Weinshilboum, Richard M.

Abstract Castration resistant prostate cancer (CRPC) is a deadly disease with a poor prognosis. Treatment options for CRPC patients involve taxanes or newly developed drugs targeting the androgen pathway or androgen synthesis, including CYP17 inhibitors. However, response to therapy varies dramatically among patients, which might be due, in part, to tumor heterogeneity. Therefore, understanding of the genomic landscape of CRPC would be important to help achieve better outcomes by developing personalized therapies. One challenge contributing to our lack of therapies for CRPC is a paucity of clinically relevant animal models predictive of response to therapy in human patients. The Prostate Cancer Medically Optimized Genome Enhanced Therapy (PROMOTE) study is a Mayo Clinic prospective trial designed for patients with metastatic CRPC who are treated with abiraterone acetate, a CYP17 inhibitor, over a 12 week period. Fresh tissue from metastatic lesions, primarily bone metastases, was obtained by needle core biopsy before and 12 weeks after initiating abiraterone acetate treatment. The samples were submitted for DNA and RNA sequencing and were also used to generate patient-derived xenograft (PDX) models. PDX models were developed by injecting biopsy samples subcutaneously or under the renal capsule in male non-obese diabetic (NOD)/severe combined immunodeficient (SCID) or NOD/SCID/IL-2γ-receptor null (NSG) mice implanted with 25mg testosterone pellets at the time of tissue implantation and 2 months later. Baseline samples from 103 unique patients as well as second samples from 61 patients post abiraterone acetate treatment were injected into approximately 600 mice. Of the samples for which pathology is currently available, 31% of the biopsies injected into mice were confirmed to include tumor tissue, which may be compared with a figure of 37.5% of samples sent for sequencing. Thus far, we have established 8 PDX models, four from baseline and four from post treatment samples. Seven models were derived from metastatic bone lesions, and one model from a liver metastasis. The tumor tissue in each model was expanded into a second of generation of mice and harvested when the tumors grew. No tumor was expanded more than four times. Genomic and transcriptomic analysis of xenograft tissue from different “generations” of xenograft expansion is ongoing and will be compared with similar data for the original biopsy. The evaluation of treatment response after exposure of these PDX models to different therapies including CYP19 inhibitors, AR blockers and taxanes is ongoing. These PDX models provide a tool for future drug testing and for studying mechanisms of resistance to therapy in CRPC. Citation Format: Fang Xie, Manish Kohli, Yin Ping, Rafael E. Jimenez, Richard M. Weinshilboum, Liewei Wang. Developing patient derived xenograft (PDX) models for metastatic castration resistant prostate cancer (CRPC) during CYP17 inhibitor therapy. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A05.

Abstract BACKGROUND: Based on prospective clinical trials, there is no evidence for differences in efficacy between the 3 aromatase inhibitors (AIs) anastrozole, exemestane, and letrozole. The purpose of this study was to identify germline genetic variants associated with response to AIs and to help identify novel mechanisms associated with drug disease efficacy. METHODS: A genome-wide association study (GWAS) was performed for 624 patients (Steroids 2015;99:32-38) to identify SNPs associated with estrogen level change in women with estrogen receptor (ER) positive breast cancer treated with anastrozole. Replication of associated SNPs was performed in a GWAS from the MA.27 trial that compared adjuvant anastrozole and exemestane treatment of post-menopausal women with ER+ breast cancer. Functional studies were subsequently performed to determine SNP effects and underlying mechanisms. RESULTS: Our initial GWAS identified SNPs within CSMD1 that were associated with changes in estrogen levels during anastrozole therapy. An additional SNP in CSMD1 was also associated with breast cancer events in CCTG MA.27. Functionally, we showed that CSMD1 regulates CYP19 expression in a SNP-, and in an anastrozole- dependent fashion. These phenomena were not observed for either letrozole or exemestane. In MA.27, an anastrozole- specific effect was also seen with the minor allele having a protective effect on time to distant metastasis (HR=0.49, p=0.00259), but this was not the case for exemestane (HR=0.71, p=0.111). Our in vitro functional studies indicated that overexpression of CSMD1 sensitized anastrozole or letrozole resistant cells to anastrozole but not to the other two AIs. The SNP in CSMD1 that was associated with increased CSMD1 and CYP19 expression levels increased anastrozole sensitivity, but not letrozole or exemestane in lymphoblastoid cell lines (LCLs) homozygous for either WT or variant CSMD1 SNP genotypes. Based on these observations, we explored whether anastrozole has additional mechanisms beyond its function as a CYP19 inhibitor. Utilizing an estrogen response element (ERE) luciferase reporter assay in a CYP19 CRISPR knockout breast cancer T47D cell line and a surface plasmon resonance (SPR) assay, we found that anastrozole can also function as an ERα agonist, and can bind to, and result in, proteasome dependent ERα degradation, especially in the presence of E2. Treatment of these CYP19 CRISPR knockout cells with anastrozole in the presence of increasing concentrations of E2 results in greater sensitivity compared with anastrozole alone, while the addition of E2, as expected, does not improve letrozole or exemestane sensitivity. These same observations were also seen in letrozole and anastrazole resistant cells. CONCLUSIONS: Our findings suggest that anastrozole might be more effective than letrozole or exemestane in patients with the CSMD1 SNP. Furthermore, anastrozole can function as an ERα agonist, binding to ERα and resulting in its degradation, especially in the presence of E2. These findings should help to make it possible to develop precision endocrine therapies for women who are candidates for AIs. Citation Format: Cairns J, Ingle J, Dudenkov T, Kalari K, Buzdar A, Kubo M, Robson M, Ellis M, Goss P, Shepherd L, Goetz M, Weinshilboum R, Wang L. CSMD1 SNPs selectively affect anastrozole response in postmenopausal breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD1-04.

