Rong Stephanie Huang

Heritable and non-genetic factors as variables of pharmacologic phenotypes in lymphoblastoid cell lines

Publicly available genetic and expression data on lymphoblastoid cell lines (LCLs) make them a unique resource for understanding the genetic underpinnings of pharmacological outcomes and disease. LCLs have been used for pharmacogenomic discovery and validation of clinical findings associated with drug response. However, variation in cellular growth rate, baseline Epstein–Barr virus (EBV) copy number and ATP levels can all be confounders in such studies. Our objective is to better define confounding variables that affect pharmacological end points in LCLs. To this end, we evaluated the effect of these three variables on drug-induced cytotoxicity in LCLs. The drugs evaluated included daunorubicin, etoposide, carboplatin, cisplatin, cytarabine, pemetrexed, 5′-deoxyfluorouridine, vorinostat, methotrexate, 6-mercaptopurine, and 5-fluorouracil. Baseline ATP or EBV copy number were not significantly correlated with cellular growth rate or drug-induced cytotoxicity. In contrast, cellular growth rate and drug-induced cytotoxicity were significantly, directly related for all drugs except vorinostat. Importantly, cellular growth rate is under appreciable genetic influence (h2=0.30–0.39) with five suggestive linkage regions across the genome. Not surprisingly, a percentage of SNPs that significantly associate with drug-induced cytotoxicity also associate with cellular growth rate (P⩽0.0001). Studies using LCLs for pharmacologic outcomes should therefore consider that a portion of the genetic variation explaining drug-induced cytotoxicity is mediated via heritable effects on growth rate.

PACdb: a database for cell-based pharmacogenomics

We have developed Pharmacogenomics And Cell database (PACdb), a results database that makes available relationships between single nucleotide polymorphisms, gene expression, and cellular sensitivity to various drugs in cell-based models to help determine genetic variants associated with drug response. The current version also supports summary analysis on differentially expressed genes between the HapMap samples of European and African ancestry, as well as queries for summary information of correlations between gene expression and pharmacological phenotypes. At present, data generated on the following anticancer agents are included: carboplatin, cisplatin, etoposide, daunorubicin, and cytarabine (Ara-C). The database is also available to assist in the investigation of the effects of potential confounding variables (e.g. cell proliferation rate) in lymphoblastoid cell lines. PACdb will be regularly updated to include more drugs and new datasets (e.g. baseline microRNA levels). PACdb will be linked into PharmGKB to benefit the next wave of pharmacogenetic and pharmacogenomic discovery.

Genome-wide Meta-analysis Identifies Variants Associated with Platinating Agent Susceptibility Across Populations

Platinating agents are used in the treatment of many cancers, yet they can induce toxicities and resistance that limit their utility. Using previously published and additional world population panels of diverse ancestry totaling 608 lymphoblastoid cell lines (LCLs), we performed meta-analyses of over 3 million single-nucleotide polymorphisms (SNPs) for both carboplatin- and cisplatin-induced cytotoxicity. The most significant SNP in the carboplatin meta-analysis is located in an intron of NBAS (neuroblastoma amplified sequence; P=5.1 × 10−7). The most significant SNP in the cisplatin meta-analysis is upstream of KRT16P2 (P=5.8 × 10−7). We also show that cisplatin-susceptibility SNPs are enriched for carboplatin-susceptibility SNPs. Most of the variants that associate with platinum-induced cytotoxicity are polymorphic across multiple world populations; therefore, they could be tested in follow-up studies in diverse clinical populations. Seven genes previously implicated in platinating agent response, including BCL2 (B-cell CLL/lymphoma 2), GSTM1 (glutathione S-transferase mu 1), GSTT1, ERCC2 and ERCC6, were also implicated in our meta-analyses.

