ESR1 mutations shape the clonal dynamics, genetic diversity and cellular states of acquired resistance to CDK4/6 inhibitors [ATAC-seq]

In estrogen receptor-positive (ER+) breast cancer, CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy (ET) are a standard first-line treatment, however, most patients eventually develop resistance. Although RB1 loss has been implicated in CDK4/6i resistance, its low prevalence suggests additional, context-dependent resistance mechanisms. To investigate the interplay between genetic evolution, clonal dynamics, and cellular plasticity during resistance, we used a high-complexity DNA barcoding system (CloneTracer) and multi-omic profiling in MCF7 models expressing wild-type or Y537S mutant ER. Barcoding revealed that resistance to palbociclib and abemaciclib is driven by the selection of rare pre-existing subclones, rather than stochastic acquisition of resistance. The Y537S mutation shaped the clonal architecture of palbociclib resistance, increasing subclonal diversity and promoting persistent ER signaling, but had less impact on abemaciclib resistance. Whole-exome sequencing uncovered distinct genetic landscapes across models, including RB1 loss in a palbociclib-resistant clone. ATAC-seq and RNA-seq analyses showed that mutant ER reprograms chromatin and transcriptional states during resistance, particularly in palbociclib-treated cells. Single-cell RNA-seq revealed further heterogeneity, with coexisting subpopulations harboring divergent resistance-associated programs within single clones. Finally, in vivo xenograft studies demonstrated site-specific clonal outgrowth in mutant ER metastases and overlap between metastatic and CDK4/6i-resistant subclones, supporting the dual role of specific populations in therapeutic resistance and tumor dissemination. Together, our findings highlight the cooperative roles of genetic drivers and cellular plasticity in resistance to CDK4/6i and suggest that strategies targeting both may be required to overcome therapeutic failure.