Histone Acetyltransferase Inhibitor Research Articles

Chromatin Condensation Delays Senescence in Human Mesenchymal Stem Cells by Safeguarding Nuclear Damages during In Vitro Expansion

Human mesenchymal stem cells (hMSCs) are multipotent cells that differentiate into adipocytes, chondrocytes, and osteoblasts. Owing to their differentiation potential, hMSCs are among the cells most frequently used for therapeutic applications in tissue engineering and regenerative medicine. However, the number of cells obtained through isolation alone is insufficient for hMSC-based therapies and basic research, which necessitates in vitro expansion. Conventionally, this is often performed on rigid surfaces such as tissue culture plates (TCPs). However, during in vitro expansion, hMSCs lose their proliferative ability and multilineage differentiation potential, rendering them unsuitable for clinical use. Although multiple approaches have been attempted to maintain hMSC stemness during prolonged expansion, finding a suitable culture system remains an unmet need. Recently, a few research groups have shown that hMSCs maintain their stemness over long passages when cultured on soft substrates. In addition, it has been shown that hMSCs cultured on soft substrates have more condensed chromatin and lower levels of histone acetylation compared to those cultured on stiff substrates. Furthermore, it has also been shown that condensing/decondensing chromatin by deacetylation/acetylation can delay replicative senescence in hMSCs during long-term expansion on TCPs. However, the mechanism by which chromatin condensation/decondensation influences nuclear morphology and DNA damage, which are strongly related to the onset of senescence, remains unknown. To answer this question, we cultured hMSCs for long duration in the presence of epigenetic modifiers, histone acetyltransferase inhibitor (HATi), which promotes chromatin condensation by preventing histone acetylation, and histone deacetylase inhibitor (HDACi), which promotes chromatin decondensation, and investigated their effects on various nuclear markers related to senescence. We found that consistent acetylation causes severe nuclear abnormalities, whereas chromatin condensation by deacetylation helps to safeguard the nucleus from damage caused by in vitro expansion.

Abstract PO3-26-04: Discovery of novel inhibitors of the epigenetic regulatory KAT6A histone acetyl transferase and their anti-tumor activity in breast cancer cells

Abstract KAT6A is a histone acetyltransferase (HAT) which directs chromatin structural rearrangement and subsequent changes in gene transcription through acetylation of histones, primarily specific lysine residues on histone H3. KAT6A is modified in cancer, including genomic amplification, overexpression, mutation, as well as genetic rearrangement resulting in the production of fusion proteins. Dysregulation of this MYST family member and its acetyl transferase activity is known to drive gene expression and tumorigenic progression in cancers. An inhibitor of KAT6A and related MYST family member KAT6B has entered phase 1 clinical trials with durable partial responses seen in a variety of tumor types, including estrogen-receptor positive (ER+) breast cancer previously treated with a CDK4/6 inhibitor (Sommerhalder, ASCO2023). Evidence of dose dependent pharmacodynamics (H3K23Ac inhibition) was observed in peripheral blood mononuclear cells and paired tumor biopsies. Isosterix has developed 2 distinct series of KAT6A selective inhibitors which engage in the active site of KAT6A in the acetyl CoA substrate binding pocket and inhibit histone acetylation activity. The kinetics of target enzyme binding and inhibition will be discussed for both series of inhibitors. These compounds have been characterized for potency and cellular activity in a variety of assays. Both series of compounds inhibit KAT6A-mediated histone acetylation with single digit nM potency in a biochemical assay as well as significant potency in the breast cancer tumor cell line ZR-75-1, inhibiting H3K23 acetylation. Using a thermal shift assay (CETSA) and mass spectrometry-based protein quantitation, direct target engagement of KAT6A within cells has been shown. These compounds reduce cell viability of a number of breast cancer cell lines with 10 days of compound administration confirming the dependence of these tumor cell lines on KAT6A acetyl transferase activity for proliferation and cell cycle progression. Compounds are orally bioavailable with excellent pharmacokinetic (PK) properties in mice including half-lives >5 hours with oral administration. Finally, Isosterix inhibitors demonstrated anti-tumor efficacy with oral administration in a ZR-75-1 mouse xenograft tumor model. Inhibitors against this novel epigenetic target demonstrate the potential for efficacy in ER+ breast cancer as well as other tumor indications through suppression of KAT6A-directed transcriptional induction and chromatin remodeling. Table. Compound Kat6A Biochemical IC50 (µM) ZR-75-1 H3K23 Acetylation EC50 (µM) Mouse PK (p.o.) Mouse PK (i.v.) Inhibitor class %F T½ (h) Cl (mL/min/kg) SL-IST-001 0.007 0.01 100 5.2 0.47 Series 1 SL-IST-002 0.007 0.005 100 6 0.48 Series 2 Citation Format: Leslie Holsinger, Roopa RaI, Rahul R. Nagawade, Benoy Pal, Samir Pawar, Pankaj Singh, Krishna Putta, Mahaboobi Jaleel, Tove Bostrom, Jakob Karen, Qianyang Huang, Alison Hannah. Discovery of novel inhibitors of the epigenetic regulatory KAT6A histone acetyl transferase and their anti-tumor activity in breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-04.

