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.

Grant Number: 1R03TR004848-01A1
NIH Institute/Center: NIH
Principal Investigator: ALAN CANTOR