Genome-Wide Identification of Genes Conferring Resistance to the Anticancer Agents Cisplatin, Oxaliplatin, and Mitomycin C

[CANCER RESEARCH 64, 3940–3948, June 1, 2004] Genome-Wide Identification of Genes Conferring Resistance to the Anticancer Agents Cisplatin, Oxaliplatin, and Mitomycin C

H. Irene Wu,1 James A. Brown,1 Mary J. Dorie,1 Laura Lazzeroni,2 and J. Martin Brown1 1Division of Radiation and Cancer Biology, Department of Radiation Oncology, and 2Department of Health Policy and Research, Stanford University School of Medicine, Stanford, California

ABSTRACT addition to the above defects in the NER pathway, disruption of other repair pathways also affect the sensitivity of cells to cisplatin, includ- Cisplatin is a crucial agent in the treatment of many solid tumors, yet ing recombinational repair (9–11), mismatch repair (MMR; Refs. many tumors have either acquired or intrinsic resistance to the drug. We have used the homozygous diploid deletion pool of Saccharomyces cerevi- 12–14), and repair by translesional synthesis (a subset of postreplica- siae, containing 4728 strains with individual deletion of all nonessential tion repair; Ref. 15). However, for genetic profiling to be maximally genes, to systematically identify genes that when deleted confer sensitivity effective a complete list of the genes affecting cisplatin sensitivity is to the anticancer agents cisplatin, oxaliplatin, and mitomycin C. We found required. The goal of the present study was to help provide such a list that deletions of genes involved in nucleotide excision repair, recombina- by performing a screen for cisplatin sensitivity with the budding yeast tional repair, postreplication repair including translesional synthesis, and Saccharomyces cerevisiae. DNA interstrand cross-link repair resulted in sensitivity to all three of the S. cerevisiae has proven to be a powerful genetic system for the agents, although with some differences between the platinum drugs and study of DNA repair because of its ease of genetic manipulation and mitomycin C in the spectrum of required translesional polymerases. Pu- tative defective repair of oxidative damage (imp2؅⌬ strain) also resulted in the high degree of functional conservation between S. cerevisiae and sensitivity to platinum and oxaliplatin, but not to mitomycin C. Surpris- human DNA repair genes (16, 17). As in humans, all three of the ingly in light of their different profiles of clinical activity, cisplatin and major complementation groups in yeast DNA repair influence plati- oxaliplatin have very similar sensitivity profiles. Finally, we identified num sensitivity. Mutation of genes within NER (RAD1, RAD10, and three novel genes (PSY1–3, “platinum sensitivity”) that, when deleted, RAD2), recombinational repair (RAD51 and RAD52), and postrepli- demonstrate sensitivity to cisplatin and oxaliplatin, but not to mitomycin cation repair (RAD6 and RAD18) confer platinum sensitivity (18–20). C. Our results emphasize the importance of multiple DNA repair path- As in humans, the loss of MMR leads to platinum resistance in yeast ways responsible for normal cellular resistance to all three of the agents. Also, the similarity of the sensitivity profiles of the platinum agents with (21), and mutations in genes in the error prone pathway in transle- that of the known DNA interstrand cross-linking agent mitomycin C, and sional synthesis (REV1, REV3, and REV7) are sensitive to cisplatin the importance of the gene PSO2 known to be involved in DNA inter- (20, 22). Finally, another repair pathway contributing to platinum strand cross-link repair strongly suggests that interstrand cross-links are resistance is the HMG-domain proteins, which bind DNA intrastrand important toxic lesions for cisplatin and oxaliplatin, at least in yeast. cross-links (23, 24). Within the HMG-domain family, the absence of the yeast IXR1 gene has been shown to cause cisplatin resistance (25), INTRODUCTION and human HMG1 has been shown to inhibit removal of platinum- DNA intrastrand cross-links (26). Genetic screens for cisplatin resist- Platinum-based chemotherapy is a vital component in the treatment ance in yeast have also found that overexpression of the transcription of many tumors including lung, testicular, ovarian, and head and neck factors Cin5 and Yap6 and disruption of the SKY1 gene confer cancers. However, despite the high efficacy of these anticancer agents, resistance to cisplatin (27, 28). However, these screens have identified drug resistance, either acquired or intrinsic, remains problematic, and genes for which the overexpression or disruption produced resistance, there is no way of predicting which individual tumors will respond to and they did not identify the known genes for which deletion causes treatment. One possible method to predict individual tumor sensitivity cisplatin sensitivity. would be to measure the protein (or mRNA levels) of genes known to The recent completion of a systematic deletion of all of the open affect sensitivity to platinum-based drugs. In support of such an reading frames (ORFs) in yeast (29) has provided a powerful new tool approach is the fact that clinical resistance to cisplatin of ovarian, for screening for genes for which deletion produces sensitivity (or lung, and gastric cancers has been reported for tumors with increased resistance) to cytotoxic agents. We and others have used this collec- levels of expression of the ERCC1 (excision repair cross-complement- tion of mutants to identify novel genes for which deletion confers ing 1) gene (1–3). The ERCC1 protein forms a heterodimer with XPF (xeroderma pigmentosum complementation group F), which performs sensitivity to UV (30), ionizing radiation (31, 32), and methyl meth- the 5Ј strand incision during nucleotide excision repair (NER; Refs. 4, ane sulfonate (MMS) (33). One of the advantages of this resource is 5) and in the removal of interstrand cross-links (6). Expression levels that the gene replacement cassette contains two molecular “bar code” of another gene involved in NER, xeroderma pigmentosum comple- tags, or unique 20-base oligonucleotide sequences, which allow for mentation group A (XPA), have also been associated with cisplatin- unique identification of the strain in a pool of all of the deletion resistant ovarian cancers (1, 7) and testicular cancer cell lines (8). In mutants by PCR amplification of the tags and subsequent hybridization to a high-density oligonucleotide array containing the corre- Received 10/2/03; revised 2/17/04; accepted 3/26/04. sponding complementary sequences (29). Grant support: Grant P01 CA67166 (to J. M. Brown) and Training Grant CA09302 Here we have used a pool of 4728 homozygous deletion strains, (to J. A. Brown) from the National Cancer Institute, Department of Health and Human representing deletion of all of the nonessential genes, to detect genes Services, and Grant R01 GM62628 from the National Institute of General Medical Sciences, Department of Health and Human Services (to L. Lazzeroni). for which deletion confers sensitivity to cisplatin, oxaliplatin, and The costs of publication of this article were defrayed in part by the payment of page mitomycin C. We chose to perform the screen with oxaliplatin, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. because it is an analog of cisplatin that has a different clinical Requests for reprints: J. Martin Brown, Division of Radiation and Cancer Biology, spectrum of activity and for which there is evidence for some differ- 269 Campus Drive, CCSR South Room 1255, Stanford University Medical Center, Stanford, CA 94305-5152. Phone: (650) 723-5881; Fax: (650) 723-7382; E-mail: ences in basic mechanisms of action (34). We chose mitomycin C [email protected]. because of the known mechanism of action of this drug in producing 3940

