Inhibition of Drp1, a mitochondrial fission GTPase disrupts mitochondrial quality control and impairs stem-like properties in Acute Myeloid Leukemia

Abstract Introduction: Mitochondrial fission is a critical aspect of mitochondrial dynamics, essential for maintaining mitochondrial function and quality control through mitophagy and reactive oxygen species (ROS) mitigation (Ni et al., Redox Biology, 2015). Mitochondrial fission and mitophagy are upregulated in acute myeloid leukemia (AML) stem cells indicating enhanced need for quality control (Pie et. al., Cell Stem Cell., 2018, Schimmer et al., Cell Stem Cell., 2018). Our previous work revealed that inhibition of ERK1/2, an upstream kinase for Drp1, a GTPase that mediates mitochondrial fission, reduces Drp1 phosphorylation, thereby suppressing its activity and impairs mitophagy and ROS mitigation in AML (Sharma et. al., Blood, 2023). Since, ERK1/2 inhibition affects targets beyond Drp1 phosphorylation, the exact interplay between Drp1, mitophagy, and stemness in AML remains unclear. Methods: Single-cell RNA sequencing (scRNA-seq) was used to compare Drp1 expression in bone marrow (BM) samples from AML patients versus healthy controls. Drp1 was conditionally knocked down via doxycycline/tetracycline inducible system. Mitochondrial length was measured using super-resolution structured illumination microscopy (OMX). Additional analyses included assessment of LC3 lipidation (autophagy) by western blot and flow cytometry, and mitochondrial ROS, mitochondrial mass, autophagic vesicles (Autophagy-CYTO ID), and mitophagy-all measured by flow cytometry. The effect of Drp1 knockdown on AML engraftment and overall survival was evaluated in NSG mice (6 per group). Results: For scRNA-seq, 17 BM samples (total 39,607 cells) from 12 AML patients (median age 67) and 3 healthy controls were integrated and annotated into 23 transcriptional cell clusters. In the Uniform Manifold Approximation and Projection (UMAP) plot, a leukemic stem cell (LSC)-like cluster overlapping with healthy hematopoietic stem cells (HSCs), along with an AML-specific cluster and a progenitor-like AML cluster, were identified. The LSC like cluster, was refractory to induction treatment with decitabine-venetoclax, and showed significantly higher Drp1 (DNM1L) expression than healthy HSCs (FC=1.46, adjusted p = 0.02). Drp1-mediated mitochondrial fission functions as a mitophagy checkpoint, crucial for maintaining mitochondrial quality. Drp1 overexpression in OCI-AML3 cells led to smaller mitochondria suggesting enhanced fission (p<0.0001) and increased LC3 lipidation (p<0.0001), indicating enhanced autophagy. In contrast, Drp1 knockdown resulted in mitochondrial elongation, indicating impaired fission, along with fewer autophagic vesicles (p<0.0001) and reduced LC3B lipidation, reflecting diminished autophagy. Conditional Drp1 knockdown significantly impaired mitophagy (p<0.0001), as detected by flow cytometry using a mitophagy specific dye that detects mitochondrial acidification upon lysosomal fusion. This was accompanied by a marked increase in mitochondrial ROS (p<0.0001), without changes in mitochondrial mass. Functionally, Drp1 knockdown reduced clonogenic capacity and colony formation (p<0.0001), highlighting the importance of mitochondrial fission, ROS mitigation, and mitophagy in maintaining leukemic stem-like properties. Additionally, Drp1 knockdown sensitized intrinsically resistant OCI-AML3 cells to venetoclax (p < 0.0001). In vivo, conditional Drp1 knockdown significantly impaired AML engraftment (p<0.01) and improved survival in NSG mice (Median survival: Sh Drp1=55 days versus Sh Control = 28 Days, p=0.0006). Conclusion: Our study demonstrates that Drp1 is critical for maintaining mitochondrial quality control and stemness in AML via regulation of mitochondrial fission and mitophagy. scRNA-seq revealed elevated Drp1 expression in an LSC-like blast cluster associated with refractory AML. Functional experiments confirmed that Drp1 knockdown disrupts mitochondrial fission, impairs mitophagy, increases mitochondrial ROS, and compromises clonogenic capacity. In vivo, conditional Drp1 knockdown significantly reduced AML engraftment and improved survival in NSG mice. These findings identify Drp1 as a critical regulator of LSC fitness and highlight mitochondrial fission as a potential vulnerability that could be exploited for therapeutic intervention in AML. To assess the translational relevance for AML therapy, ongoing experiments are evaluating the efficacy of a Drp1 GTPase inhibitor and an inhibitor targeting Drp1 recruitment to mitochondria via FIS1.