Epigenetic therapy in immune-oncology.

Epigenetic gene silencing by DNA methylation and histone methylation by EZH2 play an important role in the development of acute myeloid leukemia (AML). EZH2 catalyzes the trimethylation of histone H3-lysine 27-trimethylated (H3K27me3). These epigenetic alterations silence the expression of the genes that suppress leukemogenesis. Reversal of this gene silencing by 5-aza-2′-deoxycytidine (5-Aza-CdR), an inhibitor of DNA methylation, and by 3-deazaneplanocin-A (DZNep), an inhibitor of EZH2, results in synergistic gene reactivation and antileukemic interaction. The objective of this study is to determine if the addition of another epigenetic agent could further enhance the antileukemic action of these inhibitors of DNA and histone methylation. Vitamin C (Vit C) is reported to enhance the antineoplastic action of 5-Aza-CdR on AML cells. The mechanism responsible for this action of Vit C is due to its function as a cofactor of alpha-ketoglutarate-dependent dioxygenases (α-KGDD). The enhancement by Vit C of the catalytic activity of α-KGDD of the ten eleven translocation (TET) pathway, as well as of the Jumonji C histone demethylases (JHDMs), is shown to result in demethylation of DNA and histones, leading to reactivation of tumor suppressor genes and an antineoplastic effect. This action of Vit C has the potential to complement the antileukemic action of 5-Aza-CdR and DZNep. We observe that Vit C remarkably increases the antineoplastic activity of 5-Aza-CdR and DZNep against myeloid leukemic cells. An important step to bring this novel epigenetic therapy to clinical trial in patients with AML is the determination of its optimal dose schedule.

Epigenetic changes in cancer are well described, and it is believed aberrant DNA hypermethylation and histone deacetylation can lead to silencing of tumor suppressor genes and tumorigenesis [1]. Epigenetic therapy is progressively growing in importance as a class of therapies for cancer. Currently seven drugs are approved by the US FDA for the treatment of a variety of cancers, and target two major epigenetic systems. Those drugs that inhibit DNA methylation and those drugs that inhibit histone deacetylation (Table 1). Epigenetic therapies have demonstrated clear benefit in patients with some cancers via randomized clinical trials [2,3]. However, conclusive evidence that these drugs function via an epigenetic mechanism does not exist. In fact, studies combining epigenetic therapies have been disappointing and may suggest these agents do not act via an epigenetic target [4–6]. Azacitidine (Vidaza) and decitabine (Dacogen) are DNA methyltransferase inhibitors (DNMTi). Both are cytosine analogs that incorporate into the DNA and irreversibly inhibit DNA methyltransferase leading to a decrease in DNA methylation. Both DNMTi are approved as single-agent treatment of myelodysplastic syndrome (MDS), a hematologic malignancy closely related to acute myelogenous leukemia (AML) [7,8]. Both drugs are used at clinically low doses which are believed to take advantage of the DNA methylation inhibition properties as opposed to the cytotoxic properties of these drugs. Clinically decreases in gene-specific DNA methylation and global DNA methylation have been shown with these drugs [9,10]; however, reactivation of a specific tumor suppressor gene has not been shown to be associated with response in MDS. In addition, global DNA methylation decreases appear to be transient and rapidly return to baseline levels after the treatment is stopped or in between treatment cycles [9]. Azacitidine was compared in a randomized clinical trial to another cytosine analog, cytarabine. In this study cytarabine was given at both low doses and higher doses. Azacitidine was clearly shown to improve survival compared with both low-dose and high-dose chemotherapy [2]. Interestingly the rate of complete response (eradication of the disease and normalization of the bone marrow) was higher with intensive chemotherapy, but the clinical outcome was better with low-dose chronic azacitidine treatment [2]. Perhaps contrasting a killing the cancer strategy for intensive chemotherapy versus a modification of the phenotype by epigenetic therapy. The failure of epigenetic combination therapy for cancer and what it might be telling us about DNA methylation inhibitors

