Hypomethylating Drug Research Articles

The effect of methylation on the let-7-BCL2L1-BCL2 axis and the potential use of hypomethylating and BH3 mimetic drugs in histiocytic neoplasms.

The effect of methylation on the let-7-BCL2L1-BCL2 axis and the potential use of hypomethylating and BH3 mimetic drugs in histiocytic neoplasms.

Abstract P43: Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer

Abstract Aberrant DNA methylation is one of the critical epigenetic modifications that has become a widespread phenotype in solid tumours. Similar to haematological malignancies, many studies have shown that the DNA methylation landscape of solid tumours exhibits a pattern of either hypermethylation of the promoter regions of tumour-suppressive genes in advanced cancer or global hypomethylation in the early stages of carcinogenesis. Additionally, dysregulated DNA methylation also seems to remodel the tumour microenvironment and affect immune cells' response towards immune checkpoint inhibitors. In gastric cancer, comprehensive DNA methylation analyses have demonstrated that gastric tumours carry the CpG island methylation phenotype (CIMP), resulting in extended molecular-based subgroups such as the extremely high-methylation epigenotype (E-HME), HME, and low-methylation epigenotype (LME). Therefore, conventional histology grading and molecular subtyping are insufficient to address the multifaceted epigenome of gastric cancer. As such, current standard-of-care (SOC) therapy is not able to overcome cancer relapse and tumour metastasis. Clearly, this shows the therapeutic potential of using DNA hypomethylating drugs like DNA methyltransferase inhibitors (DNMTi) for solid tumours such as gastric cancer. In our study, we exploited the engineering-based Quadratic Phenotypic Optimization Platform (QPOP) to identify top-ranking DNMTi-based drug combinations across gastric cancer cell lines (GC-CL) and patient-derived organoids (GC-PDO). Based on QPOP analysis, we showed that a non-SOC drug combination, Docetaxel/5-Azacytidine is a frequently top-ranking. Besides, our present findings suggest that CIMP-high gastric cancer cells are more susceptible towards DNMT inhibitors through the specific reduction of DNMT1 levels. As we surveyed the effects of Docetaxel/5-Azacytidine in a CIMP-high GC-CL via Infinium methylation array, we observed robust gene methylation changes that are involved in non-canonical Wnt Signaling and EMT. On the other hand, proteomic analysis revealed a small subset of our panel of GC lines that are positive for the cancer-testis antigen, MageA4. Interestingly, the protein expression of MageA4 in some CIMP-high lines is restored upon treatment with DNMT inhibitors. This evinces that DNMTi can subject epigenetically silenced MageA4 gastric tumours to autologous T-cell receptor therapy. Taken altogether, we plan to further interrogate the mechanisms of DNMT inhibitors in sensitizing CIMP-high gastric tumours towards Taxanes by acting on the non-canonical Wnt pathway and EMT activators. Citation Format: Lissa Hooi, Vivien Koh, Dexter Kai Hao Thng, Jasmine Goh, Lim Jhin Jieh, Lee Rui Xue, Masturah Bte Mohd Abdul Rashid, Toh Tan Boon, Wei Peng Yong, Edward Kai-Hua Chow. Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P43.

Abstract B043: Dual epigenetic therapy combined with anti-PD1 rescues HMA-induced suppressive myeloid phenotype and reduces tumor growth in a PDAC model

