Background. Severe congenital neutropenia (SCN) is characterized by a critical reduction in absolute neutrophil count (ANC < 500/μl), which renders the affected individual vulnerable to recurrent, life-threatening infections. A majority of SCN cases arise due to germline mutations in the neutrophil elastase gene (ELANE). Treatment with high dose granulocyte colony stimulating factor (GCSF) increases the number of neutrophils and eliminates the risk of infection. SCN patients are at a significantly high risk of developing myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML). About 70% of the MDS/AML cases report somatic mutations in the GCSF receptor gene (CSF3R), suggesting a correlation between GCSF treatment and development of MDS/AML. The current favored mechanism for ELANE mutations promoting SCN is induction of an unfolded protein response (UPR), triggering death of neutrophil precursors resulting in neutropenia. However multiple studies of UPR activation reported a requirement of treatment with chemical UPR inducers such as tunicamycin or bortezomib to observe significant changes. The mechanism of mutant ELANE-mediated neutrophil deficit thus remains elusive. In addition, the ability of G-CSF to alleviate neutropenia and its role in progression from SCN to MDS/AML is still a confounding factor in G-CSF therapy for SCN cases.

Methods.We created a doxycycline inducible expression system of ELANE, consisting of wild type (wt) and point mutation (G185R) variant associated with SCN, in the murine myeloblast cell line 32D and the human promyelocytic leukemia cell line NB4. We determined the effect of mutant ELANE expression on cell proliferation (Trypan blue exclusion), survival (Trypan blue exclusion, AnnexinV-PI staining) and differentiation (Wright-Giemsa staining, CD11b expression). Underlying molecular changes were investigated using immunoblotting and qPCR based gene expression analysis.

Results. Induction of wt or mutant ELANEexpression with doxycycline did not have any detrimental effects on cell proliferation and survival. However, a partial block in differentiation with an accumulation of myeloid progenitors was observed in cells expression mutant ELANE. Profiling of gene expression for markers of differentiation identified reduced expression of key transcriptional factors (Cebpa, Cebpe, Cebpb, Cebpd, Gfi1, Spi1) coupled with reduced levels of other markers of differentiation (e.g., Mpo, Ltf). Increased transcript levels were also observed in some cell cycle genes (Pola1, Ccnd1). We did not observe an increase in expression of UPR genes upon mutant ELANEinduction.

Conclusion.Our data demonstrate that expression of mutant ELANE promotes an aberrant differentiation gene expression profile that leads to a partial block in differentiation. In addition, our data also suggest that mutant ELANE expression does not promote UPR. We also show a concomitant upregulation of cell proliferation genes upon mutant ELANE expression. This detour from a differentiation profile may indicate pre-leukemic underpinnings for G-CSF treatment in SCN.


No relevant conflicts of interest to declare.

Author notes


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