PS1451 THE EFFECT OF HYDROXYUREA ON DNA METHYLATION AND GENE EXPRESSION IN POLYCYTHEMIA VERA AND ESSENTIAL THROMBOCYTHEMIA
Authors: Contreras S. , Rocha A. , Duke W. , Schulz R. , Mullally A. , Harrison C. , Oakey R. 
Published: June 2019

Background:

Polycythemia vera (PV) and essential thrombocythemia (ET) are bone marrow disorder that gives rise to a high production of red blood cells and platelets leading to thrombosis and haemorrhage. Hydroxyurea is the first‐line treatment of this disease and the mechanism of action is via inhibition of the cell cycle. However, HU is also a treatment for sickle cell disease, where its therapeutic effect is manifested through the induction of foetal haemoglobin. Therefore, HU can have multiple effects on cells and we hypothesise its effects maybe mediated through epigenetic mechanisms.

Aims:

We aim to understand the mechanism of action of HU by measuring DNA methylation and gene expression in a comparative study in both human and mouse.

Methods:

To understand the effect of HU, we collected samples from PV and ET patients before HU treatment and at three, six and nine months of treatment. We measured gene expression and DNA methylation in neutrophils and CD34+ stem cells. We compared these data in a parallel comparative study using a Jak2V617F knock‐in mouse model. Accordingly, we treated this mouse model of PV with HU or vehicle for six weeks. We collected and isolated neutrophils and Lin−Sca+cKit+ (LSK) stem cells. We measured the gene expression in these cell types and a comparative analysis between the two species was performed.

Results:

We collected blood samples from PV and ET patients (n = 8) before and after HU treatment. DNA methylation was measured using the Infinium®MethylationEPICBeadChip (Illumina) at all time points in neutrophils. Subtle effects of DNA methylation were found at three and six months compared to baseline. Most changes occurred after nine months of treatment with mainly a gain of methylation at CpGs in non‐coding regions.

Comparative analysis of RNA‐seq between mouse and human was performed and overlapping genes were analysed to assess the effect of HU. At stem cell level, we found that downregulated genes were enriched for targets of transcription factors (TFs) UBTF, GATA2 and RUNX1. Upregulated genes were enriched for targets of TFs GATA1, SPI1 and MYB, and for ontologies involving neutrophil degranulation and mediated immunity. Genes CEBPA, RUNX3 and CDKN1B, that negatively regulates cell cycle progression, were upregulated in both species. However, genes CDK6, CCNB1 and CCNE1, that also control cell cycle progression, were downregulated in CD34+ cells but not in LSK cells. In neutrophils, genes TLE3, PTPN6 and ADIPOR1 were significantly downregulated in both species.

Summary/Conclusion:

Significant DNA methylation changes were identified mostly at nine months of treatment at isolated CpGs in the genome. These CpGs will be investigated to determinate its role to regulate transcription of neighbouring genes.

TFs involved in hematopoietic proliferation and development were identified as target of differentially expressed genes in CD34+ and LSK cells. Interestingly, upregulation of genes that negatively regulates cell cycle were differentially expressed but key regulators of this process were downregulated only in human CD34+ cells. In neutrophils, only three genes were significantly differentially expressed in both species. PTPN6 is involved in hematopoietic cell signalling, but the role of TLE3 and ADIPOR1 on neutrophils needs to be determinate.

Further analysis will focus on candidate genes that are directly involved in HU treatment in order to understand its mechanism of action.