Fig. 1. Distribution of the sensitivity ratios (treated:control hybridization values) for all 4728 strains treated with cisplatin, oxaliplatin, mitomycin C, or UVC irradiation.

interstrand cross-links, which is one of the suggested mechanisms by during the PCR amplification reaction. In brief, a separate UPTAG normal- which cisplatin kills cells. ization factor was calculated such that the total signal intensity of the UPTAGs Our results corroborate the importance of multiple DNA repair was equal in the control and experimental arrays. We also calculated and pathways responsible for normal cellular resistance to platinum and applied a separate DNTAG normalization factor. We calculated the back- mitomycin C, demonstrate the importance of the repair of DNA ground intensity of each array to identify those tags that fail to generate a interstrand cross-links for cisplatin and oxaliplatin sensitivity, and sufficient signal above the background to be meaningful. Of the 13,842 unassigned tags, 10,000 were chosen as a representative sampling to statisti- identify several new genes for which deletion produces sensitivity to cally model the nonspecific binding and background signal of an individual these platinum drugs. array (see Supplemental Data).3 A small subset of the unassigned tags consistently reported high levels of signal intensity indicating a high degree of MATERIALS AND METHODS specific cross-hybridization and were not used in the background calculation. An experimental:control intensity ratio was including in the data analysis only Yeast Strains. Genotypes of the parental yeast strain BY4743, construction if the signal generated in the untreated control array was at least twice the of the homozygous diploid deletion strains, and construction of the homozy- background signal. This arbitrary limit was chosen so that a maximum value gous diploid deletion pool have been described previously (35). All of the of 0.5 for the ratio of treated:control (T:C) hybridizations would be obtained if completed deletion strains are available through Research Genetics (Hunts- the treated signal was at the background level. In addition, each treated tag that ville, AL) or EUROSCARF (Frankfurt, Germany). We used a mutant pool of fell within 2 SDs of the background was flagged indicating that the measured 4728 nonessential homozygous diploid deletion strains. The parental diploid value may be an overestimation of the true ratio (i.e., the true value would strain BY4743 was used as control in survival and complementation assays. likely be lower indicating greater sensitivity). To yield a more stable estimate Drugs. Cisplatin and mitomycin C were obtained from Sigma. Oxaliplatin of the average T:C ratio with the skewed distribution of values from each was obtained from Sanofi-Sythelabo (Great Valley, PA). Cisplatin stock so- experiment (Fig. 1) we averaged the logs of the T:C ratios for each of the four lutions were prepared in yeast extract peptone dextrose (YPD), stored as tags assigned to an individual strain. Those strains that failed to have at least aliquots at Ϫ20°C, and used once; oxaliplatin and mitomycin C stock solutions two of the tags significantly above background were not called for an indi- were freshly prepared in YPD for each experiment. vidual experiment. In addition only those strains that were called in at least two Chemotherapy Treatment Assays. Deletion pool aliquots were resus- of the replicate experiments were included in further analyses. This produced pended in YPD medium, shaken at 300 rpm at 30°C, and grown to early an average rejection rate of 9.3% of the strains across all of the experiments in log-phase (A 0.2–0.3). Equivalent numbers of cells (6 ϫ 106) were then 600 this series. mock treated or treated with cisplatin, oxaliplatin, or mitomycin C for 1 or 4 h, Statistical Analyses. For each drug, we tested 4728 different deletion and shaken at 300 rpm at 30°C. Cells were then harvested, washed once with strains in several experiments per drug. Statistical procedures that simulta- buffer (10 mM Tris and 5 mM EDTA), and resuspended in YPD. A fraction of neously perform large numbers of tests have the potential to generate many the cell resuspension was added to 250 ml of YPD and grown an additional false-positive results, if done in the same manner as a single test. To avoid this 16hat30°C. Cells were then harvested by centrifugation and stored at Ϫ80°C. problem, we calculated an adjusted P for each strain, which is the probability Genomic DNA, Probe Production, Chip Hybridization, and Postscan- ning Analysis. Genomic DNA preparation, PCR, and hybridization of DNA that any of the 4728 strains would have a statistically significant result by from the treated and control cultures was performed as described previously chance alone. We obtained Ps for simultaneous two-sided tests of both sensi- (36). Each deletion strain is associated with four hybridization signals on the tivity and resistance. high-density oligonucleotide array generated in two separate PCR labeling To compute the one-sided sensitivity Ps, we ranked the geometric means for reactions, UPTAG (sense and antisense) and DNTAG (sense and antisense). all of the deletion strains within each experiment from lowest to highest and Equal numbers of cells were harvested in both the control and treated pools to computed Rj, the maximum rank achieved by deletion strain j across all of the produce equal pool label intensities. We normalized the data generated in the experimental array to that of the control array to eliminate any bias created 3 Supplemental data available at http://cbrl.stanford.edu/mbrown/Cisptresis.html. 3941

