Measuring H3K27me3 Reduction after in-Vivo Administration of Ftx-6058; A Potent Polycomb Repressive Complex 2 (PRC2) Inhibitor

FTX-6058 is an investigational drug for the treatment of Sickle Cell Disease (SCD). The molecule is a selective and potent binder of the embryonic ectoderm development protein (EED). Binding of FTX-6058 to EED inhibits PRC2, a histone methyltransferase which primarily methylates lysine 27 of histone H3. Inhibition of PRC2 results in the reduction of trimethylation at lysine 27 on histone H3 (H3K27me3). Because the level of trimethylation on lysine 27 is the immediate downstream consequence of PRC2 activity, we developed a flow cytometric assay to measure the relative level of H3K27me3 from in-vivo tissue samples. The flow cytometric assay measures the median fluorescence intensities (MFI) of H3K27me3 and total histone H3. The target engagement (TE) measurement is a ratio of H3K27me3 MFI to total Histone H3 MFI. The target cells for FTX-6058 in SCD treatment are the hematopoietic cells of the bone marrow. We demonstrate an FTX-6058 dose dependent decrease in the TE ratio of bone marrow, hematopoietic cells. Translating the TE assay from mouse pre-clinical studies to human clinical trials required use of a less invasive tissue type than bone marrow. In mice, a positive relationship between the TE ratio of hematopoietic, bone marrow cells and circulating monocytes was established. Therefore, a human monocyte TE assay for FTX-6058 was validated for human trials. Research use only (RUO) validation was completed by Q2 Solutions Laboratories. The monocyte FTX-6058 TE assay is currently being evaluated as an exploratory biomarker in Fulcrum Therapeutics' phase 1 clinical trial FIS 002-2020. Disclosures Roth: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Xie: Fulcrum Therapeutics, Inc.: Current equity holder in publicly-traded company, Ended employment in the past 24 months.

Post-mitotic accumulation of histone variant H3.3 in new cortical neurons establishes neuronal transcriptome, identity, and connectivity

Histone variants, which can be expressed outside of S-phase and deposited DNA synthesis-independently, provide replacement histones in terminally post-mitotic cells, including neurons. Histone variants can also serve active roles in gene regulation by modulating chromatin states or enabling nucleosome turnover at regulatory regions. Here, we find that newborn cortical excitatory neurons substantially accumulate the histone H3 variant H3.3 immediately post-mitosis. Co-deletion of H3.3-encoding genes H3f3a and H3f3b from new neurons abrogates this accumulation, and causes widespread disruptions in the developmental establishment of the neuronal transcriptome. These broad transcriptomic changes coincide with neuronal maturation phenotypes in acquisition of distinct neuronal identities and formation of axon tracts. Stage-dependent deletion of H3f3a and H3f3b from (1) cycling neural progenitor cells, (2) neurons immediately after terminal mitosis, or (3) several days later, reveals the first post-mitotic days as a critical window for de novo H3.3. After H3.3 accumulation within this developmental window, co-deletion of H3f3a and H3f3b from neurons causes progressive H3.3 depletion over several months without widespread transcriptional disruptions. Our study thus uncovers a key role for H3.3 in establishing neuronal transcriptome, identity, and connectivity immediately post-mitosis that is distinct from its role in maintaining total histone H3 levels over the neuronal lifespan.

A Key Silencing Histone Mark on Chromatin Is Lost When Colorectal Adenocarcinoma Cells Are Depleted of Methionine by Methionine γ-Lyase

Methionine is an essential amino acid used, beyond protein synthesis, for polyamine formation and DNA/RNA/protein methylation. Cancer cells require particularly high methionine supply for their homeostasis. A successful approach for decreasing methionine concentration is based on the systemic delivery of methionine γ-lyase (MGL), with in vitro and in vivo studies demonstrating its efficacy in cancer therapy. However, the mechanisms explaining how cancer cells suffer from the absence of methionine more significantly than non-malignant cells are still unclear. We analyzed the outcome of the human colorectal adenocarcinoma cancer cell line HT29 to the exposure of MGL for up to 72 h by monitoring cell viability, proteome expression, histone post-translational modifications, and presence of spurious transcription. The rationale of this study was to verify whether reduced methionine supply would affect chromatin decondensation by changing the levels of histone methylation and therefore increasing genomic instability. MGL treatment showed a time-dependent cytotoxic effect on HT29 cancer cells, with an IC50 of 30 µg/ml, while Hs27 normal cells were less affected, with an IC50 of >460 µg/ml. Although the levels of total histone methylation were not altered, a loss of the silencing histone mark H3K9me2 was observed, as well as a decrease in H4K20me3. Since H3K9me2/3 decorate repetitive DNA elements, we proved by qRT-PCR that MGL treatment leads to an increased expression of major satellite units. Our data indicate that selected histone methylation marks may play major roles in the mechanism of methionine starvation in cancer cells, proving that MGL treatment directly impacts chromatin homeostasis.

