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• W2914604310 abstract "•BRCA2-mutant cells rely on BER, ATR signaling, splicing, and MMEJ for survival•APEX2 and FEN1 represent potential drug targets in BRCA-mutant tumors•BRCA-mutant cells require the endonuclease function and PCNA association of APEX2•A cell-based reporter demonstrates that FEN1 participates in MMEJ BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing. We identified APEX2 and FEN1 as synthetic lethal genes with both BRCA1 and BRCA2 loss of function. BRCA2-deficient cells require the apurinic endonuclease activity and the PCNA-binding domain of Ape2 (APEX2), but not Ape1 (APEX1). Furthermore, BRCA2-deficient cells require the 5′ flap endonuclease but not the 5′-3′ exonuclease activity of Fen1, and chemically inhibiting Fen1 selectively targets BRCA-deficient cells. Finally, we developed a microhomology-mediated end-joining (MMEJ) reporter and showed that Fen1 participates in MMEJ, underscoring the importance of MMEJ as a collateral repair pathway in the context of homologous recombination (HR) deficiency. BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing. We identified APEX2 and FEN1 as synthetic lethal genes with both BRCA1 and BRCA2 loss of function. BRCA2-deficient cells require the apurinic endonuclease activity and the PCNA-binding domain of Ape2 (APEX2), but not Ape1 (APEX1). Furthermore, BRCA2-deficient cells require the 5′ flap endonuclease but not the 5′-3′ exonuclease activity of Fen1, and chemically inhibiting Fen1 selectively targets BRCA-deficient cells. Finally, we developed a microhomology-mediated end-joining (MMEJ) reporter and showed that Fen1 participates in MMEJ, underscoring the importance of MMEJ as a collateral repair pathway in the context of homologous recombination (HR) deficiency. Damage to human genetic material contributes to oncogenic transformation and threatens faithful transmission of genetic information across generations. In response to the variety of lesions in DNA caused by endogenous and exogenous sources of damage, cells have evolved a complex signal transduction pathway known as the DNA damage response (DDR). The DDR senses damage and executes a coordinated cellular response that includes lesion-appropriate repair strategies, immune system activation, cell-cycle arrest, cellular senescence, or cell death (Ciccia and Elledge, 2010Ciccia A. Elledge S.J. The DNA damage response: making it safe to play with knives.Mol. Cell. 2010; 40: 179-204Abstract Full Text Full Text PDF PubMed Scopus (2925) Google Scholar). Of the many lesions that occur in DNA, the double-strand break (DSB) is among the most detrimental. Failure to properly repair DSBs can lead to adverse outcomes such as replication fork collapse, cell death, oncogenic translocation, or loss of telomeric DNA. Three pathways exist to repair DSBs: homologous recombination (HR), classical non-homologous end-joining (NHEJ), and microhomology-mediated end-joining (MMEJ). Whereas HR utilizes information from a paired sister chromatid to seamlessly repair DSBs with high fidelity, NHEJ and MMEJ can result in sequence alterations at the repaired DSB site. However, unlike NHEJ, HR and MMEJ both act on resected DSBs (Bennardo et al., 2008Bennardo N. Cheng A. Huang N. Stark J.M. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair.PLoS Genet. 2008; 4: e1000110Crossref PubMed Scopus (608) Google Scholar), directly competing for a shared substrate (Ceccaldi et al., 2015Ceccaldi R. Liu J.C. Amunugama R. Hajdu I. Primack B. Petalcorin M.I. O’Connor K.W. Konstantinopoulos P.A. Elledge S.J. Boulton S.J. et al.Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair.Nature. 2015; 518: 258-262Crossref PubMed Scopus (492) Google Scholar, Mateos-Gomez et al., 2015Mateos-Gomez P.A. Gong F. Nair N. Miller K.M. Lazzerini-Denchi E. Sfeir A. Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination.Nature. 