Deubiquitinase Inhibitors Impair Leukemic Cell Migration Through Cofilin Oxidation and Alteration of Actin Reorganization

Abstract

Actin networks are dynamically regulated through constant depolymerization and polymerization cycles. Although the fundamental mechanisms that govern these processes have been identified, the nature and role of post-translational modifications (PTMs) of actin and actin regulatory proteins are not completely understood. Here, we employed Actin CytoFRET, a method that we developed for real time detection of fluorescence resonance energy transfer (FRET) signals generated by actin dynamics, to screen a small library of PTM-interfering compounds on a biosensor leukemic T cell line. This strategy led to the identification of small molecule inhibitors of deubiquitinating enzymes (DUBs) as potent inducers of actin polymerization and blockers of chemotactic cell migration. The examination of the underlying mechanism further revealed that the actin depolymerizing protein cofilin represents a major effector of DUB inhibitor (DUBi)-induced actin reorganization. We found that DUB blockade results in the accumulation of polyubiquitinated proteins and ROS production, associated with cofilin oxidation and dephosphorylation on serine 3, which provokes uncontrolled actin polymerization impairing cell migration. Together, our study highlights DUBs as novel regulators of actin dynamics through ROS-dependent cofilin modulation, and shows that DUBi represent attractive novel tools to impede leukemic cell migration.

Cytoskeletal dysregulation and neurodegenerative disease: Formation, monitoring, and inhibition of cofilin-actin rods

“…Deubiquitinase Inhibitors † Deubiquitinase Promote F-actin assembly independently of other pathways or through cofilin dephosphorylation (Larbret et al, 2022).…”

“…Multiple deubiquitinase (DUB) inhibitors were investigated for F-actin assembly modulated by cofilin using flow cytometry and FRET ( Larbret et al, 2022 ). Through the treatment of leukemic T cells with inhibitors WP1130 and b-AP15, DUB inhibition was found to cause a dramatic redistribution of actin to the cell membrane, preventing cell migration ( Larbret et al, 2022 ).…”

“…Multiple deubiquitinase (DUB) inhibitors were investigated for F-actin assembly modulated by cofilin using flow cytometry and FRET ( Larbret et al, 2022 ). Through the treatment of leukemic T cells with inhibitors WP1130 and b-AP15, DUB inhibition was found to cause a dramatic redistribution of actin to the cell membrane, preventing cell migration ( Larbret et al, 2022 ). By inducing DUB inhibition, actin reorganization involved a non-degradative and LIMK-independent modulation of cofilin activity, accumulation of polyubiquitinated proteins, and generation of ROS ( Larbret et al, 2022 ).…”

“…Through the treatment of leukemic T cells with inhibitors WP1130 and b-AP15, DUB inhibition was found to cause a dramatic redistribution of actin to the cell membrane, preventing cell migration ( Larbret et al, 2022 ). By inducing DUB inhibition, actin reorganization involved a non-degradative and LIMK-independent modulation of cofilin activity, accumulation of polyubiquitinated proteins, and generation of ROS ( Larbret et al, 2022 ). This response resulted in the oxidation and dephosphorylation of cofilin and may present another means of studying conditions leading to cofilin-actin rod formation.…”