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Published OnlineFirst October 4, 2012; DOI: 10.1158/1541-7786.MCR-12-0168 Molecular Cancer DNA Damage and Cellular Stress Responses Research Alkylation Sensitivity Screens Reveal a Conserved Cross-species Functionome David Svilar1,3,4, Madhu Dyavaiah7, Ashley R. Brown3, Jiang-bo Tang3,5, Jianfeng Li1,3, Peter R. McDonald1, Tong Ying Shun1, Andrea Braganza1,3, Xiao-hong Wang3, Salony Maniar6, Claudette M. St Croix6, John S. Lazo1,3, Ian F. Pollack2,3, Thomas J. Begley7, and Robert W. Sobol1,3,5 Abstract To identify genes that contribute tochemotherapy resistance in glioblastoma, weconducted a synthetic lethal screen in a chemotherapy-resistant glioblastoma-derived cell line with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored toward "druggable" targets. Select DNA repair genes in the screen were validated indepen- dently, confirming the DNA glycosylases uracil-DNA glycosylase (UNG) and A/G-specific adenine DNA glycosylase (MYH) as well as methylpurine-DNA glycosylase (MPG) to be involved in the response to high dose TMZ. The involvement of UNG and MYH is likely the result of a TMZ-induced burst of reactive oxygen species. We then compared the human TMZ sensitizing genes identified in our screen with those previously identified from alkylator screens conducted in Escherichia coli and Saccharomyces cerevisiae. The conserved biologic processes across all three species compose an alkylation functionome that includes many novel proteins not previously thought to impact alkylator resistance. This high-throughput screen, validation and cross-species analysis was then followed by a mechanistic analysis of two essential nodes: base excision repair (BER) DNA glycosylases (UNG, human and mag1, S. cerevisiae) and protein modification systems, including UBE3B and ICMT in human cells or pby1, lip22, stp22 and aim22 in S. cerevisiae. The conserved processes of BER and protein modification were dual targeted and yielded additive sensitization to alkylators in S. cerevisiae. In contrast, dual targeting of BER and protein modification genes in human cells did not increase sensitivity, suggesting an epistatic relationship. Importantly, these studies provide potential new targets to overcome alkylating agent resistance. Mol Cancer Res; 10(12); 1580–96. Ó2012 AACR. Introduction (TMZ), radiation and surgery are currently used for treat- ment of GBM, yet median survival is still less than 2 years (2– Glioblastoma multiforme (GBM) is the most common fi and aggressive primary brain tumor (1). Temozolomide 4). Chemotherapy resistance and dif culties in surgical removal contribute to poor prognosis (1). TMZ methylates several bases in DNA, including the O6 position of guanine 6 Authors' Affiliations: Departments of 1Pharmacology & Chemical Biology, (O -MeG), the N7 position of guanine (N7-MeG) and the and 2Neurosurgery, University of Pittsburgh School of Medicine; 3Univer- N3 position of adenine (N3-MeA). Two of the lesions (N7- sity of Pittsburgh Cancer Institute, Hillman Cancer Center; 4Medical Sci- entist Training Program, University of Pittsburgh/Carnegie Mellon Univer- MeG and N3-MeA) account for greater than 80% of the sity; 5Department of Human Genetics, 6Department of Environmental and DNA lesions induced by TMZ, yet result in negligible Occupational Health, University of Pittsburgh Graduate School of Public clinical toxicity because of robust repair mechanisms. These Health, Pittsburgh, Pennsylvania; and 7Nanobioscience Constellation and Systems Toxicology Laboratory, College of Nanoscale Science and Engi- lesions are predominantly repaired by the base excision repair neering, University at Albany, Albany, New York (BER) pathway (5), initiated by one of eleven lesion specific Note: Supplementary data for this article are available at Molecular Cancer DNA glycosylases (6). Research Online (http://mcr.aacrjournals.org/). Virtually all of TMZ's clinical cytotoxicity is attributable to the O6-methylguanine (O6-MeG) lesion, which accounts Current address for J.-b. Tang: Department of Cancer Biology, Abramson 6 Family Cancer Research Institute, University of Pennsylvania School of for approximately 5% of TMZ induced lesions (7). The O Medicine, Philadelphia, Pennsylvania. -MeG lesion is repaired via a direct reversal mechanism by the protein O6-methylguanine-DNA methyltransferase Current address for P.R. McDonald: High Throughput Screening Labora- 6 tory, University of Kansas, Lawrence, Kansas. (MGMT), which transfers the O -methyl group from the guanine base onto a Cys residue in the MGMT protein (8). If Current address for J.S. Lazo: Departments of Pharmacology & Chemistry, 6 University of Virginia, School of Medicine, Charlottesville, Virginia. the O -MeG lesion is not removed by MGMT, during