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Glioblastoma Cells Containing Mutations in the Cohesin Component STAG2 Are Sensitive to PARP Inhibition

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Glioblastoma Cells Containing Mutations in the Cohesin Component STAG2 Are Sensitive to PARP Inhibition Published OnlineFirst December 19, 2013; DOI: 10.1158/1535-7163.MCT-13-0749 Molecular Cancer Cancer Biology and Signal Transduction Therapeutics Glioblastoma Cells Containing Mutations in the Cohesin Component STAG2 Are Sensitive to PARP Inhibition Melanie L. Bailey1, Nigel J. O'Neil1, Derek M. van Pel2, David A. Solomon3, Todd Waldman4, and Philip Hieter1 Abstract Recent data have identified STAG2, a core subunit of the multifunctional cohesin complex, as a highly recurrently mutated gene in several types of cancer. We sought to identify a therapeutic strategy to selectively target cancer cells harboring inactivating mutations of STAG2 using two independent pairs of isogenic glioblastoma cell lines containing either an endogenous mutant STAG2 allele or a wild-type STAG2 allele restored by homologous recombination. We find that mutations in STAG2 are associated with significantly increased sensitivity to inhibitors of the DNA repair enzyme PARP. STAG2-mutated, PARP-inhibited cells accumulated in G2 phase and had a higher percentage of micronuclei, fragmented nuclei, and chromatin bridges compared with wild-type STAG2 cells. We also observed more 53BP1 foci in STAG2-mutated glioblastoma cells, suggesting that these cells have defects in DNA repair. Furthermore, cells with mutations in STAG2 were more sensitive than cells with wild-type STAG2 when PARP inhibitors were used in combination with DNA-damaging agents. These data suggest that PARP is a potential target for tumors harboring inactivating mutations in STAG2, and strongly recommend that STAG2 status be determined and correlated with therapeutic response to PARP inhibitors, both prospectively and retrospectively, in clinical trials. Mol Cancer Ther; 13(3); 724–32. Ó2013 AACR. Introduction or MCD1), and one of two possible stromal antigen Inhibition of PARP has emerged as a promising drug proteins (STAG1 or STAG2). Together, these four sub- strategy for the treatment of cancers mutated for BRCA1/2 units can encompass newly replicated sister chromatids because of its ability to selectively kill cells through syn- and hold them in close proximity (8). Beyond its well- thetic lethality (1, 2). More recently, PARP inhibitors have known function in chromosome segregation, cohesin been shown to be effective in cells with defects in other has several additional roles in the cell. Similar to other genes involved in homologous recombination and the genes sensitive to PARP inhibition, defects in cohesin DNA damage response suggesting that PARP inhibitors components affect both replication fork integrity and may be effective in treating a wider range of tumors that homologous recombination repair (7, 9, 10). Cohesin is do not have BRCA mutations (3–6). Identification of other recruited to sites of replication fork pausing and double- tumor genotypes susceptible to PARP inhibition will strand breaks (DSB) and has also been shown to pro- expand the utility of these drugs. mote replication fork restart and DNA repair through The cohesin complex, named for its role in sister its interactions with other proteins (11–13). In addition, chromatid cohesion, is well conserved across organisms because of its ability to encircle sister chromatids, the (7). In humans, the core mitotic complex consists of four cohesin complex is thought to promote error-free subunits:SMC1A,SMC3,RAD21(alsoknownasSCC1 recombination repair with the neighboring undamaged DNA strand in the S–G2 phases of the cell cycle (10). Supporting the idea that cells mutated for cohesin genes might be sensitive to PARP inhibition, we have shown Authors' Affiliations: 1Michael Smith Laboratories, University of British Columbia; 2The Centre for Drug Research and Development, Vancouver, that knockdown of three of the cohesin core components British Columbia, Canada; 3Department of Pathology, University of Cali- (SMC1, SMC3, and RAD21) can render cells sensitive to fornia San Francisco, San Francisco, California; and 4Department of the PARP inhibitor olaparib (14). Oncology, Lombardi Comprehensive Cancer Center, Georgetown Univer- STAG2 sity School of Medicine, Washington, DC Recently, the cohesin gene, , was discovered to be highly mutated in glioblastoma, Ewing sarcoma, Corresponding Author: Philip Hieter, Michael Smith Laboratories, 2185 East Mall, Room 323, University of British Columbia, Vancouver, British and melanoma cells (15). These mutations led to either Columbia, Canada V6T1Z4. Phone: 604-822-5115; Fax: 604-822-2114; truncation or functional inactivation of the STAG2 E-mail: [email protected] protein that is easily detected in cells or tissues by Note: Supplementary data for this article