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2793 APR-246 Overcomes Resistance to Asparaginase in Lymphoid Malignancies By Targeting Metabolic Cell Vulnerabilities

Program: Oral and Poster Abstracts
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
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Amira Marouf, MD, PhD1, Raphaël Liévin, MD2*, Mathilde Bonnet des Claustres3*, Matthias Titeux, PhD3*, Mathieu Simonin4*, Manon Vavasseur5*, Amandine Mainreck6*, Mehdi Latiri7*, Aurore Touzart, MD, PhD8*, Marie Bouillié9*, Olivier Pellé10*, Nicolas Gaidot11*, Mathieu Rocquet12*, Didier Bouscary, MD, PhD13*, Arnaud Jaccard, MD, PhD14*, Vahid Asnafi, MD, PhD8*, Olivier Hermine, MD, PhD15,16*, Guillaume P Andrieu, PhD17,18* and Lucile Couronné, MD, PhD19*

1INSERM U1163 Laboratory of cellular and molecular mechanisms of hematological disorders and therapeutic implications, Institut IMAGINE, Université Paris Cité, PARIS, France
2INSERM U1163 Laboratory of molecular mechanisms of hematological disorders and therapeutic implications, Institut IMAGINE, Université Paris Cité, Paris, France
3INSERM UMR 1163, Laboratory of Genetic Skin Diseases, Imagine Institute, Paris, France
4INSERM UMR_S 1151, Laboratory of Onco-Hematology, Necker Enfants-Malades Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), Université Paris Cité, Paris, Paris, France
5INSERM U1163 Laboratory of cellular and molecular mechanisms of hematological disorders and therapeutic implications, Institut IMAGINE, Paris, France
6LEAT platform – Animal Facility, Imagine Institute-SFR Necker, Paris Cite University, France., Imagine Institute-SFR Necker, Paris, France
7INSERM UMR_S 1151, Necker Enfants Malades Institute (INEM), Laboratory of Onco-Hematology, Assistance Publique-Hopitaux De Paris (AP-HP), Paris, France
8Laboratory of Onco-Hematology, Necker Enfants-Malades Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), Université Paris Cité, Paris, France
9INSERM 1163, Laboratory of cellular and molecular mechanisms of hematological disorders and therapeutic implications, Institut IMAGINE, Paris, France
10Plateforme de Cytométrie en Flux, Structure Fédérative de Recherche Necker, INSERM US24-CNRS UMS3633, Paris, France
11INSERM UMR_S 1151, Necker Enfants Malades Institute (INEM), Laboratory of Onco-Hematology, Assistance Publique-Hopitaux De Paris (AP-HP), Université Paris Cité, paris, France
12Department on Hematology, Necker Enfants Malades, Paris, France
13Hematology, Cochin Hospital, Greater Paris University Hospitals (AP-HP), Paris, France
14Hématologie Clinique, CHU de Limoges, Limoges, France
15INSERM U1163 Laboratory of cellular and molecular mechanisms of hematological disorders and therapeutic implications, Institut IMAGINE, Université Paris Cité, Paris, France
16Hopital Necker, Paris, France
17Laboratory of Onco-Hematology, Necker Children's Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France
18INSERM UMR_S 1151, Laboratory of Onco-Hematology, Institut Necker Enfants Malades (INEM), Assistance Publique-Hopitaux De Paris (AP-HP), Université Paris Cité, Paris, France
19INSERM 1163, Laboratory of cellular and molecular mechanisms of hematological disorders and therapeutic implications, Institut IMAGINE, Université Paris Cité, Paris, France

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.

Disclosures: Simonin: Clinigen: Honoraria. Jaccard: pfizer: Honoraria; Jazz Pharmacueticals: Honoraria; janssen: Honoraria; sanofi: Research Funding.

*signifies non-member of ASH