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Purpose:Both gain-of-function enhancer of zeste homolog 2 (EZH2) mutations and inactivating histone acetyltransferases mutations, such as CREBBP and EP300, have been implicated in the pathogenesis of germinal center (GC)–derived lymphomas. We hypothesized that direct inhibition of EZH2 and histone deacetyltransferase (HDAC) would be synergistic in GC-derived lymphomas.Experimental Design:Lymphoma cell lines (n = 21) were exposed to GSK126, an EZH2 inhibitor, and romidepsin, a pan-HDAC inhibitor. Synergy was assessed by excess over bliss. Western blot, mass spectrometry, and coimmunoprecipitation were performed. A SU-DHL-10 xenograft model was utilized to validate in vitro findings. Pretreatment RNA-sequencing of cell lines was performed. MetaVIPER analysis was used to infer protein activity.Results:Exposure to GSK126 and romidepsin demonstrated potent synergy in lymphoma cell lines with EZH2 dysregulation. Combination of romidepsin with other EZH2 inhibitors also demonstrated synergy suggesting a class effect of EZH2 inhibition with romidepsin. Dual inhibition of EZH2 and HDAC led to modulation of acetylation and methylation of H3K27. The synergistic effects of the combination were due to disruption of the PRC2 complex secondary to acetylation of RbAP 46/48. A common basal gene signature was shared among synergistic lymphoma cell lines and was characterized by upregulation in chromatin remodeling genes and transcriptional regulators. This finding was supported by metaVIPER analysis which also revealed that HDAC 1/2 and DNA methyltransferase were associated with EZH2 activation.Conclusions:Inhibition of EZH2 and HDAC is synergistic and leads to the dissociation of PRC2 complex. Our findings support the clinical translation of the combination of EZH2 and HDAC inhibition in EZH2 dysregulated lymphomas.

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Background: In HFpEF and PAH, men are more likely than women to develop right ventricular dysfunction (RVD) and die. Mechanisms underlying these sex differences are not well understood. In advanced HF patients, RVD is associated with RV fibrosis. Sex differences in cGMP anti-fibrotic cardioprotection have been reported. Hypothesis: Sex differences in RV response to pressure overload may be due to differential anti-fibrotic cGMP signaling in the RV. Methods: Adult male and female C57BL6/J mice underwent pulmonary artery banding (PAB, 25g) vs. thoracotomy (Sham). RV remodeling was assessed by echocardiography, pressure-volume loop recordings, morphometrics, and histology. Expression of transcripts encoding various cGMP signaling proteins ( Nppa, Nppb, Npr1, Npr2, Npr3, Gucy1a3, Gucy1b3, Pde5, Pde9a, Pkg1a ), the fetal gene program, tissue fibrosis and ECM remodeling ( Ace, Tgfβs, Smads, Ctgf, αSma, Postn, Col1α1, Lum, Igfbp5, Mmp2-3, Timp1-2) were assessed at 9wks. Results: Post-PAB survival rate did not differ between sexes (log-rank HR 1.02, 95% CI 0.30-3.48, P=NS), but median time-to-death was 20 days (IQR 15-32) for M-PAB vs. 35.5 days (IQR 10.8-41.5) for F-PAB. PAB increased RV mass in both sexes (Fulton’s index 0.45±0.07 M-PAB vs 0.21±0.01 M-Sham, P2 vs 397.7±21.6μm 2 F-PAB, PNppa, Nppb, Npr1, Npr2, Npr3, Gucy1a3, Gucy1b3, Pde5 and Pkg1a in the RV. RV transcript levels of Ace, Col1α1, Ctgf, Igfbp5, Mmp2, and Timp2 were also higher in M-PAB than F-PAB. Conclusions: Males have worse RV functional response to PAB than do females. This sex difference was also associated with increased mRNA levels of cGMP signaling mediators and upregulation of pro-hypertrophic and pro-fibrotic genes in male PAB mice. Further studies are needed to elucidate sex-dependent regulation of RV remodeling.

Purpose: Both gain-of-function enhancer of zeste homolog 2 (EZH2) mutations and inactivating histone acetyltransferases mutations, such as CREBBP and EP300, have been implicated in the pathogenesis of germinal center (GC)–derived lymphomas. We hypothesized that direct inhibition of EZH2 and histone deacetyltransferase (HDAC) would be synergistic in GC-derived lymphomas. Experimental Design: Lymphoma cell lines (n = 21) were exposed to GSK126, an EZH2 inhibitor, and romidepsin, a pan-HDAC inhibitor. Synergy was assessed by excess over bliss. Western blot, mass spectrometry, and coimmunoprecipitation were performed. A SU-DHL-10 xenograft model was utilized to validate in vitro findings. Pretreatment RNA-sequencing of cell lines was performed. MetaVIPER analysis was used to infer protein activity. Results: Exposure to GSK126 and romidepsin demonstrated potent synergy in lymphoma cell lines with EZH2 dysregulation. Combination of romidepsin with other EZH2 inhibitors also demonstrated synergy suggesting a class effect of EZH2 inhibition with romidepsin. Dual inhibition of EZH2 and HDAC led to modulation of acetylation and methylation of H3K27. The synergistic effects of the combination were due to disruption of the PRC2 complex secondary to acetylation of RbAP 46/48. A common basal gene signature was shared among synergistic lymphoma cell lines and was characterized by upregulation in chromatin remodeling genes and transcriptional regulators. This finding was supported by metaVIPER analysis which also revealed that HDAC 1/2 and DNA methyltransferase were associated with EZH2 activation. Conclusions: Inhibition of EZH2 and HDAC is synergistic and leads to the dissociation of PRC2 complex. Our findings support the clinical translation of the combination of EZH2 and HDAC inhibition in EZH2 dysregulated lymphomas.

Introduction In HFpEF as well as PAH, men are more likely than women to develop RV dysfunction and die from right heart failure. The pathophysiological mechanisms underlying these sex differences are not well understood. Some animal studies of ischemic left heart disease have suggested that sex differences in NO signaling confer cardioprotection upon females. Others have shown sex differences in left ventricular expression of NP vs NOS systems. Hypothesis We hypothesized that differential NO- vs NP- cGMP signaling within the RV underlie sex differences in its response to pressure overload. Methods Adult male and female C57BL6/J mice were subjected to pulmonary artery banding (PAB, 25gauge) vs thoracotomy (Sham) for 9 wks. Serial echocardiography was performed at 3wk intervals. Histologic and morphometric analyses, as well as RV pressure-volume loop recordings were performed. Right ventricular expression of Nppa, Nppb, Npr1, Npr2, Npr3,Gucy1a3, Gucy1b3 were assessed. Survival analysis was also performed. Results While survival post-PAB were similar between males and females (log-rank HR 2.49, 95% CI 0.488-12.66, p=0.92), time-to-death was 25.5±4.37 days vs 42 days in PAB-M vs PAB-F, respectively. For both sexes, PAB induced increase in RV mass (Fulton's index 0.47±0.03 PAB-M vs 0.29±0.01 Sham-M, p Conclusions Compared to females, males have worse RV contractile and diastolic responses to pressure overload, independent of hypertrophic and fibrotic remodeling. While transcript levels of several cGMP signaling proteins are higher in males, further studies are needed to elucidate the role of these sex differences in RV dysfunction.