EVI2B, A NOVEL C/EBPA TARGET GENE, IS REQUIRED FOR MURINE NEUTROPHILIC DIFFERENTIATION AND CONTROLS HEMATOPOIETIC STEM CELL MAINTANENCE

ABSSUB-3308

Background: Evi2b encodes a type I transmembrane protein, which is ubiquitously expressed in hematopoietic cells. Evi2b was originally identified as a common virus integration site in murine retrovirally-induced leukemias, suggesting that Evi2b might be a tumor suppressor gene or a proto-oncogene. Interestingly, we recently identifiedEVI2B as one of the genes belonging to the C/EBPα signature, suggesting it plays a role in granulocytic development. Nevertheless, little is known about the role of Evi2b in hematopoiesis and leukemic transformation.

Aims: To define the function of Evi2b in hematopoiesis. In particular, we aimed to determine the role of Evi2b in granulocytic differentiation and hematopoietic stem cell (HSC) maintenance.

Methods: K562 cells stably transfected with an inducible C/EBPα-ER fusion protein and 32D/G-CSF-R myeloid progenitor cells were used as a model for human or murine granulocytic differentiation, respectively. Distinct murine bone marrow populations were sorted according to expression of cell surface markers (LKS: lineage^- ckit⁺sca1⁺, short-term (ST)-HSC: LKS CD48⁺ CD150^-, long-term (LT)-HSC: LKS CD48^- CD150⁺). Gene expression was measured by quantitative RT-PCR. Binding of C/EBPα to human EVI2B promoter and transactivation were determined by ChIP-seq analysis and luciferase assays, respectively. Murine Evi2b silencing was mediated by 2 distinct lentiviral shRNA constructs. Colony cultures were performed using semi-solid Methocult medium. Bone marrow transplantation experiments were performed using C57Bl/NCrl congenic mice strains. LT-HSC proliferation was evaluated by single cell sorting into Terasaki plates with subsequent counting of divided cells. Cell cycle analysis was performed by Pyronin Y and Hoechst 33342 staining. Human EVI2B levels were determined using the expression data of 525 AML patient samples (GSE14468).

Results: We demonstrated that C/EBPα binds to and transactivates EVI2B promoter in a dose-dependent manner, and that activation of C/EBPα upregulates EVI2B expression. In line with these results, we observed thatEVI2B expression is downregulated in acute myeloid leukemias characterized by C/EBPα promoter hypermethylation. Next, we showed that Evi2b expression positively correlates with C/EBPα upregulation during granulocytic differentiation, and that downregulation of Evi2b leads to a block of neutrophilic differentiation in 32D/G-CSF-R cells. Since we observed that Evi2b is also abundantly expressed in ST- and LT-HSC, we investigated the role of Evi2b in HSC maintenance. Downregulation of Evi2b in murine LKS cells impairs the ability of this population to form colonies in semi-solid cultures. Further, we showed that Evi2b-silenced LKS cells present reduced ST- and LT-HSC engraftment into lethally irradiated mice. We demonstrated reduced LT-HSC cell proliferation after Evi2b silencing, and accordingly, we showed that Evi2b knockdown in the LKS population increases the percentage of cells in the G₀ quiescent cell cycle phase.

Summary/Conclusion: We have identified Evi2b as a target gene of C/EBPα, a crucial transcription factor in granulocytic differentiation. We demonstrated that Evi2b is required for proper granulocytic differentiation in 32D/G-CSF-R cells, and that Evi2b plays a critical role in HSC regulation. Altogether, our data demonstrates that Evi2b is an essential regulator of granulocytic differentiation and HSC maintenance.

Keywords: AML, C/EBP, Granulocyte, HSC

http://www.ehaweb.org/