Adult T‐cell leukemia‐lymphoma acquires resistance to DNA demethylating agents through dysregulation of enzymes involved in pyrimidine metabolism

Abstract

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.

Reprogramming of pyrimidine nucleotide metabolism supports vigorous cell proliferation of normal and malignant T cells

“…AZA-resistant (AZA-R) cells were established previously by long-term exposure to the drug. 15 The parental cell lines, as well as the AZA-R subclones, were authenticated previously using short tandem repeat profiling. 15 Cells were cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum.…”

“… 15 The parental cell lines, as well as the AZA-R subclones, were authenticated previously using short tandem repeat profiling. 15 Cells were cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum. All experiments were performed using mycoplasma-free cells.…”

“…RNA extraction, synthesis of complementary DNA, and quantification of UCK1 and UCK2 gene expression were performed as described previously. 15 …”

“… 14 Furthermore, inactivation of uridine-cytidine kinase 2 (UCK2) and deoxycytidne kinase makes cells resistant to azacitidine (AZA) and decitabine, respectively. 15 UCK proteins (UCK1 and UCK2 in humans) phosphorylate uridine and cytidine to yield monophosphorylated forms during pyrimidine nucleotide salvage synthesis. 16 Although UCK1 is expressed ubiquitously in several normal human tissues, such as the liver, kidney, muscle, lung, and heart, the expression of UCK2 is limited to the human placenta and testis.…”

Erratum

“…In [1], the following details were missing at the end of “Material and Methods” on page 1187 in the published version:…”