CancerTherapy: Preclinical

Proteasome B Subunit Pharmacogenomics: Resequencing and Functional Genomics Liewei Wang,1Shaji Kumar,2 Brooke L. Fridley,3 Krishna R. Kalari,3 Irene Moon,1Linda L. Pelleymounter,1 Michelle A.T. Hildebrandt,1Anthony Batzler,3 Bruce W. Eckloff,4 Eric D.Wieben,4 and Philip R. Greipp2

Abstract Purpose: The is a multisubunit cellular organelle that functions as a nonlysosomal threonine protease. play a critical role in the degradation of , regulating a variety of cellular processes, and they are also the target for antineoplastic proteasome inhibitors. Genetic variation in proteasome subunits could influence both proteasome function and response to drug therapy. Experimental Design:We resequenced encoding the three active proteasome h subunits using 240 DNA samples from four ethnic groups and the h5 subunit gene in 79 DNA samples from multiple myeloma patients who had been treated with the proteasome inhibitor bortezomib. Resequencing was followed by functional studies of polymorphisms identified in the coding region and 3¶-flanking region (3¶-FR) of PSMB5, the gene encoding the target for clinically useful proteasome inhibitors. Results: Resequencing of 240 DNA samples identified a series of novel ethnic-specific polymor- phisms that are not represented in public databases.The PSMB5 3¶-FR1042 G allele significantly increased during reporter gene studies, observations confirmed by genotype- phenotype correlations between single nucleotide polymorphisms (SNP) in PSMB5 and mRNA expression in the 240 lymphoblastoid cell lines from which the resequenced DNA was obtained. Studies with patient DNA samples identified additional novel PSMB5 polymorphisms, including a SNP and an insertion in the 3¶-FR. Reporter-gene studies indicated that these two novel poly- morphisms might decrease transcription. Conclusions: These results show that nonsynonymous coding SNPs in the PSMB5 gene did not show significant effects on proteasome activity, but SNPs did influence transcription. Future studies might focus on regulatory region polymorphisms.

Protein degradation regulates a variety of critical cellular It plays a critical role in degradation and is the target for processes, including cell division, signal transduction, and antineoplastic proteasome inhibitors (1–5). The 26S protea- (1–4). The proteasome is a multisubunit cellular some consists of a 20S barrel-shaped core particle and two 19S organelle that functions as a nonlysosomal threonine protease. regulatory complexes that cap the 20S core particle. The 20S core particle is a multisubunit enzyme complex that consists of four heptameric rings arranged in a7h7h7a7 fashion, surround- ing a central cavity where the catalytic sites are found (6–8). Three of the seven h subunits, h1, h2, and h5, are Authors’Affiliations: 1Division of Clinical Pharmacology, Department of Molecular 2 proteolytically active with different substrate specificities. The Pharmacology and Experimental Therapeutics and Departments of Internal h Medicine, 3Health Sciences Research, and 4Biochemistry and Molecular Biology, 1 subunit catalyzes a postglutamyl peptidyl hydrolytic-like Mayo Clinic, Mayo Medical School, Rochester, Minnesota activity; the h2 subunit catalyzes a trypic-like activity; and the Received 12/12/07; revised 1/15/08; accepted 1/28/08. h5 subunit catalyzes a chymotryptic-like activity (9). Grant support: NIH grants GM61388 (The Pharmacogenetics Research Network) Proteasome inhibitors have been tested for the treatment of and CA102701 (The Pancreatic Specialized Programs of Research a variety of types of . One of those drugs, bortezomib, Excellence), an American Society for Pharmacology and Experimental h Therapeutics-Astellas Award, and a PhRMA Foundation Center of Excellence in with activity directed mainly against the 5 subunit, has been Clinical PharmacologyAward. approved for the treatment of refractory multiple myeloma The costs of publication of this article were defrayed in part by the payment of page (10, 11). However, clinical response to bortezomib therapy charges. This article must therefore be hereby marked advertisement in accordance varies widely (12–14). Because the proteasome has critical with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research importance for cellular processes and because it is also a drug Online (http://clincancerres.aacrjournals.org/). target, it would be important to identify common sequence Requests for reprints: Liewei Wang, Division of Clinical Pharmacology, variation in genes encoding the three active h subunits and to Department of Molecular Pharmacology and Experimental Therapeutics, Mayo determine the possible functional implications of that sequence Clinic, 200 First Street Southwest, Rochester, MN 55905. Phone: 507-284-5264; @ variation. However, no systematic pharmacogenomic studies Fax: 507-507-284-4455; E-mail: wang.liewei mayo.edu. h F 2008 American Association for Cancer Research. have been done of genes encoding human proteasome doi:10.1158/1078-0432.CCR-07-5150 subunits. Therefore, in the present study, we did comprehensive

