Supplementary Figure 1 Legend from Organic Cation Transporters Are Determinants of Oxaliplatin Cytotoxicity

Supplementary Table 2 from Organic Cation Transporters Are Determinants of Oxaliplatin Cytotoxicity

Background: The ability to produce timely, clinically actionable recommendations is a cornerstone of a robust precision oncology program. This requires the assimilation of rapidly changing precision oncology data that flows asynchronously from various EMR and lab sources. A key challenge is harmonization of omics biomarkers from hospital-based and commercial testing labs, rendering these omics data interpretable and comparable between patients, labs and reports. To address this challenge, we describe an automated system leveraging a hybrid ontology and graph structure to permit the rapid harmonization and interpretation of molecular diagnostic results across multiple labs. Methods: A two-part, fully automated system to ingest and harmonize omics biomarker data from hospital-based and commercial testing labs was developed. First, an ontology-based omics lexicon provides annotation of discrete laboratory values provided electronically. The ontology model is Manchester language-based with data stored in a relational document store. Second, a graph structure serves as the knowledge model and relates the annotated lab values to 12 types of clinically informative biomarkers, including sequence and copy number variants, tumor mutational burden, MSI, gene rearrangements, wild type, and expression biomarkers. The graph structure leverages the ontology to fully describe hierarchical and categorical data associated with each biomarker. Access to the omics knowledge model uses a RESTful API and relational database architecture. Acquisition of molecular data uses custom JSON schemas and a RESTful API. Results: Feeds from 7 commercial and hospital-based labs resulted in the receipt of >12,000 molecular diagnostic reports containing >2 million individual biomarker observations, with integration of each report achieved within 2 minutes of electronic data receipt. As of Nov. 2018, 81.2% of reports had at least one biomarker observation, while the remaining reports lacked results. Within these biomarker observations, 95.8% were represented in the ontology/graph structure, resulting in successful linkage to ~22,000 distinct harmonized biomarkers out of more than 12 million in the knowledge base. Of the linked, harmonized variant biomarkers, 4% are class-level (e.g. BRAF mutation) with the remainder reflecting more specificity (e.g. BRAF V600E). Within the set of more specific biomarkers, 54% represent a protein altering variant biomarker. Conclusions: We have built an automated, scalable system using a novel ontology/graph structure to reliably and automatically harmonize and integrate omics data into a real-world oncology precision medicine platform. Proof of concept validation has been demonstrated in over 12,000 patient reports, with the results being used to enable clinical trials matching and outcomes research. Citation Format: James E. Shima, James L. Chen, Matthew J. Glick, Ryan A. Warrier, Eric C. Abruzzese, Walter C. Mankowski, Jonathan Hirsch. A hybrid ontology and graph system enabling precision oncology applications through real-time incorporation of omics biomarkers into a scalable real-world data platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1676.

e18763Background: The lack of interoperability among different electronic medical record (EMR) systems remains a challenge to fulfilling the promises of precision oncology. Typically, cancer outcomes data is stored as unstructured information in a multitude of inconsistent fields across EMRs. This prevents the integration of outcomes into the treatment decision-making process. To overcome this challenge, we leveraged the Syapse ontology, a data model that unifies biomedical data. We report our successful efforts to unify outcomes data across a large network of health systems. Methods: Health system participants included Providence St. Joseph Health, Aurora Healthcare, Henry Ford Health System, and University of Miami Sylvester Comprehensive Cancer Care. Data integration efforts were initiated with each health system between 1/2015 and 1/2018. Syapse aggregated clinical data sources and automated outcomes calculation. Source datasets within the health system included the EMR, enterprise data warehouse, tum...