Thiopurine‐related hematotoxicity in pediatric acute lymphoblastic leukemia (ALL) and inflammatory bowel diseases has been linked to genetically defined variability in thiopurine S‐methyltransferase (TPMT) activity. While gene testing of TPMT is being clinically implemented, it is unclear if additional genetic variation influences TPMT activity with consequences for thiopurine‐related toxicity. To examine this possibility, we performed a genome‐wide association study (GWAS) of red blood cell TPMT activity in 844 Estonian individuals and 245 pediatric ALL cases. Additionally, we correlated genome‐wide genotypes to human hepatic TPMT activity in 123 samples. Only genetic variants mapping to chromosome 6, including the TPMT gene region, were significantly associated with TPMT activity ( P < 5.0 × 10 ‐8 ) in each of the three GWAS and a joint meta‐analysis of 1,212 cases (top hit P = 1.2 × 10 ‐72 ). This finding is consistent with TPMT genotype being the primary determinant of TPMT activity, reinforcing the rationale for genetic testing of TPMT alleles in routine clinical practice to individualize mercaptopurine dosage.

Abstract Genetic risks in breast cancer remain only partly understood. Here, we report the results of a genome-wide association study of germline DNA from 4,658 women, including 252 women experiencing a breast cancer recurrence, who were entered on the MA.27 adjuvant trial comparing the aromatase inhibitors (AI) anastrozole and exemestane. Single-nucleotide polymorphisms (SNP) of top significance were identified in the gene encoding MIR2052HG, a long noncoding RNA of unknown function. Heterozygous or homozygous individuals for variant alleles exhibited a ∼40% or ∼63% decrease, respectively, in the hazard of breast cancer recurrence relative to homozygous wild-type individuals. Functional genomic studies in lymphoblastoid cell lines and ERα-positive breast cancer cell lines showed that expression from MIR2052HG and the ESR1 gene encoding estrogen receptor-α (ERα) was induced by estrogen and AI in a SNP-dependent manner. Variant SNP genotypes exhibited increased ERα binding to estrogen response elements, relative to wild-type genotypes, a pattern that was reversed by AI treatment. Further, variant SNPs were associated with lower expression of MIR2052HG and ERα. RNAi-mediated silencing of MIR2052HG in breast cancer cell lines decreased ERα expression, cell proliferation, and anchorage-independent colony formation. Mechanistic investigations revealed that MIR2052HG sustained ERα levels both by promoting AKT/FOXO3-mediated ESR1 transcription and by limiting ubiquitin-mediated, proteasome-dependent degradation of ERα. Taken together, our results define MIR2052HS as a functionally polymorphic gene that affects risks of breast cancer recurrence in women treated with AI. More broadly, our results offer a pharmacogenomic basis to understand differences in the response of breast cancer patients to AI therapy. Cancer Res; 76(23); 7012–23. ©2016 AACR.

