Quantification of Intracellular Decitabine-Triphosphate with A Novel, Highly Sensitive and Specific LC-MS/MS Assay in Acute Myeloid Leukemia Patients Treated with Low Dose Decitabine.
Authors: Chen Ping , Blum William , Aimiuwu Josephine , Upadhyayula Vijayasarathi , Liu Zhongfa , Liu Shujun , Pang Jiuxia , Walker Alison , Klisovic Rebecca B. , Garzon Ramiro , Grever Michael R. , Villalona-Calero Miguel , Byrd John C. , Chan Kenneth K , Marcucci Guido 
Published: October 2019

Abstract
Abstract 3782
Poster Board III-718
Low doses (5-20 mg/m2/day) of the azanucleoside decitabine (DAC) have been shown to be active in AML likely through DNA hypomethylation and re-expression of otherwise epigenetically silenced, hypermethylated genes, including tumor suppressors. Following cellular uptake, DAC rapidly undergoes phosphorylation to its active metabolite, DAC-triphosphate (DAC-TP), which is incorporated into the newly synthesized DNA, covalent binds to and sequesters DNA methyltransferases that mediate aberrant enzymatic-DNA hypermethylation. Measurement of DAC-TP remains critically important in understanding and monitoring the effect of drug treatment in order to design more effective treatment schedules. In contrast to the well-established methods for measurement of triphosphates of other nucleoside analogs (e.g. cytarabine), development of a reliable method of intracellular DAC-TP quantification has proven to be rather challenging and as yet no such method for DAC-TP has been reported. Here, we developed a non-radioactive, sensitive, and specific HPLC-MS/MS method to quantify intracellular DAC-TP that is readily applicable to intracellular quantification of DAC-TP in marrow mononuclear cells (MNCs) and peripheral blood mononuclear cells (PBMCs) from DAC-treated patients. This method uses an internal standard (IS) of 2-chloroadenosine-5'-triphosphate (ClATP), against which DAC-TP is measured; we tested the method first in vitro in DAC treated myeloid leukemia K562 cells. K562 cells were treated with 2 μM DAC for 1, 4, and 24 hours and DAC-TP, deoxynucleotides (dNTPs), and the spiked ClATP were extracted with methanol precipitation and separated through a gradient elution on a Supelcogel ODP-50 column. An ion trap mass spectrometer in a negative ion electro-spray mode was then used for detection. The multiple reaction monitor at m/z 467.1→369.0 allowed specific detection and separation of DAC-TP from other dNTPs and quantification against a known, spiked amount of ClATP. Linearity of the assay was demonstrated between 10 nM and 10 μM in K562 cell lysate with 50 nM being the lower limit of quantification (LLOQ). The within-day coefficients of variation (CVs) were found to be between 14.2-19.9% at LLOQ and 4.7-7.0% for higher concentrations. The within-day accuracy values were 83.7-115.6% for DAC-TP. Following 0.2 μM DAC treatment in K562 cells for 4 hr, the accumulation of DAC-TP peaked (∼2 pmol/106 cells) at 4 hr and was detectable at 24 hr. Next, the assay was then successfully applied to patient samples. We quantified DAC-TP in marrow MNCs collected within 3 hours from patients treated DAC at 20mg/m2 intravenously over 1 hour; we were able to detect for the first time in in vivo treated cells DAC-TP levels (0.08∼0.7 pmol/106 cells). Additionally, we were able to measure DAC-TP levels in PBMC in selected patients (0.13-1.92 pmol/106 cells). Correlation of intracellular DAC-TP with DNA hypomethylation and gene re-expression is ongoing. In conclusion, we report the first highly sensitive and specific HPLC-MS/MS analytic method for in vivo quantification of DAC-TP. This method can readily be applied for correlative studies of intracellular DAC-TP with DAC plasma pharmacokinetics, DAC-induced DNA hypomethylation/gene re-expression, and clinical outcome, and utilized for future trials based on biochemical modulation of DAC administered alone or in combination with other drugs. Supported by R01CA102031 U01-CA76576, OSU BioMedical Mass Spectrometry Laboratory and N01-CM-62207.

Disclosures:
Blum: Celgene: Research Funding.