Implication of a Chromosome 15Q15.2 Locus in Regulating UBR1 and Predisposing Smokers to MGMT Methylation in Lung Shuguang Leng1, Guodong Wu1, Leonard B

Published OnlineFirst July 16, 2015; DOI: 10.1158/0008-5472.CAN-15-0243 Cancer Prevention and Epidemiology Research

Implication of a Chromosome 15q15.2 Locus in Regulating UBR1 and Predisposing Smokers to MGMT Methylation in Lung Shuguang Leng1, Guodong Wu1, Leonard B. Collins2, Cynthia L.Thomas1, Carmen S.Tellez1, Andrew R. Jauregui3, Maria A. Picchi1, Xiequn Zhang1, Daniel E. Juri1, Dhimant Desai4, Shantu G. Amin4, Richard E. Crowell5, Christine A. Stidley5,Yushi Liu1, James A. Swenberg2, Yong Lin1, Marc G. Wathelet3, Frank D. Gilliland6, and Steven A. Belinsky1

Abstract

O6-Methylguanine-DNA methyltransferase (MGMT) is a risk prediction for MGMT methylation. We found that the DNA repair enzyme that protects cells from carcinogenic effects predisposition to MGMT methylation arising from the of alkylating agents; however, MGMT is silenced by promoter 15q15.2 locus involved regulation of the ubiquitin protein hypermethylation during carcinogenesis. A single-nucleotide ligase E3 component UBR1. UBR1 attenuation reduced turn- polymorphism (SNP) in an enhancer in the MGMT promoter over of MGMT protein and increased repair of O6-methylgua- was previously identified to be highly significantly associated nine in nitrosomethylurea-treated human bronchial epithelial with risk for MGMT methylation in lung cancer and sputum cells, while also reducing MGMT promoter activity and abol- from smokers. To further genetic investigations, a genome-wide ishing MGMT induction. Overall, our results substantiate association and replication study was conducted in two smoker reduced gene transcription as a major mechanism for predis- cohorts to identify novel loci for MGMT methylation in sputum position to MGMT methylation in the lungs of smokers, and that were independent of the MGMT enhancer polymorphism. support the importance of UBR1 in regulating MGMT homeo- Two novel trans-acting loci (15q15.2 and 17q24.3) that were stasis and DNA repair of alkylated DNA adducts in cells. Cancer identified acted together with the enhancer SNP to empower Res; 75(15); 1–10. 2015 AACR.

Introduction some (1, 2). The recovery of MGMT activity after its inactivation with alkylating agents is slow and results entirely from de novo O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA protein synthesis (3–5). repair enzyme that protects cells from carcinogenic effects of The predominant mechanism for MGMT gene inactivation in alkylating agents by removing adducts from the O6 position of carcinogenesis is hypermethylation of a CpG island within its guanine (1). This repair mechanism is conserved in all organisms promoter–enhancer region that leads to transcriptional silencing and involves transfer of methyl and other alkyl groups from the in lymphoma and several solid tumors, including brain, colon, O6 position of guanine in DNA to a cysteine residue within lung, and head and neck cancers (6). Silencing of this gene is enzymatic active site of MGMT protein. The irreversible binding associated with an increased prevalence for G:C to A:T transition of the alkyl group to the MGMT protein functionally inactivates mutations in K-ras and p53 genes in colorectal and brain tumors the enzyme and leads to a structural alteration resulting in the respectively, consistent with the important role for this gene in recognition by ubiquitin ligases and degradation by the protea- removing the highly promutagenic adduct O6-methylguanine (O6MG; refs. 7 and 8). Although alkylating adducts such as 1Lung Cancer Program, Lovelace Respiratory Research Institute, Albu- O6MG can be derived from endogenous methylation by S-ade- querque, New Mexico. 2Department of Environmental Sciences and nosylmethionine, knockouts of MGMT failed to show enhanced Engineering, University of North Carolina at Chapel Hill, Chapel Hill, tumor formation unless treated with exogenous alkylating carci- 3 North Carolina. Lung Fibrosis Program, Lovelace Respiratory nogens (9). MGMT methylation is a common event in lung Research Institute, Albuquerque, New Mexico. 4Department of Phar- macology, Penn State College of Medicine, Hershey, Pennsylvania. adenocarcinomas and associated with tumor progression (10). 5Department of Internal Medicine, University of New Mexico, Albu- Concomitant methylation of a panel of cancer-relevant genes, 6 querque, New Mexico. Keck School of Medicine, University of South- including MGMT, detected in lung epithelial cells present in ern California, Los Angeles, California. sputum predicts risk for subsequent lung cancer incidence in Note: Supplementary data for this article are available at Cancer Research moderate and heavy smokers (11, 12). In addition, MGMT Online (http://cancerres.aacrjournals.org/). methylation is also a prognostic biomarker for response of glio- Corresponding Author: Steven A. Belinsky, Lung Cancer Program, Lovelace blastoma patients to the alkylating agent temozolomide (13, 14). Respiratory Research Institute, Albuquerque, NM 87108. Phone: 505-348-9465; Genome-wide profiling of allele-specific methylation (ASM) Fax: 505-348-4990; E-mail: [email protected] using a methylation-sensitive single nucleotide polymorphism doi: 10.1158/0008-5472.CAN-15-0243 (SNP) array identified >35,000 ASM sites across the genomes of 2015 American Association for Cancer Research. multiple tissue types that were likely mediated by parental-origin