AbstractBackground6‐Mercaptopurine (6MP) and methotrexate (MTX) based maintenance therapy is a critical phase of childhood acute lymphoblastic leukemia treatment. Wide interindividual variations in drug disposition warrant frequent doses adjustments, but there is a lack of international consensus on dose adjustment guidelines.ProcedureTo identify relapse predictors, we collected 28,255 data sets on drug doses and blood counts (median: 47/patient) and analyzed erythrocyte (Ery) levels of cytotoxic 6MP/MTX metabolites in 9,182 blood samples (median: 14 samples/patient) from 532 children on MTX/6MP maintenance therapy targeted to a white blood cell count (WBC) of 1.5–3.5 × 109/l.ResultsAfter a median follow‐up of 13.8 years for patients in remission, stepwise Cox regression analysis did not find age, average doses of 6MP and MTX, hemoglobin, absolute lymphocyte counts, thrombocyte counts, or Ery levels of 6‐thioguanine nucleotides or MTX (including its polyglutamates) to be significant relapse predictors. The parameters significantly associated with risk of relapse (N = 83) were male sex (hazard ratio [HR] 2.0 [1.3–3.1], P = 0.003), WBC at diagnosis (HR = 1.04 per 10 × 109/l rise [1.00–1.09], P = 0.048), the absolute neutrophil count (ANC; HR = 1.7 per 109/l rise [1.3–2.4], P = 0.0007), and Ery thiopurine methyltransferase activity (HR = 2.7 per IU/ml rise [1.1–6.7], P = 0.03). WBC was significantly related to ANC (Spearman correlation coefficient, rs = 0.77; P < 0.001), and only a borderline significant risk factor for relapse (HR = 1.28 [95% CI: 1.00–1.64], P = 0.046) when ANC was excluded from the Cox model.ConclusionsThis study indicates that a low neutrophil count is likely to be the best hematological target for dose adjustments of maintenance therapy.

Abstract Objective: Onset and symptoms of menopause, and response to hormone therapy (HT) show large interindividual variability. SULT1A1 encodes for a highly expressed enzyme that metabolizes estrogens. We evaluated the relationship between genetic variation in SULT1A1, menopause age, symptoms, and response to HT. Methods: Women enrolled in the Kronos Early Estrogen Prevention Study at Mayo Clinic were randomized to 48 months of treatment with oral conjugated equine estrogen (n = 34), transdermal 17β-estradiol (E2) (n = 33), or placebo (n = 35). Linear regression models and ANOVA were used to test for association of SULT1A1 copy number, rs3760091, rs750155, and rs9282861 (SULT1A1 ∗ 2), with age at menopause and symptoms, levels of estrogens (estrone [E1], estrone sulfate [E1S], E2, and estradiol sulfate [E2S]), before and after HT. Results: SULT1A1 gene copy number affected the minor allele frequency for each single nucleotide polymorphisms tested. Before administration of exogenous hormones, increasing number of G alleles at rs9282861 was associated with earlier age at menopause (P = 0.014), lower frequency of night sweats (P = 0.009), and less severe insomnia (P = 0.046). After 48 months of treatment, SULT1A1 genotype was not associated with the presence of menopausal symptoms. In women randomized to oral conjugated equine estrogen, increasing number of the A allele at rs750155 was associated with lower E1S and E2S (P = 0.004 and 0.017), whereas increasing number of the C allele at rs3760091 was associated with lower E2S/E2 (P = 0.044). Conclusions: Interindividual variability in onset of menopause and symptoms before initiation of HT is explained in part by genetic variation in SULT1A1 and may represent a step toward individualizing HT treatment decisions.

