it induces a dedifferentiated, stem-like state. In addition we found that BAP1 loss led to significantly increased levels of ubiquitinated H2A. This function of BAP1 is in direct opposition to the PRC1 complex and when BAP1-deficient cells were treated with an HDAC-inhibitor, one of the targets of which is PRC1, the levels of ubiquitinated H2A were returned to normal levels. Interestingly treatment of BAP1-deficient cells with an HDAC-inhibitor restored the expression of melanocyte differentiation genes to normal levels, and shifted tumor cells towards a more differentiated phenotype. These data suggest that loss of BAP1 is contributing to a loss of melanocytic cell identity that may contribute to melanoma disease progression and points to the use of HDAC-inhibitors as a potential treatment for metastatic disease

p38 phosphorylates Rb on Ser567 by a novel, cell cycle-independent mechanism that triggers Rb-Hdm2 interaction and apoptosis

The retinoblastoma protein (Rb) inhibits both cell division and apoptosis, but the mechanism by which Rb alternatively regulates these divergent outcomes remains poorly understood. Cyclin dependent kinases (Cdks) promote cell division by phosphorylating and reversibly inactivating Rb by a hierarchical series of phosphorylation events and sequential conformational changes. The stress-regulated mitogen activated protein kinase (MAPK) p38 also phosphorylates Rb, but it does so in a cell cycle-independent manner that is associated with apoptosis rather than with cell division. Here, we show that p38 phosphorylates Rb by a novel mechanism that is distinct from that of Cdks. p38 bypasses the cell cycle-associated hierarchical phosphorylation and directly phosphorylates Rb on Ser567, which is not phosphorylated during the normal cell cycle. Phosphorylation by p38, but not Cdks, triggers an interaction between Rb and the human homologue of murine double minute 2 (Hdm2), leading to degradation of Rb, release of E2F1 and cell death. These findings provide a mechanistic explanation for how Rb regulates cell division and apoptosis through different kinases, and reveal how Hdm2 may functionally link the tumor suppressors Rb and p53

Histone deacetylase inhibitors induce growth arrest and differentiation in uveal melanoma

PURPOSE: Metastasis is responsible for the death of most cancer patients, yet few therapeutic agents are available which specifically target the molecular events that lead to metastasis. We recently showed that inactivating mutations in the tumor suppressor gene BAP1 are closely associated with loss of melanocytic differentiation in uveal melanoma and metastasis (UM). The purpose of this study was to identify therapeutic agents that reverse the phenotypic effects of BAP1 loss in UM. EXPERIMENTAL DESIGN: In silico screens were performed to identify therapeutic compounds predicted to differentiate UM cells using Gene Set Enrichment Analysis and Connectivity Map databases. Valproic acid, trichostatin A, LBH-589 and suberoylanilide hydroxamic acid were evaluated for their effects on UM cells using morphologic evaluation, MTS viability assays, BrdU incorporation, flow cytometry, clonogenic assays, gene expression profiling, histone acetylation and ubiquitination assays, and a murine xenograft tumorigenicity model. RESULTS: HDAC inhibitors induced morphologic differentiation, cell cycle exit, and a shift to a differentiated, melanocytic gene expression profile in cultured UM cells. Valproic acid inhibited the growth of UM tumors in vivo. CONCLUSIONS: These findings suggest that HDAC inhibitors may have therapeutic potential for inducing differentiation and prolonged dormancy of micrometastatic disease in UM

Histone Deacetylase Inhibitors Induce Growth Arrest and Differentiation in Uveal Melanoma

PURPOSE: Metastasis is responsible for the death of most cancer patients, yet few therapeutic agents are available which specifically target the molecular events that lead to metastasis. We recently showed that inactivating mutations in the tumor suppressor gene BAP1 are closely associated with loss of melanocytic differentiation in uveal melanoma and metastasis (UM). The purpose of this study was to identify therapeutic agents that reverse the phenotypic effects of BAP1 loss in UM. EXPERIMENTAL DESIGN: In silico screens were performed to identify therapeutic compounds predicted to differentiate UM cells using Gene Set Enrichment Analysis and Connectivity Map databases. Valproic acid, trichostatin A, LBH-589 and suberoylanilide hydroxamic acid were evaluated for their effects on UM cells using morphologic evaluation, MTS viability assays, BrdU incorporation, flow cytometry, clonogenic assays, gene expression profiling, histone acetylation and ubiquitination assays, and a murine xenograft tumorigenicity model. RESULTS: HDAC inhibitors induced morphologic differentiation, cell cycle exit, and a shift to a differentiated, melanocytic gene expression profile in cultured UM cells. Valproic acid inhibited the growth of UM tumors in vivo. CONCLUSIONS: These findings suggest that HDAC inhibitors may have therapeutic potential for inducing differentiation and prolonged dormancy of micrometastatic disease in UM

Frequent Mutation of BAP1 in Metastasizing Uveal Melanomas

Metastasis is a defining feature of malignant tumors and is the most common cause of cancer-related death, yet the genetics of metastasis are poorly understood. We used massively parallel exome sequencing coupled with Sanger re-sequencing to search for metastasis-related mutations in highly metastatic uveal melanomas of the eye. Inactivating somatic mutations were identified in the gene encoding BRCA1-associated protein 1 ( BAP1 ) on chromosome 3p21.1 in 26 of 31 (84%) metastasizing tumors, including 15 mutations causing premature protein termination, and six affecting its ubiquitin carboxy-terminal hydrolase (UCH) domains. One tumor harbored a frameshift mutation that was germline in origin, thus representing a susceptibility allele. These findings implicate loss of BAP1 in uveal melanoma metastasis and suggest the BAP1 pathway as a therapeutic target