Discovery of novel nucleoside derivatives as selective lysine acetyltransferase p300 inhibitors for cancer therapy.

P300 and CBP are two closely related histone acetyltransferases that are important transcriptional coactivators of many cellular processes. Inhibition of the transcriptional regulator p300/CBP is a promising therapeutic approach in oncology. However, there are no reported single selective p300 or CBP inhibitors to date. In this study, we designed and optimized a series of lysine acetyltransferase p300 selective inhibitors bearing a nucleoside scaffold. Most compounds showed excellent inhibitory activity against p300 with IC50 ranging from 0.18 to 9.90 µM, except for J16, J29, J40, and J48. None of the compounds showed inhibitory activity against CBP (inhibition rate < 50 % at 10 µM). Then the cytotoxicity of the compounds against a series of cancer cells were evaluated. Compounds J31 and J32 showed excellent proliferation inhibitory activity on cancer cells T47D and H520 with desirable selectivity profile of p300 over CBP. These compounds could be promising lead compounds for the development of novel epigenetic inhibitors as antitumor agents.

Characteristics of anticancer activity of CBP/p300 inhibitors - Features of their classes, intracellular targets and future perspectives of their application in cancer treatment.

Due to the contribution of highly homologous acetyltransferases CBP and p300 to transcription elevation of oncogenes and other cancer promoting factors, these enzymes emerge as possible epigenetic targets of anticancer therapy. Extensive efforts in search for small molecule inhibitors led to development of compounds targeting histone acetyltransferase catalytic domain or chromatin-interacting bromodomain of CBP/p300, as well as dual BET and CBP/p300 inhibitors. The promising anticancer efficacy in in vitro and mice models led CCS1477 and NEO2734 to clinical trials. However, none of the described inhibitors is perfectly specific to CBP/p300 since they share similarity of a key functional domains with other enzymes, which are critically associated with cancer progression and their antagonists demonstrate remarkable clinical efficacy in cancer therapy. Therefore, we revise the possible and clinically relevant off-targets of CBP/p300 inhibitors that can be blocked simultaneously with CBP/p300 thereby improving the anticancer potential of CBP/p300 inhibitors and pharmacokinetic predicting data such as absorption, distribution, metabolism, excretion (ADME) and toxicity.

Comprehensive Characterization of HATs and HDACs in Human Cancers Reveals Their Role in Immune Checkpoint Blockade.

Histone acetylation that controlled by two mutually antagonistic enzyme families, histone acetyl transferases (HATs) and histone deacetylases (HDACs), as one of major epigenetic mechanisms controls transcription and its abnormal regulation was implicated in various aspects of cancer. However, the comprehensive understanding of HDACs and HATs in cancer is still lacking. Systematically analysis through 33 cancer types based on next-generation sequence data reveals heterogeneous expression pattern of HDACs and HATs across different cancer types. In particular, HDAC10 and HDAC6 show significant downregulation in most cancers. Principal components analysis (PCA) of pan-cancer reveals significant difference of HDACs and HATs between normal tissues and normal tissue adjacent to the tumor. The abnormal expression of HDACs and HATs was partially due to CNV and DNA methylation in multiple types of cancer. Prognostic significance (AUC reached 0.736) of HDACs and HATs demonstrates a five-gene signature including KAT2A, HAT1, KAT5, CREBBP and SIRT1 in KIRC. Analysis of NCI-60 drug database reveals the cytotoxic effect of several drugs are associated with dysregulated expression of HDACs and HATs. Analysis of immune infiltration and immunotherapy reveals that KAT2B and HDAC9 are associated with immune infiltration and immunotherapy. Our analysis provided comprehensive understanding of the regulation and implication of HDACs and HATs in pan-cancer. These findings provide novel evidence for biological investigating potential individual HDACs and HATs in the development and therapy of cancer in the future.

SNORD17-mediated KAT6B mRNA 2'-O-methylation regulates vasculogenic mimicry in glioblastoma cells.