BackgroundThe inactivation of tumor suppressor genes (TSGs) by aberrant DNA methylation plays an important role in the development of malignancy. Since this epigenetic change is reversible, it is a potential target for chemotherapeutic intervention using an inhibitor of DNA methylation, such as 5-aza-2'-deoxycytidine (DAC). Although clinical studies show that DAC has activity against hematological malignancies, the optimal dose-schedule of this epigenetic agent still needs to be established.MethodsClonogenic assays were performed on leukemic and tumor cell lines to evaluate the in vitro antineoplastic activity of DAC. The reactivation of TSGs and inhibition of DNA methylation by DAC were investigated by reverse transcriptase-PCR and Line-1 assays. The in vivo antineoplastic activity of DAC administered as an i.v. infusion was evaluated in mice with murine L1210 leukemia by measurement of survival time, and in mice bearing murine EMT6 mammary tumor by excision of tumor after chemotherapy for an in vitro clonogenic assay.ResultsIncreasing the DAC concentration and duration of exposure produced a greater loss of clonogenicity for both human leukemic and tumor cell lines. The reactivation of the TSGs (p57KIP2 in HL-60 leukemic cells and p16CDKN2A in Calu-6 lung carcinoma cells) and the inhibition of global DNA methylation in HL-60 leukemic cells increased with DAC concentration. In mice with L1210 leukemia and in mice bearing EMT6 tumors, the antineoplastic action of DAC also increased with the dose. The plasma level of DAC that produced a very potent antineoplastic effect in mice with leukemia or solid tumors was > 200 ng/ml (> 1 μM).ConclusionWe have shown that intensification of the DAC dose markedly increased its antineoplastic activity in mouse models of cancer. Our data also show that there is a good correlation between the concentrations of DAC that reduce in vitro clonogenicity, reactivate TSGs and inhibit DNA methylation. These results suggest that the antineoplastic action of DAC is related to its epigenetic action. Our observations provide a strong rationale to perform clinical trials using dose intensification of DAC to maximize the chemotherapeutic potential of this epigenetic agent in patients with cancer.

Epigenetic regulation of cellular identity and function is at least partly achieved through changes in covalent modifications on DNA and histones. Much progress has been made in recent years to understand how these covalent modifications affect cell identity and function. Despite the advances, whether and how epigenetic factors contribute to memory formation is still poorly understood. In this review, we discuss recent progress in elucidating epigenetic mechanisms of learning and memory, primarily at the DNA level, and look ahead to discuss their potential implications in reward memory and development of drug addiction.

The nucleoside analog 5-aza-2'-deoxycytidine (5-aza-CdR, decitabine) is a potent inhibitor of DNA methylation in vitro. Cellular treatment with this agent induces the re-expression of methylation-silenced genes. It remains unclear to what extent this compound inhibits DNA methylation in vivo. A clinical study was designed to examine the molecular effects and toxicity of a continuous 1-week intravenous infusion of decitabine in solid tumor patients. Ten patients with refractory solid tumors were included in this study. Decitabine was administered at 2 mg/m(2)/d [DOSAGE ERROR CORRECTED] via continuous infusion for 168 hours. Quantitative polymerase chain reaction and high performance liquid chromatography were utilized to measure promoter-specific and global DNA methylation in peripheral-blood cells before and after treatment. Transient grade III/IV neutropenia (two patients) and grade II thrombocytopenia (one patient) was observed at the lowest planned dose step (2 mg/m2/d for 7 days). Nonhematologic toxicities were not observed. Quantitative polymerase chain reaction demonstrated significant MAGE-1 promoter hypomethylation by 14 days after the start of treatment in all 13 treatment cycles examined. Significant genomic DNA hypomethylation was also seen by day 14 in 11 of 13 treatment cycles analyzed. Genomic DNA methylation reverted to baseline levels by 28 to 35 days after the start of treatment, demonstrating that inhibition of DNA methylation by decitabine is transient. A 168-hour continuous infusion of decitabine is well tolerated and results in the inhibition of promoter-specific and genomic DNA methylation in vivo. This treatment schedule is suitable for evaluation of decitabine in combination with agents whose activity may be enhanced by the reversal of DNA methylation-mediated gene silencing.

