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2656 Synthetic Lipid Nanoparticles Actively Target Acute Myeloid Leukemia (AML) Cells and Induce Ferroptosis through Decreased Expression of Glutathione Peroxidase 4

Program: Oral and Poster Abstracts
Session: 604. Molecular Pharmacology and Drug Resistance: Myeloid Neoplasms: Poster II
Hematology Disease Topics & Pathways:
Research, Acute Myeloid Malignancies, AML, Translational Research, drug development, Diseases, Therapies, metabolism, Myeloid Malignancies, Biological Processes, emerging technologies, Technology and Procedures
Sunday, December 11, 2022, 6:00 PM-8:00 PM

Adam Yuh Lin, MD, PhD1, Jonathan Scott Rink, PhD1*, Eva Yang, BS1*, Sara Small, MD, PhD2, Fransheska Serrano3*, Yasmin Abaza, MD2, Jessica K. Altman, MD1, Leonidas C. Platanias, MD, PhD4, Colby Shad Thaxton, MD, PhD5* and Leo I. Gordon, MD1

1Department of Medicine, Division of Hematology/Oncology and the Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Northwestern University Feinberg School of Medicine, Chicago, IL
2Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL
3University of Buffalo, Buffalo
4Division of Hematology-Oncology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL
5International Institute for Nanotechnology, Department of Urology, Feinberg School of Medicine, Northwestern University, Chicago, IL

Introduction: Relapsed and refractory AML patients have a 30% 5-year overall survival (SEER 2012-2018). We found that certain cancer cells that are highly dependent on cholesterol uptake and express the high-affinity receptor for cholesterol-rich high-density lipoproteins (HDL), scavenger receptor type B-1 (SR-B1), also highly express the redox enzyme, glutathione peroxidase 4 (GPX4) (Rink et al 2020). GPX4 has been shown to detoxify lipid peroxides in cancer cells and prevent cell death through ferroptosis, an iron-dependent redox form of cell death. Using a synthetic lipid nanoparticle (Au-LNP) that actively targets SR-B1, we showed modulation of cancer cell cholesterol uptake and metabolism is accompanied by a reduction in GPX4 expression. As a result, Au-LNP therapy kills these cells via ferroptosis. In AML, high GPX4 expression is associated with worse overall survival (Akiyama et al. Blood 2021). Therefore, we hypothesized that high SR-B1 expression would enable Au-LNP to target AML cells, modulate cell cholesterol, and reduce GPX4 resulting in AML cell death by ferroptosis.

Methods: GEPIA is an interactive web server that houses the RNA sequencing expression data from the TCGA and the GTEx projects. We selected the LAML database and interrogated the survival data using the top 25% and bottom 25% SR-B1 expression as cutoffs. The cell lines MV4-11 (biphenotypic B-myelomonocytic AML with translocation t(4;11) and FLT3-ITD mutation), HEL (erythroblast AML), and U937 (promonocytic AML) were used for these studies. Au-LNPs were synthesized by surface-functionalizing 5nm gold (Au) nanoparticles with apolipoprotein A-I and phospholipids. SR-B1 and GPX4 levels were measured by western blot and flow cytometry. Cell viability was measured using the MTS assay, annexin V/propidium iodide staining, and a colony forming assay. Colonies were counted by an automated microscope and analyzed with Fiji. Lipid oxidation levels were measured by a C11 BODIPY flow cytometry assay. Surface biomarker changes were measured by flow cytometry. Primary AML samples were obtained by collecting the buffy coat from peripheral blood samples from 2 patients with AML and isolating CD34 positive blasts (IRB# STU00208941).

Results: In the LAML database in GEPIA, we found a significant survival detriment with high SR-B1 expression (75th percentile) when compared with low SR-B1 expression (25th percentile; p = 0.023; hazard ratio = 2.5) (Figure 1). SR-B1 expression was observed in all three AML cell lines tested (MV4-11, HEL, U937). Au-LNPs potently induced cell death in each cell line, with IC50s for MV4-11 (4.7 nM), HEL (0.8 nM), and U937 (4.6 nM) that were much lower than with cytarabine (535 nM, 259 nM, and 96 nM, respectively). In the colony forming assays, Au-LNPs significantly decreased MV4-11 colony counts compared with PBS control. By contrast, β-Hydroxy β-methylglutaryl-CoA (HMG-CoA) inhibitors, such as atorvastatin and rosuvastatin, had no effect on cell viability, with concentrations up to 50 µM. Au-LNPs reduced GPX4 expression in MV4-11 and HEL cells, leading to the accumulation of lipid peroxides by C11 BODIPY assay. Au-LNPs-induced cell death was prevented by the addition of the lipophilic antioxidant ferrostatin-1 and the iron chelator deferoxamine (DFO), supporting the hypothesis that Au-LNP-induced cell death occurred by ferroptosis. Low dose Au-LNPs increased expression of CD11c (dendritic cell marker), CD16 (monocyte marker), and CD24 (neutrophil marker) on MV4-11 cells, suggesting an increase in myeloid differentiation. Finally, we isolated CD34+ blasts from peripheral blood samples of two AML patients. Both patients’ blasts expressed SR-B1 as detected by flow cytometry, and Au-LNPs showed significant cell killing in both patient samples.

Conclusion: We have demonstrated that Au-LNP target SR-B1, expressed by both AML cell lines and in primary AML samples, and induce cell death by ferroptosis resulting from depletion of GPX4. Future studies targeting the cholesterol scavenger receptor SR-B1 and investigating relevant resistance pathways and opportunities for synergy with available agents are indicated.

Figure 1. Overall survival of AML patients in the LAML database stratified by scavenger receptor B1 expression. (Tang, Z. et al. (2017) GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res, 10.1093/nar/gkx247)

Disclosures: Abaza: Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; ALX Oncology: Research Funding; Kite: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Altman: ImmunoGen: Research Funding; Kartos Therapeutics: Research Funding; Kura Oncology: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Research Funding; Boehringer Ingelheim: Research Funding; Loxo: Research Funding; ALX Oncology Inc: Research Funding; Aprea: Research Funding; Amgen: Research Funding; Syros: Membership on an entity's Board of Directors or advisory committees; Aptos: Research Funding; Celgene: Research Funding; Astellas: Honoraria, Research Funding; Glycomimetics: Other: Data Monitoring Committee; Fujifilm: Research Funding; Biosight: Membership on an entity's Board of Directors or advisory committees, Other: reumbursement for travel, Research Funding. Gordon: Zylem: Current equity holder in private company, Current equity holder in publicly-traded company, Patents & Royalties: Patent on nanoparticles for lymphoma therapy; Ono Pharmaceuticals: Consultancy; Janssen: Other: DSMB; BMS: Research Funding.

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