Oreste E. Salavaggione

Gemcitabine Pharmacogenomics: Cytidine Deaminase and Deoxycytidylate Deaminase Gene Resequencing and Functional Genomics

Purpose: Gemcitabine is a nucleoside analogue with activity against solid tumors. Gemcitabine metabolic inactivation is catalyzed by cytidine deaminase (CDA) or, after phosphorylation, by deoxycytidylate deaminase (DCTD). We set out to study the pharmacogenomics of CDA and DCTD. Experimental Design: The genes encoding CDA and DCTD were resequenced using DNA from 60 African American and 60 Caucasian American subjects. Expression constructs were created for nonsynonymous coding single nucleotide polymorphisms (cSNP) and reporter gene constructs were created for 5′-flanking region polymorphisms. Functional genomic studies were then conducted after the transfection of mammalian cells. Results:CDA resequencing revealed 17 polymorphisms, including one common nonsynonymous cSNP, 79 A>C (Lys27Gln). Recombinant Gln27 CDA had 66 ± 5.1% (mean ± SE) of the wild-type (WT) activity for gemcitabine but without a significant decrease in level of immunoreactive protein. The apparent Km (397 ± 40 μmol/L) for the Gln27 allozyme was significantly higher than that for the WT (289 ± 20 μmol/L; P < 0.025). CDA 5′-flanking region reporter gene studies showed significant differences among 5′-flanking region haplotypes in their ability to drive transcription. There were 29 SNPs in DCTD, including one nonsynonymous cSNP, 172 A>G (Asn58Asp), in Caucasian American DNA. Recombinant Asp58 DCTD had 11 ± 1.4% of WT activity for gemcitabine monophosphate with a significantly elevated level of immunoreactive protein. No DCTD polymorphisms were observed in the initial 500 bp of the 5′-flanking region. Conclusions: These results suggest that pharmacogenomic variation in the deamination of gemcitabine and its monophosphate might contribute to variation in therapeutic response to this antineoplastic agent.

GLUTATHIONE S-TRANSFERASE OMEGA 1 AND OMEGA 2 PHARMACOGENOMICS

Glutathione S-transferase omega 1 and omega 2 (GSTO1 and GSTO2) catalyze monomethyl arsenate reduction, the rate-limiting reaction in arsenic biotransformation. As a step toward pharmacogenomic studies of these phase II enzymes, we resequenced human GSTO1 and GSTO2 using DNA samples from four ethnic groups. We identified 31 and 66 polymorphisms in GSTO1 and GSTO2, respectively, with four nonsynonymous-coding single nucleotide polymorphisms (cSNPs) in each gene. There were striking variations among ethnic groups in polymorphism frequencies and types. Expression constructs were created for all eight nonsynonymous cSNPs, as well as a deletion of codon 155 in GSTO1, and those constructs were used to transfect COS-1 cells. Quantitative Western blot analysis, after correction for transfection efficiency, showed a reduction in protein level of greater than 50% for the GSTO1 Tyr32 variant allozyme compared with wild type (WT), whereas levels for the Asp140, Lys208, Val236, and codon 155 deletion variant constructs were similar to that of the WT. For GSTO2, the Tyr130 and Ile158 variant allozymes showed 50 and 84% reductions in levels of expression, respectively, compared with WT, whereas the Ile41 and Asp142 allozymes displayed levels similar to that of WT GSTO2. Rabbit reticulocyte lysate degradation studies showed that the GSTO1 Tyr32 and the GSTO2 Tyr130, Ile158, and Asp142/Ile158 variant allozymes were degraded more rapidly than were their respective WT allozymes. These observations raise the possibility of functionally significant pharmacogenomic variation in the expression and function of GSTO1 and GSTO2.

Glutathione S-Transferase T1 and M1: Gene Sequence Variation and Functional Genomics

