OC 10.3 - A novel model of RUNX1-haploinsufficient (RUNX1Lo): Human megakaryocytes infused into mice further characterizes this defect and identifies potential therapeutics

Background: RUNX1 is a key transcriptional regulator in hematopoiesis and megakaryopoiesis. Heterozygous defects in RUNX1 underlie familial platelet disorder with associated myeloid malignancy (FPDMM). FPDMM has symptoms of mild-to-moderate thrombocytopenia and platelet defects. Also, RUNX1Lo increases the risk of myelodysplasia and acute myeloblastic leukemia.

Aims: Since RUNX1Lo animal models do not mimic FPDMM disease, rarely developing bleeding disorder or leukemia, we tried to establish a model system to understand better the underlying mechanisms of the observed phenotypes and to identify therapeutic interventions.

Methods: We have developed an ex-vivo megakaryopoiesis system beginning with healthy human CD34+-hematopoietic stem and progenitor cells and recapitulating FPDMM by knockdown of RUNX1 expression using lentiviral short-hairpin RNA interference. We then xenotransfused these human CD34+-derived megakaryocytes into immunocompromised mice for in-vivo platelet studies.

Results: In-vitro RUNX1Lo-megakaryocyte yield was reduced, and the megakaryocytes showed a marked reduction in agonist response. In-vivo murine studies of the released platelets from infused RUNX1Lo-megakaryocytes were then characterized and therapeutic intervention tested. Infused RUNX1Lo-megakaryocytes into immunocompromised NSG mice released fewer platelets/megakaryocytes (Figure 1). RUNX1Lo-platelets poorly responded to agonists. Unlike studies of infused control megakaryocytes into NSG mice – which are also homozygous for an R1326H mutation in von Willebrand factor, switching species-specific binding from mouse to human platelets – infused RUNX1Lo-megakaryocytes are defective in thrombus formation in a Rose-Bengal-photochemical carotid injury model (Figure 2). The small-molecule inhibitor RepSox that blocks the transforming-growth factor-beta pathway rescued RUNX1Lo-megakaryopoiesis in vitro, platelets/RUNX1Lo-megakaryocyte yield in vivo (Figure 1), and thrombus formation in the photochemical injury model (Figure 2).

Conclusion(s): This model recapitulates the defect in FPDMM megakaryocytes and platelets, identifies a previously unrecognized defect in thrombopoiesis, and demonstrates reversal of these defects in vivo by a drug. We believe that such studies of human RUNX1Lo-megakaryocytes may be a useful approach for the preclinical assessment of potential therapeutics.