Identification of Resistance Genes in Breast Cancer Cells Treated with Fulvestrant and Ribociclib via Retroviral Screening and Integration Site Sequencing

Around 30% of patients with hormone receptor-positive (HR+) breast cancer acquire resistance to endocrine therapy combined with cyclin-dependent kinase 4/6 inhibitors (CDK4/6i), which are first-line treatments in metastatic settings. Therefore, we aimed to identify loci associated with resistance to endocrine therapy and CDK4/6i; this was achieved using retroviral vectors, which randomly insert gene-disrupting elements into the genome, causing gene expression alterations and potentially leading to therapy resistance. ER-positive ZR75.1 breast cancer cells transduced with retroviral vectors were treated with endocrine (tamoxifen, fulvestrant) or CDK4/6i monotherapies (abemaciclib, palbociclib, ribociclib) or a combination of fulvestrant and ribociclib. DNA was extracted, and virus integration sites (VISs) were characterized according to the detection frequency and read depth using next-generation sequencing (VIS-NGS). Resistance-associated VIS loci were identified when differentially presented in treated samples compared to controls. Well-established tamoxifen resistance genes (BCAR1, BCAR3, EGFR) were detected, enabling the validation of our approach. Thirty-seven VIS loci were associated with resistance to fulvestrant and ribociclib monotherapies. Twenty of these loci were also identified as candidates for resistance to other CDK4/6i and to fulvestrant and ribociclib combination therapy, including TRPS1 and TRIM24—genes that are involved in resistance to endocrine therapy but have not yet been associated with resistance to CDK4/6i. The identification of unique and shared resistance-associated loci highlights the complexity of resistance pathways.