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The demethylating agent 5-azacytidine (AZA) has proven its efficacy in the treatment of myelodysplastic syndrome and acute myeloid leukemia. In addition, AZA can demethylateFOXP3intron 1 (FOXP3i1) leading to the generation of regulatory T cells (Treg). Here, we investigated the impact of AZA on xenogeneic graft-vs.-host disease (xGVHD) and graft-vs.-leukemia effects in a humanized murine model of transplantation (human PBMCs-infused NSG mice), and described the impact of the drug on human T cellsin vivo. We observed that AZA improved both survival and xGVHD scores. Further, AZA significantly decreased human T-cell proliferation as well as IFNγ and TNF-α serum levels, and reduced the expression of GRANZYME B and PERFORIN 1 by cytotoxic T cells. In addition, AZA significantly increased Treg frequency through hypomethylation ofFOXP3i1as well as increased Treg proliferation. The latter was subsequent to higher STAT5 signaling in Treg from AZA-treated mice, which resulted from higher IL-2 secretion by conventional T cells from AZA-treated mice itself secondary to demethylation of the IL-2 gene promoter by AZA. Importantly, Tregs harvested from AZA-treated mice were suppressive and stable over time since they persisted at high frequency in secondary transplant experiments. Finally, graft-vs.-leukemia effects (assessed by growth inhibition of THP-1 cells, transfected to express the luciferase gene) were not abrogated by AZA. In summary, our data demonstrate that AZA prevents xGVHD without abrogating graft-vs.-leukemia effects. These findings could serve as basis for further studies of GVHD prevention by AZA in acute myeloid leukemia patients offered an allogeneic transplantation. [ABSTRACT FROM PUBLISHER]

The two main treatment options for patients with higher risk of myelodysplastic syndromes (MDS) are allogeneic hematopoietic stem cell transplantation and azacitidine therapy. Of these, only allogeneic hematopoietic stem cell transplantation is curative, and azacitidine treatment is selected for patients not suitable for transplantation. Compared to conventional treatment with supportive treatment alone or with low dose cytarabine, azacitidine treatment improves survival in MDS patients, even in patients over the age of 75. Because azacitidine treatment takes 4-6 cycles until an initial response, several response-predicting scoring systems have been developed. Novel therapeutic agents and combination therapies of azacitidine with a variety of drugs are currently under study.

Recent investigations of the treatment for hematologic neoplasms have focused on targeting epigenetic regulators. The DNA methyltransferase inhibitor 5-azacytidine (AZA) has produced good results in the treatment of patients with myelodysplastic syndromes. The mechanism underlying its pharmacological activity involves many cellular processes including histone modifications, but chromatin regulation in AZA-resistant cells is still largely unknown. Therefore, we compared human leukemia cells with AZA resistance and their AZA-sensitive counterparts with regard to the response of histone modifications and their readers to AZA treatment to identify novel molecular target(s) in hematologic neoplasms with AZA resistance. We observed an a decrease of HP1γ, a methylated lysine 9 of histone H3-specific reader protein, in AZA-sensitive cells after treatment, whereas AZA treatment did not affect HP1 family proteins in AZA-resistant cells. The expression of shRNA targeting HP1γ reduced viability and induced apoptosis specifically in AZA-resistant cells, which accompanied with down-regulation of ATM/BRCA1 signaling, indicating that chromatin regulation by HP1γ plays a key role in the survival of AZA-resistant cells. In addition, the amount of HP1γ protein in AZA-sensitive and AZA-resistant cells was decreased after treatment with the bromodomain inhibitor I-BET151 at a dose that inhibited the growth of AZA-resistant cells more strongly than that of AZA-sensitive cells. Our findings demonstrate that treatment with AZA, which affects an epigenetic reader protein and targets HP1γ, or a bromodomain inhibitor is a novel strategy that can be used to treat patients with hematopoietic neoplasms with AZA resistance.

Although the tyrosine kinase inhibitor (TKI) era has brought great improvement in outcome in chronic myelogenous leukemia (CML), prognosis of accelerated phase or myeloid blast crisis patients or of de novo Philadelphia chromosome-positive acute myeloid leukemia remains poor. We conducted a retrospective study on patients with advanced phase disease treated with a TKI and azacytidine. Sixteen patients were eligible. Median age was 64.9 years, the median number of previous therapies was 2.5 lines, and median follow-up was 23.1 months. Hematologic response (HR) rate was 81.3%. Median overall survival (OS), event free survival and relapse-free survival (RFS) were 31.5, 23.3, and 32.2 months, respectively. All except one patient were treated as out-patients after the first cycle. Five patients were bridged to allogenic hematopoietic stem cells transplant. The combination of a TKI and azacytidine is a safe and efficient regiment for patients with CML patients in advanced phases.

