ESR1 mutations and CDK4/6 inhibitor choice shape clonal selection and adaptive cell states during acquired resistance

In estrogen receptor-positive (ER +) breast cancer, CDK4/6 inhibitors (CDK4/6is) combined with endocrine therapy (ET) are standard first-line treatment for metastatic disease. However, most patients eventually develop resistance. Activating ESR1 mutations are a prevalent mechanism of acquired resistance to ET and are enriched after ET plus a CDK4/6 inhibitor (CDK4/6i), but their role in the clonal evolution and adaptive mechanisms of acquired resistance to CDK4/6 inhibition, independent of ET, is unknown. In addition, whether different CDK4/6is impose distinct selective pressures and divergent resistance states remains elusive. To investigate the clonal dynamics, cell states and cellular plasticity during acquired CDK4/6i resistance in mutant versus wild-type (WT) ESR1 , we performed high-complexity DNA barcoding (ClonTracer library) with longitudinal sampling and multi-omic profiling in an isogeneic MCF7 model expressing WT ER or Y537S mutant ER . We also evaluated the clonality of the ESR1 mutations in clinical samples with CDK4/6i resistance. We showed that ESR1 mutations are enriched in clinical tumors with acquired resistance to CDK4/6is, and in paired biopsies expanded to near clonality after treatment. We demonstrated progressive clonal selection with both divergent and partially convergent evolutionary trajectories. The ESR1 mutation substantially reshapes clonal and epigenetic evolution during palbociclib resistance but had a weaker impact under abemaciclib selection. Overall, clonal evolution and cell states in palbociclib and abemaciclib resistance were distinct. Single-cell RNA-seq revealed transcriptional heterogeneity highlighting cellular plasticity during passaging of cells and selection. Finally, in vivo barcoding of mammary xenograft, local recurrences, and distant metastases demonstrated site-specific clonal outgrowth in mutant ER metastases, and partial overlap between metastatic and CDK4/6i-resistant subclones, supporting the dual role of specific populations in therapeutic resistance and metastatic colonization. High-resolution lineage tracing and multi-omic studies demonstrate that CDK4/6i resistance is shaped by clonal selection and adaptive remodeling of cell states, with the ESR1 mutation status and the specific inhibitor acting as key determinants of evolutionary trajectories. These findings suggest that both variables should be considered when designing sequential and combination treatment strategies to overcome CDK4/6i resistance.