Small-molecule inhibition of BRDT for male contraception.

A pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception.

The transcription factor BATF operates as an essential differentiation checkpoint in early effector CD8+ T cells.

The transcription factor BATF is required for the differentiation of interleukin 17 (IL-17)-producing helper T cells (TH17 cells) and follicular helper T cells (TFH cells). Here we identified a fundamental role for BATF in regulating the differentiation of effector of CD8(+) T cells. BATF-deficient CD8(+) T cells showed profound defects in effector population expansion and underwent proliferative and metabolic catastrophe early after encountering antigen. BATF, together with the transcription factors IRF4 and Jun proteins, bound to and promoted early expression of genes encoding lineage-specific transcription-factors (T-bet and Blimp-1) and cytokine receptors while paradoxically repressing genes encoding effector molecules (IFN-γ and granzyme B). Thus, BATF amplifies T cell antigen receptor (TCR)-dependent expression of transcription factors and augments the propagation of inflammatory signals but restrains the expression of genes encoding effector molecules. This checkpoint prevents irreversible commitment to an effector fate until a critical threshold of downstream transcriptional activity has been achieved.

BET bromodomain inhibition as a therapeutic strategy to target c-Myc.

MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.

Inhibition of Eicosanoid Degradation Mitigates Fibrosis of the Heart.

BACKGROUND: Organ fibrosis due to excessive production of extracellular matrix by resident fibroblasts is estimated to contribute to >45% of deaths in the Western world, including those due to cardiovascular diseases such as heart failure. Here, we screened for small molecule inhibitors with a common ability to suppress activation of fibroblasts across organ systems. METHODS: High-content imaging of cultured cardiac, pulmonary, and renal fibroblasts was used to identify nontoxic compounds that blocked induction of markers of activation in response to the profibrotic stimulus, transforming growth factor-ß1. SW033291, which inhibits the eicosanoid-degrading enzyme, 15-hydroxyprostaglandin dehydrogenase, was chosen for follow-up studies with cultured adult rat ventricular fibroblasts and human cardiac fibroblasts (CF), and for evaluation in mouse models of cardiac fibrosis and diastolic dysfunction. Additional mechanistic studies were performed with CFs treated with exogenous eicosanoids. RESULTS: Nine compounds, including SW033291, shared a common ability to suppress transforming growth factor-ß1-mediated activation of cardiac, pulmonary, and renal fibroblasts. SW033291 dose-dependently inhibited transforming growth factor-ß1-induced expression of activation markers (eg, α-smooth muscle actin and periostin) in adult rat ventricular fibroblasts and normal human CFs, and reduced contractile capacity of the cells. Remarkably, the 15-hydroxyprostaglandin dehydrogenase inhibitor also reversed constitutive activation of fibroblasts obtained from explanted hearts from patients with heart failure. SW033291 blocked cardiac fibrosis induced by angiotensin II infusion and ameliorated diastolic dysfunction in an alternative model of systemic hypertension driven by combined uninephrectomy and deoxycorticosterone acetate administration. Mechanistically, SW033291-mediated stimulation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase signaling was required for the compound to block CF activation. Of the 12 exogenous eicosanoids that were tested, only 12(S)-hydroxyeicosatetraenoic acid, which signals through the G protein-coupled receptor, GPR31, recapitulated the suppressive effects of SW033291 on CF activation. CONCLUSIONS: Inhibition of degradation of eicosanoids, arachidonic acid-derived fatty acids that signal through G protein-coupled receptors, is a potential therapeutic strategy for suppression of pathological organ fibrosis. In the heart, we propose that 15-hydroxyprostaglandin dehydrogenase inhibition triggers CF-derived autocrine/paracrine signaling by eicosanoids, including 12(S)-hydroxyeicosatetraenoic acid, to stimulate extracellular signal-regulated kinase 1/2 and block conversion of fibroblasts into activated cells that secrete excessive amounts of extracellular matrix and contribute to heart failure pathogenesis.

Substrate stiffness modulates cardiac fibroblast activation, senescence, and proinflammatory secretory phenotype.