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Specifically, cells were collected and resuspended in 4 volumes of homogenization buffer subunit), the major proteasome inhibitor therapeutic target, as [50 mmol/L Tris-HCl (pH 7.5), 250 mmol/L MgCl , 2 mmol/L ATP, well as a correlation of level of PSMB5 expression with gene 2 1 mmol/L DTT, 0.5 mmol/L EDTA] containing freshly prepared sequence variation. We also resequenced the PSMB5 gene using 0.025% digitonin. The resuspended cells were incubated on ice for DNA from patients with multiple myeloma who had been 5 min, and were then centrifuged at 20,000 g at 4jC for 15 min. The treated with bortezomib, using both tumor and germ line DNA, supernatants were separated, and protein concentrations were deter- resulting in the identification of additional novel PSMB5 mined. The cytosolic 26S proteasomes obtained in this fashion were polymorphisms. This series of studies represents a step toward used to perform enzyme activity and inhibition studies. understanding sequence variation in genes encoding the three Proteasome activity and inhibition studies. Proteasome activity and active proteasome h subunits, as well as the potential inhibition assays were done as described previously (21). Specifically, implications of that DNA sequence variation for individual cytosolic 26S proteasomes isolated as described above were incubated j differences in response to treatment with proteasome inhibitors at 37 C with assay buffer [50 mmol/L Tris-HCl (pH 7.5), 40 mmol/L KCl, 5 mmol/L MgCl , 0.5 mmol/L ATP, 1 mmol/L DTT, and and/or contribution to disease pathophysiology. 2 0.05 mg/mL bovine serum albumin], and increasing concentrations of the fluorogenic substrate Suc-LLVY-amc (0, 12.5, 25, 50, and 100 Amol/L) and fluorescence was measured with a Safire2 plate reader Materials and Methods (Tecan). For inhibition studies, concentrations of the proteasome inhibitor MG262 ranging from 10 to 60 nmol/L were premixed with DNA samples. DNA samples from 60 Caucasian American, 60 assay buffer 10 min before the addition of substrate. All assays were African American, 60 Han Chinese American, and 60 Mexican American done at least three times, and results were expressed as mean F SE for subjects (sample sets HD100CAU, HD100AA, HD100CHI, and three determinations. HD100MEX) were obtained from the Coriell Cell Repository. These Proteasome inhibitor cytotoxicity studies. Inhibitors of the protea- DNA samples have been widely used for human gene resequencing some h5 subunit are used clinically to treat cancer because of their studies (15–20). Immortalized lymphoblastoid cell lines from these cytotoxic properties. Therefore, cytotoxicity studies were done using the same subjects are available from the Coriell Institute, and those cell h5 subunit inhibitor MG262 with lymphoblastoid cells having known lines were also used in the experiments described subsequently. All of genotypes for the PSMB5 gene. Specifically, increasing concentrations these DNA samples and cell lines had been obtained and anonymized of MG262 (10, 20, 25, and 30 nmol/L) were incubated with the cell by the National Institute of General Medical Sciences before deposit, lines in 96-well plates for 3 d, followed by MTS assays as a measure of and all subjects had provided written consent for the use of their DNA cytotoxicity. Each experiment was repeated three times, and results were and cells for experimental purposes. In addition, 79 DNA samples from expressed as GI50 values, the concentration of MG262 that inhibited patients with multiple myeloma were isolated from either bone marrow growth by 50%. or peripheral blood. Specifically, these 79 clinical DNA samples were Expression array genotype-phenotype correlation. Expression array obtained from 61 multiple myeloma patients who had been treated analyses were done using Affymetrix U133 Plus 2.0 GeneChips. with the proteasome inhibitor bortezomib. These patients had also Specifically, RNA was isolated from the same lymphoblastoid cells from provided written consent for the use of their samples for research which the DNA used to resequence PSMB1, PSMB2, and PSMB5 had purposes. The clinical DNA samples included 11 paired bone marrow been obtained. Expression array data were normalized using the GCRMA and peripheral blood samples from the same patient. A total of 39 DNA method (22), and levels of PSMB5 expression in all 240 cell lines were samples were isolated from bone marrow plasma cells, and 40 samples used to perform genotype-phenotype correlation studies. Before per- were isolated from peripheral blood obtained from these patients. All of forming the genetic association studies, Hardy-Weinberg equilibrium these experiments were reviewed and approved by Mayo Clinic was analyzed for each SNP using a stratified Hardy-Weinberg equili- Institutional Review Board. brium test. Because there were significant differences among ethnic Human PSMB1, PSMB2, and PSMB5 gene resequencing. DNA groups in minor allele frequencies for a given SNP, the genotype- samples were used to perform PCR amplifications to resequence genes phenotype association analyses were done by ethnic group for SNPs encoding the three active proteasome h subunits. PCR primer sequences with a minor allele frequency of >5% within that ethnic group. The and amplification conditions are listed in Supplementary Table S1. association of each SNP with expression level was evaluated using a Resequencing was done using dye termination sequencing chemistry as linear model in which SNP genotypes were evaluated with the use of described previously (18–20). Amplicons were sequenced on both two indicators as covariates (e.g., a 2-degree-of-freedom test for each strands in the Mayo Molecular Biology Core Facility with an ABI 377 SNP). In addition to the 2-degree-of-freedom test, a 1-degree-of-freedom DNA sequencer. To exclude PCR-induced artifacts, independent test for trend was also used, with SNP genotypes coded as 0, 1, or 2 amplifications were done for samples in which a SNP was observed according to the number of rare variants present. PSMB5 expression only once or for any sample with an ambiguous chromatogram. The values were analyzed on a log 2 scale, and the data were adjusted for chromatograms were analyzed with Mutation Surveyor (SoftGenetics). gender, race, and the time of cell storage at the Coriell Institute. Transient transfection and expression. The wild-type (WT) cDNA Luciferase reporter gene assays. To create reporter gene constructs, sequence for PSMB5 was cloned into pcDNA3.1/V5-HisTOPO TA to use f500 bp of PSMB5 3¶-flanking region (3¶-FR) sequence was amplified as a template for site-directed mutagenesis done using circular PCR to from either the Coriell or the patient genomic DNA samples. Forward create variant allozyme expression constructs that encoded PSMB5 and reverse primers for these amplifications contained ACC65I and Cys24, Cys212, and Met238. The sequences of primers used to perform XhoI restriction sites, respectively, to make it possible to subclone the site-directed mutagenesis are also listed in Supplementary Table S1. amplicons into pGL-3 Basic (Promega) upstream of the firefly luciferase Sequences of all constructs were confirmed by sequencing the insert in gene open-reading frame. Specifically, reporter gene constructs were both directions. These constructs were transiently transfected into HeLa created by the amplification of a portion of the PSMB5 3¶-FR that cells using the TransFast reagent (Promega) at a charge ratio of 1:1. Cells included either SNPs or a 17-bp insertion using DNA samples that were harvested after 48 h, and proteasomes were isolated and used to contained these polymorphisms, followed by the cloning of amplicons perform proteasome activity assays. into pGL-3 Basic, with or without subsequent site-directed mutagenesis.

