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Screening of an inhibitor library targeting kinases and epigenetic regulators identified several molecules having antiproliferative synergy with extraterminal domain (BET) bromodomain (BD) inhibitors (JQ1, OTX015) in triple-negative breast cancer (TNBC). GSK2801, an inhibitor of BAZ2A/B BDs, of the imitation switch chromatin remodeling complexes, and BRD9, of the SWI/SNF complex, demonstrated synergy independent of BRD4 control of P-TEFb–mediated pause-release of RNA polymerase II. GSK2801 or RNAi knockdown of BAZ2A/B with JQ1 selectively displaced BRD2 at promoters/enhancers of ETS-regulated genes. Additional displacement of BRD2 from rDNA in the nucleolus coincided with decreased 45S rRNA, revealing a function of BRD2 in regulating RNA polymerase I transcription. In 2D cultures, enhanced displacement of BRD2 from chromatin by combination drug treatment induced senescence. In spheroid cultures, combination treatment induced cleaved caspase-3 and cleaved PARP characteristic of apoptosis in tumor cells. Thus, GSK2801 blocks BRD2-driven transcription in combination with BET inhibitor and induces apoptosis of TNBC. Implications: Synergistic inhibition of BDs encoded in BAZ2A/B, BRD9, and BET proteins induces apoptosis of TNBC by a combinatorial suppression of ribosomal DNA transcription and ETS-regulated genes.

Targeting the dysregulated BRAF–MEK–ERK pathway in cancer has increasingly emerged in clinical trial design. Despite clinical responses in specific cancers using inhibitors targeting BRAF and MEK, resistance develops often involving nongenomic adaptive bypass mechanisms. Inhibition of MEK1/2 by trametinib in patients with triple-negative breast cancer (TNBC) induced dramatic transcriptional responses, including upregulation of receptor tyrosine kinases (RTK) comparing tumor samples before and after one week of treatment. In preclinical models, MEK inhibition induced genome-wide enhancer formation involving the seeding of BRD4, MED1, H3K27 acetylation, and p300 that drives transcriptional adaptation. Inhibition of the P-TEFb–associated proteins BRD4 and CBP/p300 arrested enhancer seeding and RTK upregulation. BRD4 bromodomain inhibitors overcame trametinib resistance, producing sustained growth inhibition in cells, xenografts, and syngeneic mouse TNBC models. Pharmacologic targeting of P-TEFb members in conjunction with MEK inhibition by trametinib is an effective strategy to durably inhibit epigenomic remodeling required for adaptive resistance. Significance: Widespread transcriptional adaptation to pharmacologic MEK inhibition was observed in TNBC patient tumors. In preclinical models, MEK inhibition induces dramatic genome-wide modulation of chromatin, in the form of de novo enhancer formation and enhancer remodeling. Pharmacologic targeting of P-TEFb complex members at enhancers is an effective strategy to durably inhibit such adaptation. Cancer Discov; 7(3); 302–21. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 235

Human cytomegalovirus (HCMV) is a significant cause of disease in immune-compromised adults and immune naïve newborns. No vaccine exists to prevent HCMV infection, and current antiviral therapies have toxic side effects that limit the duration and intensity of their use. There is thus an urgent need for new strategies to treat HCMV infection. Repurposing existing drugs as antivirals is an attractive approach to limit the time and cost of new antiviral drug development. Virus-induced changes in infected cells are often driven by changes in cellular kinase activity, which led us to hypothesize that defining the complement of kinases (the kinome), whose abundance or expression is altered during infection would identify existing kinase inhibitors that could be repurposed as new antivirals. To this end, we applied a kinase capture technique, multiplexed kinase inhibitor bead-mass spectrometry (MIB-MS) kinome, to quantitatively measure perturbations in >240 cellular kinases simultaneously in cells infected with a laboratory-adapted (AD169) or clinical (TB40E) HCMV strain. MIB-MS profiling identified time-dependent increases and decreases in MIB binding of multiple kinases including cell cycle kinases, receptor tyrosine kinases, and mitotic kinases. Based on the kinome data, we tested the antiviral effects of kinase inhibitors and other compounds, several of which are in clinical use or development. Using a novel flow cytometry-based assay and a fluorescent reporter virus we identified three compounds that inhibited HCMV replication with IC50 values of 3 log decrease in virus replication. These results show the utility of MIB-MS kinome profiling for identifying existing kinase inhibitors that can potentially be repurposed as novel antiviral drugs.

