LEF-1 Transcription Factor Regulates Proliferation and Differentiation of Myeloid Progenitors in Healthy Individuals and in Patients with Severe Congenital Neutropenia (CN).

Wnt signaling pathway plays a key role in the signal transmission during cell growth, proliferation, differentiation and survival, especially in rapidly self-renewing tissues, such as bone marrow, skin, and intestinal mucosa. Regulation of target gene expression by Wnt signaling requires the presence of lymphoid enhancer factor 1 (LEF-1) and/or T-cell factors (TCFs) within the nucleus. Previously we reported 20 times lower or even absent expression of LEF-1 mRNA and protein in CD33⁺ myeloid progenitors from patients with severe congenital neutropenia (CN), as compared to healthy controls. Abolished LEF-1 expression in CD33⁺ progenitors from CN patients was accompanied by low levels of LEF-1 target genes, such as cyclin D1, c-myc, ELA2, and survivin.

In the present study we aimed to characterize the role of LEF-1 transcription factor in myeloid cells from healthy individuals and from CN patients. Expression profile of LEF-1 mRNA during myeloid differentiation was measured by laser-assisted single-cell picking and real-time quantitative RT-PCR of different myeloid precursors (myeloblasts, promyelocytes, myelocytes/metamyelocytes, and mature granulocytes) from bone marrow smears of healthy controls and CN patients. LEF-1 mRNA was predominantly expressed in promyelocytes and myelocytes/metamyelocytes from healthy donors and was dramatically down-regulated in these cell types from CN patients. To investigate the functional consequence of LEF-1 downregulation, we specifically inhibitied LEF-1 expression in HL-60 promyelocytic cells and CD34⁺ cells from healthy individuals by lentiviral transduction of anti-LEF-1 shRNA. Interestingly, inhibition of LEF-1 resulted in down-regulation of LEF-1 target genes (cyclin D1, survivin, and c-myc), hematopoietic transcription factors C/EBPα and C/EBPε , and was accompanied by dramatically reduced proliferation and increased apoptosis in both HL-60 cells and CD34⁺ progenitors. Moreover, lentiviral transduction using vector containing LEF-1-GFP reporter into CD34⁺ cells from one CN patient led to normalization of expression of LEF-1 target genes (c-myc, survivin, cyclin D1), increased expression of C/EBPα , C/EBPε , and partial restoration of myelopoiesis.

To investigate the cause of reduced LEF-1 expression in myeloid progenitors from CN patients, we sequenced the LEF-1 gene. We could not find any mutations in 15 CN patients studied. Therefore, regulatory mechanisms of LEF-1 expression, which are different from healthy controls, exist in CN. Interestingly, noggin, one of the activators of LEF-1 expression is significantly downregulated in CN patients. When exposed to noggin-containing medium, LEF-1 and LEF-1 target genes were induced in CD33⁺ cells from one CN patient, although the effect of noggin on myelopoiesis have to be analysed.Taken together, LEF-1 transcription factor regulate proliferation and differentiation of myeloid progenitors and is involved in the pathomechanism of CN.