Abstract P026: NUC-7738 alters oxidative phosphorylation and causes terminal differentiation in acute myeloid leukemia cells

Abstract

Abstract Background NUC-7738, a phosphoramidate transformation of 3’deoxyadenosine (3’dA), is specifically designed to generate the active anti-cancer metabolite 3’-deoxyadenosine monophosphate (3’-dAMP) directly in cells, bypassing key cancer resistance mechanisms of transport, activation and breakdown. NUC-7738 is currently in a Phase I clinical study to assess safety and determine the recommended dose in patients with advanced solid tumors and lymphoma. Acute myeloid leukemia (AML) cells exhibit impaired differentiation, uncontrolled proliferation and are highly dependent on mitochondrial metabolic processes for survival and chemoresistance. The aim of this study was to determine the effect of NUC-7738 on oxidative phosphorylation (OXPHOS) proteins and resulting changes in mitochondria morphology and activation of the intrinsic apoptotic pathway. Methods AML cell lines OCI-AML3 and HL-60 were treated with NUC-7738 for 48 hours. Cell differentiation was determined by morphologic modification with Giemsa stain and assessing the expression of surface markers CD11b and CD163 by flow cytometry. Immunoblotting was performed for Mitochondrial Electron Transport Chain Complex I subunit NDUFB8, Complex II subunit SDHB, Complex III subunit UQCRC2, Complex IV subunit COX II and ATP synthase subunit ATP5A. Mitochondria morphology was assessed by confocal microscopy with MitoTracker-Deep Red CMXRos dye. Annexin-V, DAPI, Bcl-2, cytochrome c, and cleaved-caspase 7 were assessed by flow cytometry to determine intrinsic apoptosis. Results NUC-7738 increased the proportion of CD11b+ and CD163+ cells in a dose-dependent manner from 6.1% to 28.7% and 9.6% to 26.9%, respectively. CD11b positivity was also increased in HL-60 cells from 14.5% to 31.1%. NUC-7738 induced morphological differentiation as indicated by decreased nucleo-cytoplasmic ratio, appearance of multi-lobed nuclei and vacuolization of the cytoplasm in OCI-AML3 cells. Decreased nucleo-cytoplasmic ratio was also evident in HL-60 cells. NUC-7738 decreased the protein expression of all 5 complexes of oxidative phosphorylation in OCI-AML3 cells at 48 hours and this metabolic disruption was also evident from distinct changes in mitochondria morphology. NUC-7738 activated the intrinsic apoptotic pathway, causing sequestration of BCL-2 and release of cytochrome c, resulting in a decrease in the percentage of live OCI-AML3 cells from 76% to 37% at 48 hours. Conclusion Through the ability of NUC-7738 to inhibit OXPHOS complexes with consequent terminal differentiation and activation of mitochondrial-mediated apoptosis, it exploits the mitochondrial characteristics of certain cancer cells, including their greater dependence on OXPHOS for survival than normal cells. NUC-7738 offers a potential treatment option for patients with a variety of malignancies. Citation Format: Akbar M. Shahid, In Hwa Um, Oliver J. Read, David J. Harrison. NUC-7738 alters oxidative phosphorylation and causes terminal differentiation in acute myeloid leukemia cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P026.