Trichostatin C Synergistically Interacts with DNMT Inhibitor to Induce Antineoplastic Effect via Inhibition of Axl in Bladder and Lung Cancer Cells.

Aberrant epigenetic modifications are fundamental contributors to the pathogenesis of various cancers. Consequently, targeting these aberrations with small molecules, such as histone deacetylase (HDAC) inhibitors and DNA methyltransferase (DNMT) inhibitors, presents a viable strategy for cancer therapy. The objective of this study is to assess the anti-cancer efficacy of trichostatin C (TSC), an analogue of trichostatin A sourced from the fermentation of Streptomyces sp. CPCC 203909. Our investigations reveal that TSC demonstrates potent activity against both human lung cancer and urothelial bladder cancer cell lines, with IC50 values in the low micromolar range. Moreover, TSC induces apoptosis mediated by caspase 3/7 and arrests the cell cycle at the G2/M phase. When combined with the DNMT inhibitor decitabine, TSC exhibits a synergistic anti-cancer effect. Additionally, protein analysis elucidates a significant reduction in the expression of the tyrosine kinase receptor Axl. Notably, elevated concentrations of TSC correlate with the up-regulation of the transcription factor forkhead box class O1 (FoxO1) and increased levels of the proapoptotic proteins Bim and p21. In conclusion, our findings suggest TSC as a promising anti-cancer agent with HDAC inhibitory activity. Furthermore, our results highlight the potential utility of TSC in combination with DNMT inhibitors for cancer treatment.

Neuroinflammation and the role of epigenetic-based therapies for Huntington's disease management: the new paradigm.

Huntington's disease (HD) is an inherited, autosomal, neurodegenerative ailment that affects the striatum of the brain. Despite its debilitating effect on its patients, there is no proven cure for HD management as of yet. Neuroinflammation, excitotoxicity, and environmental factors have been reported to influence the regulation of gene expression by modifying epigenetic mechanisms. Aside focusing on the etiology, changes in epigenetic mechanisms have become a crucial factor influencing the interaction between HTT protein and epigenetically transcribed genes involved in neuroinflammation and HD. This review presents relevant literature on epigenetics with special emphasis on neuroinflammation and HD. It summarizes pertinent research on the role of neuroinflammation and post-translational modifications of chromatin, including DNA methylation, histone modification, and miRNAs. To achieve this about 1500 articles were reviewed via databases like PubMed, ScienceDirect, Google Scholar, and Web of Science. They were reduced to 534 using MeSH words like 'epigenetics, neuroinflammation, and HD' coupled with Boolean operators. Results indicated that major contributing factors to the development of HD such as mitochondrial dysfunction, excitotoxicity, neuroinflammation, and apoptosis are affected by epigenetic alterations. However, the association between neuroinflammation-altered epigenetics and the reported transcriptional changes in HD is unknown. Also, the link between epigenetically dysregulated genomic regions and specific DNA sequences suggests the likelihood that transcription factors, chromatin-remodeling proteins, and enzymes that affect gene expression are all disrupted simultaneously. Hence, therapies that target pathogenic pathways in HD, including neuroinflammation, transcriptional dysregulation, triplet instability, vesicle trafficking dysfunction, and protein degradation, need to be developed.

A 2-week treatment with 5-azacytidine improved the hypercontractility state in prostate from obese mice: Role of the nitric oxide-cyclic guanosine monophosphate signalling pathway.

Benign prostatic hyperplasia (BPH) is characterised by increases in prostate volume and contraction. Downregulation of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway contributes to prostate dysfunctions. Previous studies in cancer cells or vessels have shown that the epigenetic mechanisms control the gene and protein expression of the enzymes involved in the production of NO and cGMP. This study is aimed to evaluate the effect of a 2-week treatment of 5-azacytidine (5-AZA), a DNA-methyltransferase inhibitor, in the prostate function of mice fed with a high-fat diet. Functional, histological, biochemical and molecular assays were carried out. Obese mice presented greater prostate weight, α-actin expression and contractile response induced by the α-1adrenoceptors agonist. The relaxation induced by the NO-donor and the protein expression of endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) were significantly decreased in the prostate of obese mice. The treatment with 5-AZA reverted the higher expression of α-actin, reduced the hypercontractility state of the prostate and increased the expression of eNOS and sGC and intraprostatic levels of cGMP. When prostates from obese mice treated with 5-AZA were incubated in vitro with inhibitors of the NOS or sGC, the inhibitory effect of 5-AZA was reverted, therefore, showing the involvement of NO and cGMP. In conclusion, our study paves the way to develop or repurpose therapies that recover the expression of eNOS and sGC and, hence, to improve prostate function in BPH.