Purpose: The glutathione S-transferases (GSTs) catalyze the glutathione conjugation of reactive electrophiles, including carcinogens and many antineoplastic drugs. GSTT1 and GSTM1 are polymorphically deleted, but the full range of genetic variation in these two genes has not yet been explored. We set out to systematically identify common polymorphisms in GSTT1 and GSTM1, followed by functional genomic studies. Experimental Design: First, multiplex PCR was used to determine GSTT1 and GSTM1 copy number in 400 DNA samples (100 each from 4 ethnic groups). Exons, splice junctions, and 5′-flanking regions (5′-FR) were then resequenced using DNA samples that contained at least one copy of GSTT1 or GSTM1. Results: Gene deletion frequencies among ethnic groups were from 33.5% to 73.5% for GSTT1 and from 50.5% to 78.0% for GSTM1. GSTT1 deletion data correlated with the results of mRNA microarray expression studies. The 18 single nucleotide polymorphisms (SNP) observed in GSTT1 included three nonsynonymous coding SNPs (cSNPs) and one single-nucleotide deletion, whereas the 51 GSTM1 SNPs included two nonsynonymous cSNPs. Two of the GSTT1 nonsynonymous cSNPs resulted in decreases in levels of immunoreactive protein to 56% and 12% of wild type (WT), whereas those in GSTM1 resulted in modest increases in protein levels. Reporter gene assays showed that one GSTT1 5′-FR haplotype, with a frequency of 32% in African-American subjects, resulted in an increase in transcription in JEG-3 cells to 351% of that for the WT sequence, and one GSTM1 5′-FR haplotype resulted in an increase in transcription in JEG-3 cells to 129% of WT. Conclusions: These observations suggest that functionally significant pharmacogenomic variation beyond GSTT1 and GSTM1 gene deletion may contribute to carcinogenesis or individual variation in antineoplastic drug therapy response.

Human phenylethanolamine N-methyltransferase pharmacogenomics: gene re-sequencing and functional genomics

Phenylethanolamine N‐methyltransferase (PNMT, EC2.1.1.28) catalyzes the N‐methylation of norepinephrine to form epinephrine. As a step toward understanding the possible contribution of inheritance to individual variation in PNMT‐catalyzed epinephrine formation, we ‘re‐sequenced’ the entire human PNMT gene, including the three exons, the introns and approximately 1 kb of the 5′‐flanking region (5′‐FR), using DNA samples from 60 African‐American (AA) and 60 Caucasian‐American (CA) subjects. Within the 3.5 kb re‐sequenced, 18 single nucleotide polymorphisms (SNPs) were observed, including four non‐synonymous coding SNPs (cSNPs) that resulted in the following alterations in encoded amino acid sequence: Asn9Ser, Thr98Ala, Arg112Cys and Ala175Thr. When constructs for the non‐synonymous cSNPs were transiently expressed in COS‐1 cells, the Ala98 allozyme displayed significantly lower levels of both activity and immunoreactive protein (p < 0.002) than did the wild‐type (WT) enzyme due, at least in part, to accelerated protein degradation by a proteasome‐mediated process. Luciferase reporter gene constructs were also created for the six common PNMT 5′‐FR haplotypes observed. Significant differences were observed among haplotypes in their ability to drive transcription. These observations raise the possibility of inherited variation in the ability to form epinephrine from norepinephrine as a result of variant PNMT polymorphisms and haplotypes.

Human Hydroxysteroid Sulfotransferase SULT2B1 Pharmacogenomics: Gene Sequence Variation and Functional Genomics

The human hydroxysteroid sulfotransferase (SULT) 2B1 gene is a member of the cytosolic SULT gene superfamily. The two SULT2B1 isoforms, SULT2B1a and SULT2B1b, are encoded by a single gene as a result of alternative transcription initiation and alternative splicing. SULT2B1b catalyzes the sulfonation of 3β-hydroxysteroid hormones and cholesterol, whereas SULT2B1a preferentially catalyzes pregnenolone sulfonation. We used a genotype-to-phenotype approach to identify and characterize common sequence variation in SULT2B1. Specifically, we resequenced all exons, splice junctions, and ∼2.5 kb of the 5′-flanking regions (FRs) for each isoform using 60 DNA samples each from African-American and Caucasian-American subjects. We observed 100 polymorphisms, including four nonsynonymous coding single nucleotide polymorphisms and one 6-base pair deletion—all within the “shared” region of the open reading frame. Functional genomic studies of the wild type (WT) and five variant allozymes for each isoform performed with a mammalian expression system showed that variant allozyme activities ranged from 64 to 88% of WT for SULT2B1a and from 76 to 98% for SULT2B1b. Relative levels of immunoreactive protein were similar to those for enzyme activity. Luciferase reporter gene constructs for 2.5 kb of the SULT2B1b 5′-FR displayed a cell line-dependent pattern of variation in activity. Finally, deletion of the proline-rich SULT2B1 carboxyl terminus resulted in intracellular protein aggregate formation and accelerated degradation of the truncated protein. These studies resulted in the identification of common SULT2B1 gene sequence variation, as well as insight into the effects of that variation on the function of this important steroid-metabolizing enzyme.

