Regulation of CD47-mediated cell death by p21-activated kinases

CD47, a cell-surface glycoprotein widely recognized as a "don't eat me" signal, also serves as a receptor capable of triggering caspase-independent cell death. While actin dynamics have been implicated in this process, the upstream signaling regulators remain poorly defined. In this study, we identify p21-activated kinases (PAKs) as critical negative regulators of CD47-mediated cell death in acute lymphoblastic leukemia (ALL). Using phospho-proteomic and Inferred Kinase Activity (INKA) analysis, we observed that CD47 ligation with the monoclonal antibody CC2C6 leads to the downregulation of actin-regulatory kinases, including PAK1, PAK2, and PAK4, in patient-derived xenograft (PDX) models and Jurkat T-ALL cells. Pharmacological inhibition of PAKs using the pan-PAK inhibitor PF-3758309 markedly synergized with CD47-antibody CC2C6 to induce cell death across multiple B- and T-ALL cell lines. This synergy extended to inhibitors of other actin regulators, including ROCK, PKD, and the Rho-family GTPases, Cdc42 and Rac1. Mechanistically, we demonstrate that hypersensitivity to CD47 ligation in a novel Jurkat substrain (Jkt75) correlates with reduced basal PAK activity and increased levels of active (dephosphorylated) cofilin. We confirm that CD47-mediated death requires dynamic F-actin remodeling, as both the actin depolymerizer cytochalasin D and the stabilizer jasplakinolide significantly attenuated cell death. Furthermore, we reveal that CD47 ligation triggers robust cell-cell aggregation, which is actin-dependent and essential for the lethal signal; preventing physical cell-cell contact through rotation or immobilization effectively abolished the death response. Our findings establish a novel PAK-actin-aggregation axis that governs CD47-mediated programmed cell death. These results suggest that targeting PAK signaling may provide a potent strategy to enhance the efficacy of CD47-based immunotherapies in refractory leukemias.