Paralog 4 HOX Genes HOXA4 and HOXB4 Expand Pro-B Cells in Vitro

Abstract 1299

HOX genes are known for their involvement in self-renewal of normal and malignant hematopoietic stem cells (HSC). Enforced expression of HOXB4 leads to HSC expansion in vitro and in vivo without leukemia development. We have recently shown that overexpression of its paralog HOXA4 also resulted in an increase of HSCs and myeloid progenitors in vitro. HOXA4 HSC are fully functional and reconstitute mouse chimeras with normal ratios of mature cells in the periphery. Interestingly, although the mature B-cell compartment is not enhanced, the overexpression of HOXA4 resulted in a 10-fold expansion of B-cell progenitors in the bone marrow (BM). Moreover, the number of more primitive WW-IC was not affected by the overexpression of HOXA4, indicating that this gene specifically expands IL-7 responsive B-cell progenitors. Based on these observations in vivo, we sought to determine if paralog 4 HOX genes can expand B-cell progenitors in vitro. To test this B220+ cells were sorted from BM of healthy wild type mice and transduced with MSCV retroviral vectors for HOXA4-GFP, HOXB4-GFP or control-GFP. Cells were cultured in B-cell specific medium and their growth in response to IL-7 was followed for three weeks. We observed a 15- and 10-fold increase of growth for HOXA4 and HOXB4 pro-B cells compared to control, respectively, within 16 days. FACS analysis confirmed the pro-B cell phenotype of all three cultures. These cells are currently being tested for their repopulation capacity of the B-cell compartment in irradiated hosts. To further investigate potential implication of these genes in oncogenic transformation HOXA4 and HOXB4 were overexpressed in E2APBX1 B-cells derived from E2APBX1 transgenic mice. We showed that the overexpression of HOXA4 or HOXB4 induced a strong expansion of E2APBX1 pro-B cell in vitro (2381- and 1090-fold difference over the control after 23 days, respectively), leading to an immortalization of the culture. Despite this huge expansion these cells were incapable to initiate leukemia upon transfer into irradiated recipients. B-CFC assays showed that the growth of HOXA4 or HOXB4 E2APBX1 pro-B cell cultures was supported by a strong expansion of B-cell progenitors (6138- and 15307-fold difference over the control after 20 days, respectively). Taken together, these results indicate that IL-7 responsive pro-B-cell progenitors are sensitive to paralog 4 HOX genes, an effect which is dramatically enhanced in the context of E2APBX1. Lack of clear oncogenic potential puts HOXA4 and –B4 forward as promising candidates to expand B-cell progenitors in vitro. These cells could potentially be used for B-cell complementation therapy in BM transplantation recipients.