Mechanistic and Therapeutic Studies using a Xenotransplanted RUNX1-Haploinsufficient Murine Model

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ABSTRACT Runt-related transcription factor 1 (RUNX1), is key to hematopoiesis. Insufficient levels of RUNX1 (RUNX1Lo) results in familial platelet (Plt) disorder associated with myeloid malignancy (FPDMM), a rare disorder with quantitative/qualitative Plt defects and an increased risk of leukemias. Understanding the basis of the Plt defects and leukemic tendency, and developing therapeutics have been hampered by a lack of models that replicate the human disease. For example, Runx1+/- mice have minimal defects in their Plts and do not demonstrate a leukemic propensity. We and others have replicated the megakaryocyte (Mks, the cells from which Plts arise) defect seen in FPDMM using human induced pluripotent stem cells (iPSCs), providing new insights into the Plt defects. My group has extended this model using short-hairpin RNA (shRNA) technology in human CD34+- hematopoietic stem and progenitor cells (HSPCs). The resulting Mks were studied in vitro and after infusion into immunocompromised mice to release Plts in a xenotransfusion model. These studies confirmed and extended the iPSC results; however, even the xenotransfusion studies are over a short window, limiting understanding of the value of potential therapeutics on the Plt defects or the basis of leukemic transformation in FPDMM. To address these needs, we are developing a RUNX1Lo human marrow xenotransplantation model in nonirradiated, immunocompromised NOD,B6.SCID/Il2rg−/−KitW41/W41 (NBSGW) mice. Initial studies show that RUNX1Lo HSPCs engraft as well as wildtype HSPCs in this model. We can detect RUNX1Lo Plts and myeloid/lymphoid cells in the peripheral blood for >20 weeks post-injection. We now propose to complete the development of this RUNX1Lo model by pursuing the following aims: Specific Aim (SA) #1: Characterize the molecular basis of FPDMM in vivo. We will characterize RUNX1Lo HSPC engraftment, lineage commitment and Mk/Plt defects. We will also examine mice for clonal evolution and malignant transformation with or without introducing a secondary oncogenic stress to define the mechanistic of the leukemic propensity in FPDMM. SA#2: Perform in vivo drug screening to reverse FPDMM pathobiology. Using the xenotransplant system developed and analyzed in SA#1, we will carry out long-term drug exposure studies on the Mks/Plts defects, HSPC engraftment, lineage commitment and on clonal evolution and leukemic transformation. At the moment, we have identified several candidate drugs that partially or fully correct the Mk defect. RepSox, a small molecule transforming growth factor receptor b1 inhibitor drug, corrects both Mk and Plt defects ,and dasatinib, a Src kinase inhibitor that may increase RUNX1-specific activity, will be the first candidate drugs to be tested. Thus, we believe that within the R03 timeframe, we will have established a RUNX1Lo xenotransplant model, developed new insights into the mechanistic basis of the observed defects in FPDMM, and examined a series of potential therapeutics. These advances will serve as preliminary data for external funding to support additional mechanistic and therapeutic studies of RUNX1Lo as well as clinical therapeutic trials in patients with FPDMM.