Covalent Targeting of ACTN4 Disrupts Cytoskeletal Control of TfR1 Recycling and Drives Ferroptosis in Acute Myeloid Leukemia

Background: Ferroptosis is a form of regulated cell death driven by iron-dependent lipid peroxidation. Its regulation has been mainly studied at the metabolic level. The role of cytoskeleton-dependent trafficking in ferroptosis remains unclear. Methods: Chemical proteomics was used to identify protein targets of pseudolaric acid B (PAB). Binding between PAB and ACTN4 was validated by CETSA, DARTS, biophysical assays, and mutagenesis. The effects on cytoskeletal organization, endosomal trafficking, iron homeostasis, and ferroptosis were evaluated using cellular and molecular experiments. Results: ACTN4 was identified as a direct target of PAB. PAB binds to lysine 273 within the actin-binding domain. This interaction disrupts the association between ACTN4 and F-actin. It leads to cytoskeletal instability and impaired endosomal trafficking. As a result, transferrin receptor 1 accumulates intracellularly instead of being recycled. This promotes iron overload and lipid peroxidation. Mutation of ACTN4 at K273 reduces PAB binding and attenuates iron accumulation. Stabilization of F-actin restores receptor distribution and partially rescues iron imbalance. Ferroptosis inhibitors suppress lipid peroxidation but do not correct cytoskeletal defects. Conclusions: PAB targets ACTN4 and disrupts cytoskeletal integrity. This impairs TfR1 trafficking and promotes iron accumulation. Cytoskeletal dysfunction acts upstream of ferroptosis. This study identifies ACTN4 as a regulator of ferroptosis and reveals a mechanism linking actin dynamics to iron metabolism.