experiments for a given drug. Low values of Rj correspond to genes that We performed 6 identical experiments with cisplatin, 3 with oxali- consistently exhibited sensitivity to the drug. We defined our adjusted P to be platin, and 5 with mitomycin C. We also reanalyzed our previous data the probability that one or more nonsensitive strains would consistently rank at (30) for UV irradiation using the same rigorous criteria (“Materials or below Rj by chance (i.e., have no rank above Rj). The Ps are computed under and Methods”) for comparison with the drug data. As we have a nonsensitivity model in which every possible permutation of the deletion reported previously for both UV and ionizing radiation exposures (30, strains is equally likely. The probabilities can be computed explicitly (see Supplementary data for details of the derivation and formulae).3 We computed 32), there was a high degree of correlation from experiment to analogous Ps for resistance genes by reversing the order in which the genes experiment in the sensitivity rankings for each of the drugs (data not were ranked. The final adjusted P for each gene is twice the lesser of the shown). This is reflected in the large number of genes that were sensitivity and resistance Ps. The adjusted P so defined is a conservative one, classified as significantly sensitive (130) or resistant (100) to cisplatin because it is calculated on the basis of having zero false positives. according to our rigorous statistical test. Table 1 shows a listing of the In every experiment, some deletion strains fail to yield “good” ratios due to rankings for the top 50 most sensitive strains for cisplatin with the levels in the control being near background, as described above. As noted rankings for oxaliplatin, mitomycin C, and UV also shown. above we only ranked genes with 2 or more “good” values in two or more Fig. 1 shows a distribution of the ratios for the four treatments for experiments. We modified the Ps for R , because strains with fewer good j all of the strains. Because our experiment was designed to identify values are more likely to have small values of R by chance. We replaced the j ϳ total number of genes tested with the smallest number of good ratios in any one sensitive strains, we gave a dose that produced only 50% cell kill in experiment, which yields a conservative P. For each gene, we also replaced the the wild-type cells. Thus, resistant strains would not be expected to total number of experiments with the number of experiments in which the gene deviate from a T:C ratio of 1.0 as much as sensitive strains. This is yielded a good ratio. Consequently, genes with bad ratios in some experiments demonstrated in the “Results,” which show a skewing of the distri- can still reach a statistically significant result but must have smaller values of bution of ratios to low rather than high values. Nonetheless, as noted Rj to do so. we found 100 strains that were resistant (P Ͻ 0.05). Of the top 50 The adjusted Ps are valid under more general assumptions than used for the most resistant, 30 were involved in protein biosynthesis and 15 were calculations. Suppose n is the total number of genes tested. The model yields of unknown function. None were involved in DNA repair. We did not conservative adjusted Ps if, roughly, the probability a nonsensitive gene falls Յ pursue individual characterization of any of these resistant strains, but in the bottom k genes by chance is no greater than k/n, for k Rj. Clonogenic Assay. The parent strain BY4743 and selected homozygous their identities are shown in the supplementary material. diploid deletion strains were tested for clonogenic survival to cisplatin treat- Table 1 shows that many of the genes in the top 50 ranking are ment. Equivalent numbers (6 ϫ 106) of early log-phase cells were suspended known to be involved in DNA repair (including 21 of the top 25 in YPD and treated with cisplatin (0.25–1.5 mM) for 4 h while being shaken at genes). It is also apparent that there is a close similarity between the 300 rpm at 30°C. Cells were then pelleted, washed once with buffer (10 mM rankings for cisplatin and oxaliplatin, as well as a similarity with Tris and 5 mM EDTA), pelleted, and resuspended in the same buffer. Cells mitomycin C and UV irradiation. were then plated at appropriate dilutions onto YPD solid medium to allow for Because of the clear importance of repair genes, we have listed accurate counting of surviving colonies (range, 50–250). Plates were incubated (Table 2) all of the nonessential genes involved in DNA repair and for 3–4 days at 30°C before counting colonies. Full survival curves were performed at least three times for each selected deletion strain. damage checkpoint response to show which of these genes, when Complementation Assays. ORFs of selected deletion strains were gener- deleted, produced sensitivity to the three drugs and UV radiation. This ated by PCR and then subcloned into the vector pRS416.GAL1 (gift of Dr. Pat table illustrates the importance to cisplatin and oxaliplatin sensitivity Brown, Department of Biochemistry, Stanford University). Genomic clones of of the NER factors 1, 2, and 3 (but not 4 or transcription-coupled MMS4, MMS2, and MUS81 were generous gifts from Dr. Steven Brill, (De- repair), DNA interstrand cross-link repair (as shown by the impor- partment of Molecular Biology and Biochemistry, Rutgers University, Piscat- tance of PSO2), postreplication repair, and to a lesser extent recom- away, NJ). Strains were transformed by the lithium acetate method with vector binational repair. Deletion of genes involved in double-strand break alone or with the construct containing the deleted ORF. Expression of PCR repair by nonhomologous end-joining, in base excision repair, and clones was achieved by use of the galactose promoter, whereas genomic clones were under control of their native promoters. Spots were grown on the (interestingly) in the DNA-damage checkpoint response did not pro- appropriate solid medium (ura-/galactose or leu-/dextrose) and assessed after duce sensitivity to cisplatin or to oxaliplatin, although deletion of the incubation for 2 days at 30°C. checkpoint genes clearly produced sensitivity to UV and to mitomycin C. The absence of genes known to be involved in DNA repair from this table is either because they are essential or because (as in the case RESULTS of RAD6, which is known to produce sensitivity to cisplatin when Sensitivity Profiles of Cisplatin, Oxaliplatin, and Mitomycin C. deleted; Ref. 20) their hybridization signals in the untreated sample In preliminary experiments with wild-type cells we established the were within the background range. concentrations of cisplatin, oxaliplatin, and mitomycin C to obtain Confirmation of Individual Sensitivities. We selected several of ϳ50% killing with a 1-h exposure to cisplatin and a 4-h exposure to the strains that appeared sensitive to cisplatin by the hybridization oxaliplatin and mitomycin C (1 mM,10mM, and 0.5 mM for the three assay and analyzed these by clonogenic survival after exposure to drugs, respectively). In each experiment with the deletion pool we different concentrations of cisplatin (Fig. 2). In this assay we used a split a pool of 4728 deletion mutants into two and treated one with the 4-h exposure to the drug to get high levels of cell kill at soluble relevant concentration of each of the three drugs, the other serving as concentrations of cisplatin. In other tests we found that the ranking of the untreated control. After the drug exposure the cells were centri- genes to a 1-h and a 4-h exposure to cisplatin was essentially identical fuged, resuspended in fresh YPD, diluted, and grown for an additional (Supplementary Data).3 In all of the cases strains that were found to 16 h before preparation of the genomic DNA. After PCR amplifica- be sensitive by the hybridization assay were also confirmed as sensi- tion of the tags and hybridization of the products to the oligonucleo- tive by clonogenic survival. The strains showing the greatest sensi- tide arrays, we obtained signal intensities from the sense and antisense tivity were those with deletions in RAD2, RAD10, and PSO2 followed tags for both the UPTAGS and DOWNTAGS for each deletion strain by RAD5 (postreplication repair) and genes involved in recombina- in the pool. Ratios of T:C were then calculated for each of these four tional repair (MUS81, MMS4, RAD57, and SAE2). Four genes previ- tags and combined with those from replicate experiments to give an ously unknown to confer sensitivity to cisplatin when deleted were overall geometric mean T:C for each strain for each of the drugs. confirmed in the clonogenic assay as moderately sensitive to cisplatin. 3942

