Summary Circadian clocks are encoded by a transcription-translation feedback loop that aligns energetic processes with the solar cycle. Here we show that genetic disruption of the clock activator BMAL1 in skeletal myotubes and fibroblasts increased levels of the hypoxia-inducible factor 1α (HIF1α) under hypoxic conditions. Bmal1−/− myotubes displayed reduced anaerobic glycolysis, mitochondrial respiration with glycolytic fuel, and transcription of HIF1α targets Phd3, Vegfa, Mct4, Pk-m, and Ldha, whereas abrogation of the clock repressors CRY1/2 stabilized HIF1α in response to hypoxia. HIF1α bound directly to core clock gene promoters and, when co-expressed with BMAL1, led to transactivation of PER2-LUC and HRE-LUC reporters. Further, genetic stabilization of HIF1α in Vhl−/− cells altered circadian transcription. Finally, induction of clock- and HIF1α-target genes in response to strenuous exercise varied according to the time-of-day in wild-type mice. Collectively, our results reveal bi-directional interactions between circadian and HIF pathways that influence metabolic adaptation to hypoxia.

Purpose Identification of predictive biomarkers is critically needed to improve selection of patients who derive the most benefit from platinum-based chemotherapy. We hypothesized that decreased expression of SMARCA4/BRG1, a known regulator of transcription and DNA repair, is a novel predictive biomarker of increased sensitivity to adjuvant platinum-based therapies in NSCLC. Experimental Design The prognostic value was tested using a gene expression microarray from the Director’s Challenge Lung Study (n=440). The predictive significance of SMARCA4 was determined using a gene expression microarray (n=133) from control and treatment arms of the JBR.10 trial of adjuvant cisplatin/vinorelbine. Kaplan-Meier method and log-rank tests were used to estimate and test the differences of probabilities in overall survival (OS) and disease-specific survival (DSS) between expression groups and treatment arms. Multivariate Cox regression models were used while adjusting for other clinical covariates. Results In the Director’s Challenge Study, reduced expression of SMARCA4 was associated with poor OS compared to high and intermediate expression (P<0.001 and P=0.009, respectively). In multivariate analysis, compared to low, high SMARCA4 expression predicted a decrease in risk of death (HR=0.6, 95% CI: 0.4–0.8, P=0.002). In the JBR.10 trial, improved five-year DSS was noted only in patients with low SMARCA4 expression when treated with adjuvant cisplatin/vinorelbine (HR=0.1, 95% CI: 0.0–0.5, P=0.002 [low]; HR 1.0, 95% CI: 0.5–2.3, P=0.92 [high]). An interaction test was highly significant (P=0.01). Conclusions Low expression of SMARCA4/BRG1 is significantly associated with worse prognosis; however, it is a novel significant predictive biomarker for increased sensitivity to platinum-based chemotherapy in NSCLC.

Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy caused by a variety of mutations, including genes encoding the cohesin complex. Recent studies have demonstrated that reduction in cohesin complex levels leads to enhanced self-renewal in hematopoietic stem and progenitors (HSPCs). We sought to delineate the molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal since this represents a critical initial step during leukemic transformation. We verified that RNAi against the cohesin subunit Rad21 causes enhanced self-renewal of HSPCs in vitro through derepression of Polycomb Repressive Complex 2 (PRC2) target genes, including Hoxa7 and Hoxa9. Importantly, knockdown of either Hoxa7 or Hoxa9 suppressed self-renewal, implying both are critical downstream effectors of reduced cohesin levels. We further demonstrate that the cohesin and PRC2 complexes interact and are bound in close proximity to Hoxa7 and Hoxa9. Rad21 depletion resulted in decreased levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters, consistent with Rad21 being critical to proper gene silencing by recruiting the PRC2 complex. Our data demonstrates that the cohesin complex regulates PRC2 targeting to silence Hoxa7 and Hoxa9 and negatively regulate self-renewal. Our studies identify a novel epigenetic mechanism underlying leukemogenesis in AML patients with cohesin mutations.

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Key Points miR-486-5p, a GATA1 regulated miR, is expressed in ML-DS and enhances their aberrant erythroid phenotype. miR-486-5p cooperates with GATA1s to promote the survival of pre-leukemic and leukemic cells.

