Dual blockade of BRD4 and ATR/WEE1 pathways exploits ARID1A loss in clear cell ovarian cancer.

ARID1A, an epigenetic tumor suppressor, is the most common gene mutation in clear-cell ovarian cancers (CCOCs). CCOCs are often resistant to standard chemotherapy and lack effective therapies. We hypothesized that ARID1A loss would increase CCOC cell dependency on chromatin remodeling and DNA repair pathways for survival. We demonstrate that combining BRD4 inhibitor (BRD4i) with DNA damage response inhibitors (ATR or WEE1 inhibitors; e.g. BRD4i-ATRi) was synergistic at low doses leading to decreased survival, and colony formation in CCOC in an ARID1A dependent manner. BRD4i-ATRi caused significant tumor regression and increased overall survival in ARID1AMUT but not ARID1AWT patient-derived xenografts. Combination BRD4i-ATRi significantly increased γH2AX, and decreased RAD51 foci and BRCA1 expression, suggesting decreased ability to repair DNA double-strand-breaks (DSBs) by homologous-recombination in ARID1AMUT cells, and these effects were greater than monotherapies. These studies demonstrate BRD4i-ATRi is an effective treatment strategy that capitalizes on synthetic lethality with ARID1A loss in CCOC.

Efficacy and safety of aerosol inhalation of recombinant human interferon α1b (IFNα1b) injection for noninfluenza viral pneumonia, a multicenter, randomized, double-blind, placebo-controlled trial.

BACKGROUND: To investigate the efficacy and safety of aerosol inhalation of recombinant human interferon α1b (IFNα1b) injection for noninfluenza viral pneumonia. METHODS: One hundred sixty-four patients with noninfluenza viral pneumonia were divided into IFNα1b and control groups. The IFNα1b group received routine treatment + aerosol inhalation of recombinant human IFNα1b injection (50 µg × 2 injections, bid). The control group received routine treatment + IFN analog (two injections, bid). Overall response rate (ORR) of five kinds clinical symptoms. Further outcomes were daily average score and the response rate of each of the symptoms above. RESULTS: A total of 163 patients were included in the full analysis set (FAS) and 151 patients were included in the per-protocol set (PPS). After 7 days of treatment, ORR of clinical symptoms was higher in IFNα1b group than that in control group for both the FAS and PPS. Moreover, after 7 days of treatment, the daily score of three efficacy indexes including expectoration, respiratory rate, and pulmonary rales were improved. The ORRs for expectoration and pulmonary rales were higher in the IFNα1b group than in the control group (P < 0.05). There were no significant differences of the ORRs for coughing, chest pain and respiratory rate between the two groups (P > 0.05). The incidence of adverse events was 6.5% (n = 5) in IFNα1b group and 3.5% (n = 3) in control group (P > 0.05). CONCLUSION: Aerosol inhalation of recombinant human IFNα1b is safe and it can improve the clinical symptoms of noninfluenza viral pneumonia.

Age Alters Chromatin Structure and Expression of SUMO Proteins under Stress Conditions in Human Adipose-Derived Stem Cells.

Adult stem cells play a critical role in tissue homeostasis and repair. Aging leads to a decline in stem cells' regenerative capacity that contributes significantly to the maintenance of organ and tissue functions. Age-dependent genomic and epigenetic modifications together play a role in the disruption of critical cellular pathways. However, the epigenetic mechanisms responsible for the decline of adult stem cell functions remain to be well established. Here, we investigated age-dependent, genome-wide alterations in the chromatin accessibility of primary human adipose-derived stem cells (ASCs) in comparison to age-matched fibroblasts via ATAC-seq technology. Our results demonstrate that aging ASCs possess globally more stable chromatin accessibility profiles as compared to aging fibroblasts, suggesting that robust regulatory mechanisms maintain adult stem cell chromatin structure against aging. Furthermore, we observed age-dependent subtle changes in promoter nucleosome positioning in selective pathways during aging, concurrent with altered small ubiquitin-related modifier (SUMO) protein expression under stress conditions. Together, our data suggest a significant role for nucleosome positioning in sumoylation pathway regulation in stress response during adult stem cell aging. The differences described here between the chromatin structure of human ASCs and fibroblasts will further elucidate the mechanisms regulating gene expression during aging in both stem cells and differentiated cells.

