HDAC Inhibitors Research Articles

Safety and preliminary efficacy of ivaltinostat, an HDAC inhibitor, plus capecitabine in patients with pretreated metastatic pancreatic adenocarcinoma (mPDAC): Results of a phase Ib study.

742 Background: Front-line chemotherapy for patients (pts) with mPDAC typically consists of mFOLFIRINOX or gemcitabine/nab-paclitaxel given until disease progression or intolerable toxicity. For pts with durable disease control on front-line treatment, maintenance therapy that can effectively delay disease progression while preserving quality of life with minimal cumulative toxicity is highly desirable. Ivaltinostat (ival) is a pan-histone deacetylase inhibitor that increases histone acetylation, suppresses cell proliferation, and promotes apoptosis. Preclinical data demonstrated synergy with 5-FU/capecitabine (cape) in mouse models. We report here results of the Phase Ib portion of a Ib/2 trial evaluating the safety and recommended phase 2 dose (RP2D) of this combination to use in a randomized study of ival + cape vs cape as maintenance therapy. Methods: Key eligibility criteria for phase 1b included unresectable or metastatic PDAC; ≥18 years old; ≥1 prior therapy; ECOG PS 0-1; and no known germline BRCA1/2 mutation. Three cohorts were enrolled, receiving ivaltinostat at 60, 125, or 250mg/m 2 iv (weekly on days 1 and 8) in combination with cape (1000mg/m 2 po BID on days 1-14) of a 21-day cycle. 1o objectives: safety, tolerability and RP2D; 2o objectives: pharmacokinetics (PK) of ival + cape. Results: A total of 28 patients were enrolled at the 60 (n=6), 125 (n=10) and 250 (n=12) mg/m 2 ival dose levels. Median prior lines of therapy: 2 (range 1-7). Median age: 67 years (range 50-83). Only one DLT (G4 thrombocytopenia) was observed at 250mg/m 2 dose level. Treatment-emergent AE profile was similar across dose levels, with common TEAEs including fatigue (n=15, 53.6%), nausea (n=14, 50.0%), palmar-plantar erythrodysesthesia syndrome (n=12, 42.9%), diarrhea (n=10, 35.7%), and constipation (n=9, 32.1%). G3 AEs included neutropenia (n=6, 21.4%), anemia (n=4, 14.3%), abdominal pain (n=3, 10.7%) and diarrhea (n=3, 10.7%). G4 AEs included 1 (3.6%) event of neutropenia and 1 (3.6%) of thrombocytopenia. No SAEs related to study drugs occurred in any of the 3 dose cohorts. PK analyses indicated dose proportional increases in exposure with increasing doses of ival. Histone H3K27 acetylation for ival showed the similar trend in optical density evaluation. Across all dose levels and prior treatments, median OS and PFS were 9.6 and 3.3 months, respectively. Conclusions: The combination of ival and cape was generally well tolerated in this heavily pretreated patient population. Ival 250 mg/m 2 is the dose selected for the ongoing randomized study of ival + cape vs cape in the maintenance setting for mPDAC pts post-FOLFIRINOX, which is expected to finish accrual in 2025. Clinical trial information: NCT05249101 .

Lactylation of HDAC1 Confers Resistance to Ferroptosis in Colorectal Cancer.

Colorectal cancer (CRC) is highly resistant to ferroptosis, which hinders the application of anti-ferroptosis therapy. Through drug screening, it is found that histone deacetylase inhibitor (HDACi) significantly sensitized CRC to ferroptosis. The combination of HDACi and ferroptosis inducers synergically suppresses CRC growth both in vivo and in vitro. Mechanically, HDACi reduces ferroptosis suppressor protein (FSP1) by promoting its mRNA degradation. Specifically, it is confirmed that HDACi specifically targets HDAC1 and promotes the H3K27ac modification of fat mass- and obesity-associated gene (FTO) and AlkB Homolog 5, RNA Demethylase (ALKBH5), which results in significant activation of FTO and ALKBH5. The activation of FTO and ALKBH5 reduces N6-methyladenosine (m6A) modification on FSP1 mRNA, leading to its degradation. Crucially, lactylation of HDAC1K412 is essential for ferroptosis regulation. Both Vorinostat (SAHA) and Trichostatin A (TSA) notably diminish HDAC1K412 lactylation in comparison to other HDAC1 inhibitors, exhibiting a consistent trend of increasing susceptibility to ferroptosis. In conclusion, the research reveals that HDACi decreases HDAC1K412 lactylation to sensitize CRC to ferroptosis and that the combination of HDACi and ferroptosis inducers can be a promising therapeutic strategy for CRC.

