BRD9 regulates normal human hematopoietic stem cell function and lineage differentiation.

Bromodomain containing protein 9 (BRD9), a member of the non-canonical BRG1/BRM-associated factor (ncBAF) chromatin remodeling complex, has been implicated as a synthetic lethal target in AML but its function in normal human hematopoiesis is unknown. In hematopoietic stem and progenitor cells (HSPC) genomic or chemical inhibition of BRD9 led to a proliferative disadvantage and loss of stem cells in vitro. Human HSPCs with reduced BRD9 protein levels produced lower numbers of immature mixed multipotent GEMM colonies in semi-solid media. In lineage-promoting culture conditions, cells with reduced BRD9 levels failed to differentiate into the megakaryocytic lineage and showed delayed differentiation into erythroid cells but enhanced terminal myeloid differentiation. HSPCs with BRD9 knock down (KD) had reduced long-term multilineage engraftment in a xenotransplantation assay. An increased number of downregulated genes in RNAseq analysis after BRD9 KD coupled with a gain in chromatin accessibility at the promoters of several repressive transcription factors (TF) suggest that BRD9 functions in the maintenance of active transcription during HSC differentiation. In particular, the hematopoietic master regulator GATA1 was identified as one of the core TFs regulating the gene networks modulated by BRD9 loss in HSPCs. BRD9 inhibition reduced a GATA1-luciferase reporter signal, further suggesting a role for BRD9 in regulating GATA1 activity. BRD9 is therefore an additional example of epigenetic regulation of human hematopoiesis.

Synergy between BRD9- and IKZF3-Targeting as a Therapeutic Strategy for Multiple Myeloma.

Progress in the treatment of multiple myeloma (MM) has resulted in improvement in the survival rate. However, there is still a need for more efficacious and tolerated therapies. We and others have shown that bromodomain-containing protein 9 (BRD9), a member of the non-canonical SWI/SNF chromatin remodeling complex, plays a role in MM cell survival, and targeting BRD9 selectively blocks MM cell proliferation and synergizes with IMiDs. We found that synergy in vitro is associated with the downregulation of MYC and Ikaros proteins, including IKZF3, and overexpression of IKZF3 or MYC could partially reverse synergy. RNA-seq analysis revealed synergy to be associated with the suppression of pathways associated with MYC and E2F target genes and pathways, including cell cycle, cell division, and DNA replication. Stimulated pathways included cell adhesion and immune and inflammatory response. Importantly, combining IMiD treatment and BRD9 targeting, which leads to the downregulation of MYC protein and upregulation of CRBN protein, was able to override IMiD resistance of cells exposed to iberdomide in long-term culture. Taken together, our results support the notion that combination therapy based on agents targeting BRD9 and IKZF3, two established dependencies in MM, represents a promising novel therapeutic strategy for MM and IMiD-resistant disease.

BRD9 degraders as chemosensitizers in acute leukemia and multiple myeloma.

Bromodomain-containing protein 9 (BRD9), an essential component of the SWI/SNF chromatin remodeling complex termed ncBAF, has been established as a therapeutic target in a subset of sarcomas and leukemias. Here, we used novel small molecule inhibitors and degraders along with RNA interference to assess the dependency on BRD9 in the context of diverse hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) model systems. Following depletion of BRD9 protein, AML cells undergo terminal differentiation, whereas apoptosis was more prominent in ALL and MM. RNA-seq analysis of acute leukemia and MM cells revealed both unique and common signaling pathways affected by BRD9 degradation, with common pathways including those associated with regulation of inflammation, cell adhesion, DNA repair and cell cycle progression. Degradation of BRD9 potentiated the effects of several chemotherapeutic agents and targeted therapies against AML, ALL, and MM. Our findings support further development of therapeutic targeting of BRD9, alone or combined with other agents, as a novel strategy for acute leukemias and MM.

PBRM1 Regulates Stress Response in Epithelial Cells.

