Abstract 2770: Phenotypic profiling of Aurora inhibitors using the OncoPanelTMplatform

Abstract

Abstract Aurora kinases (Aur) are a family of serine/threonine kinases essential for genetic stability and cell division during mitosis. In humans, activity of the three paralogs (AurA, AurB, and AurC) is regulated throughout the cell cycle, with peak activity during mitosis. Each paralog shares a conserved C-terminal catalytic domain, but differs in substrate specificity, sub-cellular localization, and function. AurA mainly controls centrosome maturation and bipolar spindle assembly; AurB phosphorylates histone H3 (Ser10), which aids in chromatin condensation and separation; AurC, whose expression is restricted to germ cells of both genders, shares similar characteristics to AurB including substrate localization, specificity, and function during mitosis. Aberrant Aur expression leads to genomic instability or aneuploidy, and overexpression of the three paralogs is found in several cancer types, including lung, colon, and gliomas. Aur inhibition induces growth arrest and apoptosis in a variety of cancer cell types. Thus, design and development of Aur inhibitors as anti-tumor agents has been widely explored in recent years, and many are under preclinical and clinical investigation. We used the Eurofins Discovery OncoPanel™ cell-based profiling platform to investigate the specific effects of AurA, AurB, and AurC inhibitors against a select panel of human cancer cell lines. To determine the mechanistic effect of Aur inhibition in cells, we measured phospho-histone H3 (Ser10) by high-content analysis. OncoPanel human tumor cell lines arrested with nocodazole were treated with Aur inhibitors for 2 hours. Phospho-histone H3 (Ser10) levels were measured and the percentage of the DMSO vehicle-treated control cell signal was calculated for each test point. Ten-point dose-response curves were fitted using a 4-parameter log-logistic model with custom curve-fitting software to determine IC50 values. The inhibitors were also evaluated for their effect on tumor cell proliferation, apoptosis, and cell cycle arrest over the same dose-response range, after a 10-day incubation. Following exposure, the cells were fixed, stained with DAPI, anti-cleaved caspase-3, and anti-phospho-histone H3 (Ser28), and imaged. Cell proliferation dose-response curves were also fitted using the same model as above. Correlations were then made between observed mechanistic and functional activity. Activity of the AurA-selective inhibitor, MLN8054, was also evaluated in the Eurofins Discovery BioMAP® Diversity PLUS human primary cell phenotypic profiling service to give a broader view of its biological activity. These studies demonstrate the combined value of using the OncoPanel and BioMAP platforms to determine mechanistic, functional, and broader biological activity of therapeutic candidates in relevant cellular models for a better translational understanding of potential therapeutic capabilities and accelerate decision-making. Citation Format: Luciano Galdieri, Justin Lipner, Steven M. Garner, Jillian Krings, Emily Hoehn, Brendan Lahm, Kaleb Collver, Kaitlyne Powers, Alastair J. King. Phenotypic profiling of Aurora inhibitors using the OncoPanelTMplatform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2770.