Mechanisms of Impaired Granulopoiesis Due to CLPB Mutations

/Abstract The goal of this project is to define the mechanisms by which impaired mitochondrial function leads to failure of neutrophil development, and how neutrophil precursors use somatic mutations to adapt to this cell- intrinsic stress. Severe Congenital Neutropenia(SCN) is an inborn disorder of granulopoiesis characterized by severe chronic neutropenia from birth, premature death secondary to infectious complications, and transformation to myeloid malignancy. Despite the significant morbidity and mortality associated with SCN, the mechanisms underlying survival, proliferation, and differentiation of neutrophil precursors remain incompletely understood. We recently identified heterozygous missense mutations in CLPB as a new and frequent cause of SCN. CLPB is a nuclear-encoded protein that resides within the mitochondrial intermembrane space where it functions as a molecular chaperone to disaggregate and facilitate re-folding of misfolded proteins. Our previous work has also demonstrated impaired mitochondrial oxidative phosphorylation in CLPB-mutant myeloid cells accompanied by increased cell death. Interestingly, these cells demonstrate an increased non-apoptotic cell death in response to mitochondria proteostasis stress and sustained activation of the mitochondrial unfolded protein response (UPRmt). Patients with SCN also develop clonal hematopoiesis that may help alleviate cell intrinsic or cell extrinsic stress; however, the mechanisms of adaptation to CLPB-mutated SCN are not defined. In this proposal, we will test the hypothesis that mutant CLPB granulocyte precursors undergo ferroptotic cell death, a form of non-apoptotic cell death that has been linked to prolonged ROS generation. We will also test the hypothesis that CLPB-induced mitochondrial stress is ameliorated by RUNX1 mutations leading to a competitive growth advantage. To this end, the following specific aims are proposed: Aim 1) To understand the link between sustained UPRmt signaling and cell death in CLPB-mutated granulocyte precursors; Aim 2) To identify the mechanisms by which RUNX1 mutations promote clonal advantage in CLPB-mutated SCN. The proposed studies will provide an understanding of the molecular pathophysiology of CLPB-mutant SCN and RUNX1-mediated clonal adaptation. Ultimately, a better understanding of normal and SCN-related granulopoiesis may suggest new therapeutic approaches to treat or prevent neutropenia in patients with SCN, and in patients receiving myeloablative chemotherapy. These studies will also provide critical knowledge on how mitochondrial stress impacts granulocyte development, and how maladaptive clonal hematopoiesis provides a growth advantage to these cells. Filling these knowledge gaps can lay the foundation for developing strategies to augment neutrophil precursor cell survival and differentiation without enhancing risk for malignant transformation. Project Number: 1R00HL177828-01 | Fiscal Year: 2025 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: JULIA WARREN | Institution: CHILDREN'S HOSP OF PHILADELPHIA, PHILADELPHIA, PA | Award Amount: $445,000 | Activity Code: R00 | Study Section: Special Emphasis Panel[ZHL1 CSR-N (O1)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R00HL17782801