Polybromo1 (PBRM1) is a chromatin remodeler subunit highly mutated in cancer, particularly clear cell renal carcinoma. PBRM1 is a member of the SWI/SNF subcomplex, PBAF (PBRM1-Brg1/Brm-associated factors), and is characterized by six tandem bromodomains. Here we establish a role for PBRM1 in epithelial cell maintenance through the expression of genes involved in cell adhesion, metabolism, stress response, and apoptosis. In support of a general role for PBRM1 in stress response and apoptosis, we observe that loss of PBRM1 results in an increase in reactive oxygen species generation and a decrease in cellular viability under stress conditions. We find that loss of PBRM1 promotes cell growth under favorable conditions but is required for cell survival under conditions of cellular stress.

LONG-NONCODING RNAs in gastroesophageal cancers.

Despite continuing improvements in multimodal therapies, gastro-esophageal malignances remain widely prevalent in the population and is characterized by poor overall and disease-free survival rates. Due to the lack of understanding about the pathogenesis and absence of reliable markers, gastro-esophageal cancers are associated with delayed diagnosis. The increasing understanding about cancer's molecular landscape in the recent years, offers the possibility of identifying 'targetable' molecular events and in particular facilitates novel treatment strategies and development of biomarkers for early stage diagnosis. At least 98% of our genome is actively transcribed into non-coding RNAs encompassing long non-coding RNAs (lncRNAs) constituted of transcripts longer than 200 nucleotides with no protein-coding capacity. Many studies have demonstrated that lncRNAs are functional genomic elements playing pivotal roles in main oncogenic processes. LncRNA can act at multiple levels developing a complex molecular network that can modulate directly or indirectly the expression of genes involved in tumorigenesis. In this review, we focus on lncRNAs as emerging players in gastro-esophageal carcinogenesis and critically assess their potential as reliable noninvasive biomarkers and in next generation targeted therapies.

Small Molecule Targeting of Specific BAF (mSWI/SNF) Complexes for HIV Latency Reversal.

The persistence of a pool of latently HIV-1-infected cells despite combination anti-retroviral therapy treatment is the major roadblock for a cure. The BAF (mammalian SWI/SNF) chromatin remodeling complex is involved in establishing and maintaining viral latency, making it an attractive drug target for HIV-1 latency reversal. Here we report a high-throughput screen for inhibitors of BAF-mediated transcription in cells and the subsequent identification of a 12-membered macrolactam. This compound binds ARID1A-specific BAF complexes, prevents nucleosomal positioning, and relieves transcriptional repression of HIV-1. Through this mechanism, these compounds are able to reverse HIV-1 latency in an in vitro T cell line, an ex vivo primary cell model of HIV-1 latency, and in patient CD4+ T cells without toxicity or T cell activation. These macrolactams represent a class of latency reversal agents with unique mechanism of action, and can be combined with other latency reversal agents to improve reservoir targeting.

Technical advances in global DNA methylation analysis in human cancers.

Prototypical abnormalities of genome-wide DNA methylation constitute the most widely investigated epigenetic mechanism in human cancers. Errors in the cellular machinery to faithfully replicate the global 5-methylcytosine (5mC) patterns, commonly observed during tumorigenesis, give rise to misregulated biological pathways beneficial to the rapidly propagating tumor mass but deleterious to the healthy tissues of the affected individual. A growing body of evidence suggests that the global DNA methylation levels could serve as utilitarian biomarkers in certain cancer types. Important breakthroughs in the recent years have uncovered further oxidized derivatives of 5mC - 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), thereby expanding our understanding of the DNA methylation dynamics. While the biological roles of these epigenetic derivatives are being extensively characterized, this review presents a perspective on the opportunity of innovation in the global methylation analysis platforms. While multiple methods for global analysis of 5mC in clinical samples exist and have been reviewed elsewhere, two of the established methods - Liquid Chromatography coupled with mass spectrometry (LC-MS/MS) and Immunoquantification have successfully evolved to include the quantitation of 5hmC, 5fC and 5caC. Although the analytical performance of LC-MS/MS is superior, the simplicity afforded by the experimental procedure of immunoquantitation ensures it's near ubiquity in clinical applications. Recent developments in spectroscopy, nanotechnology and sequencing also provide immense promise for future evaluations and are discussed briefly. Finally, we provide a perspective on the current scenario of global DNA methylation analysis tools and present suggestions to develop the next generation toolset.

