Tracking Decitabine Incorporation into Malignant Myeloid Cell DNA in vitro and in vivo by LC-MS/MS with Enzymatic Digestion

Sujatha Chilakala,Reda Mahfouz,Ye Feng,Ebrahem Quteba,Yogen Saunthararajah,Yan Xu,Lan Li

doi:10.1038/s41598-019-41070-y

Sujatha Chilakala, Reda Mahfouz + Show 5 more

Open Access

https://doi.org/10.1038/s41598-019-41070-y

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Journal: Scientific Reports	Publication Date: Mar 14, 2019
Citations: 14	License type: open-access

Affiliation: Cleveland Clinic, Cleveland State University

Abstract

The DNA hypomethylating agents decitabine and 5-azacytidine are the only two drugs approved for treatment of all subtypes of the myeloid malignancy myelodysplastic syndromes (MDS). The key to drug activity is incorporation into target cell DNA, however, a practical method to measure this incorporation is un-available. Here, we report a sensitive and specific LC-MS/MS method to simultaneously measure decitabine incorporation and DNA hypomethylation. A stable heavy isotope of 2′-deoxycytidine was used as an internal standard and one-step multi-enzyme digestion was used to release the DNA bound drug. Enzyme-released decitabine along with other mononucleosides were separated by a reverse-phase C18 column and quantified by mass spectrometry using multiple-reaction-monitoring (MRM) mode, with a lower limit of quantitation at 1.00 nM. In vitro studies demonstrated dosage and time-dependent incorporation of decitabine into myeloid leukemia cell DNA that correlated with extent of DNA hypomethylation. When applied to clinical samples serially collected from MDS patients treated with decitabine, the method again demonstrated correlation between decitabine DNA-incorporation and DNA hypomethylation. This novel assay to measure the intended molecular pharmacodynamic effect of decitabine therapy can therefore potentially provide insights into mechanisms underlying sensitivity versus resistance to therapy.

Highlights

Decitabine (5-aza-2′-deoxycytidine) is a nucleoside analog of 2′-deoxycytidine
Among the four enzymes employed, deoxyribonuclease I type II (DNase I) is an endonuclease that splits phosphodiester bonds of DNA and yields oligonucleotides with a free 3′-end hydroxyl group and a free 5′-end phosphate group; phosphodiesterase I (PDE I) is a 3′ to 5′ exonuclease that successively hydrolyzes an oligonucleotide from 5′-end to 3′-end and produce deoxynucleoside 5′-phosphate; nuclease P1 (NP1) is a 5′ to 3′ exonuclease that acts in opposite direction to PDE I, and completely hydrolyzes an oligonucleotide from 3′-end to 5′-end to produce deoxynucleoside 5′-phosphate; and alkaline phosphatase (ALP) is a hydrolase that hydrolyzes phosphate groups of deoxynucleotides to deoxynucleosides
Enzyme digestion efficiency was evaluated by comparing the DNA concentration calculated from the amount of dG produced from DNA hydrolysis with the DNA concentration measured using UV spectroscopy

Summary

Introduction

Decitabine (5-aza-2′-deoxycytidine) is a nucleoside analog of 2′-deoxycytidine. Decitabine was introduced clinically four decades ago and was approved for the treatment of patients with myelodysplastic syndrome (MDS) in 2006 in the USA1–4. The results demonstrated that this method can quantify the incorporation of decitabine into DNA, enabling quantification of the variation in the incorporation and response to treatment in different models and in vivo. This assay could be a useful tool for the purpose of understanding treatment sensitivity versus resistance and provide important guidance towards an overall goal of individualizing and optimizing therapy with this exclusive class of agent

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