Pharmacogenetics of Phase II Drug Metabolizing Enzymes Richard Weinshilboum, PI 1, Eric Wieben, Co-PI 1, Aman Buzdar 2, Christopher Chute 1, Julie Cunningham 1, Matthew Ellis 3, Peter Fasching 4, Brooke Fridley 1, Matthew Goetz 1, Paul Goss 5, James Ingle 1, Jean-Pierre Kocher 1, David Mrazek 1, Mark Robson 6, Daniel Schaid 1, Celine Vachon 1, Liewei Wang 1, Vivien Yee 7. 1 Mayo Clinic, Rochester, MN; 2 MD Anderson Cancer Center, Houston, TX; 3 Washington University, St. Louis, MO; 4 University Hospital Erlangen, Erlangen, Germany; 5 Harvard Medical School, Boston, MA; 6 Memorial Sloan Kettering, New York, NY; 7 Case Western Reserve University, Cleveland, OH Pharmacogenetics of Phase II Drug Metabolizing Enzymes Richard Weinshilboum, PI 1, Eric Wieben, Co-PI 1, Aman Buzdar 2, Christopher Chute 1, Julie Cunningham 1, Matthew Ellis 3, Peter Fasching 4, Brooke Fridley 1, Matthew Goetz 1, Paul Goss 5, James Ingle 1, Jean-Pierre Kocher 1, David Mrazek 1, Mark Robson 6, Daniel Schaid 1, Celine Vachon 1, Liewei Wang 1, Vivien Yee 7. 1 Mayo Clinic, Rochester, MN; 2 MD Anderson Cancer Center, Houston, TX; 3 Washington University, St. Louis, MO; 4 University Hospital Erlangen, Erlangen, Germany; 5 Harvard Medical School, Boston, MA; 6 Memorial Sloan Kettering, New York, NY; 7 Case Western Reserve University, Cleveland, OH This proposal represents a request for continued funding of the Mayo Clinic Pharmacogenomics Research Network (PGRN) grant Pharmacogenetics of Phase II Drug Metabolizing Enzymes. The Mayo PGRN is an integrated, multidisciplinary, pharmacogenomic research effort based on a decades-long focus at Mayo on the pharmacogenetics of phase II (conjugating) drug metabolizing enzymes. The Mayo PGRN began by applying a genotype-to-phenotype research strategy that included, sequentially, gene resequencing, functional genomic, mechanistic and translational studies. During the present funding cycle, the Mayo PGRN has also incorporated the use of genome-wide techniques and pharmacogenomic model systems, with a special emphasis on functional mechanisms responsible for genetic effects on drug response. We have used that approach to study the pharmacogenomics of the endocrine therapy of breast cancer and selective serotonin reuptake inhibitor (SSRI) therapy of depression – research that grew out of the contribution of phase II enzymes to the biotransformation of the estrogens that play such an important role in breast cancer and biotransformation of the neurotransmitters that are central to the pathophysiology and treatment of depression. Recently, we have performed pharmacogenomic genome-wide association (GWA) studies of breast cancer, and we will soon perform similar studies of the SSRI therapy of depression. We propose to continue this genome-wide focus during the next funding cycle, with both clinical and model system GWA studies of the drug therapy of breast cancer and depression, always including replication as well as functional and mechanistic studies. We also propose two Network Resources, one designed to provide access to Next Generation DNA sequencing for all PGRN Centers and the other focused on pharmacogenomic ontology. In summary, the studies in this application build on Mayo PGRN strengths in DNA sequencing and functional genomics – while incorporating genome-wide techniques – to provide insight into the role of inheritance in variation in the efficacy and side effects of drugs used to treat breast cancer and depression. Abstract SSRI PGx Breast Cancer PGx Collaborations Drs. Richard Weinshilboum, Eric Wieben, Christopher Chute, Julie Cunningham, Brooke Fridley, Matthew Goetz, James Ingle, Jean-Pierre Kocher, David Mrazek, Daniel Schaid, Celine Vachon, Liewei Wang, Vivien Yee Breast Cancer Pharmacogenomic Projects o Introduction. The Mayo PPII PGRN has performed, or is performing a series of collaborative GWAS studies of both endocrine therapy and chemotherapy of breast cancer in the adjuvant setting (see the list of collaborative studies). The major projects planned for PGRN III will build on and complement those studies to focus on the pharmacogenomics of breast events for aromatase inhibitor (AI) therapy in the adjuvant setting (MA.27) and chemotherapy in the neoadjuvant setting (GeparQuinto). o MA.27 Events. MA.27 is a large NCIC-CTG, NCI-sponsored, ~7,000 patient, adjuvant breast cancer AI trial. The events (recurrence, disease-free survival etc.) data will be available late in 2010, and a GWAS will then be performed. Those data will also be compared with GWAS data that we have generated for the NSABP- sponsored P1 and P2 breast cancer prevention trials in which we studied pharmacogenomic signals from high risk women treated with SERMs who, nevertheless, developed breast cancer. o GeparQuinto Neoadjuvant Breast Cancer Trial. GeparQuinto is a German-based neoadjuvant breast cancer chemotherapy trial sponsored by the German AGO and GBG clinical trials groups. The phenotypes will be early response and pathological complete response (pCR) as well as drug-related toxicity, especially neutropenia. Those data will be compared with our GWAS data for the German SUCCESS A trial of 3,754 women treated with standard chemotherapy vs. standard chemotherapy