Pioneering first-in-class HDAC-ROCK inhibitors as potential multitarget anticancer agents.

With the aim of simultaneously modulating the epigenetic system and the protein kinase pathway, we selected the enzyme histone deacetylase (HDAC) and the Rho-associated protein kinases (ROCK) as desired targets to develop potential multitarget anticancer agents with additional antimetastatic properties. We report here the rational design, synthesis, and biological evaluation of the first-in-class HDAC/ROCK multitarget inhibitors in pancreatic ductal adenocarcinoma (PDAC) and triple-negative breast cancer (TNBC). A molecular docking study performed with the Gold software was used to develop HDAC/ROCK multitarget inhibitors. IC50 values were determined by enzyme assays. The cytotoxicity, anti-migratory and anti-invasive properties of the inhibitors were evaluated using triple-negative breast cancer cells (MDA-MB-231 and HCC 1973) and pancreatic ductal adenocarcinoma cells (Panc-1 and MiaPaCa-2). C-9 showed significant inhibition of HDAC6, ROCK1 and ROCK2. At the same time, this compound showed strong antiproliferative effects on MDA-MB-231, MiaPaCa-2 and Panc-1 cell lines with IC50 values of 5.81 μM, 3.87 μM and 19.57 μM. In addition, it demonstrated great anti-invasive and anti-migratory effects. The findings of this study strongly suggest that the simultaneous inhibition of ROCK and HDACs holds significant potential as a promising therapeutic strategy in the advancement of cancer treatment.

Leptin drives glucose metabolism to promote cardiac protection via OPA1-mediated HDAC5 translocation and Glut4 transcription

Metabolic reprogramming, the shifting from fatty acid oxidation to glucose utilization, improves cardiac function as heart failure (HF) progresses. Leptin plays an essential role in regulating glucose metabolism. However, the crosstalk between leptin and metabolic reprogramming is poorly understood. We tested the hypothesis that leptin improves cardiac function after myocardial infarction via enhancing glucose metabolism. In the isoproterenol (ISO)-induced heart failure model in vitro, H9c2 cell apoptosis was assessed by the TUNEL and Annexin V/PI staining assay. Leptin-mediated mitochondrial fusion was performed via TEM, and glucose oxidation was explored, as well as the ECAR, OCR, and protein expression of the vital metabolic enzymes. By blocking OPA1 expression or HDAC5 inhibition, the mitochondrial dynamic and glucose metabolic were detected to evaluate the role of OPA1 and HDAC5 in leptin-stimulated glucose metabolism. In the mouse model of HF in vivo, intraperitoneal leptin administration appreciably increased glucose oxidation and preserved cardiac function 56 days after coronary artery ligation. In vitro, we identified the OPA1-dependent HDAC5 nucleus export as a crucial process in boosting glucose utilization by activating MEF2 to upregulate Glut4 expression using the RNA interference technique in H9c2 cells. In vivo, leptin promotes glucose utilization and confers heart functional and survival benefits in chronic ischemic HF. The current study provided a novel insight into the role of leptin in metabolic reprogramming and revealed potential therapeutic targets for chronic HF.

Downregulation of HDAC2 attenuates ventricular arrhythmias in a mouse model of cardiac hypertrophy through upregulation of KCHIP2 expression.

