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 the survival and maturation of myeloid precursors in the bone marrow. Currently, the only effective therapies are subcutaneous injections of human recombinant granulocyte-colony stimulating factor and hematopoietic stem cell transplantation. However, concerns regarding the risk of leukemic transformation and the unavailability of suitable marrow donors limit the application of these therapies.

We and others have previously shown that cell-permeable NE inhibitors, as well as CRISPR-assisted genetic knockout (KO) of the ELANE gene, can correct defects in cell survival and maturation in cellular models of ELANE-associated neutropenia. (PMID:26193632, PMID:36052149, PMID:28754797, PMID:39867870, PMID:31248972, PMID:35795780)

Recently, targeted protein degradation using proteolysis targeting chimeras (PROTACs) has emerged as a novel and promising strategy for eliminating disease-associated proteins. In this study, we evaluated custom-designed anti-NE PROTACs for their ability to correct mutant NE-induced defects in the myeloid differentiation of patient derived CD34⁺ bone marrow cells in vitro.

Hypothesis:

If cell-permeable NE inhibitors and genetic KO of ELANE can rescue the neutropenic phenotype in patient-derived in-vitro models of ELANE-associated neutropenia, this suggests that intracellular targeted degradation of mutant NE through PROTACs will also result in a rescue effect on impaired neutrophil production.

Methods:

Four custom-designed PROTAC molecules were synthesized by MedChemExpress's Custom Synthesis Service, targeting wild-type NE.

Bone marrow CD34⁺ cells from patients with ELANE mutations were purified, expanded, and induced to undergo myeloid differentiation using a protocol described in PMID:35795780, in the presence or absence of 1 μM of the X4587 PROTAC degrader. On day 14, cells were analyzed by flow cytometry for neutrophilic subsets (CD14⁺/CD66b⁺ and CD11b⁺/CD15⁺). Western blot analysis was used to assess NE degradation. Molecular modeling and docking simulations of the PROTAC molecules with NE were performed using the Molecular Operating Environment (MOE) software platform (Chemical Computing Group Inc., Montreal, Canada).

CD34⁺ cells from eight different patients harboring ELANE mutations (G221X, M154R, A233P, R220Q, R191S, IVS3-8 C>A, P139L, and L47P) were included in the study. CD34⁺ cells from healthy volunteers served as controls.

Results:

Untreated patient-derived cells demonstrated significant impairments in differentiation and maturation. Flow cytometry revealed impaired myeloid differentiation consistent with the hematopoietic defects seen in patients. Populations of CD14⁺/CD66b⁺ and CD15⁺/CD11b⁺ granulocytes were reduced by 2–6 fold across different experimental replicates.

Among the four PROTAC molecules tested, X4587 was effective in improving these abnormalities: CD14⁺/CD66b⁺ and CD15⁺/CD11b⁺ cell subsets increased by at least 2-fold in two patients carrying the R220Q and M154R mutations. Western blot analysis showed a 73% reduction in NE expression following X4587 treatment.

Molecular docking simulations showed that the X4587 molecule had significantly stronger binding stability—reflected by lower S-scores—with the mutant NE models M154R and R220Q (S-scores of 49 and 109, respectively) compared to the wild-type NE and other mutant models, which had a much higher average S-score of 579.

Conclusion:

Our preliminary data indicate that targeted degradation of NE via PROTAC X4587 can rescue myeloid differentiation defects in ELANE-mutated cells in lines expressing R220Q and M154R mutations. The observed effect in only these two cell lines is most likely attributable to the selective binding and degradation of mutant NE forms that closely resemble wild-type NE, as the PROTAC molecules were originally designed to target wild-type NE. This finding underscores the potential of developing mutation-specific or broadly acting PROTACs.

Future studies will aim to fine-tune the design of PROTAC molecules to either selectively target mutant NE proteins or create broadly effective degraders capable of eliminating a wider range of NE variants associated with neutropenia.

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2025
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