Abstract P499: Histone H4K20 Trimethylation Is Differentially Regulated In Heart Disease

It has been well established that many cardiac pathologies result from dynamic changes in gene expression and conversely that modulating key epigenetic factors in murine models is capable of preventing or abrogating ischemic injury and pathological remodeling. One epigenetic mechanism is the post-translational modification of histones, which are reversibly methylated on lysine (K) residues and can accept up to three methyl groups (Me1, Me2 and Me3). In the heart, significant changes in global levels of histone H3K4Me3 and H3K9Me3 have been previously reported to be upregulated and downregulated, respectively, during hypertrophy and failure in mice. However, the majority of post-translational modifications on histones have never been examined to quantify global abundance in the heart during disease. In particular, histone H4K20Me3 is important in heterochromatin formation and gene repression in non-cardiac cells but has never been evaluated in the heart. Therefore, we utilized cardiac tissue from three animal models of cardiac stress and employed western blotting and mass spectrometry to quantify the global abundance of total histone H4 and H4K20 methylation. We specifically evaluated tissue from mice subjected to LAD ligation, transverse aortic banding and isoproterenol infusion (via mini-osmotic pump). In addition, we also utilized primary neonatal cardiomyocytes treated with the hypertrophic agonist phenylephrine to quantify H4K20 methylation. Our data show that global levels of histone H4K20Me3 are differentially regulated in some models of cardiac dysfunction, but not all (i.e. isoproterenol infusion). In addition, we measured the abundance of histone methyltransferases and demethylases (via western blotting and qPCR) which are responsible for adding or removing this methyl mark in mouse cardiac tissue, and compared this to published data from human heart failure patients. These analyses allowed us to identify two enzymes, the methyltransferase Smyd5 and demethylase KDM7B, which are also differentially expressed in cardiac tissue during disease. Together these results are the first analysis of histone H4K20 methylation in the heart and suggest a novel role for this methylation site in the pathophysiology of cardiovascular disease.

Prognostic role of levels of extracellular histones H3 for diagnosis of early dysfunction of transplant after hepatic transplantation

Objective. The aim of this study was to determine the prognostic role of extracellular histones in the diagnosis of early graft dysfunction after liver transplantation.. Materials and methods. The 93 recipients undergoing LDLT were enrolled in this prospective study. Blood samples of patients were collected on postoperative day 1 and histone levels in the plasma samples were measured with Total Histone H3 sandwich ELISA kits. 19 (20.4%) subjects had early graft dysfunction (EAD) which was diagnosed on postoperative day 7 according to Ol-thoff’s criteria, based on liver function tests and coagulation profiles. Other 74 (79.6%) recipients did not have EGD. Results. Levels of circulating histones were depressed in patients with EGD (0,808±0.026, 95% (CI) 0.752-0.864) than in patients without EGD (0.820±0.017, 95% (CI) 0.786-0.854) (P=0.727). These differences were not significant. The sources of histones in the circulation are not etiologies specific and levels of total histone H 3 after 24 h of operation had not stronger predictive value with AUC 0.477 (95 % CI 0.329 to 0.625) for liver dysfunction. The AUC value of the total bilirubin (AUC 0.685, 95 % CI 0.546 to 0.825) in predicting early graft dysfunction outperformed other LFTs and was less than CRP (AUC 0.705, CI 0.573 to 0.838). The optimal cutoff value of total bilirubin obtained from the analysis of ROC curves was 4,5 and surpassed all other parameters with a sensitivity of 94.4% and a specificity of 40.7% respectively for prognoses of EGD (P=0.012). The univariate analysis determined that postoperative neutrophils level and CRP were identified as independent risk factors for early graft dysfunction. Neutrophils had a higher predictive value for liver dysfunction than any other parameter within 24 h (Odds ratio (OR) 16.3; 95% CI: 1.7-156.3, P = 0.016). Conclusion. Collectively, extracellular histone H3 levels were depressed, total bilirubin and CRP levels were elevated in patients with EGD, which can be used as early predictors for liver tissue damage and early allograft dysfunction in patients after liver transplantation.