Decrease in repolarizing K+ currents, particularly the fast component of transient outward K+ current (Ito,f), prolongs action potential duration (APD) and predisposes the heart to ventricular arrhythmia during cardiac hypertrophy. Histone deacetylases (HDACs) have been suggested to participate in the development of cardiac hypertrophy, and class I HDAC inhibition has been found to attenuate pathological remodeling. This study investigated the potential therapeutic effects of HDAC2 on ventricular arrhythmia in pressure overload-induced cardiac hypertrophy. An in vivo cardiac hypertrophic model was produced by performing transverse aortic constriction (TAC) surgery, and an in vitro cardiomyocyte hypertrophy model by stimulating neonatal rat ventricular myocytes (NRVMs) with phenylephrine (PE). HDAC2 expression was upregulated in TAC mouse hearts and in PE-stimulated cardiomyocytes. Susceptibility to ventricular arrhythmia was increased in TAC mice, while Ito,f was decreased and APD was prolonged in TAC cardiomyocytes. Heart-specific knockdown of HDAC2 (HKD) by RNA interference increased Ito,f, shortened APD and decreased susceptibility to ventricular arrhythmia. Concomitantly, HKD increased the expression of the obligatory β subunit of Ito,f, KChIP2, which is downregulated in hypertrophic hearts. The effects of HKD on KChIP2 expression, Ito,f and APD were also observed in PE-stimulated cardiomyocytes. Mechanistically, HKD increased H3K4me3 abundance and H3K4me3 enrichment at the Kcnip2 promoter in cardiomyocytes. HKD also decreased the expression of KDM5, the H3K4me3 demethylase, which resulted in H3K4me3 upregulation. While investigating the regulatory mechanisms underlying the effect of HDAC2 on KDM5 stability, we identified CNOT4 as the active KDM5 ubiquitinase in cardiomyocytes. HKD increased CNOT4 expression and CNOT4-KDM5 interactions, and thus enhanced the polyubiquitinated degradation of KDM5. HDAC2 inhibition serves as a novel therapeutic strategy for preventing cardiac hypertrophy-associated electrophysiological remodeling. Furthermore, we identified a novel signaling pathway of CNOT4-mediated KDM5 degradation contributing to the upregulation of H3K4me3-mediated KChIP2 expression in response to HDAC2 inhibition.

Synergistic antitumor effect of MK-1775 and CUDC-907 against prostate cancer.

Due to the emergence of drug resistance, androgen receptor (AR)-targeted drugs still pose great challenges in the treatment of prostate cancer, and it is urgent to explore an innovative therapeutic strategy. MK-1775, a highly selective WEE1 inhibitor, is shown to have favorable therapeutic benefits in several solid tumor models. Recent evidence suggests that the combination of MK-1775 with DNA-damaging agents could lead to enhanced antitumor efficacy. Here, our results demonstrate that MK-1775 alone could indeed inhibit proliferation and induce apoptosis in prostate cancer. Moreover, the combination of MK-1775 and a dual PI3K and HDAC inhibitor, CUDC-907, can synergistically inhibit cell proliferation and dramatically induces apoptosis in prostate cancer cells. This effect is partially mediated by DNA damage, resulting from the downregulation of DNA damage response (DDR) proteins such as CDK, CHK, and RRM1/2. Notably, the combination of MK-1775 and CUDC-907 leads to significant antitumor effects in vivo. Our findings provide a strong basis for a promising combination strategy against prostate cancer.

Discovery of Novel Hydrazide-Based HDAC3 Inhibitors as Epigenetic Immunomodulators for Cancer Immunotherapy.

Based on our previous work, a series of imidazole-based small molecules were designed and synthesized as HDAC3 inhibitors. Among them, compound SC26 showed selective HDAC3 inhibition activity with an IC50 of 53 nM (SI = 75 for HDAC3 over HDAC1). Further studies revealed that SC26 could dose-dependently induce the expression of PD-L1 in MC38 cells by activating the PD-L1 transcription. SC26 also showed high in vivo antitumor efficacy in a colorectal cancer model (50 mg/kg po, TGI = 63%). Importantly, the combination of SC26 with the PD-L1 inhibitor NP19 activated the immune system in tumor-bearing mice, enhancing the antitumor immune response (TGI = 80%, 50 + 50 mg/kg, p.o.). Collectively, we report for the first time that an HDAC3 inhibitor could upregulate PD-L1 expression in vitro and in vivo, specifically in MC38 cells and MC38-bearing tumors, and SC26 represents a novel epigenetic immunomodulator with potential applications in tumor immunotherapy.

HDAC inhibitor SAHA triggers the production of previously undescribed sesquiterpenes and undergoes biotransformation by the fungus Robillarda sessilis from Verbena officinalis L.

