Enhancement of the Antileukemic Action of the Inhibitors of DNA and Histone Methylation: 5-Aza-2'-Deoxycytidine and 3-Deazaneplanocin-A by Vitamin C.

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 Modulation of Self-Renewal Capacity of Leukemic Stem Cells and Implications for Chemotherapy.

Most patients with acute myeloid leukemia (AML) have a poor prognosis. Curative therapy of AML requires the complete eradication of the leukemic stem cells (LSCs). One aspect of LSCs that is poorly understood is their low frequency in the total population of leukemic cells in AML patients. After each cell division of LSCs, most of the daughter cells lose their capacity for self-renewal. Investigations into the role of Isocitrate dehydrogenase (IDH) mutations in AML provide some insight on the regulation of the proliferation of LSCs. The primary role of IDH is to convert isocitrate to alpha-keto-glutarate (α-KG). When IDH is mutated, it converts α-KG to 2-hydroxyglutarate (2-HG), an inhibitor of the TET pathway and Jumonji-C histone demethylases (JHDMs). The demethylating action of these enzymes removes the epigenetic gene-silencing markers, DNA methylation, H3K27me3 and H3K9me2 and can lead to the differentiation of LSCs. This enzymatic action is blocked by 2-HG in mutated IDH (mut-IDH) AML patients, who can be induced into remission with antagonists of 2-HG. These observations suggest that there exists in cells a natural enzymatic mechanism that uses demethylation to reverse epigenetic gene-silencing, leading to a loss of the self-renewal capacity of LSCs. This mechanism limits the proliferative potential of LSCs. Epigenetic agents that inhibit DNA and histone methylation exhibit a synergistic antineoplastic action on AML cells. It is possible that the therapeutic potential of this epigenetic therapy may be enhanced by demethylation enzymes, resulting in a very effective treatment for AML.

Importance of dose-schedule of 5-aza-2'-deoxycytidine for epigenetic therapy of cancer.

BACKGROUND: The 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. METHODS: Clonogenic 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. RESULTS: Increasing 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 microM). CONCLUSION: We 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.

Synergistic effect of 5-Aza-2'-deoxycytidine and genistein in combination against leukemia.

5-Aza-2'-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylation, is an effective agent for the treatment of leukemia. The aim of this study was to investigate the antileukemic activity of this epigenetic agent in combination with genistein, a nontoxic isoflavone with chemopreventive activity. The combined treatment produced a synergistic loss of clonogenicity in human myeloid (HL-60) and lymphoid (MOLT-3) leukemic cell lines. Genistein alone showed a significant antileukemic activity against murine 5-AZA-CdR-resistant cells, and this effect was enhanced when used in combination with 5-AZA-CdR. The combined treatment also produced a synergistic increase in life span of mice with L1210 leukemia. These results suggest that genistein may have the potential to increase the clinical efficacy of 5-AZA-CdR for the treatment of leukemia.

Inhibition of DNA and Histone Methylation by 5-Aza-2'-Deoxycytidine (Decitabine) and 3-Deazaneplanocin-A on Antineoplastic Action and Gene Expression in Myeloid Leukemic Cells.

