Severe congenital neutropenia (SCN) is characterized by chronic neutropenia due to a constitutional genetic defect. Over 50% of SCN cases are associated with mutations in neutrophil elastase (NE) coded by ELANE, and are characterized by agranulocytosis with promyelocytic arrest and monocytosis. The severity and prognosis of ELANE mutations is variable but if untreated, the prognosis is generally poor. G-CSF (granulocyte colony-stimulating factor) has vastly improved the management of a subgroup of SCN patients, but has been unable to reduce malignancy rates or restore normal bactericidal activity of surviving phagocytes. The absence of adequate in vitro models of human myelopoiesis, scarcity of primary hematopoietic material from SCN patients, and lack of relevant animal models that reproduce the biology of SCN have hampered progress in our understanding of this disease. Induced pluripotent stem cells (iPSC) technologies have been proposed as an alternative method to model human disease in the culture dish and in specific protocols, which would recapitulate the majority of the cellular and molecular characteristics of myelopoiesis. Based on published and preliminary data, we hypothesize that ELANE mutations are necessary and sufficient to induce SCN through impairment of the G-CSF/CSF3R granulopoietic signaling resulting in impaired progenitor/precursor survival and granulocytic differentiation. In this project, we will exploit human myeloid cell systems to analyze mechanisms by which ELANE mutations result in agranulocytosis and monocytosis. We will first analyze whether ELANE mutations are sufficient and/or necessary to induce neutropenia through state-of-the-art CRISPR/Cas9 technology and development of isogenic iPSC lines along with knock-in/silencing expression methods and use of standardized methods to generate in vitro myelopoiesis from patient and control iPSC. Second, we will analyze the impact of mutant NE upon G-CSF/CSF3R signaling pathways in myeloid progenitors and precursors, using cellular and molecular signaling analysis and rescue experiments on ex vivo generated iPSC-derived myeloid progenitors. Finally, we will analyze the consequences of ELANE mutations on non-cell-autonomous inflammatory signaling on myeloid progenitors to explore the underlying basis of monocytosis. This proposal will not only provide key insights into how ELANE mutations result in dysfunctional granulopoiesis and SCN, but also reveal molecular mechanisms controlling normal human granulopoiesis. These studies will additionally identify disease relevant signaling pathways that predict responsiveness of SCN to G-CSF therapy and provide proof-of-concept on alternative therapies for SCN.
The use of novel tools of disease modeling will allow us to understand better the mechanisms that control the formation of human granulocytes in health and disease and provide information on novel methods for therapeutic intervention in patients with too few ineffective granulocytes.
