The urinary excretion of epigenetically modified DNA as a marker of pediatric ALL status and chemotherapy response

Rafał Różalski ,Daniel Gackowski,Agnieszka Siomek-Górecka ,Kinga Linowiecka,Ewelina Zarakowska,Jan Styczyński ,Anna Labejszo,Elwira Olinska,Aleksandra Wasilow,Maciej Gawroński ,Martyna Modrzejewska,Andrzej Kołtan ,Aleksandra Skalska-Bugała ,Tomasz Dziaman,Jolanta Guz,Justyna Szpotan,Marek Foksiński ,Anna Szpila,Marta Starczak,Lidia Gackowska,Ryszard Oliński

doi:10.1038/s41598-021-00880-9

Rafał Różalski , Daniel Gackowski + Show 19 more

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https://doi.org/10.1038/s41598-021-00880-9

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Abstract

The active DNA demethylation process may be linked to aberrant methylation and may be involved in leukemogenesis. We investigated the role of epigenetic DNA modifications in childhood acute lymphoblastic leukemia (ALL) diagnostics and therapy monitoring. We analyzed the levels of 5-methyl-2′-deoxycytidine (5-mdC) oxidation products in the cellular DNA and urine of children with ALL (at diagnosis and during chemotherapy, n = 55) using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry (2D UPLC–MS/MS). Moreover, the expression of Ten Eleven Translocation enzymes (TETs) at the mRNA and protein levels was determined. Additionally, the ascorbate level in the blood plasma was analyzed. Before treatment, the ALL patients had profoundly higher levels of the analyzed modified DNA in their urine than the controls. After chemotherapy, we observed a statistically significant decrease in active demethylation products in urine, with a final level similar to the level characteristic of healthy children. The level of 5-hmdC in the DNA of the leukocytes in blood of the patient group was significantly lower than that of the control group. Our data suggest that urinary excretion of epigenetic DNA modification may be a marker of pediatric ALL status and a reliable marker of chemotherapy response.

Highlights

The active DNA demethylation process may be linked to aberrant methylation and may be involved in leukemogenesis
We found a significant decrease in a key product of active demethylation, 5-(hydroxymethyl)-2′-deoxycytidine (5-hmdC), in the leukocyte DNA of acute lymphoblastic leukemia (ALL) patients compared with that of healthy subjects
One of many possible reasons for the loss of 5-hmCyt is the decreased expression of Ten Eleven Translocation enzymes (TETs) mRNA18,21. Consistent with this supposition, we found decreases in TET3 and TET2 but increases in TET1 mRNA expression in the leukocytes of ALL patients

Summary

Introduction

The active DNA demethylation process may be linked to aberrant methylation and may be involved in leukemogenesis. We analyzed the levels of 5-methyl-2′-deoxycytidine (5-mdC) oxidation products in the cellular DNA and urine of children with ALL (at diagnosis and during chemotherapy, n = 55) using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry (2D UPLC–MS/MS). The ALL patients had profoundly higher levels of the analyzed modified DNA in their urine than the controls. Abbreviations ALL Acute lymphoblastic leukemia TET Ten-eleven translocation 5-mdC 5-Methyl-2′-deoxycytidine 5-mCyt 5-Methylcytosine 5-hmCyt 5-Hydroxymethylcytosine 5-fCyt 5-Formylcytosine 5-caCyt 5-Carboxycytosine 5-hmUra 5-Hydroxymethyluracil 2D-UPLC-MS/MS Two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry 8-oxodG 8-Oxo-7,8-dihydro-2′-deoxyguanosine 5-hmdC 5-(Hydroxymethyl)-2′-deoxycytidine. Most patients diagnosed with ALL have genetic aberrations that contribute to a higher rate of proliferation and impaired differentiation of lymphoid hematopoietic p rogenitors[2]. One reason for the observed genetic alterations may be aberrant DNA methylation that leads to changes in the expression of hematopoietic genes. In several studies, it has been observed that the cytosine methylation pattern of leukemic cells differs from that of nonleukemic c ells[3,4,5]

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