Epigenetic alterations play an important role in the development of acute myeloid leukemia (AML) by silencing of genes that suppress leukemogenesis and differentiation. One of the key epigenetic changes in AML is gene silencing by DNA methylation. The importance of this alteration is illustrated by the induction of remissions in AML by 5-aza-2'-deoxycytidine (5-AZA-CdR, decitabine), a potent inhibitor of DNA methylation. However, most patients induced into remission by 5-AZA-CdR will relapse, suggesting that a second agent should be sought to increase the efficacy of this epigenetic therapy. An interesting candidate for this purpose is 3-deazaneplanocin A (DZNep). This analog inhibits EZH2, a histone methyltransferase that trimethylates lysine 27 histone H3 (H3K27me3), a marker for gene silencing. This second epigenetic silencing mechanism also plays an important role in leukemogenesis as shown in preclinical studies where DZNep exhibits potent inhibition of colony formation by AML cells. We reported previously that 5-AZA-CdR in combination with DZNep exhibits a synergistic antineoplastic action against human HL-60 AML cells and the synergistic activation of several tumor suppressor genes. In this report, we showed that this combination also induced a synergistic activation of apoptosis in HL-60 cells. The synergistic antineoplastic action of 5-AZA-CdR plus DZNep was also observed on a second human myeloid leukemia cell line, AML-3. In addition, 5-AZA-CdR in combination with the specific inhibitors of EZH2, GSK-126, or GSK-343, also exhibited a synergistic antineoplastic action on both HL-60 and AML-3. The combined action of 5-AZA-CdR and DZNep on global gene expression in HL-60 cells was investigated in greater depth using RNA sequencing analysis. We observed that this combination of epigenetic agents exhibited a synergistic activation of hundreds of genes. The synergistic activation of so many genes that suppress malignancy by 5-AZA-CdR plus DZNep suggests that epigenetic gene silencing by DNA and histone methylation plays a major role in leukemogenesis. Targeting DNA and histone methylation is a promising approach that merits clinical investigation for the treatment of AML.

Preclinical evaluation of antineoplastic activity of inhibitors of DNA methylation (5-aza-2'-deoxycytidine) and histone deacetylation (trichostatin A, depsipeptide) in combination against myeloid leukemic cells.

During the development of leukemia, genes that suppress growth and induce differentiation can be silenced by aberrant DNA methylation and by changes in chromatin structure that involve histone deacetylation. It has been reported that a positive interaction between DNA methylation and histone deacetylation takes place to inhibit transcription. Based on this observation, our working hypothesis was that a combination of inhibitors of these processes should produce an enhancement of their antineoplastic activity on leukemic cells. The cytosine nucleoside analog, 5-aza-2'-deoxycytidine (5AZA), is a potent inhibitor of DNA methylation, which can activate tumor suppressor genes in leukemic cells that have been silenced by aberrant methylation. In clinical trials, 5AZA was demonstrated to be an active antileukemic agent. Histone deacetylase inhibitors (HDI) can also activate gene expression in leukemic cell lines by producing changes in chromatin configuration, and show antineoplastic activity in preclinical studies. In this report, we investigated the in vitro antineoplastic activity of 5AZA, alone and in combination with the HDI, trichostatin A (TSA) and depsipeptide (FR901228, depsi), on the human myeloid leukemic cell lines, HL-60 and KG1a. The results showed that the combination of 5AZA with TSA or depsi produced a greater inhibition of growth and DNA synthesis and a greater loss of clonogenicity than either agent alone. These results suggest that 5AZA used in combination with HDI may be an interesting chemotherapeutic regimen to investigate in patients with acute myeloid leukemia that is resistant to conventional chemotherapy.

Enhancement of antineoplastic action of 5-aza-2'-deoxycytidine by phenylbutyrate on L1210 leukemic cells.

Epigenetic changes, such as aberrant DNA methylation that silences tumor suppressor genes (TSGs), can play an important role in the development of leukemia. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-AZA-CdR), can reactivate these silent TSGs and is an interesting agent to investigate for therapy of leukemia. It has been reported that the effectiveness of 5-AZA-CdR to reactivate TSG can be enhanced by inhibitors of histone deacetylase (HDIs). HDIs can convert a compact chromatin structure to an open configuration that facilitates gene expression. An interesting HDI is phenylbutyrate (PB), which has shown some clinical activity for the therapy of leukemia. In this report we have investigated the antineoplastic activity of 5-AZA-CdR and PB alone and in combination on murine L1210 lymphoid leukemic cells. The in vitro treatment of 5-AZA-CdR and PB in combination produced a greater inhibition of growth, DNA synthesis, and also a greater reduction on colony formation on both L1210 and human HL-60 leukemic cells as compared to either drug alone. The combination also produced a synergistic activation of the TSG, p15CDN2B, in the L1210 cells. In mice with L1210 leukemia the combination showed enhanced antineoplastic activity. We also observed an enhancement of the antineoplastic activity of this combination in mice with L1210 leukemia. These data provide a rationale to investigate 5-AZA-CdR and PB in patients with advanced leukemia.