Abstract INTRODUCTION. We previously showed, using the PDAC mouse model KPC (KrasLSL G12D/+; p53 r172H/+; Pdx1-Cre), that sequential treatment with the DNA hypomethylating drug decitabine (5aza-dC; DAC) followed by anti-PD-1 led to increased tumor necrosis, slower tumor growth, increased tumor-infiltrating CD8+ and CD4+ T-cells (TILs), and significantly increased mean survival time. However, despite these positive outcomes, the KPC tumors eventually escaped from treatment. We identified a unique myeloid population as a specific subtype of M2-polarized putatively immunosuppressive Chil3+ macrophages (TAMs). Here we further characterize the recruitment mechansism of these cells and show that HDAC inhibition can further improve the efficacy of DAC+PD-1 treatment. METHODS. Primary macrophages were isolated from bone marrow (BMDM) or the peritoneum of 6-8 weeks mice and RAW264.7 cells were also used and exposed to conditioned media from primary KPC cell lines after HMA treatment (KPC-SN-DAC). Gene set enrichemnt analysis was done on RNASeq data from myeloid cells and validated by RT-qPCR. We used cytokine arrays to identify secreted cytokines from KPC cells. KPC cells were orthtopically injected in C57BL/6 mice, and tumor growth was followed by untrasound. Treatments were started when tumor reaced a size of 1 mm3. RESULTS. Expression profiling reveled that exposure of the myeloid cells to KPC-SN-DAC resulted in an HMA-specific M2-like gene signature. Notably DAC alone had minimal effect on myeloid cells polarization. To assess which factors might trigger this polarization we employed cytokine arrays and identified a number of candidate cytokines induced or secreted from KPC cells specifically following HMA treatment. In order to revert this unfavorable cytokine profile, we have evaluated the combination of DAC with four different HDAC inhibitors with reported immunomodulatory effects: Romidepsin (ROMI), Domatinostat (DOMA), Pracinostat (PRAC) and Entinostat (ENT). In vitro synergy studies through cell viability assays showed that ROMI, PRAC and DOMA synergized at low doses with DAC. Pofiling of supernatants from KPC cells treated with a combination of DAC+HDACis showed that, while ROMI had limited effect, PRAC and DOMA reverted DAC-induced cytokine secretion. Lastly, we tested in vivo the combination of DAC+ROMI/DOMI/PRAC+PD1 using mice orthotopically injected with KPC cells. The combination DAC+PRAC+PD1 showed reduced tumor volume compared to DAC+PD1. Moreover, PRAC+PD1 significantly improved overall survival. Interestingly, HDAC inhibition rescued the increased Chil3 expression in DAC+PD1 treated tumors. CONCLUSIONS. Our results show that paracrine rather than direct effect of hypomethylating therapy is responsible for suppressive myeloid polarization and characterize a specific effect of hypomethylating therapy on immature myeloid cells. Moreover, we have identified HDAC inhibition as a viable strategy to prevent unfavorable myeloid polarization and improve the therapeutic efficacy of the DAC+PD1 paradigm. Citation Format: Arturo Orlacchio, Stephen Muzyka, Catherine Do, Diane M. Simeone, Tamas Gonda. Dual epigenetic therapy combined with anti-PD1 rescues HMA-induced suppressive myeloid phenotype and reduces tumor growth in a PDAC model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B043.

Ectoine Globally Hypomethylates DNA in Skin Cells and Suppresses Cancer Proliferation.

Epigenetic modifications, mainly aberrant DNA methylation, have been shown to silence the expression of genes involved in epigenetic diseases, including cancer suppression genes. Almost all conventional cancer therapeutic agents, such as the DNA hypomethylation drug 5-aza-2-deoxycytidine, have insurmountable side effects. To investigate the role of the well-known DNA protectant (ectoine) in skin cell DNA methylation and cancer cell proliferation, comprehensive methylome sequence analysis, 5-methyl cytosine (5mC) analysis, proliferation and tumorigenicity assays, and DNA epigenetic modifications-related gene analysis were performed. The results showed that extended ectoine treatment globally hypomethylated DNA in skin cells, especially in the CpG island (CGIs) element, and 5mC percentage was significantly reduced. Moreover, ectoine mildly inhibited skin cell proliferation and did not induce tumorigenicity in HaCaT cells injected into athymic nude mice. HaCaT cells treated with ectoine for 24 weeks modulated the mRNA expression levels of Dnmt1, Dnmt3a, Dnmt3b, Dnmt3l, Hdac1, Hdac2, Kdm3a, Mettl3, Mettl14, Snrpn, and Mest. Overall, ectoine mildly demethylates DNA in skin cells, modulates the expression of epigenetic modification-related genes, and reduces cell proliferation. This evidence suggests that ectoine is a potential anti-aging agent that prevents DNA hypermethylation and subsequently activates cancer-suppressing genes.