GATA1 is a master transcriptional regulator of the differentiation of several related myeloid blood cell types, including erythrocytes and megakaryocytes. Germ-line mutations that cause loss of full length GATA1, but allow for expression of the short isoform (GATA1s), are associated with defective erythropoiesis in a subset of patients with Diamond Blackfan Anemia. Despite extensive studies of GATA1s in megakaryopoiesis, the mechanism by which GATA1s fails to support normal erythropoiesis is not understood. In this study, we used global gene expression and chromatin occupancy analysis to compare the transcriptional activity of GATA1s to GATA1. We discovered that compared to GATA1, GATA1s is less able to activate the erythroid gene expression program and terminal differentiation in cells with dual erythroid-megakaryocytic differentiation potential. Moreover, we found that GATA1s bound to many of its erythroid-specific target genes less efficiently than full length GATA1. These results suggest that the impaired ability of GATA1s to promote erythropoiesis in DBA may be caused by failure to occupy erythroid-specific gene regulatory elements. Global transcriptome and chromatin occupancy analysis reveal the short isoform of GATA1 is deficient for erythroid specification and gene expression ABSTRACT MethodsCell culture G1ME cells were maintained in 1% THPO-conditioned media or in media containing THPO, SCF and EPO, as described. 3 Retroviral transduction and constructsRetroviral supernatant was prepared and applied to cells, as previously described.32 MigR1 constructs containing HA-tagged GATA1 and GATA1s have been described previously.

Acute Myeloid Leukemia (AML) is a hematologic malignancy with a poor prognosis. Recent genome-wide sequencing studies have identified frequent mutations in genes encoding members of the cohesin complex. Mutations in cohesin contribute to myeloid malignancies by conferring enhanced self-renewal of hematopoietic stem and progenitor cells but the mechanisms behind this phenotype have not been fully elucidated. Of note, cohesin mutations are highly prevalent in acute megakaryocytic leukemia associated with Down syndrome (DS-AMKL), where they occur in over half of patients. Evidence suggests that cohesin mutations alter gene expression through changes in chromatin accessibility and/or aberrant targeting of epigenetic complexes. In this review we discuss the pathogenic mechanisms by which cohesin mutations contribute to myeloid malignancies.

PurposeTo develop a microRNA (miRNA)-based predictive model for prostate cancer patients of 1) time to biochemical recurrence after radical prostatectomy and 2) biochemical recurrence after salvage radiation therapy following documented biochemical disease progression post-radical prostatectomy.MethodsForty three patients who had undergone salvage radiation therapy following biochemical failure after radical prostatectomy with greater than 4 years of follow-up data were identified. Formalin-fixed, paraffin-embedded tissue blocks were collected for all patients and total RNA was isolated from 1mm cores enriched for tumor (>70%). Eight hundred miRNAs were analyzed simultaneously using the nCounter human miRNA v2 assay (NanoString Technologies; Seattle, WA). Univariate and multivariate Cox proportion hazards regression models as well as receiver operating characteristics were used to identify statistically significant miRNAs that were predictive of biochemical recurrence.ResultsEighty eight miRNAs were identified to be significantly (p<0.05) associated with biochemical failure post-prostatectomy by multivariate analysis and clustered into two groups that correlated with early (≤ 36 months) versus late recurrence (>36 months). Nine miRNAs were identified to be significantly (p<0.05) associated by multivariate analysis with biochemical failure after salvage radiation therapy. A new predictive model for biochemical recurrence after salvage radiation therapy was developed; this model consisted of miR-4516 and miR-601 together with, Gleason score, and lymph node status. The area under the ROC curve (AUC) was improved to 0.83 compared to that of 0.66 for Gleason score and lymph node status alone.ConclusionmiRNA signatures can distinguish patients who fail soon after radical prostatectomy versus late failures, giving insight into which patients may need adjuvant therapy. Notably, two novel miRNAs (miR-4516 and miR-601) were identified that significantly improve prediction of biochemical failure post-salvage radiation therapy compared to clinico-histopathological factors, supporting the use of miRNAs within clinically used predictive models. Both findings warrant further validation studies.