Group 3 medulloblastoma transcriptional networks collapse under domain specific EP300/CBP inhibition

Chemical discovery efforts commonly target individual protein domains. Many proteins, including the EP300/CBP histone acetyltransferases (HATs), contain several targetable domains. EP300/CBP are critical gene-regulatory targets in cancer, with existing high potency inhibitors of either the catalytic HAT domain or protein-binding bromodomain (BRD). A domain-specific inhibitory approach to multidomain-containing proteins may identify exceptional-responding tumor types, thereby expanding a therapeutic index. Here, we discover that targeting EP300/CBP using the domain-specific inhibitors, A485 (HAT) or CCS1477 (BRD) have different effects in select tumor types. Group 3 medulloblastoma (G3MB) cells are especially sensitive to BRD, compared with HAT inhibition. Structurally, these effects are mediated by the difluorophenyl group in the catalytic core of CCS1477. Mechanistically, bromodomain inhibition causes rapid disruption of genetic dependency networks that are required for G3MB growth. These studies provide a domain-specific structural foundation for drug discovery efforts targeting EP300/CBP and identify a selective role for the EP300/CBP bromodomain in maintaining genetic dependency networks in G3MB.

The pharmacoepigenetic paradigm in cancer treatment.

Epigenetic modifications, characterized by changes in gene expression without altering the DNA sequence, play a crucial role in the development and progression of cancer by significantly influencing gene activity and cellular function. This insight has led to the development of a novel class of therapeutic agents, known as epigenetic drugs. These drugs, including histone deacetylase inhibitors, histone acetyltransferase inhibitors, histone methyltransferase inhibitors, and DNA methyltransferase inhibitors, aim to modulate gene expression to curb cancer growth by uniquely altering the epigenetic landscape of cancer cells. Ongoing research and clinical trials are rigorously evaluating the efficacy of these drugs, particularly their ability to improve therapeutic outcomes when used in combination with other treatments. Such combination therapies may more effectively target cancer and potentially overcome the challenge of drug resistance, a significant hurdle in cancer therapy. Additionally, the importance of nutrition, inflammation control, and circadian rhythm regulation in modulating drug responses has been increasingly recognized, highlighting their role as critical modifiers of the epigenetic landscape and thereby influencing the effectiveness of pharmacological interventions and patient outcomes. Epigenetic drugs represent a paradigm shift in cancer treatment, offering targeted therapies that promise a more precise approach to treating a wide spectrum of tumors, potentially with fewer side effects compared to traditional chemotherapy. This progress marks a step towards more personalized and precise interventions, leveraging the unique epigenetic profiles of individual tumors to optimize treatment strategies.

Reverse Mechanotransduction: Driving Chromatin Compaction to Decompaction Increases Cell Adhesion Strength and Contractility.

Mechanical extracellular signals elicit chromatin remodeling via the mechanotransduction pathway, thus determining cellular function. However, the reverse pathway is an open question: does chromatin remodeling shape cells, regulating their adhesion strength? With fluidic force microscopy, we can directly measure the adhesion strength of epithelial cells by driving chromatin compaction to decompaction with chromatin remodelers. We observe that chromatin compaction, induced by performing histone acetyltransferase inhibition or ATP depletion, leads to a reduction in nuclear volume, disrupting actin cytoskeleton and focal adhesion assembly, and ultimately decreases in cell adhesion strength and traction force. Conversely, when chromatin decompaction is drived by removing the remodelers, cells recover their original shape, adhesion strength, and traction force. During chromatin decompaction, cells use depolymerized proteins to restore focal adhesion assemblies rather than neo-synthesized cytoskeletal proteins. We conclude that chromatin remodeling shapes cells, regulating adhesion strength through a reverse mechanotransduction pathway from the nucleus to the cell surface involving RhoA activation.