Table 1 Fifty most sensitive deletion strains to cisplatin and the sensitivity rank of these strains to OXAa UV, and MMC CisPtb Maxc OXAb UVb MMCb ORF Gene Function rank rank Adj pd rank rank rank YML095C RAD10 NER- NEF1-endonuclease activity, complex with Rad1p 1 9 1.2E-12 1 3 3 YER162C RAD4 NER- NEF2-damage binding, complex with Rad23p 2 8 6.0E-13 3 2 5 YMR201C RAD14 NER- NEF1-DNA damage recognition 3 8 6.0E-13 4 4 8 YMR137C PSO2 DNA interstrand crosslink repair 4 28 1.1E-09 5 127 1 YGR258C RAD2 NER- NEF3-3Ј endonuclease 5 32 2.5E-09 2 1 4 YLR032W RAD5 PRR-error free 6 40 9.4E-09 6 6 2 YGL175C SAE2 Recombinational repair 7 12 6.9E-12 8 28 105 YJL092W HPR5 DNA repair-PRR/recom rep 8 24 4.4E-10 14 8 19 YPL022W RAD1 NER- NEF1-endonuclease activity, complex with Rad10p 9 31 2.0E-09 7 5 7 YPL167C REV3 PRR-translesional synthesis 10 20 1.5E-10 9 82 193 YOR346W REV1 PRR-translesional synthesis, complex with Rev7p 11 41 1.1E-08 16 55 275 YDR386W MUS81 Recombinational repair, interacts with Rad54p 12 39 8.1E-09 11 9 22 YGL087C MMS2 PRR-error free 13 26 7.1E-10 20 12 15 YDR075W PPH3 Protein phosphatase involved in signal transduction 14 30 1.7E-09 10 92 56 YDL059C RAD59 Recombinational repair 15 34 3.6E-09 17 15 29 YIL154C IMP2Ј Transcriptional activator, resistance to oxidants 16 29 1.4E-09 15 2234 107 YLR135W SLX4 Recombinational repair 17 38 6.9E-09 24 138 435 YNL201C PSY2 Unknown 18 34 3.6E-09 19 146 61 YLR376C PSY3 Unknown 19 35 4.2E-09 33 71 99 YJR043C POL32 PRR-translesional synthesis 20 27 8.9E-10 26 44 21 YCR066W RAD18 Postreplication repair (PRR) 21 28 1.5E-07 29 13 12 YBR098W MMS4 Recombinational repair, interacts with Mus81p 22 53 5.1E-08 27 11 16 YDR076W RAD55 Recombinational repair 23 42 1.2E-06 21 24 YIL139C REV7 PRR-translesional synthesis 24 44 1.7E-08 132 43 62 YDR004W RAD57 Recombinational repair 25 47 2.5E-08 66 26 20 YIL132C CSM2 Meiotic chromosome segregation 26 48 2.8E-08 48 84 92 YNL197C WHI3 RNA binding, involved in cell size 27 71 2.9E-07 18 3280 3286 YEL037C RAD23 NER- NEF2-damage binding, complex with RAD4 28 64 1.6E-07 84 7 122 YDR078C SHU2 Unknown, but may function in DNA damage recovery 29 64 1.6E-07 77 118 70 YBR099C (MMS4) On opposite strand to MMS4 30 74 3.8E-07 121 14 18 YNR068C ?? Has secondary mutation and incorrect construction (ref 32) 31 114 5.1E-06 82 67 36 YOR039W CKB2 Casein kinase II (protein kinase CK2), regulatory subunit 32 79 5.6E-07 28 518 3873 YIR002C MPH1 Unknown 33 63 1.4E-07 119 46 90 YHL006C SHU1 Unknown, but may function in DNA damage recovery 34 62 1.3E-07 72 87 3215 YKL076C PSY1 Unknown 35 191 1.1E-04 13 48 234 YGL163C RAD54 Recombinational repair, interacts with Mus81p 36 81 3.1E-05 129 22 YDL160C DHH1 RNA turnover 37 88 4.7E-05 112 80 YOL081W IRA2 RAS protein signal transduction 38 145 2.1E-05 41 349 116 YKL048C ELM1 Serine/threonine protein kinase 39 139 1.7E-05 144 37 35 YHR025W THR1 Homoserine kinase in threonine biosynthesis 40 110 4.1E-06 88 19 30 YGL019W CKB1 Casein kinase II regulatory ␤ subunit 41 146 5.9E-04 44 128 2885 YDL066W IDP1 Isocitrate dehydrogenase (NADPϩ), mitochondrial 42 123 8.0E-06 22 194 120 YAL021C CCR4 Regulation of transcription from Pol II promoter 43 277 1.0E-03 87 51 369 YIL005W EPS1 Protein-ER quality control 44 153 3.0E-05 104 366 3467 YPR119W CLB2 G2/M-phase-specific cyclin 45 154 3.1E-05 125 76 27 YKL101W HSL1 Serine/threonine protein kinase 46 136 1.5E-05 177 93 75 YLR426W Unknown 47 154 7.7E-04 102 54 YGL012W ERG4 Ergosterol biosynthesis 48 146 2.2E-05 3594 218 39 YMR091C NPL6 Protein-nucleus import 49 157 8.5E-04 78 58 YJL165C HAL5 Cation homeostasis 50 136 1.5E-05 1253 40 241 a OXA, oxaliplatin; MMC, mitomycin C; ORF, open reading frame; CisPt, cisplatin. b The overall sensitivity rank for each treatment based on the geometric mean of all the values of treated/control for all experiments. c The maximum ranking in six identical experiments with cisplatin. d Probability by chance alone of any of the 4728 strains having a maximum rank of less than or equal to the maximum rank observed in the six replicate cisplatin experiments assuming all strains had identical sensitivities and, therefore, random rank orders in each experiment.