2015; 518: 254-257Crossref PubMed Scopus (416) Google Scholar). Several DDR genes are frequently mutated in cancer. Germline mutations in BRCA1 or BRCA2, both critical for the HR pathway, account for the majority of hereditary breast and ovarian cancers (Petrucelli et al., 2010Petrucelli N. Daly M.B. Feldman G.L. Hereditary breast and ovarian cancer due to mutations in BRCA1 and BRCA2.Genet. Med. 2010; 12: 245-259Crossref PubMed Scopus (215) Google Scholar). Women carrying mutations in one of these tumor suppressor genes have up to an 80% risk of developing breast cancer and up to a 50% risk of developing ovarian cancer (Petrucelli et al., 2010Petrucelli N. Daly M.B. Feldman G.L. Hereditary breast and ovarian cancer due to mutations in BRCA1 and BRCA2.Genet. Med. 2010; 12: 245-259Crossref PubMed Scopus (215) Google Scholar). Overall, nearly half of high-grade serous ovarian cancers (HGSOC) harbor inactivated HR genes, 30% of which are BRCA1 or BRCA2 (Bitler et al., 2017Bitler B.G. Watson Z.L. Wheeler L.J. Behbakht K. PARP inhibitors: clinical utility and possibilities of overcoming resistance.Gynecol. Oncol. 2017; 147: 695-704Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Unlike BRCA1, BRCA2 mutation also predisposes to pancreatic and prostate cancer (Roy et al., 2011Roy R. Chun J. Powell S.N. BRCA1 and BRCA2: different roles in a common pathway of genome protection.Nat. Rev. Cancer. 2011; 12: 68-78Crossref PubMed Scopus (918) Google Scholar). BRCA1 and BRCA2 share a common function in facilitating HR and play additional roles in safeguarding genomic integrity. By loading Rad51 onto DNA breaks and gaps, Brca2 prevents Mre11-dependent degradation of nascent DNA at stalled replication forks (Kolinjivadi et al., 2017Kolinjivadi A.M. Sannino V. De Antoni A. Zadorozhny K. Kilkenny M. Techer H. Baldi G. Shen R. Ciccia A. Pellegrini L. et al.Smarcal1-mediated fork reversal triggers Mre11-dependent degradation of nascent DNA in the absence of Brca2 and stable Rad51 nucleofilaments.Mol. Cell. 2017; 67: 867-881.e867Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, Schlacher et al., 2011Schlacher K. Christ N. Siaud N. Egashira A. Wu H. Jasin M. Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11.Cell. 2011; 145: 529-542Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar) and promotes HR-mediated resolution of fork stalling (Lomonosov et al., 2003Lomonosov M. Anand S. Sangrithi M. Davies R. Venkitaraman A.R. Stabilization of stalled DNA replication forks by the BRCA2 breast cancer susceptibility protein.Genes Dev. 2003; 17: 3017-3022Crossref PubMed Scopus (200) Google Scholar). Also, Brca2 protects telomere integrity (Doksani and de Lange, 2014Doksani Y. de Lange T. The role of double-strand break repair pathways at functional and dysfunctional telomeres.Cold Spring Harb. Perspect. Biol. 2014; 6: a016576Crossref PubMed Scopus (90) Google Scholar) and prevents accumulation of R-loops, which can lead to replication fork stalling and interference with transcriptional elongation (Bhatia et al., 2014Bhatia V. Barroso S.I. García-Rubio M.L. Tumini E. Herrera-Moyano E. Aguilera A. BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2.Nature. 2014; 511: 362-365Crossref PubMed Scopus (333) Google Scholar). BRCA1 and BRCA2-deficient cells are exquisitely sensitive to treatment with inhibitors of poly(ADP-ribose) polymerase (PARP) (Bryant et al., 2005Bryant H.E. Schultz N. Thomas H.D. Parker K.M. Flower D. Lopez E. Kyle S. Meuth M. Curtin N.J. Helleday T. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase.Nature. 2005; 434: 913-917Crossref PubMed Scopus (3628) Google Scholar, Farmer et al., 2005Farmer H. McCabe N. Lord C.J. Tutt A.N. Johnson D.A. Richardson T.B. Santarosa M. Dillon K.J. Hickson I. Knights C. et al.Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy.Nature. 2005; 434: 917-921Crossref PubMed Scopus (4648) Google Scholar), and this relationship has been referred to as synthetic lethality (SL). SL describes a condition in which mutation of either of two genes is viable, but simultaneous mutation of both genes is lethal (Kaelin, 2005Kaelin Jr., W.G. The concept of synthetic lethality in the context of anticancer therapy.Nat. Rev. Cancer. 