TLEs Regulate Myeloid Proliferation and Differentiation In Association with Modulation of NF-κB and Wnt Signaling

Abstract 4176

We recently identified two members of the Groucho family of co-repressors, TLE (transducin-like enhancer of split) 1 and TLE4, as candidate tumor suppressor genes from the commonly deleted del(9q) region in acute myeloid leukemia (AML). This deletion is frequently associated with presence of the t(8;21) fusion gene AML1-ETO (RUNX1-RUNX1T1) and we have previously shown loss of these TLEs cooperates with AML1-ETO to increase survival, block differentiation, and induce myeloid cell proliferation. We have also shown that the proliferation and differentiation of several myeloid cell lines is markedly affected by modulation of TLE levels. Over-expression of TLEs induced THP-1 or HL-60 myeloid cell differentiation and blocks proliferation, while knockdown of TLEs triggers cell proliferation.

We previously showed that knockdown of TLE1 and TLE4 inhibited all-trans retinoic acid (ATRA) induced differentiation of HL-60 cells. In this study we show knockdown of the TLEs also inhibits 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced differentiation of THP-1 cells, further pointing to the importance of these proteins in myeloid differentiation. Myeloid differentiation induced by TPA in THP-1 cells was associated with a transient translocation of TLE1 and TLE4 into the nucleus. We show that lowest levels of TLE1 and TLE4 are seen in the hematopoietic stem cell containing compartment of cord blood cells. We found a peak in TLE1/4 expression in early myeloid cells with lower levels in mature granulocytes. When we simultaneously knocked down both TLE1 and TLE4 we saw an expansion of the cord blood primitive progenitor population as measured by an increase of CD34+CD38- cells, 2.9 fold increase of colony forming units and 3.5 fold increase of Long-Term Culture-Initiating Cells. This data indicates low levels of TLE expression may maintain or expand undifferentiated hematopoietic progenitor cells and that a transient increase in their expression with translocation into the nucleus may trigger myeloid differentiation.

We sought to further evaluate the mechanisms by which the TLEs regulate myeloid differentiation and proliferation. Using ImageStream analysis we found that TLE expression is able to inhibit activation of NFκB as evidenced by inhibition of nuclear NFκB translocation induced by lipopolysaccharide. Expression of the TLEs also reduces both of the nuclear translocation of β-catenin and total β-catenin in THP-1 cells. TLE1 inhibits LiCl-induced activation of Wnt signaling as measured by TOPFLASH. Knockdown of TLE1 and/or TLE4 in Kasumi1 cells induced expression of plakoglobin, a mediator of Wnt signaling. Blocking ATRA-induced myeloid differentiation by knocking down both TLE1 and TLE4 is associated with down-regulation of Nab2 and C/EBPe. In addition to the deletion of TLE1 and TLE4 in a subset of AMLs, epigenetic inactivation of TLE1 has also been reported in several myeloid malignancies, including AML. NF-κB and Wnt/β-catenin pathways in particular appear to be constitutively activated in AML and are believed to contribute to hematopoietic stem cell (HSC) expansion and increased survival. The loss of TLEs may contribute to the constitutive activation of these pathways in AML.