BRCA 1 and BRCA 2 tumor suppressors protect against endogenous acetaldehyde toxicity
2017; Springer Nature; Volume: 9; Issue: 10 Linguagem: Inglês
10.15252/emmm.201607446
ISSN1757-4684
AutoresEliana MC Tacconi, Xianning Lai, Cecilia Folio, Manuela Porru, Gijs Zonderland, Sophie Badie, Johanna Michl, Irene Sechi, Mélanie Rogier, Verónica Matía, Ankita Sati Batra, Oscar M. Rueda, Peter Bouwman, Jos Jonkers, Anderson J. Ryan, Bernardo Reina‐San‐Martin, Joannie Hui, Nelson L.S. Tang, Alejandra Bruna, Annamaria Biroccio, Madalena Tarsounas,
Tópico(s)Plant Disease Resistance and Genetics
ResumoResearch Article20 July 2017Open Access Source DataTransparent process BRCA1 and BRCA2 tumor suppressors protect against endogenous acetaldehyde toxicity Eliana MC Tacconi Eliana MC Tacconi Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Xianning Lai Xianning Lai Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Cecilia Folio Cecilia Folio Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Manuela Porru Manuela Porru Area of Translational Research, Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Gijs Zonderland Gijs Zonderland Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Sophie Badie Sophie Badie Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Johanna Michl Johanna Michl Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Irene Sechi Irene Sechi Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Mélanie Rogier Mélanie Rogier Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Institut National de la Santé et de la Recherche Médicale (INSERM), U964, Illkirch, France Centre National de Recherche Scientifique (CNRS), UMR7104, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Verónica Matía García Verónica Matía García Division of Molecular Pathology and Cancer Genomics Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Ankita Sati Batra Ankita Sati Batra Cancer Research UK Cambridge Institute, Cambridge, UK Search for more papers by this author Oscar M Rueda Oscar M Rueda Cancer Research UK Cambridge Institute, Cambridge, UK Search for more papers by this author Peter Bouwman Peter Bouwman Division of Molecular Pathology and Cancer Genomics Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Jos Jonkers Jos Jonkers orcid.org/0000-0002-9264-9792 Division of Molecular Pathology and Cancer Genomics Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Anderson Ryan Anderson Ryan Department of Oncology, Lung Cancer Translational Science Research Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Bernardo Reina-San-Martin Bernardo Reina-San-Martin Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Institut National de la Santé et de la Recherche Médicale (INSERM), U964, Illkirch, France Centre National de Recherche Scientifique (CNRS), UMR7104, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Joannie Hui Joannie Hui Department of Chemical Pathology and Paediatrics, Faculty of Medicine, Chinese University of Hong Kong, Shatin, Hong Kong, China Search for more papers by this author Nelson Tang Nelson Tang Department of Chemical Pathology and Paediatrics, Faculty of Medicine, Chinese University of Hong Kong, Shatin, Hong Kong, China Search for more papers by this author Alejandra Bruna Alejandra Bruna Cancer Research UK Cambridge Institute, Cambridge, UK Search for more papers by this author Annamaria Biroccio Annamaria Biroccio Area of Translational Research, Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Madalena Tarsounas Corresponding Author Madalena Tarsounas [email protected] orcid.org/0000-0002-4273-2870 Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Eliana MC Tacconi Eliana MC Tacconi Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Xianning Lai Xianning Lai Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Cecilia Folio Cecilia Folio Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Manuela Porru Manuela Porru Area of Translational Research, Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Gijs Zonderland Gijs Zonderland Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Sophie Badie Sophie Badie Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Johanna Michl Johanna Michl Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Irene Sechi