ER Stress-Induced Expression of CCAAT/Enhancer Binding Protein Gamma (C/EBPG) May Contribute to the Block in Granulocytic Differentiation in Severe Congenital Neutropenia

Abstract 8 Severe congenital neutropenia (SCN) is a bone marrow failure syndrome characterized by an isolated block in granulocytic differentiation at the promyelocyte/myelocyte stage, and a marked propensity to develop acute myeloid leukemia. Approximately 50% of cases of SCN are due to heterozygous mutations of ELANE, encoding neutrophil elastase (NE). We and others previously published data supporting a model in which the shared feature of the many different ELANE mutations is a propensity to misfold. Accumulation of misfolded mutant NE induces endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). However, the mechanisms by which UPR activation disrupts granulocytic differentiation are not clear. We previously presented results of RNA expression profiling of promyelocytes sorted from patients with SCN (n = 9), healthy donors (n = 5), healthy donors treated with G-CSF (n = 4), or patients with Shwachman Diamond syndrome (n = 5), an unrelated congenital neutropenia syndrome. A transcriptional signature consistent with UPR activation was observed only in SCN promyelocytes. Here we report, based on an unbiased analysis of the profiling data, that one of the most dysregulated genes in SCN promyelocytes is CCAAT/enhancer binding protein gamma (CEBPG). CEBPG is a member of the CCAAT/enhancer binding protein family that includes CEBP/alpha (CEBPA), which plays an obligatory role in granulocytic differentiation. Of note, Skokowa and colleagues previously reported reduced CEBPA expression in myeloid cells from patients with SCN (Nat Med 12:191, 2006). CEPBG is known to heterodimerize with CEBPA and inhibits its transcriptional activation of target genes. Based on these data, we hypothesize that UPR-induced expression of CEBPG may contribute to the block in granulocytic differentiation in SCN through antagonism of CEBPA. We first asked whether UPR activation directly induces CEBPG mRNA expression. Bone marrow from healthy donors was treated with a variety of UPR inducers (tunicamycin, thapsigargin, or dithiothreitol) for 24 hours, and real time PCR performed on sorted promyelocytes (CD16low, CD15+ CD14− CD9e− cells) was performed. CEBPG mRNA expression was induced 2–5 fold compared with untreated cells. Of note, no difference in CEBPA mRNA expression was observed after UPR activation. However, though confirmation is required, immunoblotting of CD15+ myeloid cells after UPR activation showed decreased expression of CEBPA protein, which is consistent with a prior report suggesting that UPR activation inhibits CEBPA translation (J Cell Mol Med. 14:1509–19). Thus, UPR activation may inhibit CEBPA function through two mechanisms: increased CEBPG antagonism and decreased CEBPA translation. We next asked whether enforced expression of CEBPG was sufficient to inhibit granulocytic differentiation. CD34+ hematopoietic progenitors from healthy donors were transduced with lentivirus expressing CEBPG or vector alone; the lentivirus also expressed green fluorescent protein (GFP) to track transduced cells. Transduced (GFP+) cells were sorted and then cultured on irradiated stromal cells in the presence of G-CSF for two weeks to induce granulocytic differentiation; CEBPG mRNA expression was increased approximately 20-fold compared to control transduced cells. Enforced expression of CEBPG resulted in a significant suppression of mature neutrophil production. The percentage of mature neutrophils in CEBPG-overexpressing cultures was 19.2 ± 0.04% compared with 42.2 ± 0.7% for vector-alone transduced cultures (P = 0.02, n =4). Together, these data suggest a model in which UPR activation inhibits granulocytic differentiation, at least in part, by inducing CEBPG expression and antagonizing CEBPA function. Disclosures: Boxer: Amgen: Equity Ownership; Alexion: Speakers Bureau; NIH: Research Funding; Up to Date: Patents & Royalties.