Amy L. Stark

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.

Functional genetic screen of human diversity reveals that a methionine salvage enzyme regulates inflammatory cell death

Genome-wide association studies can identify common differences that contribute to human phenotypic diversity and disease. When genome-wide association studies are combined with approaches that test how variants alter physiology, biological insights can emerge. Here, we used such an approach to reveal regulation of cell death by the methionine salvage pathway. A common SNP associated with reduced expression of a putative methionine salvage pathway dehydratase, apoptotic protease activating factor 1 (APAF1)-interacting protein (APIP), was associated with increased caspase-1–mediated cell death in response to Salmonella. The role of APIP in methionine salvage was confirmed by growth assays with methionine-deficient media and quantitation of the methionine salvage substrate, 5′-methylthioadenosine. Reducing expression of APIP or exogenous addition of 5′-methylthioadenosine increased Salmonellae-induced cell death. Consistent with APIP originally being identified as an inhibitor of caspase-9–dependent apoptosis, the same allele was also associated with increased sensitivity to the chemotherapeutic agent carboplatin. Our results show that common human variation affecting expression of a single gene can alter susceptibility to two distinct cell death programs. Furthermore, the same allele that promotes cell death is associated with improved survival of individuals with systemic inflammatory response syndrome, suggesting a possible evolutionary pressure that may explain the geographic pattern observed for the frequency of this SNP. Our study shows that in vitro association screens of disease-related traits can not only reveal human genetic differences that contribute to disease but also provide unexpected insights into cell biology.

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 and Validation of Genetic Variants that Influence Transcription Factor and Cell Signaling Protein Levels

Many genetic variants associated with human disease have been found to be associated with alterations in mRNA expression. Although it is commonly assumed that mRNA expression changes will lead to consequent changes in protein levels, methodological challenges have limited our ability to test the degree to which this assumption holds true. Here, we further developed the micro-western array approach and globally examined relationships between human genetic variation and cellular protein levels. We collected more than 250,000 protein level measurements comprising 441 transcription factor and signaling protein isoforms across 68 Yoruba (YRI) HapMap lymphoblastoid cell lines (LCLs) and identified 12 cis and 160 trans protein level QTLs (pQTLs) at a false discovery rate (FDR) of 20%. Whereas up to two thirds of cis mRNA expression QTLs (eQTLs) were also pQTLs, many pQTLs were not associated with mRNA expression. Notably, we replicated and functionally validated a trans pQTL relationship between the KARS lysyl-tRNA synthetase locus and levels of the DIDO1 protein. This study demonstrates proof of concept in applying an antibody-based microarray approach to iteratively measure the levels of human proteins and relate these levels to human genome variation and other genomic data sets. Our results suggest that protein-based mechanisms might functionally buffer genetic alterations that influence mRNA expression levels and that pQTLs might contribute phenotypic diversity to a human population independently of influences on mRNA expression.

Population differences in platinum toxicity as a means to identify novel genetic susceptibility variants.

Objectives Clinical studies show that Asians (ASN) are more susceptible to toxicities associated with platinum-containing regimens. We hypothesized that studying ASN as an ‘enriched phenotype’ population could enable the discovery of novel genetic determinants of platinum susceptibility. Methods Using well-genotyped lymphoblastoid cell lines from the HapMap, we determined cisplatin and carboplatin cytotoxicity phenotypes (IC50s) for ASN, Caucasians (CEU), and Africans (YRI). IC50s were used in genome-wide association studies. Results ASN were most sensitive to platinums, corroborating clinical findings. ASN genome-wide association studies produced 479 single-nucleotide polymorphisms (SNPs) associating with cisplatin susceptibility and 199 with carboplatin susceptibility (P<10−4). Considering only the most significant variants (P<9.99×10−6), backwards elimination was then used to identify reduced-model SNPs, which robustly described the drug phenotypes within ASN. These SNPs comprised highly descriptive genetic signatures of susceptibility, with 12 SNPs explaining more than 95% of the susceptibility phenotype variation for cisplatin, and eight SNPs approximately 75% for carboplatin. To determine the possible function of these variants in ASN, the SNPs were tested for association with differential expression of target genes. SNPs were highly associated with the expression of multiple target genes, and notably, the histone H3 family was implicated for both drugs, suggesting a platinum-class mechanism. Histone H3 has repeatedly been described as regulating the formation of platinum-DNA adducts, but this is the first evidence that specific genetic variants might mediate these interactions in a pharmacogenetic manner. Finally, to determine whether any ASN-identified SNPs might also be important in other human populations, we interrogated all 479/199 SNPs for association with platinum susceptibility in an independent combined CEU/YRI population. Three unique SNPs for cisplatin and 10 for carboplatin replicated in CEU/YRI. Conclusion Enriched ‘platinum susceptible’ populations can be used to discover novel genetic determinants governing interindividual platinum chemotherapy susceptibility.