2005; 5: 689-698Crossref PubMed Scopus (1104) Google Scholar). PARP inhibitors have recently gained US Food and Drug Administration (FDA) approval for treatment of patients with metastatic breast cancer and a germline BRCA mutation (Robson et al., 2017aRobson M. Goessl C. Domchek S. Olaparib for metastatic germline BRCA-mutated breast cancer.N. Engl. J. Med. 2017; 377: 1792-1793Crossref PubMed Scopus (214) Google Scholar, Robson et al., 2017bRobson M. Im S.A. Senkus E. Xu B. Domchek S.M. Masuda N. Delaloge S. Li W. Tung N. Armstrong A. et al.Olaparib for metastatic breast cancer in patients with a germline BRCA mutation.N. Engl. J. Med. 2017; 377: 523-533Crossref PubMed Scopus (1636) Google Scholar) and patients with recurrent HGSOC (Bitler et al., 2017Bitler B.G. Watson Z.L. Wheeler L.J. Behbakht K. PARP inhibitors: clinical utility and possibilities of overcoming resistance.Gynecol. Oncol. 2017; 147: 695-704Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, Mirza et al., 2016Mirza M.R. Monk B.J. Herrstedt J. Oza A.M. Mahner S. Redondo A. Fabbro M. Ledermann J.A. Lorusso D. Vergote I. et al.ENGOT-OV16/NOVA InvestigatorsNiraparib maintenance therapy in platinum-sensitive, recurrent ovarian cancer.N. Engl. J. Med. 2016; 375: 2154-2164Crossref PubMed Scopus (1478) Google Scholar). However, dual depletion of PARP and BRCA2 by small interfering RNA (siRNA) does not recapitulate the potent lethality observed upon chemical inhibition of PARP (Bryant et al., 2005Bryant H.E. Schultz N. Thomas H.D. Parker K.M. Flower D. Lopez E. Kyle S. Meuth M. Curtin N.J. Helleday T. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase.Nature. 2005; 434: 913-917Crossref PubMed Scopus (3628) Google Scholar). Rather than solely exploiting a genetic SL relationship, PARP inhibitors also cause lethality by physically trapping PARP onto single-strand break (SSB) intermediates, obstructing progression of replication forks (Helleday, 2011Helleday T. The underlying mechanism for the PARP and BRCA synthetic lethality: clearing up the misunderstandings.Mol. Oncol. 2011; 5: 387-393Crossref PubMed Scopus (544) Google Scholar, Murai et al., 2012Murai J. Huang S.Y. Das B.B. Renaud A. Zhang Y. Doroshow J.H. Ji J. Takeda S. Pommier Y. Trapping of PARP1 and PARP2 by clinical PARP inhibitors.Cancer Res. 2012; 72: 5588-5599Crossref PubMed Scopus (1337) Google Scholar, Ström et al., 2011Ström C.E. Johansson F. Uhlén M. Szigyarto C.A. Erixon K. Helleday T. Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate.Nucleic Acids Res. 2011; 39: 3166-3175Crossref PubMed Scopus (223) Google Scholar) and in that sense behaving more like classical DNA damage agents to which BRCA2-mutant tumors are also sensitive. Indeed, the toxic PARP-DNA complexes formed as a result of PARP trapping are more cytotoxic than unresolved SSBs (Murai et al., 2012Murai J. Huang S.Y. Das B.B. Renaud A. Zhang Y. Doroshow J.H. Ji J. Takeda S. Pommier Y. Trapping of PARP1 and PARP2 by clinical PARP inhibitors.Cancer Res. 2012; 72: 5588-5599Crossref PubMed Scopus (1337) Google Scholar). Targeting the intrinsic genetic vulnerabilities in BRCA1- and BRCA2-deficient cells offers the opportunity to meet an urgent clinical need in the treatment of refractory or metastatic breast cancer in patients with germline BRCA mutations as well as recurrent HGSOC. Despite recent success in clinical trials, PARP inhibitor efficacy appears to be limited by inherent and acquired resistance, underscoring the need for identification of synergistic and alternative targets (Higgins and Boulton, 2018Higgins G.S. Boulton S.J. Beyond PARP-POLtheta as an anticancer target.Science. 