Glioblastoma (GBM) is a primary tumor in the intracranial compartment. Vasculogenic mimicry (VM) is a process in which a pipeline of tumor cells that provide blood support to carcinogenic cells is formed, and studying VM could provide a new strategy for clinical targeted treatment of GBM. In the present study, we found that SNORD17 and ZNF384 were significantly upregulated and promoted VM in GBM, whereas KAT6B was downregulated and inhibited VM in GBM. RTL-P assays were performed to verify the 2'-O-methylation of KAT6B by SNORD17; IP assays were used to detect the acetylation of ZNF384 by KAT6B. In addition, the binding of ZNF384 to the promoter regions of VEGFR2 and VE-cadherin promoted transcription, as validated by chromatin immunoprecipitation and luciferase reporter assays. And finally, knockdown of SNORD17 and ZNF384 combined with KAT6B overexpression effectively reduced the xenograft tumor size, prolonged the survival time of nude mice and reduced the number of VM channels. This study reveals a novel mechanism of the SNORD17/KAT6B/ZNF384 axis in modulating VM development in GBM that may provide a new goal for the comprehensive treatment of GBM.

A Therapeutically Targetable NOTCH1-SIRT1-KAT7 Axis in T-cell Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a NOTCH1-driven disease in need of novel therapies. Here, we identify a NOTCH1-SIRT1-KAT7 link as a therapeutic vulnerability in T-ALL, in which the histone deacetylase SIRT1 is overexpressed downstream of a NOTCH1-bound enhancer. SIRT1 loss impaired leukemia generation, whereas SIRT1 overexpression accelerated leukemia and conferred resistance to NOTCH1 inhibition in a deacetylase-dependent manner. Moreover, pharmacologic or genetic inhibition of SIRT1 resulted in significant antileukemic effects. Global acetyl proteomics upon SIRT1 loss uncovered hyperacetylation of KAT7 and BRD1, subunits of a histone acetyltransferase complex targeting H4K12. Metabolic and gene-expression profiling revealed metabolic changes together with a transcriptional signature resembling KAT7 deletion. Consistently, SIRT1 loss resulted in reduced H4K12ac, and overexpression of a nonacetylatable KAT7-mutant partly rescued SIRT1 loss-induced proliferation defects. Overall, our results uncover therapeutic targets in T-ALL and reveal a circular feedback mechanism balancing deacetylase/acetyltransferase activation with potentially broad relevance in cancer. SIGNIFICANCE: We identify a T-ALL axis whereby NOTCH1 activates SIRT1 through an enhancer region, and SIRT1 deacetylates and activates KAT7. Targeting SIRT1 shows antileukemic effects, partly mediated by KAT7 inactivation. Our results reveal T-ALL therapeutic targets and uncover a rheostat mechanism between deacetylase/acetyltransferase activities with potentially broader cancer relevance. This article is highlighted in the In This Issue feature, p. 1.

Celastrol inhibits lung cancer growth by triggering histone acetylation and acting synergically with HDAC inhibitors.

Alterations in histone modification have been linked to cancer development and progression. Celastrol, a Chinese herbal compound, shows potent anti-tumor effects through multiple signaling pathways. However, the involvement of histone modifications in this process has not yet been illustrated. In this study, barcode sequencing of a eukaryotic genome-wide deletion library revealed that histone modifications, especially histone acetylation associated with the NuA4 histone acetyltransferase complex, were involved in the anti-proliferation actions of celastrol. The essential roles of histone modification were verified by celastrol sensitivity tests in cells lacking specific genes, such as genes encoding the subunits of the NuA4 and Swr1 complex. The combination of celastrol and histone deacetylase inhibitors (HDACi), rather than the combination of celastrol and histone acetyltransferase inhibitors, synergistically suppressed cancer cell proliferation. In addition to upregulating H4K16 acetylation (H4K16ac), celastrol regulates H3K4 tri-methylation and H3S10 phosphorylation. Celastrol treatment significantly enhanced the suppressive effects of HDACi on lung cancer cell allografts in mice, with significant H4K16ac upregulation, indicating that a combination of celastrol and HDACi is a potential novel therapeutic approach for patients with lung cancer.

The role of protein acetylation in carcinogenesis and targeted drug discovery.

Protein acetylation is a reversible post-translational modification, and is involved in many biological processes in cells, such as transcriptional regulation, DNA damage repair, and energy metabolism, which is an important molecular event and is associated with a wide range of diseases such as cancers. Protein acetylation is dynamically regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) in homeostasis. The abnormal acetylation level might lead to the occurrence and deterioration of a cancer, and is closely related to various pathophysiological characteristics of a cancer, such as malignant phenotypes, and promotes cancer cells to adapt to tumor microenvironment. Therapeutic modalities targeting protein acetylation are a potential therapeutic strategy. This article discussed the roles of protein acetylation in tumor pathology and therapeutic drugs targeting protein acetylation, which offers the contributions of protein acetylation in clarification of carcinogenesis, and discovery of therapeutic drugs for cancers, and lays the foundation for precision medicine in oncology.