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(imprinting) or short distance cis-acting SNPs (15–18). This effort Sputum collection and measurement of MGMT promoter was further expanded by running methylation quantitative trait methylation loci (meQTL) analyses that associated genome-wide SNP array Induced and spontaneous morning sputum samples were data with methylation array data to identify short and long collected in LSC and VSC, respectively. Sputum samples were distance cis- and trans-acting germline meQTL in normal tissues washed multiple times using Saccomanno's fixative prior to long- that included lung, adipose, peripheral blood lymphocyte, and term storage at 80C. Sputum adequacy defined as the presence brain (19–25). In normal lung, 34,304 cis- and 585 trans-acting of deep lung macrophages or Curschmann's spiral (12) was meQTL were identified and enriched in CCTC-binding factor assessed by cytotechnologists and subjects with cancer cells (CTCF)-binding sites, DNase I hypersensitivity regions, and his- detected in sputum slides were removed from this study. Sputum tone marks (19). Four of five established lung cancer risk loci DNA was isolated and bisulfite converted. Methylation status for among persons with European ancestry are cis-acting meQTL (19). CpGs around the transcriptional start site of MGMT was assessed The findings of SNP/haplotype dependent ASM and meQTL using two stage methylation-specific PCR (33). Methylation status contribute to understanding genetic-epigenetic interactions and was scored as 0 (unmethylated) or 1 (methylated). Our assay can also provide functional relevance for disease risk loci discovered reproducibly detect one methylated allele in a background of from genome-wide association studies (GWAS). 10,000 unmethylated alleles (12). In addition to germline meQTL, somatic meQTL were also fi identi ed that affected the predisposition of cancer-relevant genes GWAS genotyping and quality assurance for promoter hypermethylation acquired in carcinogenesis for The HumanOmni2.5-4v1-H BeadChip (Illumina) was used to – multiple tumor types (13, 26 32). This is illustrated by our genotype 2,450,000 SNPs in 1200 Caucasian smokers from the studies and others for MGMT methylation. An enhancer SNP LSC. We removed 37 subjects due to low call rate (<95%, n ¼ 7), fi – rs16906252 located at the rst exon intron boundary of MGMT low heterozygosity (n ¼ 1), low Caucasian ancestry (<85%, n ¼ 2), fi was highly signi cantly associated with risk for MGMT methyl- and high relatedness with other samples (n ¼ 27). Furthermore, ation in colorectal cancer, pleural mesothelioma, lung adenocar- SNPs were excluded if they had a call rate of <90%, a minor allele cinoma, glioblastoma, and diffuse large B-cell lymphoma frequency (MAF) <0.008, or P < 10 8 for Hardy–Weinberg equi- – fi (13, 27, 29 32). Signi cant association was also seen for MGMT librium test, or were on Y or pseudo-autosomal region of X methylation in sputum from smokers (27). Functional charac- chromosomes. The MAF cutoff is a technical one to identify at fi terization con rmed the ASM pattern in heterozygotes for least 20 heterozygotes for accurate genotype clustering required rs16906252 with the "A" allele preferentially silenced by meth- by GenomeStudio. After quality assessment, 1,163 subjects with ylation in primary lung tumors and lung cancer cell lines (27). 1,599,980 SNPs remained in the genetic association analysis Although rs16906252 is a major determinant for acquisition of (Supplementary Table S1). The genomic coordinate is based on MGMT methylation in lung carcinogenesis, 20% of current and hg18 unless mentioned otherwise. former smokers carrying wild homozygote (G/G) of rs16906252 are methylated for MGMT in their sputum (27), suggesting that SNP selection for replication other genetic or environmental factors may contribute to MGMT Four SNPs (rs72887860, rs62287262, rs16957091, and methylation. To address this issue, a GWAS discovery and repli- rs997781) associated with risk for MGMT methylation in LSC cation study was conducted in two smoker cohorts from New (P < 10 5 in models with adjustment for rs16906252) and with Mexico to identify novel loci whose association with risk for MAF > 0.1 were selected for replication using the VSC (n ¼ 430; MGMT methylation detected in sputum was independent of the Supplementary Table S2). Rs16957091 was genotyped as the tag enhancer SNP. Subsequent hypothesis-driven studies were SNPs at chromosome 15q15.2 locus for replication. These SNPs designed based on the genetic associations detected to explore were genotyped using the TaqMan assay (Life Technologies) in the the underlying biological mechanisms. VSC.

Materials and Methods Imputation of chromosome 15 Study populations Imputation of chromosome 15 in the LSC was conducted Longitudinal cohorts that enrolled current and former smokers using BEAGLE (version 3.3.2) with phased haplotype data of were used for the GWAS discovery (the Lovelace smokers cohort, EUR populations (n for chromosomes ¼ 758, EUR.chr15.pha- LSC) and the replication (the veterans smokers cohort, VSC). The se1_release_v3.20101123) from 1,000 genomes project pilot 1 LSC began recruitment of female smokers in 2001 and expanded study as the reference panel. Masked analyses on 20% of SNPs on to include male smokers in 2004. Enrollment is restricted to chromosome 15 identified a high correlation of the observed current and former smokers ages 40 to 74 years with a minimum versus expected allelic frequencies (Pearson correlation coeffi- of 15 pack-years of smoking. The VSC began recruitment of cient ¼ 0.98). The estimated allelic dosages for SNPs (n ¼ 2 smokers in 2000 using enrollment criteria and procedures similar 165,451) with dosage R 0.3 and MAF 0.05 in the LSC and to the LSC. Most participants in the VSC are males with greater EUR reference populations were included for assessing genetic than 20 pack-years smoking history. In total, 1,675 lung cancer- association with risk for gene methylation. free subjects (1,200 subjects from LSC and 430 subjects from VSC) were included in this study. A detailed description of subject Enrichment of SNPs in ENCODE-annotated regulatory regions enrollment and follow has been provided previously (27). All The enrichment of SNPs in ENCODE-annotated regulatory samples were collected with informed consent. The study was regions within the 472-kb haplotype block at chromosome approved by the Western Institutional Review Board and New 15q15.2 locus was assessed using FunciSNP (34). Peak files Mexico VA Health Care System. included CTCF-binding sites, DNase I hypersensitivity locations,

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DNase digital genomic foot printing, and regions enriched in upstream of the luciferase coding sequence (27). Luciferase active (H3K4me3 and H3K36me3) and repressive (H3K27me3) reporter constructs that contain HAP1 (wild haplotype allele) or histone modification markers from small airway epithelial cells HAP4 (haplotype allele carrying A allele of rs16906252) were (SAEC). Known FAIRE-Seq (Formaldehyde-Assisted Isolation of transiently transfected in control HBEC4 and HBEC4 with UBR1 Regulatory Elements), DNase I hypersensitivity location, and gene KD using Lipofectamine 3000. Transfection in HBEC1 and promoter were included as well. HBEC26 was conducted using the Neon electroporation system. Cells were harvested 48 hours posttransfection and reporter activity was measured using the Dual Luciferase Assay System Stable siRNA-mediated knockdown of UBR1 in human BEC (Promega). lines Stable UBR1 knockdown (KD) cell lines were established using three immortalized human bronchial epithelial cell lines Real-time PCR for quantifying gene expression (HBEC1, HBEC4, and HBEC26; ref. 35). The pSUPER.retro. RNA was isolated from primary human BEC cultures (n ¼ puro (Oligo Engine) was modified to encode resistance to 54), normal lungs (n ¼ 6), and cell lines. TaqMan real-time PCR hygromycin (pSUPER.retro.hyg). A pair of oligonucleotides was conducted to quantify gene expression in cDNA using the were annealed and inserted into the pSUPER.retro.hyg. The delta threshold cycle method with b-actin as the endogenous 0 two oligonucleotide sequences are as follows: 5 -GATCCCCG- control. GATCGGAATCTATTAAGATTCAAGAGATCTTAATAGATTCC- GATCCTTTTTA-30 and 50-AGCTTAAAAAGGATCGGAATC- TATTAAGATCTCTTGAATCTTAATAGATTCCGATCCGGG-30). Western blot and protein expression quantification The resulting construct uses the H1 RNA promoter to express Western blot was conducted to quantify protein levels of shRNAs that are converted by endogenous enzymes to UBR1 and MGMT using antibodies from Abcam (anti-UBR1 siRNAs targeting UBR1 mRNA. These constructs were veri- antibody, ab42420) and Invitrogen (mouse anti-MGMT, fied by sequencing and transfected in GP2-293 cells along 35-7000). Protein extract (90 mg) was digested with 100 ng with a plasmid encoding the VSV-G protein. The resultant endoproteinase GluC in a 25 mL reaction mixture before being pseudotyped retroviral particles were used to transduce resolved in PAGE gels (36). Na€ve and suicide MGMT protein HBECs and drug resistant cells or clones were obtained after levels were quantified by densitometry (Image Lab; Bio-Rad) of selection with hygromycin. Control cells or clones were the individual bands. established following the same procedure except pSUPER. retro.hyg plasmid backbone was used. On average, 70% to Quantification of O6MG 80% KD was achieved at the mRNA level in all HBECs. HBECs were treated with 1 mmol/L nitrosomethylurea (MNU) Western blots suggested that minimal residual UBR1 protein in the dark for 30 minutes. Cells were harvested at 0, 24, 48, and remained in KD HBECs compared with control lines (Fig. 72 hours post-MNU treatment. O6MG was quantified in genomic 1A, not shown). DNA using HPLC-MS (37). The average amount recovered for 50 15 13 6 0 fmol positive controls ([ N5- CD3]-O -methyl-2 -deoxyguano- Promoter activity assays sine) was 47 8 fmol (n ¼ 4). The limit of detection was 1 fmol, A 1,528-bp fragment containing the entire promoter and CpG whereas limit of quantitation was 2.5 fmol per injection. Genomic island of MGMT with different haplotype alleles was previously O6MG level was expressed as the ratio between the amount of cloned into the pGL2-basic Luciferase Reporter Vector (Promega) O6MG and dG within each DNA sample. The percentage of