Cultivation of the endophytic fungus Robillarda sessilis XL-308 with SAHA resulted in the discovery of two previously undescribed cyclonerane sesquiterpenes (1 and 2), one previously unreported norcadinane type sesquiterpene (3), and seven suberanilohydroxamic acid (SAHA) derivatives (4-10). The identification of previously undescribed compounds was confirmed through NMR spectroscopic analyses, quantum chemistry calculations, and single-crystal X-ray crystallography. Among them, compounds 1 and 2 were products of the silenced genome in XL-308 that activated by SAHA. Concurrently, SAHA underwent biotransformation because of its moderate toxicity towards this fungus. It was hypothesized that the hydroxamic acid group in SAHA triggering the self-defense mechanism of XL-308 and converting SAHA into a less toxic derivative 5.

Unveiling the signal valve specifically tuning the TGF-β1 suppression of osteogenesis: mediation through a SMAD1-SMAD2 complex

BackgroundTGF-β1 is the most abundant cytokine in bone, in which it serves as a vital factor to interdict adipogenesis and osteogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, how TGF-β1 concurrently manipulates differentiation into these two distinct lineages remains elusive.MethodsTreatments with ligands or inhibitors followed by biochemical characterization, reporter assay, quantitative PCR and induced differentiation were applied to MSC line or primary BM-MSCs for signaling dissection. In vivo adipogenesis and ex vivo culture of bone explants were used to verify the functions of different SMAD complexes. Ingenuity Pathway Analysis, and analysis of transcriptomic datasets from human BM-MSCs in combination with hierarchical clustering and STRING assay were used to decipher the interplaying co-repressors. Mouse models of chronic and acute bone loss followed by biochemical assays and micro-computed tomography demonstrated the bone effects when functionally blocking the critical co-repressor HDAC1.ResultsDistinct from the TGF-β1 inhibition on adipogenesis through canonical SMAD2/3 signaling, we clarified that TGF-β1 suppresses osteogenesis by inducing the formation of previously unidentified mixed SMADs mainly composed of SMAD1 and SMAD2, in which SMAD2 recruits more TGF-β1-induced co-repressors including HDAC1, TGIF1 and ATF3, whereas SMAD1 allows directing the whole transcriptional suppression complex to the cis-elements of osteogenic genes. Depletion of the cross-activation to the mixed SMADs dismantled specifically the TGF-β1 suppression on osteogenesis without affecting its inhibition on adipogenesis. Such phenomena can be reproduced via knockdown of co-repressors such as Hdac1 or addition of HDAC1 inhibitors in TGF-β1-treated MSCs. In either the chronic or the acute bone loss model, we demonstrated that the TGF-β signaling was augmented in the bone niche during osteolysis, whereas administration of HDAC1 inhibitors significantly improved bone quality.ConclusionThis study identifies a new signal valve through which TGF-β1 can inhibit osteogenesis specifically. Functional interruption of this valve can tilt the seesaw balance of BM-MSC differentiation towards osteogenesis, highlighting the interplaying co-repressors, such as HDAC1, as promising therapeutic targets to combat diverse degenerative orthopedic diseases.

Targeting enolase 1 reverses bortezomib resistance in multiple myeloma through YWHAZ/Parkin axis

BackgroundEnolase 1 (ENO1) is a conserved glycolytic enzyme that regulates glycolysis metabolism. However, its role beyond glycolysis in the pathophysiology of multiple myeloma (MM) remains largely elusive. Herein, this study aimed to elucidate the function of ENO1 in MM, particularly its impact on mitophagy under bortezomib-induced apoptosis.MethodsThe bone marrow of clinical MM patients and healthy normal donors was used to compare the expression level of ENO1. Using online databases, we conducted an analysis to examine the correlation between ENO1 expression and both clinicopathological characteristics and patient outcomes. To investigate the biological functions of ENO1 in MM and the underlying molecular mechanisms involved, we conducted the following experiment: construction of a subcutaneous graft tumor model, co-immunoprecipitation, western blot, quantitative real-time polymerase chain reaction, immunohistochemistry, flow cytometry, and cell functional assays.ResultsENO1 was identified as an unfavorable prognostic factor in MM. ENO1 knockdown suppresses tumorigenicity and causes cell cycle arrest. Inhibition of ENO1-regulated mitophagy sensitizes tumor cells to apoptosis. ENO1 enhanced the stability of the YWHAZ protein by increasing the acetylation of lysine in YWHAZ while antagonizing its ubiquitination, which in turn promoted mitophagy. HDAC6 mediates the deacetylation of YWHAZ by deacetylating the K138 site of YWHAZ. Inhibition of HDAC6 increased YWHAZ acetylation and decreased YWHAZ ubiquitination. Furthermore, combination treatment with bortezomib and pharmaceutical agents targeting ENO1 has synergistic anti-MM effects both in vivo and in vitro.ConclusionOur data suggest that ENO1 promotes MM tumorigenesis and progression. ENO1 activates mitophagy by promoting the stability of YWHAZ and inhibits apoptosis and thus, leads to the drug resistance. ENO1-dependent mitophagy promotes MM proliferation and suppresses the level of bortezomib-induced apoptosis. Inhibition of ENO1 may represent a potential strategy to reverse the resistance of MM to bortezomib.

