TLX1/HOX11-Mediated Disruption of Hematopoietic Differentiation Programs.

The diverged TLX1/HOX11 homeobox gene is frequently transcriptionally activated in T-cell acute lymphoblastic leukemia as a result of two recurrent chromosomal translocations or by as yet unknown mechanisms. Sporadic expression of TLX1 has also been detected in some other human tumors, raising the possibility that TLX1 played a role in the etiology of these malignancies. In this context, it is noteworthy that TLX1 is aberrantly expressed in the human erythroleukemia cell line K562. We previously reported that enforced TLX1 expression immortalizes myeloid progenitors in murine bone marrow and erythroid progenitors originating from murine embryonic stem cell-derived embryoid bodies. Based on these findings, we speculated that dysregulated TLX1 expression contributes to neoplastic transformation by interfering with hematopoietic differentiation programs. Here we carried out genome-wide expression profiling on these model systems to elucidate the mechanism of TLX1-mediated differentiation arrest. Surprisingly, these investigations uncovered a latent erythroid phenotype of the TLX1⁺ bone marrow progenitor cell lines. Transcriptome comparison with murine GATA-1-null G1E-ER4 erythroblast cells (GEO Accession No. GDS568) showed nonrandom overlap (P = 2.3 × 10⁻⁵) with a set of genes that immediately respond to GATA-1 activation. Included among the genes for which a positive correlation was observed were the erythropoietin (Epo) receptor and several functionally-associated downstream signaling components such as Lyn and Dok-1, as well as the SCL and FOG-1 transcription factor genes. We confirmed the biological relevance of these findings by demonstrating that TLX1-immortalized bone marrow progenitors proliferated in response to erythropoietin, synthesizing beta-globin mRNA. We next extended this approach to iEBHX1S-4 cells, an embryoid body-derived cell line generated by conditional (doxycycline-controlled) TLX1 expression (manuscript in preparation). iEBHX1S-4 cells require IL-3 plus stem cell factor for survival and proliferation. However, when doxycycline is removed from the culture medium, the cells undergo Epo-dependent erythroid differentiation characterized by up-regulation of the TER119 surface antigen and hemoglobin synthesis. Interestingly, whereas there was statistically significant overlap of gene expression profiles between constitutive and conditional TLX1⁺ cells (P = 2.5 × 10⁻⁵), there were no significant similarities between the iEBHX1S-4 transcriptome and the G1E-ER4 dataset until 6–24 hours after release of the TLX1 differentiation block. Bioinformatics analysis of the upstream regulatory regions of the genes identified implicated transcriptional networks involving GATA-1 as well as the p53, NF-kappaB and Egr-1 transcription factors. All of these transcription factors are substrates of the acetyltransferase CREB-binding protein (CBP), suggesting that inappropriate TLX1 expression might interfere with CBP activity. In agreement with this notion, acetylation of GATA-1, a key substrate of CBP-modulated erythroid differentiation, was increased upon down-regulation of TLX1 expression in iEBHX1S-4 cells. Experiments to directly confirm TLX1 inhibition of CBP as a central facet of TLX1 transforming function are ongoing, the results of which will be presented.