Figure 1. Effect of UBR1 KD on MGMT turnover and DNA repair. A, Western blot for UBR1 KD in HBEC1. Stable transfection with pSUPER.retro.hyg expressing siRNA targeting UBR1 led to almost undetectable UBR1 protein in HBEC1. B, the ratios of na€ve versus suicide MGMT were higher in control HBECs compared with UBR1 KD lines (0.95 0.29 vs. 0.71 0.30, paired t test P ¼ 0.037), with largest difference seen in HBEC26 (0.36). C, Western blot for MGMT protein in control HBEC26 and UBR1 KD line with and without GluC digest. D, DNA repair for O6MG is slower in control HBECs than UBR1 KD lines (percentage of O6MG left unrepaired at 24 hours post-MNU treatment; 0.22 0.0049 vs. 0.11 0.02, paired t test, P ¼ 0.011). Results are summarized with mean and standard deviation based on experiments from three HBEC lines.

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O6MG left unrepaired at 24 hours post-MNU treatment was Results further standardized by taking into account the cell growth rate. GWAS discovery in LSC Comparison of the observed and expected distribution of The cytokinesis-block micronucleus assay association estimates calculated for the GWAS discovery indicated Phytohemagglutinin-stimulated exponentially growing lym- no evidence of population stratification (inflation factor ¼ 1.003; phocyte cultures established from LSC members (n ¼ 107) were Supplementary Fig. S1). GWAS discovery was conducted in 1,163 treated with 1 mmol/L MNU for 4 hours and then incubated with LSC members with methylation of MGMT detected in 296 sub- 4.5 mg/mL cytochalasin B for an additional 24 hours before jects (25.5%). Thirty-six common SNPs (MAF > 0.1) were asso- harvest (38). The dose selected was within the linear dose– ciated with risk for MGMT methylation with P < 10 5 (Supple- response range and caused obvious genotoxicity, but minimal mentary Table S2). Enhancer SNP rs16906252 (not included on cytotoxicity. The micronuclei and nucleoplasmic bridge were the HumanOmni2.5-4v1-H BeadChip) previously shown to be scored to allow the calculation of the percentage of cells with highly significantly associated with risk for MGMT methylation chromosomal damages. was genotyped in the 1,163 LSC members and the association clearly reached genome wide significance (P ¼ 4.3 10 31). SNP rs16906252 was included in the logistic regression model to allow Statistical analysis for identification of novel SNPs with associations independent of In the GWAS discovery, the genetic association was assessed in rs16906252 and this identified 12 common SNPs with P < 10 5 1,163 subjects using logistic regression with adjustment for age (Supplementary Table S2). SNPs located around the MGMT and sex. The genotype of rs16906252 was further included for promoter became nonsignificant (Ps > 0.94; Supplementary covariate adjustment in models that would identify novel SNPs Table S2 and Fig. S2), suggesting the associations seen for these whose association with risk for MGMT methylation was inde- SNPs reflected some degree of linkage disequilibrium (LD) with pendent of rs16906252. SNPs were assessed under an additive rs16906252. However, multiple associations around chromo- genetic model. Odds ratio (OR) and 95% confidence intervals some 15q15.2 locus remained statistically significant and thus (95% CI) were calculated to quantify the magnitude of the likely independent of rs16906252. association per allele. In the VSC replication, logistic regression was applied to assess the association between each individual SNP and risk for MGMT Replication in VSC methylation in 430 subjects with adjustment for age, ethnicity Four SNPs (rs72887860, rs62287262, rs16957091, and (Hispanic versus non-Hispanic White), and rs16906252. The rs997781) associated with risk for MGMT methylation in LSC P < 5 > inclusion of Hispanics (n ¼ 141) in the analysis is mainly due with 10 and MAF 0.1 were selected for replication in 430 to the sample size consideration. However, the inclusion of both members from the VSC with MGMT methylation detected in 107 ethnicities should not affect our results because the prevalence of subjects (24.9%). Rs16957091 was genotyped as the tag SNP on MGMT methylation and the distribution of the four SNPs are not chromosome 15q15.2. Associations were replicated on chromo- P ¼ different between the two major ethnicities in the VSC. Results some 15q15.2 (rs16957091, 0.016) and 17q24.3 loci P ¼ from the two stages were combined by a meta-analysis using the (rs997781, 0.016). Meta-analysis combining the results for fi inverse variance weighting method (39). these two SNPs from LSC and VSC identi ed associations of near fi Generalized linear models (GLM) were used to assess the genome-wide signi cance (Table 1). Data from the LSC and VSC associations between SNPs under the additive inheritance model cohorts were pooled to assess change in the area under the curve and gene expression (delta Ct relative to b-actin) in primary (AUC) of the receiver operating characteristic (ROC) by adding human BECs and normal lung tissues; between UBR1 KD status, rs16957091, rs997781, and rs16906252 to the basic model con- fi promoter haplotype, and MGMT promoter activity in HBEC4; taining age, sex, race, and cohort identi er only (Supplementary between UBR1 KD status and induction of MGMT expression Table S4). Adding all three SNPs into model increased the ROC P ¼ 57 upon MNU treatment; and between rs16857091 and log10 AUC by 19.6% (from 54.2% to 73.8%, 2.97 10 ). transformed percentage of cells with chromosomal damage. The GLM included design variables and variables associated with each Association based on imputed genotypes at chromosome of the outcomes (Supplementary Table S3). The paired t test was 17q24.3 and 15q15.2 loci used to compare the difference of the ratio of na€ve versus suicide Rs997781 is located within a 131-kb haplotype block MGMT and O6MG between UBR1 KD and control cell lines. All (chr17:64593108-64724190, hg18) on chromosome 17q24.3 con- statistical tests were two-sided. Statistical analyses were conducted taining ABCA6 and ABCA10 (not shown). Rs16957091 is located in SAS 9.2, R 2.14, and PLINK 1.06. within a 472-kb haplotype block (chr15:40709948-41181608,