In vitro cultures of primary cardiac fibroblasts (CFs), the major extracellular matrix (ECM)-producing cells of the heart, are used to determine molecular mechanisms of cardiac fibrosis. However, the supraphysiologic stiffness of tissue culture polystyrene (TCPS) triggers the conversion of CFs into an activated myofibroblast-like state, and serial passage of the cells results in the induction of replicative senescence. These phenotypic switches confound the interpretation of experimental data obtained with cultured CFs. In an attempt to circumvent TCPS-induced activation and senescence of CFs, we used poly(ethylene glycol) (PEG) hydrogels as cell culture platforms with low and high stiffness formulations to mimic healthy and fibrotic hearts, respectively. Low hydrogel stiffness converted activated CFs into a quiescent state with a reduced abundance of α-smooth muscle actin (α-SMA)-containing stress fibers. Unexpectedly, lower substrate stiffness concomitantly augmented CF senescence, marked by elevated senescence-associated ß-galactosidase (SA-ß-Gal) activity and increased expression of p16 and p21, which are antiproliferative markers of senescence. Using dynamically stiffening hydrogels with phototunable cross-linking capabilities, we demonstrate that premature, substrate-induced CF senescence is partially reversible. RNA-sequencing analysis revealed widespread transcriptional reprogramming of CFs cultured on low-stiffness hydrogels, with a reduction in the expression of profibrotic genes encoding ECM proteins, and an attendant increase in expression of NF-κB-responsive inflammatory genes that typify the senescence-associated secretory phenotype (SASP). Our findings demonstrate that alterations in matrix stiffness profoundly impact CF cell state transitions, and suggest mechanisms by which CFs change phenotype in vivo depending on the stiffness of the myocardial microenvironment in which they reside.NEW & NOTEWORTHY Our findings highlight the advantages and pitfalls associated with culturing cardiac fibroblasts on hydrogels of varying stiffness. The findings also define stiffness-dependent signaling and transcriptional networks in cardiac fibroblasts.

Torsional and strain dysfunction precede overt heart failure in a mouse model of dilated cardiomyopathy pathogenesis.

Detailed assessments of whole heart mechanics are crucial for understanding the consequences of sarcomere perturbations that lead to cardiomyopathy in mice. Echocardiography offers an accessible and cost-effective method of obtaining metrics of cardiac function, but the most routine imaging and analysis protocols might not identify subtle mechanical deficiencies. This study aims to use advanced echocardiography imaging and analysis techniques to identify previously unappreciated mechanical deficiencies in a mouse model of dilated cardiomyopathy (DCM) before the onset of overt systolic heart failure (HF). Mice lacking muscle LIM protein expression (MLP-/-) were used to model DCM-linked HF pathogenesis. Left ventricular (LV) function of MLP-/- and wild-type (WT) controls were studied at 3, 6, and 10 wk of age using conventional and four-dimensional (4-D) echocardiography, followed by speckle-tracking analysis to assess torsional and strain mechanics. Mice were also studied with RNA-seq. Although 3-wk-old MLP-/- mice showed normal LV ejection fraction (LVEF), these mice displayed abnormal torsional and strain mechanics alongside reduced ß-adrenergic reserve. Transcriptome analysis showed that these defects preceded most molecular markers of HF. However, these markers became upregulated as MLP-/- mice aged and developed overt systolic dysfunction. These findings indicate that subtle deficiencies in LV mechanics, undetected by LVEF and conventional molecular markers, may act as pathogenic stimuli in DCM-linked HF. Using these analyses in future studies will further help connect in vitro measurements of the sarcomere function to whole heart function.NEW & NOTEWORTHY A detailed study of how perturbations to sarcomere proteins impact whole heart mechanics in mouse models is a major yet challenging step in furthering our understanding of cardiovascular pathophysiology. This study uses advanced echocardiographic imaging and analysis techniques to reveal previously unappreciated subclinical whole heart mechanical defects in a mouse model of cardiomyopathy. In doing so, it offers an accessible set of measurements for future studies to use when connecting sarcomere and whole heart function.

Detection of early-stage lung cancer in sputum using automated flow cytometry and machine learning.

