BCR/ABL-Mediated Myeloid Expansion Is Promoted by C/EBPβ, a Regulator of Emergency Granulopoiesis,
Authors: Hayashi Yoshihiro , Hirai Hideyo , Yao Hisayuki , Yoshioka Satoshi , Satake Sakiko , Kamio Naoka , Miura Yasuo , Ashihara Eishi , Fujiyama Yoshihide , Tenen Daniel G. , Maekawa Taira 
Published: October 2019

Abstract
Abstract 3747
Chronic phase chronic myeloid leukemia (CP-CML) is characterized by the increase of myeloid cells in the peripheral blood (PB) and bone marrow (BM). We have previously shown that the C/EBPβ transcription factor is required for emergency granulopoiesis, increased proliferation and differentiation of granulocytic precursors in emergency situations such as infection (Hirai H et al., Nature Immunol. 2006). Enhanced myelopoiesis is a common feature between emergency situations and CP-CML. However, little is known about the roles of C/EBPβ in the pathogenesis of CP-CML. The aim of this study is to elucidate the regulation and function of C/EBPβ in BCR/ABL-mediated myeloid expansion.
We first assessed the expression level of C/EBPβ in hematopoietic stem cells and myeloid progenitors in BM obtained from healthy donors or CP-CML patients. The transcript of C/EBPβ is expressed at significantly higher level in common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) in CP-CML BM than those in normal BM. When BCR/ABL was retrovirally transduced into a mouse hematopoietic stem cell line, EML, C/EBPβ expression was significantly upregulated. Treatment of the EML-BCR/ABL cells with imatinib mesylate normalized the expression level of C/EBPβ. These data suggested that C/EBPβ was upregulated in response to the downstream signaling of BCR/ABL.
In order to investigate the function of C/EBPβ in BCR/ABL-mediated myeloid expansion, BCR/ABL was retrovirally introduced into BM cells obtained from 5-FU treated C/EBPβ knockout (KO) mice and their properties were compared with those of BCR/ABL-transduced BM cells from wild type (WT) mice. When the transduced cells were cultured in cytokine-free semisolid methylcellulose medium, the number and the size of the colonies of C/EBPβ KO cells were significantly smaller. Flow cytometric analysis of the colony-forming cells revealed that the BCR/ABL-transduced C/EBPβ KO BM cells gave rise to higher frequency of c-kit+ cells and lower CD11b+ cells than BCR/ABL-transduced WT BM cells (%c-kit+ cells=8.2±3.0% vs. 11.3±3.5%, p=0.002, %CD11b+ cells=75.1±2.1% vs. 90.0±4.2%, p=0.003). In addition, BCR/ABL-transduced C/EBPβ KO BM cells revealed higher replating efficiency than BCR/ABL-transduced WT BM cells.
To investigate the role of C/EBPβ in leukemogenesis, BCR/ABL-transduced BM cells from C/EBPβ KO mice or WT mice were transplanted into lethally irradiated recipient mice. In mice transplanted with BCR/ABL-transduced C/EBPβ KO cells, the increase of white blood cell count was delayed (Figure) and higher frequency of c-kit+ cells were observed in the BM at day 19 post transplantation (16.0±2.6% vs. 5.5±4.6%, p=0.01). Spleen size of mice transplanted with BCR/ABL-transduced WT cells is much larger than that of BCR/ABL-transduced C/EBPβ KO cells (Figure). The median survival of mice transplanted with BCR/ABL-transduced WT cells was 19 days. In contrast, the median survival of mice transplanted with BCR/ABL-transduced C/EBPβ KO cells was 31 days (p=0.0005).
In summary, C/EBPβ is upregulated by BCR/ABL and the absence of C/EBPβ resulted in delayed proliferation and differentiation of myeloid cells both in vitro and in vivo. Our results suggest that C/EBPβ is involved in the BCR/ABL-mediated myeloid expansion in CP-CML and that C/EBPβ can be the novel molecular target for the therapy of CML. We are currently investigating the molecular mechanisms which mediate the upregulation of C/EBPβ and the direct targets of C/EBPβ in CP-CML.

Disclosures:
No relevant conflicts of interest to declare.