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

TL;DR

This study identifies genes linked to resistance in breast cancer cells treated with fulvestrant and ribociclib using retroviral screening and sequencing.

Contribution

The study introduces novel resistance genes like TRPS1 and TRIM24 for CDK4/6i and endocrine therapy combinations.

Findings

Thirty-seven VIS loci were associated with resistance to fulvestrant and ribociclib monotherapies.

Twenty loci were linked to resistance in combination therapy, including TRPS1 and TRIM24.

The study validated known resistance genes like BCAR1, BCAR3, and EGFR.

Abstract

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.

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