Liposomal-Mitoxantrone Overcomes Acquired Resistance and Enhances Venetoclax Synergy in AML-Mechanistic Insights and Preliminary Clinical Evidence

Introduction: Liposomal drug delivery has emerged as a promising strategy to improve the efficacy and safety of chemotherapeutic agents, yet whether and how liposomal reformulation fundamentally reshapes therapeutic response and resistance evolution remains unclear. This study compared liposomal mitoxantrone with free mitoxantrone and examined their effects on antileukemic activity, hematologic toxicity, resistance development, and cell-death pathways. Methods: Liposomal and free mitoxantrone were evaluated in an MLL-AF9-driven acute myeloid leukemia mouse model by survival analysis and peripheral blood measurements. Secondary transplantation experiments were performed to evaluate the sensitivity of residual leukemia cells. Single-cell RNA sequencing was used to characterize transcriptional features of residual leukemia cells. Drug combination with venetoclax and the cell killing pattern of the two drugs were explored based on MOLM13 cell line. Exploratory clinical observations were additionally obtained from four patients who received liposomal mitoxantrone in combination with venetoclax. Results: Liposomal mitoxantrone significantly prolonged mouse survival compared with free-mitoxantrone and control groups (both p < 0.001), while causing minimal myelosuppression. In contrast, free-mitoxantrone induced marked hematologic toxicity. Secondary transplantation demonstrated that residual cells following free-mitoxantrone treatment developed resistance upon re-exposure (p < 0.001), while those from liposomal-treated mice retained drug sensitivity and conferred sustained survival benefit (both p < 0.01). Single-cell RNA sequencing revealed that free-mitoxantrone induced transcriptional heterogeneity, characterized by enhanced metabolic and immune-related pathways, and monocyte-like features, whereas liposomal-mitoxantrone preserved a proliferative state similar to untreated cells. Consistently, liposomal-mitoxantrone demonstrated stronger synergistic effects with venetoclax. Discussion: Liposomal reformulation altered the therapeutic, transcriptional, and cell-death pattern of mitoxantrone in the models. These findings suggest that liposomal mitoxantrone may reduce hematologic toxicity and limit the emergence of drug resistance.