Abstract
HOXB4 belongs to the family of homeobox transcription factors, which play a key role in hematopoietic development. The expression of HOXB4 induces a significant increase of long-term repopulating stem cells (SC) in human and mouse models, without inducing malignant transformation. So far the underlying mechanisms of the SC amplificatory impact of HOXB4 are poorly understood. In an attempt to understand the unique characteristics of HOXB4, we performed a mutational study by deleting its proline-rich region, which has been described to act as a transcriptional activation domain in many other proteins, like non-homeobox genes (e.g. p53, AP2) and other homeobox genes (e.g. HOXD4 and HOXA13). We performed in vitro and in vivo experiments transducing murine 5-FU enriched HSCs with the pMSCV-IRES-GFP based retroviral vector harbouring the HOXB4 wild-type (wt) and several mutants, including a Δproline HOXB4 mutant (ΔP), where the proline-rich sequence (50% P) between the amino acidic positions 71–120 in the exon 1 is deleted. In previous experiments, when HOXB4-ΔPro (n=14) was over expressed in 5-FU enriched progenitor cells from BM, we reported a significant decrease (75fold, p<0.05) of the 12 days Δ-CFU-S frequency, in comparison to the HOXB4-wt (n=5), while it still generated significantly more Δ-CFU-S in comparison to the GFP control (n=11) (35fold, p<0.0003). Furthermore, we performed the CRU assay by transplanting lethally irradiated C3H×C57Bl/PeB mice with serial dilutions of 5-FU isolated bone marrow progenitor cells, in order to evaluate the effect of the HOXB4-ΔPro on the competitive repopulating unit frequency. At the 16th week post transplantation we reported no significant difference in the CRU frequency between the mice receiving HOXB4wt (CRU 1/834, n=18) and the mice receiving HOXB4-Δpro (CRU 1/413, n=18) expressing transplants. However, in mice transplanted with HOXB4wt (n=12) 45.3 % of the circulating cells belonged to the transduced compartment compared to 19.2% in the HOXB4-Δpro group (n=13) (p<0.006), whereas the lineage distribution within the transduced compartment did not differ between both experimental arms 16 wks post transplant. Of note and in contrast to HOXB4wt, mice engrafted with HOXB4-ΔPro BM cells (n=9) developed myeloproliferation with a significant increase of Mac-1 and Gr-1 positive cells over time in the PB (29% Gr1 and 43% Mac1 wk 4–16 compared to 71.2% Gr1 and 86.6% Mac1 week 36–56 wk, p<0.004). The HOXB4-ΔPro mice developed acute myeloid leukemia without maturation, as confirmed by immunohistochemical analysis after a median latency time of 279 days (n=9), while the mice transplanted with HOXB4wt expressing BM cells did not develop disease after an observation for more than 466 days (n=5, p<0.05). The AML in HOXB4-ΔPro mice was readily transplantable (66.5 days for 2nd Tx, n=6; 43 days for 3rd Tx, n=4) (p<0.05 compared to 1st recipients). In order to investigate the proviral integration pattern in the transplanted mice, we performed LM-PCR. In more than five HOXB4-Δpro mice we did not find recurrent integration sites. Taken together our results demonstrate that the N-terminal proline-rich region of HOXB4 has an important function for the stem cell amplifying function of HOXB4 and that loss of this domain converts HOXB4 in a leukemogenic gene.
Variations in in vivo phosphorylation at the proline-rich domain of the microtubule-associated protein 2 (MAP2) during rat brain development