The prognosis of elderly patients with acute myeloid leukemia (AML) is poor. Intensive chemotherapy with the combination of cytarabine and anthracyclines is typically used as the first-line treatment in the elderly with newly diagnosed AML who are able to tolerate this regimen. Unfortunately, many patients are refractory to this treatment approach. The role of hypomethylating agents in the treatment of elderly patients with refractory AML has not been clearly defined. Therefore, we conducted a focused literature review to assess the role of hypomethylating agents in elderly patients with refractory AML. In addition, we present a case report of a patient with refractory AML, who was subsequently treated with azacytidine and showed an immediate response after one treatment cycle. He then proceeded to undergo nine more cycles. Ten months after the start of treatment with azacytidine, he remains in complete remission with incomplete hematologic recovery. Given the positive results noted in multiple retrospective studies and in the presented case report, large-scale, prospective studies are needed to further define the role of hypomethylating agents in the treatment of elderly patients with refractory AML., Competing Interests: The authors have declared that no competing interests exist.

Introduction: The majority of patients with acute myeloid leukemia (AML) are older and exhibit a poor prognosis even after intensive therapy. Inducing differentiation and apoptosis of leukemic blasts by DNA-hypomethylating agents, like e.g. azacytidine (AZA) and decitabine (DAC), represent well-tolerated alternative treatment approaches. Both agents show convincing response as single agents in AML. However, there is a lack of knowledge regarding molecular mechanisms and predictive biomarkers for these agents. Areas covered: This review will (i) provide an overview of the current knowledge of molecular mechanisms underlying the action of these drugs, (ii) report promising predictive biomarkers, (iii) elude on new combined treatment options, and (iv) discuss novel approaches to improve outcomes. A literature search was performed using PubMed to find recent major publications, which provide biological and clinical research about epigenetic therapy in AML patients. Expert commentary: Numerous studies have demonstrated that HMA therapy with AZA or DAC may lead to significant response rates, even in pre-treated patients. Nevertheless, there is still an unmet need to further improve outcome in elderly AML patients. Therefore, novel treatment combinations are needed and some of them, such as AZA plus venetoclax, already show promising results.

We report the case of a patient with a history of Epstein-Barr virus-positive large B-cell lymphoma, who relapsed with an angioimmunoblastic T-cell lymphoma (AITL) associated with a chronic myelomonocytic leukaemia (CMML). We performed targeted next-generation sequencing on CMML and AITL DNA, which revealed mutations of TET2, DNMT3A, SRSF2, NRAS and IDH1, thus confirming that the spectrum of AITL mutations share similarities with myeloid disorders. The frequencies of TET2/DNMT3A and SRSF2 variants could support the hypothesis that TET2/DNMT3A mutations occurred in an early progenitor cell, which later progressed to both the AITL and CMML clones. Treatment with 5-azacytidine led to the complete remission of both diseases. Thus, targeting DNA methylation abnormalities in AITL may be an alternative strategy to chemotherapy. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Toll interleukin-1 receptor 8 (also known as TIR8, SIGIRR, or IL1R8) is a transmembrane receptor that inhibits inflammation. Accordingly, genetic inactivation of this protein exacerbates chronic inflammation and inflammation-associated tumors in mice. In particular, lack of TIR8 triggers leukemia progression in a mouse model of chronic lymphocytic leukemia (CLL), supporting its role as a novel tumor restrainer. The aim of this study was to measure the amount of TIR8 mRNA and protein in CLL cells, and to analyze its regulation of expression. Circulating leukemic cells expressed lower levels of TIR8 compared to normal B-lymphocytes. Treatment of CLL cells with Azacytidine restored higher levels of TIR8 suggesting that DNA methylation may be involved in modulating TIR8 expression, with implications for novel therapeutic strategies.