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All of these reporter gene constructs were used to transfect HEK293T phism were determined unequivocally, and other haplotypes were and COS-1 cells, and luciferase activity was determined as described inferred computationally as described by Schaid et al. (25). previously (23). All of the primers used to create the reporter gene constructs are also listed in Supplementary Table S1. Results Data analysis. DNA sequence obtained during the gene resequenc- ing studies was compared with PSMB1, PSMB2, and PSMB5 genomic PSMB1, PSMB2, and PSMB5 gene resequencing. PSMB1, and cDNA genomic consensus sequences (PSMB1 NM_144662 and PSMB2, and PSMB5 were resequenced using 240 DNA samples NT_007583.11; PSMB2, NM_002794 and NT_004511.17; and PSMB5, obtained from 60 African American, 60 Caucasian American, D29011 and NT_026437.11). IC and GI values for WT and variant 50 50 60 Han Chinese American, and 60 Mexican American subjects. allozymes, as well as the luciferase activity data for WT and variant constructs, were compared by ANOVA done with the Prism program. The areas resequenced included all exons, exon-intron splice f ¶ Linkage disequilibrium among PSMB1, PSMB2, and PSMB5 poly- junctions, and 1 to 2 kb of 5 -FR for each gene. These rese- morphisms was determined by calculating D¶ values for all possible quencing data have been deposited in the NIH database pairwise combinations of polymorphisms. This method for determin- PharmGKB. Fig. 1 shows the locations of the polymorphisms ing linkage disequilibrium is independent of allele frequency (2, 24). observed, and individual polymorphisms are listed in Haplotypes for alleles that contained only one heterozygous polymor- Table 1. Twenty-six polymorphisms were identified in PSMB1,

Fig. 1. Human PSMB1, PSMB2, and PSMB5 genetic polymorphisms; a schematic representation of the (A) PSMB1,(B) PSMB2 ,and(C) PSMB5 gene structures. Arrows, locations of polymorphisms. Rectangles, exons; black rectangles, open reading frame; open rectangles, untranslated regions. AA, African American subjects; CA, Caucasian American subjects; HCA, Han Chinese American subjects; MA, Mexican American subjects. SNPs identified in PSMB5 for DNA samples from multiple myeloma (MM) patients are also listed. *, SNPs altering encoded amino acids.

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Table 1. Human PSMB1, PSMB2, and PSMB5 genetic polymorphisms

Location Nucleotide Sequence Amino acid Frequency of variant allele change change African Caucasian Han Chinese Mexican American American American American PSMB1 5¶-FR -1199 A!G 0.017 0.000 0.000 0.000 5¶-FR -1043 A!G 0.133 0.308 0.767 0.342 5¶-FR -933 C!G 0.008 0.000 0.000 0.000 5¶-FR -779 A!C 0.000 0.017 0.000 0.000 5¶-FR -767 to -769 GAT deletion 0.000 0.008 0.000 0.008 5¶-FR -696 G!T 0.000 0.000 0.008 0.000 5¶-FR -695 C!T 0.000 0.000 0.000 0.008 5¶-FR -621 A!C 0.008 0.000 0.000 0.000 5¶-FR -573 G!A 0.017 0.000 0.000 0.000 5¶-FR -213 A!C 0.008 0.000 0.000 0.000 5¶-FR -192 G!C 0.000 0.000 0.000 0.008 5¶-FR -126 C!A 0.008 0.050 0.000 0.042 5¶-FR -62 C!T 0.008 0.000 0.000 0.000 5¶-UTR -6 C!T 0.100 0.025 0.008 0.042 Exon 1 31 C!G Pro11Ala 0.300 0.383 0.800 0.417 Exon 1 73 C!T Pro25Ser 0.000 0.000 0.008 0.000 Intron 1 -27 G!A 0.000 0.033 0.000 0.025 Intron 2 49 A!G 0.233 0.550 0.200 0.475 Intron 3 15 A!G 0.008 0.042 0.000 0.042 Intron 3 23 A!G 0.000 0.000 0.000 0.008 Intron 4 35 Deletion of G 0.208 0.000 0.000 0.017 Intron 4 128 T!C 0.042 0.167 0.008 0.308 Intron 4 136 A!G 0.000 0.000 0.000 0.008 Intron 4 154 A!G 0.133 0.000 0.000 0.008 Intron 4 -30 A!G 0.000 0.000 0.017 0.000 3¶-FR 862 C!A 0.008 0.000 0.000 0.000