Confirmation of Gene Deletions in Causing Sensitivity. To con- rankings suggested a greater effect of deletion of REV3 than of REV7 firm that the sensitivities of selected strains to cisplatin were the result on sensitivity to cisplatin (ranks 10 and 24, respectively) and espe- of deletion of the ORF and not to other genetic alterations, we cially to oxaliplatin (ranks 9 and 132, respectively), we performed reintroduced and expressed the gene products in a plasmid-based, additional experiments on these mutant strains using clonogenic as- inducible expression vector, pRS416 Gal-1. The strains we selected says to test their sensitivities to the two platinum drugs (Fig. 4). These for this were those not known previously to be sensitive to cisplatin data confirm the conclusions of the hybridization assay that deletion (imp2’, pph3, yk1076, ynl201c, yil132c, and ylr376c), as well as a of REV3 produces greater sensitivity than does that of deletion of selection of those in different pathways including recombination REV7, particularly for oxaliplatin. repair (mms4 and mus81), postreplication repair (mms2), and interstrand cross-link repair (pso2). Fig. 3 shows spot tests of cisplatin sensitivity of these strains with the same number of cells spotted for DISCUSSION wild-type, the deletion strain, and the deletion strain with the reintro- Use of Genomic Screens in Yeast in the Study of duced ORF. In all of the cases the reintroduction of the appropriate Anticancer Agents ORF complemented the sensitivity of the deletion strain back to wild-type sensitivity. We performed the current studies to identify as many genes as Role of Polymerase ␨ in Cisplatin and Oxaliplatin Sensitivity. possible for which the deletion produced sensitivity to cisplatin, Polymerase ␨, a heterodimer of Rev3p and Rev7p, is a DNA polym- oxaliplatin, and mitomycin C. The Yeast Deletion Project, an under- erase involved in translesional synthesis (37). Because our sensitivity taking by an international consortium to delete all of the known ORFs 3943