Previous studies showed that downregulation of pyrimidine salvage underlies resistance against 5-azacytidine (AZA), indicating an important role for de novo pyrimidine synthesis in AZA resistance. Because de novo pyrimidine synthesis is inhibited by the immunomodulator teriflunomide and its pro-drug leflunomide, we examined the effect of combined treatment with AZA and teriflunomide on AZA resistance to develop a novel strategy to cancel and prevent AZA resistance. Teriflunomide markedly inhibited the growth of AZA-resistant human leukemia cell lines (R-U937 and R-HL-60) in comparison with their AZA-sensitive counterparts (U937 and HL-60). In the presence of a non-toxic concentration of teriflunomide (1 μM), AZA induced apoptosis in AZA-resistant cells and leukemia cells from AZA-resistant patients. AZA acted as a DNA methyltransferase 3A inhibitor in AZA-resistant cells in the presence of 1 μM teriflunomide. Although AZA-sensitive cells acquired AZA resistance after continuous treatment with AZA for 42 days, the growth of AZA-sensitive cells continuously treated with the combination of AZA and teriflunomide was significantly inhibited in the presence of AZA, demonstrating that the combined treatment prevented AZA resistance. These results suggest that combined treatment with AZA and teriflunomide can be a novel strategy to overcome AZA resistance., Competing Interests: CONFLICTS OF INTEREST K.O. received research support from Celegene KK, served as consultant and advisor of Celegene KK, and received honoraria for lecture fees from Nippon Shinyaku. The other authors have no conflicts to disclose.

Background/aim: It is necessary to select an appropriate strategy that makes cancer cells more sensitive to chemotherapeutic drug(s). The aim of this study was to further explore the mechanism(s) behind drug sensitization in the breast cancer MCF7 cell line using 5-azacytidine (AzaC) pretreatment in doxorubicin (Dox) chemotherapy., Materials and Methods: Cells were treated with either AzaC or Dox alone or in combination (AzaC+Dox) for 24 and 48 h. Cells were also pretreated with AzaC for a week before replacement with Dox at varying concentrations in another group of experiments (AzaC/Dox) for 24 and 48 h. The cells in the groups were subjected to cytochemical analyses, including 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT), intracellular reactive oxygen species (ROS) and cell death by dual staining with Hoechst33342 (Hoechst) and propidium iodide (PI). Cells were also analyzed for determining specific alterations of possible target proteins by western blot., Results: Compared to a single (AzaC and Dox) or simultaneous drug exposure (AzaC+Dox), cellular activity was significantly reduced in AzaC/Dox group of cells in a dose- and time-dependent manner (p<0.05). The clonogenic potential of cells was also significantly inhibited in AzaC/Dox compared to Dox and AzaC group after 7 days (p<0.05). The rates of ROS-generated and PI-stained cells were significantly higher in the AzaC+Dox and AzaC/Dox groups compared to those of individual drug groups at 48 h (p<0.05). These observations were accompanied by activation of ERK1/2 and p38MAPK, as well as up-regulation of BAX and P53 in the AzaC/Dox group. However, the levels of BCL-2 were down-regulated in Dox and AzaC+Dox among a different group of cells. Elevated levels of activated caspase-3 were observed only in AzaC/Dox group of cells. The levels of phospho-nuclear factor-kappa B (pNF-ĸB)(Thr) decreased with increasing dosage of Dox in different groups, while down-regulation of heat shock protein 70 (HSP70) was observed only in the AzaC+Dox group of cells., Conclusion: AzaC significantly increases the sensitivity of MCF7 cells to Dox via activation of ERK1/2, P53, BAX and caspase-3. Moreover, the inhibition of pNF-ĸB(Thr) and HSP70 might also contribute to an increase in the sensitivity of MCF7 cells to Dox., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Azacytidine (5-AZA) is the standard first-choice treatment for high-risk myelodysplasia (MDS) patients. However, the clinical outcome for those patients who interrupt treatment or whose disease failed to respond is very poor. In order to identify the cellular pathways that are modified by long-term exposure to 5-AZA, we evaluated key proteins associated with the autophagy pathway by reverse-phase microarray (RPPA). Comparing bone marrow mononucleated cells (BMMCs) obtained from 20 newly-diagnosed patients and after four 5-AZA cycles we found an increased autophagy signaling. We then evaluated ex-vivo the effect of the combination of 5-AZA with autophagy inhibitors chloroquine (CQ) and leupeptin. Since 5-AZA and CQ showed synergism due to an increase of basal autophagy after 5-AZA exposure, we adopted a sequential treatment treating BMMCs with 5 μM 5-AZA for 72 h followed by 10 μM CQ for 24 h and found increased apoptosis, associated to a reduction of G2M phase and increase in G0-G1 phase. Long-term exposure to 5-AZA induced the reduction of the autophagic marker SQSTM1/p62, reversible by CQ or leupeptin exposure. In conclusion, we identified autophagy as a compensatory pathway occurring in MDS-BM after long-term exposure to 5-AZA and we provided evidences that a sequential treatment of 5-AZA followed by CQ could improve 5-AZA efficacy, providing novel insight for tailored therapy in MDS patients progressing after 5-AZA therapy.

