Session: 605. Molecular Pharmacology and Drug Resistance: Lymphoid Neoplasms: Poster II
Hematology Disease Topics & Pathways:
Research, Lymphoid Leukemias, ALL, Combination therapy, Adult, Translational Research, Lymphomas, Non-Hodgkin lymphoma, Drug development, T Cell lymphoma, Diseases, Aggressive lymphoma, Treatment Considerations, Lymphoid Malignancies, Metabolism, Biological Processes, Study Population, Animal model
Methods: We used the two following ASNase-R cell lines: NALM6, a human acute lymphoblastic leukemia cell line, and KHYG1, a human natural killer (NK) leukemia/ lymphoma cell line. Cells were treated in vitro with either APR-246, Asparaginase, or both and incubated for 24 to 48 hours. Cell viability was determined by flow cytometry using Annexin V / Propidium Iodide (PI). Metabolites were analyzed by mass spectrometry (UPLC-MS/MS) in the supernatant and dried pellets. To validate the metabolic findings further, we evaluated intracellular GSH and lipid peroxidation levels by flow cytometry after cell-labeling with MBCI and C11- BODIPY, respectively. To assess the drug combination's effect in vivo, we established a xenograft murine model by injecting luciferase-expressing NALM6 cells into immunocompromised mice. Mice tumor burden was evaluated using an in vivo imaging system (IVIS) based on bioluminescence quantification.
Results: Using an untargeted metabolomic approach, we showed that ASNase treatment in both ASNase-R models decreased GSH levels and reduced the GSH to oxidized GSSG ratio. Specific quantification confirmed decreased GSH levels and increased mitochondrial reactive oxygen species (ROS) in ASNase-R cells. This suggests that resistance to ASNase induces metabolic vulnerability related to cellular redox imbalance. APR-246, an antioxidant-inhibitor, was then considered to target the ASNase-induced redox imbalance. Our results showed a synergistic efficacy of APR-246 and ASNase combination in vitro, especially with Erwinase. Next, we showed that tumor growth was significantly reduced in vivo in mice treated with the combination of APR-246 and Erwinase, compared to placebo, APR-246, or Erwinase as single agents. Mechanistically, APR-246 combined with Erwinase dramatically decreased GSH levels and increased lipid peroxidation levels compared to each drug alone. Given that accumulation of lipid peroxides leads to ferroptosis, an iron-dependent mechanism of cell death, we investigated whether APR-246/Erwinase-induced cytotoxicity was mediated by ferroptosis. KHYG-1 cells were treated with Erwinase associated with a ferroptosis inducer (RSL3) and/or a ferroptosis inhibitor (ferrostatin-1). Combining Erwinase with RSL3 resulted in a similar GSH decrease, lipid peroxidation accumulation, and decreased cell viability as observed with APR-246/Erwinase combination, and cell death was partially rescued by ferrostatin-1.
Conclusion: APR-246, a p53 activator, should be also considered as the first ferroptosis-inducing agent that can be used therapeutically in humans, and our results suggest its efficacy in overcoming ASNase-R in lymphoid malignancies both in vitro and in vivo. In our work, APR-246/Erwinase combination effectively disrupts the balance between ROS generation and antioxidation dependent on glutamine/GSH metabolism in ASNase-R cells and leads to cell death by ferroptosis. Prospective phase I/II studies are now required to confirm the clinical efficacy of APR-246/Erwinase combination in ASNase-R ENKTL and ALL patients.
Disclosures: Simonin: Clinigen: Honoraria. Jaccard: pfizer: Honoraria; Jazz Pharmacueticals: Honoraria; janssen: Honoraria; sanofi: Research Funding.
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