Proteolysis in Hereditary Neutropenia - Project Summary Neutropenia, defined by abnormally low neutrophil counts, compromises innate immunity and increases susceptibility to life-threatening infections. Although most cases are acquired—resulting from malignancy, chemotherapy, infections, medications, or autoimmune disease—the study of inherited forms, though rarer, offers critical insights into the core mechanisms of myelopoiesis and granulocytic differentiation. Among these, autosomal dominant, heterozygous mutations in ELANE (formerly ELA2), which encodes the neutrophil granule serine protease neutrophil elastase (NE), represent the most common cause of severe congenital neutropenia (SCN) and the primary cause of cyclic neutropenia. SCN presents at birth with lifelong neutropenia, bone marrow maturation arrest, and elevated risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In cyclic neutropenia, neutrophil counts fluctuate between zero and near-normal with a striking 21-day periodicity. Despite their clinical importance, the pathogenic mechanisms of ELANE mutations remain poorly understood, and curative treatment is currently limited to hematopoietic stem cell transplantation. Mouse models fail to recapitulate the human phenotype, highlighting the need for human systems to investigate disease biology. All known pathogenic ELANE mutations result in production of a variant NE polypeptide, which may bypass key steps of proteolytic maturation and mislocalize within developing cells. This project tests the hypothesis that ELANE mutations cause disease by disrupting the spatial or temporal control of NE activity during granulopoiesis. Using isogenic, gene-targeted human induced pluripotent stem cells (iPSCs), the proposed research will: (1) define the spatial and temporal determinants of NE pathogenicity by introducing cis-acting suppressor mutations that disrupt its processing, trafficking, and catalytic activity; (2) determine whether the NE paralogs proteinase 3 and cathepsin G function as trans-acting modifiers; and (3) test whether CD34, a critical hematopoietic surface protein with distinct properties differing between mouse and human, is an NE substrate, and whether cleavage-resistant CD34 variants can restore granulopoiesis in ELANE-mutant cells. These studies will elucidate mechanisms of protease regulation in human neutrophil development, clarify the pathogenesis of both inherited and acquired neutropenia, and identify molecular targets for potential therapeutic intervention. The proposed work aligns directly with the NIH mission to advance understanding and treatment of hematologic and immune disorders.