are available at Molecular Cancer immunohistochemistry or Western blot analysis using Therapeutics Online (http://mct.aacrjournals.org/). antibodies. Given the previous data that knockdown of doi: 10.1158/1535-7163.MCT-13-0749 cohesin components results in PARP inhibitor sensitiv- Ó2013 American Association for Cancer Research. ity (14), we wanted to determine if tumor cells with 724 Mol Cancer Ther; 13(3) March 2014 Downloaded from mct.aacrjournals.org on November 6, 2015. © 2014 American Association for Cancer Research. Published OnlineFirst December 19, 2013; DOI: 10.1158/1535-7163.MCT-13-0749 STAG2 Mutation and PARP Inhibition in Glioblastoma Cells STAG2 mutations were susceptible to PARP inhibition. inhibitors (Roche). Cells were lysed by sonication and Here, we show that glioblastoma cell lines with muta- centrifuged to remove debris. Lysates were separated by tions in STAG2 are significantly more sensitive to PARP SDS–PAGE, transferred to polyvinylidene difluoride, and inhibitors than matched, isogenic STAG2 wild-type blotted with the indicated antibodies. lines. This proliferation defect results in an accumula- For flow cytometry, cells were grown and harvested as tion of cells in G2 phase and genome instability. Fur- above, before being fixed in cold 70% ethanol. Where thermore, STAG2-mutated cell lines demonstrate an indicated, cells were first stained with pH3 antibody increased sensitivity when combinations of DNA-dam- followed by anti-rabbit conjugated to Alexa Fluor 488 aging chemotherapeutics and PARP inhibitors are used, (Jackson Immunoresearch), before being incubated with providing a therapeutic rationale for PARP inhibitors propidium iodide and RNase A. Cell-cycle analysis was either as a single agent, or in combination with other done using FlowJo. Cell lines were compared using a one- DNA-damaging agents, in STAG2-deficient tumors. tailed, matched Student t test. Error bars represent SEM. Materials and Methods Immunofluorescence Materials and cell culture Cells were grown on coverslips with and without PARP Olaparib (AZD2281), veliparib (ABT-888), and ruca- inhibitor for 3 (H4) or 4 (42MGBA) days before fixation in parib (AG014699) were purchased from Selleck Chemicals; 1:1 methanol:acetone and permeabilization in 0.1% Triton temozolomide and camptothecin were purchased from X-100. Coverslips were incubated with anti-53BP1 and anti-rabbit conjugated to Cy3 (Jackson Immunoresearch) Sigma-Aldrich. Antibodies used were anti-PAR (Trevi- 0 gen), anti-STAG2 (Santa Cruz Biotechnology), anti-SMC1, before being stained with 4 ,6-diamidino-2-phenylindole anti-SMC3, anti-pS10 Histone H3 (pH3), anti-53BP1anti- (DAPI) and viewed on a Zeiss Axioplan 2 Fluorescence GAPD, and anti-a-tubulin (all from Abcam). H4 and microscope. At least 200 cells were counted for each 42MGBA parental and STAG2 knock-in (KI) cell lines have experiment. For micronuclei, fragmented nuclei, and chromatin bridges, cell lines were compared using a been described previously (15). H4 and 42MGBA cell lines t obtained from Solomon and colleagues were from the one-tailed, matched Student test. For 53BP1 foci, cell American Type Culture Collection and DSMZ, respective- lines were compared using a Fisher exact test. ly, and were cultured in Dulbecco’s Modified Eagle Medi- Results um (DMEM) þ 10% FBS at 37 C and 5% CO for 1 to 2 2 STAG2 months at a time before reinitiation from early passage, -mutated glioblastoma cell lines are sensitive frozen stocks. Cell lines were checked regularly for the to PARP inhibition STAG2 presence or absence of STAG2 by Western blot analysis To determine whether mutation causes PARP (Supplementary Fig. S1). inhibitor sensitivity, we used two paired sets of glio- blastoma cell lines described by Solomon and collea- Cell counting experiments and clonogenic assays gues(15):H4(whichhasa25-bpinsertioninexon12of To assess cell number by nuclei counting, cells were STAG2) and 42MGBA (which has a nonsense mutation plated in a 96-well format with 6 technical replicates for in exon 20 of STAG2),whichwereeachmatchedwith each drug concentration. Twenty-four hours after plating, STAG2 KI lines that have these mutations corrected via inhibitors or dimethyl sulfoxide (DMSO) were diluted HR (H4 STAG2 KI and 42MGBA STAG2 KI, respective- into DMEM and added to wells. Cells were fixed in 3.7% ly). Using these two independent isogenic cell line pairs, paraformaldehyde after 4 to 5 days and then stained with we first looked at the proliferation of the H4 and Hoechst 33342 before nuclei were counted on a Cellomics 42MGBA cell lines in the presence of the PARP inhib- Arrayscan VTI. itor, olaparib, and found that over a range of concen- For clonogenic assays, cells were plated at single-cell trations, both the H4 and 42MGBA STAG2-mutated cell density in 6-well dishes with three replicates per drug lines showed significantly decreased cell number when concentration.
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