Gemcitabine Pharmacogenomics: Deoxycytidine Kinase and Cytidylate Kinase Gene Resequencing and Functional Genomics

Gemcitabine and other cytidine antimetabolites require metabolic activation by phosphorylation. Deoxycytidine kinase (DCK) and cytidine monophosphate kinase (CMPK) catalyze these reactions. We have applied a genotype-to-phenotype strategy to study DCK and CMPK pharmacogenomics. Specifically, we resequenced DCK and CMPK using 240 DNA samples, 60 each from African-American, Caucasian-American, Han Chinese-American, and Mexican-American subjects. We observed 28 DCK polymorphisms and 28 polymorphisms in CMPK, 33 of which were novel. Expression in COS-1 cells showed that variant allozyme enzyme activities ranged from 32 to 105% of the wild type (WT) for DCK and from 78 to 112% of WT for CMPK—with no significant differences in apparent Km values for either enzyme except for a DCK Val24/Ser122 double variant allozyme. Relative levels of DCK and CMPK immunoreactive protein in the COS-1 cells paralleled relative levels of enzyme activity and were significantly correlated for DCK (Rp = 0.89, P = 0.0004) but not for CMPK (Rp = 0.82, P = 0.095). The results of an analysis of DCK and CMPK structural models were compatible with the observed functional consequences of sequence alterations in variant allozymes. We also confirmed that the CMPK protein expressed in COS-1 cells and in a rabbit reticulocyte lysate was 196 rather than 228 amino acids in length. In summary, we determined common sequence variations in DCK and CMPK and systematically evaluated their functional implications. These gene sequence differences may contribute to variations in the metabolic activation of gemcitabine and other cytidine antimetabolites.

Structural Basis of Substrate Recognition in Thiopurine S-Methyltransferase

Thiopurine S-methyltransferase (TPMT) modulates the cytotoxic effects of thiopurine prodrugs such as 6-mercaptopurine by methylating them in a reaction using S-adenosyl- l-methionine as the donor. Patients with TPMT variant allozymes exhibit diminished levels of protein and/or enzyme activity and are at risk for thiopurine drug-induced toxicity. We have determined two crystal structures of murine TPMT, as a binary complex with the product S-adenosyl- l-homocysteine and as a ternary complex with S-adenosyl- l-homocysteine and the substrate 6-mercaptopurine, to 1.8 and 2.0 A resolution, respectively. Comparison of the structures reveals that an active site loop becomes ordered upon 6-mercaptopurine binding. The positions of the two ligands are consistent with the expected S N2 reaction mechanism. Arg147 and Arg221, the only polar amino acids near 6-mercaptopurine, are highlighted as possible participants in substrate deprotonation. To probe whether these residues are important for catalysis, point mutants were prepared in the human enzyme. Substitution of Arg152 (Arg147 in murine TPMT) with glutamic acid decreases V max and increases K m for 6-mercaptopurine but not K m for S-adenosyl- l-methionine. Substitution at this position with alanine or histidine and similar substitutions of Arg226 (Arg221 in murine TPMT) result in no effect on enzyme activity. The double mutant Arg152Ala/Arg226Ala exhibits a decreased V max and increased K m for 6-mercaptopurine. These observations suggest that either Arg152 or Arg226 may participate in some fashion in the TPMT reaction, with one residue compensating when the other is altered, and that Arg152 may interact with substrate more directly than Arg226, consistent with observations in the murine TPMT crystal structure.

Improved Response to nab-Paclitaxel Compared with Cremophor-Solubilized Paclitaxel is Independent of Secreted Protein Acidic and Rich in Cysteine Expression in Non-Small Cell Lung Cancer