Inhibition of SUMOylation enhances DNA hypomethylating drug efficacy to reduce outgrowth of hematopoietic malignancies

Combination therapies targeting malignancies aim to increase treatment efficacy and reduce toxicity. Hypomethylating drug 5-Aza-2’-deoxycytidine (5-Aza-2’) enhances transcription of tumor suppressor genes and induces replication errors via entrapment of DNMT1, yielding DNA-protein crosslinks. Post-translational modification by SUMO plays major roles in the DNA damage response and is required for degradation of entrapped DNMT1. Here, we combine SUMOylation inhibitor TAK981 and DNA-hypomethylating agent 5-Aza-2’-deoxycytidine to improve treatment of MYC driven hematopoietic malignancies, since MYC overexpressing tumors are sensitive to SUMOylation inhibition. We studied the classical MYC driven malignancy Burkitt lymphoma, as well as diffuse large B-cell lymphoma (DLBCL) with and without MYC translocation. SUMO inhibition prolonged the entrapment of DNMT1 to DNA, resulting in DNA damage. An increase in DNA damage was observed in cells co-treated with TAK981 and 5-Aza-2’. Both drugs synergized to reduce cell proliferation in vitro in a B cell lymphoma cell panel, including Burkitt lymphoma and DLBCL. In vivo experiments combining TAK981 (25 mg/kg) and 5-Aza-2’ (2.5 mg/kg) showed a significant reduction in outgrowth of Burkitt lymphoma in an orthotopic xenograft model. Our results demonstrate the potential of tailored combination of drugs, based on insight in molecular mechanisms, to improve the efficacy of cancer therapies.

Targeted Epigenetic Interventions in Cancer with an Emphasis on Pediatric Malignancies.

Over the past two decades, novel hallmarks of cancer have been described, including the altered epigenetic landscape of malignant diseases. In addition to the methylation and hyd-roxymethylation of DNA, numerous novel forms of histone modifications and nucleosome remodeling have been discovered, giving rise to a wide variety of targeted therapeutic interventions. DNA hypomethylating drugs, histone deacetylase inhibitors and agents targeting histone methylation machinery are of distinguished clinical significance. The major focus of this review is placed on targeted epigenetic interventions in the most common pediatric malignancies, including acute leukemias, brain and kidney tumors, neuroblastoma and soft tissue sarcomas. Upcoming novel challenges include specificity and potential undesirable side effects. Different epigenetic patterns of pediatric and adult cancers should be noted. Biological significance of epigenetic alterations highly depends on the tissue microenvironment and widespread interactions. An individualized treatment approach requires detailed genetic, epigenetic and metabolomic evaluation of cancer. Advances in molecular technologies and clinical translation may contribute to the development of novel pediatric anticancer treatment strategies, aiming for improved survival and better patient quality of life.

463 Implications of the ds-RNA stress response in melanoma cell lines and patient samples

Double-stranded RNA (ds-RNA) derived from repetitive DNA elements has the potential to induce an interferon response that renders resistant melanoma sensitive to immunotherapy. As a consequence, ds-RNA has the potential to add a new approach to treating melanoma. Tissue culture and molecular biology were used to induce ds-RNA formation in melanoma cell lines using hypo-methylating drugs and other stressors. The impact was measured by quantifying cytosolic ds-RNA sensors, also known as pattern recognition receptors (pPKR, MDA5, RIG1, and DAR1) using RT-qPCR and western blotting.

