Histone Deacetylase Inhibitor Research Articles

Molecular mechanism of HDAC6-mediated pyroptosis in neurological function recovery after cardiopulmonary resuscitation in rats

Brain injury after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) is the leading cause of neurological dysfunction and death. This study aimed to explore the mechanism of histone deacetylase 6 (HDAC6) in neurofunctional recovery following CA/CPR in rats. A rat model was established by CA/CPR treatment. Adenovirus-packaged sh-HDAC6 was injected into the tail vein. To evaluate the neurofunction of rats, survival time, neurofunctional scores, serum NSE/S100B, and brain water content were measured and Morris water maze test was performed. HDAC6, microRNA (miR)-138-5p, Nod-like receptor protein 3 (NLRP3), and pyroptotic factors levels were determined by real-time quantitative polymerase chain reaction or Western blot assay. HDAC6 and H3K9ac enrichment on miR-138-5p promoter were examined by chromatin immunoprecipitation. miR-138-5p-NLRP3 binding was analyzed by dual-luciferase reporter assay. NLRP3 inflammasome was activated with nigericin sodium salt. After CPR treatment, HDAC6 was highly expressed, while miR-138-5p was downregulated. HDAC6 downregulation improved neurofunction and reduced pyroptosis. HDAC6 enrichment on the miR-138-5p promoter deacetylated H3K9ac, inhibiting miR-138-5p, and promoting NLRP3-mediated pyroptosis. Downregulating miR-138-5p partially reversed the protective effect of HDAC6 inhibition after CPR. In Conclusion, HDAC6 enrichment on miR-138-5p promoter deacetylated H3K9ac, inhibiting miR-138-5p expression and promoting NLRP3-mediated pyroptosis, worsening neurological dysfunction in rats after CPR.

A Novel Class I HDAC Inhibitor, AW01178, Inhibits Epithelial-Mesenchymal Transition and Metastasis of Breast Cancer.

Epithelial-mesenchymal transition (EMT) refers to the transformation of polar epithelial cells into motile mesenchymal cells under specific physiological or pathological conditions, thus promoting the metastasis of cancer cells. Epithelial cadherin (E-cadherin) is a protein that plays an important role in the acquisition of tumor cell motility and serves as a key EMT epithelial marker. In the present study, AW01178, a small-molecule compound with potential therapeutic efficacy, was identified via in-cell Western high-throughput screening technology using E-cadherin as the target. The compound induced the upregulation of E-cadherin at both mRNA and protein levels and inhibited the EMT of breast cancer cells in vitro as well as metastasis in vivo. Mechanistically, AW01178 is a novel benzacetamide histone deacetylase inhibitor (HDACi) mainly targeting class I histone deacetylases. AW01178 promoted the transcription and expression of E-cadherin through enhancing the acetylation level of histone H3 in the E-cadherin promoter region, thereby inhibiting the metastasis of breast cancer cells. The collective findings support the potential utility of the novel HDACi compound identified in this study, AW01178, as a therapeutic drug for breast cancer and highlight its value for the future development of HDACi structures as anticancer drugs.

Pharmacological HDAC3 inhibition alters memory updating in young and old male mice.

Long-term memories are not stored in a stable state but must be flexible and dynamic to maintain relevance in response to new information. Existing memories are thought to be updated through the process of reconsolidation, in which memory retrieval initiates destabilization and updating to incorporate new information. Memory updating is impaired in old age, yet little is known about the mechanisms that go awry. One potential mechanism is the repressive histone deacetylase 3 (HDAC3), which is a powerful negative regulator of memory formation that contributes to age-related impairments in memory formation. Here, we tested whether HDAC3 also contributes to age-related impairments in memory updating using the Objects in Updated Locations (OUL) paradigm. We show that blocking HDAC3 immediately after updating with the pharmacological inhibitor RGFP966 ameliorated age-related impairments in memory updating in 18-m.o. male mice. Surprisingly, we found that post-update HDAC3 inhibition in young (3-m.o.) male mice had no effect on memory updating but instead impaired memory for the original information, suggesting that the original and updated information may compete for expression at test and HDAC3 helps regulate which information is expressed. To test this idea, we next assessed whether HDAC3 inhibition would improve memory updating in young male mice given a weak, subthreshold update. Consistent with our hypothesis, we found that HDAC3 blockade strengthened the subthreshold update without impairing memory for the original information, enabling balanced expression of the original and updated information. Together, this research suggests that HDAC3 may contribute to age-related impairments in memory updating and may regulate the strength of a memory update in young mice, shifting the balance between the original and updated information at test.