Irene Sechi Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Mélanie Rogier Mélanie Rogier Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Institut National de la Santé et de la Recherche Médicale (INSERM), U964, Illkirch, France Centre National de Recherche Scientifique (CNRS), UMR7104, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Verónica Matía García Verónica Matía García Division of Molecular Pathology and Cancer Genomics Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Ankita Sati Batra Ankita Sati Batra Cancer Research UK Cambridge Institute, Cambridge, UK Search for more papers by this author Oscar M Rueda Oscar M Rueda Cancer Research UK Cambridge Institute, Cambridge, UK Search for more papers by this author Peter Bouwman Peter Bouwman Division of Molecular Pathology and Cancer Genomics Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Jos Jonkers Jos Jonkers orcid.org/0000-0002-9264-9792 Division of Molecular Pathology and Cancer Genomics Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Anderson Ryan Anderson Ryan Department of Oncology, Lung Cancer Translational Science Research Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Bernardo Reina-San-Martin Bernardo Reina-San-Martin Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Institut National de la Santé et de la Recherche Médicale (INSERM), U964, Illkirch, France Centre National de Recherche Scientifique (CNRS), UMR7104, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Joannie Hui Joannie Hui Department of Chemical Pathology and Paediatrics, Faculty of Medicine, Chinese University of Hong Kong, Shatin, Hong Kong, China Search for more papers by this author Nelson Tang Nelson Tang Department of Chemical Pathology and Paediatrics, Faculty of Medicine, Chinese University of Hong Kong, Shatin, Hong Kong, China Search for more papers by this author Alejandra Bruna Alejandra Bruna Cancer Research UK Cambridge Institute, Cambridge, UK Search for more papers by this author Annamaria Biroccio Annamaria Biroccio Area of Translational Research, Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Madalena Tarsounas Corresponding Author Madalena Tarsounas [email protected] orcid.org/0000-0002-4273-2870 Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Author Information Eliana MC Tacconi1,‡, Xianning Lai1,‡, Cecilia Folio1, Manuela Porru2, Gijs Zonderland1, Sophie Badie1, Johanna Michl1, Irene Sechi1, Mélanie Rogier3,4,5,6, Verónica Matía García7, Ankita Sati Batra8, Oscar M Rueda8, Peter Bouwman7, Jos Jonkers7, Anderson Ryan9, Bernardo Reina-San-Martin3,4,5,6, Joannie Hui10, Nelson Tang10, Alejandra Bruna8, Annamaria Biroccio2 and Madalena Tarsounas *,1 1Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK 2Area of Translational Research, Regina Elena National Cancer Institute, Rome, Italy 3Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France 4Institut National de la Santé et de la Recherche Médicale (INSERM), U964, Illkirch, France 5Centre National de Recherche Scientifique (CNRS), UMR7104, Illkirch, France 6Université de Strasbourg, Illkirch, France 7Division of Molecular Pathology and Cancer Genomics Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands 8Cancer Research UK Cambridge Institute, Cambridge, UK 9Department of Oncology, Lung Cancer Translational Science Research Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK 10Department of Chemical Pathology and Paediatrics, Faculty of Medicine, Chinese University of Hong Kong, Shatin, Hong Kong, China ‡These authors contributed equally to this work *Corresponding author. Tel: +44 1865 617319; E-mail: [email protected] EMBO Mol Med (2017)9:1398-1414https://doi.org/10.15252/emmm.201607446 See also: AR Chaudhuri & A Nussenzweig (October 2017) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. Here, we investigate whether HR-compromised cells are sensitive to acetaldehyde, similarly to FANCD2-deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2, or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification, and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2-deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR-deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication-associated DNA damage, leading