In recent years, recurrent somatic mutations in epigenetic regulators have been identified in patients with hematological malignancies. Furthermore, chromosomal translocations in which the fusion protein partners are themselves epigenetic regulators or where epigenetic regulators are recruited/targeted by oncogenic fusion proteins have also been described. Evidence has accumulated showing that "epigenetic drugs" are likely to provide clinical benefits in several hematological malignancies, granting their approval for the treatment of myelodysplastic syndromes and cutaneous T-cell lymphomas. A large number of pre-clinical and clinical trials evaluating epigenetic drugs alone or in combination therapies are ongoing. The aim of this review is to provide a comprehensive summary of known epigenetic alterations and of the current use of epigenetic drugs for the treatment of hematological malignancies., Competing Interests: No conflict of interest to be disclosed.

Introduction: Myelodysplastic syndrome (MDS) is a heterogeneous clonal disorder characterized by deregulation of apoptosis, dysplastic features in hematopoietic precursors, peripheral blood cytopenias and an increased risk for transformation to acute leukemia. Roughly 20% of MDS are therapy related (t-MDS), and this is considered an independent adverse prognostic factor., Areas Covered: This review based on a comprehensive literature search provides an overview on the main features of t-MDS, including its epidemiology, risk factors, molecular pathogenesis, prognostic classifications and therapy., Expert Opinion: Increasing evidence points out that the most important event in t-MDS is genetic alterations in hematopoietic stem precursor cells, however, ineffective hematopoiesis may also result from abnormalities in the bone marrow microenvironment. Thus, novel views onto the processes of t-MDS are needed such as the osteohematology concept. On the other hand, the number of people living with and beyond cancer is increasing worldwide; thus, most emphasis should be placed on preventing secondary malignancies such as t-MDS. From this review, it becomes clear that we are in urgent need not only to deepen our understanding of the leukemogenesis mechanisms induced by exposure to chemotherapy and radiation but also to translate this knowledge into clinical strategies aimed at risk reduction.

Despite improvements to hematopoietic stem cell transplantation over the past several decades, further advances are necessary to achieve: improved control of toxicities like graft-versus-host disease; enhanced immunologic reconstitution posttransplantation; and reduction in relapse risk via enhancement of graft-versus-tumor responses. Achieving these disparate hematopoietic stem cell transplantation goals will likely require the introduction of novel therapeutic agents to the current armamentarium. In this article, we outline preclinical and early-phase clinical data indicating the potential of proteasome-inhibitor therapy (bortezomib), hypomethylating agent therapy (azacytidine), and histone deacetylase-inhibitor therapy (vorinostat) to help improve hematopoietic stem cell transplantation outcomes. [ABSTRACT FROM AUTHOR]

The article reports on a case of a patient with alpha-thalassemia myelodysplastic syndrome treating with 5-azacytidine therapy. After diagnosis, the patient received supportive care with two to three packed red blood cell (pRBC) transfusions. Then after 17 months, his transfusion requirement had increased to 4 pRBC units/month. 5-azacytidine was initiated at a dose of 75 mg/m2/d for 7 days for two cycles with the patient's informed consent.

DNA methylation has been studied with regard to chemotherapeutics for a number of years. The radiation field has just begun to look at this in the context of radiotherapy or radiation exposure. So far, the data suggest that radiation induces epigenetic reprogramming which indicates a purposeful response that influences the cell fate or alters the response to future exposure. Further studies may result in discovery of biomarkers for radiotherapy outcome or prediction of the degree of radiation resistance. Past and ongoing development of DNMT1 inhibitors that lead to DNA hypomethylation appear to sensitize many tumor types to radiation and may be an area with long term clinical implications., (© 2014 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.)

