ABSTRACT
PURPOSE: Inherited pathogenic variants in PALB2 are associated with increased risk of breast and pancreatic cancer. However, the functional and clinical relevance of many missense variants of uncertain significance (VUS) identified through clinical genetic testing is unclear. The ability of patient-derived germline missense VUS to disrupt PALB2 function was assessed to identify variants with potential clinical relevance. METHODS: The influence of 84 VUS on PALB2 function was evaluated using a cellular homology directed DNA repair (HDR) assay and VUS impacting activity were further characterized using secondary functional assays. RESULTS: Four (~5%) variants (p.L24S,c.71T>C; p.L35P,c.104T>C; pI944N,c.2831T>A; and p.L1070P,c.3209T>C) disrupted PALB2-mediated HDR activity. These variants conferred sensitivity to cisplatin and a poly(ADP-ribose) polymerase (PARP) inhibitor and reduced RAD51 foci formation in response to DNA damage. The p.L24S and p.L35P variants disrupted BRCA1-PALB2 protein complexes, p.I944N was associated with protein instability, and both p.I944N and p.L1070P mislocalized PALB2 to the cytoplasm. CONCLUSION: These findings show that the HDR assay is an effective method for screening the influence of inherited variants on PALB2 function, that four missense variants impact PALB2 function and may influence cancer risk and response to therapy, and suggest that few inherited PALB2 missense variants disrupt PALB2 function in DNA repair.
Subject(s)
BRCA1 Protein/genetics , Breast Neoplasms/genetics , Fanconi Anemia Complementation Group N Protein/genetics , Rad51 Recombinase/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , DNA Damage/genetics , DNA Repair/drug effects , Female , GATA3 Transcription Factor/genetics , Genetic Predisposition to Disease , Humans , Mutation, Missense/genetics , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Recombinational DNA Repair/geneticsABSTRACT
While biallelic mutations in the PALB2 tumor suppressor cause Fanconi anemia subtype FA-N, monoallelic mutations predispose to breast and familial pancreatic cancer. Although hundreds of missense variants in PALB2 have been identified in patients to date, only a few have clear functional and clinical relevance. Herein, we investigate the effects of 44 PALB2 variants of uncertain significance found in breast cancer patients and provide detailed analysis by systematic functional assays. Our comprehensive functional analysis reveals two hotspots for potentially deleterious variations within PALB2, one at each terminus. PALB2 N-terminus variants p.P8L [c.23C>T], p.Y28C [c.83A>G], and p.R37H [c.110G>A] compromised PALB2-mediated homologous recombination. At the C-terminus, PALB2 variants p.L947F [c.2841G>T], p.L947S [c.2840T>C], and most strikingly p.T1030I [c.3089C>T] and p.W1140G [c.3418T>C], stood out with pronounced PARP inhibitor sensitivity and cytoplasmic accumulation in addition to marked defects in recruitment to DNA damage sites, interaction with BRCA2 and homologous recombination. Altogether, our findings show that a combination of functional assays is necessary to assess the impact of germline missense variants on PALB2 function, in order to guide proper classification of their deleteriousness.
Subject(s)
Breast Neoplasms/genetics , Fanconi Anemia Complementation Group N Protein/genetics , Mutation, Missense/genetics , Cell Line, Tumor , Computer Simulation , DNA Damage , Female , Genetic Loci , Homologous Recombination/genetics , Humans , Kinetics , Rad51 Recombinase/metabolism , Reproducibility of ResultsABSTRACT
Partner and Localizer of BRCA2 (PALB2) has emerged as an important and versatile player in genome integrity maintenance. Biallelic mutations in PALB2 cause Fanconi anemia (FA) subtype FA-N, whereas monoallelic mutations predispose to breast, and pancreatic familial cancers. Herein, we review recent developments in our understanding of the mechanisms of regulation of the tumor suppressor PALB2 and its functional domains. Regulation of PALB2 functions in DNA damage response and repair occurs on multiple levels, including homodimerization, phosphorylation, and ubiquitylation. With a molecular emphasis, we present PALB2-associated cancer mutations and their detailed analysis by functional assays.
Subject(s)
BRCA2 Protein/metabolism , Fanconi Anemia/metabolism , Animals , BRCA2 Protein/genetics , DNA Damage/genetics , Fanconi Anemia/genetics , Female , Humans , Mutation/genetics , Ubiquitination/genetics , Ubiquitination/physiologyABSTRACT
Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are effective in cancers with defective homologous recombination DNA repair (HRR), including BRCA1/2-related cancers. A test to identify additional HRR-deficient tumors will help to extend their use in new indications. We evaluated the activity of the PARPi olaparib in patient-derived tumor xenografts (PDXs) from breast cancer (BC) patients and investigated mechanisms of sensitivity through exome sequencing, BRCA1 promoter methylation analysis, and immunostaining of HRR proteins, including RAD51 nuclear foci. In an independent BC PDX panel, the predictive capacity of the RAD51 score and the homologous recombination deficiency (HRD) score were compared. To examine the clinical feasibility of the RAD51 assay, we scored archival breast tumor samples, including PALB2-related hereditary cancers. The RAD51 score was highly discriminative of PARPi sensitivity versus PARPi resistance in BC PDXs and outperformed the genomic test. In clinical samples, all PALB2-related tumors were classified as HRR-deficient by the RAD51 score. The functional biomarker RAD51 enables the identification of PARPi-sensitive BC and broadens the population who may benefit from this therapy beyond BRCA1/2-related cancers.