Enhancement of Radiosensitivity by DNA Hypomethylating Drugs through Apoptosis and Autophagy in Human Sarcoma Cells

The targeting of DNA methylation in cancer using DNA hypomethylating drugs has been well known to sensitize cancer cells to chemotherapy and immunotherapy by affecting multiple pathways. Herein, we investigated the combinational effects of DNA hypomethylating drugs and ionizing radiation (IR) in human sarcoma cell lines both in vitro and in vivo. Clonogenic assays were performed to determine the radiosensitizing properties of two DNA hypomethylating drugs on sarcoma cell lines we tested in this study with multiple doses of IR. We analyzed the effects of 5-aza-dC or SGI-110, as DNA hypomethylating drugs, in combination with IR in vitro on the proliferation, apoptosis, caspase-3/7 activity, migration/invasion, and Western blotting using apoptosis- or autophagy-related factors. To confirm the combined effect of DNA hypomethylating drugs and IR in our in vitro experiment, we generated the sarcoma cells in nude mouse xenograft models. Here, we found that the combination of DNA hypomethylating drugs and IR improved anticancer effects by inhibiting cell proliferation and by promoting synergistic cell death that is associated with both apoptosis and autophagy in vitro and in vivo. Our data demonstrated that the combination effects of DNA hypomethylating drugs with radiation exhibited greater cellular effects than the use of a single agent treatment, thus suggesting that the combination of DNA hypomethylating drugs and radiation may become a new radiotherapy to improve therapeutic efficacy for cancer treatment.

Repurposing proscillaridin A in combination with decitabine against embryonal rhabdomyosarcoma RD cells.

Embryonal rhabdomyosarcoma (eRMS) is the most common type of rhabdomyosarcoma in children. eRMS is characterized by malignant skeletal muscle cells driven by hyperactivation of several oncogenic pathways including the MYC pathway. Targeting MYC in cancer has been extremely challenging. Recently, we have demonstrated that the heart failure drug, proscillaridin A, produced anticancer effects with specificity toward MYC expressing leukemia cells. We also reported that decitabine, a hypomethylating drug, synergizes with proscillaridin A in colon cancer cells. Here, we investigated whether proscillaridin A exhibits epigenetic and anticancer activity against eRMS RD cells, overexpressing MYC oncogene, and its combination with decitabine. We investigated the anticancer effects of proscillaridin A in eRMS RD cells in vitro. In response to drug treatment, we measured growth inhibition, cell cycle arrest, loss of clonogenicity and self-renewal capacity. We further evaluated the impact of proscillaridin A on MYC expression and its downstream transcriptomic effects by RNA sequencing. Then, we measured protein expression of epigenetic regulators and their associated chromatin post-translational modifications in response to drug treatment. Chromatin immunoprecipitation sequencing data sets were coupled with transcriptomic results to pinpoint the impact of proscillaridin A on gene pathways associated with specific chromatin modifications. Lastly, we evaluated the effect of the combination of proscillaridin A and the DNA demethylating drug decitabine on eRMS RD cell growth and clonogenic potential. Clinically relevant concentration of proscillaridin A (5nM) produced growth inhibition, cell cycle arrest and loss of clonogenicity in eRMS RD cells. Proscillaridin A produced a significant downregulation of MYC protein expression and inhibition of oncogenic transcriptional programs controlled by MYC, involved in cell replication. Interestingly, significant reduction in total histone 3 acetylation and on specific lysine residues (lysine 9, 14, 18, and 27 on histone 3) was associated with significant protein downregulation of a series of lysine acetyltransferases (KAT3A, KAT3B, KAT2A, KAT2B, and KAT5). In addition, proscillaridin A produced synergistic growth inhibition and loss of clonogenicity when combined with the approved DNA demethylating drug decitabine. Proscillaridin A produces anticancer and epigenetic effects in the low nanomolar range and its combination with decitabine warrants further investigation for the treatment of eRMS.

Epigenetic Regulation of BST-2 Expression Levels and the Effect on HIV-1 Pathogenesis.

