TLX3 ex vivo model identifies TLE4 as a co-repressor in T-ALL
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological disease originating from the malignant transformation of T-cell progenitors caused by the accumulation of genetic aberrations. One fifth of T-ALL patients are characterized by the ectopic expression of the homeobox transcription factor TLX3, which is normally not expressed in hematopoietic cells. Strikingly, this TLX3 positive subgroup of T-ALL has a high frequency of FLT3 mutations. Co-expression of TLX3 and FLT3-ITD in ex vivo pro-T cells conferred IL7 independent growth, confirming that TLX3 expression and FLT3 signaling cooperate to transform T-cells and make them less dependent on extracellular signals. After inducing TLX3 expression for 24 hours in Pro-T cells, transducin-like enhancer of split 4 (Tle4) was detected to be downregulated. Interestingly, T-ALL patients also present a strongly decreased expression of TLE4 specific to TLX3 and TLX1 subtypes. Family members of the transcriptional corepressor TLE4 have been described to interact and thus, inhibit transcription factors, through their interaction with an Engrailed-homology 1 (Eh1) domain. Generation of a point mutation in the Eh1 domain present at the N-terminus of TLX3 allowed us to show that TLE4 has a repressive effect on TLX3 activity. Together, we propose a TLX3+FLT3-ITD Pro-T cell model and use this to illustrate that low expression levels of TLE4 are favourable for the oncogenic function of TLX3. Overall design: In a ProT-CreER cell model, we introduced FTL3-ITD and TLX3 (with a V5 tag), performed 3'end RNA-sequencing and compared this with the WT ProT CreER cells. In a ProT-CrER cell model, we induced TLX3 with tamoxifen over 24h and compared them with the non-treated (DMSO) ProT cells. We performed 3'end RNA-sequencing In a ProT-CreER cell model TLX3 and TLX1 were induced with tamoxifen over 24/48h and compared with the non-induced condition (DMSO). 3'end RNA-sequencing was performed. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) with TLX3 antibody in DND-41 cells RNA sequencing of ProT-cells with overexpression of TLX3, introduction of FLT3-ITD and overexpression of TLE4, and combinations of these.