2018; 359: 1217-1218Crossref PubMed Scopus (46) Google Scholar). Thus, we sought to systematically identify BRCA2 synthetic lethal genes (B2SLs). We found BRCA2-mutant cells to be more dependent than their wild-type counterparts on several pathways including base excision repair (BER), Atr activation, splicing, and MMEJ. We identified APEX2 and FEN1 as B2SL targets, and we showed through the use of a cell-based reporter that FEN1 participates in MMEJ. To identify B2SL candidates, we established two cell lines that are isogenic except for the presence of a functional BRCA2 gene. We obtained a modified DLD-1 cell line with a homozygous deletion of BRC repeat 6 in exon 11 that also introduces a loxP site and a stop codon between BRC repeats 5 and 6, resulting in a biallelic BRCA2 truncation mutation (Hucl et al., 2008Hucl T. Rago C. Gallmeier E. Brody J.R. Gorospe M. Kern S.E. A syngeneic variance library for functional annotation of human variation: application to BRCA2.Cancer Res. 2008; 68: 5023-5030Crossref PubMed Scopus (65) Google Scholar). To this BRCA2-mutant (B2MUT) line, we introduced a full-length BRCA2 mammalian expression construct through transfection and selection for stable integrants. We isolated individual clones from these BRCA2 wild-type (B2WT) cells and characterized several clones to demonstrate restoration of functional Brca2 expression. We confirmed full-length Brca2 protein expression by western blotting (Figure 1A). Next, we observed that expression of BRCA2 enhanced the growth rate of B2MUT cells (Figure S1A) and restored their ability to form Rad51 foci in response to ionizing radiation (IR) (Figure 1B). Finally, we confirmed that expression of Brca2 restored resistance to the PARP inhibitor olaparib (Figure 1C). Using these isogenic cell lines, we performed a series of screens designed to identify B2SL genes. We screened a targeted library of 380 genes with a known or suspected role in the DDR. In each screen, B2MUT and B2WT lines were separately transduced with a lentivirus-based small hairpin RNA (shRNA) or guide RNA (gRNA) library at a low MOI, in triplicate (Figure 1D). Cell pellets were collected before and after passaging these cells for 12 population doublings (PDs), and the relative change of shRNAs or gRNAs during culture was determined by next-generation sequencing (NGS) of PCR-amplified shRNA half-hairpins (Schlabach et al., 2008Schlabach M.R. Luo J. Solimini N.L. Hu G. Xu Q. Li M.Z. Zhao Z. Smogorzewska A. Sowa M.E. Ang X.L. et al.Cancer proliferation gene discovery through functional genomics.Science. 2008; 319: 620-624Crossref PubMed Scopus (322) Google Scholar) or gRNAs from PD0 and PD12 cell pellets. To expand the generality of our results, we obtained a second near isogenic cell line pair: ovarian PEO1 B2MUT cells, which contain a hemizygous truncation mutation in BRC repeat 5, and an in vitro derived cisplatin-selected subclone C4-2 that contains a reversion mutation restoring full-length Brca2 expression (B2WT) (Sakai et al., 2009Sakai W. Swisher E.M. Jacquemont C. Chandramohan K.V. Couch F.J. Langdon S.P. Wurz K. Higgins J. Villegas E. Taniguchi T. Functional restoration of BRCA2 protein by secondary BRCA2 mutations in BRCA2-mutated ovarian carcinoma.Cancer Res. 2009; 69: 6381-6386Crossref PubMed Scopus (237) Google Scholar). Because of its evolution, the C4-2 subclone and the parental PEO1 cells are not perfectly isogenic, but the use of two nearly isogenic cell line pairs in our screens mitigates this lack of perfect isogenicity in each case. The colonic DLD-1 B2MUT cell line and the ovarian PEO1 B2MUT cell line express similar BRCA2 truncation mutants, both prematurely terminating directly in or immediately after BRC repeat 5 in exon 11 (Figure 1E). These alleles retain the N-terminal transactivation domain of Brca2 (TAD) and the first four BRC repeats, which are known to bind free Rad51 (Carreira and Kowalczykowski, 2011Carreira A. Kowalczykowski S.C. Two classes of BRC repeats in BRCA2 promote RAD51 nucleoprotein filament function by distinct mechanisms.Proc. Natl. Acad. Sci. USA. 2011; 108: 10448-10453Crossref PubMed Scopus (104) Google Scholar), but lack the DNA-binding domains and its C-terminal Rad51- binding domain (Figure 1E). We examined BRCA2 mutations reported from 2,433 breast tumors in the METABRIC targeted exome sequencing study (Pereira et al., 2016Pereira B. Chin S.F. Rueda O.M. Vollan H.K. Provenzano E. Bardwell H.A. Pugh M. Jones L. Russell R. Sammut S.J. et al.The somatic mutation profiles of 2,433 breast cancers refines their genomic and transcriptomic landscapes.Nat. Commun. 