Chlorambucil targets BRCA 1/2‐deficient tumours and counteracts PARP inhibitor resistance
2019; Springer Nature; Volume: 11; Issue: 7 Linguagem: Inglês
10.15252/emmm.201809982
ISSN1757-4684
AutoresEliana MC Tacconi, Sophie Badie, Giuliana De Gregoriis, Timo Reisländer, Xianning Lai, Manuela Porru, Cecilia Folio, John C. Moore, Arnaud Kopp, Júlia Baguña Torres, Deborah Sneddon, Marcus Green, Simon Dedic, Jonathan W. Lee, Ankita Sati Batra, Oscar M. Rueda, Alejandra Bruna, Carlo Leonetti, Carlos Caldas, Bart Cornelissen, Laurent Brino, Anderson J. Ryan, Annamaria Biroccio, Madalena Tarsounas,
Tópico(s)Cancer Genomics and Diagnostics
ResumoArticle24 May 2019Open Access Source DataTransparent process Chlorambucil targets BRCA1/2-deficient tumours and counteracts PARP inhibitor resistance Eliana MC Tacconi Eliana MC Tacconi Genome Stability and Tumorigenesis Group, Department of Oncology, 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 Genome Stability and Tumorigenesis Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Giuliana De Gregoriis Giuliana De Gregoriis Genome Stability and Tumorigenesis Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Timo Reisländer Timo Reisländer orcid.org/0000-0003-0981-8311 Genome Stability and Tumorigenesis Group, Department of Oncology, 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 Genome Stability and Tumorigenesis Group, Department of Oncology, 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, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Cecilia Folio Cecilia Folio Genome Stability and Tumorigenesis Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author John Moore John Moore Lung Cancer Translational Science Research Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Arnaud Kopp Arnaud Kopp Institut de Génétique et de Biologie Cellulaire et Moléculaire (IGBMC), Inserm U1258, CNRS (UMR 7104), Université de Strasbourg, Illkirch, France Search for more papers by this author Júlia Baguña Torres Júlia Baguña Torres Radiopharmaceuticals and Molecular Imaging Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Deborah Sneddon Deborah Sneddon Radiopharmaceuticals and Molecular Imaging Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Marcus Green Marcus Green Lung Cancer Translational Science Research Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Simon Dedic Simon Dedic Genome Stability and Tumorigenesis Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Jonathan W Lee Jonathan W Lee Genome Stability and Tumorigenesis Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Ankita Sati Batra Ankita Sati Batra Department of Oncology, Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK Search for more papers by this author Oscar M Rueda Oscar M Rueda Department of Oncology, Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK Search for more papers by this author Alejandra Bruna Alejandra Bruna Department of Oncology, Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK Search for more papers by this author Carlo Leonetti Carlo Leonetti Area of Translational Research, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Carlos Caldas Carlos Caldas Department of Oncology, Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK Search for more papers by this author Bart Cornelissen Bart Cornelissen Radiopharmaceuticals and Molecular Imaging Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Laurent Brino Laurent Brino Institut de Génétique et de Biologie Cellulaire et Moléculaire (IGBMC), Inserm U1258, CNRS (UMR 7104), Université de Strasbourg, Illkirch, France Search for more papers by this author Anderson Ryan Anderson Ryan Lung Cancer Translational Science Research Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Annamaria Biroccio Corresponding Author Annamaria Biroccio [email protected] orcid.org/0000-0003-3198-3532 Area of Translational Research, IRCCS 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 Genome Stability and Tumorigenesis Group, Department of Oncology, 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 Genome Stability and Tumorigenesis Group, Department of Oncology, 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 Genome Stability and Tumorigenesis Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Giuliana De Gregoriis Giuliana De Gregoriis Genome Stability