MITF links differentiation with cell cycle arrest in melanocytes by transcriptional activation of INK4A

Cell cycle exit is required for proper differentiation in most cells and is critical for normal development, tissue homeostasis, and tumor suppression. However, the mechanisms that link cell cycle exit with differentiation remain poorly understood. Here, we show that the master melanocyte differentiation factor, microphthalmia transcription factor (MITF), regulates cell cycle exit by activating the cell cycle inhibitor INK4A, a tumor suppressor that frequently is mutated in melanomas. MITF binds the INK4A promoter, activates p16Ink4a mRNA and protein expression, and induces retinoblastoma protein hypophosphorylation, thereby triggering cell cycle arrest. This activation of INK4A was required for efficient melanocyte differentiation. Interestingly, MITF was also required for maintaining INK4A expression in mature melanocytes, creating a selective pressure to escape growth inhibition by inactivating INK4A. These findings demonstrate that INK4A can be regulated by a differentiation factor, establish a mechanistic link between melanocyte differentiation and cell cycle exit, and potentially explain the tissue-specific tendency for INK4A mutations to occur in melanoma

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

Retinoblastoma protein prevents enteric nervous system defects and intestinal pseudo-obstruction

The retinoblastoma 1 (RB1) tumor suppressor is a critical regulator of cell cycle progression and development. To investigate the role of RB1 in neural crest–derived melanocytes, we bred mice with a floxed Rb1 allele with mice expressing Cre from the tyrosinase (Tyr) promoter. TyrCre(+);Rb1(fl/fl) mice exhibited no melanocyte defects but died unexpectedly early with intestinal obstruction, striking defects in the enteric nervous system (ENS), and abnormal intestinal motility. Cre-induced DNA recombination occurred in all enteric glia and most small bowel myenteric neurons, yet phenotypic effects of Rb1 loss were cell-type specific. Enteric glia were twice as abundant in mutant mice compared with those in control animals, while myenteric neuron number was normal. Most myenteric neurons also appeared normal in size, but NO-producing myenteric neurons developed very large nuclei as a result of DNA replication without cell division (i.e., endoreplication). Parallel studies in vitro found that exogenous NO and Rb1 shRNA increased ENS precursor DNA replication and nuclear size. The large, irregularly shaped nuclei in NO-producing neurons were remarkably similar to those in progeria, an early-onset aging disorder that has been linked to RB1 dysfunction. These findings reveal a role for RB1 in the ENS

Multimodal imaging of sarcoid choroidal granulomas

BACKGROUND: Enhanced-depth imaging optical coherence tomography (EDI-OCT) provides high-resolution imaging of the choroid. Herein, we report multimodal imaging, including EDI-OCT, of a case of sarcoid choroidal granulomas. FINDINGS: A 63-year-old female with biopsy-supported sarcoidosis presented with unilateral multifocal choroidal granulomas. Enhanced-depth imaging optical coherence tomography (EDI-OCT) demonstrated a homogenous hyporeflective choroidal lesion with choriocapillaris thinning and sparing of the surrounding choroid. The patient was started on oral steroids with a weekly taper schedule. Within 5 weeks, the choroidal lesions had clinically resolved with return of normal-appearing choroidal architecture on EDI-OCT. Indocyanine green angiography, however, demonstrated hypofluoresence at the sites of choroidal granulomas 11 months after the clinical resolution, suggesting a longstanding choroidal perfusion deficit undetected by OCT. CONCLUSIONS: Choroidal imaging via EDI-OCT provides detailed morphologic information of sarcoid granulomas and can accurately demonstrate structural resolution of the lesions

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.Fil: Kuznetsov, Jeffim N.. University of Miami; Estados UnidosFil: Agüero, Tristán Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. University of Miami; Estados UnidosFil: Owens, Dawn A.. University of Miami; Estados UnidosFil: Kurtenbach, Stefan. University of Miami; Estados UnidosFil: Field, Matthew G.. University of Miami; Estados UnidosFil: Durante, Michael A.. University of Miami; Estados UnidosFil: Rodriguez, Daniel A.. University of Miami; Estados UnidosFil: King, Mary Lou. University of Miami; Estados UnidosFil: Harbour, J. William. University of Miami; Estados Unido

Oncogenic mutations in GNAQ occur early in uveal melanoma.

PURPOSE: Early/initiating oncogenic mutations have been identified for many cancers, but such mutations remain unidentified in uveal melanoma (UM). An extensive search for such mutations was undertaken, focusing on the RAF/MEK/ERK pathway, which is often the target of initiating mutations in other types of cancer. METHODS: DNA samples from primary UMs were analyzed for mutations in 24 potential oncogenes that affect the RAF/MEK/ERK pathway. For GNAQ, a stimulatory α(q) G-protein subunit which was recently found to be mutated in uveal melanomas, re-sequencing was expanded to include 67 primary UMs and 22 peripheral blood samples. GNAQ status was analyzed for association with clinical, pathologic, chromosomal, immunohistochemical and transcriptional features. RESULTS: Activating mutations at codon 209 were identified in GNAQ in 33/67 (49%) primary UMs, including 2/9 (22%) iris melanomas and 31/58 (54%) posterior UMs. No mutations were found in the other 23 potential oncogenes. GNAQ mutations were not found in normal blood DNA samples. Consistent with GNAQ mutation being an early or initiating event, this mutation was not associated with any clinical, pathologic or molecular features associated with late tumor progression. CONCLUSIONS: GNAQ mutations occur in about half of UMs, representing the most common known oncogenic mutation in this cancer. The presence of this mutation in tumors at all stages of malignant progression suggests that it is an early event in UM. Mutations in this G-protein provide new insights into UM pathogenesis and could lead to new therapeutic possibilities

ASXL1 interacts with the cohesin complex to maintain chromatid separation and gene expression for normal hematopoiesis

ASXL1 is frequently mutated in a spectrum of myeloid malignancies with poor prognosis. Loss of Asxl1 leads to myelodysplastic syndrome-like disease in mice; however, the underlying molecular mechanisms remain unclear. We report that ASXL1 interacts with the cohesin complex, which has been shown to guide sister chromatid segregation and regulate gene expression. Loss of Asxl1 impairs the cohesin function, as reflected by an impaired telophase chromatid disjunction in hematopoietic cells. Chromatin immunoprecipitation followed by DNA sequencing data revealed that ASXL1, RAD21, and SMC1A share 93% of genomic binding sites at promoter regions in Lin-cKit+ (LK) cells. We have shown that loss of Asxl1 reduces the genome binding of RAD21 and SMC1A and alters the expression of ASXL1/cohesin target genes in LK cells. Our study underscores the ASXL1-cohesin interaction as a novel means to maintain normal sister chromatid separation and regulate gene expression in hematopoietic cells

Co-Targeting FASN and mTOR Suppresses Uveal Melanoma Growth

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

Clinical evidence framework for Bayesian networks

There is poor uptake of prognostic decision support models by clinicians regardless of their accuracy. There is evidence that this results from doubts about the basis of the model as the evidence behind clinical models is often not clear to anyone other than their developers. In this paper, we propose a framework for representing the evidence-base of a Bayesian network (BN) decision support model. The aim of this evidence framework is to be able to present all the clinical evidence alongside the BN itself. The evidence framework is capable of presenting supporting and conflicting evidence, and evidence associated with relevant but excluded factors. It also allows the completeness of the evidence to be queried. We illustrate this framework using a BN that has been previously developed to predict acute traumatic coagulopathy, a potentially fatal disorder of blood clotting, at early stages of trauma care