Epigenetic therapy of acute myeloid leukemia using 5-aza-2'-deoxycytidine (decitabine) in combination with inhibitors of histone methylation and deacetylation.

BACKGROUND: The silencing of tumor suppressor genes (TSGs) by aberrant DNA methylation occurs frequently in acute myeloid leukemia (AML). This epigenetic alteration can be reversed by 5-aza-2'-deoxcytidine (decitabine, 5-AZA-CdR). Although 5-AZA-CdR can induce complete remissions in patients with AML, most patients relapse. The effectiveness of this therapy may be limited by the inability of 5-AZA-CdR to reactivate all TSGs due to their silencing by other epigenetic mechanisms such as histone methylation or chromatin compaction. EZH2, a subunit of the polycomb repressive complex 2, catalyzes the methylation of histone H3 lysine 27 (H3K27) to H3K27me3. 3-Deazaneplanocin-A (DZNep), an inhibitor of methionine metabolism, can reactivate genes silenced by H3K27me3 by its inhibition of EZH2. In a previous report, we observed that 5-AZA-CdR, in combination with DZNep, shows synergistic antineoplastic action against AML cells. Gene silencing due to chromatin compaction is attributable to the action of histone deacetylases (HDAC). This mechanism of epigenetic gene silencing can be reversed by HDAC inhibitors such as trichostatin-A (TSA). Silent TSGs that cannot be reactivated by 5-AZA-CdR or DZNep have the potential to be reactivated by TSA. This provides a rationale for the use of HDAC inhibitors in combination with 5-AZA-CdR and DZNep to treat AML. RESULTS: The triple combination of 5-AZA-CdR, DZNep, and TSA induced a remarkable synergistic antineoplastic effect against human AML cells as demonstrated by an in vitro colony assay. This triple combination also showed a potent synergistic activation of several key TSGs as determined by real-time PCR. The triple combination was more effective than the combination of two agents or a single agent. Microarray analysis showed that the triple combination generated remarkable changes in global gene expression. CONCLUSIONS: Our data suggest that it may be possible to design a very effective therapy for AML using agents that target the reversal of the following three epigenetic "lock" mechanisms that silence gene expression: DNA methylation, histone methylation, and histone deacetylation. This approach merits serious consideration for clinical investigation in patients with advanced AML.

Epigenetic action of decitabine (5-aza-2'-deoxycytidine) is more effective against acute myeloid leukemia than cytotoxic action of cytarabine (ARA-C).

Treatment of elderly patients with acute myeloid leukemia (AML) with standard cytarabine (ARA-C) chemotherapy can achieve some complete responses (CR), but the median overall survival is less than one year. New approaches should be investigated. The inhibitor of DNA methylation, 5-aza-2'-deoxycytidine (decitabine, DAC), shows effectiveness in these patients, but was not approved by the US Federal Drug Administration. This decision was based on a clinical trial where DAC showed a median survival of 7.0 months as compared to standard ARA-C therapy or supportive care of 5.0 months. However, the difference was not statistically significant. Preclinical data indicate that DAC is much more effective against human AML than ARA-C. The key question is should these preclinical data also be used in the evaluation of new drugs for the clinical treatment of AML? The delayed epigenetic action of DAC is very different than the acute cytotoxic action of ARA-C and should be taken into account in the design clinical trials and evaluation of the response.

Synergistic antileukemic action of a combination of inhibitors of DNA methylation and histone methylation.