Utilizing epigenetic regulators to improve HSC–based lentiviral gene therapy

AbstractThe curative benefits of autologous and allogeneic transplantation of hematopoietic stem cells (HSCs) have been proven in various diseases. However, the low number of true HSCs that can be collected from patients and the subsequent in vitro maintenance and expansion of true HSCs for genetic correction remains challenging. Addressing this issue, we here focused on optimizing culture conditions to improve ex vivo expansion of true HSCs for gene therapy purposes. In particular, we explored the use of epigenetic regulators to enhance the effectiveness of HSC–based lentiviral (LV) gene therapy. The histone deacetylase inhibitor quisinostat and bromodomain inhibitor CPI203 each promoted ex vivo expansion of functional HSCs, as validated by xenotransplantation assays and single-cell RNA sequencing analysis. We confirmed the stealth effect of LV transduction on the loss of HSC numbers in commonly used culture protocols, whereas the addition of quisinostat or CPI203 improved the expansion of HSCs in transduction protocols. Notably, we demonstrated that the addition of quisinostat improved the LV transduction efficiency of HSCs and early progenitors. Our suggested culture conditions highlight the potential therapeutic effects of epigenetic regulators in HSC biology and their clinical applications to advance HSC-based gene correction.

Histone modifications in head and neck squamous cell carcinoma.

Head and neck cancer is the main cause of cancer death worldwide, with squamous cell carcinoma (HNSCC) being the second most frequent subtype. HNSCC poses significant health threats due to its high incidence and poor prognosis, underscoring the urgent need for advanced research. Histone modifications play a crucial role in the regulation of gene expression and influencing various biological processes. In the context of HNSCC, aberrant histone modifications are increasingly recognized as critical contributors to its development and pathologic progression. This review demonstrates the molecular mechanisms, by which histone modifications such as acetylation, methylation, phosphorylation, and ubiquitination, impact the pathogenesis of HNSCC. The dysregulation of histone-modifying enzymes, including histone acetyltransferases (HATs), histone deacetylases (HDACs), and histone methyltransferases (HMTs), is discussed for its role in altering chromatin structure and gene expression in HNSCC. Moreover, we will explore the potential of targeting histone modifications as a therapeutic strategy, highlighting current preclinical and clinical studies that investigate histone deacetylase inhibitors (HDIs) and other epigenetic drugs, referring to the completed and ongoing clinical trials on those medications.

Targeting histone deacetylase in cardiac diseases.

Histone deacetylases (HDAC) catalyze the removal of acetylation modifications on histones and non-histone proteins, which regulates gene expression and other cellular processes. HDAC inhibitors (HDACi), approved anti-cancer agents, emerge as a potential new therapy for heart diseases. Cardioprotective effects of HDACi are observed in many preclinical animal models of heart diseases. Genetic mouse models have been developed to understand the role of each HDAC in cardiac functions. Some of the findings are controversial. Here, we provide an overview of how HDACi and HDAC impact cardiac functions under physiological or pathological conditions. We focus on in vivo studies of zinc-dependent classical HDACs, emphasizing disease conditions involving cardiac hypertrophy, myocardial infarction (MI), ischemic reperfusion (I/R) injury, and heart failure. In particular, we review how non-biased omics studies can help our understanding of the mechanisms underlying the cardiac effects of HDACi and HDAC.

Histone Deacetylases in Retinoblastoma.

