Small Molecule Inhibitor of Neutrophil Elastase and Severe Congenital Neutropenia.
Authors: Aprikyan Andrew A , Makaryan Vahagn , Totrov Maxim , Abagyan Ruben , Dale David C. 
Published: October 2019

Abstract
Abstract 552
Severe congenital neutropenia (SCN) is a rare heritable hematopoietic disorder characterized by maturation arrest at the promyelocytes, recurring severe infections, and evolution to leukemia. Heterozygous mutations in the neutrophil elastase (NE or ELANE) gene (sporadic or autosomal-dominant SCN) or homozygous mutations in the HAX1 gene (autosomal-recessive SCN) are associated with similar clinical phenotype and a block of myeloid differentiation or “maturation arrest” in the marrow. We and others reported that human myeloid progenitor cells expressing mutant elastase exhibit impaired cell survival (Aprikyan et al, 2003, Massullo et al, 2005, Kollner et al, 2006, Grenda et al, 2007). The hetero- and homozygous deletion of NE as well as the knock-in of mutant NE identified in SCN/AML patient failed to produce severe neutropenia phenotype in mice. Thus, the pathomechanism of severe neutropenia remains largely unclear due to the lack of cellular or animal model of SCN with characteristic block of myeloid differentiation and accelerated apoptosis.
We established a cellular model of SCN with inducible tet-regulated expression of del.145-152 NE mutant (identified in SCN/AML patients) in human promyelocytic tet-off HL60 cells. The ratio of normal/mutant NE products in these cells is approximately 1:1, which is similar to that expected in SCN patients with heterozygous NE mutation. Expression of mutant NE in the promyelocytic cells results in a characteristic block of myeloid differentiation with ∼70% decline in differentiated neutrophils which is similar to that observed in SCN, whereas induced expression of control wild type NE has no effect on myeloid differentiation. Reduced production of myeloid cells and accelerated apoptosis are also observed upon DMSO or retinoic acid induced granulocytic differentiation of the cells in response to mutant, but not wild type NE expression. Thus, this cellular model of SCN appears to closely recapitulate the human phenotype. Expression of mutant NE resulted in approximately 40% increase in total NE-specific proteolytic activity, suggesting that mutant elastase exhibits at least some proteolytic activity. To date there are more than 50 heterozygous mutations in the NE gene have been identified in pre-leukemic SCN patients. Molecular modeling of the NE tertiary structure revealed that these mutations predominantly affect the N-glycosylation sites or the binding pocket of neutrophil elastase. Importantly, the active site of the mutant protease appears to be intact, which suggests that NE-specific small molecule inhibitors may be useful in preventing accelerated apoptosis and the characteristic block of myeloid differentiation of myeloid progenitor cells. Screening this cellular model of SCN, we identified a proprietary cell-penetrant elastase-specific small molecule inhibitor (compound A, Merck, USA), which inhibits the proteolytic activity of NE by more than 80%. When treated with compound A, control cells with induced expression of wtNE exhibit normal myeloid differentiation and production of myeloid cells, similar to that in untreated cells. These data suggest that human NE is dispensable and that accelerated apoptosis and impaired myeloid differentiation in SCN is due to a gain-of-function effect of pro-apoptotic mutant elastase. Importantly, treatment of human promyelocytic cells expressing del.145-152 mutant NE with this small molecule inhibitor restores impaired production of myeloid cells and improves myeloid differentiation to near normal levels, thus neutralizing the pro-apoptotic effect of mutant NE. These data suggest that small molecule inhibitors of NE may represent a promising therapy in severe congenital neutropenia. We have examined the effect of the inhibitor on bone marrow cells from an SCN patient positive for NE mutation. At the time of bone marrow aspiration the patient was on G-CSF and the patient's freshly isolated bone marrow CD33+ progenitor cells exhibited ∼21% apoptosis, which was gradually increased reaching 43% at 3 days of culture. However, daily treatment of SCN cells with compound A preserved the cell survival rate at the initial value resulting in approximately 2-fold reduction in apoptotic cell death at 3 days of culture. These data demonstrate that the small molecule inhibitor of NE and its analogs should be considered for clinical trials in patients with SCN that is attributable to mutant NE.

Disclosures:
Dale: Merck: Research support; Amgen: Consultancy, Research Funding, Speaker.