DNA methylation and histone methylation are both involved in epigenetic regulation of gene expression and their dysregulation can play an important role in leukemogenesis. Aberrant DNA methylation has been reported to silence the expression of tumor suppressor genes in leukemia. Overexpression of the histone methyltransferase, EZH2, a subunit of the polycomb group repressive complex 2 (PRC2), was observed to promote oncogenesis. This is due to aberrant gene silencing by the trimethylation of histone H3 lysine 27 (H3K27me3) by EZH2. Since both these epigenetic silencing events are reversible, they are interesting targets for chemotherapeutic intervention by using an inhibitor of DNA methylation, such as 5-aza-2'-deoxcytidine (5-AZA-CdR), and 3-deazaneplanocin-A (DZNep), an inhibitor of the EZH2. Human HL-60 and murine L1210 leukemic cells exposed in vitro to 5-AZA-CdR and DZNep in combination showed a synergistic loss of clonogenicity in a colony assay as compared to each agent alone. This positive chemotherapeutic interaction was also observed in mice with L1210 leukemia. Quantitative PCR showed that the combination also produced a remarkable synergistic activation of the tumor suppressor genes, CDKN1A and FBXO32. Microarray analysis showed that 5-AZA-CdR plus DZNep produced a synergistic activation of >150 genes. Our results indicate that 5-AZA-CdR plus DZNep can reactivate target genes that are silenced by two distinct epigenetic mechanisms leading to a loss of the proliferative potential of leukemic cells.

3-Deazauridine enhances the antileukemic action of 5-aza-2'-deoxycytidine and targets drug-resistance due to deficiency in deoxycytidine kinase.

New approaches should be sought to treat high-risk acute lymphoblastic leukemia (ALL). Since aberrant DNA methylation plays an important role in leukemogenesis of ALL, it can be targeted by 5-aza-2'-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylation. 5-AZA-CdR is a prodrug that is activated by deoxycytidine kinase (DCK). Leukemic cells lacking DCK are drug-resistant. In a previous phase I study, we reported that 5-AZA-CdR could induce remissions in ALL. However, some patients developed drug-resistance due to deficiency in DCK. These observations aroused our interest in 3-deazauridine (3-DU), a CTP synthetase inhibitor that is effective against leukemic cells deficient in DCK. In this report, we observed that 3-DU enhanced the in vitro antineoplastic action of 5-AZA-CdR on human leukemic cells by increasing its incorporation into DNA. Using an optimized dose-schedule we showed that this combination could cure some mice bearing L1210 leukemia, even in the presence of a subpopulation of drug-resistant (L1210/ARA-C) leukemic cells lacking DCK. 3-DU alone also cured some mice with L1210/ARA-C leukemia. In a pilot study on 3 relapsed patients with advanced ALL, the combination of 5-AZA-CdR and 3-DU produced a marked reduction in leukemic blasts, confirming our preclinical observations. Furthermore, after several treatments with these agents all three patients developed drug-resistance to 5-AZA-CdR as determined by an in vitro drug sensitivity test. In two patients we showed by enzymatic analysis that the drug-resistance was due to deficiency in DCK. Our preclinical and clinical results provide a strong rationale to further investigate the combination of 5-AZA-CdR and 3-DU for the treatment of advanced ALL.

Inhibition of cytidine deaminase by zebularine enhances the antineoplastic action of 5-aza-2'-deoxycytidine.

Cytidine (CR) deaminase is a key enzyme in the catabolism of cytosine nucleoside analogues, since their deamination results in a loss of their pharmacological activity. In this report we have investigated the importance of CR deaminase with respect to the antineoplastic action of inhibitors of DNA methylation, 5-aza-2'-deoxycytidine (5-AZA-CdR) and zebularine. Zebularine has a dual mechanism of action, since it can also inhibit CR deaminase. The objective of our study was to investigate the importance of zebularine as an inhibitor of CR deaminase with respect to the antineoplastic action of 5-AZA-CdR. Using an in vitro clonogenic assay, we investigated the antineoplastic action of 5-AZA-CdR and zebularine, alone and in combination on wild type 3T3 murine fibroblasts and corresponding V5 cells transduced with CR deaminase gene to express a very high level of CR deaminase activity. The V5 cells were much less sensitive to 5-AZA-CdR than the wild type 3T3 cells. The addition of zebularine significantly enhanced the antineoplastic action of 5-AZA-CdR on V5 cells, but not 3T3 cells. Enzymatic analysis on CR deaminase purified from the V5 cells showed that zebularine is a competitive inhibitor of the deamination of 5-AZA-CdR. These in vitro observations are in accord with our in vivo study in mice with L1210 leukemia, which showed that zebularine increased the antileukemic activity of 5-AZA-CdR. Pharmacokinetic analysis also showed that zebularine increased the plasma level of 5-AZA-CdR during an i.v. infusion in mice. Our results indicate that the major mechanism by which zebularine enhances the antineoplastic action of 5-AZA-CdR is by inhibition of CR deaminase. These findings provide a rationale to investigate 5-AZA-CdR in combination with zebularine in patients with advanced leukemia.