Abstract 3217: Discovery of novel inhibitors of the epigenetic regulator Kat6a histone acetyl transferase and characterization of their anti-tumor activity

Abstract KAT6A and its paralog KAT6B are histone acetyltransferases (HATs) which direct chromatin structural rearrangement and subsequent changes in gene transcription through acetylation of histones, primarily specific lysine residues on histone H3. KAT6A is modified in cancer, including genomic amplification, overexpression, mutation, as well as genetic rearrangement, resulting in the production of fusion proteins. Dysregulation of this MYST family member and its acetyl transferase activity is known to drive gene expression and tumorigenic progression in cancers. An inhibitor of KAT6A and KAT6B entered phase 1 clinical trials (NCT04606446). With continuous daily dosing, durable partial responses were seen in estrogen-receptor positive (ER+) breast cancer patients who had progressed on a prior endocrine therapy and CDK4/6 inhibitor. In that study, evidence of dose dependent pharmacodynamics (H3K23Ac inhibition) was observed in peripheral blood mononuclear cells and paired tumor biopsies (Sommerhalder, ASCO2023). Isosterix has developed a series of structurally diverse small molecule KAT6A-selective inhibitors which engage at the active site in the acetyl CoA substrate binding pocket. Lead compounds are selective for KAT6A and have significant potency and cellular activity in a variety of assays; they inhibit KAT6A-mediated histone acetylation in a biochemical assay as well as pharmacodynamic dose-dependent inhibition of histone acetylation in the breast cancer tumor cell line ZR-75-1. These compounds reduce cell viability of ZR-75-1 in 10 days with continuous compound administration, confirming the dependence of this cell line on KAT6A acetyl transferase activity for proliferation and cell cycle progression. Lead inhibitors are orally bioavailable with excellent pharmacokinetic properties in rodents. They demonstrate dose-dependent anti-tumor efficacy with minimal weight loss with continuous daily dosing in mouse xenograft tumor models. Inhibitors against this novel epigenetic target demonstrate the potential for efficacy in not only ER+ breast cancer but also other tumor indications which exhibit transcriptional induction dependent on KAT6A-directed chromatin remodeling. Selection of a lead candidate is planned for early 2024 with IND enabling studies to follow. Citation Format: Leslie J. Holsinger, Roopa Rai, Rahul R. Nagawade, Benoy K. Pal, Samir G. Pawar, Krishna Anand Putta, Somashekar S.C., Mahaboobi Jaleel, Jianhong Wang, Alison L. Hannah. Discovery of novel inhibitors of the epigenetic regulator Kat6a histone acetyl transferase and characterization of their anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3217.

Abstract 5327: Interleukin-1 upregulates dual oxidase 2 expression and ROS production in human pancreatic cancer cells

Abstract Chronic pancreatitis increases the risk of developing pancreatic ductal adenocarcinoma (PDAC) and is associated with enhanced expression of the NADPH oxidase (NOX) isoform dual oxidase 2 (DUOX2). Interleukin-1 (IL-1) is a major upstream pro-inflammatory cytokine secreted by malignant or microenvironmental cells that supports pancreatic inflammation, tumorigenesis, invasiveness, and tumor heterogeneity. Previously, our laboratory demonstrated that the expression of DUOX2, one of seven NADPH oxidase (NOX) family members, is significantly enhanced by a wide variety of pro-inflammatory cytokines, and that DUOX2-related ROS production may contribute to the development of an oxidative, pro-angiogenic microenvironment in PDAC. In the current studies, we have shown that DUOX2 is upregulated and expressed along with its maturation factor, DUOXA2, in several human pancreatic cancer cell lines following exposure to both IL-1α and IL-1ß, two major agonists of IL-1 signaling. Administration of the anti-inflammatory antibody anakinra (an IL-1R1 antagonist) either in conjunction with or after prolonged treatment with IL-1 in vitro ablated induction of DUOX2/DUOXA2 and of IL-8 (CXCL8), a chemokine essential for neutrophil recruitment and a potent promoter of angiogenesis. Increased expression of other NOX family members following IL-1 exposure was not observed. Using CRISPR/Cas9-mediated DUOX2 knockout models, we report that DUOX2 is the major contributor to IL-1-mediated ROS production in PDAC cells. In a panel of PDAC lines, siRNA knockdown of proximal IL-1 signaling nodes, such as MYD88, significantly decreased expression of DUOX2/DUOXA2 and of CXCL8. However, knockdown of IRAK1 or IRAK4, two critical signaling kinases downstream of MYD88, as well as known downstream mediators of IL-1-related transcription (such as NF-κB, STAT1, MEK/ERK), did not affect IL-1-induced DUOX2/DUOXA2 upregulation, suggesting that alternative pathways of IL-1 signaling might be involved in the enhancement of DUOX2 expression by the cytokine. Because IL-1 signaling is known to affect various acetylation/methylation events in epithelial cells, we evaluated the effect of administering the histone acetyltransferase (HAT) inhibitor A-485 with IL-1; we found that A-485 significantly diminished cytokine-induced DUOX2/DUOXA2 upregulation; however, CXCL8 induction was unchanged. These studies suggest that epigenetic modulation of PDAC cells by IL-1 could contribute to DUOX2-induced ROS production and ROS-related inflammatory stress in the PDAC microenvironment. Citation Format: David J. Mallick, Yongzhong Wu, Mariam M. Konaté, Becky Diebold, Smitha Antony, Jennifer L. Meitzler, Guojian Jiang, Jiamo Lu, Krishnendu Roy, James H. Doroshow. Interleukin-1 upregulates dual oxidase 2 expression and ROS production in human pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5327.