Comprehensive genetic analysis of cytarabine sensitivity in a cell-based model identifies polymorphisms associated with outcome in AML patients.

A whole-genome approach was used to investigate the genetic determinants of cytarabine-induced cytotoxicity. We performed a meta-analysis of genome-wide association studies involving 523 lymphoblastoid cell lines (LCLs) from individuals of European, African, Asian, and African American ancestry. Several of the highest-ranked single-nucleotide polymorphisms (SNPs) were within the mutated in colorectal cancers (MCC) gene. MCC expression was induced by cytarabine treatment from 1.7- to 26.6-fold in LCLs. A total of 33 SNPs ranked at the top of the meta-analysis (P < 10(-5)) were successfully tested in a clinical trial of patients randomized to receive low-dose or high-dose cytarabine plus daunorubicin and etoposide; of these, 18 showed association (P < .05) with either cytarabine 50% inhibitory concentration in leukemia cells or clinical response parameters (minimal residual disease, overall survival (OS), and treatment-related mortality). This count (n = 18) was significantly greater than expected by chance (P = .016). For rs1203633, LCLs with AA genotype were more sensitive to cytarabine-induced cytotoxicity (P = 1.31 × 10(-6)) and AA (vs GA or GG) genotype was associated with poorer OS (P = .015), likely as a result of greater treatment-related mortality (P = .0037) in patients with acute myeloid leukemia (AML). This multicenter AML02 study trial was registered at www.clinicaltrials.gov as #NCT00136084.

Mixed Effects Modeling of Proliferation Rates in Cell-Based Models: Consequence for Pharmacogenomics and Cancer

The International HapMap project has made publicly available extensive genotypic data on a number of lymphoblastoid cell lines (LCLs). Building on this resource, many research groups have generated a large amount of phenotypic data on these cell lines to facilitate genetic studies of disease risk or drug response. However, one problem that may reduce the usefulness of these resources is the biological noise inherent to cellular phenotypes. We developed a novel method, termed Mixed Effects Model Averaging (MEM), which pools data from multiple sources and generates an intrinsic cellular growth rate phenotype. This intrinsic growth rate was estimated for each of over 500 HapMap cell lines. We then examined the association of this intrinsic growth rate with gene expression levels and found that almost 30% (2,967 out of 10,748) of the genes tested were significant with FDR less than 10%. We probed further to demonstrate evidence of a genetic effect on intrinsic growth rate by determining a significant enrichment in growth-associated genes among genes targeted by top growth-associated SNPs (as eQTLs). The estimated intrinsic growth rate as well as the strength of the association with genetic variants and gene expression traits are made publicly available through a cell-based pharmacogenomics database, PACdb. This resource should enable researchers to explore the mediating effects of proliferation rate on other phenotypes.

Genome-Wide Local Ancestry Approach Identifies Genes and Variants Associated with Chemotherapeutic Susceptibility in African Americans

