A potential drug to overcome the venetoclax resistance challenge in AML: Mitoxantrone hydrochloride liposome

Abstract The venetoclax combined with azacitidine regimen has greatly improved the prognosis of acute myeloid leukemia (AML). However, approximately 42% of patients receiving the combined regimen exhibit resistance or experience relapse. What's more, patients who fail of venetoclax-based regimen have an exceptionally poor prognosis, with a median survival of < 3 months and no effective salvage therapies currently available. To overcome venetoclax resistance in AML, our team previously screened an FDA-approved compound library using our established venetoclax-resistant AML cell lines (MV4-11-VR and MOLM-13-VR) and identified multiple TOP2 inhibitors as effective against the resistant cells. Here, we further calculated the synergy index between those TOP2 inhibitors and venetoclax in MV4-11-VR and MOLM-13-VR via SynergyFinder, demonstrating that mitoxantrone exhibited the highest synergy score with venetoclax. We then evaluated liposomal mitoxantrone hydrochloride (Lipo-MIT), a novel liposomal-encapsulated formulation with mitoxantrone as the pharmacologically active ingredient, which is reported to have superior safety and efficacy compared to conventional mitoxantrone formulations. Interestingly, we found that Lipo-MIT exhibited a higher synergy index with venetoclax in MV4-11-VR cells. Additionally, Lipo-MIT could enhance venetoclax sensitivity across multiple AML cell lines and overcome venetoclax resistance in MV4-11-VR and MOLM-13-VR. Flow cytometry apoptosis assays and mitochondrial membrane potential measurements confirmed that Lipo-MIT combined with venetoclax induced apoptosis in AML cells. The results were validated by Western blotting, that the combination induced a decrease in apoptosis-related proteins, including the key venetoclax resistance protein MCL-1. To further investigate the mechanism by which Lipo-MIT overcomes venetoclax resistance in AML, we performed transcriptomic analysis of control, Lipo-MIT, venetoclax, and combination groups. The results showed that both Lipo-MIT and the combination groups exhibited suppression of DNA replication and DNA damage repair pathways, consistent with the function of TOP2 inhibitors. Subsequent Western blot analysis revealed elevated expression of DNA replication and damage repair-related proteins (e.g., TOP2A/TOP2B and ATR/ATM) in venetoclax-resistant cells compared to parental cells. We next established AML xenografts in NOD/SCID mice using luciferase-labeled MOLM-13 cells. Bioluminescent imaging showed that Lipo-MIT combined with venetoclax significantly suppressed AML progression and flow cytometry analysis also confirmed a markedly decreased leukemia burden in bone marrow. No significant differences in body weight were observed between groups, and the combination therapy significantly prolonged the survival of AML mice. Finally, we designed a phase I clinical study to assess the safety and preliminary efficacy of the Lipo-MIT combined with venetoclax and azacitidine (MAV) regimen in patients with relapsed or refractory AML. Preliminary results were presented at the EHA 2025 Congress (Abstract PS1516). Notably, among 10 patients with prior venetoclax therapy, 6 (60%) achieved complete remission after one cycle. At the cutoff date of May 2025, the median follow-up time was 7.6 months (range 1.5–10.5 months). The 6-month relapse-free survival (RFS) and overall survival (OS) rates were 83.3% and 72.7%, respectively. Our study suggests that the MAV regimen may serve as a potential salvage therapy for AML patients who have failed venetoclax-based treatment. A phase II clinical trial is currently underway to further validate the efficacy and safety of the MAV regimen.