Enhancement of antineoplastic action of 5-aza-2'-deoxycytidine by zebularine on L1210 leukemia.

Tumor suppressor genes that have been silenced by aberrant DNA methylation are potential targets for reactivation by novel chemotherapeutic agents. The potent inhibitor of DNA methylation and antileukemic agent, 5-aza-2'-deoxycytidine (5-AZA-CdR, Decitabine), can reactivate silent tumor suppressor genes. One hindrance to the curative potential of 5-AZA-CdR is its rapid in vivo inactivation by cytidine deaminase (CD). An approach to overcome this obstacle is to use 5-AZA-CdR in combination with zebularine (Zeb), a potent inhibitor of CD. Zeb also possesses independent antineoplastic activity due to its inhibition of DNA methylation. We tested the capacity of 5-AZA-CdR and Zeb alone and in combination to inhibit growth and colony formation of different leukemic cell lines. 5-AZA-CdR and Zeb in combination produced a greater inhibition of growth against murine L1210 lymphoid leukemic cells, and a greater reduction in colony formation by L1210 and human HL-60 myeloid leukemic cells, than either agent alone. The ability of these agents to reactivate the tumor suppressor gene, p57KIP2, was also tested using RT-PCR. The combination produced a synergistic reactivation of p57KIP2 in HL-60 leukemic cells. A methylation-specific PCR assay showed that this combination also induced a significantly greater demethylation level of the p57KIP2 promoter than either drug alone. The in vivo antineoplastic activity of the agents was evaluated in mice with L1210 leukemia. A greater increase in survival time of mice with L1210 leukemia was observed with the combination than with either agent alone using three different dose schedules. The enhanced activity observed with 5-AZA-CdR plus Zeb in both murine and human leukemic cells lines provides a rationale for the clinical investigation of these drugs in patients with advanced leukemia. The probable mechanism of this drug interaction involves inhibition of CD by Zeb and the complementary inhibition of DNA methylation by both agents.

Antineoplastic action of 5-aza-2'-deoxycytidine and phenylbutyrate on human lung carcinoma cells.

Current chemotherapy of advanced non-small cell lung cancer produces only a modest increase in survival time. New approaches are needed to improve its effectiveness. During tumorigenesis, silencing of tumor suppressor genes can occur by aberrant methylation. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-AZA-CdR), can reactivate the expression of these genes. Nucleosomes containing unacetylated positively charged histones bind tightly to DNA producing a compact configuration, which inhibits transcription. Phenylbutyrate (PB), an inhibitor of histone deacetylase (HDAC), increases histone acetylation, neutralizing its positive charge and resulting in DNA with a more open structure, which favors transcription. It has been reported that 5-AZA-CdR in combination with HDAC inhibitor can increase the expression of silent tumor suppressor genes. The objective of our study was to determine if these agents, in combination, produce an enhancement of their antitumor activity. We evaluated the antineoplastic activity of 5-AZA-CdR and PB alone or in combination on human A549 and Calu-6 lung carcinoma cell lines by inhibition of DNA synthesis and clonogenic assays. 5-AZA-CdR and PB in combination produced a greater inhibition of DNA synthesis than either agent alone. Also, in a clonogenic assay the combination of these drugs showed a significant synergistic antitumor effect. These results provide a rationale to investigate the combination of 5-AZA-CdR and PB in patients with advanced lung cancer.