to checkpoint activation, G2/M arrest, and cell death. Acetaldehyde-arrested replication forks require BRCA2 and FANCD2 for protection against MRE11-dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2-deficient tumors and ex vivo in patient-derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP-ribose) polymerase (PARP) inhibitors. The work presented here therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2-deficient cells and tumors. Synopsis Treatment with acetaldehyde or with the alcohol-deterrent disulfiram, which enhances acetaldehyde levels, selectively eliminates BRCA1/2-deficient cells and tumors. Increasing cellular acetaldehyde might thus benefit cancer patients with BRCA1/2 mutations. Acetaldehyde and disulfiram increased the levels of RPA foci and decreased replication fork progression, leading to accumulation of replication-associated DNA damage specifically in BRCA2-deficient cells. The Aldh2 gene encodes an aldehyde dehydrogenase with key roles in endogenous acetaldehyde detoxification. Aldh2 gene deletion or its point mutation E487K associated with the ethanol-induced flushing syndrome in humans causes proliferation arrest in cells lacking BRCA1/2 expression. Growth of BRCA1/2-defective tumors, including those that have acquired resistance to PARP inhibitors, is suppressed by acetaldehyde treatment. Introduction BRCA1 and BRCA2 germ line mutations increase breast and ovarian cancer susceptibility in heterozygous carriers (Roy et al, 2012). More recently, BRCA2 mutations have been linked to predisposition to prostate and pancreatic cancers (Sandhu et al, 2013; Mateo et al, 2015; Waddell et al, 2015). At cellular level, BRCA1 and BRCA2 proteins play critical functions in genome integrity. In response to DNA damage induced by exogenous agents (e.g., ionizing radiation), they initiate HR reactions for double-strand break (DSB) repair and in response to replication stress they act to protect and restart replication forks stalled at sites of DNA damage. In both settings, BRCA1 and BRCA2 promote loading of RAD51 recombinase onto single-stranded DNA generated at DSBs and stalled replication forks. In addition to their role in HR repair, BRCA1, BRCA2, and RAD51 have been assigned key functions in the FA pathway of interstrand cross-link (ICL) repair (Howlett et al, 2002; Domchek et al, 2013; Ameziane et al, 2015; Wang et al, 2015). ICLs represent one of the most deleterious types of DNA damage, known to obstruct both replication and transcription (Kottemann & Smogorzewska, 2013). Consistent with this role, BRCA1/2-mutated cells and tumors are hypersensitive to ICL-inducing chemo-therapeutic agents, including cisplatin and mitomycin C (Deans & West, 2011). The eukaryotic genome is under continuous genotoxic attack from cell-intrinsic sources, one of the most potent being endogenous aldehydes. Acetaldehyde, an intermediate in the metabolic processing of alcohol, inflicts DNA damage particularly in the form of base damage, DNA–protein crosslinks, and ICLs (Lorenti Garcia et al, 2009). Highly reactive aldehydes are metabolized to less toxic substrates by at least 19 different aldehyde dehydrogenase (ALDH) enzymes (Koppaka et al, 2012). ALDH2 in particular has been implicated in the breakdown of acetaldehyde to acetate, an obligatory step in alcohol metabolism. Disulfiram (Antabuse) is an alcohol-aversive drug metabolized to products that inhibit ALDH1A1 and ALDH2 (Chen et al, 2014). By inhibiting ALDH activity, disulfiram causes acetaldehyde accumulation in the blood, culminating in unpleasant and potentially serious physiological symptoms including flushing, nausea, anxiety, blurred vision, and difficulty breathing, which are intended as a deterrent to prevent alcohol consumption. The discovery that FANCD2-deficient cells are hypersensitive to acetaldehyde provided the first indication that acetaldehyde-induced DNA damage requires the FA pathway for accurate repair (Langevin et al, 2011). BRCA1 and BRCA2 act in concert with the FA pathway during ICL repair to stabilize replication forks stalled at ICL sites and/or to repair resulting DNA lesions (Ceccaldi et al, 2016; Michl et al, 2016b). However, FANCD2 plays unique roles in BRCA1- or