RBBP6 Interaction with the p53–MDM2 Complex Supports Plerixafor-Based Anticancer Targeting

The Retinoblastoma Binding Protein 6 (RBBP6) is a multidomain protein with an E3 ubiquitin ligase motif (RING) and negatively regulates p53 stability. While its role in cell proliferation, cellular signalling, and RNA processing is well established, the subcellular distribution and functional relevance of its association with p53 in cancer remain incompletely understood. In this study, we investigated the subcellular localization and interactions of RBBP6, p53, and MDM2 in a normal human (HEK293) and cancer cell lines. Confocal microscopy revealed that in HEK293 cells, RBBP6 localizes predominantly to nuclear speckles, whereas p53 is largely cytoplasmic, suggesting compartmental separation under basal conditions. In lung adenocarcinoma A549 cells, RBBP6 exhibited increased cytoplasmic localization and p53 accumulated in the nucleus, with some co-localization. Co-immunoprecipitation experiments performed in HEK293 and MCF-7 breast cancer cells confirmed the formation of a tripartite RBBP6/p53/MDM2 complex, demonstrating that this interaction occurs in normal and malignant cells. We further examined the functional consequences of targeting this axis using plerixafor, an FDA-approved CXCR4 antagonist previously predicted in silico to bind the RBBP6 RING finger domain. Plerixafor induced apoptosis in A549 cells at concentrations not toxic to HEK293 cells. The apoptotic effect was coupled with downregulation of p21, without changes in RBBP6 or ERK expression. Since the RBBP6/p53/MDM2 interaction influences p53 tumor suppressor activity, disrupting this axis could restore p53 function and inhibit tumor growth. The p21 perturbation suggests that plerixafor may directly or indirectly interfere with the p53 pathway, providing a basis for potential therapeutic repurposing in cancers with dysregulated p53 pathways. This work provides mechanistic insight into RBBP6-mediated p53 regulation and supports the potential repurposing of plerixafor for cancers characterized by dysregulation of the p53/MDM2/RBBP6 axis.