Differential Expression of ERG Isoforms in Acute Myeloid and T-Lymphoblastic Leukemia Is Regulated by DNA-Methylation.

The oncogenic ETS transcription factor ERG is involved in various cellular pathways including developmental regulation, proliferation, and differentiation. In hematopoiesis ERG plays a specific role during normal T-cell differentiation showing high expression levels in stem cells and down regulation in the progenitor compartment. In this regard, it is intriguing that aberrant expression of ERG was found in a subset of patients with acute T-lymphoblastic leukemia (T-ALL) and was associated with an inferior outcome. Furthermore, high level ERG expression was of adverse prognostic significance in patients with newly diagnosed acute myeloid leukemia (AML), thus highlighting ERG’s potential role in myeloid as well as T-lineage leukemogenesis. ERG3 (NM_182918) and ERG2 (NM_004449) represent the main isoforms and show abundant expression in myeloid and lymphoid hematopoietic progenitor cells. The expression pattern of specific ERG isoforms in acute leukemias has yet to be investigated. To further elucidate the nature of aberrant ERG expression we have determined the existence and transcriptional regulation of ERG isoforms in pretreatment bone marrow samples of adult T-ALL (n=21) and AML (n=20) patients as well as in normal CD34+ hematopoietic cells of healthy volunteers (n=5). 5′RACE revealed the presence of a new ERG isoform (ERG3Δex12) characterized by expression of exon 5 and absence of exon 12. Expression of ERG3Δex12 was verified by RT-PCR in AML, T-ALL, and CD34+ cells. In addition, real-time RT-PCR showed concomitant expression of the two main isoforms ERG2 and ERG3 in AML and normal CD34+ cells. In contrast, T-ALL patients lacked expression of ERG isoforms harboring exon 4 (ERG2). Promoter analyses of ERG2 and ERG3 revealed the presence of two CpG islands in the ERG2 promoter region, whereas no CpG island was predicted in the ERG3 promoter. Bisulfit conversion of genomic DNA and sequencing of cloned PCR products revealed a significantly higher degree of methylation of CpG island 2 in T-ALL samples (median: 86.4%, range: 16.0 – 98.8%) as compared to AML (median: 38.1%, range: 10.9 – 60.7%; P-value=0.0002 - two sided T-test). As for CpG island 1, CD34+ cells had the lowest rate of methylation in CpG island 2 (median: 7.7%, range: 2.4 – 20.7%). Thus, the differential expression of ERG isoforms is mediated by epigenetic silencing of exon 4 containing transcripts in T-ALL. In conclusion, the identification of the new ERG isoform (ERG3Δex12) suggests the association with different partners as the central exons, including exon 12, guide the interaction with different proteins. Furthermore, the distinct expression of specific ERG transcripts controlled by methylation adds to the complexity of ERG directed downstream pathways in different leukemic subtypes.