Retinoblastoma, a pediatric ocular malignancy, presents significant challenges in comprehending its molecular underpinnings and targeted therapeutic approaches. The dysregulated activity of histone deacetylases (HDACs) has been associated with retinoblastoma pathogenesis, influencing critical cellular processes like cell cycle regulation or retinal ganglion cell apoptosis. Through their deacetylase activity, HDACs exert control over key tumor suppressors and oncogenes, influencing the delicate equilibrium between proliferation and cell death. Furthermore, the interplay between HDACs and the retinoblastoma protein pathway, a pivotal aspect of retinoblastoma etiology, reveals a complex network of interactions influencing the tumor microenvironment. The examination of HDAC inhibitors, encompassing both established and novel compounds, offers insights into potential approaches to restore acetylation balance and impede retinoblastoma progression. Moreover, the identification of specific HDAC isoforms exhibiting varying expression in retinoblastoma provides avenues for personalized therapeutic strategies, allowing for interventions tailored to individual patient profiles. This review focuses on the intricate interrelationship between HDACs and retinoblastoma, shedding light on epigenetic mechanisms that control tumor development and progression. The exploration of HDAC-targeted therapies underscores the potential for innovative treatment modalities in the pursuit of more efficacious and personalized management strategies for this disease.

Single cell proteomics by mass spectrometry reveals deep epigenetic insight into the actions of an orphan histone deacetylase inhibitor.

Epigenetic programming has been shown to play a role in nearly every human system and disease where anyone has thought to look. However, the levels of heterogeneity at which epigenetic or epiproteomic modifications occur at single cell resolution across a population remains elusive. While recent advances in sequencing technology have allowed between 1 and 3 histone post-translational modifications to be analyzed in each single cell, over twenty separate chemical PTMs are known to exist, allowing thousands of possible combinations. Single cell proteomics by mass spectrometry (SCP) is an emerging technology in which hundreds or thousands of proteins can be directly quantified in typical human cells. As the proteins detected and quantified by SCP are heavily biased toward proteins of highest abundance, chromatin proteins are an attractive target for analysis. To this end, I applied SCP to the analysis of cancer cells treated with mocetinostat, a class specific histone deacetylase inhibitor. I find that 16 PTMs can be confidently identified and localized with high site specificity in single cells. In addition, the high abundance of histone proteins allows higher throughput methods to be utilized for SCP than ever described. While quantitative accuracy suffers when analyzing more than 700 cells per day, 9 histone proteins can be measured in single cells analyzed at even 3,500 cells per day, a throughput 10-fold greater than any previous report. In addition, the unbiased global approach utilized herein identifies a previously uncharacterized response to this drug through the S100-A8/S100-A9 protein complex partners. This response is observed in nearly every cell of the over 1,000 analyzed in this study, regardless of the relative throughput of the method utilized. While limitations exist in the methods described herein, current technologies can easily improve upon the results presented here to allow comprehensive analysis of histone PTMs to be performed in any mass spectrometry lab. All raw and processed data described in this study has been made publicly available through the ProteomeXchange/MASSIVE repository system as MSV000093434.

DIPG-28. CHEMOGENOMIC PROFILING OF HDAC ISOFORMS REVEALS A PARADIGM OF ESSENTIALITY WITH THERAPEUTICALLY RELEVANT IMPLICATIONS IN H3K27-ALTERED DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse midline glioma (DMG), H3K27-altered, is a lethal pediatric brain tumor driven by epigenetic dysregulation. Histone deacetylase (HDAC) inhibitors have been identified as a promising drug class; however, the precise mechanism of efficacy for these agents in DMG has not been fully explored. Furthermore, their translational potential has been limited by off-target effects and poor CNS penetration of clinically available drugs. METHODS We investigated the genetic and chemical dependencies of HDAC isoforms in DMG cell lines using CRISPR/Cas9 screening and an HDAC inhibitor toolbox. We identified an HDAC inhibitor termed “Compound 26” with class I isoform selectivity and excellent CNS penetration (brain/plasma AUC of >7). Effects of Compound 26 on DMG cells was evaluated in-vitro through assessment of cytotoxicity, proliferation and apoptosis. Mechanistic studies were undertaken using bulk RNAseq and HiChIP (H3K27ac, H3K27me3). Efficacy of Compound 26 in-vivo was determined using an aggressive orthotopic xenograft model. RESULTS We confirmed that HDAC class I isoforms are dependencies in DMG cell lines, with HDAC2 representing a selective dependency that is highly expressed in patient tumors. Compound 26 is cytotoxic in DMG cells, reducing proliferation through cell cycle arrest. Differential expression analysis confirmed profound effects on cycling programs with Compound 26 treatment. Furthermore, analysis of genome structure using HiChIP revealed global increases in H3K27ac contacts, with notable spreading along key cell cycle gene bodies such as p16 (CDKN2A). Interestingly, treatment with Compound 26 also significantly increased global H3K27me3 contacts, which is canonically decreased in H3K27-altered DMG. Most importantly, Compound 26 was well tolerated, effectively crossed the blood-brain barrier, and significantly extended survival in an aggressive DMG-orthotopic xenograft model. CONCLUSIONS This study comprehensively outlines the reliance of DMG cells on HDAC class I isoforms, elucidates novel mechanisms for this dependency, and highlights a compelling new agent to bypass prior translational pitfalls