HIV-1 must overcome host antiviral restriction factors for efficient replication. We hypothesized that elevated levels of bone marrow stromal cell antigen 2 (BST-2), a potent host restriction factor that interferes with HIV-1 particle release in some human cells and is antagonized by the viral protein Vpu, may associate with viral control. Using cryopreserved samples, from HIV-1 seronegative and seropositive Black women, we measured in vitro expression levels of BST-2 mRNA using a real-time PCR assay and protein levels were validated by Western blotting. The expression level of BST-2 showed an association with viral control within two independent cohorts of Black HIV infected females (r=-0.53, p=0.015, [n =21]; and r=-0.62, p=0.0006, [n=28]). DNA methylation was identified as a mechanism regulating BST-2 levels, where increased BST-2 methylation results in lower expression levels and associates with worse HIV disease outcome. We further demonstrate the ability to regulate BST-2 levels using a DNA hypomethylation drug. Our results suggest BST-2 as a factor for potential therapeutic intervention against HIV and other diseases known to involve BST-2.

Distinct Responses of Arabidopsis Telomeres and Transposable Elements to Zebularine Exposure.

Involvement of epigenetic mechanisms in the regulation of telomeres and transposable elements (TEs), genomic regions with the protective and potentially detrimental function, respectively, has been frequently studied. Here, we analyzed telomere lengths in Arabidopsis thaliana plants of Columbia, Landsberg erecta and Wassilevskija ecotypes exposed repeatedly to the hypomethylation drug zebularine during germination. Shorter telomeres were detected in plants growing from seedlings germinated in the presence of zebularine with a progression in telomeric phenotype across generations, relatively high inter-individual variability, and diverse responses among ecotypes. Interestingly, the extent of telomere shortening in zebularine Columbia and Wassilevskija plants corresponded to the transcriptional activation of TEs, suggesting a correlated response of these genomic elements to the zebularine treatment. Changes in lengths of telomeres and levels of TE transcripts in leaves were not always correlated with a hypomethylation of cytosines located in these regions, indicating a cytosine methylation-independent level of their regulation. These observations, including differences among ecotypes together with distinct dynamics of the reversal of the disruption of telomere homeostasis and TEs transcriptional activation, reflect a complex involvement of epigenetic processes in the regulation of crucial genomic regions. Our results further demonstrate the ability of plant cells to cope with these changes without a critical loss of the genome stability.

A DNA Hypomethylating Drug Alters the Tumor Microenvironment and Improves the Effectiveness of Immune Checkpoint Inhibitors in a Mouse Model of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer that has proven refractory to immunotherapy. Previously, treatment with the DNA hypomethylating drug decitabine (5-aza-dC; DAC) extended survival in the KPC-Brca1 mouse model of PDAC. Here we investigated the effects of DAC in the original KPC model and tested combination therapy with DAC followed by immune checkpoint inhibitors (ICI). Four protocols were tested: PBS vehicle, DAC, ICI (anti-PD-1 or anti-VISTA), and DAC followed by ICI. For each single-agent and combination treatment, tumor growth was measured by serial ultrasound, tumor-infiltrating lymphoid and myeloid cells were characterized, and overall survival was assessed. Single-agent DAC led to increased CD4+ and CD8+ tumor-infiltrating lymphocytes (TIL), PD1 expression, and tumor necrosis while slowing tumor growth and modestly increasing mouse survival without systemic toxicity. RNA-sequencing of DAC-treated tumors revealed increased expression of Chi3l3 (Ym1), reflecting an increase in a subset of tumor-infiltrating M2-polarized macrophages. While ICI alone had modest effects, DAC followed by either of ICI therapies additively inhibited tumor growth and prolonged mouse survival. The best results were obtained using DAC followed by anti-PD-1, which extended mean survival from 26 to 54 days (P < 0.0001). In summary, low-dose DAC inhibits tumor growth and increases both TILs and a subset of tumor-infiltrating M2-polarized macrophages in the KPC model of PDAC, and DAC followed by anti-PD-1 substantially prolongs survival. Because M2-polarized macrophages are predicted to antagonize antitumor effects, targeting these cells may be important to enhance the efficacy of combination therapy with DAC plus ICI. SIGNIFICANCE: In a pancreatic cancer model, a DNA hypomethylating drug increases tumor-infiltrating effector T cells, increases a subset of M2 macrophages, and significantly prolongs survival in combination with immune checkpoint inhibitors.See related commentary by Nephew, p. 4610.