The Cancer Genome Atlas (TCGA) identified four integrated clusters for endometrial cancer (EC): POLE, MSI, CNL and CNH. We evaluated differences in gene expression profiles of obese and non-obese women with EC and examined the association of body mass index (BMI) within the clusters identified in TCGA.TCGA RNAseq data was used to identify genes related to increasing BMI among ECs. The POLE, MSI and CNL clusters were composed mostly of endometrioid EC. Patient BMI was compared between these three clusters with one-way ANOVA. Association between gene expression and BMI was also assessed while adjusting for confounding effects of potential confounding factors. p-Values testing the association between gene expression and BMI were adjusted for multiple hypothesis testing over the 20,531 genes considered.Mean BMI was statistically different between the ECs in the CNL (35.8) versus POLE (29.8) cluster (p=0.006) and approached significance for the MSI (33.0) versus CNL (35.8) cluster (p=0.05). 181 genes were significantly up- or down-regulated with increasing BMI in endometrioid EC (q-value0.01), including LPL, IRS-1, IGFBP4, IGFBP7 and the progesterone receptor. DAVID functional annotation analysis revealed significant enrichment in "cell cycle" (adjusted p-value=1.5E-5) and "DNA metabolic processes" (adjusted p-value=1E-3) for the identified genes.Obesity related genes were found to be upregulated with increasing BMI among endometrioid ECs. Patients with POLE tumors have the lowest median BMI when compared to MSI and CNL. Given the heterogeneity among endometrioid EC, consideration should be given to abandoning the Type I and II classification of EC tumors.

Significance The mammalian circadian clock is controlled by a transcription-translation feedback loop consisting of transcriptional activators circadian locomotor output cycles kaput (CLOCK)–brain and muscle Arnt-like protein-1 (BMAL1), which function as a complex at E/E'-box elements, and repressors Cryptochrome 1 (CRY1)/CRY2 and PER1/PER2. CRYs repress upon binding as CRY–CLOCK–BMAL1–E-box complexes. Period proteins (PERs) repress by removing the heterotrimeric complexes from the E-box. We report here that in the Cry1 promoter, the CRY1–CLOCK–BMAL1–E-box complex represses a transcriptional activator acting in cis, and removal of the heterotrimeric complex by PER2 de-represses the transcriptional activator. ChIP-seq and RNA-seq experiments identified other genes also de-repressed by PER2. These data clarify the role of PER2 and reveal the level of complexity in regulation of Cry1 and other circadian-controlled genes.

In DAE (DNA After Enrichment)-seq experiments, genomic regions related with certain biological processes are enriched/isolated by an assay and are then sequenced on a high-throughput sequencing platform to determine their genomic positions. Statistical analysis of DAE-seq data aims to detect genomic regions with significant aggregations of isolated DNA fragments ("enriched regions") versus all the other regions ("background"). However, many confounding factors may influence DAE-seq signals. In addition, the signals in adjacent genomic regions may exhibit strong correlations, which invalidate the independence assumption employed by many existing methods. To mitigate these issues, we develop a novel Autoregressive Hidden Markov Model (AR-HMM) to account for covariates effects and violations of the independence assumption. We demonstrate that our AR-HMM leads to improved performance in identifying enriched regions in both simulated and real datasets, especially in those in epigenetic datasets with broader regions of DAE-seq signal enrichment. We also introduce a variable selection procedure in the context of the HMM/AR-HMM where the observations are not independent and the mean value of each state-specific emission distribution is modeled by some covariates. We study the theoretical properties of this variable selection procedure and demonstrate its efficacy in simulated and real DAE-seq data. In summary, we develop several practical approaches for DAE-seq data analysis that are also applicable to more general problems in statistics.

For reasons unknown, temporomandibular disorder (TMD) can manifest as localized pain or in conjunction with widespread pain. We evaluated relationships between cytokines and TMD without or with widespread palpation tenderness (TMD−WPT or TMD+WPT, respectively) at protein, transcription factory activity, and gene levels. Additionally, we evaluated the relationship between cytokines and intermediate phenotypes characteristic of TMD and WPT. In a case-control study of 344 females, blood samples were analyzed for levels of 22 cytokines and activity of 48 transcription factors. Intermediate phenotypes were measured by quantitative sensory testing and questionnaires asking about pain, health, and psychological status. Single nucleotide polymorphisms (SNPs) coding cytokines and transcription factors were genotyped. TMD−WPT cases had elevated protein levels of proinflammatory cytokine monocyte chemotactic protein (MCP-1) and antiinflammatory cytokine interleukin (IL)-1ra, whereas TMD+WPT cases had elevated levels of proinflammatory cytokine IL-8. MCP-1, IL-1ra, and IL-8 were differentially associated with experimental pain, self-rated pain, self-rated health, and psychological phenotypes. TMD−WPT and TMD+WPT cases had inhibited transcription activity of the antiinflammatory cytokine transforming growth factor β1 (TGFβ1). Interactions were observed between TGFβ1 and IL-8 SNPs: an additional copy of the TGFβ1 rs2241719 minor T allele was associated with twice the odds of TMD+WPT among individuals homozygous for the IL-8 rs4073 major A allele, and half the odds of TMD+WPT among individuals heterozygous for rs4073. These results demonstrate how pro- and antiinflammatory cytokines contribute to the pathophysiology of TMD and WPT in genetically susceptible people. Furthermore, they identify MCP-1, IL-1ra, IL-8, and TGFβ1 as potential diagnostic markers and therapeutic targets for pain in patients with TMD.