Microglial forkhead box O3a deficiency attenuates LPS-induced neuro-inflammation and depressive-like behaviour through regulating the expression of peroxisome proliferator-activated receptor-γ.

Depression is closely linked with microglial activation and neuro-inflammation. Peroxisome proliferator-activated receptor-γ (PPAR-γ) plays an important role in M2 activation of microglia. Forkhead box (FOX) O3a has been implicated in the regulation of mood-relevant behaviour. However, little is known about the inflammatory mechanisms of in the microglia of the brain. Here, we have investigated the role of microglial FOXO3a/PPAR-γ in the development of depression. The effect of FOXO3a on microglia inflammation was analysed in vitro and in lipopolysaccharide (LPS)-induced depression-like behaviours in vivo. ChIP-seq and Dual-luciferase reporter assays were used to confirm the interaction between FOXO3a and PPAR-γ. Behavioural changes were measured, while inflammatory cytokines, microglial phenotype and morphological properties were determined by ELISA, qRT-PCR, western blotting and immunostaining. Overexpression of FOXO3a significantly attenuated expression of PPAR-γ and enhanced the microglial polarization towards the M1 phenotype, while knockdown of FOXO3a had the opposite effect. FOXO3a binds to the promoters of PPAR-γ and decreases its transcription activity. Importantly, deacetylation and activation of FOXO3a regulate LPS-induced neuro-inflammation by inhibiting the expression of PPAR-γ in microglia cells, supporting the antidepressant potential of histone deacetylase inhibitors. Microglial FOXO3a deficiency in mice alleviated LPS-induced neuro-inflammation and depression-like behaviours but failed to reduce anxiety behaviour, whereas pharmacological inhibition of PPAR-γ by GW9662 restored LPS-induced microglial activation and depressive-like behaviours in microglial FOXO3a-deficient mice. FOXO3a/PPAR-γ axis plays an important role in microglial activation and depression, identifying a new therapeutic avenue for the treatment of major depression.

Recent Advances in Molecular and Genetic Research on Uveal Melanoma.

Uveal melanoma (UM), a distinct subtype of melanoma, presents unique challenges in its clinical management due to its complex molecular landscape and tendency for liver metastasis. This review highlights recent advancements in understanding the molecular pathogenesis, genetic alterations, and immune microenvironment of UM, with a focus on pivotal genes, such as GNAQ/11, BAP1, and CYSLTR2, and delves into the distinctive genetic and chromosomal classifications of UM, emphasizing the role of mutations and chromosomal rearrangements in disease progression and metastatic risk. Novel diagnostic biomarkers, including circulating tumor cells, DNA and extracellular vesicles, are discussed, offering potential non-invasive approaches for early detection and monitoring. It also explores emerging prognostic markers and their implications for patient stratification and personalized treatment strategies. Therapeutic approaches, including histone deacetylase inhibitors, MAPK pathway inhibitors, and emerging trends and concepts like CAR T-cell therapy, are evaluated for their efficacy in UM treatment. This review identifies challenges in UM research, such as the limited treatment options for metastatic UM and the need for improved prognostic tools, and suggests future directions, including the discovery of novel therapeutic targets, immunotherapeutic strategies, and advanced drug delivery systems. The review concludes by emphasizing the importance of continued research and innovation in addressing the unique challenges of UM to improve patient outcomes and develop more effective treatment strategies.