Chidamide, Decitabine, Cytarabine, Aclarubicin, and Granulocyte Colony-stimulating Factor Therapy for Patients with Relapsed/Refractory Acute Myeloid Leukemia: A Retrospective Study from a Single-Center.

OBJECTIVE: Preclinical evidence and clinical trials have suggested synergistic effects of epigenetic modifiers in combination with cytotoxic agents for the treatment of leukemia. However, their efficacy in patients with relapsed/refractory acute myeloid leukemia (R/R AML) remains unclear. METHODS: Clinical data of R/R AML patients who received a CDCAG regimen (chidamide, decitabine, cytarabine, aclarubicin, and granulocyte colony-stimulating factor) from July 1, 2018 to October 31, 2021 at our center were retrospectively assessed, and the safety and efficacy of the CDCAG regimen were evaluated. Patients were followed up until November 30, 2021, with a median follow-up of 21.6 months (95% CI: 10.0-33.2 months). RESULTS: A total of 67 patients were enrolled. Two patients died within 3 weeks after the initiation, and therefore only 65 patients underwent the assement for clinical response and survival. It was found that 56.9% patients achieved complete remission with a median overall survival (OS) of 9.6 months. The median OS of responders was 25.9 months, while that of non-responders was 5.0 months (P<0.0001). Patients with gene mutations had a superior overall response rate (ORR) (80.4% vs. 45.5%, P=0.043) compared to those without gene mutations. The presence of DNA methyltransferase 3 A (DNMT3A), ten-eleven translocation-2 (TET2), and isocitrate dehydrogenase 1/2 (IDH1/2) mutations did not affect the response rate (88.2% vs. 68.9%, P=0.220) and reflected a better OS (not attained vs. 9.0 months, P=0.05). The most common non-hematologic adverse events were pulmonary infection (73.1%), followed by febrile neutropenia (23.9%) and sepsis (19.4%). CONCLUSIONS: The CDCAG regimen was effective and well-tolerated in R/R AML patients, increasing the potential for allogeneic hematopoietic stem cell transplantation. Moreover, patients with DNMT3A, TET2, and IDH1/2 mutations might benefit from this regimen.

Characterisation of miRNA Expression in Dental Pulp Cells during Epigenetically-Driven Reparative Processes.

Within regenerative endodontics, exciting opportunities exist for the development of next-generation targeted biomaterials that harness epigenetic machinery, including microRNAs (miRNAs), histone acetylation, and DNA methylation, which are used to control pulpitis and to stimulate repair. Although histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) induce mineralisation in dental pulp cell (DPC) populations, their interaction with miRNAs during DPC mineralisation is not known. Here, small RNA sequencing and bioinformatic analysis were used to establish a miRNA expression profile for mineralising DPCs in culture. Additionally, the effects of a HDACi, suberoylanilide hydroxamic acid (SAHA), and a DNMTi, 5-aza-2'-deoxycytidine (5-AZA-CdR), on miRNA expression, as well as DPC mineralisation and proliferation, were analysed. Both inhibitors increased mineralisation. However, they reduced cell growth. Epigenetically-enhanced mineralisation was accompanied by widespread changes in miRNA expression. Bioinformatic analysis identified many differentially expressed mature miRNAs that were suggested to have roles in mineralisation and stem cell differentiation, including regulation of the Wnt and MAPK pathways. Selected candidate miRNAs were demonstrated by qRT-PCR to be differentially regulated at various time points in mineralising DPC cultures treated with SAHA or 5-AZA-CdR. These data validated the RNA sequencing analysis and highlighted an increased and dynamic interaction between miRNA and epigenetic modifiers during the DPC reparative processes.

