C/EBPα and C/EBPε Induce Monocytic Differentiation of Myelomonocytic Cells with MLL Chimeric Fusion Gene.
Authors: Matsushita Hiromichi , Nakajima Hideaki , Nakamura Yoshihiko , Tsukamoto Hideo , Tanaka Yumiko , Asai Satomi , Nosaka Tetsuya , Ando Kiyoshi , Miyachi Hayato 
Published: October 2019

Abstract
Mixed lineage leukemia (MLL) gene, located on chromosome segment 11q23, is involved in various types of hematological malignancies including infant and secondary leukemias. To date, more than 40 partners have been reported in the recurrent translocations with 11q23, and the resultant fusion genes are believed to contribute to leukeomogenesis. In acute myeloid leukemia (AML), the incidence of MLL rearrangements is higher in subtypes with monocytic components, namely M4, M5a and M5b. Furthermore, several MLL fusion genes are known to cause differentiation arrest at the myelo-monocytic stage when they are expressed in murine hematopietic cells. Recent studies have revealed that the myeloid transcription factors, CCAAT/enhancer binding proteins (C/EBPs), regulate not only granulocytic but also monocytic differentiation. Accordingly, we investigated the roles of C/EBPα and C/EBPε in myelomonocytic leukemia with MLL fusion genes. The protein expression of C/EBPα and C/EBPε was induced in retinoic acid-mediated monocytic differentiation of human AML cell lines with MLL-AF9, THP-1 and MOLM-14. Furthermore, 9-cis Retinoic acid upregulated the mRNA levels of Cebpa and Cebpe in monocytic differentiation of an IL-3-dependent murine cell line carrying MLL-SEPT6, HF-6. When HF-6 cells were retrovirally infected with inducible forms of C/EBPα (C/EBPα-ER) or C/EBPε (C/EBPε-ER), mature monocytes were markedly induced. Flow cytometric analysis revealed the increased intensity of Mac-1 and Gr-1 with C/EBPα or C/EBPε induction. In addition, their cell growth was severely impaired. This growth inhibition was believed to be partly due to induction of apoptosis. There were no apparent differences in monocytic differentiation induction and growth inhibition between C/EBPα and C/EBPε. The overexpression of C/EBPα or C/EBPε in HF-6 cells was found to repress the gene expressions of Myc, Hoxa7 and Hoxa9, and induced CdknIa and SfpiI in quantitative RT-PCR analysis. The transcriptional level of C/EBPε was up-regulated by the induction of C/EBPα, but not vice versa. Since FLT3-ITD has been detected in 20 to 30% of AML cases and is known to cause a more immature type of AML with MLL-SEPT6 in vivo analysis, we subsequently introduced internal tandem duplication of FLT3 (FLT3-ITD) into HF-6 cells. These cells could proliferate without IL-3. Overexpression of C/EBPα and C/EBPε in these cells, however, inhibited the cellular proliferation and induced monocytic differentiation, as was seen in the original HF-6 cells. MOLM-14 cells, also possessing FLT3-ITD, have recently reported to differentiate by overexpression of C/EBPα (J Exp Med2006: 203: 371–381). Taken together, these findings suggest that not only C/EBPα but C/EBPε alone can induce monocytic differentiation of myelomonocytic leukemia cells with MLL fusion gene, regardless of the existence of FLT3-ITD, and that this process is accompanied by down-regulation of the genes which are related to MLL-induced AML leukemogenesis.