Quizartinib enhances conditioning for hematopoietic stem cell transplantation via transcriptional suppression of DNA repair pathways

Abstract Background: Quizartinib, a FLT3 inhibitor, has been shown to induce quiescence in healthy hematopoietic stem and progenitor cells (HSPCs), affording protection from S-phase-specific chemotherapies. In this study we investigated whether quizartinib can be combined with other chemotherapies and radiotherapy for therapeutic benefit. Methods C57BL/6 mice were treated with quizartinib (30 mg/kg) or vehicle by oral gavage and subsequently exposed to DNA-damaging chemotherapies or total body irradiation (TBI) at 2–6 hours later. HSPCs were analyzed via flow cytometry (including phospho-flow) and RNA-sequencing at defined timepoints post-treatment. To assess HSC transplantation efficacy, mice were conditioned with 700 or 1000 Rads of TBI, transplanted with 2 million CD45.1 bone marrow (BM) cells, and monitored for donor cell engraftment for 4 months. Results Quizartinib synergized with DNA damaging chemotherapeutic agents (cyclophosphamide, carboplatin, cisplatin, temozolomide, and mitoxantrone) to deplete HSPC populations. Flow cytometry revealed significant reductions in myeloid and multipotent progenitors, as well as short- and long-term HSCs in quizartinib-primed mice compared to controls. When combined with 5.5 Gy TBI (half a standard conditioning dose), quizartinib resulted in profound myeloablation. Even at 3 Gy, quizartinib maintained potent depletion of total BM, progenitor, and stem cells, with sensitization persisting when dosed 24 hours prior to TBI. To explore underlying mechanisms, we performed RNA-sequencing of sorted LSK and LK cells 20 hours post-treatment. Quizartinib downregulated genes critical for DNA damage repair, including Brca1, Brca2, Rad51, and Nbn, in both murine (LSK cells) and human (CD34+) HSPCs. Ionizing radiation exposure in quizartinib-primed HSPCs caused elevated and prolonged γ-H2AX and phospho-ATM signaling, indicating impaired DNA repair and persistence of DNA damage. We next evaluated the effect of quizartinib upon hematopoeitic stem cell transplant (HSCT) by treating mice with quizartinib, or vehicle, followed by 700–1000 Rads TBI and BM transplantation. Quizartinib treated mice exhibited enhanced donor-derived multilineage reconstitution compared to vehicle treated mice suggesting that quizartinib enhances myeloablative conditioning by augmenting host HSPC clearance. Conclusions Quizartinib sensitizes HSPCs to DNA-damaging therapies via downregulation of DNA repair genes and impaired resolution of DNA damage, resulting in enhanced HSPC depletion. This quizartinib-driven sensitization demonstrated improved engraftment after HSCT in murine models. These results provide a rationale for incorporating quizartinib into reduced-intensity, myeloablative conditioning regimens to improve transplant outcomes while minimizing non-hematopoietic toxicity.