Abstract 2212: Cellular genetics approaches to defining toxicity pathways.
Authors: Frick Amber , Thomas Rusty , Richards Kristy , Damania Blossom , Fedoriw Yuri , Parks Bethany , Chan Emmanuel , Wiltshire Tim 
Published: August 2014

Background: Significant, nonspecific cytotoxic adverse effects that complicate non-Hodgkin lymphoma treatment vary between patients and are likely due to complex genetic differences between individuals. Our ultimate goal is to identify genetic biomarkers that signal adverse clinical outcomes, maximizing patient safety and minimizing drug cost by identifying patients most likely to have an advantageous risk/benefit ratio for a particular therapy. We are particularly interested in the investigation of the novel anti-lymphoma agents BEZ235, a dual PI3K/mTOR inhibitor, and selumetinib, a MEK inhibitor. Our study is innovative in that we are identifying genetic biomarkers using a strategy that can be employed during preclinical development.

Methods: Genome-wide association analysis requires diversity of both the genotype and phenotype among individuals in a mapping population. Thus, we are taking a model organism approach to evaluate pharmacotherapeutic response in a panel of isogenic mice based on priority strains from the Jackson Laboratory's Mouse Phenome Diversity Panel. Association mapping in this fixed and inbred population utilizes existing dense maps of SNP genotype information, providing precision (greater than 1 to 2 Mb) in localizing quantitative trait loci using SNPster and EMMA algorithms.

We have developed a cellular genetics screening approach with robust, replicable, multiplexed assays to accurately describe toxicity response using flow cytometry. Our population of immune cells was derived from splenocytes isolated from 35 strains of mice using standard procedures. Of note, the splenocytes were not stimulated to avoid confounding effects, as identification of genes would be attributed to cellular stimulation rather than associated with toxicity. Cells at a density of 100,000 cells per well with 100 μl media were incubated with anti-lymphoma 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.

Results: Phenotypes have been quantified using flow cytometry, yielding interstrain variation for measured endpoints for different immune function cells. The heritability for viability of T-cells at the 1 μM concentration of doxorubicin, idarubicin, BEZ235, and selumetinib is respectively 86, 83, 56, and 57%.

Conclusion: Using this approach, we aim to identify genetic determinants of cellular response to drugs, including candidate genes and cellular toxicity pathways for future validation in human studies.

Citation Format: Amber Frick, Rusty Thomas, Kristy Richards, Blossom Damania, Yuri Fedoriw, Bethany Parks, Emmanuel Chan, Tim Wiltshire. Cellular genetics approaches to defining toxicity pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2212. doi:10.1158/1538-7445.AM2013-2212