Transcriptional and Cell Cycle Alterations Mark Aging of Primary Human Adipose-Derived Stem Cells.

Adult stem cells play a critical role in the maintenance of tissue homeostasis and prevention of aging. While the regenerative potential of stem cells with low cellular turnover, such as adipose-derived stem cells (ASCs), is increasingly recognized, the study of chronological aging in ASCs is technically difficult and remains poorly understood. Here, we use our model of chronological aging in primary human ASCs to examine genome-wide transcriptional networks. We demonstrate first that the transcriptome of aging ASCs is distinctly more stable than that of age-matched fibroblasts, and further, that age-dependent modifications in cell cycle progression and translation initiation specifically characterize aging ASCs in conjunction with increased nascent protein synthesis and a distinctly shortened G1 phase. Our results reveal novel chronological aging mechanisms in ASCs that are inherently different from differentiated cells and that may reflect an organismal attempt to meet the increased demands of tissue and organ homeostasis during aging. Stem Cells 2017;35:1392-1401.

Discovery of Genetic Variation on Chromosome 5q22 Associated with Mortality in Heart Failure.

Failure of the human heart to maintain sufficient output of blood for the demands of the body, heart failure, is a common condition with high mortality even with modern therapeutic alternatives. To identify molecular determinants of mortality in patients with new-onset heart failure, we performed a meta-analysis of genome-wide association studies and follow-up genotyping in independent populations. We identified and replicated an association for a genetic variant on chromosome 5q22 with 36% increased risk of death in subjects with heart failure (rs9885413, P = 2.7x10-9). We provide evidence from reporter gene assays, computational predictions and epigenomic marks that this polymorphism increases activity of an enhancer region active in multiple human tissues. The polymorphism was further reproducibly associated with a DNA methylation signature in whole blood (P = 4.5x10-40) that also associated with allergic sensitization and expression in blood of the cytokine TSLP (P = 1.1x10-4). Knockdown of the transcription factor predicted to bind the enhancer region (NHLH1) in a human cell line (HEK293) expressing NHLH1 resulted in lower TSLP expression. In addition, we observed evidence of recent positive selection acting on the risk allele in populations of African descent. Our findings provide novel genetic leads to factors that influence mortality in patients with heart failure.

Differential expression of PDE5 in failing and nonfailing human myocardium.

BACKGROUND: Recognizing that inhibitors of phosphodiesterase type 5 (PDE5) are increasingly employed in patients with pulmonary hypertension and right ventricular (RV) failure, we examined PDE5 expression in the human RV and its impact on myocardial contractility. METHODS AND RESULTS: Tissue extracts from the RV of 20 patients were assayed for PDE5 expression using immunoblot and immunohistochemical staining. Tissues were selected from groups of nonfailing organ donors and transplant recipients with endstage ischemic cardiomyopathy or idiopathic dilated cardiomyopathy. Among dilated cardiomyopathy patients, subgroups with mild or severe RV dysfunction and prior left ventricular assist devices were analyzed separately. Our results showed that PDE5 abundance increased more than 4-fold in the RVs of the ischemic cardiomyopathy compared with the nonfailing group. In dilated cardiomyopathy, PDE5 upregulation was more moderate and varied with the severity of RV dysfunction. Immunohistochemical staining confirmed that cardiac myocytes contributed to the upregulation in the failing hearts. In functional studies, PDE5 inhibition produced little change in developed force in RV trabeculae from nonfailing hearts but produced a moderate increase in RV trabeculae from failing hearts. CONCLUSIONS: Our results showed the etiology- and severity-dependent upregulation of myocyte PDE5 expression in the RV and the impact of this upregulation on myocardial contractility. These findings suggest that RV PDE5 expression could contribute to the pathogenesis of RV failure, and direct myocardial responses to PDE5 inhibition may modulate the indirect responses mediated by RV afterload reduction.

Differential regulation of PDE5 expression in left and right ventricles of feline hypertrophy models.