Fusobacterium nucleatum determines the expression of amphetamine-induced behavioral responses through an epigenetic phenomenon.

Amphetamines (AMPHs) are psychostimulants commonly used for the treatment of neuropsychiatric disorders. They are also misused (AMPH use disorder; AUD), with devastating outcomes. Recent studies have implicated dysbiosis in the pathogenesis of AUD. However, the mechanistic roles of microbes in AUD are unknown. Fusobacterium nucleatum ( Fn ) is a bacterium that increases in abundance in both rats and humans upon AMPH exposure. Fn releases short-chain fatty acids (SCFAs), bacterial byproducts thought to play a fundamental role in the gut-brain axis as well as the pathogenesis of AUD. We demonstrate that in gnotobiotic Drosophila melanogaster, colonization with Fn or dietary supplementation of the SCFA butyrate, a potent inhibitor of histone deacetylases (HDACs), enhances the psychomotor and rewarding properties of AMPH as well as its ability to promote male sexual motivation. Furthermore, solely HDAC1 RNAi targeted inhibition recapitulates these enhancements, pointing to a specific process underlying this Fn phenomenon. Of note is that the expression of these AMPH behaviors is determined by the increase in extracellular dopamine (DA) levels that result from AMPH-induced reversal of DA transporter (DAT) function, termed non-vesicular DA release (NVDR). The magnitude of AMPH-induced NVDR is dictated, at least in part, by DAT expression levels. Consistent with our behavioral data, we show that Fn , butyrate, and HDAC1 inhibition enhance NVDR by elevating DAT expression. Thus, the participation of Fn in AUD stems from its ability to release butyrate and inhibit HDAC1. These data offer a microbial target and probiotic interventions for AUD treatment.

Acetylation-enhanced Sp1 transcriptional activity suppresses Mlph expression

Melanosome transport is regulated by major proteins, including Rab27a, Melanophilin (Mlph), and Myosin Va (Myo-Va), that form a tripartite complex. Mutation of these proteins causes melanosome aggregation around the nucleus. Among these proteins, Mlph is a linker between Rab27a and Myo-Va. There are some studies about the regulation of Mlph transcriptional expression. However, its regulation by post-translational modifications remains unclear. In this study, inhibition of HDACs by SAHA and TSA disrupted melanosome transport, causing melanosome aggregation. Specifically, we identified a novel mechanism in which HDAC5 regulates Mlph expression via Sp1. Knockdown of HDAC5 increased the acetylation of Sp1 and the binding to the Mlph promoter, thereby modulating its expression. This study highlights the crucial role of HDAC5 in melanosome transport through its interaction with Sp1. These findings suggest that HDAC5-mediated deacetylation is pivotal in the post-translational modification of melanosome transport, providing insights into the molecular mechanisms underlying this process.

A phase I study of MLN4924 and belinostat in relapsed/refractory acute myeloid leukemia or myelodysplastic syndrome

