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PACSIN2 regulates mercaptopurine cytotoxicity and cellular biomechanics through Rac1 modulation in intestinal
Alessia Norbedo1, Giulia Zudeh2, Donatella Fiducioso3
1Department of Medicine Surgery and Health Sciences, University of Trieste, Trieste, Italy.
Introduction:
Mercaptopurine (MP) is used to treat pediatric acute lymphoblastic leukemia and inflammatory bowel disease (IBD). The Protein Kinase C and Casein Kinase Substrate in Neurons 2 (PACSIN2) rs2413739 polymorphism is an expression-quantitative trait locus associated with reduced PACSIN2 expression in gastrointestinal tissues and thiopurine-related gastrointestinal toxicity. PACSIN2 interacts with Rac1, a key thiopurine target protein involved in cytoskeletal regulation.
Methods:
LS180 intestinal cells with stable PACSIN2 knockdown (KD) or transient overexpression were used to assess the role of PACSIN2 in regulating Rac1 under basal conditions and after MP exposure. Rac1 levels and activity were measured by Western blot and G-LISA assay, respectively. Cytotoxicity was evaluated by MTT assay following exposure to MP and the Rac1 inhibitor NSC23766. Cell stiffness was assessed by atomic force microscopy, and cytoskeletal organization was examined by immunofluorescence.
Results:
PACSIN2 KD cells exhibited elevated Rac1 expression and activity, and increased sensitivity to MP, which was further augmented by the Rac1 inhibitor NSC23766. Conversely, PACSIN2 overexpression reduced Rac1 levels and conferred MP resistance. PACSIN2 depletion decreased cell stiffness affecting the cytoskeletal architecture, particularly impairing tubulin levels and actin distribution. MP exposure reduced cell stiffness and decreased both Rac1 expression and activity across all tested cell lines, with more pronounced effects in LS180 KD cells. Altered cytoskeletal organization, especially actin redistribution, correlated with changes in cellular biomechanical properties and drug sensitivity.
Conclusion:
These findings suggest that PACSIN2 modulates MP response by regulating Rac1 levels and cytoskeletal integrity, providing a molecular basis for the clinical association between PACSIN2 and thiopurine-related gastrointestinal toxicity. These insights may contribute to the development of personalized approaches to optimize thiopurine therapy in pediatric patients.