This is a review of the enzymatic mechanism of DNA methyltransferase Dnmt1 and analysis of its implications on regulation of DNA methylation in mammalian cells and design of novel mechanism-based inhibitors. The methylation reaction by Dnmt1 has different phases that depend on DNA substrate and allosteric regulation. Consequently, depending on the phase, the differences in catalytic rates between unmethylated and pre-methylated DNA can vary between 30-40 fold, 3-6 fold or only 1 fold. The allosteric site and the active site can bind different molecules. Allosteric activity depends on DNA sequence, methylation pattern and DNA structure (single stranded vs. double stranded). Dnmt1 binds poly(ADP-ribose) and some RNA molecules. The results on kinetic preferences, allosteric activity and binding preference of Dnmt1 are combined together in one comprehensive model mechanism that can address regulation of DNA methylation in cells; namely, inhibition of DNA methylation by poly(ADP-ribose), RNA-directed DNA methylation by methylated and unmethylated non-coding RNA molecules, and transient interactions between Dnmt1 and genomic DNA. Analysis of reaction intermediates showed that equilibrium between base-flipping and base-restacking events can be the key mechanism in control of enzymatic activity. The two events have equal but opposite effect on accumulation of early reaction intermediates and methylation rates. The accumulation of early reaction intermediates can be exploited to improve the current inhibitors of Dnmt1 and achieve inhibition without toxic modifications in genomic DNA. [1,2-dihydropyrimidin-2-one]-5-methylene-(methylsulfonium)-adenosyl is described as the lead compound.

Background:Osteoarthritis (OA) remains one of the most common osteopathy for centuries, which can be attributed to multiple risk factors including mechanical and biochemical ones. More and more studies verified that inflammatory cytokines play important roles in the progression of OA, such as tumor necrosis factor-alpha (TNF-α). In this study, we aimed to investigate the relationship between epigenetic manifestations of TNF-? and the pathogenesis of OA.Methods:Totally, 37 OA patients’ cartilage was collected through the knee joint and 13 samples of articular cartilage as healthy control was collected through traumatic amputation. Real-time PCR, Western blot and ELISA analysis were performed to observe the expression of target genes and proteins in collected samples.Results:Compared with the healthy control group, TNF-? was over-expressing in cartilage which was collected from OA patients. DNA hypomethylation, histone hyperacetylation and histone methylation were observed in the TNF-? promoter in OA compared with normal patients, and we also studied series of enzymes associated with epigenetics. The results showed that by increasing DNA methylation and decreasing histone acetylation in the TNF-? promoter, and TNF-? over-expression in OA cartilage was suppressed, histone methylation has no significant correlation with OA.Conclusion:In conclusion, the changes of epigenetic status regulate TNF-α expression in the cells, which are pivotal to the OA disease process. These results may give us a better understanding of OA and may provide new therapeutic options.

A major question facing the use of epigenetic therapies in cancer is specificity in modulating gene expression. In addition, combined targeting of DNA and histone methylation remains largely unexplored despite the promising synergistic effects observed from combining DNA methyltransferase inhibitors with HDAC inhibitors. To address these questions, we performed RNA-seq, DNA methylation analysis and ChIP-seq (H3K4me2, H3K9me2, and H3K27me3) to study the effects of inhibitors of DNA methyltransferases (DAC), histone deacetylases (Depsi), histone demethylases (KDM1A inhibitor S2101), and histone methylases (EHMT2 inhibitor UNC0638 and EZH2 inhibitor GSK343) in three different cancer models (colon cancer, breast cancer, and leukemia). In colon cancer cells (YB5), DAC affected 3% of the transcriptome and 93% of the effect was gene upregulation. DAC had a greater effect on genes expressed in normal tissues and silenced in cancer (443 genes) compared to genes that do not change in cancer (194 genes). 90% of DAC targets genes showed no promoter DNA methylation in normal colon but gained methylation in cancer. Depsi changed the expression of 35% of the transcriptome and showed little specificity for gene upregulation or silenced genes. S2101, UNC0638, and GSK343 had limited effects on their own (<1.5% of the transcriptome), but UNC0638 and GSK343 preferentially targeted genes with H3K9me2 or H3K27me3, respectively. DAC combined with histone methylation inhibitors led to synergistic gene upregulation while still maintaining specificity for DNA methylated and silenced genes. These synergistic genes had limited overlap, indicating the possibility to target distinct sets of genes based on different epigenetic therapy combinations. IPA analysis demonstrated that these genes are enriched in cancer pathways, and consistent with this analysis, the combination therapies were able to decrease cancer cell proliferation more effectively than monotherapy. Broadly similar results were seen with genome wide studies in both breast cancer (MCF7) and leukemia (HL-60) cells. These results demonstrate that DNA methyltransferase inhibitors preferentially target cancer relevant genes, and can be combined with inhibitors targeting histone methylation for synergistic effects while still maintaining specificity. Citation Format: Takahiro Sato, Matteo Cesaroni, Shoghag Panjarian, Anthony Tran, Jozef Madzo, Yasuyuki Okamoto, Hanghang Zhang, Xiaowei Chen, Jaroslav Jelinek, Jean-Pierre J. Issa. Target specificity of epigenetic therapy in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2657.