PSMB2 5¶-FR -1041 A!G 0.033 0.000 0.000 0.000 5¶-FR -976 to -975 Deletion of TA 0.033 0.000 0.000 0.000 5¶-FR -859 T!G 0.000 0.008 0.000 0.000 5¶-FR -826 (TC)n n = 2 0.782 1.000 0.917 0.967 n = 3 0.182 0.000 0.083 0.017 n = 4 0.036 0.000 0.000 0.000 5¶-FR -716 A!G 0.125 0.000 0.008 0.017 5¶-FR -707 to -706 Deletion of CT 0.092 0.000 0.017 0.008 5¶-FR -555 G!A 0.000 0.008 0.000 0.000 5¶-FR -477 G!T 0.192 0.917 0.208 0.492 5¶-FR -425 G!A 0.117 0.000 0.000 0.017 5¶-FR -405 T!C 0.000 0.008 0.000 0.000 5¶-FR -381 G!C 0.000 0.000 0.008 0.000 5¶-FR -224 G!A 0.008 0.000 0.000 0.000 5¶-FR -197 C!A 0.033 0.000 0.000 0.000 5¶-FR -146 Deletion of A 0.025 0.000 0.000 0.000 5¶-UTR -109 Deletion of T 0.017 0.000 0.000 0.000 Intron 1 20 G!T 0.008 0.000 0.000 0.000 Intron 1 43 G!C 0.008 0.000 0.000 0.000 Intron 1 -27 T!C 0.008 0.000 0.000 0.000 Exon 2 133 C!T 0.000 0.000 0.008 0.000 Intron 2 57 A!C 0.008 0.000 0.000 0.000 Intron 2 123 C!G 0.000 0.000 0.000 0.008 Intron 2 145 G!A 0.000 0.008 0.000 0.008 Intron 3 6 T!G 0.000 0.000 0.000 0.017

(Continued on the following page)

including two nonsynonymous cSNPs; 40 polymorphisms, but the subunits (9). The vast majority of these polymorphisms no nonsynonymous SNPs, were identified in PSMB2; and 21 were not available in public databases. For example, 21 of 26 polymorphisms were identified in PSMB5, including three polymorphisms that we identified in PSMB1, 36 of 40 poly- nonsynonymous cSNPs. None of the nonsynonymous cSNPs morphisms in PSMB2, and 14 of 21 in PSMB5—including two changed amino acids located within the catalytic sites for any of novel nonsynonymous cSNPs—were not publicly available.

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Table 1. Human PSMB1, PSMB2, and PSMB5 genetic polymorphisms (Cont’d)

Location Nucleotide Sequence Amino acid Frequency of variant allele change change African Caucasian Han Chinese Mexican American American American American Intron 3 7446 A!G 0.000 0.000 0.000 0.008 Intron 3 7763 to 7766 Deletion of CAGA 0.008 0.000 0.000 0.000 Intron 3 7772 C!G 0.092 0.000 0.017 0.008 Intron 3 7776 C!T 0.092 0.000 0.017 0.008 Intron 3 7937 T!C 0.092 0.000 0.017 0.008 Intron 3 -68 C!T 0.017 0.000 0.000 0.000 Intron 3 -30 T!C 0.008 0.000 0.000 0.000 Intron 4 30 Deletion of G 0.017 0.000 0.000 0.000 Intron 4 65 T!C 0.058 0.000 0.000 0.000 Intron 4 72 T!C 0.125 0.000 0.017 0.008 Intron 4 93 G!A 0.117 0.000 0.008 0.017 Intron 4 -26 T!C 0.000 0.025 0.000 0.008 3¶-UTR 611 A!G 0.092 0.000 0.017 0.008 3¶-UTR 641 T!C 0.033 0.000 0.000 0.000 3¶-UTR 683 G!A 0.000 0.000 0.008 0.000 3¶-UTR 684 C!T 0.092 0.000 0.017 0.008 3¶-FR 758 T!C 0.008 0.000 0.000 0.000

Location Nucleotide Sequence Amino acid Frequency of variant allele change change African Caucasian Han Chinese Mexican Multiple American American American American myeloma samples PSMB5 5¶-FR -693 C!T 0.117 0.658 0.825 0.717 0.538 5¶-FR -534 G!A 0.067 0.000 0.000 0.000 0.013 5¶-FR -438 G!A 0.008 0.000 0.000 0.000 0.000 5¶-FR -384 T!C 0.000 0.000 0.000 0.000 0.000 5-FR -310 T!C 0.158 0.000 0.000 0.000 0.013 5¶-FR -187 C!G 0.008 0.000 0.000 0.000 0.000 5¶-FR -178 to -177 Insertion of C 0.000 0.000 0.042 0.000 0.000 5¶-FR -57 G!T 0.000 0.000 0.000 0.008 0.000 Exon 1 70 C!T Arg24Cys 0.025 0.083 0.000 0.017 0.114 Intron 1 230 A!T 0.000 0.008 0.000 0.000 0.000 Intron 1 269 G!T 0.000 0.000 0.008 0.000 0.000 Intron 1 331 C!G 0.000 0.008 0.000 0.000 0.000 Exon 3 588 G!C 0.000 0.000 0.008 0.000 0.000 Exon 3 635 A!GTyr212Cys 0.000 0.000 0.000 0.008 0.000 Exon 3 712 G!AVal238Met 0.000 0.000 0.008 0.000 0.000 3¶-UTR 821 C!T 0.008 0.000 0.000 0.000 0.000 3¶-UTR 847 G!A 0.075 0.000 0.000 0.000 0.013 3¶-UTR 938 to 939 AT deletion 0.000 0.000 0.008 0.000 0.000 3¶-FR 1042 G!A 0.200 0.683 0.932 0.792 0.608 3¶-FR 1094 G!A 0.000 0.000 0.000 0.000 0.006 3¶-FR 1103 G!A 0.175 0.533 0.825 0.700 0.487 3¶-FR 1209 Insertion of 0.000 0.000 0.000 0.000 0.013 GAGAAGGAGAGAGAGGC 3¶-FR 1131 to 1133 AGT deletion 0.008 0.025 0.000 0.000 0.000

NOTE: Polymorphism locations, alterations in nucleotide and amino acid sequences, and minor allele frequencies for the polymorphisms observed during the gene resequencing studies are listed for each of the four ethnic groups studied. The least common allele in African American samples has been designated as the minor allele. Polymorphisms identified in the PSMB5 gene in multiple myeloma patient samples were also listed. The table also indicates whether the polymorphism is represented in dbSNP. Highlighted and bold-faced SNPs are found in dbSNP. Polymorphisms in exons and UTRs and FRs have been numbered with respect to the ‘‘A’’ in the ‘‘ATG’’ translation initiation codon, with positive numbers located 3- and negative numbers 5- to that position. Nucleotides located within introns are numbered based on their distance from splice junctions, using negative or positive numbers, respectively, for distance to 5¶- and 3¶-splice sites.

