Abstract 4710: The polymeric AMD3100 based drug PAMD-Ch17 induces its anti-leukemic effects in a CXCR4 independent mechanism

Abstract Background: Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are devastating blood cancers associated with 5-year survival rates of ~50% each in adults. We recently developed a new class of polymeric drugs called PAMDs based on the CXCR4 inhibitor AMD3100 (plerixafor). PAMDs have the potential to be more effective and less toxic therapeutics than the standard of care high-dose chemotherapy. We tested one variant (PAMD-Ch17), and found that unlike AMD3100, PAMD-Ch17 has several novel anti-leukemic effects. To understand the potential mechanism(s) for these new activities, we performed the following study. Methods: To test the role of CXCR4 in PAMD-Ch17’s activities, we generated CXCR4 knockout cells via Crispr/Cas9. We used PrestoBlue, 7-AAD, and Annexin V staining to assay viability and apoptosis. To identify pathways deregulated by PAMD-Ch17, we performed whole transcriptomic sequencing (RNA-Seq). To investigate if PAMD-Ch17 mediates its activities by inducing oxidative stress, we performed SeaHorse assays, and dihydroethidium (DHE), Mitosox, and Bodipy C11 staining. To investigate whether PAMD induces programmed cell death pathways, we used ferrostatin and deferoxamine to inhibit ferroptosis, necrostatin-1 against necroptosis, and Z-VAD-FMK against apoptosis. Results: We found that PAMD-Ch17 induces cell death in a dose dependent manner in human AML and ALL cell lines and, mouse primary leukemia cells, but not healthy mouse bone marrow cells. Surprisingly, PAMD-Ch17 induces equivalent levels of cell death in both wild type and CXCR4 knockout Jurkat cells, indicating that the only known target of the polymer is not required for its anti-leukemic effects. By RNA-Seq, we found altered expression of genes related to mitochondrial function, suggesting that PAMD-Ch17 could be targeting the mitochondria. To test this, we performed SeaHorse assays and found that PAMD-Ch17 induces a significant decrease in mitochondrial respiration. Consistent with this, we found that PAMD-Ch17 induces a significant increase in superoxide production, as well as lipid peroxidation. Using inhibitors of programmed cell death pathways, we found that preventing necroptosis, apoptosis, or ferroptosis caused only subtle effects on PAMD-Ch17 induced cell death, implying that the polymer’s anti-leukemic activity is likely not entirely dependent on these pathways. Conclusions: These results indicate that PAMD-Ch17 induces cell death in leukemia cells but not healthy bone marrow via a CXCR4 independent mechanism. We also found that PAMD-Ch17 induces reactive oxygen species, potentially explaining its selectivity for leukemia cells compared to healthy blood cells. Continued investigation of PAMD-Ch17 will not only further development of this new class of drugs, but also provide much needed insight into leukemia biology. Citation Format: Calvin Lam, Jogdeo Chinmay, Ekta Kapoor, Siyuan Tang, Svetlana Romanova, David Oupicky, Katherine Hyde. The polymeric AMD3100 based drug PAMD-Ch17 induces its anti-leukemic effects in a CXCR4 independent mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4710.