Solute carriers are eukaryotic membrane proteins that control the uptake and efflux of solutes, including essential cellular compounds, environmental toxins, and therapeutic drugs. Solute carriers can share similar structural features despite weak sequence similarities. Identification of sequence relationships among solute carriers is needed to enhance our ability to model individual carriers and to elucidate the molecular mechanisms of their substrate specificity and transport. Here, we describe a comprehensive comparison of solute carriers. We link the proteins using sensitive profile-profile alignments and two classification approaches, including similarity networks. The clusters are analyzed in view of substrate type, transport mode, organism conservation, and tissue specificity. Solute carrier families with similar substrates generally cluster together, despite exhibiting relatively weak sequence similarities. In contrast, some families cluster together with no apparent reason, revealing unexplored relationships. We demonstrate computationally and experimentally the functional overlap between representative members of these families. Finally, we identify four putative solute carriers in the human genome. The solute carriers include a biomedically important group of membrane proteins that is diverse in sequence and structure. The proposed classification of solute carriers, combined with experiment, reveals new relationships among the individual families and identifies new solute carriers. The classification scheme will inform future attempts directed at modeling the structures of the solute carriers, a prerequisite for describing the substrate specificities of the individual families.

Apical reabsorption from the urine has been shown to be important for such processes as the maintenance of critical metabolites in the blood and the excretion of nephrotoxic compounds. The solute carrier (SLC) transporter OAT4 (SLC22A11) is expressed on the apical membrane of renal proximal tubule cells and is known to mediate the transport of a variety of xenobiotic and endogenous organic anions. Functional characterization of genetic variants of apical transporters thought to mediate reabsorption, such as OAT4, may provide insight into the genetic factors influencing the complex pathways involved in drug elimination and metabolite reclamation occurring in the kidney. Naturally occurring genetic variants of OAT4 were identified in public databases and by resequencing DNA samples from 272 individuals comprising 4 distinct ethnic groups. Nine total nonsynonymous variants were identified and functionally assessed using uptake of three radiolabeled substrates. A nonsense variant, R48Stop, and three other variants (R121C, V155G, and V155M) were found at frequencies of at least 2% in an ethnic group specific fashion. The L29P, R48Stop, and H469R variants displayed a complete loss of function, and kinetic analysis identified a reduced V(max) in the common nonsynonymous variants. Plasma membrane levels of OAT4 protein were absent or reduced in the nonfunctional variants, providing a mechanistic reason for the observed loss of function. Characterization of the genetic variants of reabsorptive transporters such as OAT4 is an important step in understanding variability in tubular reabsorption with important implications in innate homeostatic processes and drug disposition.

Membrane transporters play crucial roles in the cellular uptake and efflux of an array of small molecules including nutrients, environmental toxins, and many clinically used drugs. We hypothesized that common genetic variation in the proximal promoter regions of transporter genes contribute to observed variation in drug response. A total of 579 polymorphisms were identified in the proximal promoters (-250 to +50 bp) and flanking 5' sequence of 107 transporters in the ATP Binding Cassette (ABC) and Solute Carrier (SLC) superfamilies in 272 DNA samples from ethnically diverse populations. Many transporter promoters contained multiple common polymorphisms. Using a sliding window analysis, we observed that, on average, nucleotide diversity (pi) was lowest at approximately 300 bp upstream of the transcription start site, suggesting that this region may harbor important functional elements. The proximal promoters of transporters that were highly expressed in the liver had greater nucleotide diversity than those that were highly expressed in the kidney consistent with greater negative selective pressure on the promoters of kidney transporters. Twenty-one promoters were evaluated for activity using reporter assays. Greater nucleotide diversity was observed in promoters with strong activity compared to promoters with weak activity, suggesting that weak promoters are under more negative selective pressure than promoters with high activity. Collectively, these results suggest that the proximal promoter region of membrane transporters is rich in variation and that variants in these regions may play a role in interindividual variation in drug disposition and response.