Identification of genetic variants and gene expression relationships associated with pharmacogenes in humans

Objectives The very important pharmacogenes (VIPs) were selected by Pharmacogenetic Research Network (National Institutes of Health-PGRN) owing to their significant effects on drug treatment both at the pharmacokinetic and pharmacodynamic levels. Our objective was to identify single nucleotide polymorphisms (SNPs) that potentially affected the expression of these genes or potential SNP–gene interactions involved to improve our understanding of genetic effects on drug therapy. Basic methods Gene expression was evaluated in 176 International HapMap lymphoblastoid cell lines derived from CEU (CEPH, Utah residents with ancestry from northern and western Europe; n=87) and YRI (Yoruba in Ibadan, Nigeria; n=89) using Affymetrix GeneChip Human Exon 1.0 ST arrays (Affymetrix Laboratory, Affymetrix Inc., Santa Clara, California, USA) with interrogation of greater than 17 000 human genes. Genome-wide association was performed between over two million publicly available HapMap SNPs and gene expression. Main results The expression of two PGRN-VIPs (GSTT1 and GSTM1) are significantly associated with SNPs within 2.5 Mb of the genes; whereas the expression of three and ten PGRN-VIPs are significantly associated with distant-acting SNPs in CEU and YRI, respectively. In addition, three and four PGRN-VIPs harbor SNPs that are distantly associated with other gene expressions in CEU and YRI, respectively. Principal conclusion Using this information, one may identify genetic variants that are significantly associated with the expression of any set of genes of interest; or evaluate potential gene–gene interaction through SNP expression relationships.

Genetic Variation That Predicts Platinum Sensitivity Reveals the Role of miR-193b* in Chemotherapeutic Susceptibility

Platinum agents are the backbone of cancer chemotherapy. Recently, we identified and replicated the role of a single nucleotide polymorphism (SNP, rs1649942) in predicting platinum sensitivity both in vitro and in vivo. Using the CEU samples from the International HapMap Project, we found the same SNP to be a master regulator of multiple gene expression phenotypes, prompting us to investigate whether rs1649942-mediated regulation of miRNAs may in part contribute to variation in platinum sensitivity. To these ends, 60 unrelated HapMap CEU I/II samples were used for our discovery-phase study using high-throughput genome-wide miRNA and gene expression profiling. Examining the relationships among rs1649942, its gene expression targets, genome-wide miRNA expression, and cellular sensitivity to carboplatin and cisplatin, we identified 2 platinum-associated miRNAs (miR-193b* and miR-320) that inhibit the expression of 5 platinum-associated genes (CRIM1, IFIT2, OAS1, KCNMA1, and GRAMD1B). We further replicated the relationship between the expression of miR-193b*, CRIM1, IFIT2, KCNMA1, and GRAMD1B, and platinum sensitivity in a separate HapMap CEU III dataset. We then showed that overexpression of miR-193b* in a randomly selected HapMap cell line results in resistance to both carboplatin and cisplatin. This relationship was also found in 7 ovarian cancer cell lines from NCI60 dataset and confirmed in an OVCAR-3 that overexpression of miR-193b* leads to increased resistance to carboplatin. Our findings highlight a potential mechanism of action for a previously observed genotype-survival outcome association. Further examination of miR-193b* in platinum sensitivity in ovarian cancer is warranted. Mol Cancer Ther; 11(9); 2054–61. ©2012 AACR.

Chemotherapeutic-induced apoptosis: a phenotype for pharmacogenomics studies.

Aim To determine whether cellular apoptosis is a suitable phenotypic trait for pharmacogenomics studies by evaluating caspase 3/7-mediated activity in lymphoblastoid cell lines after treatment with six chemotherapeutic agents: 5′-deoxyfluorouridine, pemetrexed, cytarabine, paclitaxel, carboplatin, and cisplatin. Materials and methods Using monozygotic twin pair and sibling pair lymphoblastoid cell lines, we identified conditions for measurement of caspase 3/7 activity in lymphoblastoid cell lines. Genome-wide association studies were performed with over 2 million single nucleotide polymorphisms (SNPs) and cisplatin-induced apoptosis in HapMap CEU cell lines (n=77). Results Although treatment with 5′-deoxyfluorouridine and pemetrexed for up to 24 h resulted in low levels of apoptosis or interindividual variation in caspase-dependent cell death; paclitaxel, cisplatin, carboplatin, and cytarabine treatment for 24 h resulted in 9.4-fold, 9.1-fold, 7.0-fold, and 6.0-fold increases in apoptosis relative to control, respectively. There was a weak correlation between caspase activity and cytotoxicity (r2=0.03–0.29) demonstrating that cytotoxicity and apoptosis are two distinct phenotypes that may produce independent genetic associations. Estimated heritability (h2) for apoptosis was 0.57 and 0.29 for cytarabine (5 and 40 &mgr;mol/l, respectively), 0.22 for paclitaxel (12.5 nmol/l), and 0.34 for cisplatin (5 &mgr;mol/l). In the genome-wide association study using the HapMap CEU panel, we identified a significant enrichment of cisplatin-induced cytotoxicity SNPs within the significant cisplatin-induced apoptosis SNPs and an enrichment of expression quantitative trait loci (eQTL). Among these eQTLs, we identified several eQTLs with known function related to apoptosis and/or cytotoxicity. Conclusion Our study identifies apoptosis as a phenotype for pharmacogenomic studies in lymphoblastoid cell lines after treatment with paclitaxel, cisplatin, carboplatin, and cytarabine that may have utility for discovering biomarkers to predict response to certain chemotherapeutics.

