Quantification of the Active Decitabine-Triphosphate (DAC-TP) Metabolite: A Novel Pharmacoanalytical Endpoint for Optimization of Hypomethylating Therapy in Acute Myeloid Leukemia (AML)

Abstract 3578

Decitabine (DAC) is successfully used for treatment of patients (pts) with myelodysplastic syndromes and AML. Following cellular uptake, DAC is thought to be activated to DAC-TP and incorporated into DNA. The DAC-TP/DNA complex binds and inactivates DNA methyltransferases (DNMTs), thereby leading to hypomethylation and re-expression of epigenetically silenced tumor suppressor genes and ultimately anti-leukemia activity. However, direct evidence of in vivo DAC-TP occurrence in DAC-treated pts has been difficult to demonstrate due to a lack of suitable validated analytical methodology. Thus, we developed and validated a sensitive and specific LC-MS/MS method for quantification of DAC-TP. The assay exhibited excellent accuracy and precision. The accuracy values were 83.7–109.4%, as determined by calculating the percentage of measured DAC-TP relative to the respective nominal concentrations (50, 500 and 5,000 nM) of the quality control samples. The within-day coefficients of variation (CVs) were 19.9 % (n=6) at 50 nM and 4.7–7.0 % between 500–5,000 nM; the between-day CVs (n=3) were 15.2 % at 50 nM and 7.5–10.2 % between 500–5,000 nM.

Following DAC treatment, we detected DAC-TP in parental and DAC-resistant MV4–11, and in THP-1 and FDC-P1/Kit^mut cells (in vitro); and in bone marrow (BM) and spleen of normal and FDC-P1/Kit^mut-driven AML mice (in vivo). DAC-TP reached peak levels (0.8, 1.4 and 0.5 pmol/10⁶ cells) in 1–4 hours and declined to 20 % of its peak concentration after 24 hours incubation with 2.5 μM DAC in MV4–11, THP-1 and FDC-P1/Kit^mut cells, respectively. Inhibition of hENT1 that mediates DAC transport into the cells and dCK that phosphorylates DAC into DAC-TP by NBTI and 2-thio-2′-deoxycytidine, respectively, significantly inhibited DAC-TP accumulation in AML cells. DAC-TP decay was instead blocked by tetrahydrouridine (THU)-induced inhibition of CDA, the catabolizing enzyme for cytidine and deoxycytidine and analogs. Consistent with these results, low dCK and hENTs but not CDA expression were detected in DAC-resistant MV4–11 cells, which showed 60 % decrease in DAC-TP levels as compared to their parental counterparts. DAC/DAC-TP-mediated downregulation of DNMT proteins (preferentially DNMT1 and DNMT3a) was also demonstrated in the AML cells even at DAC-TP concentrations as low as 0.1–1.3 pmol/10⁶ cells in vitro after 4 hours DAC incubation. In the in vivo experiments, DAC-TP levels in leukemic mice were comparable to that in normal C57BL/6 mice, 0.3 pmol/10⁶ cells in BM and 199.2 pmol/g tissue in spleen at 4-hours and 0.2 pmol/10⁶ cells in BM and 165.3 pmol/g tissue in spleen at 24-hours following an i.v. bolus of 6.5 mg/kg DAC. In BM of leukemic mice, not only DNMT1 and DNMT3a but also DNMT3b protein expression reduced 80 % (DNMT3a) or diminished (DNMT1 and DNMT3b).

The clinical applicability of this method was proven by measuring DAC-TP level in BM and blood mononuclear cells (PBMC) from AML pts treated with a 10-day regimen of DAC given 20 mg/m²/day i.v. over 1 hour. In BM samples, the mean DAC-TP levels were 0.8 ± 0.6 (Day 1) and 0.9 ± 0.5 pmol/10⁶ cells (Day∼5) in complete responsive (CR) pts (n=4); and 0.4 ± 0.3 (Day 1) and 0.12 ± 0.02 pmol/10⁶ cells (Day∼5) in non-responsive (NR) pts (n=3). In PBMC samples, the mean DAC-TP levels were 0.5 ± 0.2 (Day 1) and 1.2 ± 0.4 pmol/10⁶ cells (Day∼5) in CR pts (n=3); and 0.02 ± 0.02 (Day 1) and 0.21 ± 0.04 pmol/10⁶ cells (Day∼5) in NR pts (n=3). These data suggested that higher levels are seemingly associated with clinical response, but a larger number of pts need to be tested. In conclusion, monitoring the intracellular concentration of DAC-TP is feasible, and DAC-TP levels correlate with DNMT downregulation and may serve as a novel pharmacological endpoint for designing more effective DAC-based regimens.