2016; 7: 11479Crossref PubMed Scopus (933) Google Scholar) and found that mutations in the BRC repeat region of exon 11 are not uncommon, with ∼16% of functionally impactful mutations occurring in this region (Table S1; Figure S1B). We screened our CRISPR-Cas9-based library, but not our shRNA-based libraries, in this ovarian isogenic cell line pair (Figure S1C). We analyzed our screens by MAGeCK (Li et al., 2014Li W. Xu H. Xiao T. Cong L. Love M.I. Zhang F. Irizarry R.A. Liu J.S. Brown M. Liu X.S. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens.Genome Biol. 2014; 15: 554Crossref PubMed Scopus (874) Google Scholar) and edgeR (Robinson et al., 2010Robinson M.D. McCarthy D.J. Smyth G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.Bioinformatics. 2010; 26: 139-140Crossref PubMed Scopus (20834) Google Scholar), relying on edgeR analysis to calculate false discovery rates (FDRs) (Figures 2A–2F; Tables S2, S3, S4, S5, and S6). Depletion of B2SL genes inhibits growth in B2MUT cells more than B2WT cells, yielding a B2MUT/B2WT ratio that is negative on a log2 scale (Figures 2A–2C); this value can also be negative for genes that promote growth of B2WT cells more than B2MUT cells (Figures 2D and 2F). Overall, the magnitude of effects we observed was more potent with our CRISPR library than our shRNA library, and the CRISPR library produced larger fold changes in the euploid colonic background than the aneuploid ovarian background. Analysis of our screens identified POLQ as a B2SL gene (Figures 2A and 2F). POLQ encodes polymerase theta (Polθ), a large A-family DNA polymerase that plays an important role in MMEJ and is known to be synthetic lethal with HR pathway mutants (Ceccaldi et al., 2015Ceccaldi R. Liu J.C. Amunugama R. Hajdu I. Primack B. Petalcorin M.I. O’Connor K.W. Konstantinopoulos P.A. Elledge S.J. Boulton S.J. et al.Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair.Nature. 2015; 518: 258-262Crossref PubMed Scopus (492) Google Scholar, Mateos-Gomez et al., 2015Mateos-Gomez P.A. Gong F. Nair N. Miller K.M. Lazzerini-Denchi E. Sfeir A. Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination.Nature. 2015; 518: 254-257Crossref PubMed Scopus (416) Google Scholar). Thus, POLQ serves as a positive control for BRCA2 synthetic lethality SL. We also found SF3B2 to be a B2SL candidate. SF3B2 is part of the U2 small nuclear ribonucleoprotein particle (snRNP) that assembles with other snRNP components to form the spliceosome (Cretu et al., 2016Cretu C. Schmitzová J. Ponce-Salvatierra A. Dybkov O. De Laurentiis E.I. Sharma K. Will C.L. Urlaub H. Lührmann R. Pena V. Molecular architecture of SF3b and structural consequences of its cancer-related mutations.Mol. Cell. 2016; 64: 307-319Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). While SF3B2 loss is toxic to both B2MUT and B2WT cells, B2MUT cells appear to be more reliant on its function than B2WT cells (Figures 2A and 2D). When we rescreened 50 hits from our shRNA library in a secondary screen, we observed that SF3B2 and POLQ validated as B2SL candidates (Figures S2A and S2B) and that our secondary screen shRNA sublibrary enriched for B2SL candidates overall (Figures S2C and S2F). Screening our CRISPR library in the DLD-1 isogenic lines confirmed POLQ and also found FEN1, APEX2, UBE2A, CLSPN, and DCLRE1C to be B2SLs (Figures 2B and 2E). Screening in the PEO1 isogenic lines identified POLQ, FEN1, XRCC1, and RHNO1 as strong B2SL hits (Figures 2C and 2F). As we are most interested in genes that broadly generalize as SL, we compared our ovarian and colonic CRISPR screens and found that POLQ, FEN1, and APEX2 score in both (Figures 2G and 2H). While POLQ serves as a positive control, FEN1 and APEX2 represent unknown B2SL genes and potential drug targets in BRCA-deficient tumors. Notably, several of our B2SL hits demonstrated stronger SL than PARP1 in both ovarian and colonic cells, including FEN1, APEX2, POLQ, XRCC1, and UBE2A. This observation underscores the well-established finding that PARP inhibitors are effective in BRCA2-deficient cells not only because of a mild genetic interaction between PARP1 and BRCA2 but also because of the phenomenon of PARP trapping (Ström et al., 2011Ström C.E. Johansson F. Uhlén M. Szigyarto C.A. Erixon K. Helleday T. Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate.Nucleic Acids Res. 2011; 39: 3166-3175Crossref PubMed Scopus (223) Google Scholar, Murai et al., 2012Murai J. Huang S.Y. Das B.B. Renaud A. Zhang Y. Doroshow J.H. Ji J. Takeda S. Pommier Y. Trapping of PARP1 and PARP2 by clinical PARP inhibitors.Cancer Res. 2012; 72: 5588-5599Crossref PubMed Scopus (1337) Google Scholar). We noticed a striking phenomenon in both screens: every RAD51 paralog (RAD51, RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) exhibited a strongly positive genetic interaction (GI) score in both cell line contexts (Figures 2G and 2H), as did PALB2. This GI score (the average log2 fold change in B2MUT versus B2WT cells) reflects not only a detrimental effect in B2WT cells from the loss of these HR components but also a surprising enhancement of B2MUT cell growth (Figures 2E and 2F). Similarly, CRISPR-based knockout of BRCA2 itself (with gRNAs targeting the expressed portion of its truncation mutants) enhances growth of B2MUT cells (Figures 2E and 2F). These observations suggest that the residual BRCA2 truncation mutants present in our ovarian and colonic B2MUT cells exert a detrimental effect involving RAD51 paralogs. To validate this result, we employed a multicolor competition assay (MCA). As previously described (Smogorzewska et al., 2007Smogorzewska A. Matsuoka S. Vinciguerra P. McDonald 3rd, E.R. Hurov K.E. Luo J. Ballif B.A. Gygi S.P. Hofmann K. D’Andrea A.D. Elledge S.J. Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair.Cell. 2007; 129: 289-301Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar), we mixed GFP-labeled B2MUT cells with E2-Crimson-labeled B2WT cells, infected the mixture with individual gRNAs, and monitored the relative change in percent GFP+ cells by fluorescence-activated cell sorting (FACS) (Figure S2G). We individually tested the relative effect of two gRNAs to RAD51, RAD51C, RAD51D, and the N-terminal portion of BRCA2 in B2MUT versus B2WT cells, utilizing our colonic isogenic cell line pair. Consistent with our screen results, all of the gRNAs tested resulted in an enhanced ratio of B2MUT to B2WT cells relative to negative control gRNAs (Figure S2H). It is surprising that depletion of RAD51 and its paralogs suppresses the detrimental effects of BRCA2 mutation relative to wild-type, suggesting the possibility that these two similar BRCA2 truncation mutants may behave in an unusual manner. To ask if this phenotype is specific to mutants truncated near BRC repeat 5, we performed MCAs in a pair of isogenic pancreatic CAPAN-1 cells that express a BRCA2 allele truncated in BRC repeat 8 or a full-length revertant (C5-2 cells) (Sakai et al., 2008Sakai W. Swisher E.M. Karlan B.Y. Agarwal M.K. Higgins J. Friedman C. Villegas E. Jacquemont C. Farrugia D.J. Couch F.J. et al.Secondary mutations as a mechanism of cisplatin resistance in BRCA2-mutated cancers.Nature. 2008; 451: 1116-1120Crossref PubMed Scopus (814) Google Scholar). In these cells, the same individual gRNAs targeting RAD51, RAD51C, RAD51D, and the N-terminal portion of BRCA2 no longer result in an enhancement of B2MUT cell growth relative to B2WT cells but instead act oppositely, being more toxic in B2MUT cells (Figure S2H). Thus, the toxic effect of Rad51 depletion in cells expressing Brca2-mutant truncation protein terminating early in BRC repeat 5 does not extend to cells that possesses a Brca2-mutant protein with a nearly intact BRC repeat region and is therefore likely to be an allele-specific phenomenon. From the analysis of our screens, several general themes emerged. Overall, BER scores potently as SL (Figure 3A). Almost every member of this pathway scores as SL, except for the partially redundant glycosylases, which initiate repair through this pathway by removing damaged sugars; their absence would not be expected to lead to nicks or DSBs that could result in an increased need for HR (Figure 3B). Similarly, several members of