Amber Frick

Design of biologically active heparan sulfate and heparin using an enzyme-based approach

Covering: up to August 2008. Heparan sulfate (HS) is a highly sulfated polysaccharide that plays essential physiological and pathophysiological roles. Heparin, a special form of HS, is a commonly used anticoagulant drug. The biosynthesis of HS involves numerous enzymes, including sulfotransferases, glycosyl transferases and an epimerase. It is widely believed that unique sulfation patterns is critical for elucidating the function-structure relationship of this important class of biomolecules. The chemical syntheses of such sulfated saccharides, especially molecules larger than an octasaccharide, are extremely difficult. Therefore, employing HS biosynthetic enzymes to synthesize HS that has the desired biological functions offers an attractive alternative. This review presents the recent progress on this approach. In addition, we discuss the mechanism used by HS sulfotransferases to recognize specific sulfated saccharide sequences. 186 References are cited.

Immune cell-based screening assay for response to anticancer agents: applications in pharmacogenomics.

Background Interpatient variability in immune and chemotherapeutic cytotoxic responses is likely due to complex genetic differences and is difficult to ascertain in humans. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at examining interstrain differences in viability on normal, noncancerous immune cells following chemotherapeutic cytotoxic insult. Drug effects were investigated by comparing selective chemotherapeutic agents, such as BEZ-235 and selumetinib, against conventional cytotoxic agents targeting multiple pathways, including doxorubicin and idarubicin. Methods Splenocytes were isolated from 36 isogenic strains of mice using standard procedures. Of note, the splenocytes were not stimulated to avoid attributing responses to pathways involved with cellular stimulation rather than toxicity. Cells were incubated with compounds on a nine-point logarithmic dosing scale ranging from 15 nM to 100 μM (37°C, 5% CO2). At 4 hours posttreatment, cells were labeled with antibodies and physiological indicator dyes and fixed with 4% paraformaldehyde. Cellular phenotypes (eg, viability) were collected and analyzed using flow cytometry. Dose-response curves with response normalized to the zero dose as a function of log concentration were generated using GraphPad Prism 6. Results Phenotypes were quantified using flow cytometry, yielding interstrain variation for measured endpoints in different immune cells. The flow cytometry assays produced over 16,000 data points that were used to generate dose-response curves. The more targeted agents, BEZ-235 and selumetinib, were less toxic to immune cells than the anthracycline agents. The calculated heritability for the viability of immune cells was higher with anthracyclines than the novel agents, making them better suited for downstream genetic analysis. Conclusion Using this approach, we identify cell lines of variable sensitivity to chemotherapeutic agents and aim to identify robust, replicable endpoints of cellular response to drugs that provide the starting point for identifying candidate genes and cellular toxicity pathways for future validation in human studies.

Identifying genes that mediate anthracyline toxicity in immune cells

The role of the immune system in response to chemotherapeutic agents remains elusive. The interpatient variability observed in immune and chemotherapeutic cytotoxic responses is likely, at least in part, due to complex genetic differences. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at identifying genes underlying these chemotherapeutic cytotoxic effects on immune cells. Using genome-wide association studies (GWAS), we identified four genome-wide significant quantitative trait loci (QTL) that contributed to the sensitivity of doxorubicin and idarubicin in immune cells. Of particular interest, a locus on chromosome 16 was significantly associated with cell viability following idarubicin administration (p = 5.01 × 10−8). Within this QTL lies App, which encodes amyloid beta precursor protein. Comparison of dose-response curves verified that T-cells in App knockout mice were more sensitive to idarubicin than those of C57BL/6J control mice (p < 0.05). In conclusion, the cellular screening approach coupled with GWAS led to the identification and subsequent validation of a gene involved in T-cell viability after idarubicin treatment. Previous studies have suggested a role for App in in vitro and in vivo cytotoxicity to anticancer agents; the overexpression of App enhances resistance, while the knockdown of this gene is deleterious to cell viability. Further investigations should include performing mechanistic studies, validating additional genes from the GWAS, including Ppfia1 and Ppfibp1, and ultimately translating the findings to in vivo and human studies.