BACKGROUND: Though long known to affect smooth muscle biology, recent studies indicate that phosphodiesterase 5 (PDE5) is also expressed in myocardium. Recognizing that the regulation of PDE5 in hypertrophy is not well understood, we assessed the response of PDE5 expression and the level of cGMP-dependent kinase I (cGKI) in the left and right ventricles of feline hypertrophy models. METHODOLOGY/PRINCIPAL FINDINGS: Using a cDNA library of feline aortic smooth muscle cells, we identified and cloned PDE5 cDNA for the first time in this species. The sequence shares 98% identity with its human orthologue at the amino acid level. E. coli expression of the cloned allele allowed selection of antibodies with appropriate specificity, facilitating the analysis of PDE5 expression in feline models created by selective proximal aortic (Ao) or pulmonary artery (PA) banding that resulted in hypertrophy of the left ventricle (LV) and right ventricle (RV), respectively. We demonstrated that PDE5 expression responded differentially with a decreased expression in the LV and an increased expression in the RV in the Ao-banded model. Similarly, in the PA-banded model, LV showed reduced expression while the RV expression was unaltered. In addition, the expression of cGKI was significantly decreased in the RV of Ao-banded group, correlating inversely with the increase in PDE5 expression. CONCLUSIONS/SIGNIFICANCE: The differential regulation of PDE5 and cGKI expression suggests that the mechanisms involved in hypertrophy could be different in RV vs. LV. Reciprocal PDE5 and cGKI expression in the RV of Ao-banded model suggests functional significance for PDE5 up-regulation.

Apoptosis signal-regulating kinase 1 attenuates atrial natriuretic peptide secretion.

Atrial natriuretic peptide (ANP) is an endogenous peptide hormone that is synthesized and secreted by the myocardium in health and disease. Although the bioactivity of this molecule has been studied extensively, cellular mechanisms governing its processing and secretion are not fully understood. Through a yeast two-hybrid screen of a cDNA library made from tissue of a failing human heart, we have discovered that the precursor of ANP, natriuretic peptide precursor (NPPA), physically interacts with the N-terminus of apoptosis signal-regulating kinase 1 (ASK1), a kinase believed to be involved in the pathogenesis of heart failure. We demonstrated that NPPA is a substrate of ASK1 in an in vitro kinase assay. Indirect immunofluorescence microscopy shows that, when expressed in Hela cells, ASK1 and NPPA exhibit distinct, but overlapping, staining patterns, suggesting partial colocalization in cells. Additionally, coexpressing wild-type ASK1 with NPPA in Hela cells led to reduced levels of NPPA in the culture medium, suggesting that ASK1 negatively impacts NPPA processing and/or secretion. This negative effect was less pronounced when a dominant-negative allele of ASK1 with deficient kinase activity was coexpressed with NPPA. Because both ASK1 and ANP are associated with pathologic cardiac hypertrophy, their interaction may have pathophysiological and therapeutic relevance.

Increased cardiac myocyte progenitors in failing human hearts.

BACKGROUND: Increasing evidence, derived mainly from animal models, supports the existence of endogenous cardiac renewal and repair mechanisms in adult mammalian hearts that could contribute to normal homeostasis and the responses to pathological insults. METHODS AND RESULTS: Translating these results, we isolated small c-kit+ cells from 36 of 37 human hearts using primary cell isolation techniques and magnetic cell sorting techniques. The abundance of these cardiac progenitor cells was increased nearly 4-fold in patients with heart failure requiring transplantation compared with nonfailing controls. Polychromatic flow cytometry of primary cell isolates (<30 microm) without antecedent c-kit enrichment confirmed the increased abundance of c-kit+ cells in failing hearts and demonstrated frequent coexpression of CD45 in these cells. Immunocytochemical characterization of freshly isolated, c-kit-enriched human cardiac progenitor cells confirmed frequent coexpression of c-kit and CD45. Primary cardiac progenitor cells formed new human cardiac myocytes at a relatively high frequency after coculture with neonatal rat ventricular myocytes. These contracting new cardiac myocytes exhibited an immature phenotype and frequent electric coupling with the rat myocytes that induced their myogenic differentiation. CONCLUSIONS: Despite the increased abundance and cardiac myogenic capacity of cardiac progenitor cells in failing human hearts, the need to replace these organs via transplantation implies that adverse features of the local myocardial environment overwhelm endogenous cardiac repair capacity. Developing strategies to improve the success of endogenous cardiac regenerative processes may permit therapeutic myocardial repair without cell delivery per se.

