Development of Bone Marrow Organoid System to Study RUNX1-Familial Platelet Disorder with Associated Myeloid Malignancy (RUNX1-FPD/MM) - ABSTRACT RUNX1 Familial Platelet Disorder with Propensity to Develop Myeloid Malignancy (RUNX1-FPD/MM) is a rare genetic disorder due to germline heterozygous loss-of-function mutations in the gene encoding the key hematopoietic transcription factor RUNX1. Affected individuals have thrombocytopenia, platelet dysfunction, autoinflammatory symptoms, early clonal hematopoiesis (CH), and a high risk of developing myelodysplastic syndrome (MDS) and leukemia (~35-45% lifetime risk with median onset age of 33 years). Unfortunately, the mechanisms that predispose to early CH and hematologic malignancy remain poorly understood. Moreover, no interventions have been identified to reduce the risk of leukemia development. A major obstacle in the field has been the lack of practical and faithful experimental models to study RUNX1-FPD/MM and do high-throughput drug screens. Mice are not as sensitive to RUNX1 haploinsufficiency as humans, and mouse models do not develop leukemia. Non-human primate models show greater phenotypic similarity with human disease but are costly and not amenable to high throughput analysis. CRISPR-gene editing of human primary CD34+ cell has been attempted to knock-in heterozygous patient mutations, but challenges remain in avoiding alteration of the wild type allele. shRNA models have been generated to knock-down RUNX1 to ~50% levels. However, this represents the cell population average and not necessarily levels in individual cells. Availability of primary patient samples is limited by the rarity of the disease, and xenotransplantation of these samples to make long-lived models has been challenging. Human induced pluripotent stem cell (hIPSC) lines have been valuable but have only been examined in 2D cultures whose conditions are tailored to specific lineages. 3D organoid culture systems have been developed for many solid organs and have served as valuable experimental models since they recapitulate the complex microenvironment including stromal elements, contain more physiologic cytokine/chemokine levels, and are amenable to drug screening/testing. Very recently, 3D organoid culture systems have been established for the human bone marrow (BM). The objective of this 1-year pilot proposal is to develop BM organoid system to study RUNX1-FPD/MM. This will involve generating BM organoids from two hIPSC lines derived from patients with RUNX1-FPD (a splice-site acceptor mutation and gene deletion) along with isogenic gene corrected controls. Comparative analysis will be performed to determine the extent to which the BM organoids recapitulate RUNX1-FPD/MM phenotype. Proof-of-principle experiments will be performed to assess their utility in drug testing. Lastly, chimeric organoids containing small clones with an additional somatic mutation in BCOR, a common occurrence during CH in RUNX1-FPD/MM patients, will be generated and used to determine if the organoids can be used to study clonal dynamics in this disorder. The successful outcome of this study will be the development of a new tractable experimental system to study human RUNX1-FPD/MM in a more physiologic setting and one that is amenable to future drug development.
