Abstract 5339: Single cell analysis enables tracking the evolution of resistance to CDK4/6 inhibitors in ER+ breast cancer

Abstract The combination of CDK4/6 inhibitors (CDK4/6i) with endocrine therapy prolongs survival of patients with ER+/HER2- metastatic breast cancer. Nearly all patients with advanced disease treated with this combination eventually progress, highlighting a need for therapeutic interventions that prevent the emergence of drug resistance. Drug tolerant persister (DTP) cells represent a reservoir of surviving cells from which drug-resistant clones eventually emerge. We hypothesized that targeting breast cancer cells in a DTP state that emerge under selective pressure from CDK4/6i would prevent the emergence of drug resistance. First, we aimed to delineate genomic and epigenomic features of DTPs under continuous CDK4/6i + antiestrogen therapy. We treated MCF7 and T47D ER+ breast cancer cells with palbociclib + estrogen deprivation long-term (4 weeks). Cells that survived long-term treatment exhibited characteristics of DTPs, such as reversible sensitivity to CDK4/6i + estrogen deprivation, reversible cell cycle arrest, and reversible transcriptional programs associated with the DTP state. To translate these findings to patients, we examined gene expression data from residual tumors after neoadjuvant treatment with palbociclib and the aromatase inhibitor anastrozole in the NeoPalAna trial and found that these tumors showed a similar enrichment of gene signatures associated with DTPs compared to tumors before treatment. To elucidate with DTPs give rise to drug resistant clones, we barcoded MCF7 and T47D cells with a lineage and RNA recovery (LARRY) barcode library with ∼315, 000 diverse barcodes, such that we can track them during long-term treatment. Barcoded cells were treated with palbociclib plus estrogen deprivation for 0 h, 72 h, and 4 weeks and then subjected to single-cell RNA-seq; 101 and 15 clones that were initially arrested in G0/G1 eventually expanded at 4 weeks, while 376 and 27 clones were eliminated during treatment of MCF7 and T47D cells, respectively. Comparing the expanding clones with the exhausted clones, we identified 50 marker genes for DTP cells in each cell line. We are currently using a CRISPR-Cas9 targeted knockout screen to identify which of these genes are causal to DTP survival. We will then compare results from these single cell approaches with RNA microarray analysis from residual tumors after neoadjuvant treatment in the NeoPalAna trial. In summary, a small subset of ER+ breast cancer cells survive long-term treatment with CDK4/6i plus estrogen deprivation and exhibit reversible transcriptomic features of DTPs. Single-cell RNA-seq analysis in barcoded breast cancer cells revealed genes significantly upregulated in expanding clones that show features of the DTP state. We proposed therapeutic targeting of these genes may reduce survival of drug-tolerant cells and prevent the emergence of drug resistance. Citation Format: Yuki Matsunaga, Emma Aleksandrovic, Hima M. Patel, Dhivya R. Sudhan, Khushi Ahuja, Dan Ye, Chang-Ching A. Lin, Lei Guo, Cynthia X. Ma, Jeon Lee, Siyuan Zhang, Carlos L. Arteaga, Ariella B. Hanker. Single cell analysis enables tracking the evolution of resistance to CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5339.