Abstract 564: Therapeutic targeting of JAK/STAT signaling and histone acetyltransferase activity in post-myeloproliferative neoplasm secondary AML

Abstract Post-myeloproliferative neoplasm secondary Acute Myeloid Leukemia (Post-MPN sAML) is an aggressive and lethal hematologic malignancy arising from myeloproliferative neoplasms (MPN). Hyperactivation of JAK/STAT signaling was identified in 50-90% of MPNs - underscoring its role in driving MPN pathogenesis and transformation to post-MPN sAML. Ruxolitinib, a Type-I JAK2 inhibitor approved for the treatment of myelofibrosis, was tested in a Phase II clinical trial in post-MPN sAML. The trial revealed that ruxolitinib improved the quality of life in patients but was not disease-modifying. Thus, there is an urgent need to find synergistic drug combinations capable of complementing the therapeutic activity of JAK2 inhibitors in post-MPN sAML. Here, we demonstrate that combination treatment of ruxolitinib and CBP30, a bromodomain inhibitor of histone acetyltransferases (HAT) CBP and p300, enhances the therapeutic activity of ruxolitinib in post-MPN sAML. Genome-wide CRISPR loss-of-function screens in JAK2-mutated HEL cells determined that knockout of CBP sensitizes leukemic cells to JAK2 inhibitors. Consistently, functional validation through CRISPR/Cas9 demonstrated that genetic deletion of CBP but not of p300 renders HEL cells sensitive to ruxolitinib treatment. Pharmacologic inhibition of JAK/STAT signaling and HAT activity through ruxolitinib and CBP30 treatment in post-MPN sAML lines HEL and SET2 decreases proliferation and induces apoptosis and cell cycle arrest. Mechanistically, the synergism between ruxolitinib and CBP30 elicits a unique transcriptome profile in HEL with significant downregulation of E2F and MYC targets. In addition, epigenetic profiling revealed that the synergism induces global changes in H3K27ac signals with downregulation and upregulation of H3K27ac signals in predicted STAT5/BCL3 and GATA-bounded regions, respectively. Remarkably, combination treatment of ruxolitinib and CBP30 in HEL luciferase NSG mouse xenografts showed significant leukemia regression and decrease in spleen size compared to mice treated with vehicle or single agents. Overall, these results substantiate the potential of targeting JAK/STAT signaling and histone acetyltransferase activity in the treatment of post-MPN sAML. Citation Format: Kalay Bertulfo, Koen Debackere, Hannah Miller, Cindy Ma, Ryan Najac, Adolfo Ferrando, Teresa Palomero. Therapeutic targeting of JAK/STAT signaling and histone acetyltransferase activity in post-myeloproliferative neoplasm secondary AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 564.

Electroacupuncture regulates histone acetylation of Bcl-2 and Caspase-3 genes to improve ischemic stroke injury

BackgroundImbalances between Bcl-2 and caspase-3 are significant evidence of apoptosis, which is considered an influential factor in rapidly occurring neuronal cell death and the decline of neurological function after stroke. Studies have shown that acupuncture can reduce poststroke brain cell damage via either an increase in Bcl-2 or a reduction in caspase-3 exposure. The current study aimed to investigate whether acupuncture could modulate Bcl-2 and caspase-3 expression through histone acetylation modifications, which could potentially serve as a neuroprotective mechanism. MethodsThis study used TTC staining, Nissl staining, Clark neurological system score, and Evans Blue (EB) extravasation to evaluate neurological damage following stroke. The expression of Bcl-2/caspase-3 mRNA was detected by real-time fluorescence quantification of PCR (real-time PCR), whereas the protein expression levels of Bcl-2, Bax, caspase-3, and cleaved caspase-3 were assessed using western blotting. TUNEL staining of the ischemic cortical neurons determined apoptosis in the ischemic cortex. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities, along with the protein performance of AceH3, H3K9ace, and H3K27ace, were detected to evaluate the degree of histone acetylation. The acetylation enrichment levels of H3K9 and K3K27 in the Bcl-2/caspase-3 gene were assessed using Chromatin Immunoprecipitation (ChIP) assay. ResultsOur data demonstrated that electroacupuncture (EA) exerts a significant neuroprotective effect in middle cerebral artery occlusion (MCAO) rats, as evidenced by a reduction in infarct volume, neuronal damage, Blood-Brain Barrier (BBB) disruption, and decreased apoptosis of ischemic cortical neurons. EA treatment can promote the mRNA and protein expression of the Bcl-2 gene in the ischemic brain while reducing the mRNA and protein expression levels of caspase-3 and effectively decreasing the protein expression levels of Bax and cleaved caspase-3. More importantly, EA treatment enhanced the level of histone acetylation, including Ace-H3, H3K9ace, and H3K27ace, significantly enhanced the occupancy of H3K9ace/H3K27ace at the Bcl-2 promoter, and reduced the enrichment of H3K9ace and H3K27ace at the caspase-3 promoter. However, the Histone Acetyltransferase inhibitor (HATi) treatment reversed these effects. ConclusionsOur data demonstrated that EA mediated the expression levels of Bcl-2 and caspase-3 in MCAO rats by regulating the occupancy of acetylated H3K9/H3K27 at the promoters of these two genes, thus exerting a cerebral protective effect in ischemic reperfusion (I/R) injury.

