Mechanisms Contributing to the Dysregulation of miRNA-124 in Pulmonary Hypertension

Chronic pulmonary hypertension (PH) is a fatal disease characterized by the persistent activation of pulmonary vascular cells that exhibit aberrant expression of genes including miRNAs. We and others reported that decreased levels of mature microRNA-124 (miR-124) plays an important role in modulating the activated phenotype of pulmonary vascular cells and HDAC inhibitors (HDACi) can restore the levels of mature miR-124 and reverse the persistently activated phenotype of PH vascular cells. In this study, we sought to determine the mechanisms contributing to reduced levels of miRNAs, as well as how HDACi restores the levels of reduced miRNA in PH vascular cells. We found that pulmonary artery fibroblasts isolated from IPAH patients (PH-Fibs) exhibit reduced levels of mature miR-124 and several other miRNAs including let-7i, miR-224, and miR-210, and that these reduced levels can be restored by HDACi. Using miR-124 expression in human PH-Fibs as a model, we determined that reduced miR-124 gene transcription, not decreased expression of miRNA processing genes, is responsible for reduced levels of mature miR-124 in human PH-Fibs. Using both DNase I Sensitivity and chromatin immunoprecipitation assays, we found that the miR-124-1 gene exhibits a more condensed chromatin structure in human PH-Fibs, compared to corresponding controls. HDACi relaxed miR-124-1 chromatin structure, evidenced by increased levels of the open chromatin mark H3K27Ac, but decreased levels of closed chromatin mark H3K27Me3. Most importantly, the delivery of histone acetyltransferase (HAT) via CRISPR-dCas9-HAT and guiding RNAs to the promoter of the miR-124-1 gene increased miR-124-1 gene transcription. Thus, our data indicate epigenetic events play important role in controlling miR-124 and likely other miRNA levels and epigenetic regulators such as HDACs appear to be promising therapeutic targets for chronic PH.

HebbPlot: An intelligent tool for learning and visualizing chromatin mark signatures

Histone modifications play important roles in gene regulation, heredity, imprinting, and many human diseases. The histone code is complex, consisting of about 100 marks. Biologists need computational tools for characterizing general signatures representing the distributions of tens of chromatin marks around thousands of regions. To this end, we developed a software tool called HebbPlot, which utilizes a Hebbian neural network to learn such signatures. HebbPlot presents a signature as a digitized image, which can be easily interpreted. We validated HebbPlot in six case studies. HebbPlot is applicable to a wide array of studies, facilitating the deciphering of the histone code.

Pre-Clinical Efficacy of Combined Therapy with LSD1 Antagonist SP-2509 and Pan-Histone Deacetylase Inhibitor Against AML Blast Progenitor Cells

Abstract 868 LSD1 (KDM1A) is an FAD-dependent histone demethylase, with homology to amine oxidases. LSD1 demethylates di- and mono-methylated lysine (K) 4 on histone H3, reducing the permissive H3K4Me3 chromatin mark for gene expression. LSD1 forms a complex with the histone deacetylases (HDAC) 1 and 2 and with the co-repressor CoREST, which stimulates the activity of LSD1 toward nucleosomes. While high LSD1 expression may be an effector of blocked differentiation and confers poor prognosis in AML, LSD1 inhibition induces the expression of myeloid–differentiation associated genes and attenuates growth of AML blast progenitor cells (BPCs). Recently, LSD1 was shown to sustain the in vivo leukemogenic potential of MLL-AF9 expressing leukemia stem cells. Also, co-treatment with the LSD1 inhibitor tranylcypromine (TCP) and all-trans retinoic acid (ATRA) was shown to diminish the engraftment of primary AML BPCs in vivo in NOD-SCID-γIL-2 receptor deficient (NSG) mice. Previous studies have shown that HDAC inhibitors attenuate the levels of LSD1 through Sp1 inhibition. SP-2509 is a potent and selective FAD-binding pocket, non-MAOA and MAOB, inhibitor with an IC50 of 13 nM for LSD1. In the present studies, we determined the chromatin effects and anti-AML efficacy of SP-2509 alone and in combination with the pan-HDAC inhibitor panobinostat (PS) (Novartis Pharmaceuticals) in cultured (HL-60, OCI-AML3, MV4-11, MOLM13, THP1 and SKM1 cells) and primary human AML BPCs. Treatment with SP-2509 (250 to 1000 nM) dose-dependently increased the levels of H3K4Me2 & Me3 chromatin mark, and chromatin immunoprecipitation followed by QPCR analyses showed an increase in the H3K4Me3 mark on the gene promoters of KLF4, HMOX1, p57 and p21 in AML BPCs. SP-2509 treatment attenuated the binding of LSD1 with CoREST, accompanied with increased levels of p16, p21 and p27 in AML BPCs. Consistent with this, treatment with SP-2509 inhibited the suspension and colony growth of AML BPCs regardless of whether they expressed MLL fusion oncoproteins. Knockdown of LSD1 by shRNA also inhibited the suspension and colony growth of AML blast progenitor cells. SP-2509 also induced C/EBPα expression and features of morphologic differentiation in the cultured and primary AML BPCs. Following tail vein infusion and establishment of AML by OCI-AML3 or MOLM13 cells in NOD/SCID mice, treatment with SP-2509 (25 mg/kg b.i.w. via IP injection) for three weeks demonstrated improved survival of the mice compared to the vehicle control treated mice (p <0. 001). We have previously reported that treatment with PS depleted polycomb repressive complex proteins EZH2, SUZ12 and BMI1 but also reduced LSD1 expression in AML cells. Co-treatment with PS enhanced SP-2509-induced chromatin effects and differentiation of AML cells. Also, PS and SP-2509 synergistically induced apoptosis of the cultured AML OCI-AML3, MOLM13 and MV4-11cells (combination indices, CI <1.0). Additionally, co-treatment with SP-2509 sensitized AML cells to ATRA-induced differentiation. Notably, co-treatment with SP-2509 and PS also induced significantly greater loss of viability of primary AML BPCs but not of normal CD34+ cells. SP-2509 treatment (15 mg/kg b.i.w. IP) also dramatically improved survival of NSG mice with established human AML following tail-vein injection of primary AML blasts. Survival was further significantly improved upon co-treatment with SP-2509 and PS (5 mg/kg IP, MWF) (p < 0.001). Mice did not experience any toxicity or weight loss. Taken together, these findings demonstrate promising pre-clinical activity of combined therapy with SP-2509 and PS, warranting further in vivo development and testing of SP-2509 against human AML. Disclosures: Sharma: Salarius Pharmaceuticals: Equity Ownership.