The second messenger, cGMP, mediates a host of cellular responses to various stimuli, resulting in the regulation of many critical physiologic functions. The existence of specific cGMP transporters on the plasma membrane that participate in the regulation of cGMP levels has been suggested in a large number of studies. In this study, we identified a novel plasma membrane transporter for cGMP. In particular, we showed that hOAT2 (SLC22A7), a member of the solute carrier (SLC) superfamily, was a facilitative transporter for cGMP and other guanine nucleotides. hOAT2, which is ubiquitously expressed at high levels in many cell types, was previously thought to primarily transport organic anions. Among purine and pyrimidine nucleobases, nucleosides, and nucleotides, hOAT2 showed the greatest preference for cGMP, which transported cGMP with a K m value of 88 +/- 11 mu M and exhibited between 50- and 100-fold enhanced uptake over control cells. Our data revealed that hOAT2 is a bidirectional facilitative transporter that can control both intracellular and extracellular levels of cGMP. In addition, we observed that a common alternatively spliced variant of hOAT2 demonstrated a complete loss of transport function as a result of a low expression level on the plasma membrane. We conclude that hOAT2 is a highly efficient, facilitative transporter of cGMP and may be involved in cGMP signaling in many tissues. Our study suggests that hOAT2 represents a potential new drug target for regulating cGMP levels.

The human concentrative nucleoside transporter 2 (CNT2) plays an important role in the absorption, disposition, and biological effects of endogenous nucleosides and nucleoside analog drugs. We identified genetic variation in the basal promoter region of CNT2 and characterized the function of the variants. We screened DNA from an ethnically diverse population and identified five basal promoter variants in CNT2. Three major haplotypes in the CNT2 basal promoter region were identified and were found at different allele frequencies in various ethnic groups. The common promoter variants and haplotypes were constructed and characterized for their promoter activity using luciferase reporter assays. One polymorphic variant, rs2413775 (-146T>A), with an allele frequency >20% in all populations, showed a gain of function in luciferase activity. Furthermore, in vivo mouse promoter assays of these nucleotide variants using the hydrodynamic tail vein injection, leading to their expression in the liver, demonstrated similar results. Transcription factor binding site (TFBS) analysis indicated this variant alters a hepatic nuclear factor (HNF) 1 TFBS. Electrophoretic mobility shift assay demonstrated stronger binding of HNF1alpha and weaker binding of HNF1beta to the -146T and -146A regions, whereas the single nucleotide polymorphism (SNP), -146A, exhibited enhanced binding to both HNF1alpha and HNF1beta, consistent with its greater activity in reporter assays. The data collectively suggest that the common variant, -146T>A, in the proximal promoter of CNT2 may result in an enhanced transcription rate of the gene and, thus, expression levels of CNT2. This SNP may play a role in variation in the pharmacokinetics and pharmacological effects of nucleoside analogs.

Abstract Although the platinum-based anticancer drugs cisplatin, carboplatin, and oxaliplatin have similar DNA-binding properties, only oxaliplatin is active against colorectal tumors. The mechanisms for this tumor specificity of platinum-based compounds are poorly understood but could be related to differences in uptake. This study shows that the human organic cation transporters (OCT) 1 and 2 (SLC22A1 and SLC22A2) markedly increase oxaliplatin, but not cisplatin or carboplatin, accumulation and cytotoxicity in transfected cells, indicating that oxaliplatin is an excellent substrate of these transporters. The cytotoxicity of oxaliplatin was greater than that of cisplatin in six colon cancer cell lines [mean ± SE of IC50 in the six cell lines, 3.9 ± 1.4 μmol/L (oxaliplatin) versus 11 ± 2.0 μmol/L (cisplatin)] but was reduced by an OCT inhibitor, cimetidine, to a level similar to, or even lower than that of, cisplatin (29 ± 11 μmol/L for oxaliplatin versus 19 ± 4.3 μmol/L for cisplatin). Structure-activity studies indicated that organic functionalities on nonleaving groups coordinated to platinum are critical for selective uptake by OCTs. These results indicate that OCT1 and OCT2 are major determinants of the anticancer activity of oxaliplatin and may contribute to its antitumor specificity. They also strongly suggest that expression of OCTs in tumors should be investigated as markers for selecting specific platinum-based therapies in individual patients. The development of new anticancer drugs, specifically targeted to OCTs, represents a novel strategy for targeted drug therapy. The results of the present structure-activity studies indicate specific tactics for realizing this goal. (Cancer Res 2006; 66(17): 8847-57)