Why do we not have more drugs approved for MDS? A critical viewpoint on novel drug development in MDS.

Approval of new agents to treat higher risk (HR) myelodysplastic syndrome (MDS) has stalled since the approval of DNA methyltransferase inhibitors (DNMTi). In addition, the options for patients with lower risk (LR) MDS who have high transfusion needs and do not harbor ring sideroblasts or 5q- syndrome are limited. Here, we review the current treatment landscape in MDS and identify areas of unmet need, such as treatment after failure of erythropoiesis-stimulating agents or DNMTis, TP53-mutated disease, and MDS with potentially targetable mutations. We discuss how our understanding of MDS pathogenesis can inform therapy development, including treating HR-MDS similarly to AML and pursuing therapies to address splicing factor mutations and dysregulated inflammation. We then bring a critical lens to current methodology of MDS studies and propose solutions to improve the efficiency and yield of these clinical trials, including using the most meaningful response metrics and expanding enrollment.

DNA methylation subtypes guiding prognostic assessment and linking to responses the DNA methyltransferase inhibitor SGI-110 in urothelial carcinoma.

BACKGROUND: At present, the extent and clinical relevance of epigenetic differences between upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB) remain largely unknown. Here, we conducted a study to describe the global DNA methylation landscape of UTUC and UCB and to address the prognostic value of DNA methylation subtype and responses to the DNA methyltransferase inhibitor SGI-110 in urothelial carcinoma (UC). METHODS: Using whole-genome bisulfite sequencing (n = 49 samples), we analyzed epigenomic features and profiles of UTUC (n = 36) and UCB (n = 9). Next, we characterized potential links between DNA methylation, gene expression (n = 9 samples), and clinical outcomes. Then, we integrated an independent UTUC cohort (Fujii et al., n = 86) and UCB cohort (TCGA, n = 411) to validate the prognostic significance. Furthermore, we performed an integrative analysis of genome-wide DNA methylation and gene expression in two UC cell lines following transient DNA methyltransferase inhibitor SGI-110 treatment to identify potential epigenetic driver events that contribute to drug efficacy. RESULTS: We showed that UTUC and UCB have very similar DNA methylation profiles. Unsupervised DNA methylation classification identified two epi-clusters, Methy-High and Methy-Low, associated with distinct muscle-invasive statuses and patient outcomes. Methy-High samples were hypermethylated, immune-infiltrated, and enriched for exhausted T cells, with poor clinical outcome. SGI-110 inhibited the migration and invasion of Methy-High UC cell lines (UMUC-3 and T24) by upregulating multiple antitumor immune pathways. CONCLUSIONS: DNA methylation subtypes pave the way for predicting patient prognosis in UC. Our results provide mechanistic rationale for evaluating SGI-110 in treating UC patients in the clinic.

PBRM1 Deficiency Sensitizes Renal Cancer Cells to DNMT Inhibitor 5-Fluoro-2'-Deoxycytidine.

PBRM1 is a tumor suppressor frequently mutated in clear cell renal cell carcinoma. However, no effective targeted therapies exist for ccRCC with PBRM1 loss. To identify novel therapeutic approaches to targeting PBRM1-deficient renal cancers, we employed a synthetic lethality compound screening in isogenic PBRM1+/+ and PBRM1-/- 786-O renal tumor cells and found that a DNMT inhibitor 5-Fluoro-2'-deoxycytidine (Fdcyd) selectively inhibit PBRM1-deficient tumor growth. RCC cells lacking PBRM1 show enhanced DNA damage response, which leads to sensitivity to DNA toxic drugs. Fdcyd treatment not only induces DNA damage, but also re-activated a pro-apoptotic factor XAF1 and further promotes the genotoxic stress-induced PBRM1-deficient cell death. This study shows a novel synthetic lethality interaction between PBRM1 loss and Fdcyd treatment and indicates that DNMT inhibitor represents a novel strategy for treating ccRCC with PBRM1 loss-of-function mutations.