and Tumorigenesis Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Timo Reisländer Timo Reisländer orcid.org/0000-0003-0981-8311 Genome Stability and Tumorigenesis Group, Department of Oncology, 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 Genome Stability and Tumorigenesis Group, Department of Oncology, 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, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Cecilia Folio Cecilia Folio Genome Stability and Tumorigenesis Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author John Moore John Moore Lung Cancer Translational Science Research Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Arnaud Kopp Arnaud Kopp Institut de Génétique et de Biologie Cellulaire et Moléculaire (IGBMC), Inserm U1258, CNRS (UMR 7104), Université de Strasbourg, Illkirch, France Search for more papers by this author Júlia Baguña Torres Júlia Baguña Torres Radiopharmaceuticals and Molecular Imaging Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Deborah Sneddon Deborah Sneddon Radiopharmaceuticals and Molecular Imaging Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Marcus Green Marcus Green Lung Cancer Translational Science Research Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Simon Dedic Simon Dedic Genome Stability and Tumorigenesis Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Jonathan W Lee Jonathan W Lee Genome Stability and Tumorigenesis Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Ankita Sati Batra Ankita Sati Batra Department of Oncology, Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK Search for more papers by this author Oscar M Rueda Oscar M Rueda Department of Oncology, Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK Search for more papers by this author Alejandra Bruna Alejandra Bruna Department of Oncology, Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK Search for more papers by this author Carlo Leonetti Carlo Leonetti Area of Translational Research, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Carlos Caldas Carlos Caldas Department of Oncology, Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK Search for more papers by this author Bart Cornelissen Bart Cornelissen Radiopharmaceuticals and Molecular Imaging Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Laurent Brino Laurent Brino Institut de Génétique et de Biologie Cellulaire et Moléculaire (IGBMC), Inserm U1258, CNRS (UMR 7104), Université de Strasbourg, Illkirch, France Search for more papers by this author Anderson Ryan Anderson Ryan Lung Cancer Translational Science Research Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Annamaria Biroccio Corresponding Author Annamaria Biroccio [email protected] orcid.org/0000-0003-3198-3532 Area of Translational Research, IRCCS 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 Genome Stability and Tumorigenesis Group, Department of Oncology, 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, Sophie Badie1,‡, Giuliana De Gregoriis1,‡, Timo Reisländer1,‡, Xianning Lai1, Manuela Porru2, Cecilia Folio1, John Moore3, Arnaud Kopp4, Júlia Baguña Torres5, Deborah Sneddon5, Marcus Green3, Simon Dedic1, Jonathan W Lee1, Ankita Sati Batra6, Oscar M Rueda6, Alejandra Bruna6, Carlo Leonetti2, Carlos Caldas6, Bart Cornelissen5, Laurent Brino4, Anderson Ryan3, Annamaria Biroccio *,2 and Madalena Tarsounas *,1 1Genome Stability and Tumorigenesis Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK 2Area of Translational Research, IRCCS Regina Elena National Cancer Institute, Rome, Italy 3Lung Cancer Translational Science Research Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK 4Institut de Génétique et de Biologie Cellulaire et Moléculaire (IGBMC), Inserm U1258, CNRS (UMR 7104), Université de Strasbourg, Illkirch, France 5Radiopharmaceuticals and Molecular Imaging Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK 6Department of Oncology, Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +39 06 5266 2569 2545; E-mail: [email protected] *Corresponding author. Tel: +44 1865 617319; E-mail: [email protected] EMBO Mol Med (2019)11:e9982https://doi.org/10.15252/emmm.201809982 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 Due to compromised homologous recombination (HR) repair, BRCA1- and BRCA2-mutated tumours accumulate DNA damage