Allele frequencies for the nonsynonymous cSNPs differed PSMB5 Val238Met polymorphism was observed only in a Han greatly among ethnic groups (Table 1). For example, the Chinese American subject (Table 1). It should also be noted PSMB5 Arg24Cys polymorphism was present in all ethnic that we arbitrarily selected the least common allele in the groups except Han Chinese American; PSMB5 Try212Cys was African American data as the variant allele but, in several cases, observed only in one Mexican American subject; and the that was the most common allele in other populations. All of

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Table 2. PSMB1, PSMB2, and PSMB5 haplotypes

PSMB1

Observed Haplotype African Caucasian Han Chinese Mexican 5¶-FR 5¶-FR 5¶-FR 5¶-FR 5¶-FR 5¶UTR or inferred designation American American American American (-1199) (-1043) (-779) (-573) (-126) (-6) o *1A 0.184 A A A G C C o *1B 0.183 0.386 0.192 0.167 A A A G C C o *1C 0.117 A A A G C C o *1D 0.067 A A A G C C i *1E 0.083 0.025 0.033 A A A G C T i *1F 0.033 0.156 0.300 A A A G C C i *1G 0.033 0.025 A A A G A C o *2A 0.150 0.050 0.025 0.050 A A A G C C o *2B 0.116 0.300 0.733 0.333 A G AG C C i *2C 0.017 G AAA CC i *2D 0.017 A A C GC C i *2E 0.014 0.025 A A A G C C i *2F 0.017 A G AG C C i *2 and *3 0.008 A A A G C C

PSMB2

Observed Haplotype African Caucasian Han Chinese Mexican 5¶-FR 5¶-FR 5¶-FR 5¶-FR 5¶-FR 5-FR or inferred designation American American American American (-1041) (-826) (-716) (-707) (-477) (-425)

(TC)n o *1A 0.316 0.075 0.725 0.450 A 2 A I G G o *1B 0.123 0.867 0.158 0.467 A 2 A I T G i *1C 0.091 0.025 A 3 A I G G o *1D 0.083 A 2 G IG G o *1E 0.075 0.017 A 2 A I G A o *1F 0.050 A 2 A I G G i *1G 0.026 0.017 A 3 A D GG i *1H 0.025 A 3 A DT G o *1I 0.018 A 4 A I G G i *1J 0.017 G 2AIG G i *1K 0.017 A 3 A D G A i *1L 0.014 A 2 G IG G i *1M 0.014 A 2 G IG G o *1N 0.025 A 2 A I T G o *1O 0.042 A 3 A I T G i *1P 0.017 A 2 G IG G

PSMB5

Observed Haplotype African Caucasian Han Chinese Mexican 5¶-FR 5¶-FR 5¶-FR 5¶-FR Exon 1 Intron 1 or inferred designation American American American American (-693) (-534) (-310) (-178) (70) *2 (269) o *1A 0.537 0.081 0.067 0.166 C G T D C G o *1B 0.139 C G C DC G o *1C 0.108 0.489 0.761 0.683 T GTDC G i *1D 0.065 C A TD C G o *1E 0.057 0.020 C G T D C G o *1F 0.025 0.142 0.105 0.083 C G T D C G o *1G 0.025 T GTDC G o *1H 0.144 0.025 T GTDC G o *1I 0.039 T GTI CG i *2A 0.022 0.075 0.008 C G T D T G i *2B 0.003 C G C D T G i *2C 0.008 0.008 C G T D T G i *3A 0.008 T GTDC G i *4A 0.008 T GTDC T

NOTE: Nucleotide positions are numbered as described in Table 1. Variant nucleotides compared to the reference sequence, that is, the most common sequence in African American subjects, are highlighted as white on black. Initial haplotype designations (*1, *2, *3, etc.) were made on the basis of amino acids that vary, with the WT sequence designated *1. Subsequent assignments (letter designations) were made within ethnic groups, based on decreasing frequencies. o, unequivocal haplotypes; i, inferred haplotypes; I, insertion; D, deletion. the polymorphisms identified during the resequencing effort adjusted for the number of alleles studied. Two standard were in Hardy-Weinberg equilibrium (P > 0.05). measures of nucleotide diversity are p, average heterozygosity We also used our gene resequencing data to calculate per site, and u, a population mutation measure that is nucleotide diversity, a quantitative measure of genetic variation, theoretically equal to the neutral mutation parameter (26).