Abstract Background: Rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) therapy is standard of care for patients with newly diagnosed DLBCL. A majority of patients respond to R-CHOP, however ∼9% will have primary refractory disease and outcomes in these patients are extremely poor. While there is some knowledge of the pharmacogenomic (PGx) determinants of individual agents within R-CHOP, little is known about the PGx determinants of combination therapy. Our study aims to elucidate germline PGx determinants of drug bioavailability and undesirable side chain reactions in DLBCL patients treated with R-CHOP having potential to influence refractory disease. Methods: Newly diagnosed, DLBCL patients treated with R-CHOP between 2002 and 2012 with germline DNA and genotyping data were selected from the Molecular Epidemiology Resource within the Iowa/Mayo Lymphoma SPORE. Refractory disease was defined as either i) a best response of stable or progressive disease or ii) progressive disease by the end of the planned number of cycles of R-CHOP. PGx candidates within known pharmacokinetics and pharmacodynamics (PD) pathways for 11 genes (1,040 single nucleotide polymorphisms (SNPs)) were selected for study (CPT 2012;92(4):414-7). Odds ratios (ORs) and 95% confidence intervals (CI) were used to estimate the association of SNPs with refractory disease, controlling for age, gender, and age-adjusted International Prognostic Index. Results: The median age at diagnosis of 424 of DLBCL patients treated with R-CHOP was 62 years (range 18-92); 45.7% were female; and 58.4% had Ann Arbor Stage III-IV. By the end of initial therapy 40 patients had primary refractory disease. Of the 1,040 SNPs evaluated, 120 (12%) were significantly associated with refractory disease at the 5% significance threshold suggesting novel associations with drug metabolism. The top SNP from each PGx candidate gene are shown in Table, with ALDH5A1 and CYP2B6 showing the most significant associations. Conclusions: While needing replication, we identified associations within two PGx candidate genes that are potentially functionally related to cyclophosphamide PD: alleles within CYP2B6 may have relevance in R-CHOP outcomes due to potential competition with other drug metabolizing enzymes shared with prednisone and vincristine biotransformation in the liver, and alleles withinALDH5A1 may impact a deactivating side chain reaction in B lymphocytes. Table: R-CHOP PGx candidates with known side chain reactionsPGx candidaten SNPsTop SNPChrOR* (95% CI)P-valueMAFABCB1365rs3480093571.86 (1.14 – 3.04)1.32E-020.29ADH1A32rs122996841.66 (0.60 - 4.53)3.27E-010.038ALDH3A116rs2228100171.26 (0.71 - 2.22)4.28E-010.24ALDH5A1192rs946103466.30 (2.43 - 16.4)1.55E-040.030CBR110rs2835266212.10 (0.90 - 4.89)8.76E-020.052CBR356rs45527331212.12 (0.91 - 4.98)8.29E-020.050CYP2B6107rs1038376192.38 (1.44 - 3.94)7.74E-040.27CYP3A421rs2898858372.32 (0.80 - 6.78)1.23E-010.028CYP3A531rs778032871.69 (0.68 - 4.16)2.57E-010.054SLC22A16193rs11217403561.80 (1.06 - 3.03)2.78E-020.21SOD116rs1041740211.35 (0.83 - 2.19)2.26E-010.32Chr=chromosome, MAF=minor allele frequency, *Adjusted for age, sex, and age-adjusted International Prognostic Index Citation Format: Matthew K. Breitenstein, Susan L. Slager, Anne J. Novak, Matthew J. Maurer, Carrie A. Thompson, Umar Farooq, Brian K. Link, Thomas M. Habermann, Curtis L. Olswold, Andrew L. Feldman, Grzegorz S. Nowakowski, Stephen M. Ansell, Thomas E. Witzig, Richard M. Weinshilboum, Liewei Wang, James R. Cerhan. Pharmacogenomic of refractory response to R-CHOP therapy in diffuse large B-cell lymphoma (DLBCL) patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B143.

Objective The study aimed to define thresholds of clinically significant change in 17‐item Hamilton Depression Rating Scale (HDRS‐17) scores using the Clinical Global Impression‐Improvement (CGI‐I) Scale as a gold standard. Methods We conducted a secondary analysis of individual patient data from the Pharmacogenomic Research Network Antidepressant Medication Pharmacogenomic Study, an 8‐week, single‐arm clinical trial of citalopram or escitalopram treatment of adults with major depression. We used equipercentile linking to identify levels of absolute and percent change in HDRS‐17 scores that equated with scores on the CGI‐I at 4 and 8 weeks. Additional analyses equated changes in the HDRS‐7 and Bech‐6 scale scores with CGI‐I scores. Results A CGI‐I score of 2 (much improved) corresponded to an absolute decrease (improvement) in HDRS‐17 total score of 11 points and a percent decrease of 50–57%, from baseline values. Similar results were observed for percent change in HDRS‐7 and Bech‐6 scores. Larger absolute (but not percent) decreases in HDRS‐17 scores equated with CGI‐I scores of 2 in persons with higher baseline depression severity. Conclusions Our results support the consensus definition of response based on HDRS‐17 scores ( > 50% decrease from baseline). A similar definition of response may apply to the HDRS‐7 and Bech‐6. Copyright © 2016 John Wiley & Sons, Ltd.