Abstract PD3-01: Single cell analysis identifies genes that enable survival of drug tolerant persisters upon treatment with CDK4/6 inhibitors

Abstract CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy prolong survival in ER+/HER2- metastatic breast cancer, but nearly all patients eventually progress underscoring the need of strategies that prevent drug resistance. Drug tolerant persister (DTP) cells are a heterogeneous reservoir of surviving cells from which drug-resistant clones eventually emerge. We hypothesized that targeting breast cancer DTPs upon selective pressure from CDK4/6i would prevent the emergence of drug resistance. First, we identified genomic and epigenomic features of DTPs under continuous therapy. We treated MCF7 and T47D ER+ breast cancer cells with the CDK4/6i palbociclib 250nM + estrogen deprivation (E2dep) long-term (4 weeks). Cells that survived long-term treatment exhibited reversible sensitivity to CDK4/6i + E2dep and reversible cell cycle arrest. Whole-exome sequencing did not reveal known genomic alterations associated with resistance to CDK4/6i. In bulk RNA-seq of MCF7 DTP cells, cell cycle-related gene signatures were downregulated upon treatment, while interferon response and KRAS signatures were upregulated. MCF7 persisters were characterized by enrichment in senescence, diapause, chemotherapy-induced stressed gene signatures, while MYC target signatures were downregulated; all these gene signatures were reversed upon drug washout. To translate these findings to patients, we examined RNA expression data from residual tumors after neoadjuvant treatment with palbociclib and the aromatase inhibitor anastrozole in the NeoPalAna trial. Results were completely consistent with those observed in MCF7 DTPs. To elucidate which 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 + E2dep for up to 4 weeks and then subjected to scRNA-seq; 101 and 15 clones that were initially arrested in G0/G1 eventually expanded at 4 weeks whereas 376 and 27 clones were eliminated during treatment of MCF7 and T47D cells, respectively. Comparison between expanding and exhausted clones identified genes upregulated in DTPs. To identify genes required for DTP survival, we performed a targeted CRISPR-Cas9 knockout screen using a custom library comprising the top 25 DTP marker candidate genes from each cell line, totaling 47 genes, including 3 shared between both cell lines. The library also included previously reported DTP-associated genes such as GPX4, KDM5A, KDM5B, ALDH1A1, ALDH1A2, and IGF1R, as well as RB as a negative control and PLK as a positive control. Cas9-expressing T47D and MCF7 cells were transduced with a lentiviral sgRNA library. Cells were treated with palbociclib + E2dep or 0.1% DMSO for 4 weeks. Enrichment or depletion of sgRNAs in treated and control cells was analyzed. HES1, a transcriptional repressor and downstream target of the Notch pathway, was among the top three genes with the most negative differential beta scores in both T47D and MCF7 cells, suggesting essentiality for DTP survival. In summary, a small subset of ER+ breast cancer cells survives long-term treatment with CDK4/6i + E2dep and exhibits reversible transcriptomic features of DTPs. scRNA-seq analysis of barcoded breast cancer cells revealed genes significantly upregulated in DTP clones that expanded after initial cell cycle arrest. A CRISPR-Cas9 screen of these genes revealed HES1 as a potentially essential gene for DTP survival. We propose therapeutically targeting DTP-associated genes such as HES1 may delay or prevent resistance to CDK4/6i in breast cancer. Citation Format: Y. Matsunaga, E. Aleksandrovic, H. Patel, D. Sudhan, K. Ahuja, D. Ye, C. A. Lin, L. Guo, C. X. Ma, J. Lee, S. Zhang, C. L. Arteaga, A. B. Hanker. Single cell analysis identifies genes that enable survival of drug tolerant persisters upon treatment with CDK4/6 inhibitors abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PD3-01.