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Table 2. PSMB1, PSMB2, and PSMB5 haplotypes (Cont’d)

PSMB1

Exon 1 Exon 1 Intron 1 Intron 2 Intron 3 Intron 4 Intron 4 Intron 4 Intron 4 (31) *2 (73) *3 (-27) (49) (15) (35) (128) (154) (-30) CC G A A I T A A CC G G AITAA CC G A A D T G A CC G A A D TAA CC G A A I T A A CC G G AIC AA CC G A G ITAA G CGAA I TAA G CGAA I TAA G CGAA I TAA G C A AA I TAA G C A AA I TAA G CG A A I T A G GT GAA I TAA

PSMB2

5¶-FR 5¶-FR 5¶-UTR Intron Intron 3 Intron 3 Intron 3 Intron Intron Intron Intron Intron 3¶-UTR 3¶-UTR 3¶-UTR (-197) (-146) (-109) 3 (6) (7772) (7776) (7937) 4 (30) 4 (65) 4 (72) 4 (93) 4 (-26) (611) (641) (684)

CI IT C C T ITTGTATC CI IT C C T ITTGTATC CI IT C C T ITTGTATC CI IT C C T ITTA TATC CI IT C C T ITTGTATC CI IT C C T IC TG T ATC CI IT GTCITC GTG T T CI IT GTCITC GTG T T CI IT C C T ITTGTATC AD IT C C T ITC GTAC C CI IT GTCITC GTG T T CID TC C T ITTA TATC CI IT C C T D TTA TATC CI IT C C T ITTGC ATC CI IT C C T ITTGTATC CI IG CCTITTA TATC

PSMB5

Exon 3 (635) *4 Exon 3 (712) *5 3¶UTR (847) 3¶-FR (1042) 3¶-FR (1103) 3¶-FR (1131)

AGGGGI AGGGGI AGGAA I AGA GG I AGGAA I AGGA GI AGGAAD AGGGGI AGGAA I AGGGGI AGGGGI AGGA GI G GGAA I A A G AA I

These values for PSMB1, PSMB2, and PSMB5 are listed in population (Supplementary Table S2). Under conditions of Supplementary Table S2. For all three genes, DNA from African neutrality, Tajima’s D should equal zero; none of these values American subjects showed greater apparent diversity in differed significantly from zero in any of the populations sequence than DNA obtained from other ethnic groups, studied. probably reflecting the greater antiquity of these sequences PSMB1, PSMB2, and PSMB5 haplotype and linkage disequi- (27). In addition, values for Tajima’s D, a test of the neutral librium analyses. Haplotype and pairwise linkage disequilib- mutation hypothesis (28), were also estimated for each rium analyses were done for all three genes because it is

www.aacrjournals.org 3509 Clin Cancer Res 2008;14(11) June 1, 2008 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. CancerTherapy: Preclinical becoming increasingly clear that the determination of haplo- Proteasome activity and inhibitor studies. Because the h5 type may be more helpful than the assay of individual SNPs for subunit is the major therapeutic target for proteasome use in association studies (29). A total of 14 haplotypes were inhibitors used in the clinic (10, 11), and because three identified for PSMB1, including 6 observed and 8 inferred nonsynonymous cSNPs were observed in that gene with minor haplotypes; 16 haplotypes were identified in PSMB2, including allele frequencies that differed among ethnic groups, functional 8 observed and 8 inferred; and there were 14 PSMB5 characterization of those three nonsynonymous cSNPs was haplotypes, 8 of which were observed (Table 2). Haplotype done after transient transfections of HeLa cells. HeLa cells have frequencies also differed among ethnic groups. African Amer- been used extensively to study proteasome activity (21, 30). In ican subjects had more unique haplotypes than other ethnic addition, lymphoblastoid cell lines that naturally expressed groups. For example, four unique PSMB1 haplotypes were these nonsynonymous cSNPs, either homozygous or heterozy- observed for African American subjects, including one that was gous, were also used to perform functional studies. Specifically, the most common haplotype in this ethnic group, with a proteasomes isolated from HeLa cells transfected with four frequency of f18%; however, that haplotype was not observed different constructs and from lymphoblastoid cell lines were in other ethnic groups (Table 2). Similar observations were used to perform assays with the fluorogenic substrate Suc-LLVY- made for PSMB5, with African American subjects having three amc to measure the chymotrypsin-like activity of the h5 haplotypes that were unique when compared with the other subunit. There were no significant differences among the four ethnic groups (Table 2). Haplotype designations were assigned allozymes (WT plus three variants) studied with regard to levels based on the amino acid sequences of the encoded allozyme, of enzyme activity with either type of cell extract (data not with the WT amino acid sequence designated as *1. Subsequent shown). number designations referred to variant haplotypes that Because the h5 subunit is the target for clinically used contained nonsynonymous cSNPs, beginning at the NH2 proteasome inhibitors, we also did inhibition studies using a terminus and proceeding to the COOH terminus of the specific h5 proteasome inhibitor, MG262. Those experiments encoded protein. Letters were then added for haplotypes that were done with proteasomes isolated from HeLa cells included variant nucleotides that did not alter amino acid transiently transfected with constructs for PSMB5 variant sequence, ranked from most common to least common. allozymes, as well as proteasomes isolated from lymphoblas- Linkage disequilibrium analysis was also done for all pairwise toid cells that naturally expressed these allozymes. Because the combinations of SNPs in these three genes by calculating D¶ PSMB5 WT gene sequence for the African American samples values (2, 24). D¶ values can range from +1.0, when two was different from that for the other three ethnic groups, we polymorphisms are maximally associated, to zero, when they used two WT samples, one for African American and the other are randomly associated. However, no clearly defined haplo- for the remaining ethnic groups. Concentrations of MG262 that type blocks were observed in any of these relatively short genes ranged from 10 to 60 nmol/L were tested, but IC50 values did when those data were displayed graphically (data not shown). not differ significantly among proteasomes isolated from either

Fig. 2. MG262 inhibition studies and cytotoxicity studies. MG262 was used to perform (A) inhibition studies with cell extracts from HeLa cells transfected with expression constructs for PSMB5 WTand variant allozymes, (B) inhibition studies with lymphoblastoid cell extracts from cells expressing WTand variant PSMB5 allozymes, and (C) cytotoxicity studies with lymphoblastoid cells expressing WTand variant allozymes. Columns, mean for three independent determinations; bars, SE.