BRCA2-deficient cells by limiting replication and providing an independent mechanism for fork protection (Kais et al, 2016; Michl et al, 2016a). Thus, FANCD2 inactivation is lethal to BRCA1/2-deficient cells and tumors. In spite of the significant functional interactions between FANCD2 and BRCA1/2 in DNA repair and replication, whether BRCA1 and BRCA2 are also required in response to acetaldehyde-induced DNA damage similarly to FANCD2 has not yet been investigated. Here, we demonstrate for the first time the striking and specific vulnerability of BRCA1/2-deficient human and mouse cells to acetaldehyde. Disulfiram is also toxic in this context, as a result of ALDH2 inhibition. We provide evidence that replication-associated DNA damage and MRE11-dependent fork degradation trigger acetaldehyde sensitivity in human cells lacking BRCA2. This further explains tumor growth inhibition by acetaldehyde in BRCA1/2-deficient tumors, including those that have acquired resistance to PARP inhibitors. Therefore, our data provide a rationale for the use of drugs that increase endogenous acetaldehyde in treatment of BRCA1/2-compromised cancers, in response to the need for novel effective therapies targeting this tumor subset. Results HR-deficient cells are hypersensitive to acetaldehyde Mouse and chicken cells lacking FANCD2 are hypersensitive to exogenous acetaldehyde (Langevin et al, 2011), consistent with its ability to inflict DNA damage through DNA crosslinking (Lorenti Garcia et al, 2009). We thus investigated whether BRCA1 and BRCA2 deficiencies could also predispose cells to acetaldehyde sensitivity. First, we attempted to recapitulate acetaldehyde sensitivity in human colorectal adenocarcinoma DLD1 cells in which we deleted FANCD2 gene using the CRISPR/Cas9 system (Michl et al, 2016a). This deletion sensitized cells not only to cisplatin (Fig EV1A), a crosslinking agent in clinical use (Deans & West, 2011), but also to acetaldehyde treatment (Fig EV1B). Next, we investigated a potential role for BRCA2 in the cellular response to acetaldehyde by evaluating the impact of acetaldehyde on the viability of BRCA2-deleted DLD1 human cells (Horizon Discovery; Fig 1A). The PARP inhibitor olaparib was used as an additional control, based on its established ability to kill BRCA2-deficient cells (Bryant et al, 2005; Farmer et al, 2005). Incubation in the presence of acetaldehyde led to highly specific reduction in the viability of BRCA2-deficient DLD1 cells, comparable to the effect of olaparib treatment. Acetaldehyde also caused a significant reduction in survival of DLD1 cells lacking BRCA2 using clonogenic assays, in which olaparib and cisplatin were used as controls (Appendix Fig S1A–C). Importantly, acetaldehyde was toxic to BRCA2-deleted cells in spite of the FA pathway remaining active, as demonstrated by FANCD2 ubiquitylation (Fig EV1C). Click here to expand this figure. Figure EV1. Acetaldehyde toxicity to human FANCD2-deleted human cells and FANCD2 ubiquitylation in BRCA2-deleted cells A, B. Human DLD1 cells in which FANCD2 was deleted with CRISPR/Cas9 and control cells were incubated with the indicated concentrations of cisplatin (A) or acetaldehyde (B) for 6 days before processing for dose-dependent viability assays. Graphs are representative of two independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment. Inset, Western blot detection of FANCD2 expression. SMC1 was used as a loading control. C. BRCA2-proficient (+BRCA2) or BRCA2-deficient (−BRCA2) DLD1 cells were incubated with 4 mM acetaldehyde for 48 h before being processed for immunoblotting as indicated. D. H1299 cells expressing a DOX-inducible BRCA2 shRNA were grown in the presence or absence of DOX and transfected with control or FANCD2 siRNA before being processed for immunoblotting as indicated. DOX, doxycycline. Download figure Download PowerPoint Figure 1. BRCA1- and BRCA2-deficient human cells are hypersensitive to acetaldehyde A. BRCA2-proficient (+BRCA2) or BRCA2-deficient (−BRCA2) human DLD1 cells were incubated with the indicated concentrations of olaparib or acetaldehyde for 6 days before processing for dose-dependent viability assays. Graphs are representative of three independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment. Cell extracts prepared at the time of acetaldehyde addition were immunoblotted as indicated. SMC1 was used as a loading control. B, C. Human H1299 cells expressing DOX-inducible BRCA2 or BRCA1 shRNAs were grown in the presence or absence of DOX and incubated with the indicated concentrations of olaparib or acetaldehyde for 6 days, before processing for dose-dependent viability assays. Graphs are representative of three independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment. Cell extracts prepared at the time of acetaldehyde addition were immunoblotted as indicated. SMC1 was used as a loading control. DOX, doxycycline. Download figure Download PowerPoint Using RAD51 accumulation into nuclear foci as readout for HR activation, we addressed whether HR is required for the repair of DNA damage induced by acetaldehyde. We observed a marked induction of RAD51 foci in acetaldehyde-treated BRCA2-proficient DLD1 cells (Fig EV2A and B). Thus, acetaldehyde treatment invokes HR repair, which could explain the reduced survival of BRCA2-deleted cells in the presence of this compound. Click here to expand this figure. Figure EV2. Acetaldehyde and disulfiram induce RAD51 foci in BRCA2-proficient DLD1 cells Human DLD1 cells, BRCA2-proficient (+BRCA2) were incubated with acetaldehyde (4 mM) or disulfiram (10 μM) for 96 h prior to processing for immunofluorescence staining with anti-RAD51 antibody (green). DNA was counter-stained with DAPI (blue). Scale bar, 10 μm. Quantification of percentage of cells with 10 or more RAD51 foci in cells treated as in (A). At least 100 nuclei were quantified for each treatment. Ac, acetaldehyde; Di, disulfiram. Error bars represent SD of two independent experiments. P-values were calculated using an unpaired two-tailed t-test. Download figure Download PowerPoint To address whether acetaldehyde toxicity can be extended to other cell lines, we examined the response to this compound in human H1299 non-small cell lung carcinoma cells expressing a doxycycline (DOX)-inducible BRCA2 shRNA (BRCA2shDOX; Fig 1B). DOX addition inhibited BRCA2 expression assessed by immunoblotting. We observed a profound reduction in the viability of BRCA2-deficient H1299 cells upon treatment with acetaldehyde, similar to the effect of olaparib. Acetaldehyde was also toxic to H1299 human cells expressing a DOX-inducible shRNA against BRCA1 (BRCA1shDOX; Fig 1C). Olaparib sensitivity characteristic of these cells was used as a control. Further supporting the effect on cells lacking BRCA1 tumor suppressor, acetaldehyde also targeted specifically Brca1F/− MEFs in which Brca1-gene was deleted using Cre recombinase (Appendix Fig S2A). In contrast, MEFs lacking 53BP1, a DNA damage response factor known to promote non-homologous end joining (NHEJ; Bouwman et al, 2010; Bunting et al, 2010), were not affected by acetaldehyde treatment (Appendix Fig S2B). Moreover, RAD51 depletion in DLD1 and H1299 human cells led to acetaldehyde hypersensitivity to a similar extent as olaparib and cisplatin (Appendix Fig S3A and B). These results clearly demonstrate specific acetaldehyde toxicity to HR-compromised cells. Disulfiram, an ALDH inhibitor, targets BRCA1- and BRCA2-deficient cells Acetaldehyde is a product of physiological cell metabolism (Fig 2A), processed to acetate by ALDH enzymes, among which ALDH2 is best-characterized in human cells (Chen et al, 2014). Disulfiram, an ALDH inhibitor with high specificity for ALDH1A1 and ALDH2, is known to increase endogenous acetaldehyde levels, which represents the basis of its clinical use as an alcohol deterrent (Koppaka et al, 2012). In a viability screen for FDA-approved drugs that kill specifically BRCA2-deficient cells, we identified disulfiram among the high-scoring hits (our unpublished results). Moreover, we observed elevated levels of RAD51 foci upon disulfiram treatment of BRCA2-deleted DLD1 cells (Fig EV2A and B), indicating that DNA damage elicited by this drug requires HR repair. These results provided a strong rationale for investigating the impact of disulfiram on the viability of human cells lacking BRCA2. Disulfiram treatment led to a striking reduction in the viability of the BRCA2-deficient DLD1 