the ATR/CHK1 signaling pathway were B2SL, except for some of the more essential components such as ATR, ATRIP, and RPA1, which did not score using the CRISPR library (Figure 3A). Known components of MMEJ scored more strongly as B2SL than NHEJ components, perhaps implying that the majority of detrimental unresolved DSBs addressed by these auxiliary pathways occurs during S phase. The strikingly potent positive GI score we observe for BRCA2 and RAD51 paralogs does not extend to proteins functioning in earlier or later steps of HR but primarily the components involved in Rad51 loading. Finally, although SF3B2 and SF1 perform as a B2SL in the shRNA screen (Figure 3A; Table S5), they do not score in the CRISPR screen, likely due to essentiality. We further validated some of these pathways using chemical inhibitors. We tested two spliceosome inhibitors in MCAs, spliceostatin-A and sudemycin D6, both of which inhibit the U2 component SF3B1 (Effenberger et al., 2016Effenberger K.A. Urabe V.K. Prichard B.E. Ghosh A.K. Jurica M.S. Interchangeable SF3B1 inhibitors interfere with pre-mRNA splicing at multiple stages.RNA. 2016; 22: 350-359Crossref PubMed Scopus (56) Google Scholar). Both drugs caused a dose-dependent depletion of B2MUT cells relative to B2WT cells, supporting the hypothesis that inhibition of the U2 spliceosome component is SL with BRCA2 loss of function (Figures 3C and 3D). Similarly, because several Atr signaling components, such as RHNO1, CLSPN, and TOPBP1, emerged from our CRISPR screens, we tested the Atr inhibitor VE-821 for synthetic lethality. VE-821 exhibited dose-dependent selective inhibition of B2MUT cell growth (Figure 3E), confirming that B2MUT cells are more dependent on Atr activation than B2WT cells. Finally, we tested whether B2MUT cells are more dependent on competent function of the BER pathway by increasing the load of damage that must be repaired by BER. We introduced deoxyuridine monophosphate (dUMP) directly into our cell culture medium, which increases misincorporation of uracil into DNA (Tsuchimoto et al., 2001Tsuchimoto D. Sakai Y. Sakumi K. Nishioka K. Sasaki M. Fujiwara T. Nakabeppu Y. Human APE2 protein is mostly localized in the nuclei and to some extent in the mitochondria, while nuclear APE2 is partly associated with proliferating cell nuclear antigen.Nucleic Acids Res. 2001; 29: 2349-2360Crossref PubMed Scopus (131) Google Scholar). dUMP also caused a dose-dependent selective depletion of B2MUT versus B2WT cells in our MCA, supporting our hypothesis that B2MUT cells are more dependent on BER than B2WT cells (Figure 3F). APEX2 encodes Ape2, an apurinic (AP) endonuclease responsible for the second step of BER; after base removal by a lesion-appropriate glycosylase, Ape2 hydrolyzes the phosphodiester backbone immediately 5′ to the AP site to create an SSB. In addition to its AP endonuclease activity, Ape2 also possesses 3′ phosphodiesterase activity and 3′-5′ exonuclease activity, executed from the same ExoIII-like nuclease domain. Human cells rely on two ExoIII-family type II endonucleases to generate a nick upstream of AP sites during BER, encoded by APEX1 and APEX2. While APEX2 scored strongly as a B2SL gene, APEX1 scored weakly. Ape1 and Ape2 both contain an ExoIII-like endonuclease domain with 29% sequence identity and high sequence similarity (Hadi et al., 2002Hadi M.Z. Ginalski K. Nguyen L.H. Wilson 3rd, D.M. Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III.J. Mol. Biol. 2002; 316: 853-866Crossref PubMed Scopus (104) Google Scholar), but Ape1 also contains a separate, distinct N-termin" @default.
• W2914604310 created "2019-02-21" @default.
• W2914604310 creator A5006215755 @default.
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• W2914604310 date "2019-03-01" @default.
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• W2914604310 title "Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets" @default.
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• W2914604310 doi "https://doi.org/10.1016/j.molcel.2018.12.008" @default.
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