Beneficial effect of sodium butyrate, a histone deacetylase inhibitor, on the honey bee’s (Apis mellifera) immune response and oxidative status

By investigating the intriguing process of epigenetic changes, mainly acetylation and deacetylation of histones, we can further expand our knowledge of the development and ageing of an organism. This challenging task is made more difficult with the implication of nutritional and environmental impacts on epigenetic mechanisms. Hence, the potential use of chemicals as therapeutics that promote health and increase lifespan could bring upon a new era in natural sciences. One of the most interesting histone deacetylase inhibitors is sodium butyrate (SB). Honey bees (Apis mellifera L.) are the most frequently used model organism in ageing studies due to their extreme phenotypic plasticity in regard to lifespan and their tremendous economic importance and role in maintaining biodiversity. In this study, we showed that supplementation with SB on honey bees increased lifespan in the range of concentrations from 5 mM to 60 mM, with the highest effect at 10 mM and 20 mM. Furthermore, we showed that 10-day supplementation with those two SB concentrations up-regulated vitellogenin, histone deacetylase HDAC1 and HDAC3 isoforms (but not Sirt 1), and immune-related gene expression, as well as improved the oxidative status of honey bees by increasing antioxidative capacity, reduced glutathione (GSH) levels, protein thiol groups, and lowering lipid peroxidation. The activity of the antioxidative enzymes superoxide dismutase (SOD) and glutathione S-transferase (GST) was also affected, but no changes in catalase (CAT) activity were recorded. These findings could lead to improvements in beekeeping practice.

HIV-1 latency reversal agent boosting is not limited by opioid use.

The opioid epidemic may impact the HIV-1 reservoir and its reversal from latency in virally suppressed people with HIV (PWH). We studied forty-seven PWH and observed that lowering the concentration of HIV-1 latency reversal agents (LRA), used in combination with small molecules that do not reverse latency, synergistically increases the magnitude of HIV-1 re-activation ex vivo, regardless of opioid use. This LRA boosting, which combines a Smac mimetic or low-dose protein kinase C agonist with histone deacetylase inhibitors, can generate significantly more unspliced HIV-1 transcription than phorbol 12-myristate 13-acetate (PMA) with ionomycin (PMAi), the maximal known HIV-1 reactivator. LRA boosting associated with greater histone acetylation in CD4+ T cells and modulated T cell activation-induced markers and intracellular cytokine production; Smac mimetic-based boosting was less likely to induce immune activation. We found that HIV-1 reservoirs in PWH contain unspliced and polyadenylated (polyA) virus mRNA, the ratios of which are greater in resting than total CD4+ T cells and can correct to 1:1 with PMAi exposure. Latency reversal results in greater fold-change increases to HIV-1 poly(A) mRNA than unspliced message. Multiply spliced HIV-1 transcripts and virion production did not consistently increase with LRA boosting, suggesting the presence of a persistent post-transcriptional block. LRA boosting can be leveraged to probe the mechanisms of an effective cellular HIV-1 latency reversal program.

Synergistic cytotoxicity of histone deacetylase and poly-ADP ribose polymerase inhibitors and decitabine in pancreatic cancer cells: Implications for novel therapy.