Table 2 Average sensitivity rankings of the four agents to deletions of DNA repair and damage checkpoint genesa Gene Gene function CisPtb OXA MMC UV RAD1 NER- NEF1-endonuclease activity, complex with RAD10 9 7 7 5 RAD10 NER- NEF1-endonuclease activity, complex with RAD1 1 1 3 3 RAD14 NER- NEF1-DNA damage recognition 3 4 8 4 RAD4 NER- NEF2-damage binding, complex with RAD23 2 3 5 2 RAD23 NER- NEF2-damage binding, complex with RAD4 28 84 122 7 RAD2 NER- NEF3-3Ј endonuclease 5 2 4 1 RAD7 NER- NEF4-ATP dependent damage sensor-complex with RAD16 160 549 57 10 RAD16 NER- NEF4-ATP dependent damage sensor-complex with RAD7 572 659 335 99 RAD26 NER-transcription coupled repair 378 575 1636 202 RAD28 NER-transcription coupled repair 1825 2132 2642 785 PSO2 DNA interstrand crosslink repair 4 5 1 127 RAD18 Postreplication repair (PRR) 21 29 12 13 RAD5 PRR-error free 6 6 2 6 MMS2 PRR-error free 13 20 15 12 REV1 PRR-translesional synthesis 11 16 275 55 REV3 PRR-translesional synthesis 10 9 193 82 REV7 PRR-translesional synthesis 24 132 62 43 POL32 PRR-error free 20 26 21 44 RAD30 PRR-translesional synthesis 579 292 1243 50 HPR5 DNA repair-PRR/recombinational repair 8 14 19 8 SAE2 Recombinational repair, Meiotic DNA dsb processing 7 8 105 28 MUS81 Recombinational repair, interacts with Rad54p 12 11 22 9 MMS4 Recombinational repair, interacts with Mus81p 22 27 16 11 RAD51 Recombinational repair 114 264 27 RAD54 Recombinational repair, interacts with Mus81p 36 129 22 RAD55 Recombinational repair 15 21 24 RAD57 Recombinational repair 23 66 20 26 RAD59 Recombinational repair 25 17 29 15 SLX4 Recombinational repair 17 24 435 138 YKU70 Double-strand break repair by non homologous end joining 273 253 1439 221 YKU80 Double-strand break repair by non homologous end joining 395 467 2114 825 LIF1 Double-strand break repair by non homologous end joining 2950 2825 598 1685 DNL4 Double-strand break repair by non homologous end joining 2491 3306 2833 2675 APN1 Base excision repair 117 162 1742 RAD27 Base excision repair 159 702 61 UNG1 Base excision repair 3768 1185 2711 3017 MAG1 Base excision repair 3025 1324 3464 2352 OGG1 Base excision repair 1863 577 1043 951 NTG1 Base excision repair 472 231 1706 177 NTG2 Base excision repair 2699 1740 2784 4029 APN2 Base excision repair 2579 2218 2882 3190 RAD9 DNA damage checkpoint 124 157 6 18 RAD24 DNA damage checkpoint 694 372 17 33 RAD17 DNA damage checkpoint 420 322 9 17 MEC3 DNA damage checkpoint 388 319 10 21 DDC1 DNA damage checkpoint 439 320 11 25 TEL1 DNA damage checkpoint 986 760 2360 233 IMP2Ј Other DNA repair 16 15 107 2234 CSM2 Other DNA repair 26 48 92 84 a The ranking of sensitivities is from 1–4728 with 1 being the most sensitive. As a rough guide, rankings of 1–50 and 51–100 would be considered to be sensitive and mildly sensitive, respectively. b CisPt, cisplatin; OXA, oxaliplatin; MMC, mitomycin C. in yeast, has produced a powerful resource to enable such a screen to of sensitivities to any agent, it is somewhat artificial to categorize be performed (29). The value of such a screen in yeast relies on the strains into having reduced, normal, or increased sensitivities. For this high conservation of genes from yeast to humans, particularly those reason we have highlighted the top 50 most sensitive strains as involved in repairing DNA (16, 17). The other major advantage of this determined by the hybridization assay (Table 1), because these have resource is the fact that DNA extracted from a pool of all of the the most dramatic impact on drug resistance. However, all of the deletion mutants can be hybridized to a high density oligonucleotide results can be accessed in the Supplementary Data.3 array to allow the relative abundances of each strain in the pool, and, We selected 18 strains for individual testing in cell survival assays hence, their relative sensitivities, to be determined rapidly and quan- identified as sensitive in the hybridization assay, and we confirmed titatively (38). We have previously used this technique to identify the sensitivities of all of these strains. We were also able to show in genes involved in the resistance of cells to UV and X-irradiation (30, 10 of 10 of these sensitive strains that the sensitivity of the strain 32). In the present study we introduced a new method for data analysis could be attributed to deletion of the specific gene, because introduc- that allowed us to better identify the strains where low abundance in tion of the gene in the deletion strain abrogated the sensitivity of that the pool could affect their apparent sensitivity analyzed by the DNA strain. These data provide a measure of confidence that the hybrid- hybridization technique. Our analysis of multiple experiments con- ization assay with the deletion pool can identify with some accuracy firmed the high reproducibility of this system in identifying sensitive genes involved in cisplatin sensitivity. strains. On the basis of this close agreement between multiple exper- One limitation of the present system is that essential genes cannot iments we performed a rigorous statistical analysis to identify sensi- be interrogated. Although inactivation of the human orthologs of these tive and resistant stains and found 130 deletion strains that were genes is unlikely in human cancers (because the cells would not be sensitive to cisplatin with the criterion of zero false positives at the expected to be viable), hypomorphic mutations in these genes could 95% level of confidence and 100 deletion strains that were resistant at lead to a phenotype. One way to investigate this would be to examine the same level of confidence. However, because there is a continuum the sensitivity of strains with heterozygous deletion of these genes. 3944

