The Epistatic Relationship between BRCA2 and the Other RAD51 Mediators in Homologous Recombination

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BRCA1, RAD52, SFR1, SWS1, and XRCC3 genes in the brca2-null cells. All the resulting double-mutant cells displayed a phenotype that was very similar to that of the brca2-null cells. We suggest that BRCA2 might thus serve as a platform to recruit various RAD51 mediators at the appropriate position at the DNA–damage site.</p

The Epistatic Relationship between BRCA2 and the Other RAD51 Mediators in Homologous Recombination

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BRCA1, RAD52, SFR1, SWS1, and XRCC3 genes in the brca2-null cells. All the resulting double-mutant cells displayed a phenotype that was very similar to that of the brca2-null cells. We suggest that BRCA2 might thus serve as a platform to recruit various RAD51 mediators at the appropriate position at the DNA–damage site

The balance between B55α and Greatwall expression levels predicts sensitivity to Greatwall inhibition in cancer cells

The Greatwall kinase inhibits PP2A-B55 phosphatase activity during mitosis to stabilise critical Cdk1-driven mitotic phosphorylation. Although Greatwall represents a potential oncogene and prospective therapeutic target, our understanding of the cellular and molecular consequences of chemical Greatwall inactivation remains limited. To address this, we introduce C-604, a highly selective Greatwall inhibitor, and characterise both immediate and long-term cellular responses to the chemical attenuation of Greatwall activity. We demonstrate that Greatwall inhibition causes systemic destabilisation of the mitotic phosphoproteome, premature mitotic exit and pleiotropic cellular pathologies. Importantly, we show that the cellular and molecular abnormalities associated with reduced Greatwall activity are specifically dependent on the B55α isoform, rather than other B55 variants, underscoring PP2A-B55α phosphatases as key mediators of the cytotoxic effects of Greatwall-targeting agents in human cells. Additionally, we establish that sensitivity to Greatwall inhibition varies in different cell line models and that dependency on Greatwall activity reflects the balance between Greatwall and B55α expression levels. Our findings highlight Greatwall dependency as a cell-specific vulnerability and propose the B55α-to-Greatwall expression ratio as a predictive biomarker of cellular responses to Greatwall-targeted therapeutics

ニワトリ Bリンパ サイボウカブ オ リヨウシタ ケミカル ジェネティックス ニ ヨル Cyclin dependent kinase イソンテキナ サイボウ ブンレツ シュウキ セイギョ キコウ ノ カイセキ

京都大学0048新制・課程博士博士(医学)甲第14471号医博第3316号新制||医||973(附属図書館)UT51-2009-D183京都大学大学院医学研究科分子医学系専攻(主査)教授 玉木 敬二, 教授 篠原 隆司, 教授 長田 重一学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

Abstract 1764: Overexpression of the DNA repair regulator PARI in pancreatic cancers promotes genomic instability and tumorigenesis.

Abstract Inappropriate homologous recombination (HR) causes genomic instability and cancer. We recently showed that the protein PARI, containing a UvrD-like helicase domain, is a novel PCNA interacting partner, required for preservation of genome stability in human and DT40 chicken cells (Moldovan et al, Molecular Cell 2012). Using cell-based and biochemical assays, we showed that PARI restricts unscheduled recombination by interfering with the formation of RAD51-DNA HR structures. Thus, we proposed that PARI is a long sought-after factor that suppresses inappropriate recombination events at mammalian replication forks. We recently found that PARI is specifically overexpressed in pancreatic cancer cells (O'Connor et al, Cancer Research, in revision). PARI up-regulation results in DNA repair deficiency and genomic instability. Importantly, PARI knockdown compromises the proliferation of pancreatic cancer cells in vitro. PARI depletion results in an altered cell cycle, defined by S-phase accumulation, perturbed DNA replication, and activation of the DNA damage response pathway in the absence of exogenous DNA damage. Moreover, we found that in pancreatic cancer cells, PARI overexpression results in DNA damage tolerance by promoting replication of damaged DNA. Finally, using a xenograft tumor model in nude mice we show that PARI knockdown reduces pancreatic tumor growth in vivo. Thus, our work suggests that PARI is a potential target for pancreatic ductal adenocarcinoma therapy. Citation Format: Kevin O'Connor, Donniphat Dejsuphong, Alan D'Andrea, George-Lucian Moldovan. Overexpression of the DNA repair regulator PARI in pancreatic cancers promotes genomic instability and tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1764. doi:10.1158/1538-7445.AM2013-1764</jats:p