PBRM1 Regulates the Expression of Genes Involved in Metabolism and Cell Adhesion in Renal Clear Cell Carcinoma.

Polybromo-1 (PBRM1) is a component of the PBAF (Polybromo-associated-BRG1- or BRM-associated factors) chromatin remodeling complex and is the second most frequently mutated gene in clear-cell renal cell Carcinoma (ccRCC). Mutation of PBRM1 is believed to be an early event in carcinogenesis, however its function as a tumor suppressor is not understood. In this study, we have employed Next Generation Sequencing to profile the differentially expressed genes upon PBRM1 re-expression in a cellular model of ccRCC. PBRM1 re-expression led to upregulation of genes involved in cellular adhesion, carbohydrate metabolism, apoptotic process and response to hypoxia, and a downregulation of genes involved in different stages of cell division. The decrease in cellular proliferation upon PBRM1 re-expression was confirmed, validating the functional role of PBRM1 as a tumor suppressor in a cell-based model. In addition, we identified a role for PBRM1 in regulating metabolic pathways known to be important for driving ccRCC, including the regulation of hypoxia response genes, PI3K signaling, glucose uptake, and cholesterol homeostasis. Of particular novelty is the identification of cell adhesion as a major downstream process uniquely regulated by PBRM1 expression. Cytoskeletal reorganization was induced upon PBRM1 reexpression as evidenced from the increase in the number of cells displaying cortical actin, a hallmark of epithelial cells. Genes involved in cell adhesion featured prominently in our transcriptional dataset and overlapped with genes uniquely regulated by PBRM1 in clinical specimens of ccRCC. Genes involved in cell adhesion serve as tumor suppressor and maybe involved in inhibiting cell migration. Here we report for the first time genes linked to cell adhesion serve as downstream targets of PBRM1, and hope to lay the foundation of future studies focusing on the role of chromatin remodelers in bringing about these alterations during malignancies.

A Study of Alterations in DNA Epigenetic Modifications (5mC and 5hmC) and Gene Expression Influenced by Simulated Microgravity in Human Lymphoblastoid Cells.

Cells alter their gene expression in response to exposure to various environmental changes. Epigenetic mechanisms such as DNA methylation are believed to regulate the alterations in gene expression patterns. In vitro and in vivo studies have documented changes in cellular proliferation, cytoskeletal remodeling, signal transduction, bone mineralization and immune deficiency under the influence of microgravity conditions experienced in space. However microgravity induced changes in the epigenome have not been well characterized. In this study we have used Next-generation Sequencing (NGS) to profile ground-based "simulated" microgravity induced changes on DNA methylation (5-methylcytosine or 5mC), hydroxymethylation (5-hydroxymethylcytosine or 5hmC), and simultaneous gene expression in cultured human lymphoblastoid cells. Our results indicate that simulated microgravity induced alterations in the methylome (~60% of the differentially methylated regions or DMRs are hypomethylated and ~92% of the differentially hydroxymethylated regions or DHMRs are hyperhydroxymethylated). Simulated microgravity also induced differential expression in 370 transcripts that were associated with crucial biological processes such as oxidative stress response, carbohydrate metabolism and regulation of transcription. While we were not able to obtain any global trend correlating the changes of methylation/ hydroxylation with gene expression, we have been able to profile the simulated microgravity induced changes of 5mC over some of the differentially expressed genes that includes five genes undergoing differential methylation over their promoters and twenty five genes undergoing differential methylation over their gene-bodies. To the best of our knowledge, this is the first NGS-based study to profile epigenomic patterns induced by short time exposure of simulated microgravity and we believe that our findings can be a valuable resource for future explorations.

