Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy of CD4+ T-cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Most HTLV-1-infected individuals are asymptomatic and only 3-5% of carriers develop ATL. Here, we describe the contribution of aberrant DNA methylation to ATL leukemogenesis. HTLV-1-infected T-cells and their uninfected counterparts were separately isolated based on CADM1 and CD7 expression status, and differentially methylated positions (DMPs) specific to HTLV-infected T-cells were identified through genome-wide DNA methylation profiling. Accumulation of DNA methylation at hypermethylated DMPs correlated strongly with ATL development and progression. In addition, we identified 22 genes down-regulated due to promoter hypermethylation in HTLV-1-infected T-cells, including THEMIS, LAIR1, and RNF130, which negatively regulate T-cell receptor (TCR) signaling. Phosphorylation of ZAP-70, a transducer of TCR signaling, was dysregulated in HTLV-1-infected cell lines but was normalized by re-expression of THEMIS. Therefore, we hypothesized that DNA hypermethylation contributes to growth advantages in HTLV-1-infected cells during ATL leukemogenesis. To test this idea, we investigated the anti-ATL activities of OR-1200 and OR-2100 (OR21), novel decitabine (DAC) prodrugs with enhanced oral bioavailability. Both DAC and OR21 inhibited cell growth, accompanied by global DNA hypomethylation, in xenograft tumors established by implantation of HTLV-1-infected cells. OR21 was less hematotoxic than DAC, whereas tumor growth inhibition was almost identical between the two compounds, making it suitable for long-term treatment of ATL patient-derived xenograft mice. Our results demonstrate that regional DNA hypermethylation is functionally important for ATL leukemogenesis and an effective therapeutic target.

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Discontinuation of tyrosine kinase inhibitors (TKIs) is now a feasible therapeutic goal for patients with chronic phase chronic myeloid leukemia (CML-CP). Whereas approximately half of patients experience molecular relapse, after resuming with any TKI; the majority re-achieve a deep molecular response (DMR). It is unclear whether such patients who re-achieve a durable DMR can discontinue TKI safely again. Here, we retrospectively assessed first, second, and third attempts to stop TKIs in patients with CML-CP. At the first attempt, 28 out of a total of 53 patients achieved sustained treatment-free remission (TFR; 53.4%; 95% confidence interval [CI], 39.0%-65.9%). Subsequently, 10 of 25 patients attempted a second TKI discontinuation, and in all cases, this was after receiving second-generation TKIs.Four of 10 patients successfully achieved TFR (37.5%; 95% CI, 9.9%-65.9%). All patients who relapsed at the second TKI discontinuation attempt were readministered TKIs, and soon achieved at least a major molecular remission. All six second relapse patients had a loss of MR 4.5 at 3 months after TKI discontinuation. These findings suggest that second and third attempts to successfully stop TKI treatment are feasible in patients with CML-CP.

Adult T‐cell leukemia‐lymphoma (ATL) is an aggressive neoplasm derived from T‐cells transformed by human T‐cell lymphotropic virus‐1 (HTLV‐1). Recently, we reported that regional DNA hypermethylation in HTLV‐1‐infected T‐cells reflects the disease status of ATL and the anti‐ATL effects of DNA demethylating agents, including azacitidine (AZA), decitabine (DAC) and a new DAC prodrug, OR‐2100 (OR21), which we developed. Here, to better understand the mechanisms underlying drug resistance, we generated AZA‐, DAC‐ and OR21‐resistant (AZA‐R, DAC‐R and OR21‐R, respectively) cells from the ATL cell line TL‐Om1 and the HTLV‐1‐infected cell line MT‐2 via long‐term drug exposure. The efficacy of OR21 was almost the same as that of DAC, indicating that the pharmacodynamics of OR21 were due to release of DAC from OR21. Resistant cells did not show cellular responses observed in parental cells induced by treatment with drugs, including growth suppression, depletion of DNA methyltransferase DNMT1 and DNA hypomethylation. We also found that reduced expression of deoxycytidine kinase (DCK) correlated with lower susceptibility to DAC/OR21 and that reduced expression of uridine cytidine kinase2 (UCK2) correlated with reduced susceptibility to AZA. DCK and UCK2 catalyze phosphorylation of DAC and AZA, respectively; reconstitution of expression reversed the resistant phenotypes. A large homozygous deletion in DCK and a homozygous splice donor site mutation in UCK2 were identified in DAC‐R TL‐Om1 and AZA‐R TL‐Om1, respectively. Both genomic mutations might lead to loss of protein expression. Thus, inactivation of UCK2 and DCK might be a putative cause of phenotypes that are resistant to AZA and DAC/OR21, respectively.