cells compared to wild-type control cells (Fig 2B and Appendix Fig S1D). FACS-based ALDEFLUOR™ assays (Garaycoechea et al, 2012) showed similar inhibition of ALDH activity by disulfiram in BRCA2-proficient and BRCA2-deficient DLD1 cells (Fig EV3). This excludes the possibility that the marked sensitivity of BRCA2-deficient cells to disulfiram could be due to more effective ALDH inhibition in these cells. Figure 2. BRCA1- and BRCA2-deficient human cells are hypersensitive to disulfiram A. Schematic representation of sources of acetaldehyde and its cellular catabolism. ALDH2, a disulfiram target, is also indicated. B. BRCA2-proficient (+BRCA2) or BRCA2-deficient (−BRCA2) human DLD1 cells were incubated with the indicated concentrations of disulfiram for 6 days, before processing for dose-dependent viability assays. Graphs are representative of three independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment. C, D. Human H1299 cells expressing DOX-inducible BRCA2 or BRCA1 shRNAs were grown in the presence or absence of DOX and incubated with the indicated concentrations of disulfiram for 6 days, before processing for dose-dependent viability assays. Graphs are representative of three independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment. DOX, doxycycline. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. ALDEFLUOR™ assay in human DLD1 cells treated with disulfiram Quantification of ALDH activity relative to internal DEAB control in BRCA2-proficient (+BRCA2) and BRCA2-deficient (−BRCA2) human DLD1 cells treated with DMSO or disulfiram (10 μM) for 4 days. Error bars represent SD of two independent experiments. P-values were calculated using a one-sample t-test. Download figure Download PowerPoint Incubation of BRCA2shDOX H1299 cells in the presence of disulfiram also led to specific elimination of BRCA2-deficient (+DOX) relative to BRCA2-proficient (−DOX) cells (Fig 2C). Paradoxically, we observed an upturn in cell survival by increasing disulfiram concentration. This could be due to drug aggregate assembly at high concentrations, which cannot penetrate the cellular membrane and therefore lower drug efficiency. Furthermore, BRCA1 depletion using a DOX-inducible shRNA (BRCA1shDOX) in H1299 cells elicited disulfiram sensitivity (Fig 2D). The striking and specific disulfiram toxicity to cells lacking BRCA1 or BRCA2 recapitulates the effects of exogenous acetaldehyde. Acute replication stress induced by acetaldehyde and disulfiram in BRCA2-deficient cells The genotoxic potential of acetaldehyde is mediated in part by its ability to cause ICLs (Lorenti Garcia et al, 2009), which arrest replication and inflict replication-associated DSBs. We therefore investigated the possibility that acetaldehyde accumulation, either directly administered or indirectly, mediated by disulfiram addition, could trigger replication stress. RPA sub-nuclear foci mark regions of exposed single-stranded DNA and are commonly used as a readout for replication stress (Zeman & Cimprich, 2014). Indeed, immunofluorescence (IF) analysis of RPA foci revealed a marked induction specifically in BRCA2-deficient DLD1 cells upon treatment with either 10 μM disulfiram or 4 mM acetaldehyde (Fig 3A and B). In contrast, treatment of BRCA2-proficient cells with either compound resulted only in a small increase in the frequency of cells with 10 or more RPA foci. In order to directly evaluate the level of replication stress induced by acetaldehyde or disulfiram, we performed DNA fiber analyses, which allowed quantification of replication fork progression. We observed that replication track length was specifically and significantly reduced in the absence of BRCA2, upon treatment with either disulfiram or acetaldehyde (Fig 3C and Appendix Table S1). Figure 3. Elevated replication stress and G2/M accumulation in BRCA2-deficient human cells treated with disulfiram or acetaldehyde Representative images of BRCA2-proficient (+BRCA2) and BRCA2-deficient (−BRCA2) human DLD1 cells incubated with 10 μM disulfiram or 4 mM acetaldehyde for 4 days prior to processing for immunofluorescence staining with anti-RPA antibody (green). DNA was counter-stained with D
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