Table 1. Associations between four SNPs and risk for MGMT methylation in LSC and VSC LSC VSC Meta-analysis SNP Location Allelea MAFb OR (95% CI) Pc OR (95% CI) Pd OR (95% CI) P rs72887860 Chr3:61999431 G/A 0.14 1.87 (1.42–2.47) 7.5 106 0.91 (0.57–1.45) 0.69 rs62287262 Chr4:6601119 A/G 0.40 0.58 (0.47–0.72) 1.4 106 1.20 (0.84–1.73) 0.32 rs16957091 Chr15:40804718 T/G 0.23 1.72 (1.36–2.17) 4.5 106 1.63 (1.10–2.41) 0.016 1.70 (1.39–2.07) 2.5 107 rs997781 Chr17:64629283 G/A 0.29 1.64 (1.33–2.03) 4.2 106 1.57 (1.09–2.25) 0.016 1.63 (1.36–1.95) 2.1 107 aSecond allele is the minor allele. bMAF was assessed in LSC. cLogistic regression with adjustment for age, sex, and rs16906252. dLogistic regression with adjustment for age, ethnicity (Hispanic vs. non-Hispanic White), and rs16906252.

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Figure 2. Imputation of chromosome 15q15.2. Imputation was conducted using phased genotype data of EUR populations from 1,000 Genomes project. Genetic associations are plotted for 941 variants (MAF 0.05) within the region of Chr15: 40304748– 41304718 (hg18) that include 758 imputed variants (dosage R2 is 0.3). Circles, genotyped SNPs; squares, imputed SNPs; purple circle, rs16957091. Degree of LD with rs16957091 is schemed as the gradient of red to dark blue color, with red as high LD (R2 ¼ 0.8–1) and blue as low LD (R2 ¼ 0–0.2). No SNPs in LD (R2 > 0.2) with rs16957091 can be detected outside of this region on chromosome 15.

hg18) on chromosome 15q15.2 that contains STARD9, CDAN1, (pairwise Pearson correlation coefficients ¼ 0.46–0.67, Ps TTBK2, and UBR1 (Fig. 2). Genotype imputation successfully 0.0005). However, STARD9 expression was not correlated with (dosage R2 0.3) estimated the allelic dosage for 126,145 the other three genes within the same haplotype block (Ps > 0.07) common SNPs (MAF 0.05) on chromosome 15 that were possibly due to genomic recombination detected within STARD9. not genotyped by the Illumina HumanOmni2.5M chip. Associ- A significant association was observed in human BECs (Ptrend ¼ ation analyses based on imputation results on chromosome 15 0.018) and replicated in normal lung tissues (Ptrend ¼ 0.018) with identified five SNPs associated with risk for MGMT methylation the variant allele (G) of rs16957091 associated with reduction in with P-values slightly lower than that seen for rs16957091, UBR1 gene expression (Fig. 3A). The effect of rs16957091 on although all five SNPs are in almost perfect LD with rs16957091 STARD9, CDAN1, and TTBK2 gene expression was not statistically (R2s 0.95; Fig. 2). Imputation at chromosome 17q24.3 did not significant (Ps > 0.32). find any associations that were more significant than that seen Experiments were designed to exam whether rs16957091– for rs997781. UBR1 expression correlation was caused by sequence-dependent allele-specific gene expression. Nine SNPs were annotated to any Rs16957091 affects UBR1 expression UBR1 exons or 50 or 30 untranslated regions with MAF ranged from The enrichment of SNPs in ENCODE-annotated regulatory 0 to 0.12. LD analysis between rs16957091 and each of these nine regions within the 472-kb haplotype block (chr15:40709948- SNPs using 1,000 Genomes CEU population identified low LD 41181608, hg18) at chromosome 15q15.2 locus was assessed between rs16957091 and rs3917223 (R2 ¼ 0.34). The LD esti- using FunciSNP with data from SAEC. Multiple chromatin fea- mates between rs16957091 and each of the other eight SNPs are tures surrounding STARD9, CDAN1, TTBK2, and UBR1 were very low (R2 ¼ 0–0.13). Sequencing genomic DNA and cDNA identified to overlap with SNPs in LD with rs16957091 (n ¼ containing rs3917223 from seven BECs heterozygous for 108, R2 0.2, P-values by Benjamini–Hochberg adjustment rs16957091 and rs3917223 did not identify an allele-specific <0.01; Supplementary Fig. S3). Thus, the effect of rs16957091 gene expression pattern for rs3917223. on the expression of these four genes was assessed in 54 human primary BEC cultures and 59 normal lung tissues. Among the four Reduced UBR1 expression affects MGMT turnover and DNA genes within this block, UBR1 has the highest gene expression in repair human primary BECs (Fig. 3A). A significant correlation of gene UBR1 contains binding sites recognizing internal degrons of expression among UBR1, CDAN1, and TTBK2 was identified MGMT protein and the UBR1/RAD6-dependent N-end rule