Epigenetics in Male Germ Cells

Histone deacetylase inhibitors in myelodysplastic syndrome

A central challenge in the development of epigenetic cancer therapy is the ability to direct selectivity in modulating gene expression for disease-selective efficacy. To address this issue, we characterized by RNA-seq, DNA methylation, and ChIP-seq analyses the epigenetic response of a set of colon, breast, and leukemia cancer cell lines to small-molecule inhibitors against DNA methyltransferases (DAC), histone deacetylases (Depsi), histone demethylases (KDM1A inhibitor S2101), and histone methylases (EHMT2 inhibitor UNC0638 and EZH2 inhibitor GSK343). We also characterized the effects of DAC as combined with the other compounds. Averaged over the cancer cell models used, we found that DAC affected 8.6% of the transcriptome and that 95.4% of the genes affected were upregulated. DAC preferentially regulated genes that were silenced in cancer and that were methylated at their promoters. In contrast, Depsi affected the expression of 30.4% of the transcriptome but showed little selectivity for gene upregulation or silenced genes. S2101, UNC0638, and GSK343 affected only 2% of the transcriptome, with UNC0638 and GSK343 preferentially targeting genes marked with H3K9me2 or H3K27me3, respectively. When combined with histone methylase inhibitors, the extent of gene upregulation by DAC was extended while still maintaining selectivity for DNA-methylated genes and silenced genes. However, the genes upregulated by combination treatment exhibited limited overlap, indicating the possibility of targeting distinct sets of genes based on different epigenetic therapy combinations. Overall, our results demonstrated that DNA methyltransferase inhibitors preferentially target cancer-relevant genes and can be combined with inhibitors targeting histone methylation for synergistic effects while still maintaining selectivity. Cancer Res; 77(2); 470-81. ©2016 AACR.

<div>Abstract<p>A central challenge in the development of epigenetic cancer therapy is the ability to direct selectivity in modulating gene expression for disease-selective efficacy. To address this issue, we characterized by RNA-seq, DNA methylation, and ChIP-seq analyses the epigenetic response of a set of colon, breast, and leukemia cancer cell lines to small-molecule inhibitors against DNA methyltransferases (DAC), histone deacetylases (Depsi), histone demethylases (KDM1A inhibitor S2101), and histone methylases (EHMT2 inhibitor UNC0638 and EZH2 inhibitor GSK343). We also characterized the effects of DAC as combined with the other compounds. Averaged over the cancer cell models used, we found that DAC affected 8.6% of the transcriptome and that 95.4% of the genes affected were upregulated. DAC preferentially regulated genes that were silenced in cancer and that were methylated at their promoters. In contrast, Depsi affected the expression of 30.4% of the transcriptome but showed little selectivity for gene upregulation or silenced genes. S2101, UNC0638, and GSK343 affected only 2% of the transcriptome, with UNC0638 and GSK343 preferentially targeting genes marked with H3K9me2 or H3K27me3, respectively. When combined with histone methylase inhibitors, the extent of gene upregulation by DAC was extended while still maintaining selectivity for DNA-methylated genes and silenced genes. However, the genes upregulated by combination treatment exhibited limited overlap, indicating the possibility of targeting distinct sets of genes based on different epigenetic therapy combinations. Overall, our results demonstrated that DNA methyltransferase inhibitors preferentially target cancer-relevant genes and can be combined with inhibitors targeting histone methylation for synergistic effects while still maintaining selectivity. <i>Cancer Res; 77(2); 470–81. ©2016 AACR</i>.</p></div>

