CBFA2T3 Enhances Transcriptional Activity of RUNX1: Implication in Leukemia

Pre-B acute lymphoblastic leukemia (BCP-ALL) is the most common type of pediatric cancer. Many expression-profiling studies have uncovered genes deregulated in BCP-ALL compared to normal pre-B cells. Yet, the roles of the deregulation of those genes in the onset and maintenance of BCP-ALL are still unclear. We were interested in two genes, CBFA2T3 (Core-Binding Factor, Runt Domain, Alpha Subunit 2; Translocated To, 3) and RUNX1 (Runt Related Transcription Factor 1), which are specifically overexpressed in BCP-ALL characterized by the presence of ETV6-RUNX1 fusion gene. CBFA2T3 is a hematopoietic corepressor. Alteration of its function is implicated in leukemia. RUNX1 is a transcription factor, whose activation or repression activity depends on cofactors. RUNX1 deregulation is also involved in many hematological disorders. Our goal was to explore the role of these two genes in BCP-ALL. We demonstrated the major role of CBFA2T3 in the control of RUNX1 activity.

Based on our patients' data and public database, we not only confirmed that CBFA2T3 and RUNX1 transcripts were overexpressed in ETV6-RUNX1 BCP-ALL, but also found that their expression levels were positively correlated. Expression of EVT6-RUNX1 in Nalm6 cells, a BCP-ALL cell line that normally does not express the ETV6-RUNX1 fusion protein, showed an overexpression of CBFA2T3 and RUNX1 proteins, confirming the role of the ETV6-RUNX1 fusion protein as a priming event in CBFA2T3 and RUNX1 expression.

To further determine the role of RUNX1 in the control of transcription in the context of BCP-ALL, we performed chromatin immunoprecipitation followed by sequencing (ChIP-Seq) with anti-RUNX1 in Nalm6 cells and in REH cells, a ETV6-RUNX1 BCP-ALL cell line. Notably, we observed several significant enrichments of RUNX1 binding at CBFA2T3 and RUNX1 promoters in both cell lines. Our work on the promoters of these two genes was carried out with luciferase reporter assay. The luciferase results demonstrated that RUNX1 controlled the transcription of CBFA2T3 and RUNX1 and showed that CBFA2T3 enhanced the transcriptional activity of RUNX1.

As CBFA2T3 is neither a transcription factor, nor a DNA binding protein, we suspected that CBFA2T3 could directly modulate RUNX1 activity. Chromatin immunoprecipitation of RUNX1 coupled to mass spectrometry identified a putative interaction between CBFA2T3 and RUNX1 in BCP-ALL lymphoblasts. Using Proximity Ligation Assay, we next confirmed the protein interaction between CBFA2T3 and RUNX1, as well as the interaction between CBFA2T3 and the fusion protein ETV6-RUNX1 in two BCP-ALL cell lines and, more importantly, in BCP-ALL patients' cells. Finally, we identified the CBFA2T3 protein domain responsible for the interaction with RUNX1, thanks to coimmunoprecipitation assay with truncated CBFA2T3. Using this CBFA2T3 domain, we next performed luciferase assay and were able to inhibit the effect of full length CBFA2T3 on the transcriptional activity of RUNX1.

To conclude, this work highlights the interaction between CBFA2T3 with RUNX1 and ETV6-RUNX1. We identified a regulatory loop between CBFA2T3 and RUNX1 that could explain their deregulation in patients. Our finding reveals a novel molecular mechanism of control of RUNX1 involving CBFA2T3 and RUNX1 itself. It may also open new perspectives to understand the mechanisms implicated in acute myeloid leukemia with AML1-MTG16 (RUNX1-CBFA2T3) fusion protein.

This work is supported by Ligue Régionale contre le cancer, Région Bretagne, Rennes Métropole, French Research Ministry, the société française de lutte contre les cancers et les leucémies de l'enfant et de l'adolescent, the Fédération Enfants et Santé, the Société Française de Biochimie et Biologie Moléculaire, the Société Française d'Hématologie, Mrs. M-Dominique Blanc-Bert and the Marie Curie Actions of the European Union FP7 under REA grant agreement n°291851.