Chemotherapeutic agents are used in the treatment of many cancers, yet variable resistance and toxicities among individuals limit successful outcomes. Several studies have indicated outcome differences associated with ancestry among patients with various cancer types. Using both traditional SNP-based and newly developed gene-based genome-wide approaches, we investigated the genetics of chemotherapeutic susceptibility in lymphoblastoid cell lines derived from 83 African Americans, a population for which there is a disparity in the number of genome-wide studies performed. To account for population structure in this admixed population, we incorporated local ancestry information into our association model. We tested over 2 million SNPs and identified 325, 176, 240, and 190 SNPs that were suggestively associated with cytarabine-, 5′-deoxyfluorouridine (5′-DFUR)-, carboplatin-, and cisplatin-induced cytotoxicity, respectively (p≤10−4). Importantly, some of these variants are found only in populations of African descent. We also show that cisplatin-susceptibility SNPs are enriched for carboplatin-susceptibility SNPs. Using a gene-based genome-wide association approach, we identified 26, 11, 20, and 41 suggestive candidate genes for association with cytarabine-, 5′-DFUR-, carboplatin-, and cisplatin-induced cytotoxicity, respectively (p≤10−3). Fourteen of these genes showed evidence of association with their respective chemotherapeutic phenotypes in the Yoruba from Ibadan, Nigeria (p<0.05), including TP53I11, COPS5 and GAS8, which are known to be involved in tumorigenesis. Although our results require further study, we have identified variants and genes associated with chemotherapeutic susceptibility in African Americans by using an approach that incorporates local ancestry information.

The Role of Gene Body Cytosine Modifications in MGMT Expression and Sensitivity to Temozolomide

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is known to play a role in sensitivity to temozolomide. Promoter hypermethylation of MGMT is commonly used to predict low expression levels of MGMT in gliomas, despite observed discordance between promoter methylation and protein levels. Here, we investigated the functional role of gene body cytosine modification in regulating levels of MGMT gene expression and sensitivity to temozolomide. In 91 human glioblastoma samples, we observed significant variation in MGMT expression levels in patients with an unmethylated promoter, with higher levels of gene body cytosine modification correlating with higher gene expression levels. Furthermore, inducing hypomethylation across the MGMT gene body with decitabine corresponded with decreased levels of MGMT gene expression in lymphoblastoid and glioblastoma cell lines, indicating an important functional role for gene body cytosine modifications in maintaining gene expression. We reasoned that the decrease in MGMT expression induced by decitabine may render resistant glioblastoma cell lines more sensitive to temozolomide. Consistent with this reasoning, we found that the MGMT-expressing glioblastoma cell lines exhibiting an unmethylated MGMT promoter that were pretreated with decitabine became significantly more sensitive to temozolomide. Overall, our results suggest a functional role for gene body cytosine modification in regulating gene expression of MGMT and indicate that pretreating patients whose tumors have an unmethylated MGMT promoter with decitabine before temozolomide treatment may increase their response to therapy. Mol Cancer Ther; 13(5); 1334–44. ©2014 AACR.

Protein quantitative trait loci identify novel candidates modulating cellular response to chemotherapy.

Annotating and interpreting the results of genome-wide association studies (GWAS) remains challenging. Assigning function to genetic variants as expression quantitative trait loci is an expanding and useful approach, but focuses exclusively on mRNA rather than protein levels. Many variants remain without annotation. To address this problem, we measured the steady state abundance of 441 human signaling and transcription factor proteins from 68 Yoruba HapMap lymphoblastoid cell lines to identify novel relationships between inter-individual protein levels, genetic variants, and sensitivity to chemotherapeutic agents. Proteins were measured using micro-western and reverse phase protein arrays from three independent cell line thaws to permit mixed effect modeling of protein biological replicates. We observed enrichment of protein quantitative trait loci (pQTLs) for cellular sensitivity to two commonly used chemotherapeutics: cisplatin and paclitaxel. We functionally validated the target protein of a genome-wide significant trans-pQTL for its relevance in paclitaxel-induced apoptosis. GWAS overlap results of drug-induced apoptosis and cytotoxicity for paclitaxel and cisplatin revealed unique SNPs associated with the pharmacologic traits (at p<0.001). Interestingly, GWAS SNPs from various regions of the genome implicated the same target protein (p<0.0001) that correlated with drug induced cytotoxicity or apoptosis (p≤0.05). Two genes were functionally validated for association with drug response using siRNA: SMC1A with cisplatin response and ZNF569 with paclitaxel response. This work allows pharmacogenomic discovery to progress from the transcriptome to the proteome and offers potential for identification of new therapeutic targets. This approach, linking targeted proteomic data to variation in pharmacologic response, can be generalized to other studies evaluating genotype-phenotype relationships and provide insight into chemotherapeutic mechanisms.