The hypomethylating agent Decitabine causes a paradoxical increase in 5-hydroxymethylcytosine in human leukemia cells.

The USFDA approved "epigenetic drug", Decitabine, exerts its effect by hypomethylating DNA, demonstrating the pivotal role aberrant genome-wide DNA methylation patterns play in cancer ontology. Using sensitive technologies in a cellular model of Acute Myeloid Leukemia, we demonstrate that while Decitabine reduces the global levels of 5-methylcytosine (5mC), it results in paradoxical increase of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) levels. Hitherto, the only biological mechanism known to generate 5hmC, 5fC and 5caC, involving oxidation of 5mC by members of Ten-Eleven-Translocation (TET) dioxygenase family, was not observed to undergo any alteration during DAC treatment. Using a multi-compartmental model of DNA methylation, we show that partial selectivity of TET enzymes for hemi-methylated CpG dinucleotides could lead to such alterations in 5hmC content. Furthermore, we investigated the binding of TET1-catalytic domain (CD)-GFP to DNA by Fluorescent Correlation Spectroscopy in live cells and detected the gradual increase of the DNA bound fraction of TET1-CD-GFP after treatment with Decitabine. Our study provides novel insights on the therapeutic activity of DAC in the backdrop of the newly discovered derivatives of 5mC and suggests that 5hmC has the potential to serve as a biomarker for monitoring the clinical success of patients receiving DAC.

Quantification of 5-methylcytosine, 5-hydroxymethylcytosine and 5-carboxylcytosine from the blood of cancer patients by an enzyme-based immunoassay.

BACKGROUND: Genome-wide aberrations of the classic epigenetic modification 5-methylcytosine (5mC), considered the hallmark of gene silencing, has been implicated to play a pivotal role in mediating carcinogenic transformation of healthy cells. Recently, three epigenetic marks derived from enzymatic oxidization of 5mC namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), have been discovered in the mammalian genome. Growing evidence suggests that these novel bases possess unique regulatory functions and may play critical roles in carcinogenesis. METHODS: To provide a quantitative basis for these rare epigenetic marks, we have designed a biotin-avidin mediated enzyme-based immunoassay (EIA) and evaluated its performance in genomic DNA isolated from blood of patients diagnosed with metastatic forms of lung, pancreatic and bladder cancer, as well as healthy controls. The proposed EIA incorporates spatially optimized biotinylated antibody and a high degree of horseradish-peroxidase (HRP) labeled streptavidin, facilitating signal amplification and sensitive detection. RESULTS: We report that the percentages of 5mC, 5hmC and 5caC present in the genomic DNA of blood in healthy controls as 1.025±0.081, 0.023±0.006 and 0.001±0.0002, respectively. We observed a significant (p<0.05) decrease in the mean global percentage of 5hmC in blood of patients with malignant lung cancer (0.013±0.003%) in comparison to healthy controls. CONCLUSION: The precise biological roles of these epigenetic modifications in cancers are still unknown but in the past two years it has become evident that the global 5hmC content is drastically reduced in a variety of cancers. To the best of our knowledge, this is the first report of decreased 5hmC content in the blood of metastatic lung cancer patients and the clinical utility of this observation needs to be further validated in larger sample datasets.

Real-time dynamics of methyl-CpG-binding domain protein 3 and its role in DNA demethylation by fluorescence correlation spectroscopy.