Curcumin-Induced DNA Demethylation in Human Gastric Cancer Cells Is Mediated by the DNA-Damage Response Pathway.

Curcumin, a natural polyphenol antioxidant extracted from the root of turmeric (Curcuma longa), can induce apoptosis and DNA demethylation in several types of cancer cells. However, the mechanism of its anticancer potentials and DNA demethylation effects and the potential relationships between these outcomes have not been clearly elucidated. In the present study, the effects of curcumin on the proliferation, colony formation, and migration of human gastric cancer cells (hGCCs) were explored. Reactive oxygen species (ROS) levels, mitochondrial damage, DNA damage, and apoptosis of curcumin-treated hGCCs were analyzed. Changes in the expression of several genes related to DNA damage repair, the p53 pathway, cell cycle, and DNA methylation following curcumin treatment were also evaluated. We observed that curcumin inhibited the proliferation, colony formation, and migration of hGCCs in a dose- and time-dependent fashion. A high concentration of curcumin elevated ROS levels and triggered mitochondrial damage, DNA damage, and apoptosis of hGCCs. Further, curcumin-induced DNA demethylation of hGCCs was mediated by the damaged DNA repair-p53-p21/GADD45A-cyclin/CDK-Rb/E2F-DNMT1 axis. We propose that the anticancer effect of curcumin could largely be attributed to its prooxidative effect at high concentrations and ROS elevation in cancer cells. Moreover, we present a novel mechanism by which curcumin induces DNA demethylation of hGCCs, suggesting the need to further investigate the demethylation mechanisms of other DNA hypomethylating drugs.

Coactivation of NF-κB and Notch signaling is sufficient to induce B-cell transformation and enables B-myeloid conversion

AbstractNF-κB and Notch signaling can be simultaneously activated in a variety of B-cell lymphomas. Patients with B-cell lymphoma occasionally develop clonally related myeloid tumors with poor prognosis. Whether concurrent activation of both pathways is sufficient to induce B-cell transformation and whether the signaling initiates B-myeloid conversion in a pathological context are largely unknown. Here, we provide genetic evidence that concurrent activation of NF-κB and Notch signaling in committed B cells is sufficient to induce B-cell lymphomatous transformation and primes common progenitor cells to convert to myeloid lineage through dedifferentiation, not transdifferentiation. Intriguingly, the converted myeloid cells can further transform, albeit at low frequency, into myeloid leukemia. Mechanistically, coactivation of NF-κB and Notch signaling endows committed B cells with the ability to self renew. Downregulation of BACH2, a lymphoma and myeloid gene suppressor, but not upregulation of CEBPα and/or downregulation of B-cell transcription factors, is an early event in both B-cell transformation and myeloid conversion. Interestingly, a DNA hypomethylating drug not only effectively eliminated the converted myeloid leukemia cells, but also restored the expression of green fluorescent protein, which had been lost in converted myeloid leukemia cells. Collectively, our results suggest that targeting NF-κB and Notch signaling will not only improve lymphoma treatment, but also prevent the lymphoma-to-myeloid tumor conversion. Importantly, DNA hypomethylating drugs might efficiently treat these converted myeloid neoplasms.

Identification of microRNAs modulated by DNA hypomethylating drugs in extranodal NK/T-cell lymphoma

To identify epigenetically silenced miRNAs and to investigate their influences on predictive target oncogenes in extranodal natural killer/T-cell lymphoma (NKTCL). Decitabine treatment was performed to evaluate methylated miRNAs in NKTCL cells. The relationship between a given miRNA and its target mRNA was validated using 24 tumor tissues. miR-379, miR-134, miR-20b, miR-376a, miR-654-3p, miR-143, miR-181c, miR-1225-5p, miR-1246, and miR-1275 were epigenetically silenced in SNK6 cells. miR-134, miR-376a, miR-143 and miR-181c significantly affected cellular viability. PDGFRα was regulated by miR-34a and miR-181c. miR-143, miR-20b and miR34a regulated STAT3 expression. miR-20b and miR-143 expression showed inverse correlations with STAT3 mRNA expression in NKTCL tissues. K-RAS was regulated by miR-181c. Downregulation of cell viability by salirasib treatment was identified. miRNAs were downregulated by DNA methylation, and several microRNAs affected the viability of NKTCL cells. miR-34a and miR-181c may be involved in the oncogenic progression of NKTCL through the regulation of PDGFRα, STAT3, and K-RAS.