PurposeRelapsed and/or refractory acute myeloid leukemia and high-risk myelodysplastic syndrome continue to have a poor prognosis with limited treatment options despite advancements in rational combination and targeted therapies. Belinostat (an HDAC inhibitor) and Pevonedistat (a NEDD8 inhibitor) have each been independently studied in hematologic malignancies and have tolerable safety profiles with limited single-agent activity. Preclinical studies in AML cell lines and primary AML cells show the combination to be highly synergistic, particularly in high-risk phenotypes such as p53 mutant and FLT-3-ITD positive cells. Here, we present the safety, pharmacokinetics and pharmacodynamics of belinostat and pevonedistat in a dose escalation Phase I study in AML and High-Risk MDS.MethodsEighteen patients (16 with AML, 2 with MDS) were treated at 5 dose levels (belinostat 800–1000 mg/m2, pevonedistat 20–50 mg/m2). Safety and tolerability were assessed according to protocol defined dose limiting toxicities (DLTs). Correlative pharmacokinetic and pharmacodynamic analyses were performed.ResultsNo dose limiting toxicities were noted. Most Grade 3 or 4 toxicities were hematologic in nature. The best response was stable disease in four patients, and complete remission in one patient who qualified as an exceptional responder. Pharmakokinetic studies revealed no association between drug exposure and best response. Pharmacodynamic RT-PCR studies demonstrated post-treatment increases in several proteins, including quantitative increases in the oxidative stress protein NQO1, ferroptosis protein SLC7A11, and GSR, linked to glutathione metabolism and oxidative stress, as did the anti-oxidants SRXN1 and TXNRD1.ConclusionsPatterns of post-treatment changes in correlative pharmacodynamic parameters may suggest possible mechanistic changes in the DNA damage response, oxidative damage, and ferroptosis pathways. The combination of pevonedistat plus belinosat is safe in an adult relapsed and/or refractory AML/High-Risk MDS population with modest but notable activity in this heavily treated, high risk population. Our findings also raise the possibility that certain extremely poor prognosis AML patients may respond to a regimen combining two targeted agents that have little or no activity when administered individually.Trial registrationClinicalTrials.gov ID NCT03772925, first posted 12/12/2018; CTEP Identifier 10246.

P53 mediated regulation of LINE1 retrotransposon derived R-loops.

Long Interspersed Nuclear Element 1 (LINE1/L1) retrotransposons, which comprise 17% of the human genome, typically remain inactive in healthy somatic cells but are reactivated in several cancers. We previously demonstrated that p53 silences L1 transposons in human somatic cells, potentially acting as a tumor-suppressive mechanism. However, the precise molecular mechanisms underlying p53-mediated repression of L1 and its life cycle intermediates remain unclear. In this study, we used DRIP-sequencing experiments to investigate RNA-DNA hybrids, which are key intermediates formed during L1 retrotransposition. Our findings reveal that L1 mRNA-genomic DNA (cis L1 R-loops) and L1 mRNA-complementary DNA (trans L1 R-loops) hybrids, are de-repressed in p53-/- cells. This increase is synergistic with L1 activation by HDAC inhibitors (HDACi). However, treatment with a reverse transcriptase inhibitor reduces this accumulation, indicating that retrotransposition activity plays a significant role in R-loop accumulation. Interestingly, in p53 wild-type cells, hyperactivated L1 transposons are re-silenced upon HDACi withdrawal. L1 resilecing in wt cells coincided with the recruitment of repressive marks, specifically H3K9me3 and H3K27me3, simultaneously preventing the addition of activating marks like H3K4me3, and H3K9ac at the L1 5'UTR. Mechanistically, we demonstrate that p53 cooperates with histone methyltransferases SETDB1 and G9A to deposit H3K9me3 marks at the L1 promoter, thereby silencing transposons. This study is the first to reveal novel roles of p53 in preventing the formation of L1-derived RNA-DNA hybrids (R-loops) and re-silencing of hyperactivated L1 elements by co-operating with histone methyltransferases, underscoring its critical role in maintaining genomic stability.

Molecular Mediators of Neutrophil Primary Granule Release Following Acute Ischemic Stroke and their Associated Epigenetic Modulation by HDAC2.