Hen Egg Lysozyme Alleviates Static Mechanical Pain Via NRF1-Parkin-TACAN Signaling Axis in Sensory Neurons.

Mechanical allodynia impinges on the life quality of patients. Hen Egg Lysozyme (HEL) is a substance extracted from eggs that is commonly used to inhibit bacterial activity. The role of HEL in regulating and treating pain is unclear. Here, we find that HEL selectively attenuates static mechanical allodynia of mice induced by complete Freund's adjuvant (CFA), spinal nerve ligation (SNL) and chemotherapeutic agent. RNA-seq screening reveals that CFA significantly reduces the expression of Parkin in dorsal root ganglion (DRG) neurons of mice, while pre-administration of HEL increases the expression of Parkin and remits the static mechanical allodynia induced by Parkin-siRNA. Moreover, HEL increases the interaction between nuclear respiratory factor 1 (NRF1) and histone acetyltransferase P300 and then enhances the NRF1 mediated histone acetylation in prkn promoter region in DRGs of mice. Further, Parkin interacts with mechanotransducing ion channel TACAN (Tmem120a) and knockdown of Parkin significantly increases the membrane trafficking of TACAN in sensory neurons of mice. While pre-administration of HEL inhibits the increased membrane trafficking of TACAN in sensory neurons of mice induced by Parkin-siRNA. In addition, pre-given of HEL also significantly attenuates the static mechanical allodynia induced by overexpression of TACAN in mice, and the effect of HEL can be blocked by Parkin-siRNA. This indicates that HEL increases the expression of Parkin through epigenetic mechanisms and then decreases TACAN membrane trafficking in sensory neurons to relieve static mechanical hypersensitivity. Therefore, we reveal a novel function of HEL, which is a potential substance for the treatment of static mechanical pain.

Epigenetic modifications control loss of adhesion and aggressiveness of cancer stem cells derived from head and neck squamous cell carcinoma with intrinsic resistance to cisplatin.

OBJECTIVES: The aims of this study were to investigate the epigenetic mechanisms and biological changes implicated in intrinsic and acquired resistance to cisplatin, a chemotherapy commonly used to treat head and neck squamous cell carcinoma. DESIGN: Intrinsic resistance (IR) was established in CAL-27 and acquired resistance (AR) in SCC-9 cell lines. Changes in the phenotype were evaluated by immunofluorescence, colony assay, invasion and spheres formation. Epigenetic regulation were assessed by quantitative PCR and western blot. RESULTS: Changes DNA damage accumulation, and a decrease of reactive oxygen species in cisplatin-resistant cell lines suggest a protection mechanism against cell death. Increases in aggressiveness, observed by clonogenic and invasive potentials, were more pronounced on the CAL-27 IR cell line. Cancer stem cells (CSC) were increased in cisplatin-resistant cells, and the administration of cisplatin increases CSC accumulation in CAL-27 IR. The loss of adhesion was noticed in CSC from IR cells. The upregulation of the genes HDAC2, HDAC9, SIRT1, KAT2B, KAT6A, KAT6B, and BRD4, the HDAC1 nuclear distribution and the decrease of the acetylated proteins H3K9, H3K36, H3K79, and H4K5 indicate that the IR mobilizes epigenetic modifications in acetylation levels, favoring the aggressiveness phenotype. Therefore, the treatment of CSC derived from CAL-27 IR with the histone deacetylase inhibitor, Vorinostat, partially recovered the CSC adhesion ability by up-regulating the levels of FAK, ß3 integrin, and Vinculin proteins. CONCLUSIONS: Our findings indicate that intrinsic-resistant cells are regulated by epigenetic modifications, which could be a potential target to treat resistant head and neck squamous cell carcinoma.

Abnormal expression of histone acetylases in CD8+ T cells of patients with severe aplastic anemia.

INTRODUCTION: We aimed to investigate the balance between the mRNA levels of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in CD8+ T cells of patients with severe aplastic anemia (SAA). METHODS: Twenty untreated SAA patients, 18 remission SAA patients (R-SAA), and 22 normal controls were evaluated. The mRNA expression levels of HATs, HDACs, and IFNG in CD8+ T cells were measured by real-time quantitative reverse transcription polymerase chain reaction. RESULTS: Histone acetylase EP300 and CREBBP mRNA levels were significantly elevated in CD8+ T cells of SAA patients compared with the normal controls (both p < 0.05). No significant differences were observed in HDAC1 and HDAC7 mRNA between SAA patients and the normal controls. There was an obvious positive correlation between IFNG and EP300 (r = 0.5126, p < 0.01), and CREBBP (r = 0.4663, p < 0.05), respectively, in SAA and R-SAA patients. In addition, EP300 and CREBBP mRNA levels were clearly correlated with clinical parameters of peripheral blood and bone marrow in those patients. CONCLUSION: Our findings suggest that EP300 and CREBBP are increased in CD8+ T cells of SAA patients and are correlated with disease severity. The imbalances in HATs and HDACs may play a role in activating CD8+ T cells to promote the immune pathogenesis of SAA.

