Molecular and functional consequences of the Cdc42 C81Y mutation in immune-associated malignancy

Cell division control protein 42 homolog (Cdc42) is a small Rho GTPase that cycles between active GTP-bound and inactive GDP-bound states to regulate cytoskeletal dynamics and immune signalling. Pathogenic variants of Cdc42 have been linked to rare immunological disorders, including a recently described C81Y mutation associated with primary immunodeficiency (PID) and Hodgkin lymphoma. Although C81Y shares phenotypic similarities with the non-oncogenic C81F variant, it is uniquely associated with thrombocytopenia, chronic inflammation, and malignancy, suggesting distinct molecular consequences that remain poorly characterised. In this study, we first assessed the impact of the C81Y mutation expressed in HEK293T cells using biochemical approaches. Cdc42C81Y exhibited reduced interaction with canonical downstream effectors, particularly WASp and N-WASp, indicating attenuation of classical Cdc42 signalling outputs. To gain mechanistic insight into these biochemical findings, we next applied molecular modelling approaches. Computational analyses revealed that the C81Y mutation alters nucleotide-dependent conformational dynamics, stabilizes the GDP-bound state, and modifies surface electrostatic properties, consistent with impaired effector engagement. Functional assays in HEK293T cells demonstrated that Cdc42C81Y enhanced cellular migration without significantly affecting proliferation and was associated with increased secretion of IL-6 and IL-10. Together, these data suggest that the Cdc42C81Y mutation disrupts canonical Cdc42 signalling while promoting context-dependent cellular and inflammatory responses. This study provides mechanistic insight into how Cdc42C81Y may contribute to PID-associated malignancy and underscores its role at the intersection of cytoskeletal regulation and immune dysregulation.