BACKGROUND: Low-dose spiral computed tomography (LDCT) may not lead to a clear treatment path when small to intermediate-sized lung nodules are identified. We have combined flow cytometry and machine learning to develop a sputum-based test (CyPath Lung) that can assist physicians in decision-making in such cases. METHODS: Single cell suspensions prepared from induced sputum samples collected over three consecutive days were labeled with a viability dye to exclude dead cells, antibodies to distinguish cell types, and a porphyrin to label cancer-associated cells. The labeled cell suspension was run on a flow cytometer and the data collected. An analysis pipeline combining automated flow cytometry data processing with machine learning was developed to distinguish cancer from non-cancer samples from 150 patients at high risk of whom 28 had lung cancer. Flow data and patient features were evaluated to identify predictors of lung cancer. Random training and test sets were chosen to evaluate predictive variables iteratively until a robust model was identified. The final model was tested on a second, independent group of 32 samples, including six samples from patients diagnosed with lung cancer. RESULTS: Automated analysis combined with machine learning resulted in a predictive model that achieved an area under the ROC curve (AUC) of 0.89 (95% CI 0.83-0.89). The sensitivity and specificity were 82% and 88%, respectively, and the negative and positive predictive values 96% and 61%, respectively. Importantly, the test was 92% sensitive and 87% specific in cases when nodules were < 20 mm (AUC of 0.94; 95% CI 0.89-0.99). Testing of the model on an independent second set of samples showed an AUC of 0.85 (95% CI 0.71-0.98) with an 83% sensitivity, 77% specificity, 95% negative predictive value and 45% positive predictive value. The model is robust to differences in sample processing and disease state. CONCLUSION: CyPath Lung correctly classifies samples as cancer or non-cancer with high accuracy, including from participants at different disease stages and with nodules < 20 mm in diameter. This test is intended for use after lung cancer screening to improve early-stage lung cancer diagnosis. Trial registration ClinicalTrials.gov ID: NCT03457415; March 7, 2018.

Hyperglycemia Induces Trained Immunity in Macrophages and Their Precursors and Promotes Atherosclerosis.

BACKGROUND: Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. METHODS: Bone marrow-derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) Ldlr-/- mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. RESULTS: In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow-derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) Ldlr-/- mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow-derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. CONCLUSIONS: Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

GATA6 regulates aging of human mesenchymal stem/stromal cells.

Cellular reprogramming forcing the expression of pluripotency markers can reverse aging of cells, but how molecular mechanisms through which reprogrammed cells alter aging-related cellular activities still remains largely unclear. In this study, we reprogrammed human synovial fluid-derived mesenchymal stem cells (MSCs) into induced pluripotent stem cells (iPSCs) using six reprogramming factors and reverted the iPSCs back to MSCs, as an approach to cell rejuvenation. Using the parental and reprogrammed MSCs as control nonrejuvenated and rejuvenated cells, respectively, for comparative analysis, we found that aging-related activities were greatly reduced in reprogrammed MSCs compared with those in their parental lines, indicating reversal of cell aging. Global transcriptome analysis revealed differences in activities of regulatory networks associated with inflammation and proliferation. Mechanistically, we demonstrated that, compared with control cells, the expression of GATA binding protein 6 (GATA6) in reprogrammed cells was attenuated, resulting in an increase in the activity of sonic hedgehog signaling and the expression level of downstream forkhead box P1 (FOXP1), in turn ameliorating cellular hallmarks of aging. Lower levels of GATA6 expression were also found in cells harvested from younger mice or lower passage cultures. Our findings suggest that GATA6 is a critical regulator increased in aged MSCs that controls the downstream sonic hedgehog signaling and FOXP1 pathway to modulate cellular senescence and aging-related activities.

Condensin II protein dysfunction impacts mitochondrial respiration and mitochondrial oxidative stress responses.

The maintenance of mitochondrial respiratory function and homeostasis is essential to human health. Here, we identify condensin II subunits as novel regulators of mitochondrial respiration and mitochondrial stress responses. Condensin II is present in the nucleus and cytoplasm. While the effects of condensin II depletion on nuclear genome organization are well studied, the effects on essential cytoplasmic and metabolic processes are not as well understood. Excitingly, we observe that condensin II chromosome-associated protein (CAP) subunits individually localize to different regions of mitochondria, suggesting possible mitochondrial-specific functions independent from those mediated by the canonical condensin II holocomplex. Changes in cellular ATP levels and mitochondrial respiration are observed in condensin II CAP subunit-deficient cells. Surprisingly, we find that loss of NCAPD3 also sensitizes cells to oxidative stress. Together, these studies identify new, and possibly independent, roles for condensin II CAP subunits in preventing mitochondrial damage and dysfunction. These findings reveal a new area of condensin protein research that could contribute to the identification of targets to treat diseases where aberrant function of condensin II proteins is implicated.

Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation.

RATIONALE: Small molecule inhibitors of the acetyl-histone binding protein BRD4 have been shown to block cardiac fibrosis in preclinical models of heart failure (HF). However, since the inhibitors target BRD4 ubiquitously, it is unclear whether this chromatin reader protein functions in cell type-specific manner to control pathological myocardial fibrosis. Furthermore, the molecular mechanisms by which BRD4 stimulates the transcriptional program for cardiac fibrosis remain unknown. OBJECTIVE: We sought to test the hypothesis that BRD4 functions in a cell-autonomous and signal-responsive manner to control activation of cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart. METHODS AND RESULTS: RNA-sequencing, mass spectrometry, and cell-based assays employing primary adult rat ventricular fibroblasts demonstrated that BRD4 functions as an effector of TGF-ß (transforming growth factor-ß) signaling to stimulate conversion of quiescent cardiac fibroblasts into Periostin (Postn)-positive cells that express high levels of extracellular matrix. These findings were confirmed in vivo through whole-transcriptome analysis of cardiac fibroblasts from mice subjected to transverse aortic constriction and treated with the small molecule BRD4 inhibitor, JQ1. Chromatin immunoprecipitation-sequencing revealed that BRD4 undergoes stimulus-dependent, genome-wide redistribution in cardiac fibroblasts, becoming enriched on a subset of enhancers and super-enhancers, and leading to RNA polymerase II activation and expression of downstream target genes. Employing the Sertad4 (SERTA domain-containing protein 4) locus as a prototype, we demonstrate that dynamic chromatin targeting of BRD4 is controlled, in part, by p38 MAPK (mitogen-activated protein kinase) and provide evidence of a critical function for Sertad4 in TGF-ß-mediated cardiac fibroblast activation. CONCLUSIONS: These findings define BRD4 as a central regulator of the pro-fibrotic cardiac fibroblast phenotype, establish a p38-dependent signaling circuit for epigenetic reprogramming in heart failure, and uncover a novel role for Sertad4. The work provides a mechanistic foundation for the development of BRD4 inhibitors as targeted anti-fibrotic therapies for the heart.

Mediator kinase inhibition further activates super-enhancer-associated genes in AML.

Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes. BRD4 and CDK7 are positive regulators of SE-mediated transcription. By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo, and disproportionately induces upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the transcription factors CEBPA, IRF8, IRF1 and ETV6 (refs 6-8). The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types, and can be pharmacologically targeted as a therapeutic approach to AML.

Ectopic protein interactions within BRD4-chromatin complexes drive oncogenic megadomain formation in NUT midline carcinoma.

To investigate the mechanism that drives dramatic mistargeting of active chromatin in NUT midline carcinoma (NMC), we have identified protein interactions unique to the BRD4-NUT fusion oncoprotein compared with wild-type BRD4. Using cross-linking, affinity purification, and mass spectrometry, we identified the EP300 acetyltransferase as uniquely associated with BRD4 through the NUT fusion in both NMC and non-NMC cell types. We also discovered ZNF532 associated with BRD4-NUT in NMC patient cells but not detectable in 293T cells. EP300 and ZNF532 are both implicated in feed-forward regulatory loops leading to propagation of the oncogenic chromatin complex in BRD4-NUT patient cells. Adding key functional significance to our biochemical findings, we independently discovered a ZNF532-NUT translocation fusion in a newly diagnosed NMC patient. ChIP sequencing of the major players NUT, ZNF532, BRD4, EP300, and H3K27ac revealed the formation of ZNF532-NUT-associated hyperacetylated megadomains, distinctly localized but otherwise analogous to those found in BRD4-NUT patient cells. Our results support a model in which NMC is dependent on ectopic NUT-mediated interactions between EP300 and components of BRD4 regulatory complexes, leading to a cascade of misregulation.

Drosophila Condensin II subunit Chromosome-associated protein D3 regulates cell fate determination through non-cell-autonomous signaling.

