Defective sister chromatid cohesion is synthetically lethal with impaired APC/C function

ARTICLE Received 21 Nov 2014 | Accepted 19 Aug 2015 | Published 1 Oct 2015 DOI: 10.1038/ncomms9399 OPEN Defective sister chromatid cohesion is synthetically lethal with impaired APC/C function Job de Lange1, Atiq Faramarz1, Anneke B. Oostra1, Renee X. de Menezes2, Ida H. van der Meulen3, Martin A. Rooimans1, Davy A. Rockx1, Ruud H. Brakenhoff4, Victor W. van Beusechem3, Randall W. King5, Johan P. de Winter1,z & Rob M.F. Wolthuis1 Warsaw breakage syndrome (WABS) is caused by defective DDX11, a DNA helicase that is essential for chromatid cohesion. Here, a paired genome-wide siRNA screen in patientderived cell lines reveals that WABS cells do not tolerate partial depletion of individual APC/C subunits or the spindle checkpoint inhibitor p31comet. A combination of reduced cohesion and impaired APC/C function also leads to fatal mitotic arrest in diploid RPE1 cells. Moreover, WABS cell lines, and several cancer cell lines with cohesion defects, display a highly increased response to a new cell-permeable APC/C inhibitor, apcin, but not to the spindle poison paclitaxel. Synthetic lethality of APC/C inhibition and cohesion defects strictly depends on a functional mitotic spindle checkpoint as well as on intact microtubule pulling forces. This indicates that the underlying mechanism involves cohesion fatigue in response to mitotic delay, leading to spindle checkpoint re-activation and lethal mitotic arrest. Our results point to APC/C inhibitors as promising therapeutic agents targeting cohesion-defective cancers. 1 Department of Clinical Genetics, section Oncogenetics, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. 2 Department of Epidemiology and Biostatistics, VU University Medical Center, De Boelelaan 1118, 1081 HV Amsterdam, The Netherlands. 3 Department of Medical Oncology, RNA Interference Functional Oncogenomics Laboratory, VU University Medical Center, De Boelelaan 1118, 1081 HV Amsterdam, The Netherlands. 4 Department of Otolaryngology—Head and Neck Surgery, VU University Medical Center, De Boelelaan 1118, 1081 HV Amsterdam, The Netherlands. 5 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA. Correspondence and requests for materials should be addressed to J.D.L. (email: ) or to R.M.F.W. (email: ). zDeceased. NATURE COMMUNICATIONS | 6:8399 | DOI: 10.1038/ncomms9399 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 1 ARTICLE C NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9399 ell division requires the duplication of all chromosomes, followed by their segregation as two identical sister chromatids into two new daughter cells. Sister chromatid cohesion holds sister chromatids together until their proper separation is initiated at the metaphase-to-anaphase transition. Pairing of sister chromatids is achieved by a huge ring-shaped protein complex named cohesin, which consists of Smc1, Smc3, Rad21 (Scc1 in yeast) and either SA1 or SA2 (Scc3 in yeast). Besides keeping sister chromatids paired during early stages of mitosis, cohesin’s DNA tethering capacity facilitates multiple additional processes in the cell, such as DNA repair, ribosome biogenesis, regulation of gene transcription and initiation of DNA replication1. Defects in the cohesion network are the cause of several rare genetic diseases named cohesinopathies. These include Cornelia de Lange Syndrome (CdLS, caused by mutations in NIPBL, Smc1A, Smc3, Rad21 or HDAC8 (refs 2–5)), Roberts Syndrome (RBS, caused by ESCO2 mutations6,7) and Warsaw Breakage Syndrome (WABS, caused by DDX11 mutations8). Although it is not clear whether these predispositions are linked to an increased cancer risk, mutations in genes encoding cohesin subunits and regulators have been reported in a substantial number of human tumours9–15. Cohesion defects may thus form a new hall mark of cancer that could be exploited in therapy. When cells enter mitosis, the bulk of cohesin is removed from chromosome arms during prophase, in a manner dependent on phosphorylation of cohesin subunits by mitotic kinases and the cohesion antagonist Wapl (reviewed in ref. 16). However, centromeres are protected against loss of cohesion by Sgo1, which attracts a phosphatase to prevent phosphorylation of the Wapl antagonist Sororin, and SA2 (refs 17–21). During prometaphase, the kinetochores of paired sister chromatids attach to the mitotic spindle and subsequently come under tension of spindle pulling forces. Resisting spindle pulling forces is an important function of sister chromatid cohesion, preventing premature sister chromatid separation until the last pair of sister chromatids becomes bioriented on the mitotic spindle. The occurrence of prematurely separated sister chromatids which lose microtubule-kinetochore attachments activates the spindle assembly checkpoint (SAC)22. Continuous arrest of cells in the SAC may lead to cell death or highly aneuploid daughter cells23. The SAC is an evolutionary conserved signalling cascade that acts in prometaphase and keeps cyclin B1-Cdk1 active during the process of chromosome biorientation24,25. Proper attachment of all the paired sister chromatids to the spindle and their alignment to the cell equator is a stochastic process that can take roughly up to 1 h in normal cells. Maintenance of cyclin B1-Cdk1 activity during this phase is essential to keep the mitotic state until biorientation is complete. Simultaneously, Separase, a Rad21 protease, must be kept inactivated to protect centromere cohesion. The SAC is kept activate by kinetochores that are not properly attached to spindle microtubules, stimulating production of the mitotic checkpoint complex (MCC), composed of BubR1, Bub3, Mad2 and Cdc20 (ref. 26). The MCC blocks the anaphase promoting complex or cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase, so that three of its substrates remain stable for multiple hours: Securin, which blocks Separase27, cyclin B1, which keeps Cdk1 active to keep cells in mitosis28, and geminin, which blocks premature DNA replication licensing29. Achievement of proper attachment and centromere tension silences the SAC, activating APC/C-Cdc20. This leads to degradation of securin to release Separase, cleaving the cohesin subunit Rad21 and allowing chromatid separation to opposite spindle poles. Cyclin B1 degradation occurs at the same time and causes inactivation of Cdk1, initiation of cytokinesis and mitotic exit30. Geminin is also degraded, preparing cells for DNA replication29. 2 SAC silencing may involve multiple mechanisms, such as tension-sensitive kinetochore phosphorylations31, activation of phosphatases that antagonize certain mitotic kinases32 and dynein-microtubule-mediated stripping of SAC proteins from kinetochores upon microtubule attachment33. Furthermore, p31comet promotes the release of Mad2 from the MCC, thereby initiating Cdc20 release downstream of kinetochore (...truncated)