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ratio of na€ve versus suicide MGMT decreased with increasing concentrations of MNU treatment with almost no active MGMT left at the highest MNU concentration (not shown). Interest- ingly, without MNU treatment, protease V8 cutting is still detected supporting formation of endogenous alkylating DNA adducts as a part of normal cellular metabolism in these cells (1, 42), although the assay specificity for detecting suicide MGMT may not be perfect due to its potential binding to other proteins. The ratio of na€ve versus suicide MGMT was higher in control HBECs compared with UBR1 KD lines (0.95 vs. 0.71, paired t test P ¼ 0.037; Fig. 1B) with the largest difference seen in HBEC26 (0.36; Fig. 1C), suggesting accumulation of suicide MGMT in UBR1 KD lines. UBR1 knockout cells are resistant to alkylating agent (MNNG) induced genotoxicity probably because UBR1 targets both alkylated and unmodified MGMT (41). Thus, HBECs were treated with 1 mmol/L MNU and O6MG was quantified in genomic DNA recovered from cells at 0, 24, 48, and 72 hours post-MNU treatment. On average, 78% of O6MG adducts were repaired within 24 hours in control HBECs. In contrast, 89% O6MG were repaired within 24 hours in UBR1 KD lines (Fig. 1D). Rates of repair within 24 hours differed significantly between control and UBR1 KD (paired t test, P ¼ 0.011; Fig. 1D). The modest but statistically significant effect of UBR1 KD on MGMT degradation and repair of O6-methylguanine may reflect the complexity of constitutive and exogenous alkylating agent-accelerated degradation of MGMT. In addition to the UBR1/RAD6-dependent N-end rule pathway, the UFD4/UBC4-dependent ubiquitin fusion degradation pathway functions as an enhancer of the processivity of MGMT polyubiquitylation by the N-end rule pathway through physical interaction between the HECT-type UFD4 E3 and the RING-typeUBR1E3(40,41). Because rs16957091 reduced UBR1 expression and UBR1 KD was associated with faster DNA repair of O6MG induced by Figure 3. Rs16957091 affects expression of UBR1 and DNA repair capacity towards MNU MNU, we further assessed whether rs16957091 is associated treatment. A, G allele of rs16957091 is associated with reduced gene with better repair in cultured lymphocytes collected from LSC expression of UBR1 in primary human BECs (n ¼ 54; P ¼ 0.018) and normal subjects (n ¼ 107). A significant inverse association (P ¼ lungs (n ¼ 59; P ¼ 0.018). Y axis was truncated between 0.0075 and 0.015. B, 0.036; Fig. 3B) between the number of rs16957091 G alleles G allele of rs16957091 is associated with lower percentage of cells with and percentage of cells with chromosomal damage was chromosomal damage (n ¼ 107, P ¼ 0.036), indicating better repair towards observed, suggesting that the G allele associated with reduced MNU treatment. Results are summarized with mean and standard error. UBR1 expression is associated with greater DNA repair of MNU- induced DNA damage. pathway is a major mechanism targeting suicide MGMT protein by alkylating agents and also unmodified MGMT for subse- UBR1 expression levels affect MGMT transcription quent degradation via the 26S proteasome in eukaryotes (40, Reduced gene transcription is a major mechanism predisposing 41). Recovery of MGMT activity after its inactivation results MGMT for promoter hypermethylation in multiple tissue types entirely from de novo protein synthesis (4). Our previous (13, 27); therefore, a luciferase reporter assay was used to assess studies demonstrated reduced gene transcription associated whether UBR1 KD would result in reduced MGMT promoter with the enhancer SNP rs16906252 as a major mechanism activity. Consistent with our previous studies and others, HAP4 predisposing MGMT for promoter hypermethylation in the (rs16906252 carrier) was associated with a 35% (P < 0.0001) and lungs of smokers (27). Thus, the lower expression of UBR1 22% (P ¼ 0.0019) reduction in promoter activity compared with associated with rs16957091 variant allele could result in slower HAP1 (wild type) in control and UBR1 KD HBEC1 lines, respec- turnover of the MGMT protein under normal physiological tively (Fig. 4A). Excitingly, UBR1 KD reduced HAP1 promoter conditions and/or following repair in response to challenge by activity by 25% compared with control line (P ¼ 0.0009). Reduc- the alkylating agents, that in turn may lead to reduced MGMT tion of promoter activity (12%) in UBR1 KD line was also transcription. observed for HAP4 (P ¼ 0.089), although to a lesser degree that UBR1 stable KD cell lines were established from three immor- likely stemmed from the inefficient transcription regulatory talized HBECs (HBEC1, HBEC4, and HBEC26) to test this hypoth- mechanism due to carrying rs16906252. The combination esis. A protease V8 digestion method (36) was used to distinguish of UBR1 KD and HAP4 reduced promoter activity by 50% (P < na€ve and suicide MGMT in HBEC1 treated with different MNU 0.0001) compared with wild-type UBR1 and HAP1. The reduction concentrations (0, 0.5, 1, and 2.5 mmol/L) for 30 minutes. The of HAP1 promoter activity in the UBR1 KD HBEC4 line was

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Chromosome 15q15.2 for MGMT Promoter Hypermethylation

Figure 4. Effect of UBR1 KD on MGMT promoter activity and induction of MGMT expression following MNU treatment. A, both UBR1 KD and HAP4 (rs16906252 carrier) reduced MGMT promoter activity in HBEC4. The combination of UBR1 KD and HAP4 reduced promoter activity by almost 50% (P < 0.0001) compared with wild-type UBR1 and HAP1. The reduction of HAP1 promoter activity in UBR1 KD HBEC4 lines was replicated in HBEC1 (reduced by 23%; P ¼ 0.0046) and in HBEC26 (reduced by 28%; P ¼ 2.5 108). B, MNU treatment at a noncytotoxic dose (1 mmol/L) induced MGMT transcription in control HBEC4 and HBEC26 at 24 and 48 hours post-MNU treatment. UBR1 KD largely abolished this induction in HBEC4 and HBEC26. Although no induction was observed in HBEC1, control line still had higher MGMT expression than UBR1 KD line (P ¼ 0.086). Results are summarized with mean and standard deviation.

replicated in HBEC1 (reduced by 23%, P ¼ 0.0046) and HBEC26 Discussion (reduced by 28%, P ¼ 2.5 10 8; Fig. 4A). These findings suggest Our GWAS study identified cis- and trans-acting SNPs that affect that impaired MGMT turnover due to UBR1 KD may lead to silencing of a DNA repair gene (MGMT) by promoter hyper- reduced MGMT transcription, an important factor in the predis- methylation acquired during lung carcinogenesis. Two novel position for promoter hypermethylation of MGMT. trans-acting loci at chromosome 15q15.2 and 17q24.3 were The recovery of MGMT activity after its inactivation by identified as being associated with risk for MGMT methylation alkylating agent results entirely from de novo protein synthesis detected in sputum and their effects were independent of the and this rate differs between pulmonary cell types in the rat enhancer SNP rs16906252. These three SNPs together improved (4, 43). Furthermore, a large induction of MGMT measured in the ROC AUC by 20% in a logistic regression that explored steady-state RNA has been observed in rat liver cell lines >24 determinants for risk for MGMT methylation in current and hours post-alkylating agent treatment and was due to activation former smokers. Thus, SNPs affecting the predisposition of of the MGMT promoter (3, 5). Strong induction of MGMT MGMT silencing by promoter hypermethylation, together with expression in steady state RNA was also observed in control SNPs affecting methylation of other important cancer-relevant HBEC4 and HBEC26 at 24 and 48 hours post-MNU treatment genes acquired during carcinogenesis may lead to the establish- (1 mmol/L), but not in control HBEC1 (Fig. 4B). In contrast, ment of a polygenic marker that could improve current risk only minimal induction was observed in HBEC4 and HBEC26 prediction models for these cancers (18). with UBR1 KD at 48 hours post-MNU treatment (Fig. 4B). GLM MGMT methylation status is a validated prognostic biomarker was conducted to assess the effect of UBR1 status (KD vs. WT) for the response of glioblastoma patients to the alkylating agent and time point (24 hours vs. 48 hours) on induction of MGMT temozolomide (14). Our previous study and others also sug- expression in HBEC4 and HBEC26. MGMT induction at 24 and gested that MGMT methylation status may affect the response of 48 hours post-MNU treatment was not statistically different in lung tumors to temozolomide treatment (27, 44). The assessment either cell lines (Ps > 0.11). However, statistically significant of MGMT promoter activity using HEBC4 identified a 50% differences were identified for MGMT induction between con- reduction in luciferase reporter activity for the combination of trolandUBR1KDinHBEC4(leastsquaremeansandstandard UBR1 KD and HAP4 (rs16906252 carrier) compared with wild- error, 1.68 0.068 vs. 1.17 0.068, P ¼ 0.0057) and HBEC26 type UBR1 and HAP1 (non-rs16906252 carrier). This functional (least square means and standard error, 1.43 0.067 vs. 1.13 assessment strongly supports the epidemiological findings that 0.067, P ¼ 0.032) in the GLM with adjustment for assay batch subjects carrying variant alleles for cis- (rs16906252) and trans- and time point (24 hours vs. 48 hours).

