Abstract PO5-23-10: Development of a long-term in vitro assay able to identify compounds that can overcome resistance to CDK4/6 inhibitor plus endocrine therapy in ER+ HER2- breast cancer
Authors: Kronstadt Stephanie , Umbreit Neil , Niederst Matt , Janssens Rens 
Published: May 2024

Abstract
CDK4/6 inhibitor plus endocrine therapy (ET) has emerged as the standard of care first line therapy in ER+ HER2- breast cancer. While this combination has shown the ability to extend overall survival, nearly all patients acquire resistance and progress after 2-3 years. Subsequently, interest has turned to identifying a third partner compound that can be integrated into the CDK4/6i plus ET backbone regimen with the goal of overcoming resistance and driving a deeper response. However, a comprehensive assay-based workflow proficient in identifying compounds with the most promising potential is lacking. To fill this need, we set out to develop a high-throughput in vitro assay with relevant multi-faceted readouts to assess the efficacy of potential triplet combinations using 12 human ER+ breast cancer lines. For this assay, cells were engineered to express nuclear RFP to enable accurate cell counting using basic imaging techniques in 384 well plates. Each third partner compound was tested at two different doses and, in addition to the triplet combination (CDK4/6i + ET + third partner), single agent as well as double agent combinations were investigated. The assay was imaged and re-dosed every 5 days until day 15. On day 15, the compounds were washed out and the cells underwent a 25-day drug holiday to monitor relapse. Duplicate plates of all conditions were monitored and imaged every two hours for 24 hours for live cell staining of apoptosis markers. Additional duplicate plates were fixed and stained on days 5 and 15 for markers of senescence and cell cycle. Images were subsequently fed into a python-based algorithm to determine the percentage of cell population expressing the various markers and phenotypic correlates were extracted. We are looking for triplet combinations that induce a deeper cell cycle arrest, senescence, and/or cell death compared with the CDK4/6 inhibitor plus ET only treatment. Our drug holiday results will help elucidate the treatments able to continue to induce suppression once washed out, indicating treatment options that may induce long-lasting, possibly irreversible effects on the cells. Finally, by extracting phenotypic correlates from brightfield images we hope to cluster the various compounds in their effects across cell lines of diverse backgrounds to help understand the best way to benefit multiple patient populations. Overall, the assay will be able to identify and differentiate various therapeutic combinations based on cell count, senescence, and cell death. The format of this assay enables depth of analysis not accessible using other techniques such as FACS, including full dose-response curves, combination matrices, and longitudinal analyses. Additionally, the assay can be modified to evaluate any number of cellular markers and can be useful across a number of applications. The approach developed here will be foundational in discovering next-step strategies for patients with therapy resistance.
Citation Format: Stephanie Kronstadt, Neil Umbreit, Matt Niederst, Rens Janssens. Development of a long-term in vitro assay able to identify compounds that can overcome resistance to CDK4/6 inhibitor plus endocrine therapy in ER+ HER2- breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-23-10.