cellular replication the mis-pairing of O6-MeG with thy- Corresponding Author: Robert W. Sobol, Hillman Cancer Center, Uni- versity of Pittsburgh Cancer Institute, Research Pavilion, Suite 2.6a, 5117 mine is detected by the mismatch repair enzymes, triggering Centre Avenue, Pittsburgh, Pennsylvania 15213-1863. Phone: 412-623- apoptosis signaling and cytotoxicity (9). However, 5-year 7764; Fax: 412-623-7761; E-mail: [email protected] survival rates still remain low in TMZ-treated patients (3, 4), doi: 10.1158/1541-7786.MCR-12-0168 and TMZ resistance and/or recurrence with chemotherapy Ó2012 American Association for Cancer Research. resistant tumors is common. Resistant cells can harbor 1580 Mol Cancer Res; 10(12) December 2012 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst October 4, 2012; DOI: 10.1158/1541-7786.MCR-12-0168 A Cross-species Alkylation Functionome mutations in mismatch repair proteins such as mutS homo- were from Greiner Bio-One (GmbH). OptiMEM, EMEM, log 6 (MSH6; ref. 10) or have elevated expression of MGMT phosphate buffered saline (PBS), and Hoechst 33342 were (as in the T98G cell line; ref. 11). Earlier endeavors to from Invitrogen. The shRNA vectors used for stable KD cell enhance TMZ efficacy by using MGMT inhibitors to line creation were obtained as glycerol stocks from Sigma- prevent the repair of O6-MeG lesions have not shown an Aldrich and the UPCI lentiviral core facility (http://www. increase in sensitivity or efficacy in clinical trials (12), upci.upmc.edu/vcf/lenti.cfm). TMZ was from the National especially in TMZ-resistant GBM (13). Cancer Institute Developmental Therapeutics Program Because GBM tumors can be resistant to TMZ therapy (Bethesda, MD). Puromycin was from Clontech Laborato- and recurring tumors may acquire resistance to the O6-MeG ries, and gentamycin was obtained from Irvine Scientific. We lesion (14, 15), we explored novel mechanisms of TMZ used the following primary antibodies: UNG antibody resistance in tumor cells independent of the O6-MeG lesion. (#MBS200056) was from MyBioSource and proliferating To discover genes that sensitize cells to TMZ, we undertook cell nuclear antigen (PCNA) antibody (#sc-56) was from a synthetic lethal screen using Ambion's Silencer Human Santa Cruz. Secondary antibodies: goat anti-mouse horse- Druggable Genome siRNA library, the chemotherapeutic radish peroxidase (GAM-HRP) conjugates were from Bio- agent TMZ and the T98G cell line, a TMZ-resistant cell line Rad. Signal generation substrates were from Bio-Rad and derived from a GBM tumor (16). We determined that Thermo Fisher Scientific. All electrophoresis reagents were several biologic pathways were enriched for TMZ-sensitizing from Bio-Rad. genes, including proteins involved in BER, response to DNA damage, cellular proliferation, and protein modification. Cell lines and culture conditions Many similar biologic processes were also highly enriched Cell line and culture conditions were as previously in previous DNA alkylation screens conducted in Saccha- described (20–22). Briefly, the T98G glioblastoma cell line romyces cerevisiae and Escherichia coli (17, 18). We combined was purchased from American Type Culture Collection the selected hits from all 3 species and conducted a functio- (ATCC; obtained in 2004) and cultured in EMEM with nome analysis to identify significantly enriched biologic 10% heat-inactivated FBS, nonessential amino acids, sodium processes conserved across all 3 organisms (19). This allowed pyruvate, antibiotic/antimycotic, and gentamycin (16). The us to create a cross-species network representing the shared LN428 cells were cultured in alpha minimum essential media alkylation response from bacteria, yeast, and humans and with 10% heat-inactivated FBS, antibiotic/antimycotic, and shows an alkylation functionome that includes many novel gentamicin as we have described (23). The T98G cell line was proteins not previously thought to impact alkylation tested for cross species contamination and authenticated by resistance. Our identification of evolutionarily conserved RADIL cell check services as of November 16, 2012 (after the mechanisms affecting TMZ sensitivity suggests that although completion of the siRNA screen). The genetic profile pro- many proteins and processes impact sensitivity to alkylators, vided by RADIL was checked against the ATCC STR loci several critical survival pathways can be targeted to improve database to confirm the identity of the T98G cell line. chemotherapy efficacy. Simultaneous inhibition of both DNA repair and protein modification processes in yeast Pathway analysis and network visualization yielded greater sensitivity to alkylating agents than inhibition Several different programs were used to analyze
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