Effective oral hypomethylating drugs in intermediate-risk or high-risk myelodysplasia: a breakthrough?

Effective oral hypomethylating drugs in intermediate-risk or high-risk myelodysplasia: a breakthrough?

Novel Therapeutic Targeting of UHRF1 Restores 5'-Azacytidine Response in Resistant Acute Myeloid Leukemia

DNA hypermethylation plays a pivotal role in the pathogenesis of acute myeloid leukemia (AML). Most of the recurrent driver mutations and chromosomal translocations in AML involve genes encoding chromatin modifiers and DNA methylation relevant enzymes. Hypo-methylating drugs such as 5-Azacytidine (AZA) that target DNMTs prolong overall survival in AML patients. However, their long term treatments lead to emergence of acquired therapy resistance mostly through unknown mechanisms and hence there is an urgent need for alternate therapeutics to address AZA resistance in AML patients. Recently, it has been shown that AZA resistant leukemic cells are relatively quiescent with higher expression of many components of DNA methylation machinery that also includes UHRF1 (ubiquitin-like with PHD and ring finger domains 1). UHRF1 is a key epigenetic modulator that regulates DNA methylation and gene expression. It is a multi-domain nuclear protein with an SRA (SET-and-RING-associated) domain to recognize hemi-methylated DNA immediately after replication. It plays a crucial role in the maintenance of DNA methylation by recruiting DNMT1 to replication sites and facilitates methylation on newly synthesized DNA strand. UHRF1 is frequently overexpressed in multiple human neoplasms including AML and in the absence of UHRF1, hematopoietic stem cells undergo erythroid-biased differentiation at the expense of self-renewal capacity. Despite UHRF1 being key a therapeutic target against AML, specific, and cell-permeable inhibitors of UHRF1 have not been identified yet.In this study, we hypothesized that targeting UHRF1 using novel small molecule inhibitor will interfere with DNMT1-dependent DNA methylation at newly synthesized DNA strand, which may further synergize with antiproliferative effect of classical DNMT inhibitors in AML cells. In this study, we used in silico strategy to discover novel putative UHRF1 inhibitors by screening NCI compound database. For in vitro validation, we have first purified the SRA domain of UHRF1 followed by analysis of total DNA methylation levels using 5'-methyl cytosine (5mC) dot blot in the presence of each inhibitor. After a series of stringent in vitro and cell based assays we have identified lead compound 20 (C20) as a potent UHRF1 inhibitor which suppresses DNA methylation without affecting DNMTs in leukemic cells. Specificity of C20 against SRA domain was further established by isothermal titration calorimetry (ITC). We next found that C20 treatment significantly decreased UHRF1 and DNMT1 foci formation in the nucleus of mouse embryonic fibroblast and stem cells. Based on the its critical role in DNA methylation and enhanced expression in resistant cells, we assumed that AZA resistance in AML may be mediated by UHRF1 and C20 might restore AZA sensitivity by attenuating enhanced UHRF1 activity. To validate this, we pretreated AZA resistant leukemic cells (HL60R) with suboptimal dose of C20 followed by AZA treatment. Interestingly, we found a synergistic increase in antiproliferative effect by flow cytometry and colony formation assay. By analyzing the surface expression of myeloid differentiation markers, we found that C20 treatment promotes differentiation and decreases quiescent leukemic cell population. In conclusion, we report a novel UHRF1 inhibitor as a sensitizer of resistant AML cells towards AZA treatment potentially by promoting differentiation, suggesting a novel combination approach for future clinical evaluations. DisclosuresNo relevant conflicts of interest to declare.