Histone deacetylase inhibitors (HDACi) can modulate the acetylation status of proteins, influencing the genomic instability exhibited by cancer cells. Poly (ADP ribose) polymerase (PARP) inhibitors (PARPi) have a direct effect on protein poly (ADP-ribosyl)ation, which is important for DNA repair. Decitabine is a nucleoside cytidine analogue, which when phosphorylated gets incorporated into the growing DNA strand, inhibiting methylation and inducing DNA damage by inactivating and trapping DNA methyltransferase on the DNA, thereby activating transcriptionally silenced DNA loci. We explored various combinations of HDACi and PARPi +/- decitabine (hypomethylating agent) in pancreatic cancer cell lines BxPC-3 and PL45 (wild-type BRCA1 and BRCA2) and Capan-1 (mutated BRCA2). The combination of HDACi (panobinostat or vorinostat) with PARPi (talazoparib or olaparib) resulted in synergistic cytotoxicity in all cell lines tested. The addition of decitabine further increased the synergistic cytotoxicity noted with HDACi and PARPi, triggering apoptosis (evidenced by increased cleavage of caspase 3 and PARP1). The 3-drug combination treatments (vorinostat, talazoparib, and decitabine; vorinostat, olaparib, and decitabine; panobinostat, talazoparib, and decitabine; panobinostat, olaparib, and decitabine) induced more DNA damage (increased phosphorylation of histone 2AX) than the individual drugs and impaired the DNA repair pathways (decreased levels of ATM, BRCA1, and ATRX proteins). The 3-drug combinations also altered the epigenetic regulation of gene expression (NuRD complex subunits, reduced levels). This is the first study to demonstrate synergistic interactions between the aforementioned agents in pancreatic cancer cell lines and provides preclinical data to design individualized therapeutic approaches with the potential to improve pancreatic cancer treatment outcomes.

Nanoparticle delivery of Tat synergizes with classical latency reversal agents to express HIV antigen targets.

Limited cellular levels of the HIV transcriptional activator Tat are one contributor to proviral latency that might be targeted in HIV cure strategies. We recently demonstrated that lipid nanoparticles containing HIV tat mRNA induce HIV expression in primary CD4 T cells. Here, we sought to further characterize tat mRNA in the context of several benchmark latency reversal agents (LRAs), including inhibitor of apoptosis protein antagonists (IAPi), bromodomain and extra-Terminal motif inhibitors (BETi), and histone deacetylase inhibitors (HDACi). tat mRNA reversed latency across several different cell line models of HIV latency, an effect dependent on the TAR hairpin loop. Synergistic enhancement of tat mRNA activity was observed with IAPi, HDACi, and BETi, albeit to variable degrees. In primary CD4 T cells from durably suppressed people with HIV, tat mRNA profoundly increased the frequencies of elongated, multiply-spliced, and polyadenylated HIV transcripts, while having a lesser impact on TAR transcript frequencies. tat mRNAs alone resulted in variable HIV p24 protein induction across donors. However, tat mRNA in combination with IAPi, BETi, or HDACi markedly enhanced HIV RNA and protein expression without overt cytotoxicity or cellular activation. Notably, combination regimens approached or in some cases exceeded the latency reversal activity of maximal mitogenic T cell stimulation. Higher levels of tat mRNA-driven HIV p24 induction were observed in donors with larger mitogen-inducible HIV reservoirs, and expression increased with prolonged exposure time. Combination LRA strategies employing both small molecule inhibitors and Tat delivered to CD4 T cells are a promising approach to effectively target the HIV reservoir.

Trichostatin A Promotes Cytotoxicity of Cisplatin, as Evidenced by Enhanced Apoptosis/Cell Death Markers.

Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, promotes the cytotoxicity of the genotoxic anticancer drug cisplatin, yet the underlying mechanism remains poorly understood. Herein, we revealed that TSA at a low concentration (1 μM) promoted the cisplatin-induced activation of caspase-3/6, which, in turn, increased the level of cleaved PARP1 and degraded lamin A&C, leading to more cisplatin-induced apoptosis and G2/M phase arrest of A549 cancer cells. Both ICP-MS and ToF-SIMS measurements demonstrated a significant increase in DNA-bound platinum in A549 cells in the presence of TSA, which was attributable to TSA-induced increase in the accessibility of genomic DNA to cisplatin attacking. The global quantitative proteomics results further showed that in the presence of TSA, cisplatin activated INF signaling to upregulate STAT1 and SAMHD1 to increase cisplatin sensitivity and downregulated ICAM1 and CD44 to reduce cell migration, synergistically promoting cisplatin cytotoxicity. Furthermore, in the presence of TSA, cisplatin downregulated TFAM and SLC3A2 to enhance cisplatin-induced ferroptosis, also contributing to the promotion of cisplatin cytotoxicity. Importantly, our posttranslational modification data indicated that acetylation at H4K8 played a dominant role in promoting cisplatin cytotoxicity. These findings provide novel insights into better understanding the principle of combining chemotherapy of genotoxic drugs and HDAC inhibitors for the treatment of cancers.