Antitumor Effect of Simvastatin in Combination With DNA Methyltransferase Inhibitor on Gastric Cancer via GSDME-Mediated Pyroptosis.

Gasdermin E (GSDME) is one of the executors of pyroptosis, a type of programmed lytic cell death, which can be triggered by caspase-3 activation upon stimulation. Silenced GSDME expression due to promoter hypermethylation is associated with gastric cancer (GC), which is confirmed in the present study by bioinformatics analysis and methylation-specific PCR (MSP) test of GC cell lines and clinical samples. GC cell lines and mouse xenograft models were used to investigate the pyroptosis-inducing effect of the common cholesterol-depleting, drug simvastatin (SIM), allied with upregulating GSDME expression by doxycycline (DOX)- inducible Tet-on system or DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR). Cell viability assessment and xenograft tumour growth demonstrated that the tumour inhibition effects of SIM can be enhanced by elevated GSDME expression. Morphological examinations and assays measuring lactate dehydrogenase (LDH) release and caspase-3/GSDME protein cleavage underlined the stimulation of pyroptosis as an important mechanism. Using short hairpin RNA (shRNA) knockdown of caspase-3 or GSDME, and caspase-3-specific inhibitors, we provided evidence of the requirement of caspase-3/GSDME in the pyroptosis process triggered by SIM. We conclude that reactivating GSDME expression and thereby inducing cancer cell-specific pyroptosis could be a potential therapeutic strategy against GC.

Drugging the Epigenome: Overcoming Resistance to Targeted and Immunotherapies in Melanoma.

This past decade has seen tremendous advances in understanding the molecular pathogenesis of melanoma and the development of novel effective therapies for melanoma. Targeted therapies and immunotherapies that extend survival of patients with advanced disease have been developed; however, the vast majority of patients experience relapse and therapeutic resistance over time. Moreover, cellular plasticity has been demonstrated to be a driver of therapeutic resistance mechanisms in melanoma and other cancers, largely functioning through epigenetic mechanisms, suggesting that targeting of the cancer epigenetic landscape may prove a worthwhile endeavor to ensure durable treatment responses and cures. Here, we review the epigenetic alterations that characterize melanoma development, progression, and resistance to targeted therapies as well as epigenetic therapies currently in use and under development for melanoma and other cancers. We further assess the landscape of epigenetic therapies in clinical trials for melanoma and provide a framework for future advances in epigenetic therapies to circumvent the development of therapeutic resistance in melanoma.

Pembrolizumab plus azacitidine in patients with chemotherapy refractory metastatic colorectal cancer: a single-arm phase 2 trial and correlative biomarker analysis.

