Abstract
Mechanisms-of-resistance to decitabine and 5-azacytidine, mainstay treatments for myeloid malignancies, require investigation and countermeasures. Both are nucleoside analog pro-drugs processed by pyrimidine metabolism into a deoxynucleotide analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1). Here, upon serial analyses of DNMT1 levels in patients’ bone marrows on-therapy, we found DNMT1 was not depleted at relapse. Showing why, bone marrows at relapse exhibited shifts in expression of key pyrimidine metabolism enzymes in directions adverse to pro-drug activation. Further investigation revealed the origin of these shifts. Pyrimidine metabolism is a network that senses and regulates deoxynucleotide amounts. Deoxynucleotide amounts were disturbed by single exposures to decitabine or 5-azacytidine, via off-target depletion of thymidylate synthase and ribonucleotide reductase respectively. Compensating pyrimidine metabolism shifts peaked 72–96 h later. Continuous pro-drug exposures stabilized these adaptive metabolic responses to thereby prevent DNMT1-depletion and permit exponential leukemia out-growth as soon as day 40. The consistency of the acute metabolic responses enabled exploitation: simple treatment modifications in xenotransplant models of chemorefractory leukemia extended noncytotoxic DNMT1-depletion and leukemia control by several months. In sum, resistance to decitabine and 5-azacytidine originates from adaptive responses of the pyrimidine metabolism network; these responses can be anticipated and thus exploited.
Highlights
These authors contributed : Xiaorong Gu, Rita Tohme
We extended protein level analyses to additional pyrimidine metabolism enzymes playing nodal roles in nucleotide balance: thymidylate synthase (TYMS) that is the major mediator of deoxythymidine triphosphate (dTTP) production [34,35,36], and subunits RRM1 and RRM2A of the ribonucleotide reductase complex that converts RNA molecules, such as 5azacytidine, into DNA molecules such as decitabine
Decitabine and 5-azacytidine are processed by pyrimidine metabolism into the DNA methyltranseferase 1 (DNMT1)-depleting nucleotide AzadCTP (“Supplementary Discussion”), and here we found that expression changes in pyrimidine metabolism enzymes was how malignant cells avoided DNMT1-depletion to resist decitabine or 5-azacytidine in vitro, in mice and in patients
Summary
The deoxycytidine analog pro-drug decitabine and the cytidine analog pro-drug 5-azacytidine can increase lifespans of patients with myeloid malignancies, shown by randomized trials in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) (reviewed in [1]) Both pro-drugs are processed by pyrimidine metabolism into a deoxycytidine triphosphate (dCTP) analog, Aza-dCTP, that depletes the key epigenetic regulator DNA methyltransferase 1 (DNMT1) from dividing cells [2]. AML cell lines resistant to >50 μM of 5-azacytidine contained inactivating mutations in UCK2 [19], and sensitivity was restored by transfection with an expression vector for UCK2 [19] Despite such in vitro data, contributions of altered DCK and/or UCK2 to clinical relapse have been minimally investigated: one study of 14 decitabine-treated patients measured DCK expression in peripheral blood or bone marrow at relapse versus diagnosis, with inconclusive results [20]; another study of eight decitabine-treated patients did find that DCK expression was significantly decreased at relapse [21]. The consistency and predictability of metabolic reconfiguration enabled anticipation, out-maneuvering and even exploitation: simple, practical treatment modifications preserved the favorable therapeutic index of noncytotoxic DNMT1depletion and markedly improved efficacy in preclinical in vivo models of aggressive chemo-refractory AML
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