Clin Cancer Res 2008;14(11) June 1, 2008 3510 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Proteosome Pharmacogenomics transfected cells or from cells that naturally expressed these expression using seven SNPs with minor allele frequencies allozymes (Fig. 2A and B). >5% in at least one ethnic group. One SNP at position 1042 in Proteasome inhibitors are used clinically because of their the PSMB5 3¶-FR showed a significant correlation with level of cytotoxic properties; therefore, we also did MG262 cytotoxicity expression for all cell lines, with G at that position being studies with the lymphoblastoid cell lines from which the associated with elevated expression (Fig. 3B). However, this proteasomes with known PSMB5 genotypes had been isolated. association was statistically significant only in CA subjects, GI50 values calculated from the cytotoxicity data also failed to perhaps because of differences in allele frequencies among show significant differences among cells expressing these ethnic groups, with P = 0.00038 for normalized data adjusted allozymes (Fig. 2C). All of these functional genomic results for covariates. The P value for CA subjects remained significant indicated that the three nonsynonymous PSMB5 cSNPs did not even after correction for multiple comparisons (P = 0.012). The seem to have significant effects on proteasome enzyme activity, G nucleotide at position 1042 was the WT sequence in the inhibition by MG262, or MG262-induced cytotoxicity. How- African American population, but G was the variant sequence ever, a h5 proteasome inhibitor, bortezomib, has been in the other three populations studied (see Table 1). This approved by the Food and Drug Administration to treat polymorphism was located very close to the area of PSMB5 that refractory multiple myeloma. To determine whether there shares >70% sequence identity with the mouse gene sequence. might be additional sequence variation in the PSMB5 gene in Therefore, we created luciferase constructs that contained the patients with multiple myeloma that might influence response 1042 SNP as well as the unique 3¶-FR polymorphisms identified to proteasome inhibitor therapy, we also resequenced PSMB5 in the patient samples to test their possible effect on using 79 DNA samples obtained from 61 patients with multiple transcription. myeloma who had been treated with bortezomib. Those Luciferase reporter gene assays. Luciferase reporter gene samples included both DNA isolated from bone marrow constructs were created; these constructs contained the two myeloma cells and from peripheral blood. novel 3¶-FR polymorphisms that we had identified in samples PSMB5 resequencing using DNA from multiple myeloma from patients with multiple myeloma as well as the SNP at patients. The same primers that had been used to amplify nucleotide 1042 that was associated with level of mRNA PSMB5 from Coriell Institute DNA samples were used to expression in the lymphoblastoid cell lines. WT and these three amplify the gene using DNA from multiple myeloma patients. variant constructs were transiently transfected into HEK293T A total of 9 PSMB5 polymorphisms were identified in these and COS-1 cells with Renilla luciferase (Promega) as a control 79 DNA samples (Table 1; Fig. 1). When we compared the for transfection efficiency. Patterns of luciferase activity for resequencing results for 11 individuals for whom we had two variant constructs were very similar in the two cell lines studied DNA samples, one from bone marrow myeloma cells and the (Fig. 4A and B). Luciferase activity was significantly elevated in other isolated from peripheral blood, no differences in PSMB5 both cell lines after transfection with the construct containing a sequence were observed between the two sources of DNA. G at position 1042 (P < 0.001), consistent with the results of However, we observed novel PSMB5 polymorphisms in these the genotype-phenotype correlation study in the lymphoblas- DNA samples, including a 3¶-FR 1094 G/A polymorphism and toid cell lines. These results were compatible with the an insertion of GAGAAGGAGAGAGAGGC at nucleotide 1209, conclusion that the SNP at nucleotide 1042 can influence also located in the 3¶-FR of the gene, neither of which had been PSMB5 expression. The two polymorphisms identified in identified in the 240 Coriell DNA samples that we had samples from patients with multiple myeloma, 1094A and resequenced (Table 1). The 1094 G/A polymorphism was the insertion at position 1209, showed slightly decreased present in one DNA sample extracted from bone marrow activity in both cell lines when compared with the WT sequence myeloma cells, and the nucleotide 1209 insertion was present (Fig. 4A and B); however, those differences were not statistically in peripheral blood DNA obtained from the two patients. Both significant. of these novel polymorphisms were located in an area that shared 73% sequence identity with the mouse gene, indicating Discussion that it might be of functional significance. Therefore, we created luciferase reporter gene constructs that contained these two The proteasome plays a major role in the degradation of polymorphisms to test their possible effect on the regulation of proteins that regulate critical cellular processes and it is also the transcription. We also attempted to determine whether a target for antineoplastic proteasome inhibitors such as borte- genotype-phenotype correlation might exist among SNPs in zomib (1, 3, 4, 10, 31). Because of the importance of the the PSMB5 gene and clinical response to bortezomib therapy of proteasome and because it is a drug target, we set out to multiple myeloma as the phenotype. However, perhaps determine whether sequence variation in genes encoding the because of the relatively small number of samples, no three active proteasome h subunits might influence proteasome significant associations were observed. activity or expression. This sequence variation might also PSMB5 genotype-phenotype correlation for expression. To contribute to individual variation in disease pathophysiology determine whether any of the SNPs that we had identified in or response to drug therapy. The proteasome inhibitor PSMB5 might be associated with the level of , bortezomib is used to treat refractory multiple myeloma and RNA isolated from the same 240 lymphoblastoid cells from non–small cell lung cancer. There are large variations in which DNA had been isolated to perform the gene resequenc- response to this drug (12–14). For example, only f35% to ing was used to perform expression array studies using 50% of multiple myeloma patients respond to bortezomib Affymetrix U133 Plus 2.0 GeneChips. PSMB5 expression varied therapy (32). Obviously, multiple factors can influence drug f2-fold among these 240 cell lines (Fig. 3A). We then response, including genetic factors, age, gender, and environ- correlated PSMB5 genotype in these cells with level of mental factors. In the treatment of cancer, both tumor DNA

