PB2204: THE ROLE OF HES6 IN NORMAL HUMAN HEMATOPOIESIS

Background: The Notch signaling pathway is an important regulator of normal hematopoiesis and is often involved in leukemia. Some members of the HES gene family of transcription factors, like HES1 and HES4, are known Notch targets and have important roles in hematopoiesis. HES6, on the other hand, does not seem to be Notch dependent and has been poorly studied in the hematopoietic system. Interestingly, in the context of neurogenesis, it has been shown that HES6 can inhibit HES1 function, raising the hypothesis that HES6 might influence canonical Notch signaling activity. Within the hematopoietic system, HES6 has been described to be overexpressed in BPDCN, a very rare and aggressive hematological malignancy of plasmacytoid dendritic cells (pDCs). Also other studies have suggested the involvement of HES6 in cancer, including glioma, colorectal cancer, prostate cancer and uveal melanoma, and this mostly relates to a possible role for HES6 in cell proliferation. Aims: Given that HES6 is poorly studies within the hematopoeitic system, this study aims to unravel the role of HES6 in normal human hematopoiesis. Methods: We analyzed published datasets to examine the expression pattern of HES6 in different blood cell types. Through shRNA-mediated knockdown, we studied the impact of HES6 knockdown on the differentiation of CD34+ hematopoietic stem cells from cord blood to various hematopoietic lineages using in vitro stromal co-cultures and colony-forming unit assays. These experiments were analyzed using flow cytometry for the evaluation of differentiation, and cell counts for the evaluation of cell proliferation. Results: HES6 is highly expressed in megakaryocyte-erythroid progenitors (MEPs) and to a lesser extent in NK cells, pDCs and T cells. When exploring the expression in developing T cells, we observed that HES6 expression is inversely correlated with the expression of HES1. Considering the high HES6 expression in MEPs, we particularly studied the role of this gene during erythroid/megakaryocyte development. In our stromal cultures, HES6 expression remained high in developing erythroblasts and was low in megakaryocytes. Upon HES6 knockdown in CD34+ cells, a significant reduction in the number of megakaryocytes (50-85%) and erythroblasts (75-85%) was observed. The effect of HES6 knockdown in the was significant, with a 55-80%.reduction of colony formation. The number of cells per colony was also reduced by approximately 50%. HES6 knockdown during the development of other lineages also appears to impact cell proliferation but this effect is less obvious and needs further exploration. Summary/Conclusion: Consistent with the high expression of HES6 in MEPs, we observed significantly reduced cell proliferation upon HES6 knockdown during erythroid/mekaryocyte differentiation, with the most distinct effect on erythroblasts. HES6 knockdown also seems to impact the differentiation potential of precursor cells towards the myeloid lineages, as shown through colony-forming assays. Although the impact of HES6 on other hematopoietic lineages needs to be explored further, our results do provide the first clear insights into the functional role of HES6 in human hematopoiesis.