Phase I trial of chidamide, an oral HDAC inhibitor, in combination with oral etoposide in patients with refractory/recurrent neuroblastoma.

10034 Background: Chidamide, an oral subtype-selective histone deacetylase (HDAC) inhibitor, has been shown to be effective in adult patients with hematological tumors. However, there is no data on its safety and efficacy in pediatric cancer patients. Combination of HDAC inhibitors and etoposide have shown synergistic anti-tumor effects in preclinical studies of neuroblastoma. We conducted a phase I trial in pediatric patients with recurrent or refractory neuroblastoma to determine the maximum tolerable dose (MTD) of chidamide combined with oral etoposide treatment. Methods: Daily oral etoposide 35 mg/m2/dose was administered on days 1–21 in combination with escalating doses of chidamide (14 and 17 mg/m2/d) twice weekly in each 28-day cycle using the standard 3 + 3 design. Patients with response or stable disease after two cycles would maintain treatment until disease progression or unacceptable toxicity occurred. Chidamide pharmacokinetic testing was performed. Results: 31 patients were enrolled in this study. The median age was 7 years (range 3 – 18). 14 patients were included in the dose escalation phase (Ia), 17 patients in the expansion cohort (Ib). The MTD of chidamide was 14 mg/m2/d, with dose limiting neutropenia and thrombocytopenia observed at 17 mg/m2/d. Median number of cycles completed was 2.5 (range 1–11). A total of 19 patients (61.3%) experienced treatment-related grade 3/4 hematologic toxicity, and no grade 3/4 non-hematological toxicity was observed. No objective responses were seen. Conclusions: In children with refractory/recurrent neuroblastoma, chidamide is well-tolerated at 14 mg/m2/d twice weekly when combining with oral etoposide 35 mg/m2/d daily. Prolonged disease stability achieved in patients with minimal residual diseases deserves further investigation. Clinical trial information: NCT05338541 .

TORSEL, a 4EBP1-based mTORC1 live-cell sensor, reveals nutrient-sensing targeting by histone deacetylase inhibitors

BackgroundMammalian or mechanistic target of rapamycin complex 1 (mTORC1) is an effective therapeutic target for diseases such as cancer, diabetes, aging, and neurodegeneration. However, an efficient tool for monitoring mTORC1 inhibition in living cells or tissues is lacking.ResultsWe developed a genetically encoded mTORC1 sensor called TORSEL. This sensor changes its fluorescence pattern from diffuse to punctate when 4EBP1 dephosphorylation occurs and interacts with eIF4E. TORSEL can specifically sense the physiological, pharmacological, and genetic inhibition of mTORC1 signaling in living cells and tissues. Importantly, TORSEL is a valuable tool for imaging-based visual screening of mTORC1 inhibitors. Using TORSEL, we identified histone deacetylase inhibitors that selectively block nutrient-sensing signaling to inhibit mTORC1.ConclusionsTORSEL is a unique living cell sensor that efficiently detects the inhibition of mTORC1 activity, and histone deacetylase inhibitors such as panobinostat target mTORC1 signaling through amino acid sensing.

Synergistic cytotoxicity of histone deacetylase, poly-ADP ribose polymerase inhibitors and decitabine in breast and ovarian cancer cells: Therapeutic implications.