ATRX limits the accessibility of histone H3-occupied HSV genomes during lytic infection

Histones are rapidly loaded on the HSV genome upon entry into the nucleus of human fibroblasts, but the effects of histone loading on viral replication have not been fully defined. We showed recently that ATRX is dispensable for de novo deposition of H3 to HSV genomes after nuclear entry but restricted infection through maintenance of viral heterochromatin. To further investigate the roles that ATRX and other histone H3 chaperones play in restriction of HSV, we infected human fibroblasts that were systematically depleted of nuclear H3 chaperones. We found that the ATRX/DAXX complex is unique among nuclear H3 chaperones in its capacity to restrict ICP0-null HSV infection. Only depletion of ATRX significantly alleviated restriction of viral replication. Interestingly, no individual nuclear H3 chaperone was required for deposition of H3 onto input viral genomes, suggesting that during lytic infection, H3 deposition may occur through multiple pathways. ChIP-seq for total histone H3 in control and ATRX-KO cells infected with ICP0-null HSV showed that HSV DNA is loaded with high levels of histones across the entire viral genome. Despite high levels of H3, ATAC-seq analysis revealed that HSV DNA is highly accessible, especially in regions of high GC content, and is not organized largely into ordered nucleosomes during lytic infection. ATRX reduced accessibility of viral DNA to the activity of a TN5 transposase and enhanced accumulation of viral DNA fragment sizes associated with nucleosome-like structures. Together, these findings support a model in which ATRX restricts viral infection by altering the structure of histone H3-loaded viral chromatin that reduces viral DNA accessibility for transcription. High GC rich regions of the HSV genome, especially the S component inverted repeats of the HSV-1 genome, show increased accessibility, which may lead to increased ability to transcribe the IE genes encoded in these regions during initiation of infection.

Cardiac Gq Receptors and Calcineurin Activation Are Not Required for the Hypertrophic Response to Mechanical Left Ventricular Pressure Overload

RationaleGq-coupled receptors are thought to play a critical role in the induction of left ventricular hypertrophy (LVH) secondary to pressure overload, although mechano-sensitive channel activation by a variety of mechanisms has also been proposed, and the relative importance of calcineurin- and calmodulin kinase II (CaMKII)-dependent hypertrophic pathways remains controversial.ObjectiveTo determine the mechanisms regulating the induction of LVH in response to mechanical pressure overload.Methods and ResultsTransgenic mice with cardiac-targeted inhibition of Gq-coupled receptors (GqI mice) and their non-transgenic littermates (NTL) were subjected to neurohumoral stimulation (continuous, subcutaneous angiotensin II (AngII) infusion for 14 days) or mechanical pressure overload (transverse aortic arch constriction (TAC) for 21 days) to induce LVH. Candidate signaling pathway activation was examined. As expected, LVH observed in NTL mice with AngII infusion was attenuated in heterozygous (GqI+/–) mice and absent in homozygous (GqI–/–) mice. In contrast, LVH due to TAC was unaltered by either heterozygous or homozygous Gq inhibition. Gene expression of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and α-skeletal actin (α-SA) was increased 48 h after AngII infusion or TAC in NTL mice; in GqI mice, the increases in ANP, BNP and α-SA in response to AngII were completely absent, as expected, but all three increased after TAC. Increased nuclear translocation of nuclear factor of activated T-cells c4 (NFATc4), indicating calcineurin pathway activation, occurred in NTL mice with AngII infusion but not TAC, and was prevented in GqI mice infused with AngII. Nuclear and cytoplasmic CaMKIIδ levels increased in both NTL and GqI mice after TAC but not AngII infusion, with increased cytoplasmic phospho- and total histone deacetylase 4 (HDAC4) and increased nuclear myocyte enhancer factor 2 (MEF2) levels.ConclusionCardiac Gq receptors and calcineurin activation are required for neurohumorally mediated LVH but not for LVH induced by mechanical pressure overload (TAC). Rather, TAC-induced LVH is associated with activation of the CaMKII-HDAC4-MEF2 pathway.

