FLT3L-based conjugate targets chemoresistant leukemia stem cells via cell cycle re-entry in Acute Myeloid Leukemia Free

Acute myeloid leukemia (AML) is a genetically heterogeneous hematopoietic malignancy with poor prognosis. Standard cytotoxic chemotherapy causes significant toxicity due to its non-selective action on both malignant and healthy cells, while disease relapse is often driven by a subset of therapy-resistant leukemic cells. Among these, leukemia stem cells (LSCs) and therapy-induced senescence-like cells represent major barriers to curative treatment, as both reside in non-proliferative or quiescent states and can evade cytotoxic agents. Strategies that induce cell-cycle re-entry to sensitize these refractory populations have shown potential but require more selective and efficient delivery approaches. FLT3 is a receptor tyrosine kinase frequently mutated in AML, playing a critical role in leukemogenesis. Its high surface expression on AML blasts makes FLT3 an attractive target for antibody-drug conjugates (ADCs), which enable selective delivery of cytotoxic agents to malignant cells while sparing normal tissues.

In this study, we developed a ligand-drug conjugate, FL-Fc-DM1, which combines a FLT3 ligand-Fc fusion protein for receptor engagement and cell cycle progression with the cytotoxic microtubule inhibitor mertansine (DM1). FL-Fc-DM1 retained FLT3-binding capacity and bioactivity, induced receptor internalization, and transiently activated downstream ERK and AKT signaling in AML cells. These effects promoted AML cell progression from G1 into S and G2/M phases, sensitizing quiescent cells to DM1-induced mitotic catastrophe. FL-Fc-DM1 outperformed unconjugated DM1 in vitro, remained effective in the presence of physiological FLT3L, induced FLT3 internalization, and activated the p53 pathway. Cytarabine treatment induces a senescence-like phenotype in AML characterized by G1 arrest and upregulation of senescence-associated genes, which confer resistance. FL-Fc-DM1 reversed this phenotype by reinitiating cell cycling, reducing β-galactosidase activity, and enhancing apoptosis when used alone or following cytarabine exposure. In primary AML samples ex vivo, FL-Fc-DM1 inhibited cell proliferation, induced cell apoptosis, overcame cell cycle arrest, and inhibited colony formation. In cytarabine-pretreated cells, FL-Fc-DM1 induced apoptosis and reversed therapy-induced senescence. Mechanistically, FL-Fc-DM1 upregulated pro-apoptotic genes while increasing anti-apoptotic MCL1, suggesting compensatory survival signaling. Co-treatment with the MCL1 inhibitor AZD-5991 or the BCL2 inhibitor venetoclax synergistically enhanced cell death, particularly in FLT3-ITD-positive AML cells. In vivo, FL-Fc-DM1 significantly reduced leukemic burden and prolonged survival in HCD-57 transformed by oncogenic hFLT3-ITD and multiple AML PDX models, including FLT3 wild-type and FLT3-ITD subtypes. It markedly reduced spleen size, human CD45⁺ infiltration in liver and spleen, and systemic leukemia infiltration. Neither FL-Fc nor DM1 alone showed comparable efficacy. Serial transplantation and limiting dilution assays demonstrated that FL-Fc-DM1 selectively depleted LSCs, impairing leukemia-initiating potential.

Importantly, at therapeutically relevant concentrations, FL-Fc-DM1 selectively suppressed colony formation of AML-derived CD34⁺ cells while sparing healthy donor CD34⁺ hematopoietic stem cells ex vivo. Consistently, in a humanized mouse model reconstituted with healthy human CD34⁺ cells, FL-Fc-DM1 treatment preserved normal hematopoiesis, immune cell populations, and body weight, demonstrating minimal off-target toxicity and excellent tolerability, suggesting a favorable safety profile for clinical application.In conclusion, FL-Fc-DM1 is a promising therapeutic strategy that exploits ligand-induced cell cycle re-entry to selectively target chemoresistant AML cells and LSCs through mitotic disruption, offering a novel approach for treating relapsed/refractory AML.