APR-246 Overcomes Resistance to Asparaginase in Lymphoid Malignancies By Targeting Metabolic Cell Vulnerabilities

Introduction: Cancer cells rely on metabolic reprogramming to meet the increased bioenergetic and biosynthetic demand required for tumor initiation and progression. L-asparaginase (ASNase), an enzymatic drug depleting plasmatic asparagine (ASN), is the only clinically approved targeted therapy of a specific amino acid addiction that clearly demonstrated efficacy in hematological malignancies such as acute lymphoblastic leukemia (ALL) and extranodal NK/T cell lymphoma (ENKTL). Despite the benefits of ASNase, refractory forms are observed in 40% of the patients, either by immunization against the enzyme or by metabolic adaptation to ASN deprivation. APR-246 was originally known to reactivate transcriptional activity by refolding p53 mutants. Additional p53-independent effects have been reported, including oxidative stress increase, particularly by decreasing glutathione (GSH) levels through direct covalent binding to cysteine residues. Due to its effect on antioxidant defense systems, APR-246 might represent an efficient anti-cancer therapeutic strategy in ASNase-resistant (ASNase -R) cells by targeting metabolic cell vulnerabilities. This study evaluated APR-246 as a new candidate to overcome ASNase resistance in lymphoid malignancies treated with ASNase-containing regimens as frontline therapy. 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.