A cellular genetics approach identifies gene-drug interactions and pinpoints drug toxicity pathway nodes

New approaches to toxicity testing have incorporated high-throughput screening across a broad-range of in vitro assays to identify potential key events in response to chemical or drug treatment. To date, these approaches have primarily utilized repurposed drug discovery assays. In this study, we describe an approach that combines in vitro screening with genetic approaches for the experimental identification of genes and pathways involved in chemical or drug toxicity. Primary embryonic fibroblasts isolated from 32 genetically-characterized inbred mouse strains were treated in concentration-response format with 65 compounds, including pharmaceutical drugs, environmental chemicals, and compounds with known modes-of-action. Integrated cellular responses were measured at 24 and 72 h using high-content imaging and included cell loss, membrane permeability, mitochondrial function, and apoptosis. Genetic association analysis of cross-strain differences in the cellular responses resulted in a collection of candidate loci potentially underlying the variable strain response to each chemical. As a demonstration of the approach, one candidate gene involved in rotenone sensitivity, Cybb, was experimentally validated in vitro and in vivo. Pathway analysis on the combined list of candidate loci across all chemicals identified a number of over-connected nodes that may serve as core regulatory points in toxicity pathways.

In vitro and in vivo mouse models for pharmacogenetic studies.

The identification of causative genes underlying biomedically relevant phenotypes, particularly complex multigenic traits, is of vital interest to modern medicine. Using genome-wide association analysis, many studies have successfully identified thousands of loci (called quantitative trait loci or QTL), some of these associating with drug response phenotypes. However, the determination and validation of putative genes has been much more challenging. The actions of drugs, both efficacious and deleterious, are complex phenotypes that are controlled or influenced in part by genetic mechanisms.Investigation for genetic correlates of complex traits and pharmacogenetic traits is often difficult to perform in human studies due to cost, availability of relevant sample population, and limited ability to control for environmental effects. These challenges can be circumvented with the use of mouse models for pharmacogenetic studies. In addition, the mouse can be treated at sub- and supratherapeutic doses and subjected to invasive procedures, which can facilitate measures of drug response phenotypes, making identification of pharmacogenetically relevant genes more feasible. The availability of multiple mouse genetic and phenotypic resources is an additional benefit to using the mouse for pharmacogenetic studies.Here, we describe the contribution of animal models, specifically the mouse, towards the field of pharmacogenetics. In this chapter, we describe different mouse models, including the knockout mouse, recombinant mouse inbred strains, in vitro mouse cell-based assays, as well as novel experimental approaches like the Collaborative Cross recombinant mouse inbred panel, which can be applied to preclinical pharmacogenetics research. These approaches can be used to assess drug response phenotypes that are difficult to model in humans, thereby facilitating drug discovery, development, and application.

Dysopsonin Activity of Serum DNA-Binding Proteins Favorable for Gene Delivery

Naked DNA is regarded as the safest and simplest method of gene delivery. However, normally intravenously injected naked plasmid DNA is rapidly eliminated from the blood. It has been hypothesized that opsonins, a category of serum DNA-binding proteins (SDBPs), label the injected plasmid DNA as foreign so that it may be recognized and rapidly removed from the bloodstream by liver nonparenchymal cells. Contrary to the hypothesis, our data indicate that some SDBPs across multiple species may have important dysopsonin properties, acting to reduce liver uptake. Formation of SDBP and DNA complexes was observed by agarose gel electrophoresis. An in vivo study involving hepatic artery and portal vein occlusion in a mouse model confirmed the activity of serum diminishing liver uptake of DNA. Data using hydrodynamic gene transfer in the mouse liver and in situ transfection in the mouse lung revealed that serum proteins bound to DNA do not affect the biological activity of the plasmid DNA. We have identified several SDBPs with potential dysopsonin properties. The SDBPs with dysopsonin properties and DNA complexes may be further modified and ultimately be developed into a novel DNA carrier system favorable for systemic gene delivery.