Anserine, a Histidine-Containing Dipeptide, Suppresses Pressure Overload-Induced Systolic Dysfunction by Inhibiting Histone Acetyltransferase Activity of p300 in Mice.

Anserine, an imidazole dipeptide, is present in the muscles of birds and fish and has various bioactivities, such as anti-inflammatory and anti-fatigue effects. However, the effect of anserine on the development of heart failure remains unknown. We cultured primary cardiomyocytes with 0.03 mM to 10 mM anserine and stimulated them with phenylephrine for 48 h. Anserine significantly suppressed the phenylephrine-induced increases in cardiomyocyte hypertrophy, ANF and BNP mRNA levels, and histone H3K9 acetylation. An in vitro histone acetyltransferase (HAT) assay showed that anserine directly suppressed p300-HAT activity with an IC50 of 1.87 mM. Subsequently, 8-week-old male C57BL/6J mice were subjected to transverse aortic constriction (TAC) and were randomly assigned to receive daily oral treatment with anserine-containing material, Marine Active® (60 or 200 mg/kg anserine) or vehicle for 8 weeks. Echocardiography revealed that anserine 200 mg/kg significantly prevented the TAC-induced increase in left ventricular posterior wall thickness and the decrease in left ventricular fractional shortening. Moreover, anserine significantly suppressed the TAC-induced acetylation of histone H3K9. These results indicate that anserine suppresses TAC-induced systolic dysfunction, at least in part, by inhibiting p300-HAT activity. Anserine may be used as a pharmacological agent for human heart failure therapy.

HDAC2 counteracts vascular calcification by activating autophagy in chronic kidney disease.

Vascular calcification is a major risk factor for cardiovascular disease mortality, with a significant prevalence in chronic kidney disease (CKD). Pharmacological inhibition of histone acetyltransferase has been proven to protect against from vascular calcification. However, the role of Histone Deacetylase 2 (HDAC2) and molecular mechanisms in vascular calcification of CKD remains unknown. An invivo model of CKD was established using mouse fed with a high adenine and phosphate diet, and an invitro model was produced using human aortic vascular smooth muscle cells (VSMCs) stimulated with β-glycerophosphate (β-GP). HDAC2 expression was found to be reduced in medial artery of CKD mice and β-GP-induced VSMCs. Overexpression of HDAC2 attenuated OPN and OCN upregulation, α-SMA and SM22α downregulation, and calcium deposition in aortas of CKD. The invitro results also demonstrated that β-GP-induced osteogenic differentiation was inhibited by HDAC2. Furthermore, we found that HDAC2 overexpression caused an increase in LC3II/I, a decrease in p62, and an induction of autophagic flux. Inhibition of autophagy using its specific inhibitor 3-MA blocked HDAC2's protective effect on osteogenic differentiation in β-GP-treated VSMCs. Taken together, these results suggest that HDAC2 may protect against vascular calcification by the activation of autophagy, laying out a novel insight for the molecular mechanism in vascular calcification of CKD.

Rescuing lung development through embryonic inhibition of histone acetylation.

A major barrier to the impact of genomic diagnosis in patients with congenital malformations is the lack of understanding regarding how sequence variants contribute to disease pathogenesis and whether this information could be used to generate patient-specific therapies. Congenital diaphragmatic hernia (CDH) is among the most common and severe of all structural malformations; however, its underlying mechanisms are unclear. We identified loss-of-function sequence variants in the epigenomic regulator gene SIN3A in two patients with complex CDH. Tissue-specific deletion of Sin3a in mice resulted in defects in diaphragm development, lung hypoplasia, and pulmonary hypertension, the cardinal features of CDH and major causes of CDH-associated mortality. Loss of SIN3A in the lung mesenchyme resulted in reduced cellular differentiation, impaired cell proliferation, and increased DNA damage. Treatment of embryonic Sin3a mutant mice with anacardic acid, an inhibitor of histone acetyltransferase, reduced DNA damage, increased cell proliferation and differentiation, improved lung and pulmonary vascular development, and reduced pulmonary hypertension. These findings demonstrate that restoring the balance of histone acetylation can improve lung development in the Sin3a mouse model of CDH.