Regulation of Toll-like receptor 4-induced proasthmatic changes in airway smooth muscle function by opposing actions of ERK1/2 and p38 MAPK signaling.

Activation of Toll-like receptors (TLRs) on immune surveillance cells in the lung has been implicated in the pathobiology of allergic asthma, a condition associated with altered airway smooth muscle (ASM) contractility. Because ASM is known to directly respond to various proasthmatic stimuli, the potential role of TLR signaling in ASM in regulating airway expression of the proasthmatic phenotype was investigated. Cultured human ASM cells were found to express TLR4 and TLR9 mRNA transcripts and, whereas TLR9 stimulation had little effect, TLR4 activation with LPS elicited significant increases in IL-6 release and evoked proasthmatic-like changes in the constrictor and relaxation responsiveness of isolated rabbit ASM tissues. Complementary studies further demonstrated that the ASM responses to LPS were associated with activation of the ERK1/2 and p38 MAPK signaling pathways, IKK-mediated activation of NF-kappaB, and coupling of phosphorylated ERK1/2 with the p65 subunit of NF-kappaB. Moreover, the induced NF-kappaB activity and changes in ASM responsiveness were prevented in LPS-exposed ASM that were pretreated with inhibitors of ERK1/2 signaling, whereas inhibition of p38 MAPK augmented the proasthmatic responses to LPS. Finally, activation of p38 MAPK with anisomycin prevented both the LPS-induced stimulation of ERK1/2-mediated NF-kappaB activity and associated changes in ASM responsiveness. Collectively, these data support the novel concept that TLR4 activation in ASM elicits changes in ASM function that are regulated by opposing effects of MAPK signaling, wherein LPS-induced ERK1/2 activation mediates NF-kappaB-dependent proasthmatic-like changes in ASM function, whereas coactivation of p38 MAPK serves to homeostatically downregulate the proasthmatic effects of ERK1/2 activation.

Proasthmatic effects and mechanisms of action of the dust mite allergen, Der p 1, in airway smooth muscle.

BACKGROUND: House dust mite allergen exposure is a key risk factor for the development of allergic asthma. Beyond provoking immune cell-mediated allergic responses, house dust mite allergens were recently shown to exert direct effects on airway structural cells secondary to their intrinsic protease activities. OBJECTIVE: This study tested the hypothesis that house dust mite allergen exposure can produce changes in airway responsiveness through a direct effect on airway smooth muscle (ASM). METHODS: Isolated rabbit ASM tissues were exposed to the house dust mite allergen, Der p 1, and induced changes in ASM responsiveness and activation of mitogen-activated protein kinase (MAPK) signaling pathways were examined under different experimental conditions. RESULTS: The observations demonstrated the following: (1) Der p 1 exposure elicited enhanced constrictor responses and impaired relaxation responses in the ASM tissues, (2) these proasthmatic-like effects of Der p 1 were attributed to its intrinsic cysteine protease activity, and (3) the induced changes in ASM responsiveness were associated with activation of both the extracellular signal-regulated kinase (ERK) 1/2 and the p38 MAPK signaling pathways. Additionally, specific blockade of ERK1/2 signaling was found to prevent the Der p 1-induced changes in ASM responsiveness, whereas inhibition of p38 MAPK signaling enhanced the proasthmatic-like action of Der p 1, with the latter effect a result of augmented activation of ERK1/2. CONCLUSION: These findings are the first to demonstrate that the dust mite allergen, Der p 1, can directly elicit changes in ASM responsiveness that are associated with activation of MAPK signaling, wherein proasthmatic effects induced by Der p 1 are attributed to activation of ERK1/2, whereas coactivation of p38 MAPK exerts a homeostatic action by negatively regulating ERK1/2 signaling.