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Leng et al.

(rs16957091) acting SNPs that regulate MGMT transcription had accumulation of unmodified MGMT resulting from UBR1 KD the highest risk for MGMT methylation (OR, 17.9; 95% CI, 10.7– (41). More importantly, stable UBR1 KD reduced MGMT pro- 30.1; P ¼ 5.8 10 28; Supplementary Table S5). Furthermore, moter activity and abolished the induction of MGMT expression these two SNPs should also augment MGMT methylation status in HBEC models treated with noncytotoxic MNU. These findings for the better stratification of patients for alkylating therapy due to suggest that compromised MGMT degradation may not be able to their association with reduced MGMT transcription in MGMT provide an efficient positive feedback to the MGMT transcrip- unmethylated cells. tional machinery under normal physiological conditions or in The mechanism for trans-acting meQTL is largely unknown. response to alkylating agent exposure. Thus, our findings not only Among 585 trans-acting meQTL identified in normal lungs, substantiate the reduced gene transcription as a major determi- approximately 196 trans-acting meQTL were mediated by meth- nant for MGMT methylation in the lungs of smokers, but also ylation levels of CpG sites nearby the trans-acting SNPs (19). support the importance of the UBR1-mediated N-end rule path- Ontology classification of genes (n ¼ 115) in the region of the way in regulating MGMT homeostasis and DNA repair towards proximal CpG sites mediating the trans-acting associations iden- alkylated DNA adducts. tified GTPase activity related genes, genes regulating transcription, The mechanism for how rs16957091, located 223 kb from and genetic imprinting as the top three categories involved in UBR1 regulates its expression was explored by assessing the DNA methylation regulation. A unique mechanism underlying enrichment of rs16957091 and SNPs in moderate and high LD the predisposition of MGMT methylation in lungs of smokers with it (R2 0.2) in ENCODE-annotated regulatory regions involving the regulation of UBR1 expression by rs16957091 surrounding UBR1 using SAEC data. Multiple chromatin features within the chromosome 15q15.2 locus was elucidated (Fig. 5). surrounding UBR1 were identified to overlap with SNPs (n ¼ 22) Because reduced gene transcription is a major mechanism pre- in LD with rs16957091 (Supplementary Fig. S3), suggesting that a disposing MGMT for promoter hypermethylation in multiple combination of variant alleles of these SNPs may form a genomic tissue types (13, 27), three HBEC lines with stable UBR1 KD backbone associated with a particular active or repressive chro- were established to assess whether reduced UBR1 expression matin conformation that affects UBR1 expression. In addition, could affect the MGMT transcription. The functional validity of SNP rs68062403 located at þ178 relative to the transcription start UBR1 KD was confirmed by showing compromised degradation site of UBR1 is in moderate LD (R2 ¼ 0.25) with rs16957091. A of suicide MGMT or unmodified MGMT and increased DNA search of the ChIP-seq database identified multiple transcription repair towards MNU induced O6MG in UBR1 KD lines compared factors (POLR2A, YY1, and TAF1) overlapping with the region with control HBECs. These effects by UBR1 are consistent with its where rs68062403 resides in A549, a lung carcinoma cell line. role of binding to the internal degrons of suicide MGMT protein Furthermore, rs68062403 is located within an open chromatin and even unmodified MGMT for subsequent degradation via the region likely to be enriched in gene regulatory elements in SAEC 26S proteasome in eukaryotes (1, 40, 41). In addition, greater (Supplementary Fig. S3) and A549 (not shown). Thus, the asso- DNA repair towards O6MG in UBR1 KD lines may be due to the ciation seen in this study could be due to the LD with SNP

Figure 5. Conceptual diagram for cis- and trans- acting SNPs for MGMT promoter hypermethylation. The UBR1/RAD6- mediated N-end rule pathway regulates MGMT homeostasis under normal MGMT turnover and degradation of suicide MGMT upon alkylating agent challenge. Reduced UBR1 expression due to SNPs at 15q15.2 locus likely induces the accumulation of unmodified MGMT that is associated with better DNA repair towards alkylating agent induced DNA adducts. In addition, the compromised degradation of unmodified MGMT and suicide MGMT is associated with reduced MGMT promoter activity and expression induction, although the molecular mechanism is still unknown. This study also verified MGMT enhancer SNP (rs16906252) as the driver cis- acting SNP for MGMT methylation through reducing MGMT transcription. Thus, both cis-andtrans-acting SNPs could affect the MGMT transcriptional machinery that predisposes MGMT for promoter hypermethylation in lungs of smokers.

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Chromosome 15q15.2 for MGMT Promoter Hypermethylation

rs68062403 that in turn may affect the binding of a transcription Administrative, technical, or material support (i.e., reporting or organizing factor. data, constructing databases): C.L. Thomas, M.A. Picchi, D.E. Juri, D. Desai, S. Our studies provide a proof-of-concept for a unique mecha- A. Belinsky Study supervision: S. Leng, S.A. Belinsky nism underlying the predisposition of MGMT methylation in Other (statistical analysis): G. Wu lungs of smokers that involves the regulation of MGMT homeo- Other (primarily worked with Dr. Belinsky and Shuguang Leng on the DNA stasis by the UBR1/RAD6-mediated N-end rule pathway (Fig. 5). adduct data, in collaboration with Leonard Collins in the mass spectrometry The cis- and trans-acting SNPs affecting MGMT methylation facility): J.A. Swenberg should have strong clinical translational significance in terms of fi their utility in cancer risk assessment and patient strati cation for Acknowledgments alkylating agent chemotherapy. The author thank Mr. Randall P. Willink, Ms. Kieu C. Do, and Ms. Mabel T. Padilla from LRRI for assistance in conducting methylation-specific PCR and Disclosure of Potential Conflicts of Interest Western blot assay, and Mr. Thomas J. Gagliano from LRRI for the scientific No potential conflicts of interest were disclosed. editing of all figures and conceptualization of the diagram.