<div>Abstract<p>A central challenge in the development of epigenetic cancer therapy is the ability to direct selectivity in modulating gene expression for disease-selective efficacy. To address this issue, we characterized by RNA-seq, DNA methylation, and ChIP-seq analyses the epigenetic response of a set of colon, breast, and leukemia cancer cell lines to small-molecule inhibitors against DNA methyltransferases (DAC), histone deacetylases (Depsi), histone demethylases (KDM1A inhibitor S2101), and histone methylases (EHMT2 inhibitor UNC0638 and EZH2 inhibitor GSK343). We also characterized the effects of DAC as combined with the other compounds. Averaged over the cancer cell models used, we found that DAC affected 8.6% of the transcriptome and that 95.4% of the genes affected were upregulated. DAC preferentially regulated genes that were silenced in cancer and that were methylated at their promoters. In contrast, Depsi affected the expression of 30.4% of the transcriptome but showed little selectivity for gene upregulation or silenced genes. S2101, UNC0638, and GSK343 affected only 2% of the transcriptome, with UNC0638 and GSK343 preferentially targeting genes marked with H3K9me2 or H3K27me3, respectively. When combined with histone methylase inhibitors, the extent of gene upregulation by DAC was extended while still maintaining selectivity for DNA-methylated genes and silenced genes. However, the genes upregulated by combination treatment exhibited limited overlap, indicating the possibility of targeting distinct sets of genes based on different epigenetic therapy combinations. Overall, our results demonstrated that DNA methyltransferase inhibitors preferentially target cancer-relevant genes and can be combined with inhibitors targeting histone methylation for synergistic effects while still maintaining selectivity. <i>Cancer Res; 77(2); 470–81. ©2016 AACR</i>.</p></div>

Is Cladribine, a Treatment for Chronic Lymphocytic Leukemia, a DNA Methylation Inhibitor?.

Somatostatin (SMS), binds to its specific receptors (SSTRs) and transduces growth inhibitory, anti-secretory and apoptotic signals. Several human cancers express SSTRs, including prostate cancer, and therefore SMS is of interest for anti-cancer therapy. DNA methylation and histone modifications are involved in normal cell development, gene imprinting and human carcinogenesis. Reversing DNA methylation is an attractive therapeutic possibility, since epigenetic modifications change gene expression without changing the gene function. DNA methylation inhibitors such as 5-aza-2'-deoxycytidine (5'-aza, decitabine) have been used to treat several types of haematological malignancies. Histone deacetylase inhibitors such as trichostatin (TSA), are a new class of 'targeted anti-cancer agents'. TSA and decitabine can induce growth arrest, apoptosis or terminal differentiation in a variety of solid and haematological cancers in advanced disease patients. In the present study, the LNCaP cell line (prostate cancer) was incubated with SMS or Somadex (an SMS polymer conjugate) for three days, 1 nM per day, and the untreated cells were the negative control. For DNA demethylation, cells were grown in the presence of 2.5 microM 5-aza for 120 h, and re-fed with 5-aza-containing fresh medium at day 3. The total incubation time with 5-aza was 120 h. TSA at 1.0 microM was added into the cultured cells for 24 h. The combined treatment of 5-aza and TSA was performed by incubating the cells with 5-aza for 120 h followed by a 24-h exposure to TSA. Using cDNA obtained from these cell lines, the difference in the expression level of SSTR mRNA transcripts before and after 5-aza and TSA treatments was analyzed by RT-PCR. An increased induction of mRNA expression of the five SSTR subtypes was observed in the LNCaP cells when incubated with SMS/Somadex (dose-dependent). The inhibition of DNA methylation and histone acetylation resulted in the up-regulation of SSTR5 mRNA expression. The results demonstrate a positive feedback loop between SMS and its receptors. This regulation pathway may enhance the anti-tumor activity of somatostatin. To benefit from this effect in a clinical setting, the dose, dose frequency and pan affinity of the SMS derivative are important factors. The epigenetic manipulation with DNA methylation or histone deacetylase inhibitors, combined with SMS, may offer a novel alternative for the treatment of advanced prostate cancer.