Myelodysplastic syndrome (MDS) is a disorder of hematopoietic stem cells (HSCs) that is often treated with DNA methyltransferase 1 (DNMT1) inhibitors (5-azacytidine [AZA], 5-aza-2'-deoxycytidine), suggesting a role for DNA methylation in disease progression. How DNMT inhibition retards disease progression and how DNA methylation contributes to MDS remain unclear. We analyzed global DNA methylation in purified CD34+ hematopoietic progenitors from MDS patients undergoing multiple rounds of AZA treatment. Differential methylation between MDS phenotypes was observed primarily at developmental regulators not expressed within the hematopoietic compartment and was distinct from that observed between healthy hematopoietic cell types. After AZA treatment, we observed only limited DNA demethylation at sites that varied between patients. This suggests that a subset of the stem cell population is resistant to AZA and provides a basis for disease relapse. Using gene expression data from patient samples and an in vitro AZA treatment study, we identified differentially methylated genes that can be activated following treatment and that remain silent in the CD34+ stem cell compartment of high-risk MDS patients. Haploinsufficiency in mice of one of these genes (NR4A2) has been shown to lead to excessive HSC proliferation, and our data suggest that suppression of NR4A2 by DNA methylation may be involved in MDS progression., (©AlphaMed Press.)

We have previously demonstrated proteasomal degradation of DNMT1 in mammalian cells following treatment with several DNA hypomethylating agents. Here, we demonstrate dose-dependent degradation of Dnmt1 in mouse embryonic stem (ES) cells expressing catalytic site mutant (cys-ser), confirming that the covalent bond formation between Dnmt1 and decitabine-incorporated DNA is not essential for this process. DNMT1o, the oocyte-specific isoform that lacks the N-terminal 118-amino acid domain, did not undergo decitabine-mediated degradation, which further proves the requirement of multiple domains including nuclear localization signal, KEN box, and BAH domains for this process. Analysis of glycerol density gradient fractions of micrococcal nuclease-digested nuclei showed that both nucleosomal and nucleoplasmic DNMT1 are degraded upon decitabine treatment. Among different inhibitors tested, the inhibitors of the proteasomal pathway and several protein kinases impeded decitabine-induced DNMT1 degradation. The maximal effect caused by inhibiting protein kinase C (PKC) persuaded us to investigate further its role in decitabine-mediated DNMT1 degradation. Blockage of the degradation process after treatment with rottlerin, an inhibitor of PKCδ, or after siRNA-mediated depletion of PKCδ, indicated that this protein kinase is involved in decitabine-mediated depletion of DNMT1. PKCδ interacted with and phosphorylated DNMT1 in vitro. Moreover, rottlerin inhibited both basal and decitabine-induced phosphorylation of DNMT1. These studies provide substantial evidence that decitabine-induced degradation of the maintenance methyltransferase DNMT1 does not require covalent bond formation with the substrate and also elucidate its underlying molecular mechanism.

Neuroendocrine tumors (NETs) hypersecrete neuropeptides that cause debilitating symptoms of carcinoid syndrome, including cardiac abnormalities. Surgical resection is the only potentially curative treatment for NETs; however, 90% of NE cancer patients are not candidates for surgery due to extensive hepatic sites involved with NETs. Recently, DNA methyltransferase inhibitors (DNMTI) such as azacytidine (AzaC) have shown efficacy in clinical treatments of hematological malignancies, but effects on NETs are not well-studied. We hypothesized that this novel class of drugs inhibits NET cell growth and decreases NE markers. Three carcinoid types-human midgut (CDNT2.5), pulmonary (H727), and gastrointestinal (BON)- were treated with AzaC (0-100uM) over 6 days. MTT Assays were used to measure cellular proliferation. Western blots were performed with antibodies against chromogranin A (CgA), Neuron-Specific Enolase (NSE), and Cyclin B1. Flow cytometric data was collected from AzaC-treated CNDT2.5 cells for DNA cell cycle analysis. Results showed that treatment of CDNT2.5, H727, and BON carcinoid cells with AzaC resulted in a dose-dependent reduction in tumor cell proliferation. Flow cytometric analysis showed that AzaC-treated cells accumulate in the G2 Phase of cell cycle. AzaC treatment led to: significant decreases in CgA and NSE, indicating that AzaC inhibits neuroendocrine markers; and significant increases in the levels of Cyclin B1, further supporting the flow cytometric data and conclusion that AzaC induces G2/M arrest. The data indicate that AzaC suppresses cell growth in three different carcinoid types, reduces neuroendocrine markers in CNDT2.5 cells, and inhibits cell proliferation by inducing G2/M phase arrest. The results suggest that DNMTIs may be a novel class of therapeutic agents that can effectively control tumor growth and the release of bioactive peptides in patients with NETs.