Non-Canonical Senescence Phenotype in Resistance to CDK4/6 Inhibitors in ER-Positive Breast Cancer

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) have transformed the treatment landscape for estrogen receptor-positive (ER+) breast cancer, yet re-sistance remains a major clinical challenge. Although CDK4/6i induce G1 ar-rest and therapy-induced senescence (TIS), the exact nature of this senescent state and its contribution to resistance are not well understood. To explore this, we developed palbociclib- (2PR, 9PR, TPR) and abemaciclib- (2AR, 9AR, TAR) resistant ER+ breast cancer sublines through prolonged drug exposure over six months. Resistant cells demonstrated distinct phenotypic alterations, including cellular senescence, reduced mitochondrial membrane potential, and impaired glycolytic activity. Cytokine profiling and Enzyme-Linked Im-munosorbent Assay (ELISA) validation revealed a non-canonical Senes-cence-Associated Secretory Phenotype (SASP) characterized by elevated Growth/differentiation factor 15 (GDF-15) and serpin E1 (Plasminogen acti-vator inhibitor-1, PAI-1), and absence of classical pro-inflammatory interleu-kins including IL-1α and IL-6. IL-8 levels were significantly elevated, but no association with epithelial-mesenchymal transition (EMT) was observed. Re-sistant cells preserved their epithelial morphology, showed no upregulation of EMT markers, and lacked Aldehyde dehydrogenase 1-positive (ALDH1+) stem-like populations. Additionally, Regulated Upon Activation, Normal T-cell Expressed, and Secreted (RANTES) was strongly upregulated in palbo-ciclib-resistant cells. Together, these findings identify a distinct, non-canonical senescence phenotype associated with CDK4/6i resistance and may provide a foundation for identifying new vulnerabilities in resistant ER+ breast cancers through targeting SASP-related signaling.