Dna Methyltransferase Inhibitors

Gene silencing due to hypermethylation is the most common epigenetic modification in human malignancies.2 Unlike genetic changes involving mutations or deletions, this modification is potentially reversible, making it attractive as a therapeutic target. Hyper-methylation of the chromatin is caused by an increase in DNA methyltransferase I activity; this enzyme covalently links a methyl group to the 5-position of cytosine of CpG residues typically found in gene promoter regions (CpG

islands). Normally, unmethylated CpG islands are protected from hypermethylation. This protection is lost in neoplastic cells due to the increase in DNA methyltrans-ferase activity. The methylated promoters bind specific proteins, which then recruit transcriptional corepressors. These complexes lead to transcriptional silencing through chromatin remodeling. DNA hypermethylation of promoter regions of tumor suppressor genes leads, ultimately, to uncontrolled cell proliferation. Several of the DNA methyltransferase inhibitors are S-phase specific, making inhibitors of these agents the logical agents for cancer therapy. In the late 1960s, 5-azacytidine (Aza-C) was developed as an antitumor agent for the treatment of AML.3 Aza-C, an analog of cytosine, is incorporated into the DNA but, unlike cytosine, cannot be methylated at position 5. In addition, Aza-C binds directly to and inhibits methyltransferase. The drug also exerts cytotoxic and cell-differentiating properties by unknown mechanisms. Most recently, as described below, Aza-C has received attention as a therapeutic agent for MDS, leading to the recent full FDA approval for Aza-C on May 19, 2004, for MDS patients of all subtypes. This is the very first agent to be fully approved by the FDA for the treatment of MDS.

The pivotal phase III study for Aza-C was a randomized controlled Cancer and Leukemia Group B (CALGB) trial carried out in 191 patients with MDS of all subtypes.4 Half of the patients received Aza-C 75 mg/m2 subcutaneously for 7 days every 4 weeks for a minimum of 16 weeks, and half received only SC. A 60% overall response was found in patients on the treatment arm (7% complete response (CR), 16% partial response (PR), and 37% with hematologic improvement (HI)), with tri-lineage responses in 23% of patients. Only 5% of those on the observation arm showed hematologic improvement (P < 0.001). Furthermore, when a subset of 49 patients from the observation arm crossed over to the treatment arm, a 47% response was achieved, once again with 10% achieving a CR, 4% a PR, and 33% with clinical improvement. The median duration of response was 15 months, but the most striking observation in this study was the significantly longer median time to transformation to acute leukemia and longer median survival in patients receiving treatment: 21 months, versus 12 months for the observation-only patients. Patients initially treated with Aza-C showed a median survival of 18 months in comparison to 14 months for the early crossover arm, and 11 months for those crossed over after 6 months or not crossed over at all.

Aza-C is a pro-drug for 5-aza-2'-deoxycitadine (decitabine, DAC), a pyrimidine nucleoside analog that has completed a phase III clinical trial. Aza-C is first converted to DAC before incorporation into DNA. It then binds and irreversibly inhibits DNA methyltrans-ferase. DAC is a strong inhibitor of DNA methylation, and like Aza-C has been used for the treatment of high-risk MDS. In a phase II trial by Wijermans et al., 66 MDS patients received DAC 45 mg/m2/day every 8 h for 3 days every 6 weeks, for a maximum of 6 cycles.5

The overall response rate was 49%, with a 64% response rate in those with an IPSS high-risk score. There were 34 CRs, with a median response duration of 31 weeks. However, there was an 8% toxic death rate, with a delayed onset of cytopenias. In a subsequent study using the same drug regime, 16 out of 50 patients with clonal cytogenetic abnormalities developed normal karyotypes after treatment with DAC,6 responses lasting for a median of 7.5 months. This study also noted correlation of cytogenetic responses to survival.

Saba et al. have recently shown responses and better tolerance in a phase III multicenter trial of 170 MDS patients treated with DAC.7 DAC was administered at 15 mg every 8 h for 3 days every 6 weeks. The response rate by International Working Group (IWG) criteria was 35% (10% CR, 15% PR, and 10% HI) for DAC versus 0% for SC (P < 0.001). Time to response was 100 days, and the median duration was estimated at >9 months. There were no significant differences in mortality rates on treatment, and no treatment-related deaths. As expected, grade III-IV toxicity (mainly hematologic) occurred more in DAC patients than in SC patients. Most patients tolerated the treatment well; discontinuation due to toxicity as primary reason was reported in 9% of DAC patients.

The correlation between methylation status and clinical response, however, is not yet clear. The suppressor genes p15 and p16 inhibit cyclin-dependent kinases and are frequently hypermethylated in hematologic neo-plasias, including AML and MDS. Bone marrow mononuclear cells from patients with high-risk MDS were examined for p15/INK-4B and p16 methylation status during treatment with DAC.8 Hypermethylation of the 5' p15 region was detected in 15 of 23 (65%) patient samples examined. After treatment, 9 of 12 patient samples showed a decrease in methylation, which was associated with clinical responses in these patients. However, response to the drug was also seen in patients without hypermethylation. Thus, DAC may work by multiple mechanisms, and direct clinical correlation to methyla-tion status cannot be assumed at this time.

More recently, a phase I trial of prolonged exposure to lower doses of DAC was conducted in 50 patients, mostly with AML/MDS.9 Patients were given four different doses of DAC: 5 mg, 10 mg, 15 mg, and 20 mg/m2/day i.v. over 1 h for 10 days. All doses were well tolerated and responses were noted even in the lowest doses. There was a 65% response rate at the 15 mg/m2 dose with no increase in response after dose escalation or prolongation to 15 and 20 days. In this study, there was no correlation noted between p15 methylation at baseline or after therapy and response to DAC. Hence, DAC appears to be an effective therapy for MDS with manageable toxicity, and its role in light of the recent FDA approval for Aza-C remains to be determined.

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