Authors' Contributions Conception and design: S. Leng, C.S. Tellez, D. Desai, C.A. Stidley, Y. Liu, Y. Lin, Grant Support M.G. Wathelet, F.D. Gilliland, S.A. Belinsky This work was primarily supported by the National Cancer Institute grant Development of methodology: S. Leng, L.B. Collins, C.S. Tellez, A.R. Jauregui, R01 CA097356 (S.A. Belinsky). The State of New Mexico as a direct appropri- S.G. Amin, Y. Liu, J.A. Swenberg, M.G. Wathelet, S.A. Belinsky ation from the Tobacco Settlement Fund to S.A. Belinsky through collaboration Acquisition of data (provided animals, acquired and managed patients, with the University of New Mexico provided initial support to establish the LSC. provided facilities, etc.): S. Leng, L.B. Collins, C.S. Tellez, R.E. Crowell, Additional support was provided by NIH/NCI P30 CA118100 and NIEHS P30- S.A. Belinsky ES10126. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, The costs of publication of this article were defrayed in part by the payment of advertisement computational analysis): S. Leng, C.S. Tellez, X. Zhang, C.A. Stidley, Y. Liu, page charges. This article must therefore be hereby marked in S.A. Belinsky accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Writing, review, and/or revision of the manuscript: S. Leng, C.S. Tellez, A.R. Jauregui, M.A. Picchi, D. Desai, S.G. Amin, C.A. Stidley, M.G. Wathelet, Received January 26, 2015; revised May 22, 2015; accepted May 22, 2015; F.D. Gilliland, S.A. Belinsky published OnlineFirst July 16, 2015.

References 1. Pegg AE. Multifaceted roles of alkyltransferase and related proteins in DNA 10. Pulling LC, Divine KK, Klinge DM, Gilliland FD, Kang T, Schwartz AG, et al. repair, DNA damage, resistance to chemotherapy, and research tools. Promoter hypermethylation of the O6-methylguanine-DNA methyltrans- Chem Res Toxicol 2011;24:618–39. ferase gene: more common in lung adenocarcinomas from never-smokers 2. Srivenugopal KS, Yuan XH, Friedman HS, Ali-Osman F. Ubiquitination- than smokers and associated with tumor progression. Cancer Res dependent proteolysis of O6-methylguanine-DNA methyltransferase in 2003;63:4842–8. human and murine tumor cells following inactivation with O6-benzyl- 11. Belinsky SA. Gene-promoter hypermethylation as a biomarker in lung guanine or 1,3-bis(2-chloroethyl)-1-nitrosourea. Biochemistry 1996; cancer. Nat Rev Cancer 2004;4:707–17. 35:1328–34. 12. Belinsky SA, Liechty KC, Gentry FD, Wolf HJ, Rogers J, Vu K, et al. Promoter 3. Fritz G, Tano K, Mitra S, Kaina B. Inducibility of the DNA repair hypermethylation of multiple genes in sputum precedes lung cancer gene encoding O6-methylguanine-DNA methyltransferase in mam- incidence in a high-risk cohort. Cancer Res 2006;66:3338–44. malian cells by DNA-damaging treatments.MolCellBiol1991; 13. McDonald KL, Rapkins RW, Olivier J, Zhao L, Nozue K, Lu D, et al. The T 11:4660–8. genotype of the MGMT C>T (rs16906252) enhancer single-nucleotide 4. Pegg AE, Wiest L, Mummert C, Stine L, Moschel RC, Dolan ME. Use of polymorphism (SNP) is associated with promoter methylation and longer antibodies to human O6-alkylguanine-DNA alkyltransferase to study the survival in glioblastoma patients. Eur J Cancer 2013;49:360–8. content of this protein in cells treated with O6-benzylguanine or N-methyl- 14. Weller M, Stupp R, Reifenberger G, Brandes AA, van den Bent MJ, Wick W, N0-nitro-N-nitrosoguanidine. Carcinogenesis 1991;12:1679–83. et al. MGMT promoter methylation in malignant gliomas: ready for 5. Grombacher T, Mitra S, Kaina B. Induction of the alkyltransferase (MGMT) personalized medicine? Nat Rev Neurol 2010;6:39–51. gene by DNA damaging agents and the glucocorticoid dexamethasone and 15. Schalkwyk LC, Meaburn EL, Smith R, Dempster EL, Jeffries AR, Davies MN, comparison with the response of base excision repair genes. Carcinogenesis et al. Allelic skewing of DNA methylation is widespread across the genome. 1996;17:2329–36. Am J Hum Genet 2010;86:196–212. 6. Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG. Inactivation of 16. Kerkel K, Spadola A, Yuan E, Kosek J, Jiang L, Hod E, et al. Genomic surveys the DNA repair gene O6-methylguanine-DNA methyltransferase by pro- by methylation-sensitive SNP analysis identify sequence-dependent allele- moter hypermethylation is a common event in primary human neoplasia. specific DNA methylation. Nat Genet 2008;40:904–8. Cancer Res 1999;59:793–7. 17. Paliwal A, Temkin AM, Kerkel K, Yale A, Yotova I, Drost N, et al. 7. Esteller M, Toyota M, Sanchez-Cespedes M, Capella G, Peinado MA, Comparative anatomy of chromosomal domains with imprinted and Watkins DN, et al. Inactivation of the DNA repair gene O6-methylgua- non-imprinted allele-specific DNA methylation. PLoS Genet 2013;9: nine-DNA methyltransferase by promoter hypermethylation is associated e1003622. with G to A mutations in K-ras in colorectal tumorigenesis. Cancer Res 18. Tycko B. Mapping allele-specific DNA methylation: a new tool for max- 2000;60:2368–71. imizing information from GWAS. Am J Hum Genet 2010;86:109–12. 8. Yin D, Xie D, Hofmann WK, Zhang W, Asotra K, Wong R, et al. DNA repair 19. Shi J, Marconett CN, Duan J, Hyland PL, Li P, Wang Z, et al. Characterizing gene O6-methylguanine-DNA methyltransferase: promoter hypermethy- the genetic basis of methylome diversity in histologically normal human lation associated with decreased expression and G:C to A:T mutations of lung tissue. Nat Commun 2014;5:3365. p53 in brain tumors. Mol Carcinog 2003;36:23–31. 20. Drong AW, Nicholson G, Hedman AK, Meduri E, Grundberg E, Small KS, 9. Sakumi K, Shiraishi A, Shimizu S, Tsuzuki T, Ishikawa T, Sekiguchi M. et al. The presence of methylation quantitative trait loci indicates a direct Methylnitrosourea-induced tumorigenesis in MGMT gene knockout mice. genetic influence on the level of DNA methylation in adipose tissue. PLoS Cancer Res 1997;57:2415–8. One 2013;8:e55923.

www.aacrjournals.org Cancer Res; 75(15) August 1, 2015 OF9

Leng et al.