Fig. 2. Cell survival curves determined by clonogenic assay (see “Materials and Methods”) for cisplatin cytotoxicity (4-h exposure) for 16 strains showing sensitivity to cisplatin in the hybridization assay. All of the strains were diploid with homozygous deletion of the indicated genes. BY4743 is the wild-type diploid strain from which all of the deletion strains have been constructed. The strains are categorized into different DNA repair groups or unknowns. Each cell survival curve shows pooled data from three to four experiments per strain.

in the screen. This was particularly the case for the endonucleases performing the incisions on both sides of the lesion, the Rad1p- Rad10p complex, and Rad2p (XPF-ERCC1 and XPG in humans). Strains with deletions of the genes involved in damage recognition, RAD14 and RAD4, were also equally sensitive to all three of the drugs and UV irradiation. Deletion of RAD23, which encodes a protein that forms a complex with Rad4p, produced somewhat less sensitivity to cisplatin than to UV and less still for oxaliplatin and mitomycin C. These data suggest less importance of Rad23p than Rad4p in recog- nizing the bulky adducts caused by cisplatin, oxaliplatin, or mitomycin C compared with UV. An even greater loss of sensitivity compared with UV was seen for strains with deletion of RAD7 and RAD16. Deletion of RAD26 and RAD28, the S. cerevisiae homologs of the Cockayne’s syndrome B and A genes, did not produce any sensitivity to cisplatin or to UV, consistent with earlier reports (42, 43). The involvement of NER in the response of cells to these three cross-linking anticancer drugs very likely reflects the role of intrastrand cross-links in killing cells by these agents. However, for some of

Fig. 3. Spot tests of sensitivity to cisplatin for 10 sensitive deletion strains and the same strains with the deleted open reading frame (ORF) reintroduced on an expression vector. The spots have the same number of cells plated for wild-type, the deletion strain, and the deletion strain with the ORF reintroduced.

At the present time there is no comparable resource to perform such a screen with mammalian cells. However, it has been argued that gene expression profiling after exposure to DNA-damaging agents could identify the genes that might be important for the survival of the cells to that agent (39, 40). Unfortunately, we and others have shown using yeast that there is no relationship between the genes that are induced after a range of DNA-damaging agents and the genes that are neces- sary for survival to those same agents (33, 36, 41).

Multiple DNA-Repair Pathways Participate in Platinum and Mitomycin C Resistance. Fig. 4. Cell survival curves determined by clonogenic assays for cisplatin (4-h exposure) and oxaliplatin (6-h exposure) for strains with deletions of REV3 and REV7. NER. Many of the strains with deletions of genes involved in NER Each curve shows pooled data for two experiments each for cisplatin and oxaliplatin, with were found to be sensitive to cisplatin, oxaliplatin, and mitomycin C the concurrently tested wild-type strain BY4743. 3945

