Abstract GS3-06: Rb Functions as a Transcriptional Activator of ER Targets Following CDK4/6 Inhibition in Luminal Breast Cancer

Abstract CDK4/6 inhibitors induce durable responses in estrogen receptor-positive (ER+) breast cancer, primarily via activation of the tumor suppressor Rb. While Rb is classically viewed as a repressor of E2F-dependent cell cycle genes, evidence from the pre-genomic era suggested broader roles in gene regulation. Here, we identify a previously unrecognized function of Rb as a transcriptional activator in luminal breast cancer, with key implications for endocrine sensitivity and resistance. Using CUT&RUN, we mapped genome-wide Rb binding in ER+ breast cancer cell lines (MCF7, ZR-75-1) and a patient-derived xenograft (PDX) following abemaciclib or palbociclib treatment. CDK4/6 inhibition led to Rb hypo-phosphorylation and a large increase in chromatin association. As expected, Rb localized to E2F-regulated promoters, repressing canonical cell cycle genes. Unexpectedly, drug-induced hypo-phosphorylated Rb also bound thousands of non-promoter sites that gained H3K27ac upon CDK4/6 inhibition, and these regions were associated with transcriptional upregulation of nearby genes—many enriched for estrogen receptor targets. H3K27ac HiChIP showed that CDK4/6 inhibition caused Rb to localize to enhancer-rich regulatory hubs, many looping to ER target gene promoters. Notably, 60% of these Rb-associated hubs overlapped ER-bound loci. Canonical ER target promoters (e.g., CCND1, TFF1) and luminal markers (e.g., KRT18, KRT19) were loop-connected to Rb-bound enhancers and transcriptionally induced following treatment, suggesting that hypo-phosphorylated Rb directly engages enhancers to promote ER-driven transcription. Consistent with this, bulk RNA-seq revealed that estrogen response gene sets were among the top upregulated pathways in cell lines and PDX tumors after CDK4/6 inhibition. Transcriptomic analysis from the POP trial (N=44) showed that short-term palbociclib monotherapy induced consistent upregulation of estrogen response gene sets, supporting the clinical relevance of Rb-mediated transcriptional activation. Functional validation using CRISPR-Cas9 deletion of an Rb-bound enhancer looping to the KRT18 promoter impaired its induction by abemaciclib. RB1 knockout suppressed estrogen response gene upregulation genome-wide, confirming functional dependence on Rb. To further understand Rb-ER interaction, we studied isogenic MCF7-derived models harboring wild-type or mutant ESR1. In the wild-type setting, Rb activation by CDK4/6 inhibitors enhanced ER-responsive gene expression and reinforced luminal identity, explaining synergy with endocrine therapy. In ESR1-mutant cells, which exhibit ligand-independent ER activity, the Rb-associated enhancer program persisted despite estrogen deprivation (mimicking aromatase inhibition). This sustained expression of estrogen-responsive genes (including CCND1, encoding cyclin D1) offers a potential explanation for resistance to CDK4/6 inhibitor plus AI combinations in ESR1-mutant tumors. Finally, immunoprecipitation of Rb from nuclear lysates followed by mass spectrometry confirmed an interaction between Rb and ER after CDK4/6 inhibition, along with increased association of Rb with chromatin modifiers KDM5A and KDM5B. Knockdown of either KDM5A or KDM5B suppressed CDK4/6 inhibitor-induced, Rb-dependent estrogen response gene expression, implicating these demethylases as functional mediators linking hypo-phosphorylated Rb to ER-driven transcription. These findings redefine Rb as a dual-function regulator: repressing E2F-driven cell cycle genes while promoting estrogen-responsive transcription via enhancer engagement. This paradigm reshapes our understanding of Rb biology and highlights a mechanistic basis for CDK4/6 inhibitor activity, synergy with endocrine therapy, and resistance in ESR1-mutant tumors. Citation Format: A. C. Watt, A. Ahn, C. Blyth, J. Dixon-Douglas, K. Ambani, R. Coulson, M. Taylor, K. Chan, C. Dietrich, B. E. Russ, S. Ramm, C. A. Mahendra, K. Lu, N. Pires, E. Lim, S. Chandarlapaty5, F. Andre, S. Goel. Rb Functions as a Transcriptional Activator of ER Targets Following CDK4/6 Inhibition in Luminal Breast Cancer [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 GS3-06.