plus gemcitabine in the adjuvant setting. Breast Cancer Resources o Human Variation Panel lymphoblastoid cell lines (LCLs). The Human Variation Panel cell lines consist of 100 LCLs from African-American, 100 from Han Chinese-American and 100 from European-American subjects for which, for each cell line, we have: ~1.3 million genome-wide SNPs 54,000 Affymetrix expression array probe set data microRNA data Multiple drug-response phenotype (cytotoxicity) datasets These cell lines have proven valuable for Hypothesis Generation (e.g., Dr. Liewei Wangs studies of gemcitabine mechanism of action) and for Hypothesis Testing (e.g., our successful identification of the TCLIA gene as the basis for the effects of GWAS SNPs associated with AI-induced musculoskeletal pain. o Functional Genomic Validation. The Mayo PPII PGRN has placed an emphasis on the critical importance of functional validation of any signals identified during our GWAS studies, both clinical GWAS and cell line-based GWAS. We have always attempted to move beyond biomarkers to: Functional validation Mechanistic insight SSRI and SNRI Pharmacogenomic Projects o Introduction. The Mayo PPII PGRN has performed and is performing both pharmacogenomic GWA studies and complementary pharmacometabolomic studies of SSRI response in patients with major depressive disorder (MDD). Some of those projects are also described in the subsequent list of collaborative studies. During the PGRN III funding cycle, we plan to build on those results by also studying SNRI response in MDD patients who fail to remit during SSRI therapy and to expand our preliminary pharmacometabolomic studies described under Collaborations to include assays with additional metabolomic platforms beyond the GC-MS/MS platform used in our initial project. o SSRI and SNRI Response Pharmacogenomics. We have already performed a preliminary GWAS using DNA samples from the initial 529 Mayo PGRN SSRI-treated MDD patients. During the upcoming PGRN III funding cycle, we will continue recruitment to this study (which currently has recruited over 600 patients) and add a secondary SNRI treatment phase for the >50% of MDD patients who fail to remit with SSRI therapy. o SSRI and SNRI Pharmacometabolomics. An important feature of these studies includes the use of samples from these MDD patients for pharmacometabolomic analysis. Specifically, GC-MS/MS, coulomb array and LC-MS/MS platforms will all be used to identify metabolomic signatures for SSRI/SNRI response. Those signatures will be mapped to pathways and focused genotyping of genes encoding proteins in those pathways will be performed. This strategy of pharmacometabolomics-informed pharmacogenomics has already been applied successfully in a preliminary study in which glycine was identified by the use of metabolomics – allowing a pharmacogenomic study of genes encoding glycine synthesizing and degrading proteins. A SNP in the glycine dehydrogenase (GLDC) gene was identified using DNA from the Mayo PGRN SSRI study and was replicated using DNA from the NIMH-supported STAR*D study. This same strategy will be used in future SSRI/SNRI pharmacometabolomic studies. o Functional Genomic Validation. Just as for our breast cancer pharmacogenomic studies, we plan to replicate all SSRI/SNRI SNP signals, but also to functionally validate them using cell line-based systems (Human Variation Panel LCLs) and to pursue underlying mechanisms responsible for our observations. RIKEN – Collaborative Clinical GWA Studies o Mayo PGRN Aromatase Inhibitor (AI) Phenotype Study. ~850 women with breast cancer treated with anastrozole in the adjuvant setting with (a) hormone concentrations before and after drug, (b) drug and drug metabolite data, (c) breast density and bone mineral density before and after drug (in collaboration with Indiana University). o NCIC-CTG-NCI MA.27 AI Study. ~7,000 patients enrolled in an AI adjuvant therapy study. In this case, musculoskeletal pain after AI therapy was the phenotype. JCO in press. o NSABP SERM Breast Cancer Prevention P1 and P2 Studies. ~600 cases of breast cancer occurrence and two matched controls per case from the ~30,000 women studied. Genotyping and initial analysis complete. o Mayo PGRN SSRI Response Study in MDD. ~500 MDD patients with QIDS-C scores and SSRI drug and metabolite levels. Genotyping and initial analysis complete. o MA.27 AI Fragility Fracture GWAS. GWAS of AI-related fragility fractures in breast cancer patients treated with AIs in the adjuvant setting. Approved for genotyping. o International SSRI Consortium. GWA study of SSRI response utilizing samples from an international consortium. Approved for genotyping. AGO and German Breast Group (GBG) o The Mayo PGRN has established active collaborations with two German breast cancer groups – the AGO and the GBG. Studies with both of these groups are ongoing or planned, in this case addressing the pharmacogenomics of breast cancer chemotherapy. NIH Metabolomics Research Network o Pharmacometabolomics of SSRI Response. The Mayo PGRN has interacted with the Metabolomics Research Network and has completed an initial pharmacometabolomics-informed pharmacogenomics project that resulted in a manuscript that is currently under review.