BACKGROUND: DNA mismatch repair proficient (pMMR) metastatic colorectal cancer (mCRC) is not responsive to pembrolizumab monotherapy. DNA methyltransferase inhibitors can promote antitumor immune responses. This clinical trial investigated whether concurrent treatment with azacitidine enhances the antitumor activity of pembrolizumab in mCRC. METHODS: We conducted a phase 2 single-arm trial evaluating activity and tolerability of pembrolizumab plus azacitidine in patients with chemotherapy-refractory mCRC (NCT02260440). Patients received pembrolizumab 200 mg IV on day 1 and azacitidine 100 mg SQ on days 1-5, every 3 weeks. A low fixed dose of azacitidine was chosen in order to reduce the possibility of a direct cytotoxic effect of the drug, since the main focus of this study was to investigate its potential immunomodulatory effect. The primary endpoint of this study was overall response rate (ORR) using RECIST v1.1., and secondary endpoints were progression-free survival (PFS) and overall survival (OS). Tumor tissue was collected pre- and on-treatment for correlative studies. RESULTS: Thirty chemotherapy-refractory patients received a median of three cycles of therapy. One patient achieved partial response (PR), and one patient had stable disease (SD) as best confirmed response. The ORR was 3%, median PFS was 1.9 months, and median OS was 6.3 months. The combination regimen was well-tolerated, and 96% of treatment-related adverse events (TRAEs) were grade 1/2. This trial was terminated prior to the accrual target of 40 patients due to lack of clinical efficacy. DNA methylation on-treatment as compared to pre-treatment decreased genome wide in 10 of 15 patients with paired biopsies and was significantly lower in gene promoter regions after treatment. These promoter demethylated genes represented a higher proportion of upregulated genes, including several immune gene sets, endogenous retroviral elements, and cancer-testis antigens. CD8+ TIL density trended higher on-treatment compared to pre-treatment. Higher CD8+ TIL density at baseline was associated with greater likelihood of benefit from treatment. On-treatment tumor demethylation correlated with the increases in tumor CD8+ TIL density. CONCLUSIONS: The combination of pembrolizumab and azacitidine is safe and tolerable with modest clinical activity in the treatment for chemotherapy-refractory mCRC. Correlative studies suggest that tumor DNA demethylation and immunomodulation occurs. An association between tumor DNA demethylation and tumor-immune modulation suggests immune modulation and may result from treatment with azacitidine. Trial registration ClinicalTrials.gov, NCT02260440. Registered 9 October 2014, https://clinicaltrials.gov/ct2/show/NCT02260440 .

Recent progress in DNA methyltransferase inhibitors as anticancer agents.

DNA methylation mediated by DNA methyltransferase is an important epigenetic process that regulates gene expression in mammals, which plays a key role in silencing certain genes, such as tumor suppressor genes, in cancer, and it has become a promising therapeutic target for cancer treatment. Similar to other epigenetic targets, DNA methyltransferase can also be modulated by chemical agents. Four agents have already been approved to treat hematological cancers. In order to promote the development of a DNA methyltransferase inhibitor as an anti-tumor agent, in the current review, we discuss the relationship between DNA methylation and tumor, the anti-tumor mechanism, the research progress and pharmacological properties of DNA methyltransferase inhibitors, and the future research trend of DNA methyltransferase inhibitors.

5-Azacitidine and Trichostatin A induce DNA damage and apoptotic responses in tongue squamous cell carcinoma: An in vitro study.

OBJECTIVE: The present in vitro study aims to investigate the potential use of epigenetic inhibitors as treatment modalities in tongue squamous cell carcinoma. DESIGN: The human tongue squamous cell carcinoma cell line (CAL-27) was cultured and exposed to varying concentrations of 5-Azacitidine (5-Aza) or Trichostatin A (TSA) in the culture medium. The cell apoptosis was evaluated using Annexin V/PI by flow cytometry. To evaluate DNA damage response, γH2AX foci analysis was performed using immunofluorescence. Single cell gel electrophoresis (SCGE) was applied to measure DNA strand breaks. Gene expression was assessed by quantitative real-time PCR. RESULTS: The results showed that 5-Aza and TSA had apoptotic effects on the SCC cell line at concentrations of 50-200 µM and 0.5-5 µM, respectively. Immunofluorescence analysis showed increased expression of γH2AX, the marker of DNA damage response after treatment of 5-Aza and TSA that was associated with increased DNA strand breaks. The expressions of urokinase plasminogen activator, its receptor and matrix metalloproteinase-2, were significantly reduced in TSA- and 5-Aza-treated cells. CONCLUSIONS: Our results showed that 5-Aza and TSA increase apoptotic and DNA damage response in squamous cell carcinoma cell line while reducing the expression of tumor invasion genes that further indicating the potential therapeutic value of two epigenetic modifiers in squamous cell carcinoma.

Epigenetic regulation of dental pulp stem cells and its potential in regenerative endodontics.

