Epor Over-Expression In TEL-AML1 Positive ALL: Critical Roles for DNA Methylation, GATA-2 or MicroRNAs?

Abstract 3619

Erythropoietin (Epo) and its receptor (EpoR) play vital roles in regulating red blood cell production. However, aberrant over-expression of EpoR has been reported in a wide range of tumours and hematological malignancies, particularly acute lymphoblastic leukemia (ALL) with the chromosomal translocation TEL-AML1 (ETV6/RUNX1). Around 25% of B-cell ALLs contain this chromosomal translocation which is associated with a favourable, prognosis but the role of EpoR is unknown.

We have investigated whether methylation, transcription factors or miRNA regulation are implicated in the mechanism of the specific EpoR up-regulation associated with the TEL-AML1 rearrangement using two human B precursor cell line models. The pre-B REH cell line is TEL-AML1 positive whereas the pre-B NALM-6 cell line is TEL-AML1 negative; in addition the erythroleukemia cell line UT-7, which constitutively over- expresses EpoR was used as a positive control. Sequencing confirmed that the EpoR in these cell lines was wild type. Quantitative RT-PCR (RQ-PCR) of EpoR mRNA levels showed ~25 fold higher expression in REH cells compared to NALM-6 cells, verified by western blot analysis. RQ-PCR analysis of 20 pediatric ALL patient samples confirmed a 14-fold up-regulation (p<0.05) of EpoR in TEL-AML1 positive patients compared to hyper-diploid ALL patients without the translocation. Furthermore, the EpoR is functional in REH cells as serum starvation followed by stimulation with 10 U/ml Epo clearly activated the JAK2/STAT5, PI3K/Akt and Ras/ERK downstream signaling pathways within 15 min. In contrast, NALM-6 cells showed no activation of the signaling pathways in response to Epo.

DNA methylation is an epigenetic marker which can directly impact on gene transcription, with aberrant methylation reported in many cancers. The methylation status of 18 CpG sites over 342 bp within the EpoR promoter encompassing the GATA and Sp1 binding sites was analysed by pyrosequencing in REH and NALM-6 cells. Fifteen of the 18 CpG sites showed significantly higher (p<0.001) levels of methylation in the NALM-6 cells (13-71%) compared to REH (2.4-10%) and UT-7 (1.8-8.5%) cells. The 15 significantly different sites had an average of 4.1% methylation in REH and UT-7 cells and 36.6% methylation in NALM-6 cells, showing a difference of 32.5% between the REH and NALM-6 cells. Comparable levels of methylation were seen at three CpG sites (7, 8 and 18) located near the Sp1 binding site. Treatment of NALM-6 cells with the IC₅₀ dose (0.5 μmol) of Decitabine resulted in a regional decrease in methylation but no overall increase in EpoR expression whereas treatment of REH cells with Decitabine had no effect on methylation or expression. This suggests that methylation status of the EpoR promoter is altered by the presence of the TEL- AML1 fusion protein but may not be the critical factor in the up-regulation of EpoR expression.

The GATA family of zinc-finger proteins have well characterised roles in hematopoiesis and EpoR regulation. Measurement of the expression of all six GATA transcription factors, by RQ-PCR showed that only GATA-2 was significantly up-regulated in REH cells (p<0.001) compared to the TEL-AML1 negative NALM-6 cell line with a Ct difference of 8.6 (~388 fold change). The up-regulation of GATA-2 was recapitulated in 10 TEL-AML1 positive patients and 10 hyper-diploid ALL patients who showed an average 28-fold difference in expression (p<0.0001). An increase in GATA-2 protein levels was also evident in REH cells using western blot analysis.

Furthermore, initial studies have indicated differential expression, between the REH and NALM-6 cell lines, in miRNA whose targets have been predicted (www.diana.pcbi.upenn.edu) to be EpoR and/or GATA-2. Further investigation of the modulation of EpoR expression by methylation, GATA-2 or miRNAs, will lead to a greater understanding of of the factors regulating the expression of EpoR and the potential for alternative forms of therapeutic intervention in patients with TEL-AML1 ALL.