Background: The secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that is produced by tumor and/or neighboring stroma. SPARC expression is thought to facilitate the intracellular accumulation of nanoparticle albumin-bound paclitaxel (nab-paclitaxel, abraxane [ABX]). Gene hypermethylation is a common mechanism for loss of SPARC expression in non-small cell lung cancer (NSCLC). We aim to demonstrate the role of SPARC expression as biomarker for treatment selection using ABX in NSCLC and to evaluate the presence of synergistic antitumor effect when a demethylating agent is combined with ABX. Methods: We analyzed the SPARC messenger RNA expression and SPARC gene methylation status in 13 NSCLC cell lines and 22 minimally passaged patient-derived (PD) NSCLC tumors using real-time (RT) polymerase chain reaction. The effect of ABX on tumor growth was compared with cremophor-solubilized paclitaxel (taxol) in severe combined immunodeficiency mice bearing SPARC-positive PD xenografts. The effect of pretreatment with a demethylating agent, 5-Aza-2′-deoxycytidine (DEC) in SPARC-negative tumors was assessed. Results: SPARC expression was weak to absent in 62% of established NSCLC cell lines and 68% of PD NSCLC tumor xenografts. SPARC expression could be up-regulated/restored by DEC treatment in both SPARC-negative cell lines and PD xenografts in vitro and in vivo. ABX demonstrated better antitumor efficacy than equitoxic dose of taxol in SPARC-expressing xenografts and some SPARC-negative xenografts. At equimolar doses in vitro, there was similar increased cytotoxicity on DEC pretreatment with either ABX or taxol in SPARC-negative cell lines. At equitoxic doses, there was similar additive antitumor activity of DEC with either ABX or taxol in SPARC-negative PD xenografts. Conclusion: Endogenous SPARC status is somewhat uncorrelated with response to ABX in NSCLC. The greater antitumor effect of ABX compared with equitoxic dose of taxol observed in SPARC-expressing NSCLC tumors can also be seen in some SPARC-negative tumors. DEC pretreatment similarly enhanced antitumor activity with either ABX or taxol in SPARC-negative tumors.

Canine red blood cell thiopurine S-methyltransferase: companion animal pharmacogenetics.

Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurine drugs. In humans, a common genetic polymorphism for TPMT is a major factor responsible for individual variation in the toxicity and therapeutic efficacy of these drugs. Dogs (Canis familiaris) are also treated with thiopurine drugs and, similar to humans, they display large individual variations in thiopurine toxicity and efficacy. We set out to determine whether dogs might also display genetically determined variation in TPMT activity. As a first step, we observed that canine red blood cell (RBC) TPMT activity in samples from 145 dogs varied over a nine-fold range. That variation was not associated with either the age or sex of the animal. Subsequently, we cloned the canine TPMT cDNA and gene. The canine cDNA encoded a protein that was 81.2% identical to the enzyme encoded by the most common TPMT allele in humans. A genotype-phenotype correlation analysis was performed by resequencing the canine gene using DNA samples from 39 animals selected for high, low or intermediate levels of RBC TPMT activity. We observed nine polymorphisms in these 39 DNA samples, including three insertion/deletion events and six single nucleotide polymorphisms (SNPs), one of which was a nonsynonymous cSNP (Arg97Gln). However, when the variant allozyme at codon 97 was expressed in COS-1 cells, it did not display significant differences in either basal levels of TPMT activity or in substrate kinetics compared with the wild-type allozyme. Six of the nine canine TPMT polymorphisms were associated with 67% of the variation in level of RBC TPMT activity in these 39 blood samples. When those six SNPs were assayed using DNA from all 145 animals studied, 40% of the phenotypic variance in the entire population sample could be explained by these polymorphisms. Therefore, inheritance is a major factor involved in the regulation of variation in RBC TPMT in the dog, just as it is in humans. These observations represent a step towards the application of pharmacogenetic and pharmacogenomic principles to companion animal drug therapy.

Thiopurine methyltransferase activity in red blood cells of dogs

Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of thiopurine medications such as azathioprine. In humans, activity varies widely among individuals, primarily because of genetic polymorphisms. Low TPMT activity increases the risk of myelosuppression from azathioprine and 6-mercaptopurine, whereas high TPMT activity is associated with poor drug efficacy. The purpose of this study was to determine whether dogs also show a wide range of TPMT activity. Heparinized blood samples were obtained from 177 dogs associated with a veterinary teaching hospital. Red blood cell (RBC) TPMT activity was measured by means of a modification of a radiochemical method as established for use in people. TPMT activity varied across a 9-fold range (7.9-71.8 U of RBC per milliliter; median, 21.7). Variation in TPMT activity was not associated with age, sex, or neutering status. Giant Schnauzers had much lower TPMT activity (7.9-20 U of RBC per milliliter; median, 13.1; P < .001) than did other breeds, and Alaskan Malamutes had much higher TPMT activity (22.7-71.8 U of RBC per milliliter; median, 36.0; P < .001) than did other breeds. Such variations in TPMT activity in the canine population and within groups of related dogs could affect thiopurine drug toxicity and efficacy in canine patients.