Regenerative endodontics (RE) therapy means physiologically replacing damaged pulp tissue and regaining functional dentin-pulp complex. Current clinical RE procedures recruit endogenous stem cells from the apical papilla, periodontal tissue, bone marrow and peripheral blood, with or without application of scaffolds and growth factors in the root canal space, resulting in cementum-like and bone-like tissue formation. Without the involvement of dental pulp stem cells (DPSCs), it is unlikely that functional pulp regeneration can be achieved, even though acceptable repair can be acquired. DPSCs, due to their specific odontogenic potential, high proliferation, neurovascular property, and easy accessibility, are considered as the most eligible cell source for dentin-pulp regeneration. The regenerative potential of DPSCs has been demonstrated by recent clinical progress. DPSC transplantation following pulpectomy has successfully reconstructed neurovascularized pulp that simulates the physiological structure of natural pulp. The self-renewal, proliferation, and odontogenic differentiation of DPSCs are under the control of a cascade of transcription factors. Over recent decades, epigenetic modulations implicating histone modifications, DNA methylation, and noncoding (nc)RNAs have manifested as a new layer of gene regulation. These modulations exhibit a profound effect on the cellular activities of DPSCs. In this review, we offer an overview about epigenetic regulation of the fate of DPSCs; in particular, on the proliferation, odontogenic differentiation, angiogenesis, and neurogenesis. We emphasize recent discoveries of epigenetic molecules that can alter DPSC status and promote pulp regeneration through manipulation over epigenetic profiles.

Targeting Epigenetic Modifiers Can Reduce the Clonogenic Capacities of Sézary Cells.

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL) in which the human Telomerase Reverse Transcriptase (hTERT) gene is re-expressed. Current available treatments do not provide long-term response. We previously reported that Histone deacetylase inhibitors (HDACi, romidespin and vorinostat) and a DNA methyltransferase inhibitor (DNMTi, 5-azacytidine) can reduce hTERT expression without altering the methylation level of hTERT promoter. Romidepsin and vorinostat are approved for CTCL treatment, while 5-azacytidine is approved for the treatment of several hematological disorders, but not for CTCL. Here, using the soft agar assay, we analyzed the functional effect of the aforementioned epidrugs on the clonogenic capacities of Sézary cells. Our data revealed that, besides hTERT downregulation, epidrugs' pressure reduced the proliferative and the tumor formation capacities in Sézary cells in vitro.

Oncolytic Virotherapy: The Cancer Cell Side.

Cell autonomous immunity genes mediate the multiple stages of anti-viral defenses, including recognition of invading pathogens, inhibition of viral replication, reprogramming of cellular metabolism, programmed-cell-death, paracrine induction of antiviral state, and activation of immunostimulatory inflammation. In tumor development and/or immunotherapy settings, selective pressure applied by the immune system results in tumor immunoediting, a reduction in the immunostimulatory potential of the cancer cell. This editing process comprises the reduced expression and/or function of cell autonomous immunity genes, allowing for immune-evasion of the tumor while concomitantly attenuating anti-viral defenses. Combined with the oncogene-enhanced anabolic nature of cancer-cell metabolism, this attenuation of antiviral defenses contributes to viral replication and to the selectivity of oncolytic viruses (OVs) towards malignant cells. Here, we review the manners by which oncogene-mediated transformation and tumor immunoediting combine to alter the intracellular milieu of tumor cells, for the benefit of OV replication. We also explore the functional connection between oncogenic signaling and epigenetic silencing, and the way by which restriction of such silencing results in immune activation. Together, the picture that emerges is one in which OVs and epigenetic modifiers are part of a growing therapeutic toolbox that employs activation of anti-tumor immunity for cancer therapy.

Learning against the Background of DNA Methyltransferase Inhibition Leads to the Formation of Memory That Is Resistant to Reactivation and Impairment.

The involvement of DNA methylation in the mechanisms of formation of conditioned food aversion memory was studied on Helix lucorum snails. The dynamics of aversion formation in snails injected with DNA methyltransferase inhibitor RG108 did not differ from that in control snails. The memory was retained for more than one month after training following RG108 injection and the duration of memory persistence did not differ from that in control animals. However, the characteristics of memory in control and experimental snails differed significantly. In control snails, injections of glutamate NMDA-receptor antagonist or protein synthesis inhibitor before memory retrieval caused disorders in the memory reconsolidation and development of amnesia 2 days after training. By contrast, injections of these substances before retrieval to snails trained against the background of RG108 treatment caused no memory disorders. We hypothesized that inhibition of DNA methylation processes led to the formation of strong memory, not reactivated after retrieval and not transformed into a labile state sensitive to amnesic agents.

