Aberrant Regional DNA Hypermethylation As a Preventive and Therapeutic Target for Adult T-Cell Leukemia-Lymphoma

Aberrant DNA methylation profiles in various types of cancer have highlighted the importance of DNA methylation in human carcinogenesis and opened the prospect of targeting aberrant DNA methylation with demethylating agents as a therapy for cancer. Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy derived from CD4 (+) T-cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Although it has been estimated that 20 million people are infected with HTLV-1 worldwide, most HTLV-1 infected individuals have no symptoms and only 3-7% of HTLV-1 positive carriers develop ATL in their life time. HTLV-1 infected cells undergo multistep leukemogenesis and there are four clinical subtypes of ATL, smoldering type, chronic type, acute type, and lymphoma type. It takes several decades to acquire aggressive phenotypes through the accumulation of genetic and epigenetic abnormalities. In this study, we aimed to elucidate the contribution of aberrant DNA methylation to ATL leukemogenesis and the anti-ATL effects of DNA demethylating agents.

Since the expression profiles of CADM1 and CD7 in CD4 (+) T-cells reflect ATL disease progression, fractions of HTLV-1 infected cells and normal T-cells were isolated from ATL patients, HTLV-1 carriers, and healthy volunteers using the expression statuses of CADM1 and CD7. Comprehensive genome-wide profiling of DNA methylation was performed by quantitative array-based methylation analysis at the single-CpG-site level using the Infinium HumanMethylation450 BeadChip array. Anti-ATL effects of four DNA demethylating agents were investigated in in vitro experiments using ATL-related cell lines and a xenograft mouse model.

Unsupervised hierarchical clustering analysis was first conducted using the DNA methylation profiles of 20,000 CpG probes, which were randomly picked from 470,870 probes. Global DNA hypomethylation was detected in HTLV-1 infected cells at the asymptomatic carrier stage. To identify differentially methylated positions (DMPs) that specifically reflect ATL disease status, the DNA methylation profiles of a normal cell subpopulation were compared with those of a HTLV-1 infected subpopulation. We identified 12,025 hypermethylated and 33,581 hypomethylated DMPs that were specific to the HTLV-1 infected subpopulation. Importantly, the methylation profiles of hypermethylated DMPs, but not those of hypomethylated DMPs, were different between the aggressive and indolent types of ATL and could be used to distinguish them. Therefore, we next extracted and analyzed 1,207 hypermethylated CpG sites located in TSS200 CpG islands (CGIs) in hypermethylated DMPs, since TSS200 CGIs are widely recognized to regulate gene expression in a methylation dependent manner. We found the DNA methylation profiles of 1,207 probes tended to correlate with ATL disease status.

Since regional DNA hypermethylation appears to be associated with ATL disease progression, we tested the anti-leukemia activities of two novel decitabine prodrugs, OR-21 and OR-12, and compared their efficacies with those of clinically available AZA and DAC. OR-21 and OR-12 showed enhanced oral bioavailability and sustained release of decitabine and decitabine 5'-monophosphate, respectively. In ATL-related T-cell lines cultured in the presence of each DNA demethylating compound, DAC and OR-21 significantly suppressed cell growth and decreased DNA methylation at LINE-1 repeat regions, which are used as a biomarker for the monitoring of global changes in DNA methylation.

To establish a xenograft mouse model, cells of the HTLV-1-transformed T-cell line, MT-2, were inoculated into the subcutaneous tissue of immunodeficient Balb/c Rag-2^-/- Jak3^-/- mice, which lack mature T and B lymphocytes and NK cells. A visible tumor appeared 10 days after inoculation and DAC or OR-21 was injected intraperitoneally twice a week, because they were rapidly degraded in the stomach acidic environment and could not be administered orally. The AUC-guided dosing of OR21 (3.39mg/kg) suppressed tumor growth, which was comparable with that obtained with 1.25 mg/kg DAC, while OR21 showed less hematotoxicity than DAC.