The Deletion of NHR1 Region of the AML1-ETO Protein Significantly Decreases Its Ability To Promote Proliferation and Self-Renewal of Early Hematopoietic Cells in Culture.

Although the transcriptional product of the (8;21) chromosomal translocation, the AML1-ETO protein, has numerous well established effects on the behavior of human hematopoietic cells, the specific cellular pathways perturbed by it are only partly known. Recently AML1-ETO has been shown to bind E-proteins (members of the class I basic helix-loop-helix family) via its NHR1 (nervy homology region one) domain, thus causing a replacement of the co-activator p300/CBP complex by the co-repressor hystone deacytelase complexes and abrogating the E-protein induced transcriptional activation (Zhang et al, Science 2004). The removal of amino acids 93 to 189 from the NHR1 domain (the ΔNHR1 mutant form) has completely reversed this inhibitory effect, and thus we have studied the biological effects of this deletion.

To assess the difference in self-renewal capacity between cells transduced with the full length and the ΔNHR1 forms of AML1-ETO, CD34+ cells were plated into methylcellulose culture medium. On days 14 and 28 myeloid and erythroid colonies were scored, and the cells harvested and re-plated. The scoring on day 14 showed no significant difference in either the overall number or the type of colonies, with both the full-length AML1-ETO and the ΔNHR1 mutant showing a decreased erythroid colony formation, thus indicating a preserved negative effect on hematopoietic cell differentiation. However, on days 28 and 42 cells expressing the full-length AML1-ETO scored a significantly higher number of colonies than cells expressing the ΔNHR1 mutant, whereas cells transduced with the empty MIGR1 vector were unable to form any colonies after second re-plating.

To assess the difference in growth potential, we have plated cells containing each of the constructs in IMD medium supplemented with 20% BIT and cytokines (SCF, FLT-3L, IL-6, TPO). Following one week in culture the cells were harvested, counted and then serially re-plated for five weeks. We observed a significantly higher number of cells expressing the full-length AML1-ETO as compared to the ΔNHR1 mutant at each weekly time point. Cells growing in the liquid culture were also weekly re-plated into methylcellulose culture medium. In this assay the loss of NHR1 domain significantly diminished the ability of AML1-ETO to maintain colony-forming progenitors in culture. To assess the difference in transcriptional activity of the different constructs, we have measured the expression of p21, and several other potential AML1-ETO target genes, by Real Time PCR. The expression of p21 was increased 14-fold by the full-length AML1-ETO, but only 2-fold by the ΔNHR1 mutant.

We conclude that partial deletion of the NHR1 lowers the self-renewal capacity of transduced hematopoietic cells, decreases the proliferative advantage conferred by AML1-ETO, undermines its ability to maintain colony-forming units, and affects the transcriptional activation of AML1-ETO target genes. As the loss of NHR1 abolishes the E-protein induced transcriptional activation without affecting the binding of AML1-ETO to DNA, our findings identify an additional mechanism of action for AML1-ETO, independent of its dominant-negative effect on AML1 target genes.