Small-molecule CBP/p300 histone acetyltransferase inhibition mobilizes leukocytes from the bone marrow via the endocrine stress response

Mutations of the CBP/p300 histone acetyltransferase (HAT) domain can be linked to leukemic transformation in humans, suggestive of a checkpoint of leukocyte compartment sizes. Here, we examined the impact of reversible inhibition of this domain by the small-molecule A485. We found that A485 triggered acute and transient mobilization of leukocytes from the bone marrow into the blood. Leukocyte mobilization byA485 was equally potent as, but mechanistically distinct from, granulocyte colony-stimulating factor (G-CSF), which allowed for additive neutrophil mobilization when both compounds were combined. These effects were maintained in models of leukopenia and conferred augmented host defenses. Mechanistically, activation of the hypothalamus-pituitary-adrenal gland (HPA) axis by A485 relayed shifts in leukocyte distribution through corticotropin-releasing hormone receptor 1 (CRHR1) and adrenocorticotropic hormone (ACTH), but independently of glucocorticoids. Our findings identify a strategy for rapid expansion of the blood leukocyte compartment via a neuroendocrine loop, with implications for the treatment of human pathologies.

Balanced spatiotemporal arrangements of histone H3 and H4 posttranslational modifications are necessary for meiotic prophase I chromosome organization.

Dynamic nuclear architecture and chromatin organizations are the key features of the mid-prophase I in mammalian meiosis. The chromatin undergoes major changes, including meiosis-specific spatiotemporal arrangements and remodeling, the establishment of chromatin loop-axis structure, pairing, and crossing over between homologous chromosomes, any deficiencies in these events may induce genome instability, subsequently leading to failure to produce gametes and infertility. Despite the significance of chromatin structure, little is known about the location of chromatin marks and the necessity of their balance during meiosis prophase I. Here, we show a thorough cytological study of the surface-spread meiotic chromosomes of mouse spermatocytes for H3K9,14,18,23,27,36, H4K12,16 acetylation, and H3K4,9,27,36 methylation. Active acetylation and methylation marks on H3 and H4, such as H3K9ac, H3K14ac, H3K18ac, H3K36ac, H3K56ac, H4K12ac, H4K16ac, and H3K36me3 exhibited pan-nuclear localization away from heterochromatin. In comparison, repressive marks like H3K9me3 and H3K27me3 are localized to heterochromatin. Further, taking advantage of the delivery of small-molecule chemical inhibitors methotrexate(heterochromatin enhancer), heterochromatin inhibitor, anacardic acid (histone acetyltransferase inhibitor), trichostatin A (histone deacetylase inhibitor), IOX1 (JmjC demethylases inhibitor), and AZ505 (methyltransferase inhibitor) in seminiferous tubules through the rete testis route, revealed that alteration in histone modifications enhanced the centromere mislocalization, chromosome breakage, altered meiotic recombination and reduced sperm count. Specifically, IOX1 and AZ505 treatment shows severe meiotic phenotypes, including altering chromosome axis length and chromatin loop size via transcriptional regulation of meiosis-specific genes. Our findings highlight the importance of balanced chromatin modifications in meiotic prophase I chromosome organization and instability.

Altered histone acetylation patterns in pancreatic cancer cell lines induce subtype‑specific transcriptomic and phenotypical changes.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at advanced tumor stages with chemotherapy as the only treatment option. Transcriptomic analysis has defined a classical and basal‑like PDAC subtype, which are regulated by epigenetic modification. The present study aimed to determine if drug‑induced epigenetic reprogramming of pancreatic cancer cells affects PDAC subtype identity and chemosensitivity. Classical and basal‑like PDAC cell lines PaTu‑S, Capan‑1, Capan‑2, Colo357, PaTu‑T, PANC‑1 and MIAPaCa‑2, were treated for a short (up to 96h) and long (up to 30weeks) period with histone acetyltransferase (HAT) and histone deacetylase (HDAC) inhibitors. The cells were analyzed using gene expression approaches, immunoblot analysis, and various cell assays to assess cell characteristics, such as proliferation, colony formation, cell migration and sensitivity to chemotherapeutic drugs. Classical and basal‑like PDAC cell lines showed pronounced epigenetic regulation of subtype‑specific genes through acetylation of lysine 27 on Histone H3 (H3K27ac). Moreover, classical cell lines revealed a significantly decreased expression of HDAC2 and increased total levels of H3K27ac in comparison with the basal‑like cell lines. Following HAT inhibitor treatment, classical cell lines exhibited a loss of epithelial marker gene expression, decreased chemotherapy response gene score and increased cell migration invitro, indicating a tumor‑promoting phenotype. HDAC inhibitor treatment, however, exerted minimal reprogramming effects in both subtypes. Epigenetic reprogramming of classical and basal‑like tumor cells did not have a major impact on gemcitabine response, although the gemcitabine transporter gene SLC29A1 (solute carrier family 29 member 1) was epigenetically regulated.

