The Effect of Neutrophil Elastase Inhibitors on in-Vitro Myeloid Differentiation of CD34+ Cells of Patients with ELANE Associated Neutropenia

Background: Mutations in ELANE are the most common cause of both cyclic and severe congenital neutropenia. ELANE encodes neutrophil elastase (NE), a tissue specific serine protease expressed primarily in neutrophils. Expression of the mutant protein impairs survival and maturation of myeloid precursors in bone marrow. Currently, the only effective therapies are human recombinant granulocyte stimulating factor (rGCSF) subcutaneous injections and hematopoietic stem cell transplantation (HSCT). Concerns about the risk of leukemic transformation and unavailability of suitable marrow donors limit application of these therapies. We previously showed that cell permeable inhibitors of NE, especially an inhibitor developed my Merck, MK-0339, can correct the defect in cell survival and maturation in cellular models of HL60 cells and induced pluripotent stem cells (iPSC). (Makaryan et al. J Leukoc Biol. 2017;102:1143, Makaryan et al, J Cell Immunol, 2022, 4, 19 - 28). Recently, two promising strategies of genetic editing have been described: single- or double allele genetic knock out of ELANE (Nasri et al, DOI: 10.3324/haematol. 2019.221804, Sabo et al, https://doi.org/10.1016/j.omtm.2022.06.002).

Hypothesis: If cell permeable, selective NE inhibitors at a clinically achievable concentration can correct the defect of myeloid differentiation and maturation of CD34+ cells derived from patients with ELANE neutropenia, this will be a strong evidence to support a clinical trial of these compounds.

Methods: Bone marrow CD34+ cells from a patient with the M154R ELANE mutation were enriched using RosetteSep™ Progenitor Cell Enrichment Cocktail (Stemcell Technologies) and purified by density gradient centrifugation using Lymphoprep (Stemcell Technologies). The purified CD34+ cells were expanded for 4 days in StemSpan™ SFEM II media, supplemented with StemSpan™ CD34+ Expansion Supplement. Expanded CD34+ cells were further purified using StemSep™ Human CD34 Positive Cocktail and pushed towards myeloid differentiation using a modified protocol adapted from Nasri et al (Nasri et al, ibid) in the presence or absence of NE inhibitors. We used Merck developed MK-0339, Sivelestat (MedChemExpress) and GW311616 (MedChemExpress) cell permeable NE inhibitors. On day 14, cells were analyzed by flow cytometry for monocytic (CD14⁺/CD66b^-) and neutrophilic (CD66b⁺), (CD11b⁺/CD15⁺/CD16⁺) subsets. Differentiated cells were also evaluated under light microscope after Kwik-Diff (ThermoFisher) staining. Healthy volunteer CD34+ cells were used as a control.

Results: Untreated patient cells demonstrated significant impairment in differentiation and maturation. Flow cytometry showed block of myeloid differentiation and 5-fold greater monocytes compared to control, consistent with the hematopoietic defect in SCN patients. Populations of CD66b+, CD15+/CD11b+ and CD16+/CD15+ cells in patient cells were decreased by 68%, 65.7% and 98.1% respectively. Only MK-0339 inhibitor improved these cellular abnormalities: the subset of CD66b+ and CD15+/CD11b+ positive cells increased by 40% and 23.3%, respectively. Sivelestat had no effect on differentiation of CD34+ cells. Interestingly, GW311616 had a negative effect on differentiation, decreasing the subset of CD66b+ cells by about 70%. Studies are in progress for additional patients harboring different ELANE mutations.

Conclusions: The results with this initial patient, together with our previous studies using HL60 cells, indicate that some, but not all, selective NE inhibitors can correct the cellular abnormalities in ELANE associated neutropenia. We are investigating the effectiveness of this potential therapeutic approach across a diversity of mutations and other cell permeable NE inhibitors. Current evidence suggests that a clinical trial of MK-0339 is justified if there is supportive data on the safety of administration of this compound.

Kelley:X4 Pharmaceuticals, Inc.: Research Funding.