and genomic rearrangements conducive of tumour progression. To identify drugs that target specifically BRCA2-deficient cells, we screened a chemical library containing compounds in clinical use. The top hit was chlorambucil, a bifunctional alkylating agent used for the treatment of chronic lymphocytic leukaemia (CLL). We establish that chlorambucil is specifically toxic to BRCA1/2-deficient cells, including olaparib-resistant and cisplatin-resistant ones, suggesting the potential clinical use of chlorambucil against disease which has become resistant to these drugs. Additionally, chlorambucil eradicates BRCA2-deficient xenografts and inhibits growth of olaparib-resistant patient-derived tumour xenografts (PDTXs). We demonstrate that chlorambucil inflicts replication-associated DNA double-strand breaks (DSBs), similarly to cisplatin, and we identify ATR, FANCD2 and the SNM1A nuclease as determinants of sensitivity to both drugs. Importantly, chlorambucil is substantially less toxic to normal cells and tissues in vitro and in vivo relative to cisplatin. Because chlorambucil and cisplatin are equally effective inhibitors of BRCA2-compromised tumours, our results indicate that chlorambucil has a higher therapeutic index than cisplatin in targeting BRCA-deficient tumours. Synopsis BRCA1/2-deficient tumours accumulate DNA damage and genomic rearrangements conducive for tumour progression, which is exploited in the clinic by targeted therapies against the BRCA1/2-mutated tumour subset. Chlorambucil is identified as the most effective drug in eliminating BRCA2-deficient cells. The bi-functional alkylator chlorambucil was specifically toxic to BRCA1/2-deficient cells and tumours, but not to wild type controls. Chlorambucil effectively eliminated cisplatin-resistant and olaparib-resistant BRCA1/2-deficient cells and tumours. Mechanistically, chlorambucil toxicity is mediated by accumulation of replication-associated DNA damage, similarly to cisplatin. ATR, FANCD2 and SNM1A nuclease are determinants of cellular sensitivity to both drugs. Chlorambucil is substantially less toxic to normal cells and tissues than cisplatin. Introduction BRCA1 and BRCA2 germline mutations have been associated with approximately 25% of the familial cases of breast and ovarian cancer (Futreal et al, 1994; Miki et al, 1994; Wooster et al, 1995); therefore, BRCA1 and BRCA2 represent classical tumour suppressor genes (Lord & Ashworth, 2016). In addition, somatic BRCA1 and BRCA2 mutations, as well as their epigenetic inactivation, have been unravelled in a significant proportion of the sporadic cancers, by recent comprehensive genome sequencing studies (Cancer Genome Atlas Research Network, 2011; Cancer Genome Atlas Network, 2012; Curtis et al, 2012; Ali et al, 2014; Pereira et al, 2016). Thus, the subset of patients affected by BRCA1/2 mutations appears to be greater than initially anticipated. BRCA1 and BRCA2 play essential roles in DNA replication and DSB repair (Michl et al, 2016). Both factors promote HR, a DNA repair pathway active during S/G2 phases of the cell cycle, which also provides a mechanism for the re-start of stalled replication forks. Consequently, BRCA1 or BRCA2 abrogation confers exquisite sensitivity to DNA damage-inducing drugs, in particular those inflicting cytotoxic DNA crosslinks (i.e. platinum drugs and DNA alkylators), which interfere with DNA replication. Sensitivity of BRCA1/2-mutated tumours to platinum compounds has been validated in multiple pre-clinical and clinical studies (Byrski et al, 2009, 2010; Silver et al, 2010; Tutt et al, 2018). Cisplatin and its derivatives are widely used chemotherapeutic drugs, which inflict complex DNA lesions in the form of intra- and inter-strand crosslinks (ICLs; Deans & West, 2011). Similar lesions are induced by DNA-alkylating agents (Fu et al, 2012), which include mono-functional (e.g. mitomycin C, nimustine) or bifunctional alkylators (e.g. chlorambucil, cyclophosphamide, melphalan), some showing specific toxicity against BRCA1/2-deficient cells and tumours (Evers et al, 2010; Vollebergh et al, 2014; Pajic et al, 2017). Interestingly, cisplatin induces primarily intrastrand crosslinks (Jamieson & Lippard, 1999), whilst bifunctional alkylators cause mainly ICLs, which represent the most potent type of cytotoxic DNA