The pattern of the Drosophila melanogaster adult wing is heavily influenced by the expression of proteins that dictate cell fate decisions between intervein and vein during development. dSRF (Blistered) expression in specific regions of the larval wing disc promotes intervein cell fate, whereas EGFR activity promotes vein cell fate. Here, we report that the chromatin-organizing protein CAP-D3 acts to dampen dSRF levels at the anterior/posterior boundary in the larval wing disc, promoting differentiation of cells into the anterior crossvein. CAP-D3 represses KNOT expression in cells immediately adjacent to the anterior/posterior boundary, thus blocking KNOT-mediated repression of EGFR activity and preventing cell death. Maintenance of EGFR activity in these cells depresses dSRF levels in the neighboring anterior crossvein progenitor cells, allowing them to differentiate into vein cells. These findings uncover a novel transcriptional regulatory network influencing Drosophila wing vein development, and are the first to identify a Condensin II subunit as an important regulator of EGFR activity and cell fate determination in vivo.

Glucocorticoids enhance muscle endurance and ameliorate Duchenne muscular dystrophy through a defined metabolic program.

Classic physiology studies dating to the 1930s demonstrate that moderate or transient glucocorticoid (GC) exposure improves muscle performance. The ergogenic properties of GCs are further evidenced by their surreptitious use as doping agents by endurance athletes and poorly understood efficacy in Duchenne muscular dystrophy (DMD), a genetic muscle-wasting disease. A defined molecular basis underlying these performance-enhancing properties of GCs in skeletal muscle remains obscure. Here, we demonstrate that ergogenic effects of GCs are mediated by direct induction of the metabolic transcription factor KLF15, defining a downstream pathway distinct from that resulting in GC-related muscle atrophy. Furthermore, we establish that KLF15 deficiency exacerbates dystrophic severity and muscle GC-KLF15 signaling mediates salutary therapeutic effects in the mdx mouse model of DMD. Thus, although glucocorticoid receptor (GR)-mediated transactivation is often associated with muscle atrophy and other adverse effects of pharmacologic GC administration, our data define a distinct GR-induced gene regulatory pathway that contributes to therapeutic effects of GCs in DMD through proergogenic metabolic programming.

EZH2 synergizes with BRD4-NUT to drive NUT carcinoma growth through silencing of key tumor suppressor genes.

NUT carcinoma (NC) is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of pro-growth genes. BET bromodomain inhibitors (BETi) impede BRD4-NUT's ability to activate genes and are thus a promising treatment but limited as monotherapy. The role of gene repression in NC is unknown. Here, we demonstrate that EZH2, which silences genes through establishment of repressive chromatin, is a dependency in NC. Inhibition of EZH2 with the clinical compound tazemetostat (taz) potently blocked growth of NC cells. Epigenetic and transcriptomic analysis revealed that taz reversed the EZH2-specific H3K27me3 silencing mark, and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4- NUT-regulated genes. CDKN2A was identified as the only gene amongst all taz-derepressed genes to confer resistance to taz in a CRISPR-Cas9 screen. Combined EZH2 inhibition and BET inhibition synergized to downregulate cell proliferation genes resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In pre-clinical models, combined taz and BETi synergistically blocked growth and prolonged survival of NC-xenografted mice, with all mice cured in one cohort. STATEMENT OF SIGNIFICANCE: Identification of EZH2 as a dependency in NC substantiates the reliance of NC tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary chromatin regulatory pathways to maintain NC growth. In particular, repression of CDKN2A expression by EZH2 provides a mechanistic rationale for combining EZH2i with BETi for the clinical treatment of NC.

EZH2 Cooperates with BRD4-NUT to Drive NUT Carcinoma Growth by Silencing Key Tumor Suppressor Genes.

NUT carcinoma is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of progrowth genes. BET bromodomain inhibitors (BETi) are a promising treatment for NUT carcinoma that can impede BRD4-NUT's ability to activate genes, but the efficacy of BETi as monotherapy is limited. Here, we demonstrated that enhancer of zeste homolog 2 (EZH2), which silences genes through establishment of repressive chromatin, is a dependency in NUT carcinoma. Inhibition of EZH2 with the clinical compound tazemetostat potently blocked growth of NUT carcinoma cells. Epigenetic and transcriptomic analysis revealed that tazemetostat reversed the EZH2-specific H3K27me3 silencing mark and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4-NUT-regulated genes. Indeed, H3K27me3 and H3K27ac domains were found to be mutually exclusive in NUT carcinoma cells. CDKN2A was identified as the only gene among all tazemetostat-derepressed genes to confer resistance to tazemetostat in a CRISPR-Cas9 screen. Combined inhibition of EZH2 and BET synergized to downregulate cell proliferation genes, resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In preclinical models, combined tazemetostat and BETi synergistically blocked tumor growth and prolonged survival of NUT carcinoma-xenografted mice, with complete remission without relapse in one cohort. Identification of EZH2 as a dependency in NUT carcinoma substantiates the reliance of NUT carcinoma tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary, chromatin regulatory pathways to maintain NUT carcinoma growth. SIGNIFICANCE: Repression of tumor suppressor genes, including CDKN2A, by EZH2 provides a mechanistic rationale for combining EZH2 and BET inhibitors for the clinical treatment of NUT carcinoma. See related commentary by Kazansky and Kentsis, p. 3827.