e15193 Background: Innovative strategies are needed for breast and ovarian cancer. We explored the synergistic cytotoxicity of histone deacetylase inhibitors (HDACi), poly(ADP-ribose) polymerase inhibitors (PARPi), and decitabine (DAC, D) in breast and ovarian cancer cells. The drugs affect epigenetic regulation and DNA repair, making them promising anticancer therapies. Methods: Breast (MDAMB231 and MCF-7) and ovarian (HEY-T30 and SKOV-3) cells were exposed to HDACi (panobinostat, Pano, P; vorinostat; Vori, V), PARPi (talazoparib, TLZ, T; olaparib, Ola, O) and DAC. Synergism was assessed by MTT and clonogenic assays. Changes in DNA damage response/repair markers were assessed with Western blotting. Results: The combinations of HDACi, PARPi, and DAC had synergistic effects in all cell lines, evidenced by combination index <1. Clonogenic assay confirmed sensitivity of all cell lines to the 3-drug combinations (Pano, TLZ, DAC; Pano, Ola, DAC; Vori, TLZ, DAC; Vori, Ola, DAC). Cell proliferation was inhibited by 50% - 70%, and Annexin V positivity was 51% - 58% in all cells exposed to the combinations. Western blots showed inhibited protein PARylation, cleaved caspase 3 and PARP1 (suggestive of apoptosis), and down-regulated c-MYC. The 3-drug combinations induced more DNA damage (increased phosphorylation of ƔH2AX) than the individual drugs, impaired DNA repair (decreased ATM, BRCA1, ATRX proteins; Artemis), and altered gene expression (reduced NuRD complex subunits). DNA damage caused by HDACis and DAC may confer increased cellular dependence on protein PARylation, increasing sensitivity to combined HDACi, PARPi, and DAC. The P values comparing the inhibitory effects of each drug combination with that of individual drugs (MTT assay) are shown in Table ( P ≤ .05, statistically significant). Conclusions: The results support exploring HDACi, PARPi, and DAC combinations to overcome drug resistance and improve patient outcomes, and carefully designed clinical trials are warranted in breast and ovarian cancer. [Table: see text]

PRM1201 effectively inhibits colorectal cancer metastasis via shaping gut microbiota and short- chain fatty acids

BackgroundPRM1201 is a traditional medicine with beneficial effects against colorectal cancer (CRC) metastasis. However, the underlying mechanism of this action remains to be determined. HypothesisRemodeling microbiota and short-chain fatty acids (SCFAs) metabolism might be a potential mechanism to explain the anti-metastatic action of PRM1201, as this gut-microbiota dependent effect involves downregulation of histone deacetylation and EMT. MethodsTo investigate this possibility, clinical specimens were sequenced and the correlation between the anti-metastatic efficacy of PRM1201 and the restoration of SCFA-producing bacteria was studied. To obtain solid causal evidence, a mouse metastasis model was established to detect the influence of PRM1201 on cancer metastasis. Specifically, 16S amplicon sequencing, ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) analysis, and bacterial manipulation were used to examine the gut microbiota-driven anti-metastatic action of PRM1201. ResultsClinical data showed that PRM1201 increased both the number of SCFA-producing bacteria and generation of SCFAs in the feces of CRC patients. A positive correlation between the anti-metastatic efficacy of PRM1201 and the restoration of SCFAs observed. The animal experiments demonstrated that PRM1201 effectively blocked CRC metastasis in a dose-dependent manner. PRM1201 treatment modulated the composition of gut microbiota, and promoted the proliferation of beneficial SCFAs producers such as Akkermansia, Lachnospiraceae_NK4A136_group and Blautia, while simultaneously reducing the abundance of pathogenic bacteria like Escherichia-Shigella. In addition, PRM1201 led to augmentation of SCFAs content. Further results indicated that the anti-cancer metastatic mechanism of PRM1201 was linked to inhibition of histone deacetylation and suppression of epithelial-to-mesenchymal transition (EMT) in metastatic lesions. Microbiota depletion treatment and fecal microbiota transplantation (FMT) underscored the microbiota-dependent nature of this phenomenon. Moreover, this anti-colorectal cancer metastatic effect and mechanism of total SCFAs and single SCFA were also confirmed. ConclusionIn summary, PRM1201 exerts its anti-metastatic effects by modulating SCFA-producing bacteria and enhancing the production of SCFAs. Furthermore, the prebiotic-like actions of PRM1201, along with the PRM1201-treated bacteria, function as inhibitors of histone deacetylases (DHACs) thereby effectively suppressing EMT events.