lesion (McHugh et al, 2001). Although alkylating agents display similar selectivity to cisplatin in targeting BRCA1/2-deficiencies, they have largely been abandoned for clinical use in breast and ovarian cancers, due to early sub-optimal results in non-stratified patient populations (Williams et al, 1985). Small molecule inhibitors of poly(ADP-ribose) polymerase (PARP) are currently at the forefront of clinical research for the treatment of BRCA-compromised breast, ovarian and prostate tumours (Mateo et al, 2015; Mirza et al, 2016; Robson et al, 2017; Litton et al, 2018). PARP inhibitors induce DNA damage indirectly (Lord & Ashworth, 2017) by immobilising PARP enzymes to DNA ends and suppressing their ability to PARylate various substrates (Murai et al, 2012; Pascal & Ellenberger, 2015). In spite of the fact that platinum drugs and PARP inhibitors show initially good responses in the clinic, most patients acquire resistance to these drugs (Rottenberg et al, 2007; Sakai et al, 2008; Shafee et al, 2008; Tutt et al, 2010; Norquist et al, 2011; Ter Brugge et al, 2016). Thus, there is a clear necessity for identifying new drugs or drug combinations that can target BRCA1/2-deficient cells and tumours. Here, we report the screen of a chemical library containing 1,280 drugs approved for clinical use by the US Food and Drug Administration (FDA). The highest scoring hit in our screen was chlorambucil, a bifunctional alkylator routinely used in chemotherapeutic regimens against CLL (Goede et al, 2014; Jain & O'Brien, 2015). We demonstrate that chlorambucil has high selective toxicity against human cells and xenograft tumours with compromised BRCA1/2 function. Mechanistically, chlorambucil acts by inducing replication stress and DSBs in actively replicating cells. Although similar to cisplatin in targeting BRCA-deficient tumours, chlorambucil shows substantially lower toxicity to normal cells and tissues. Our results suggest that the clinical use of chlorambucil in the BRCA1/2-deficient subset of cancer patients should be re-evaluated. Results Pharmacological screen for drugs that selectively eliminate BRCA2-deficient cells In order to identify drugs in clinical use that can target specifically BRCA2-deficient cells, we performed a viability screen using the Prestwick chemical library (http://www.prestwickchemical.com/libraries-screening-lib-pcl.html) containing 1,280 FDA-approved drugs. Since all drugs are suitable for human testing, any compounds identified in this screen could rapidly be repurposed for the treatment of BRCA1/2-mutated patients. We conducted two independent screens, each in triplicate, at drug concentration of 5 μM (Dataset EV1, Appendix Fig S1) using hamster BRCA2-deficient VC8 cells and control BRCA2-complemented cells (Kraakman-van der Zwet et al, 2002). We demonstrated previously (Chaikuad et al, 2014; Zimmer et al, 2016) that these BRCA2-deficient cells are hypersensitive to PARP inhibitors, ERK1/2 inhibitors and pyridostatin, when compared to BRCA2-proficient counterparts. A similar chemical library screen aiming to identify drugs that target BRCA2-deficiency was previously performed (Evers et al, 2010) using Brca2−/− mouse mammary tumour-derived cell lines and the LO-PAC®1280 Sigma library of pharmacologically active compounds (Dataset EV1). The chemical composition of this library was different from that of the Prestwick library used here, with the two libraries having approximately 25% compounds in common. Among the top scoring hits in our Prestwick library screens (Dataset EV1, Appendix Fig S1), we identified chlorambucil, a bifunctional alkylating agent used in the past for the treatment of breast and ovarian cancer (Williams et al, 1985; Senn et al, 1997), irinotecan, a topoisomerase I inhibitor in use for the treatment of cancer patients with BRCA1 mutations (Kennedy et al, 2004), and disulfiram, an aldehyde dehydrogenase inhibitor used in the clinic as an alcohol deterrent. Our group has recently characterised disulfiram as an agent specifically toxic to BRCA1/2-deficient cells and tumours, with significant therapeutic potential (Tacconi et al, 2017). Given that our screens were conducted in hamster cells, we validated chlorambucil and irinotecan in BRCA2-deficient human cells. Human colorectal adenocarcinoma BRCA2−/− DLD1 cells (Zimmer