MSACL 2019 EU Abstract

Tandem Mass Spectrometry-based Analysis Reveal Relationship between Active DNA Demethylation and Krebs Cycle in AML and MDS

Justyna Szpotan, Martyna Modrzejewska, Kinga Linowiecka, Agnieszka Siomek-Górecka, Daniel Gackowski, Ryszard Oliński, Marek Foksiński
Department of Clinical Biochemistry, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Poland

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Justyna Szpotan (Presenter) Collegium Medicum in Bydgoszcz >> POSTER (PDF)

Presenter Bio: I graduated with BSc and MSc degrees in biotechnology from the Technical University of Lodz, Poland in 2014 and 2015, respectively. I conducted research at University of Natural Resources and Life Sciences in Vienna, Austria, as an Erasmus transfer student. The main purpose of my study was characterization of genomic stability in long term cell lines.
Currently, I am a third-year PhD student in Clinical Biochemistry, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Poland. My research focus on metabolism and epigenetic interplay in cancer diseases especially acute leukemias. I use tandem mass spectrometry in identifying and quantifying 5-methylcythosine, derivatives of active demethylation pathway and metabolites. I hope that these research contribute to the implementation of novel diagnostic systems or biomarkers for acute leukemias.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION: The most dynamic process which regulates DNA methylation is recently discovered active demethylation. It involves ten-eleven translocation (TET) enzymes to catalyze stepwise oxidation of 5-methylcytosine (5-mCyt) to 5-hydroxymethylcytosine (5-hmCyt) and further demethylation products 5-formylcytosine (5-fCyt) and 5-carboxylcytosine (5-caCyt). Mutations targeting TET genes are frequently observed in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Mutations in genes: succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) are also found in acute leukemias. These mutations result in accumulation of the succinate (SA), fumarate (FA), 2-oxoglutarate (2-OG) and R-2-hydroxyglutarate (R-2HG). It may deregulate the activity of TET enzymes and, in turn, DNA demethylation. Although the oncogenic mechanism of these mutations remains still under investigation, determine of these metabolites could be relevant for diagnosis, prognosis and treatment of a subset of patients with AML and MDS.

OBJECTIVES: The main objective of this study is to find out the relationship between the level of 5-mCyt and the derivatives of active DNA demethylation process and the level of metabolites: SA, FA and 2-OG/2-HG in plasma/urine of patients developing AML and MDS.

METHODS: In this study we have examined 3 groups: healthy controls, patients with AML and MDS at diagnosis de novo. In all groups we have measured the level of epigenetic DNA modifications in leukocytes using isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) and plasma/ urine concentrations of SA, FA and 2-OG using UPLC- MS/MS method. The level of R-2HG in urine and plasma has been measured using 2D-UPLC-MS/MS after derivatization with DATAN (Di-O-acetyl-L-tartaric anhydride).

RESULTS: Our preliminary research has shown two 5-hmCyt subpopulations in AML patient cohorts with higher and lower level of 5-hmCyt compare to healthy controls. Besides, we have observed reverse correlation between global level of 5-hmCyt and 2-hydroxyglutarates. We have noticed a few extreme values of 2-HGs in the urine of patients with AML and MDS. The level of D-2-HG and D/L 2HG ratio are notably increased 10-100-fold in AML (22%) and MDS (10%) patients in urine.
CONCLUSION: These research have shown relationship between the level of the derivates of active DNA demethylation process and metabolites. Larger studies need to be performed to revealed how the concentrations of SA, FA, 2-OG and R-2HG influence key enzymes of active DNA demethylation pathway.