Salinomycin Nanoparticles Induce Ferroptosis and Synergize with the BCL-2 Inhibitor Venetoclax to Promote AML Cell Death

Background: Salinomycin, is a polyether ionophore antibiotic used in poultry farming which was found to target cancer stem cells. Its anticancer effects are pleiotropic, but include ferroptosis, the toxic accumulation of labile iron pools in mitochondria, potentially in lysozymes. Without encapsulation in nanoparticles, it is highly toxic to normal cells. We aimed to determine if salinomycin enclosed in a nanoparticle (SAL-NP) might inhibit leukemia cells without untoward effect on normal stem cells, and if so, what drugs would be optimally combined with this agent in both in vivo and in vitro systems using human leukemia cell lines and primary AML blasts from patients. Methods: SAL-NP were obtained from the Hillstream Biosciences (Bridgewater, New Jersey) and used at concentrations ranging from 0.25 to 2 uM (based on SAL). We treated human AML cell lines (MOLM-14, MOLM-13, MV4-11, HL-60, and THP-1) including venetoclax-resistant (VEN-R) THP-1 cells as well as primary AML blasts from patients with SAL-NP alone and in combination with decitabine, midostaurin, S63845 (MCL-1 inhibitor) and venetoclax and measured cell growth by trypan blue exclusion assay, cell death by propidium iodide (PI) staining and colony formation. Dynamic BH3-profiling (DBP), a functional assay that measures changes in cancer cells' proximity to apoptosis and their dependencies on individual anti-apoptotic proteins upon drug treatment, was performed in MV4-11 AML cells treated with SAL-NPs or VEN. To define the mechanism underlying the anti-AML effect we measured PARP and Caspase 3-cleavage, c-MYC and GPX4 by western blotting, labile iron pools, and generation of reactive oxygen species (ROS). We assessed mitochondrial integrity by scanning electron microscopy. We inoculated SCID mice subcutaneously with 2 x 106 of luciferase labeled Mv4-11 cells and after one week treated with SAL-NP at 5 mg/kg IV twice a week for 3 weeks and assessed leukemia growth by bioluminescence and overall survival plotted by Kaplan-Meier curves. Results: IC 50s for cell growth and colony formation in a variety of AML cells including VEN-R and primary AML blasts ranged from 0.25-2 uM SAL-NP. Cell death was significantly induced by SAL-NP at similar concentrations. SAL-NP treatment of SCID mice (n=6) inoculated with MV4-11 cells resulted in significant tumor growth inhibition and prolongation of survival without apparent toxicity. Dynamic BH3 profiling revealed an increased dependency on BCL-2 and BFL-1. Additionally, the mechanism of action of SAL-NP appeared to also be through ferroptosis based on induction of labile iron pools, increase in reactive oxygen species, inhibition of GPX4 levels and induction of transferrin receptor (TfR-1) expression. We reasoned that the distinct mechanisms of induction of cell killing (apoptosis for VEN and ferroptosis for SAL-NP) might lead to synergistic activity. Indeed, SAL-NP and VEN synergistically impaired cell growth, promoted cell death, and disrupted mitochondrial anatomy and membrane potential without appreciable effects on normal PBMCs. SAL-NP in combination with VEN substantially reduced c-MYC and GPX4 levels and significantly induced cleavage of Caspase-3 and PARP compared with either agent alone. Conclusion: SAL-NP is a promising anti-AML agent. Its mechanism of action may involve the promotion of ferroptosis and increased dependency on BCL-2, which may account for the apparent activity in VEN-R cells and suggests the potential clinical benefit of combining SAL-NP with BCL-2 inhibitors.