Low-dose hypomethylating agent decitabine in combination with aclacinomycin and cytarabine achieves a better outcome than standard FLAG chemotherapy in refractory/relapsed acute myeloid leukemia patients with poor-risk cytogenetics and mutations.

BackgroundRelapsed and refractory acute myeloid leukemia (RR-AML) still poses major treatment concerns. Current treatments include high doses of cytarabine or fludarabine in combination with cytarabine and G-CSF (FLAG), but provide mixed results. Low-dose decitabine, a hypomethylating drug, in combination with aclarubicin and cytarabine (DAC) has shown safety and efficacy in the treatment of AML; however, clinical data are limited for the treatment of RR-AML.MethodsIn this study, we retrospectively compared the response and safety of DAC vs FLAG for RR-AML patients.ResultsFor the 35 patients with RR-AML enrolled in this study, the overall response rates reached 100% and 55.6% in the DAC group and FLAG group, respectively (P=0.002). Complete response rates after DAC and FLAG treatment were 64.7% and 33.3%, respectively (P=0.002). Median overall survival (95% CI) of the DAC treatment group was significantly higher than for the FLAG group (median not achieved vs 16.8 months, P=0.021).ConclusionDAC treatment was also more effective in those patients with poor prognosis, suggesting that DAC resulted in a better outcome for RR-AML treatment. In conclusion, in our study, DAC therapy provided more safety and effectiveness and lower toxicity in the treatment of RR-AML compared to FLAG therapy.

Decitabine induces regulatory T cells, inhibits the production of IFN-gamma and IL-17 and exerts preventive and therapeutic efficacy in rodent experimental autoimmune neuritis

Guillain-Barré syndrome (GBS) is an immune-mediated acute disorder of the peripheral nervous system. Despite treatment, there is an associated mortality and severe disability in 9 to 17% of the cases. Decitabine (DAC) is a hypomethylating drug used in myelodisplastic syndrome, that has been shown to exert immunomodulatory effects. We have evaluated the effects of DAC in two rodent models of GBS, the Experimental Allergic Neuritis (EAN). Both prophylactic and therapeutic treatment with DAC ameliorated the clinical course of EAN, increasing the numbers of thymic regulatory T cells and reducing the production of proinflammmatory cytokines. Our data suggest the possible use of decitabine for the treatment of GBS.

Nucleosidic DNA demethylating epigenetic drugs - A comprehensive review from discovery to clinic.

DNA methylation plays a pivotal role in the etiology of cancer by mediating epigenetic silencing of cancer-related genes. Since the relationship between aberrant DNA methylation and cancer has been understood, there has been an explosion of research at developing anti-cancer therapies that work by inhibiting DNA methylation. From the discovery of first DNA hypomethylating drugs in the 1980s to recently discovered second generation pro-drugs, exceedingly large number of studies have been published that describe the DNA hypomethylation-based anti-neoplastic action of these drugs in various stages of the pre-clinical investigation and advanced stages of clinical development. This review is a comprehensive report of the literature published in past 40 years, on so far discovered nucleosidic DNA methylation inhibitors in chronological order. The review will provide a complete insight to the readers about the mechanisms of action, efficacy to demethylate and re-express various cancer-related genes, anti-tumor activity, cytotoxicity profile, stability, and bioavailability of these drugs. The review further presents the far known mechanisms of primary and secondary resistance to azanucleoside drugs. Finally, the review highlights the ubiquitous role of DNA hypomethylating epi-drugs as chemosensitizers and/or priming agents, and recapitulate the combinatorial cancer preventive effects of these drugs with other epigenetic agents, conventional chemo-drugs, or immunotherapies. This comprehensive review analyzes the beneficial characteristics and drawbacks of nucleosidic DNA methylation inhibitors, which will assist the pre-clinical and clinical researchers in the design of future experiments to improve the therapeutic efficacy of these drugs and circumvent the challenges in the path of successful epigenetic therapy.