Integrative analyses of genetic variation, epigenetic regulation, and the transcriptome to elucidate the biology of platinum sensitivity

BackgroundUsing genome-wide genetic, gene expression, and microRNA expression (miRNA) data, we developed an integrative approach to investigate the genetic and epigenetic basis of chemotherapeutic sensitivity.ResultsThrough a sequential multi-stage framework, we identified genes and miRNAs whose expression correlated with platinum sensitivity, mapped these to genomic loci as quantitative trait loci (QTLs), and evaluated the associations between these QTLs and platinum sensitivity. A permutation analysis showed that top findings from our approach have a much lower false discovery rate compared to those from a traditional GWAS of drug sensitivity. Our approach identified five SNPs associated with 10 miRNAs and the expression level of 15 genes, all of which were associated with carboplatin sensitivity. Of particular interest was one SNP (rs11138019), which was associated with the expression of both miR-30d and the gene ABCD2, which were themselves correlated with both carboplatin and cisplatin drug-specific phenotype in the HapMap samples. Functional study found that knocking down ABCD2 in vitro led to increased apoptosis in ovarian cancer cell line SKOV3 after cisplatin treatment. Over-expression of miR-30d in vitro caused a decrease in ABCD2 expression, suggesting a functional relationship between the two.ConclusionsWe developed an integrative approach to the investigation of the genetic and epigenetic basis of human complex traits. Our approach outperformed standard GWAS and provided hints at potential biological function. The relationships between ABCD2 and miR-30d, and ABCD2 and platin sensitivity were experimentally validated, suggesting a functional role of ABCD2 and miR-30d in sensitivity to platinating agents.

High expression of miR-532-5p, a tumor suppressor, leads to better prognosis in ovarian cancer both in vivo and in vitro

Ovarian cancer is the leading cause of death for gynecologic cancers, ranking fifth overall for cancer-related death among women. The identification of biomarkers and the elucidation of molecular mechanisms for improving treatment options have received extensive efforts in ovarian cancer research. miRNAs have high potential to act as both ovarian cancer biomarkers and as critical regulators of ovarian tumor behavior. We comprehensively analyzed global mRNA, miRNA expression, and survival data for ovarian cancer from The Cancer Genome Atlas (TCGA) to pinpoint miRNAs that play critical roles in ovarian cancer survival through their effect on mRNA expression. We performed miRNA overexpression and gene knockdown experiments to confirm mechanisms predicted in our bioinformatics approach. We established that overexpression of miR-532-5p in OVCAR-3 cells resulted in a significant decrease in cell viability over a 96-hour time period. In the TCGA ovarian cancer dataset, we found 67 genes whose expression levels were negatively correlated with miR-532-5p expression and correlated with patient survival, such as WNT9A, CSNK2A2, CHD4, and SH3PXD2A. The potential miR-532-5p–regulated gene targets were found to be enriched in the Wnt pathway. Overexpression of miR-532-5p through miRNA mimic caused downregulation of CSNK2A2, CHD4, and SH3PXD2A in the OVCAR-3 cell line. We have discovered and validated the tumor-suppressing capabilities of miR-532-5p both in vivo through TCGA analysis and in vitro through ovarian cancer cell lines. Our work highlights the potential clinical importance of miR-532-5p expression in ovarian cancer patients. Mol Cancer Ther; 15(5); 1123–31. ©2016 AACR.

