Small Molecule Inhibitor of Neutrophil Elastase Restores Impaired Production of Human Myeloid Cells Observed in Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) is a rare hematopoietic disease characterized by maturation arrest at the promyelocytes, recurring severe infections, and evolution to leukemia. Heterozygous mutations in either the neutrophil elastase (NE, ELA2) gene (sporadic or autosomal-dominant SCN) or homozygous mutations in the HAX1 gene (autosomal-recessive SCN) are associated with a similar clinical phenotype and similar block of myeloid differentiation in the marrow. Most studies now indicate that the maturation arrest in SCN is due to accelerated apoptosis of myeloid progenitors triggered by the mutant gene products. The hetero and homozygous deletion of NE in mice as well as the knock-in of mutant NE identified in SCN/AML patient failed to produce severe neutropenia phenotype in mice. We established a cellular model of SCN with inducible expression of del.145–152 NE mutant in human promyelocytic tet-off HL60 cells. Ratio of normal /mutant NE products in these cells is approximately 1:1, similar to that expected in SCN patients with heterozygous NE mutation. Expression of mutant NE in promyelocytic cells resulted in a characteristic block of myeloid differentiation with ∼70% decline in differentiated neutrophils which is similar to that observed in SCN. Cell growth reduction and accelerated apoptosis were also observed in these cells in response to mutant NE expression. Thus, this SCN model appears to closely recapitulate the human phenotype. Analysis of the proteolytic activity of cells expressing mutant elastase revealed approximately 40% increase in total NE-specific activity in response to mutant NE expression compared with controls, suggesting that mutant elastase exhibits at least some proteolytic activity. To date we identified more than 40 heterozygous mutations in the NE gene in pre-leukemic SCN patients, however, the pathomechanism remains unclear. Molecular modeling of the NE tertiary structure revealed that these mutations predominantly affect the N-glycosylation sites or the binding pocket of elastase. Importantly, the active site of the mutant protease appears to be intact, which suggested that NE-specific small molecule inhibitors may be useful in preventing accelerated apoptosis and the block of myeloid differentiation of myeloid cells. Examining this SCN model, we identified a proprietary cell-permeable elastase-specific small molecule inhibitor (compound A, Merck, USA), which inhibited the proteolytic activity of the NE by more than 80%. Our studies indicate that treatment of human promyelocytic cells expressing del.145–152 mutant NE with this small molecule inhibitor restored the impaired production of myeloid cells and improved myeloid differentiation to near normal level. Importantly, the compound A did not impair the growth rate of control cells with normal NE expression. These data suggest that NE-specific small molecule inhibitor and its analogs should be considered in clinical trials in patients with SCN that is attributable to mutant NE.