PALB2 HOMODIMERIZATION: INSIGHTS FROM MISSENSE VARIANTS

INTRODUCTION AND OBJECTIVE: Approximately 10% of all cancer cases have a hereditary origin, the majority caused by dysfunctional mutations in genes that play a role in the DNA damage response (DDR). PALB2 was identified in the early 2000s and readily associated with hereditary predisposition to breast and pancreatic cancer. It encodes a homonymous protein that plays a key role in Homologous Recombination (HR)-mediated DDR together with BRCA1 and BRCA2. Mechanistically, PALB2 is recruited to DNA damaged sites through its heterodimerization with BRCA1, which was shown to be regulated by post-translational modifications in PALB2, such as phosphorylation and ubiquitination. PALB2 interaction with BRCA1 is mediated by its coiled-coil domain, which also promotes its homodimerization. The identification of PALB2 variants in the population has increased in recent years, with different studies seeking to functionally analyze the impact of these alterations in HR proficiency. Little is still known about the effect of these mutations in PALB2 homodimerization. Previous reports proposed a control for HR based on the balance between PALB2 homodimerization and its heterodimerization with BRCA1; however, the mechanisms that modulate these interactions have not yet been totally elucidated. Here, we attempt to functionally characterize PALB2 genetic variants regarding their homodimerization proficiency and evaluate the modulating mechanisms of PALB2 homodimerization. MATERIAL AND METHOD: PALB2 homodimerization was evaluated by Bimolecular Fluorescence Complementation (BiFC) assay and by co-immunoprecipitation. PALB2 mutations were generated by site-directed mutagenesis. RESULTS AND CONCLUSION: We are assessing multiple PALB2 missense variants located within its coiled-coil domain and adjacent regions for their ability to form homodimers. Preliminary data suggest reduced homodimerization capacity for p.L24A and p.L35P variants, which is consistent with prior findings. In contrast, both the p.K18R and p.Y28C exhibited distinct homodimerization proficiency compared to previously described reports. Four variants, p.K30N, p.L32V, p.R37H and p.H46Y, displayed a wild-type-like phenotype and the remaining variants, p.L169I and p.S319Y, showed an increased homodimerization ability compared to the wild type protein. Using PALB2 phosphomimetic and phosphodeficient mutants targeting ATR and CDK sites, we uncover that PALB2 phosphorylation status modulates its homodimerization in an BRCA1-dependent manner, as it was only observed in BRCA1-proficient cells. Our study seeks to understand the relationship between PALB2 homodimerization and genomic integrity maintenance and functionally characterize PALB2 variants according to their homodimerization proficiency.