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Chordoma is a rare bone tumor with genetic risk factors largely unknown. We conducted a whole-exome sequencing (WES) analysis of germline DNA from 19 familial chordoma cases in five pedigrees and 137 sporadic chordoma patients and identified 17 rare germline variants in PALB2 and BRCA2, whose products play essential roles in homologous recombination (HR) and tumor suppression. One PALB2 variant showed disease cosegregation in a family with four affected people or obligate gene carrier. Chordoma cases had a significantly increased burden of rare variants in both genes when compared to population-based controls. Four of the six PALB2 variants identified from chordoma patients modestly affected HR function and three of the 11 BRCA2 variants caused loss of function in experimental assays. These results, together with previous reports of abnormal morphology and Brachyury expression of the notochord in Palb2 knockout mouse embryos and genomic signatures associated with HR defect and HR gene mutations in advanced chordomas, suggest that germline mutations in PALB2 and BRCA2 may increase chordoma susceptibility. Our data shed light on the etiology of chordoma and support the previous finding that PARP-1 inhibitors may be a potential therapy for some chordoma patients.

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.

BRCA1 is a major tumor suppressor that functions in the accurate repair of DNA double-strand breaks via homologous recombination (HR). Nonsense mutations in

Genetic testing for BRCA1, a DNA repair protein, can identify carriers of pathogenic variants associated with a substantially increased risk for breast and ovarian cancers. However, an association with increased risk is unclear for a large fraction of BRCA1 variants present in the human population. Most of these variants of uncertain clinical significance lead to amino acid changes in the BRCA1 protein. Functional assays are valuable tools to assess the potential pathogenicity of these variants. Here, we systematically probed the effects of substitutions in the C terminus of BRCA1: the N- and C-terminal borders of its tandem BRCT domain, the BRCT-[N-C] linker region, and the α1 and α′1 helices in BRCT-[N] and -[C]. Using a validated transcriptional assay based on a fusion of the GAL4 DNA-binding domain to the BRCA1 C terminus (amino acids 1396–1863), we assessed the functional impact of 99 missense variants of BRCA1. We include the data obtained for these 99 missense variants in a joint analysis to generate the likelihood of pathogenicity for 347 missense variants in BRCA1 using VarCall, a Bayesian integrative statistical model. The results from this analysis increase our understanding of BRCA1 regions less tolerant to changes, identify functional borders of structural domains, and predict the likelihood of pathogenicity for 98% of all BRCA1 missense variants in this region recorded in the population. This knowledge will be critical for improving risk assessment and clinical treatment of carriers of BRCA1 variants.

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.

BRCA1 gene encodes an 1863 amino-acid protein comprising a RING finger domain in the N-terminus and two tandem BRCT domains (tBRCT) in C-terminal region. When fused to a heterologous DNA-binding domain, BRCA1 C-terminal region is capable of activating transcription. In addition, BRCA1 is associated with the RNA polymerase II holoenzyme (RNA pol II), a number of transcription factors and regulators. As BRCA1 cancer-associated mutations located in the C-terminal region of the protein lead to an impaired transcriptional activation phenotype, the ability to trigger transcription seems to be an important feature of BRCA1 tumor suppression function. BARD1 (BRCA1 associated RING domain 1) also encloses a N-terminal RING finger and a C-terminal tBRCT domains. The BRCA1/BARD1 heterodimer was found to ubiquitinate RNA pol II leading to a reduced transcription initiation. Recently, our group described a protein interaction network that identified putative interacting partners of seven different proteins enclosing the tBRCT domain. Among other hits, Cyclin-Dependent Kinase 9 (CDK9) was identified as a common interaction partner of BRCA1 and BARD1 tBRCTs. CDK9 is a component of the positive transcription elongation factor b complex (P-TEFb), which is involved in co-transcriptional histone modification, mRNA processing, mRNA export and transcriptional elongation. The interaction between constitutive CDK9 and BARD1 was confirmed through co-immunoprecipitation (Co-IP) assay using HEK293FT cells nuclear extracts. We also demonstrated that CDK9 isoforms were able to interact with both BARD1 N- and C-terminal regions in a GST pulldown assay. In addition, the BRCA1/CDK9 constitutive interaction was confirmed by Co-IP assay in HeLa cells. We showed that, differently from BARD1, BRCA1 tBRCT interacts only with the 42kDa isoform of CDK9. In order to investigate CDK9 role in BRCA1 mediated transcription, the kinase expression was downregulated (by shRNA methodology) in HeLa cells, resulting in a two-fold increase in transcription (observed in a luciferase reporter assay). On the other hand, the super expression of CDK9 42kDa isoform leads to the suppression of BRCA1 mediated transcription. These data suggest that the CDK9 42kDa isoform modulates BRCA1 mediated transcription through its tBRCT domain. A cisplatin resistance phenotype was also observed in CDK9 downregulated cells only in a BRCA1 competent context. Now, we are extending our approach investigating the role of CDK9/BRCA1 complex in gene expression control and DNA damage repair. A better understanding of these functions will help to unravel the BRCA1 mediated transcription and its role as a tumor suppressor. Citation Format: Thales Nepomuceno, Vanessa Fernandes, Thiago Gomes, Giuliana De Gregoris, Renato Carvalho, Guilherme Kurtz, Álvaro Monteiro, Marcelo Alex Carvalho. Characterization of CDK9/BRCA1 complex in DNA damage response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3025. doi:10.1158/1538-7445.AM2015-3025