ATR Inhibitor Synergizes PARP Inhibitor Cytotoxicity in Homologous Recombination Repair Deficiency TK6 Cell Lines

The inhibition of poly(ADP-ribose) polymerases (PARPs) and ataxia telangiectasia and Rad3-related (ATR) would be an alternative approach for cancer treatments. The aim of this study is to investigate the synergy of the different combinations of PARP inhibitors (olaparib, talazoparib, or veliparib) and ATR inhibitor AZD6738. A drug combinational synergy screen that combines olaparib, talazoparib, or veliparib with AZD6738 was performed to identify the synergistic interaction, and the combination index was calculated to verify synergy. TK6 isogenic cell lines with defects in different DNA repair genes were used as a model. Cell cycle analysis, micronucleus induction, and focus formation assays of serine-139 phosphorylation of the histone variant H2AX demonstrated that AZD6738 diminished G2/M checkpoint activation induced by PARP inhibitors and allowed DNA damage-containing cells to continue dividing, leading to greater increases in micronuclei as well as double-strand DNA breaks in mitotic cells. We also found that AZD6738 was likely to potentiate cytotoxicity of PARP inhibitors in homologous recombination repair deficiency cell lines. AZD6738 sensitized more genotypes of DNA repair-deficient cell lines to talazoparib than to olaparib and veliparib, respectively. The combinational approach of PARP and ATR inhibition to enhance response to PARP inhibitors could expand the utility of PARP inhibitors to cancer patients without BRCA1/2 mutations.</jats:p

Compensatory Functions and Interdependency of the DNA-Binding Domain of BRCA2 with the BRCA1–PALB2–BRCA2 Complex

Abstract BRCA1, BRCA2, and PALB2 are key players in cellular tolerance to chemotherapeutic agents, including camptothecin, cisplatin, and PARP inhibitor. The N-terminal segment of BRCA2 interacts with PALB2, thus contributing to the formation of the BRCA1–PALB2–BRCA2 complex. To understand the role played by BRCA2 in this complex, we deleted its N-terminal segment and generated BRCA2ΔN mutant cells. Although previous studies have suggested that BRCA1–PALB2 plays a role in the recruitment of BRCA2 to DNA-damage sites, BRCA2ΔN mutant cells displayed a considerably milder phenotype than did BRCA2−/− null-deficient cells. We hypothesized that the DNA-binding domain (DBD) of BRCA2 might compensate for a defect in BRCA2ΔN that prevented stable interaction with PALB2. To test this hypothesis, we disrupted the DBD of BRCA2 in wild-type and BRCA2ΔN cells. Remarkably, although the resulting BRCA2ΔDBD cells displayed a moderate phenotype, the BRCA2ΔN+ΔDBD cells displayed a very severe phenotype, as did the BRCA2−/− cells, suggesting that the N-terminal segment and the DBD play a substantially overlapping role in the functionality of BRCA2. We also showed that the formation of both the BRCA1–PALB2–BRCA2 complex and the DBD is required for efficient recruitment of BRCA2 to DNA-damage sites. Our study revealed the essential role played by both the BRCA1–PALB2–BRCA2 complex and the DBD in the functionality of BRCA2, as each can compensate for the other in the recruitment of BRCA2 to DNA-damage sites. This knowledge adds to our ability to accurately predict the efficacy of antimalignant therapies for patients carrying mutations in the BRCA2 gene. Cancer Res; 74(3); 797–807. ©2013 AACR.</jats:p