ETV6/RUNX1 Dysregulates Genes with RUNX1 Binding Site Via Mechanism Reversible by Histone Deacetylase Inhibitors.

RUNX1 is implicated in over 30 different translocations in human acute leukaemia. RUNX1 can either activate or repress transcription of key regulators of cell growth and differentiation through binding to promoters or enhancer elements. The ETV6/RUNX1 (TEL/AML1) fusion resulting from t(12;21) translocation is the most common chromosomal aberration in paediatric cancers (25% of ALL). The ETV6 part of the fusion protein contains domains interacting with the mSin3, N-CoR and HDAC-3 corepressors. A part of the RUNX1 gene involved in the fusion carries DNA-binding domain. RUNX1 regulates haematopoietic myeloid cell differentiation and transcriptional activation but the role in lymphoid development is not yet fully understood. We hypothesize that ETV6/RUNX1 causes pathological differentiation block in lymphoid cells via repression of target genes of RUNX1 by histone deacetylation. Therefore, we studied the role of histone deacetylase inhibitors (HDACi). We have previously confirmed specific effect of HDACi (valproate-VPA, Trichostatin A-TSA) on ETV6/RUNX1 leukaemic cells in comparison with lymphoblastic leukaemias with different mechanism of leukaemogenesis (BCR/ABL and PDGFRβ/ETV6). To prove the direct effect of HDACi on ETV6/RUNX1 in vitro, we utilized a target gene of RUNX1, granzyme B (GZMB). To determine whether ETV6/RUNX1 represses GZMB via direct interaction with RUNX1-binding site at GZMB promoter, luciferase activity was measured in HeLa cells transfected with pcDNA3.1-ETV6/RUNX1Myc and compared with HeLa with pcDNA3.1 empty vector. Cells were transfected with pGZMB-luc or pGL3-basic to normalize the luciferase activity (pGZMB-luc/pGL3-basic). Fold change of ~3 RLU indicated that GZMB was downregulated by ETV6/RUNX1. To test the direct effect of HDACi on ETV6/RUNX1, after incubation of HeLa cells with VPA and TSA, luciferase activity was monitored again. Repression activity was reduced in treated transfected HeLa cells to 53% after VPA administration and 49% after TSA administration when compared to untreated cells. We subsequently used the effect of HDACi on ETV6/RUNX1-positive leukaemic cells for identification of (ETV6/)RUNX1 target genes in the lymphoid progenitor cells. Expression profiling analysis confirmed changes in the expression of group of genes differentially expressed in ETV6/RUNX1-positive and -negative leukaemic cells. In selected group of genes with known role in the cell cycle regulation (JunD, ACK1, PDGFRB and TCF4) expression changes were confirmed by quantitative expression analysis (qRT-PCR). In established HeLa model direct interaction of ETV6/RUNX1 with promoter sites of these genes will be confirmed. This work shows for the first time direct transcription repression by ETV6/RUNX1 on GZMB gene model and further it shows reversibility of this effect by HDACi. These data support our hypothesis that histone deacetylase inhibitors affect ETV6/RUNX1-positive cells by direct disruption of its repression activity, and that treatment with HDACi may release pathological differentiation block caused by ETV6/RUNX1 chimeric transcription factor.