Abstract 553: Drp1 knockdown impairs mitophagy and sensitizes AML cells to venetoclax by inducing apoptosis

Abstract Background: Mitochondrial structural dynamic of fission and fusion impacts mitochondrial function as well as mitochondrial quality control by facilitating mitophagy and ROS mitigation. Mitophagy as well as the expression of DRP1, a dynamin related protein driving mitochondrial fission, are increased in acute myelogenous leukemia (AML) resistant to Bcl-2 inhibitor venetoclax. Our previous findings demonstrated that ERK1/2 inhibition reduced the phosphorylation of Drp1, a dynamin-like GTPase involved in mitochondrial fission, and overcame resistance to venetoclax in acute myeloid leukemia (AML). While Drp1-driven mitochondrial fission acts as a mitophagy checkpoint, the precise relationship between Drp1 and mitophagy is not fully elucidated in AML. Methods: Effect of doxycycline induced Drp1 knock down on viable cell count and apoptosis in AML cells was determined by bead-based annexin-V assay. Mitochondrial length was assessed using super resolution imaging with OMX Blaze. Mitochondrial ROS levels, mitochondrial mass, LC3 lipidation (autophagosome maturation), mitophagy and autophagosome content were determined using flowcytometry. Results: Mitochondrial fission driven by Drp1 acts as a mitophagic checkpoint to regulate mitochondrial quality. Drp1 overexpression in OCIAML3 cells resulted in smaller mitochondria and higher LC3 lipidation, while knockdown increased mitochondrial length with a decrease in autophagic vesicles and LC3B lipidation suggesting decreased autophagy. Conditional knockdown of Drp1 inhibited mitophagy (p<0.0001), as detected with flowcytometry using the mitophagy dye that detects mitochondrial acidification resulting from fusion of mitochondria to lysosomes. Additionally, Drp1 knockdown significantly increased mitochondrial ROS with no change in mitochondrial mass. Knockdown of Drp1 significantly inhibited the clonogenic potential of AML cells, leading to a marked reduction in colony formation compared to control cells supporting that mitochondrial fission, ROS mitigation and mitophagy are crucial for preserving the stem-like properties of leukemia stem cells in AML. Moreover, conditional knockdown of Drp1 sensitized OCIAML3 cells to venetoclax (p<0.0001) as well as Idarubicin (p<0.0001) as demonstrated by a reduction in viable cell count and an increase in apoptosis. Conclusion: Drp1 knockdown reduces mitochondrial fission and mitophagy in AML cells and sensitizes AML cells to venetoclax. These results further elucidate the role of Drp1 in mitochondria and offer new mechanistic insights into the interplay between mitophagy and mitochondrial fission in AML, highlighting the potential therapeutic benefit of co-targeting Drp1 in venetoclax-based regimens to eliminate leukemia stem cells. Ongoing studies are investigating the molecular mechanisms and in vivo efficacy of Drp1 knockdown in AML. Citation Format: Priyanka Sharma, Hiroki Akiyama, M. Anna Zal, Bing Z. Carter, Michael Andreeff, Gautam Borthakur. Drp1 knockdown impairs mitophagy and sensitizes AML cells to venetoclax by inducing apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 553.