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

Abstract

Abstract 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.