Implementing Clinical Pharmacogenomics in the Classroom: Student Pharmacist Impressions of an Educational Intervention Including Personal Genotyping

Pharmacogenomics provides a personalized approach to pharmacotherapy by using genetic information to guide drug dosing and selection. However, partly due to lack of education, pharmacogenomic testing has not been fully implemented in clinical practice. With pharmacotherapy training and patient accessibility, pharmacists are ideally suited to apply pharmacogenomics to patient care. Student pharmacists (n = 222) participated in an educational intervention that included voluntary personal genotyping using 23andMe. Of these, 31% of students completed both pre- and post-educational interventions to evaluate their attitudes and confidence towards the use of pharmacogenomics data in clinical decision making, and 55% of this paired subset obtained personal genotyping. McNemar’s test and the Wilcoxon signed-rank test were used to analyze responses. Following the educational intervention, students regardless of genotyping were more likely to recommend personal genotyping (36% post-educational intervention versus 19% pre-educational intervention, p = 0.0032), more confident in using pharmacogenomics in the management of drug therapy (51% post-educational intervention versus 29% pre-educational intervention, p = 0.0045), and more likely to believe that personalized genomics would have an important role in their future pharmacy career (90% post-educational intervention versus 51% pre-educational intervention, p = 0.0072) compared to before receiving the educational intervention. This educational intervention positively influenced students’ attitudes and confidence regarding pharmacogenomics in the clinical setting. Future studies will examine the use of next-generation sequencing assays that selectively examine pharmacogenes in the education of student pharmacists.

Genetic Testing in Clinical Settings

Genetic testing is used for screening, diagnosis, and prognosis of diseases consistent with a genetic cause and to guide drug therapy to improve drug efficacy and avoid adverse effects (pharmacogenomics). This In Practice review aims to inform about DNA-related genetic test availability, interpretation, and recommended clinical actions based on results using evidence from clinical guidelines, when available. We discuss challenges that limit the widespread use of genetic information in the clinical care setting, including a small number of actionable genetic variants with strong evidence of clinical validity and utility, and the need for improving the health literacy of health care providers and the public, including for direct-to-consumer tests. Ethical, legal, and social issues and incidental findings also need to be addressed. Because our understanding of genetic factors associated with disease and drug response is rapidly increasing and new genetic tests are being developed that could be adopted by clinicians in the short term, we also provide extensive resources for information and education on genetic testing.

Abstract A17: Cellular genomics approaches to defining toxicity pathways of chemotherapeutic agents in immune cells

Background: While the role of the immune system in cancer development is known, its role in response to chemotherapeutic agents remains elusive. Interpatient variability in immune and chemotherapeutic cytotoxic responses is likely due to complex genetic differences. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at examining novel mechanisms underlying these chemotherapeutic cytotoxic responses on immune cells. Drug effects were investigated by comparing more selective chemotherapeutic agents such as BEZ235 and selumetinib against conventional cytotoxic agents, including doxorubicin and idarubicin. Our ultimate goal is to identify genetic biomarkers to determine which patients are most likely to have an advantageous risk-benefit ratio for a particular therapy. Methods: Splenocytes were isolated from 35 isogenic strains of mice using standard procedures. Of note, the splenocytes were not stimulated to avoid attributing genes to cellular stimulation rather than toxicity. Cells at a density of 100,000 cells per well with 100 μl media were incubated with compounds on a 10-point logarithmic dosing scale ranging from 0 to 100 μM (37°C, 5% CO2). At 4 hours post-treatment, cells were labeled with antibodies and physiological indicator dyes and fixed with 4% paraformaldehyde. Cellular phenotypes (e.g., viability, mitochondrial membrane potential, and caspase activity) were collected with the BD Biosciences FACSCanto II flow cytometer and analyzed with Flow Jo version X. Dose response curves with response normalized to the zero dose as a function of log concentration were subsequently generated using GraphPad Prism 5. SNPster and EMMA algorithms were used to perform genome-wide association mapping, providing precision (1 to 2 Mb) in localizing quantitative trait loci. Potential candidate genes for validation studies were prioritized using genome-wide significance, spleen expression data, haplotype structure, biological relevance, etc. Validation of the candidate gene App consisted of isolating splenocytes from App knockout mice with a C57BL/6J background and comparing their response to idarubicin with control C57BL/6J mice. Results: Phenotypes were quantified using flow cytometry, yielding interstrain variation for measured endpoints in different immune cells. Our flow cytometry assay produced nearly 16,000 data points that were used to generate dose response curves. The more targeted agents, BEZ-235 and selumetinib, were less toxic to immune cells than the anthracycline agents. Also, heritability for the viability of immune cells was higher (approximately 70 to 90%) for anthracyclines than the novel agents (approximately 35 to 60%), making them ideal for genetic analysis. Using genome-wide association studies, we identified loci that contributed to the sensitivity of doxorubicin and idarubicin in immune cells. We identified multiple QTL containing 35 promising candidate genes. Of particular interest, App encoding for amyloid beta precursor protein was identified under a peak on chromosome 16 (p = 5.01x10 -8 ) in T-cells exposed to idarubicin. Dose response curves verified that T-cells in App knockout mice were more sensitive to idarubicin than those in C57BL/6J control mice (p = 0.01). Conclusion: Using a cellular screening approach, we identified and subsequently validated a gene candidate encoding for amyloid beta precursor protein in T-cells exposed to idarubicin. The literature has suggested a role for App in in vitro and in vivo cytotoxicity to anticancer agents; the overexpression of App enhances resistance, while the knockdown of this gene is deleterious to cell viability. In the future, we aim to perform mechanistic studies in primary and immortalized immune cells and, ultimately, to translate our findings to in vivo and human studies. Citation Format: Amber D. Frick, Kristy Richards, Yuri Fedoriw, Russell Thomas, Timothy Wiltshire. Cellular genomics approaches to defining toxicity pathways of chemotherapeutic agents in immune cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A17.