Genome-wide discovery of genetic variants affecting tamoxifen sensitivity and their clinical and functional validation

BACKGROUND Beyond estrogen receptor (ER), there are no validated predictors for tamoxifen (TAM) efficacy and toxicity. We utilized a genome-wide cell-based model to comprehensively evaluate genetic variants for their contribution to cellular sensitivity to TAM. DESIGN Our discovery model incorporates multidimensional datasets, including genome-wide genotype, gene expression, and endoxifen-induced cellular growth inhibition in the International HapMap lymphoblastoid cell lines (LCLs). Genome-wide findings were further evaluated in NCI60 cancer cell lines. Gene knock-down experiments were performed in four breast cancer cell lines. Genetic variants identified in the cell-based model were examined in 245 Caucasian breast cancer patients who underwent TAM treatment. RESULTS We identified seven novel single-nucleotide polymorphisms (SNPs) associated with endoxifen sensitivity through the expression of 10 genes using the genome-wide integrative analysis. All 10 genes identified in LCLs were associated with TAM sensitivity in NCI60 cancer cell lines, including USP7. USP7 knock-down resulted in increasing resistance to TAM in four breast cancer cell lines tested, which is consistent with the finding in LCLs and in the NCI60 cells. Furthermore, we identified SNPs that were associated with TAM-induced toxicities in breast cancer patients, after adjusting for other clinical factors. CONCLUSION Our work demonstrates the utility of a cell-based model in genome-wide identification of pharmacogenomic markers.BACKGROUND Beyond estrogen receptor (ER), there are no validated predictors for tamoxifen (TAM) efficacy and toxicity. We utilized a genome-wide cell-based model to comprehensively evaluate genetic variants for their contribution to cellular sensitivity to TAM. DESIGN Our discovery model incorporates multidimensional datasets, including genome-wide genotype, gene expression, and endoxifen-induced cellular growth inhibition in the International HapMap lymphoblastoid cell lines (LCLs). Genome-wide findings were further evaluated in NCI60 cancer cell lines. Gene knock-down experiments were performed in four breast cancer cell lines. Genetic variants identified in the cell-based model were examined in 245 Caucasian breast cancer patients who underwent TAM treatment. RESULTS We identified seven novel single-nucleotide polymorphisms (SNPs) associated with endoxifen sensitivity through the expression of 10 genes using the genome-wide integrative analysis. All 10 genes identified in LCLs were associated with TAM sensitivity in NCI60 cancer cell lines, including USP7. USP7 knock-down resulted in increasing resistance to TAM in four breast cancer cell lines tested, which is consistent with the finding in LCLs and in the NCI60 cells. Furthermore, we identified SNPs that were associated with TAM-induced toxicities in breast cancer patients, after adjusting for other clinical factors. CONCLUSION Our work demonstrates the utility of a cell-based model in genome-wide identification of pharmacogenomic markers.

Peripheral blood IRF1 expression as a marker for glucocorticoid sensitivity.

Objective Despite of the common usage of glucocorticoids (GCs), a significant portion of asthma patients exhibit GC insensitivity. This could be mediated by diverse mechanisms, including genomics. Recent work has suggested that measuring changes in gene expression may provide more predictive information about GC insensitivity than baseline gene expression alone, and that expression changes in peripheral blood may be reflective of those in the airway. Methods We performed in silico discovery using gene expression omnibus (GEO) data that evaluated GC effect on gene expression in multiple tissue types. Subsequently, candidate genes whose expression levels are affected by GC were examined in cell lines and in primary cells derived from human airway and blood. Results Through gene expression omnibus analysis, we identified interferon regulator factor 1 (IRF1), whose expression is affected by GC treatment in airway smooth muscle cells, normal human bronchial epithelial (NHBE) cells, and lymphoblastoid cell lines (LCLs). Significant IRF1 downregulation post GC exposure was confirmed in two cultured airway epithelial cell lines and primary NHBE cells (P<0.05). We observed large interindividual variation in GC-induced IRF1 expression changes among primary NHBE cells tested. Significant downregulation of IRF1 was also observed in six randomly selected LCLs (P<0.05), with variable degrees of downregulation among different samples. In peripheral blood mononuclear cells obtained from healthy volunteers, variable downregulation of IRF1 by GC was also shown. NFKB1, a gene whose expression is known to be downregulated by GC and the degree of downregulation of which is reflective of GC response, was used as a control in our study. IRF1 shows more consistent downregulation across tissue types when compared with NFKB1. Conclusion Our results suggest that GC-induced IRF1 gene expression changes in peripheral blood could be used as a marker to reflect GC response in the airway.