21. Fraser HB, Lam LL, Neumann SM, Kobor MS. Population-specificity of 33. Leng S, Liu Y, Thomas CL, Gauderman WJ, Picchi MA, Bruse SE, et al. Native human DNA methylation. Genome Biol 2012;13:R8. American ancestry affects the risk for gene methylation in the lungs of 22. Gibbs JR, van der Brug MP, Hernandez DG, Traynor BJ, Nalls MA, Hispanic smokers from New Mexico. Am J Respir Crit Care Med Lai SL, et al. Abundant quantitative trait loci exist for DNA 2013;188:1110–6. methylation and gene expression in human brain. PLoS Genet 34. Coetzee SG, Rhie SK, Berman BP, Coetzee GA, Noushmehr H. FunciSNP: 2010;6:e1000952. an R/bioconductor tool integrating functional non-coding data sets 23. Heyn H, Moran S, Hernando-Herraez I, Sayols S, Gomez A, Sandoval J, et al. with genetic association studies to identify candidate regulatory SNPs. DNA methylation contributes to natural human variation. Genome Res Nucleic Acids Res 2012;40:e139. 2013;23:1363–72. 35. Ramirez RD, Sheridan S, Girard L, Sato M, Kim Y, Pollack J, et al. Immor- 24. Heyn H, Sayols S, Moutinho C, Vidal E, Sanchez-Mut JV, Stefansson OA, talization of human bronchial epithelial cells in the absence of viral et al. Linkage of DNA methylation quantitative trait loci to human cancer oncoproteins. Cancer Res 2004;64:9027–34. risk. Cell Rep 2014;7:331–8. 36. Ayi TC, Oh HK, Lee TK, Li BF. A method for simultaneous identification of 25. Zhang D, Cheng L, Badner JA, Chen C, Chen Q, Luo W, et al. Genetic control human active and active-site alkylated O6-methylguanine-DNA methyl- of individual differences in gene-specific methylation in human brain. Am J transferase and its possible application for monitoring human exposure to Hum Genet 2010;86:411–9. alkylating carcinogens. Cancer Res 1994;54:3726–31. 26. Boumber YA, Kondo Y, Chen X, Shen L, Guo Y, Tellez C, et al. An Sp1/Sp3 37. Sharma V, Collins LB, Clement JM, Zhang Z, Nakamura J, Swenberg JA. binding polymorphism confers methylation protection. PLoS Genet Molecular dosimetry of endogenous and exogenous O(6)-methyl-dG and 2008;4:e1000162. N7-methyl-G adducts following low dose [D3]-methylnitrosourea expo- 27. Leng S, Bernauer AM, Hong C, Do KC, Yingling CM, Flores KG, et al. The sures in cultured human cells. Chem Res Toxicol 2014;27:480–2. A/G allele of rs16906252 predicts for MGMT methylation and is selectively 38. Duan H, Leng S, Pan Z, Dai Y, Niu Y, Huang C, et al. Biomarkers measured silenced in premalignant lesions from smokers and in lung adenocarci- by cytokinesis-block micronucleus cytome assay for evaluating genetic nomas. Clin Cancer Res 2011;17:2014–23. damages induced by polycyclic aromatic hydrocarbons. Mutat Res 28. Ronneberg JA, Tost J, Solvang HK, Alnaes GI, Johansen FE, Brendeford 2009;677:93–9. EM, et al. GSTP1 promoter haplotypes affect DNA methylation levels 39. Egger M, Smith GD, Altman DG. Systematic reviews in health care: meta- and promoter activity in breast carcinomas. Cancer Res 2008;68: analysis in context. London: BMJ; 2001. xviii, 487 p. p. 5562–71. 40. Hwang CS, Shemorry A, Auerbach D, Varshavsky A. The N-end rule 29. Hawkins NJ, Lee JH, Wong JJ, Kwok CT, Ward RL, Hitchins MP. MGMT pathway is mediated by a complex of the RING-type Ubr1 and HECT- methylation is associated primarily with the germline C>T SNP type Ufd4 ubiquitin ligases. Nat Cell Biol 2010;12:1177–85. (rs16906252) in colorectal cancer and normal colonic mucosa. Mod 41. Hwang CS, Shemorry A, Varshavsky A. Two proteolytic pathways regulate Pathol 2009;22:1588–99. DNA repair by cotargeting the Mgt1 alkylguanine transferase. Proc Natl 30. Kristensen LS, Nielsen HM, Hager H, Hansen LL. Methylation of MGMT in Acad Sci U S A 2009;106:2142–7. malignant pleural mesothelioma occurs in a subset of patients and is 42. Nakamura J, Mutlu E, Sharma V, Collins L, Bodnar W, Yu R, et al. The associated with the T allele of the rs16906252 MGMT promoter SNP. Lung endogenous exposome. DNA Repair 2014;19:3–13. Cancer 2011;71:130–6. 43. Belinsky SA, Dolan ME, White CM, Maronpot RR, Pegg AE, Anderson MW. 31. Kristensen LS, Treppendahl MB, Asmar F, Girkov MS, Nielsen HM, Kjeldsen Cell specific differences in O6-methylguanine-DNA methyltransferase TE, et al. Investigation of MGMT and DAPK1 methylation patterns in activity and removal of O6-methylguanine in rat pulmonary cells. diffuse large B-cell lymphoma using allelic MSP-pyrosequencing. Sci Rep Carcinogenesis 1988;9:2053–8. 2013;3:2789. 44. Pietanza MC, Kadota K, Huberman K, Sima CS, Fiore JJ, Sumner DK, et al. 32. Ogino S, Hazra A, Tranah GJ, Kirkner GJ, Kawasaki T, Nosho K, et al. MGMT Phase II trial of temozolomide in patients with relapsed sensitive or germline polymorphism is associated with somatic MGMT promoter refractory small cell lung cancer, with assessment of methylguanine-DNA methylation and gene silencing in colorectal cancer. Carcinogenesis methyltransferase as a potential biomarker. Clin Cancer Res 2012; 2007;28:1985–90. 18:1138–45.

OF10 Cancer Res; 75(15) August 1, 2015 Cancer Research

Implication of a Chromosome 15q15.2 Locus in Regulating UBR1 and Predisposing Smokers to MGMT Methylation in Lung

Shuguang Leng, Guodong Wu, Leonard B. Collins, et al.

Cancer Res Published OnlineFirst July 16, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-0243

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2021/03/04/0008-5472.CAN-15-0243.DC1

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