Abstract 5560: A high-throughput cellular genetics approach to identifying genes associated with sorafenib response and toxicity

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Sorafenib is an oral multikinase inhibitor, approved for hepatocellular, renal and thyroid carcinomas, which decreases tumor angiogenesis and proliferation. The antitumor efficacy and toxicity profiles of sorafenib vary among patients. No predictive biomarkers of sorafenib activity exist to help guide clinicians. Novel pathways and targets of sorafenib activity remain to be identified. We aimed to identify novel genes associated with sorafenib activity by using an in vitro methodology based upon mouse genomics. Methods: We profiled primary mouse embryonic fibroblasts (MEFs) from 32 inbred strains for sorafenib cytotoxicity utilizing high content imaging and simultaneous evaluation of cell health parameters. The 32 strains have been genomically characterized previously (PMID: 21623374). MEF cells were treated with varying concentrations (0-300 µM) of sorafenib, incubated for 24 h or 72 h, and then fixed and stained. Nuclear staining was used to assess sorafenib cytotoxicity and establish our cell viability phenotype. Dose response curves were generated from data, and EC50 values for each strain were identified using a Brain-Cousens model. Genome-wide association mapping, using the SNPster algorithm, was performed on cell viability EC50 values to identify quantitative trait loci (QTLs) associated with sorafenib cytotoxicity. Approximately 277,000 single nucleotide polymorphisms were tested, and genomic loci with p-values < 3.5x10-5 were selected for additional analyses. Results: Interstrain EC50 variability among the 32 MEF strains was observed after 24 h (21-121 µM) and 72 h (17-32 µM) sorafenib incubations. We identified three total peaks associated with cell viability: two on chromosome 13 (23 Mb apart; p = 3.4x10-5 and = 1.6x10-5, respectively), and one on chromosome 4 (p = 2.2x10-5). From these three peaks, we have identified candidate genes that may underlie variability in sorafenib cytotoxicity. A total of 16 genes expressed in MEF cells at mRNA level are present in these QTLs. Of particular interest, we identified one locus that contains Nfyc, a gene that encodes the C subunit of the NF-Y transcription factor. This transcription factor complex is conserved between humans and mice. In humans, NF-Y regulates MYC signaling and DNA-dependent transcription of PDGFR-β (a primary target of sorafenib) (PMID: 12167641). Conclusions: Our innovative high-throughput cellular genetics approach has identified three regions with genetic loci potentially associated with sorafenib cytotoxicity. This approach is capable of identifying robust interstrain cellular differences in sorafenib activity. Functional validation of Nyfc and other promising candidates should be conducted. Citation Format: Daniel J. Crona, Oscar Suzuki, O. Joseph Trask, Amber Frick, Bethany Parks, Tim Wiltshire, Federico Innocenti. A high-throughput cellular genetics approach to identifying genes associated with sorafenib response and toxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5560. doi:10.1158/1538-7445.AM2014-5560