Integrative analysis of HIF binding and transactivation reveals its role in maintaining histone methylation homeostasis.

Adaptation to hypoxia is mediated through a coordinated transcriptional response driven largely by hypoxia-inducible factor 1 (HIF-1). We used ChIP-chip and gene expression profiling to identify direct targets of HIF-1 transactivation on a genome-wide scale. Several hundred direct HIF-1 targets were identified and, as expected, were highly enriched for proteins that facilitate metabolic adaptation to hypoxia. Surprisingly, there was also striking enrichment for the family of 2-oxoglutarate dioxygenases, including the jumonji-domain histone demethylases. We demonstrate that these histone demethylases are direct HIF targets, and their up-regulation helps maintain epigenetic homeostasis under hypoxic conditions. These results suggest that the coordinated increase in expression of several oxygen-dependent enzymes by HIF may help compensate for decreased levels of oxygen under conditions of cellular hypoxia.

Discordant Genome Assemblies Drastically Alter the Interpretation of Single-Cell RNA Sequencing Data Which Can Be Mitigated by a Novel Integration Method.

Advances in sequencing and assembly technology have led to the creation of genome assemblies for a wide variety of non-model organisms. The rapid production and proliferation of updated, novel assembly versions can create vexing problems for researchers when multiple-genome assembly versions are available at once, requiring researchers to work with more than one reference genome. Multiple-genome assemblies are especially problematic for researchers studying the genetic makeup of individual cells, as single-cell RNA sequencing (scRNAseq) requires sequenced reads to be mapped and aligned to a single reference genome. Using the Astyanax mexicanus, this study highlights how the interpretation of a single-cell dataset from the same sample changes when aligned to its two different available genome assemblies. We found that the number of cells and expressed genes detected were drastically different when aligning to the different assemblies. When the genome assemblies were used in isolation with their respective annotations, cell-type identification was confounded, as some classic cell-type markers were assembly-specific, whilst other genes showed differential patterns of expression between the two assemblies. To overcome the problems posed by multiple-genome assemblies, we propose that researchers align to each available assembly and then integrate the resultant datasets to produce a final dataset in which all genome alignments can be used simultaneously. We found that this approach increased the accuracy of cell-type identification and maximised the amount of data that could be extracted from our single-cell sample by capturing all possible cells and transcripts. As scRNAseq becomes more widely available, it is imperative that the single-cell community is aware of how genome assembly alignment can alter single-cell data and their interpretation, especially when reviewing studies on non-model organisms.

Monocytes transition to macrophages within the inflamed vasculature via monocyte CCR2 and endothelial TNFR2.

Monocytes undergo phenotypic and functional changes in response to inflammatory cues, but the molecular signals that drive different monocyte states remain largely undefined. We show that monocytes acquire macrophage markers upon glomerulonephritis and may be derived from CCR2+CX3CR1+ double-positive monocytes, which are preferentially recruited, dwell within glomerular capillaries, and acquire proinflammatory characteristics in the nephritic kidney. Mechanistically, the transition to immature macrophages begins within the vasculature and relies on CCR2 in circulating cells and TNFR2 in parenchymal cells, findings that are recapitulated in vitro with monocytes cocultured with TNF-TNFR2-activated endothelial cells generating CCR2 ligands. Single-cell RNA sequencing of cocultures defines a CCR2-dependent monocyte differentiation path associated with the acquisition of immune effector functions and generation of CCR2 ligands. Immature macrophages are detected in the urine of lupus nephritis patients, and their frequency correlates with clinical disease. In conclusion, CCR2-dependent functional specialization of monocytes into macrophages begins within the TNF-TNFR2-activated vasculature and may establish a CCR2-based autocrine, feed-forward loop that amplifies renal inflammation.