Novel berberine derivatives as p300 histone acetyltransferase inhibitors in combination treatment for breast cancer

Adenoviral E1A binding protein p300 (EP300 or p300) and its similar paralog, cyclic-AMP response element binding protein (CBP), are important histone acetyltransferases (HAT) and transcriptional co-activators in epigenetics, participating in numerous cellular pathways including proliferation, differentiation and apoptosis. The overexpression or dysregulation of p300/CBP is closely related to oncology-relevant disease. The inhibition of p300 HAT has been found to be a potential drug target. Berberine has been reported to show anticancer activity and synergistic effect in combination with some of the clinical anticancer drugs via modulation of various pathways. Here, the present study sought to discover more chemotypes of berberine derivatives as p300 HAT inhibitors and to examine the combination of these novel analogues with doxorubicin for the treatment of breast cancer. A series of novel berberine derivatives with modifications of A/B/D rings of berberine have been designed, synthesized and screened. Compound 7b was found to exhibit inhibitory potency against p300 HAT with IC50 values of 1.51 μM. Western blotting proved that 7b decreased H3K27Ac and interfered with the expression of oncology-relevant protein in MCF-7 cells. Further bioactive evaluation showed that combination of compound 7b with doxorubicin could significantly inhibit tumor growth and invasion in vitro and in vivo.

Effects of histone acetyltransferase (HAT) and histone deacetylase (HDAC) inhibitors on proliferative, differentiative, and regenerative functions of Toll-like receptor 2 (TLR-2)-stimulated human dental pulp cells (hDPCs).

This in vitro study aimed to modify TLR-2-mediated effects on the paracrine, proliferative, and differentiation potentials of human dental pulp-derived cells using histone acetyltransferase (HAT) and histone deacetylase (HDAC) inhibitors. Cell viability was assessed using the XTT assay. Cells were either treated with 10μg/ml Pam3CSK4 only, or pre-treated with valproic acid (VPA) (3mM), trichostatin A (TSA) (3μM), and MG-149 (3μM) for a total of 4h and 24h. Control groups included unstimulated cells and cells incubated with inhibitors solvents only. Transcript levels for NANOG, OCT3-4, FGF-1 and 2, NGF, VEGF, COL-1A1, TLR-2, hβD-2 and 3, BMP-2, DSPP, and ALP were assessed through qPCR. After 24h, TSA pre-treatment significantly upregulated the defensins and maintained the elevated pro-inflammatory cytokines, but significantly reduced healing and differentiation genes. VPA significantly upregulated the pro-inflammatory cytokine levels, while MG-149 significantly downregulated them. Pluripotency genes were not significantly affected by any regimen. At the attempted concentrations, TSA upregulated the defensins gene expression levels, and MG-149 exerted a remarkable anti-inflammatory effect; therefore, they could favorably impact the immunological profile of hDPCs. Targeting hDPC nuclear function could be a promising option in the scope of the biological management of inflammatory pulp diseases.

Histone H3 Acetylation Is Involved in Retinoid Acid-Induced Neural Differentiation through Increasing Mitochondrial Function.

Histone acetylation and mitochondrial function contribute importantly to neural differentiation, which is critically associated with neurodevelopmental disorders such as Down Syndrome (DS). However, whether and how histone acetylation regulates mitochondrial function and further affects neural differentiation has not been well described. In this study, when treated with retinoid acid (RA), the human neuroblastoma SH-SY5Y cell line was used as a neural differentiation model. We found that the acetylation of histone H3, especially H3 lysine 14 acetylation (H3K14ac), and mitochondrial function, including biogenesis and electron transport chain, were enhanced during neural differentiation. Specific inhibition of histone acetyltransferases (HATs) induced neural differentiation deficits, accompanied by downregulation of mitochondrial function. Furthermore, RA receptors (RARs) interacting with HATs were involved in the increased H3K14ac and the enhanced mitochondrial function during the neural differentiation process. Finally, receptor-interacting protein 140 (RIP140), a co-repressor of RARs, was also involved in regulating histone acetylation. RIP140 overexpression inhibited histone acetylation and mediated negative feedback on target genes which are involved in RA signaling. These findings evidenced that when interacting with RARs which had been negatively regulated by RIP140, RA promoted neural differentiation by promoting H3K14ac and enhanced mitochondrial function. This provides a molecular foundation for further investigations into abnormal neural development.