High concentrations of neutrophil degranulation products in the plasma and thrombi are poor prognostic indicators in patients with acute ischemic stroke (AIS). This study aimed to identify candidate effectors capable of mediating neutrophil degranulation post-AIS, and to reveal their underlying epigenetic mechanisms. Microarrays and ChIP-seq were applied to analyze the neutrophils of patients with AIS. Cerebral ischemia was induced in C57/BL6 mice by middle cerebral artery occlusion (MCAO). Lipopolysaccharide was used to induce inflammation in HL-60 Cells. Protein and mRNA levels were assessed using flow cytometry, ELISA, western blotting, and RT-PCR. Degranulation was identified as a significant pathway in the neutrophils of patients with AIS, while Rho GTPase and the SNARE complex also showed importance. HDAC2 differentially binds to genes involved in neutrophil degranulation in patients with AIS. SYT9, SH3BP1, and STXBP1 were identified in two sequencing experiments, for which HDAC2 bound to their promoter, intron, and upstream regions, respectively. Consistently, candidate degranulation effectors and products showed substantially increased expression and co-localization in the neutrophils of thrombi obtained from patients with middle cerebral artery stenosis with poor prognosis, a mouse model of MCAO, and an HL-60 cell-based model of inflammation. Knockdown of SYT9, SH3BP1, and STXBP1 impaired primary granule release in vitro, whereas HDAC2 activity was decreased following LPS induction and ischemic stroke in mice. Furthermore, HDAC2 inhibition upregulated SYT9, SH3BP1, and STXBP1. Our findings suggest that these three molecules may be indispensable in the process of neutrophil degranulation following AIS, and are targeted by HDAC2, paving the way for the development of new drugs.

Cytotoxic mechanisms of pemetrexed and HDAC inhibition in non-small cell lung cancer cells involving ribonucleotides in DNA

The cytotoxic mechanisms of thymidylate synthase inhibitors, such as the multitarget antifolate pemetrexed, are not yet fully understood. Emerging evidence indicates that combining pemetrexed with histone deacetylase inhibitors (HDACi) may enhance therapeutic efficacy in non-small cell lung cancer (NSCLC). To explore this further, A549 NSCLC cells were treated with various combinations of pemetrexed and the HDACi MS275 (Entinostat), and subsequently assessed for cell viability, cell cycle changes, and genotoxic markers. Proteomic alterations were analyzed using label-free shotgun and targeted LC–MS/MS. MS275 enhanced the sensitivity of A549 cells to pemetrexed, but only when administered following prior treatment with pemetrexed. Both HeLa (p53 negative) and A549 (p53 positive) showed robust activation of γH2AX upon treatment with this combination. Importantly, CRISPR/Cas9 knockout of the uracil-DNA glycosylase UNG did not affect γH2AX activation or sensitivity to pemetrexed. Proteomic analysis revealed that MS275 altered the expression of known pemetrexed targets, as well as several proteins involved in pyrimidine metabolism and DNA repair, which could potentiate pemetrexed cytotoxicity. Contrary to the conventional model of antifolate toxicity, which implicates futile cycles of uracil incorporation and excision in DNA, we propose that ribonucleotide incorporation in nuclear and mitochondrial DNA significantly contributes to the cytotoxicity of antifolates like pemetrexed, and likely also of fluorinated pyrimidine analogs. HDAC inhibition apparently exacerbates cytotoxicity of these agents by inhibiting error-free repair of misincorporated ribonucleotides in DNA. The potential of HDACis to modulate pyrimidine metabolism and DNA damage responses offers novel strategies for improving NSCLC outcomes.

Liver oval cells in response to HDAC1 inhibitor trichostatin A: immunohistochemical characterization using OV-6 hepatic expression.

Liver regeneration is intricate, involves many cells, and necessitates extended research. This study aimed to investigate the response of liver oval cells (bipotent liver progenitors) to the epigenetic modifier trichostatin A (TSA), an HDAC1 inhibitor, and to develop a scoring system for assessing the response of these cells. Three groups of equally divided rats (n=24) were selected: control (A, dimethyl sulfoxide treated); oval cell induction (B, acetylaminofluorene [2-AAF] to block hepatocyes/carbon tetrachloride [CCL4] to induce oval cell response); and epigenetic modulation (C, TSA post 2-AAF/CCL4 injury). The oval cell response was quantified using immunoreactivity to the OV-6 antibody, and the ductular response was measured by calculating the bile duct (BD) to portal vein (PV) ratio and the percentage of individual oval cells in liver sections. The expression level of HDAC1 was also analyzed. The administration of TSA significantly enhanced oval cell proliferation and the ductular response (6.13±0.28). The control group exhibited limited immunoreactivity to OV-6, while group B showed significant induction of ductular response with distinct morphology (4.13±0.28). The expression levels of HDAC1 were elevated in both the oval cell induction group and the epigenetic modulation group compared to the control group. This study developed a precise method for quantifying liver oval cells and analyzing their response to TSA. TSA administration enhanced oval cell proliferation, suggesting its significance in regulating hepatic progenitor cell dynamics. The findings support the use of epigenetic modifiers in liver regeneration and propose a scoring system for assessing the response of liver oval cells.