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lines. That study identified one SNP, a polymorphic nucleotide at position 1042 in the 3¶-FR of PSMB5 (near a region conserved in the mouse) that was significantly associated with level of expression in the lymphoblastoid cell lines (Fig. 3B). These genotype-phenotype association results were supported by data from reporter gene assays (Fig. 4). Finally, because patients with refractory multiple myeloma are treated with bortezomib (13, 33), we also resequenced PSMB5 using 79 DNA samples from both tumor cells and peripheral blood obtained from patients with multiple myeloma who had been treated with this drug. We were not able to detect significant associations between SNPs in PSMB5 and response to bortezomib therapy in this group of patients, although these results must be viewed as preliminary because of the relatively small number of samples studied and the heterogeneous nature of this disease. However, resequencing of these 79 patient DNA samples resulted in the identification of two additional novel PSMB5 polymorphisms, both located in the 3¶-FR of the gene in the same conserved region where the functionally significant 1042 SNP was located. Therefore, we created luciferase reporter gene constructs to test the possible implications for transcription of these two novel polymor- phisms together with the nucleotide 1042 SNP. As anticipated on the basis of the genotype-phenotype correlation analysis done with the lymphoblastoid cell lines, a G at nucleotide 1042 significantly increased luciferase activity (Fig. 4). Although the Fig. 3. PSMB5 expression and association with the A1042G SNP in 240 1094 SNP and the insertion at nucleotide 1209 that we had lymphoblastoid cell lines. A, PSMB5 expression in 240 lymphoblastoid cells. identified by resequencing DNA samples from the patients PSMB5 expression was assayed with Affymetrix U133 Plus2 GeneChips. showed slightly decreased luciferase activity when compared Expression levels were normalized with GCRMA. Columns, individual cell line; colors, ethnic groups. B, PSMB5 A1042G SNP genotypes were correlated with corresponding normalized expression array data for each of the 240 cell lines shown in A. Columns, mean; bars SE. and germ line DNA are important for explaining variation in response to drug therapy. Therefore, we resequenced the genes encoding the three active proteasome h subunits using DNA samples from four ethnic groups and characterized the functional effects of sequence variation in PSMB5, the gene encoding the h5 subunit, the major clinical target for proteasome inhibitors (10, 31). We identified a series of SNPs in genes encoding the three active proteasome h subunits (Table 1), and the majority of these SNPs were not available in any public database. The h5 subunit of the proteasome has chymotryptic-like protease activity, and this subunit is the target for clinical proteasome inhibitors such as bortezomib (9, 10, 13, 31, 33). Therefore, we focused our functional genomic studies, including activity assays, inhibition studies, and cytotoxicity studies done with the specific proteasome inhibitor, MG262, on three non- synonymous cSNPs in the PSMB5 gene (Fig. 2A-C). All of these results were compatible with the conclusion that these non- synonymous cSNPs in PSMB5 did not significantly affect proteasome function. Obviously, we cannot rule out the possibility that these polymorphisms might alter the of specific substrates, including endogenous substrates, or the response to inhibitors other than MG262. However, genetic polymorphisms can also influence transcription, RNA splicing, Fig. 4. PSMB5 3¶-FR luciferase reporter gene studies. Reporter gene studies and mRNA stability. Therefore, we next turned our attention to were done with (A)COS-1and(B) HEK293Tcells transfected with reporter gene transcription. Specifically, we did genotype-phenotype correla- constructs containingWTsequence as well as variant sequence at nucleotides 1042 and 1094 and the insertion at position 1209. Luciferase activity was corrected for tion analyses using expression array data (Fig. 3A) and our Renilla luciferase activity and is expressed as a percent of theWTactivity. Columns, PSMB5 gene resequencing data for all 240 lymphoblastoid cell mean for three independent experiments; bars, SE.

ClinCancerRes2008;14(11)June1,2008 3512 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Proteosome Pharmacogenomics with the WT sequence, those differences were not statistically variation in these important genes and represent a step toward significant (Fig. 4). future translational pharmacogenomic studies of patients In summary, we have done a comprehensive series of studies treated with proteasome inhibitors. of the pharmacogenomics of genes encoding the three active proteasome subunits. Those experiments resulted in the identification of a large number of novel SNPs and haplotypes Disclosure of Potential Conflicts of Interest in both control subjects and patients with multiple myeloma No potential conflicts of interest were disclosed. that were not represented in the HapMap or other public databases. Functional characterization of nonsynonymous cSNPs and SNPs in the 3¶-FR of the PSMB5 gene showed that Acknowledgments the common nucleotide 1042 SNP in the 3¶-FR of the gene significantly increased transcription, confirming the results of We thank Luanne Wussow for her assistance with the preparation of the manuscript. genotype-phenotype correlation studies between SNPs and The PSMB1, PSMB2, and PSMB5 gene sequence data reported in this article PSMB5 expression in 240 lymphoblastoid cell lines. These have been deposited in the NIH-supported database PharmGKB with accession results significantly increase our knowledge of common genetic numbers PS205879, PS205881,and PS205880.

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Liewei Wang, Shaji Kumar, Brooke L. Fridley, et al.

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