Histone Acetyltransferase and Deacetylase Inhibitors—New Aspects and Developments

Epigenetic processes modulate gene transcription and genomic stability, ensuring proper cell development and differentiation [...]

Therapeutic Targeting of JAK/STAT Signaling and Histone Acetyltransferase Activity in Post-Myeloproliferative Neoplasm Secondary AML

Post-myeloproliferative neoplasm secondary Acute Myeloid Leukemia (Post-MPN sAML) is an aggressive and lethal hematologic malignancy arising from myeloproliferative neoplasms (MPN). Hyperactivation of JAK/STAT signaling is present in 50-90% of MPNs, underscoring its role in driving MPN pathogenesis and transformation to acute sAML. Ruxolitinib, a Type-I JAK2 inhibitor approved for the treatment of myelofibrosis, was tested in a Phase II clinical trial in post-MPN sAML. Despite enhancing patients' quality of life, ruxolitinib did not exert major improvements on disease outcome (Eghtedar et al, 2012). Thus, there is an urgent need to find synergistic drug combinations capable of complementing the therapeutic activity of JAK2 inhibitors in post-MPN sAML. Here, we demonstrate that combination treatment of ruxolitinib and CBP30, a bromodomain inhibitor of histone acetyltransferases (HAT) CREBBP and p300, enhances the therapeutic activity of ruxolitinib in post-MPN sAML using high throughput genetic screens, in vitro functional assays, transcriptomic and epigenetic profiling and mouse xenograft in vivo experiments. To identify druggable determinants of JAK2 inhibitor response in post-MPN sAML, we performed genome-wide loss-of-function CRISPR screens in sAML HEL cells treated with four different JAK2 inhibitors, including ruxolitinib. We discovered that depletion of the histone acetyl transferase CREBBP sensitizes HEL cells to JAK2 inhibition (Figure 1A). Pharmacological inhibition of JAK/STAT signaling and HAT activity using a combination of ruxolitinib and CREBBP inhibitors significantly reduced the proliferation of sAML cells HEL and SET2 (Figure 1B). Among the CREBBP inhibitors, CBP30 synergizes best with ruxolitinib having Chou-Talalay combination indices of 0.152 and 0.704 in HEL and SET2, respectively. Moreover, combination treatment of ruxolitinib and CBP30 markedly increased apoptosis and induced cell cycle arrest at G1 in HEL. To mechanistically characterize the synergism between ruxolitinib and CBP30, we performed ChIP-seq to profile STAT5 binding and H3K27ac levels in HEL cells treated with DMSO, ruxolitinib, CBP30 and their combination. STAT5 binding profiles did not reveal major differences in ruxolitinib versus ruxolitinib + CBP30 treated cells, suggesting that the drug combination does not significantly alter STAT5 binding. Conversely, we noticed striking global changes in H3K27ac levels in the combination compared with DMSO or single agents, indicating that the synergistic effect between ruxolitinib and CBP30 is mediated by substantial changes in transcriptional regulation. Notably, analysis of H3K27ac downregulated and upregulated sites identified enrichments in motifs recognized by FOS/SMAD4 and GATA transcription factors, respectively. These TFs are involved in the regulation of myeloid differentiation implicating a synergistic regulation of relevant myeloid differentiation pathways by the combination of JAK2 and CREBBP inhibitors in sAML. Furthermore, ruxolitinib + CBP30 treatment in HEL elicits a unique transcriptome profile with expression deregulation of genes associated with cell cycle, DNA repair, HAT activity and TGF-beta signaling as determined by DAVID functional annotation analysis. Lastly, to evaluate the synergistic effects of ruxolitinib and CBP30 in vivo, we treated luciferase-expressing HEL sAML mouse xenografts with vehicle, ruxolitinib (80 mg/kg), CBP30 (30 mg/kg) and ruxolitinib (80 mg/kg) + CBP30 (30 mg/kg). Combination of ruxolitinib and CBP30 displayed significant decrease in leukemia burden after 4 weeks of treatment as monitored by bioluminescence imaging and quantification. Consistently, mice treated with both drugs also showed remarkable decrease in their white blood cell count and spleen size compared with single-agent-treated and vehicle-treated mice. Collectively, our results substantiate the combinatorial therapeutic targeting of JAK/STAT signaling and HAT activity as a potential treatment approach for post-MPN sAML.