Cardiac Gq receptors and calcineurin activation are not required for the hypertrophic response to mechanical left ventricular pressure overload

RationaleGq-coupled receptors are thought to play a critical role in the induction of left ventricular hypertrophy (LVH) secondary to pressure overload, although mechano-sensitive channel activation by a variety of mechanisms has also been proposed, and the relative importance of calcineurin- and calmodulin kinase II (CaMKII)-dependent hypertrophic pathways remains controversial.ObjectiveTo determine the mechanisms regulating the induction of LVH in response to mechanical pressure overload.Methods and ResultsTransgenic mice with cardiac-targeted inhibition of Gq-coupled receptors (GqI mice) and their non-transgenic littermates (NTL) were subjected to neurohumoral stimulation (continuous, subcutaneous angiotensin II (AngII) infusion for 14 days) or mechanical pressure overload (transverse aortic arch constriction (TAC) for 21 days) to induce LVH. Candidate signalling pathway activation was examined. As expected, LVH observed in NTL mice with AngII infusion was attenuated in heterozygous (GqI+/-) mice and absent in homozygous (GqI-/-) mice. In contrast, LVH due to TAC was unaltered by either heterozygous or homozygous Gq inhibition. Gene expression of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and α-skeletal actin (α-SA) was increased 48 hours after AngII infusion or TAC in NTL mice; in GqI mice, the increases in ANP, BNP and α-SA in response to AngII were completely absent, as expected, but all three increased after TAC. Increased nuclear translocation of nuclear factor of activated T-cells c4 (NFATc4), indicating calcineurin pathway activation, occurred in NTL mice with AngII infusion but not TAC, and was prevented in GqI mice infused with AngII. Nuclear and cytoplasmic CaMKIIδ levels increased in both NTL and GqI mice after TAC but not AngII infusion, with increased cytoplasmic phospho- and total histone deacetylase 4 (HDAC4) and increased nuclear myocyte enhancer factor 2 (MEF2) levels.ConclusionCardiac Gq receptors and calcineurin activation are required for neurohumorally mediated LVH but are not required for LVH induced by mechanical pressure overload (TAC); the latter is mediated by activation of the CaMKII-HDAC4-MEF2 pathway.

The Prognostic Significance of Global Histones Deacetylation in Colorectal Cancer Patients Treated with Fluoropyrimdine Therapy

To study the impact of fluoropyrimidine (FP)- therapy on the acetylation level of histones (H3 and H4), and its correlation to treatment outcome in colorectal cancer (CRC) patients. Total histone protein was extracted, and the level of global histones (H3 and H4) acetylation was determined in the peripheral blood of 66 CRC patients before treatment with FP therapy, and also in 48 and 32 of those patients after 3 and 6 months of treatment, respectively. Clinicopathological stratification of patients was conducted for subgroup analysis. After three years of follow up, event-free survival (EFS) and the hazard of recurrence and progression were determined by Kaplan –Meier and COX regression analyses, respectively. Baseline CRC patients showed global H3 hyperacetylation by 160%, but H4 hypoacetylation by 87% relative to healthy control. FP therapy significantly reduced global H3 and H4 acetylation levels especially in subgroups of CRC patients > 45 years, females, with right colon tumours, with normal baseline levels of CEA and CA19.9, with negative lymph nodes and negative metastasis, and also in the patients who showed no signs of recurrence or progression after three years of follow up. Survival analyses showed decreased median EFS time and increased the hazard of recurrence and progression in patients with high CEA (HR= 3.38, P= 0.023), positive metastasis (HR= 1.16, P<0.001), and H4 hypoacetylation <87% (HR= 1.55, P=0.014). FP therapy-induced reduction in the global level of histones acetylation declared in subgroups of CRC patients with excellent prognostic features.

Reduced Activity of HDAC3 and Increased Acetylation of Histones H3 in Peripheral Blood Mononuclear Cells of Patients with Rheumatoid Arthritis

Aberrant histone acetylation and deacetylation are increasingly thought to play important roles in the pathogenesis of rheumatoid arthritis (RA). However, limited data from studies about the activity of histone deacetylases (HDACs) and histone acetyltransferase (HAT) in RA are controversial. Those conflicting results may be caused by sample size, medication, and age- and sex-matched controls. The aim of this study is to investigate the expression and activity of class I HDACs (1–3.8) and their effects on histone acetylation in peripheral blood mononuclear cells (PBMCs) from RA patients. The expression of class I HDACs in PBMCs from RA patients was decreased in both mRNA and protein levels in comparison with HCs. The nuclear HAT activities were dramatically increased. Further, we found HDAC3 activity to be the most significantly reduced in overall reduction of HDACs in the RA group. The extent of total histone H3, but not H4, acetylation in PBMCs from RA patients was increased compared to that in healthy controls (HCs) (p<0.01). In RA PBMCs, the activity and expression of class I HDACs are decreased, which is accompanied with enhanced HAT activity. An altered balance between HDAC and HAT activity was found in RA PBMCs.