et al, 2016; Fig 1A) were hypersensitive to both drugs, when compared with BRCA2+/+ DLD1 cells. Olaparib and cisplatin were used as controls for selective targeting of BRCA2-deficient cells. Moreover, spheroid cultures established from BRCA2−/− DLD1 cells recapitulated the chlorambucil sensitivity observed in 2D cultures (Fig 1B). Figure 1. Chlorambucil sensitivity of BRCA2-deficient human cells and spheroids A. Dose-dependent viability assays of BRCA2-proficient (+BRCA2) or BRCA2-deficient (−BRCA2) human DLD1 cells treated with drugs at the indicated concentrations for 6 days. B. Human spheroids established from BRCA2-proficient (+BRCA2) or BRCA2-deficient (−BRCA2) DLD1 cells were incubated with 1.25 µM olaparib or 0.5 µM chlorambucil over the indicated period of time. Data information: (A, B) Graphs represent average values obtained from three independent experiments, each performed in triplicate. Error bars represent SEM. Download figure Download PowerPoint Chlorambucil is toxic to BRCA1-deficient tumour cells, including those that acquired olaparib resistance To address the efficacy of chlorambucil against other HR-deficient cells, we assessed the response to this drug in BRCA1-deficient human cells. RPE1 cells immortalised by hTERT overexpression and TP53 knockout, which carry a BRCA1 CRISPR/Cas9-mediated deletion (Zimmermann et al, 2018), were hypersensitive to chlorambucil, as well as to olaparib and cisplatin used as controls (Fig 2A). Figure 2. Chlorambucil sensitivity of BRCA1-deficient human and mouse cells, including those that have acquired olaparib resistance A. Dose-dependent viability assays of BRCA1-proficient (+BRCA1) or BRCA2-deficient (−BRCA1) human RPE1-hTERT and TP53-deleted cells treated with drugs at the indicated concentrations for 6 days. B, C. Dose-dependent viability assays of Brca1+/+ and Brca1−/− mouse mammary tumour-derived cell lines treated with drugs at the indicated concentrations for 6 days. Data information: (A–C) Graphs represent average values obtained from three independent experiments, each performed in triplicate. Error bars represent SEM. Download figure Download PowerPoint Moreover, we tested chlorambucil in cellular models in which BRCA1 gene inactivation is associated with olaparib resistance. Olaparib sensitivity characteristic of Brca1-deleted mouse mammary tumour-derived cells is abrogated upon loss of 53BP1 (Fig 2B; Bouwman et al, 2010; Tacconi et al, 2017). Nevertheless, these cells remained hypersensitive to cisplatin and chlorambucil. Notably, Brca1−/−53bp1−/− cells were more sensitive to cisplatin than Brca1−/− cells. A similar trend was previously reported in mouse embryonic fibroblasts (Bunting et al, 2012), suggesting that BRCA1/53BP1-deficient, olaparib-resistant tumours may be also more responsive to cisplatin in the clinic. This is indeed the case as demonstrated by a recent clinical trial in which patients with BRCA1/2 mutated, PARP inhibitor-resistant ovarian cancers showed a robust response to platinum-based therapies (Ang et al, 2013). To generate a second model of olaparib resistance, we inactivated REV7 using two different shRNAs in Brca1-deleted mouse cells, as previously described (Xu et al, 2015). Cells lacking both REV7 and BRCA1 were less sensitive to olaparib than BRCA1-deficient; however, they were effectively eliminated by cisplatin and chlorambucil treatments (Fig 2C). Thus, chlorambucil, similarly to cisplatin, can eliminate BRCA1-deficient cells that developed PARP inhibitor resistance via 53BP1 or REV7 inactivation. Cisplatin-resistant BRCA2-deficient cells derived from human tumours are targeted by chlorambucil To further investigate the therapeutic potential of chlorambucil, we tested its effect in cell lines established from BRCA2-compromised human tumours. Capan-1 cells derived from a pancreatic adenocarcinoma carry a C-terminal BRCA2 truncation, which impairs RAD51 nuclear localisation (Chen et al, 1998). Capan-1 cells showed significantly higher sensitivity to chlorambucil, as well as to cisplatin and olaparib, when compared to MIA PaCa-2 pancreatic cancer cells with normal BRCA2 expression (Fig 3A). Figure 3. Chlorambucil sensitivity of BRCA2-deficient human tumour-derived cell lines, including those that have acquired cisplatin resistance A. Dose-dependent viability