the proteins it could also reflect their involvement in interstrand DNA Damage Checkpoints. A major difference between the two cross-link repair (see below). platinum drugs and mitomycin C was the sensitivity of mutants with Repair of DNA Interstrand Cross-Links. Repair of DNA inter- deletion of genes in the DNA-damage checkpoint response including strand cross-links is a complex process that involves genes that are in RAD9, RAD24, RAD17, MEC3, and DCC1. All of these genes when other DNA repair pathways, namely NER, homologous recombina- deleted produced sensitivity to mitomycin C but not to either cisplatin tion, and postreplication repair (22, 44). Thus, the fact that yeast with or oxaliplatin. This is somewhat in contrast to a previous report (20) deletions in any of these three pathways are sensitive to cisplatin, showing similar, although marginal, sensitivities of rad9, rad17, and oxaliplatin, and mitomycin C does not constitute evidence that inter- mec3 mutants to cisplatin and mitomycin C. However, it is in agree- strand cross-links are important for the killing to these agents. How- ment with the work of Grossmann et al. (22), who showed that ever, one nonessential gene, PSO2/SNM1, has been demonstrated to cisplatin did not cause an S-phase arrest and who found no sensitivity be involved in interstrand cross-link repair without being involved in of the checkpoint mutants rad9, rad17, and rad24 and slight sensi- NER, postreplication repair, or recombination repair. We found that tivity to cisplatin of the mec3 mutant. deletion of this gene conferred exquisite sensitivity to cisplatin, ox- Other Genes Conferring Sensitivity to Cisplatin. The deletion aliplatin, and mitomycin C but little or no sensitivity to UV as strain imp2’ showed sensitivity by the hybridization assay to cisplatin, reported earlier (22). In clonogenic survival assays of the individual which we confirmed by clonogenic survival. This gene has been strains the pso2 deletion was equally sensitive as strains with deletion reported to encode a transcriptional activator that is involved in the of either of the DNA endonucleases encoding genes involved in NER protection of yeast against oxidative damage produced by bleomycin (RAD2 and RAD10). This is evidence for the involvement of DNA and other oxidants (46). Although the sensitivity of imp2’ mutants to interstrand cross-links in the killing of yeast cells by these three platinum drugs has not been reported previously, there are data in the chemotherapeutic drugs. A human homologue of PSO2, hSNM1, has literature showing that some of the toxic side effects of cisplatin can ␣ been identified and characterized (45). Mice with homozygous dele- be abrogated using antioxidants such as -tocopherol (47, 48). tion of the gene (mSNM1Ϫ/Ϫ mice) are viable and are sensitive to The deletion strain pph3 was sensitive to cisplatin, oxaliplatin, and mitomycin C. A similar sensitivity of mSNM1Ϫ/Ϫ ES cells to mito- mitomycin C. Again, this is a novel finding, although this strain has mycin C but not to cisplatin was also observed (45). been reported previously to be sensitive to MMS (41). This gene Postreplication Repair. Deletion mutants in all of the components encodes a protein serine/threonine phosphatase related to PP2A phosphatases. These are involved in many signal transduction pathways of postreplication repair were sensitive to cisplatin, although not and have also been reported to be involved in DNA repair (49). equally so for oxaliplatin and mitomycin C. The two major proteins in We also identified three unknown genes that were sensitive both to postreplication repair, Rad6p and Rad18p, form a heterodimer that cisplatin and oxaliplatin and less so to mitomycin C and UV modifies proteins by conjugation to ubiquitin. The pathway then (YKL076C, YNL201C, and YLR376C, which we have named PSY1, divides into two error-free branches, both involving the Ubc13p/ 2, and 3, respectively). All three were shown to be moderately Mms2p heterodimer and a third pathway, which is error prone and sensitive to cisplatin by clonogenic assay with the psy2 strain being involves translational synthesis by Rev1p and the heterodimer Rev3p/ the most sensitive. Experiments are under way to determine the Rev7p. Because their hybridization intensities were at background function of these genes. levels we could not obtain data for the sensitivity of the rad6 and ubc13 deletion mutants, but rad6 deletion strains have been reported Cisplatin and Oxaliplatin Have Similar but Not Identical to be as sensitive to cisplatin as rad18 strains (20). Of interest is the Sensitivity Profiles sensitivity of the rev1 and rev3 deletion strains to cisplatin and oxaliplatin but not to mitomycin C. This is in agreement with earlier Oxaliplatin is a third generation platinum anticancer drug with studies (20). One of the few strains that showed a different sensitivity differences from cisplatin that include a different toxicity profile as to cisplatin and oxaliplatin was the rev7 mutant, which was sensitive well as activity against platinum-resistant cells and tumors (50, 51). to cisplatin but much less so for oxaliplatin. This is somewhat sur- Despite that fact that oxaliplatin forms covalent adducts with DNA prising, because Rev3p and Rev7p form a heterodimer making up that have a similar sequence and region specificity to those formed by polymerase ␨ (37). This suggests that Rev3p may be able to function cisplatin (52) they are more cytotoxic than the adducts formed by independently of Rev7p in repairing cisplatin lesions. The strains with cisplatin (51). However, the mechanisms for the higher specific tox- deletion of components of the two error-free pathways of postrepli- icity of the DNA adducts and for the lack of cross-resistance with cation repair (rad5, pol32, and MMS2) showed equal sensitivity to cisplatin are not understood, but there are differences between the two cisplatin, oxaliplatin, and mitomycin C. In agreement with earlier drugs both in translesional synthesis (53, 54) and in MMR (55, 56). In studies (22) we found that yeast deficient in DNA polymerase pol-eta our analysis we found that loss of the stimulatory component of the ␨ (Rad30p), which is involved in translesional synthesis past UV- replicative bypass polymerase , Rev7p, conferred greater sensitivity induced photoproducts, was not sensitive to cisplatin. to cisplatin than it did to oxaliplatin (Table 2; Fig. 4). However, the Recombinational Repair. Mutants in recombinational repair that significance of this is unclear, because loss of the catalytic subunit of ␨ showed sensitivity to both cisplatin and oxaliplatin included the polymerase , Rev3p, produced equal sensitivity to the two drugs (Fig. deletion strains sae2, mus81, mms4, slx4, rad55, and rad59. Strains 4). The possibility that defective MMR does not result in oxaliplatin with deletions of RAD54 and RAD57 showed sensitivity to cisplatin resistance is especially intriguing, given the activity of oxaliplatin in but only modest sensitivity to oxaliplatin. Several members of this colon cancers, where aberrant MMR is common and cisplatin is group had hybridization signals too close to background levels to be ineffective. However, because our study was designed to detect sensitivity and not resistance, we were not able to detect any MMR gene able to assess their sensitivity including rad50, Mre11, Xrs2, and deletions that differentiated the two drugs. rad51. Nonhomologous End-Joining and Base Excision Repair. As ex- Conclusions pected from the literature, mutants in double-strand break repair by nonhomologous end-joining and mutants in base excision repair In this screen using a pool of homozygous deletion mutants of all showed no sensitivity to any of the drugs or to UV radiation. nonessential genes in diploid yeast we identified Ͼ130 and 100 genes 3946

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H. Irene Wu, James A. Brown, Mary J. Dorie, et al.

Cancer Res 2004;64:3940-3948.

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