Minocycline suppresses lipogenesis via inhibition of p300 histone acetyltransferase activity in human SZ95 sebocytes.

BACKGROUND: Minocycline is a second-generation tetracycline drug, which is widely used to treat diverse infectious and inflammatory diseases such as acne vulgaris. The effects of minocycline on acne vulgaris have been mainly attributed to its anti-inflammatory effect; however, its sebum-regulating effect and the relevance to epigenetic regulation in human sebaceous glands remain uninvestigated. OBJECTIVES: To identify the potential underlying epigenetic mechanism of sebum-inhibitory effects of minocycline in human SZ95 sebocytes. METHODS: The quantity of lipid droplets and the expression of key lipogenic genes were analysed in minocycline-treated SZ95 sebocytes. To examine whether the sebum-inhibitory effects of minocycline are relevant to histone acetylation, we analysed the effects of minocycline on p300 HAT and total HDAC activity. To elucidate the functional implication of p300 HAT inhibition by minocycline in sebocytes, we assessed the effect of p300 knockdown, inhibition and overexpression on lipid accumulation in SZ95 sebocytes. RESULTS: Minocycline suppressed the insulin and liver X receptor agonist-induced lipid accumulation and the expression of the key lipogenic transcription factor sterol regulatory element-binding protein 1 (SREBP1) and its downstream genes, fatty acid synthase (FAS) and acetyl-CoA carboxylase α (ACCα). Minocycline inhibited p300 HAT activity in a concentration-dependent manner, but demonstrated no effect on global HDAC activity, resulting in a significant decrease in histone acetylation. Downregulation of p300 by knockdown or inhibition significantly suppressed SREBP1 expression, histone acetylation and lipid accumulation, whereas p300 overexpression enhanced these effects. Moreover, p300 overexpression rescued minocycline-inhibited SREBP1 expression and lipid synthesis. CONCLUSIONS: Our findings revealed a novel sebum-regulating effect of minocycline. Moreover, as p300 HAT is a key epigenetic regulator of sebaceous lipogenesis, its inhibitors could be used for the treatment of acne vulgaris.

Histone Acetyltransferases in Cancer: Guardians or Hazards?

Histone acetyltransferases (HATs) catalyzing N-epsilon-lysine or N-alpha-terminal acetylation on histone and non-histone substrates are important epigenetic regulators controlling gene expression and chromatin structure. Deregulation of these enzymes by genetic or epigenetic alterations accompanied by defects in gene transcription have been implicated in oncogenesis. Therefore, these enzymes are considered promising therapeutic targets, offering new horizons for epigenetic cancer therapy. However, recent observations suggest that these enzymes function as both oncogenes and tumor suppressors. In this review, we present the current evidence demonstrating that individual HATs can either prevent cancer cell proliferation or drive malignant transformation depending on the molecular context and cancer type. We therefore advocate that future therapeutic interventions targeted toward these enzymes should carefully consider the fact that HATs commonly have a two-sided role in carcinogenesis.

Recombinant human hyaluronidase facilitated subcutaneous immunoglobulin treatment in pediatric patients with primary immunodeficiencies: long-term efficacy, safety and tolerability.

AIM: To assess the long-term efficacy, safety and tolerability of recombinant human hyaluronidase-facilitated subcutaneous infusion of immunoglobulin (Ig) (fSCIG; HYQVIA(®); IGHy) in children aged <18 years. PATIENTS & METHODS: Patients with primary immunodeficiency diseases were included in the studies. IGHy was administered every 3 or 4 weeks. RESULTS: Validated acute serious bacterial infections were reported at 0.08/patient-year (four pneumonia episodes in three patients). No serious adverse drug reaction (ADR) was reported, and rates of local and systemic ADRs were low (0.09/infusion and 0.1/infusion). Infection rates were low (3.02/patient-year) with sustained Ig trough levels (median: 1009 mg/dl). Of 674 IGHy infusions, 97.2% required no change of administration due to ADR, in most (82.5%) with one infusion site. No patient developed neutralizing anti-rHuPH20 antibodies. Postpivotal study, 100% of patients aged <14 years or their caregivers and 85.7% of patients aged 14 to <18 years expressed preference for IGHy compared with Ig administered intravenously or Ig administered subcutaneously. CONCLUSION: These studies, with the longest (maximum: 3.3 years) duration of any reported Ig replacement trials in children with primary immunodeficiency diseases, showed low infection, local and systemic reaction rates along with well-tolerated infusions given in a single site.