Recent Advances in the Clinical Translation of Small-Cell Lung Cancer Therapeutics.

Small-cell lung cancer (SCLC) is a recalcitrant form of cancer, representing 15% of lung cancer cases globally. SCLC is classified within the range of neuroendocrine pulmonary neoplasms, exhibiting shared morphologic, ultrastructural, immunohistochemical, and molecular genomic features. It is marked by rapid proliferation, a propensity for early metastasis, and an overall poor prognosis. The current conventional therapies involve platinum-etoposide-based chemotherapy in combination with immunotherapy. Nonetheless, the rapid emergence of therapeutic resistance continues to pose substantial difficulties. The genomic profiling of SCLC uncovers significant chromosomal rearrangements along with a considerable mutation burden, typically involving the functional inactivation of the tumor suppressor genes TP53 and RB1. Identifying biomarkers and evaluating new treatments is crucial for enhancing outcomes in patients with SCLC. Targeted therapies such as topoisomerase inhibitors, DLL3 inhibitors, HDAC inhibitors, PARP inhibitors, Chk1 inhibitors, etc., have introduced new therapeutic options for future applications. In this current review, we will attempt to outline the key molecular pathways that play a role in the development and progression of SCLC, together with a comprehensive overview of the most recent advancements in the development of novel targeted treatment strategies, as well as some ongoing clinical trials against SCLC, with the goal of improving patient outcomes.

Discovery of WDR5-MLL1 and HDAC Dual-Target Inhibitors for the Treatment of Acute Myeloid Leukemia.

Targeting the WDR5-MLL1 protein-protein interaction (PPI) is considered to be an effective approach for the treatment of MLL-rearranged leukemia. However, interfering with WDR5-MLL1 PPI reduces methylated H3K4 levels and induces a decline in acetylated H3 levels, which may contribute to the suboptimal cellular efficacy of WDR5 inhibitors. We observed that cotreatment with WDR5-MLL1 PPI and HDAC inhibitors augmented the antiproliferative effect in MV-4-11 cells. Thus, a series of dual-target inhibitors was developed by merging the pharmacophores of the WDR5 and HDAC inhibitors. Among the developed inhibitors, compound 32d displayed an 89-fold increase in antiproliferative efficacy and induced potent cell apoptosis by impeding the DNA damage repair signaling pathway. Furthermore, the administration of 30 mg/kg of compound 32d was well tolerated, inhibiting MV-4-11 xenograft growth by 87.1%. Our investigation established the therapeutic effectiveness of the developed WDR5-MLL1/HDAC dual-target inhibitor against acute myeloid leukemia, providing a valuable tool for further exploration of crosstalk between the two targets.

H3K9 post-translational modifications regulate epiblast/primitive endoderm specification in rabbit blastocysts

Post-translational modifications of histone H3 on lysine 9, specifically acetylation (H3K9ac) and tri-methylation (H3K9me3), play a critical role in regulating chromatin accessibility. However, the role of these modifications in lineage segregation in the mammalian blastocyst remains poorly understood. We demonstrate that di- and tri-methylation marks, H3K9me2 and H3K9me3, decrease during cavitation and expansion of the rabbit blastocyst. Notably, H3K9me3 levels are particularly low in inner cell mass cells at the onset of blastocyst formation but increase again just before gastrulation. Conversely, H3K9ac is abundant in early blastocyst stages but decreases during the transition from the inner cell mass to the epiblast. These distinct distribution patterns correlate with high expression levels of methyltransferases (EHMT1, EHMT2, SETDB1) and deacetylases (HDAC1, HDAC2, HDAC5) in expanding blastocysts. Functionally, inhibiting H3K9me2/3 through an EHMT1/2 inhibitor disrupts primitive endoderm segregation, whereas enhancing histone acetylation (including H3K9ac) using a class I HDAC inhibitor promotes epiblast expansion at the expense of the primitive endoderm. These modifications impact the expression of genes associated with pluripotency and lineage determination, underscoring the importance of H3K9 modifications in embryonic cell fate decisions.