Chidamide, decitabine, cytarabine, aclarubicin, and granulocyte colony-stimulating factor (CDCAG) in patients with relapsed/refractory acute myeloid leukemia: a single-arm, phase 1/2 study.

BACKGROUND: Epigenetic mechanisms play an important role in the chemoresistance of acute myeloid leukemia (AML). The clinical response to epigenetic modifier-based chemotherapy in patients with relapsed/refractory AML (r/r AML) is unclear. This multicenter clinical trial evaluated the safety and efficacy of epigenetic modifiers (chidamide and decitabine) in combination with aclarubicin, cytarabine, and granulocyte colony-stimulating factor (G-CSF) in patients with r/r AML. RESULTS: Adult patients with r/r AML were treated with chidamide, decitabine, cytarabine, aclarubicin, and G-CSF (CDCAG). The primary measures were overall response (OR), overall survival (OS), and safety. Next-generation sequencing was performed to analyze the correlation between gene mutations and response. A total of 93 patients with r/r AML were enrolled. Overall, 24 patients had a complete remission (CR) and 19 patients achieved CR with incomplete blood count recovery (CRi). The overall response rate (ORR) was 46.2%. The overall survival of these 43 patients who achieved CR/CRi was significantly longer than that of patients who failed to achieve remission (563 vs 152 days, P < 0.0001). Of the patients with mutations in epigenetic and transcription factor-related genes, but without internal tandem duplications in FMS-like tyrosine kinase3 (FLT3-ITDs), 55.6% achieved CR/CRi, whereas the ORR was 28.2% for patients with mutations in other genes. CONCLUSIONS: The CDCAG regimen was well tolerated and effective in r/r AML. Patients with epigenetic and transcription factor-related gene mutations, but without FLT3-ITD mutations, may benefit from this regimen. TRIAL REGISTRATION: Clinical Trials, NCT02886559 . Registered 01 September 2016.

5-aza-2'-Deoxycytidine Induces a RIG-I-Related Innate Immune Response by Modulating Mitochondria Stress in Neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is one of the most common malignant solid tumors to occur in children, characterized by a wide range of genetic and epigenetic aberrations. We studied whether modifications of the latter with a 5-aza-2'-deoxycytidine (decitabine, Dac) DNA methyltransferase inhibitor can provide a therapeutic advantage in NB. METHODS: NB cells with or without MYCN amplification were treated with Dac. We used flow cytometry to measure cell apoptosis and death and mitochondrial reactive oxygen species (mtROS), microarray to analyze gene expression profile and bisulfite pyrosequencing to determine the methylation level of the DDX58/RIG-I promoter. Western blot was used to detect markers related to innate immune response and apoptotic signaling, while immunofluorescent imaging was used to determine dsRNA. We generated mtDNA depleted ρ0 cells using long-term exposure to low-dose ethidium bromide. RESULTS: Dac preferentially induced a RIG-I-predominant innate immune response and cell apoptosis in SK-N-AS NB cells, significantly reduced the methylation level of the DDX58/RIG-I promoter and increased dsRNA accumulation in the cytosol. Dac down regulated mitochondrial genes related to redox homeostasis, but augmented mtROS production. ρ0 cells demonstrated a blunted response in innate immune response and apoptotic cell death, as well as greatly diminished dsRNA. The response of NB cells to CDDP and poly(I:C) was potentiated by Dac in association with increased mtROS, which was blunted in ρ0 cells. CONCLUSIONS: This study indicates that Dac effectively induces a RIG-I-related innate immune response and apoptotic signaling primarily in SK-N-AS NB cells by hypomethylating DDX58/RIG-I promoter, elevated mtROS and increased dsRNA. Dac can potentiate the cytotoxic effects of CDDP and poly(I:C) in NB cells.