Comparative evaluation of the efficacy of 2% lidocaine containing 1:200,000 epinephrine with and without hyaluronidase (75 IU) in patients with irreversible pulpitis.

INTRODUCTIONS: The purpose of this study was to determine the anesthetic efficacy of lidocaine containing epinephrine compared with lidocaine containing epinephrine plus hyaluronidase (75 IU) when performing an inferior alveolar nerve block. METHODS: Patients complaining of pain in the mandibular posterior teeth were selected. Based on their chief complaint, proper clinical and radiographic examinations were performed. Among them, 40 subjects diagnosed with irreversible pulpitis were selected. The inferior alveolar nerve block was induced using 3 mL 2% lidocaine with epinephrine. Hyaluronidase (75 IU) or a placebo was injected 30 minutes after the beginning of pulpal anesthesia (randomized and double-blind trial). The duration of the effect in the pulpal and gingival tissues was evaluated by the response to painful electrical stimuli applied to the adjacent premolar and by mechanical stimuli (pinprick) to the buccal gingiva, respectively. RESULTS: In both pulpal and gingival tissues, the duration of the anesthetic effects with hyaluronidase was longer than with placebo. CONCLUSIONS: Hyaluronidase increased the duration of the effects of lidocaine in inferior alveolar nerve blocks.

Histone deacetylase inhibitors: new treatment options for inflammatory joint disease?

Histone deacetylase inhibitors (HDIs) are a new class of compounds that are being developed for the treatment of malignancies such as cutaneous T-cell lymphoma. HDIs inhibit the removal of acetyl groups from histones. The histone acetylation process is dependent on two enzymes, histone acetyl transferase (HAT) and histone deacetylase (HDAC), and regulates the expression of genes, including those encoding cell survival or apoptosis. In addition to regulating cell growth, HDIs exert anti-inflammatory effects by controlling the production of anti-inflammatory cytokines; modulating the function of cells such as T cells, monocytes-macrophages, chondrocytes, and osteoclasts; and modulating angiogenesis. In several animal models of arthritis, HDIs improve the clinical manifestations and prevent damage to the bone and cartilage. In humans, the only relevant data available so far come from studies of HAT and HDAC expression in the synovial membrane of patients with rheumatoid arthritis. HDIs may hold promise for the treatment of inflammatory joint disease.

Randomized sibling-oocyte study using recombinant human hyaluronidase versus bovine-derived Sigma hyaluronidase in ICSI patients.

BACKGROUND: The enzyme hyaluronidase from bovine origin is commonly used for oocyte-cumulus cell removal in ICSI. A recombinant human hyaluronidase (rHuPH20) has been introduced as a quality-controlled and safe alternative. METHODS: In order to validate its effectiveness, a non-inferiority trial was started on sibling cumulus-oocyte complexes (135 ICSI patients). Oocyte denudation involved enzyme incubation under Pasteur pipetting, followed by further mechanical stripping. Primary end-points were oocyte intactness after ICSI and fertilization rate. Secondary end-points were embryo development and positive hCG. RESULTS: Oocyte intactness after ICSI was 89.6% and 92.9% with rHuPH20 and bovine hyaluronidase, respectively [absolute difference -3.3% (-7.4 to 10.7)]. The fertilization rate was 73.9% after rHuPH20 and 77.1% after bovine hyaluronidase treatment [absolute difference -3.2% (-8.3 to 1.8)]. Embryo development was similar in both treatment groups up till Day 5. Positive hCGs were equally distributed over mixed transfers 21/45 (46.7%) and transfers of only embryos from rHuPH20 treatment 17/35 (48.6%) or transfers of only embryos from bovine hyaluronidase treatment 22/48 (45.8%). CONCLUSIONS: Our results indicate that rHuPH20 is not inferior to bovine hyaluronidase for oocyte denudation, with regard to oocyte survival and fertilization. rHuPH20 treatment of human oocytes is compatible with good embryo development, with positive hCG results and with live birth.