With unprecedented development in technology, epigenetics is recognized as a substantial and flexible regulatory pathway for phenotyping. Cytosine methylation and its subsequent oxidization have attracted significant attention due to their direct impact on gene regulation, in association with methyl-CpG-binding domain proteins (MBDs) and transcription related factors. In this study we record the dynamics of DNA demethylation using the recombinant MBD3-GFP protein in living cells under hypoxia and Decitabine treatment using Fluorescence Correlation Spectroscopy (FCS) by monitoring the diffusion dynamics of MBD3. Our study shows a DNA-replication-independent decrease of 5-methylcytosine (5mC)/5-hydroxymethylcytosine (5hmC) under hypoxia vs. a dependent decrease under Decitabine treatment. Further, we define a significantly faster diffusion of MBD3 in the nucleus as a precursory event for active demethylation rather than the Decitabine induced passive demethylation. By monitoring the diffusion of bound and unbound MBD3 in the nucleus we were able to identify and characterize hypoxia-sensitive cells from insensitive/tolerant cells, as well as the respective contribution to active demethylation in a time-dependent manner. Last, we quantitatively describe the concurrent decreasing trend in all of the three oxidized products of 5mC, which points to the potential involvement of ten-eleven-translocation proteins (TETs) in hypoxia induced active demethylation. Overall, for the first time we correlate the dynamic process of DNA demethylation with the biophysical properties of the corresponding DNA binding proteins in live single cells by single molecule spectroscopy.

Probing site-exclusive binding of aqueous QDs and their organelle-dependent dynamics in live cells by single molecule spectroscopy.

Understanding the biophysical and chemical interactions of nanoprobes and their fate upon entering live cells is critical for developing fundamental insights related to intracellular diagnostics, drug delivery and targeting. In this article we report herein a single molecule analysis procedure to quantitate site-specific exclusive membrane binding of N-acetyl-L-cysteine (NAC)-capped cadmium telluride (CdTe) quantum dots (QDs) in A-427 lung carcinoma cells (k(eq) = 0.075 ± 0.011 nM(-1)), its relative intracellular distribution and dynamics using fluorescence correlation spectroscopy (FCS) combined with scanning confocal fluorescence lifetime imaging (FLIM). In particular, we demonstrate that the binding efficacy of QDs to the cell membrane is directly related to their size and the targeting of QDs to specific membrane sites is exclusive. We also show that QDs are efficiently internalized by endocytosis and enclosed within the endosome and organelle-dependent diffusion dynamics can be monitored in live cells.

Antimicrobial sensitivity of Escherichia coli to alumina nanoparticles.

Metal oxide nanoparticles (NPs) are known to possess strong antimicrobial properties. Aluminum oxide NPs have wide-range applications in industrial as well as personal care products. In the absence of prior reports on the antimicrobial properties of alumina NPs for a wide concentration range, the principal objective of the present work was to study the growth-inhibitory effect of alumina NPs over a wide concentration range (10-1000 microg/mL) on an environmentally relevant gram-negative model microorganism, Escherichia coli. The mean diameter of the NPs was determined to be 179 nm in aqueous dispersion used for this study, and surface area was determined to be 21.23 m(2)/g. The concentration of 1000 microg/mL was found to be moderately inhibitory for bacteria. Almost negligible dependence of growth rate on the concentration of the NPs was observed. The extracellular protein content was found to be slightly lower in case of cells interacting with 1000 microg/mL alumina than the uninteracted control cells. Fourier transform-infrared studies established differences in structure between interacted and uninteracted cells. Alumina NPs showed a mild growth-inhibitory effect, only at very high concentrations, which might be due to surface charge interactions between the particles and cells. Free-radical scavenging properties of the particles might have prevented cell wall disruption and drastic antimicrobial action. This laboratory-scale study suggests that alumina NPs may only exhibit mild toxicity toward microorganisms in the environment. FROM THE CLINICAL EDITOR: Metal oxide NPs, inluding aluminum oxide NPs, are known to possess strong antimicrobial properties. The study demonstrated a mild to moderate growth-inhibitory effect of alumina NPs over a wide concentration range (10-1000 microg/mL) on Escherichia coli. Almost negligible dependence on the concentration was observed.