assays of BRCA2-deficient (Capan-1) or BRCA2-proficient (MIA PaCa-2) human pancreatic carcinoma-derived cells treated with drugs at the indicated concentrations for 6 days. B. Dose-dependent viability assays of BRCA2-deficient (PEO1) or BRCA2-proficient (C4-2) human ovarian tumour-derived cells treated with drugs at the indicated concentrations for 6 days. C. BRCA2-deficient (PEO1) or BRCA2-proficient (C4-2) human ovarian tumour-derived cells were infected with lentiviruses expressing control or CHD4 shRNAs, followed by selection with puromycin for 72 h. Dose-dependent viability assays were performed on cells treated with drugs at the indicated concentrations for 6 days. Data information: (A, B) Graphs represent average values obtained from three independent experiments, each performed in triplicate. Error bars represent SEM. (C) Error bars represent SEM of three technical replicates. Source data are available online for this figure. Source Data for Figure 3 [emmm201809982-sup-0003-SDataFig3.pdf] Download figure Download PowerPoint As a second tumour-derived model, we used PEO1 cells established from a human ovarian tumour carrying a N-terminal BRCA2 truncation, which abrogates HR repair. C4-2 cells, in which wild-type BRCA2 was restored by treatment with cisplatin (Sakai et al, 2009), were used as a control. Viability assays demonstrated that PEO1 cells were hypersensitive to chlorambucil, in contrast to C4-2 cells (Fig 3B). Notably, PEO1 cells, as well as other human cell lines lacking BRCA1 or BRCA2 (DLD1 BRCA2−/−, HCT116 BRCA2−/− and RPE1 BRCA1−/−), showed sensitivity to melphalan, another bifunctional alkylator (Appendix Fig S2). These results support the efficacy of other bifunctional alkylators against BRCA1/2-deficient cells, in agreement with previous studies (Evers et al, 2010). Loss of the chromatin remodelling factor CHD4 confers resistance to cisplatin in BRCA2-deficient PEO1 cells, through unknown, HR-independent mechanisms that confer DNA damage tolerance (Guillemette et al, 2015). We recapitulated this observation by inhibiting CHD4 expression in PEO1 BRCA2-deficient cells (Fig 3C). Lentiviral shRNA-mediated CHD4 depletion increased resistance of PEO1 cells to cisplatin, whilst it had no effect on the cisplatin response of BRCA2-proficient C4-2 cells. Importantly, chlorambucil effectively eliminated both cisplatin-sensitive and cisplatin-resistant BRCA2-deficient PEO1 cells. These results suggest a potential clinical use for chlorambucil in targeting BRCA2-deficient tumours which acquired cisplatin resistance. Chlorambucil induces replication stress and DNA damage accumulation in BRCA2-deficient cells Alkylating agents can inflict DNA lesions in the form of intra- and inter-strand DNA crosslinks, with a bias towards the latter (Deans & West, 2011). HR repair is an obligatory step in ICL resolution. In cells with compromised HR repair, ICLs interfere with DNA replication, leading to DSB accumulation and cell death (Michl et al, 2016). We therefore addressed the possibility that chlorambucil toxicity to BRCA2-deficient cells is due to ICL-inflicted DNA replication and DSB repair defects. The response of BRCA2-proficient and BRCA2-deficient DLD1 cells to chlorambucil was evaluated using time course experiments and immunoblotting for checkpoint activation markers (Fig 4A). RPA phosphorylation at Ser33, a marker for ATR activation and replication stress (Zeman & Cimprich, 2014), was induced in BRCA2-proficient cells following exposure to 1 μM chlorambucil for 48 h. In contrast, BRCA2-deficient cells, with intrinsic defects in replication fork progression and stability (Zimmer et al, 2016), showed detectable levels of RPA Ser33 phosphorylation even in the absence of any treatment (0 h), and these were markedly increased upon incubation with 1 μM chlorambucil from 16 h onwards. BRCA2-deficient cells also showed elevated levels of KAP1 Ser824 phosphorylation, a signature of ATM-dependent checkpoint activation and DNA damage accumulation. Phosphorylated KAP1 was detected from 24 h of treatment with chlorambucil in BRCA2-deficient cells. As RPA phosphorylation occurs earlier (16 h), this suggests that replication stress may precede DSB formation in response to chlorambucil. Cleaved PARP, an a
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