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Background Despite the remarkable activity of CDK4/6 inhibitors (CDK4/6i) in the treatment of estrogen receptor positive (ER+) metastatic breast cancer (BC), most patients eventually develop resistance to these drugs. The ctDNA analysis of the PALOMA-3 trial showed that the estrogen receptor (ER) mutation Y537S is a potential mechanism of acquired resistance to the combination of endocrine therapy (ET) with CDK4/6i. To date, the role of the ER mutations in the clonal evolution and the mechanisms of acquired resistance to CDK4/6i is unknown. Moreover, it is not known if the development of resistance to CDK4/6i in the presence or absence of ER mutations is due to the expansion of pre-existing resistant clones or to the de novo acquisition of resistance mechanisms. Methods To explore the clonal evolution and the mechanisms of resistance to CDK4/6i in ER-wild type (ER-WT) and ER-mutant (ER-Mut) BC, we transduced doxycycline (DOX)-inducible Y537S ER-Mut MCF7 cells with the ClonTracer library, a high-complexity DNA barcode library, and cultured the barcoded cells without DOX (MCF7), or with DOX to induce the expression of the Y537S ER mutation (MCF7-YS). To develop Palbociclib (Palbo)-resistant (PDR) and Abemaciclib (Abema)-resistant (ABR) cell models, the barcoded MCF7 and MCF7-YS cells were passaged in culture with increasing concentrations of Palbo and Abema until the acquisition of resistance. The clonal dynamics and the molecular characteristics of the PDR and ABR models were investigated by barcode sequencing, whole-exome sequencing (WES), bulk and single cell RNA sequencing (RNAseq) and protein analyses. Finally, using an ER-Mut barcoded mice model, we compared the in vitro clonal evolution of ER-Mut CDK4/6i-resistant cells with the in vivo clonal evolution of ER-Mut metastases. Results The analysis of the barcodes revealed that during the acquisition of resistance to either Palbo or Abema there is a strong clonal selection of pre-existing resistant clones. The PDR clones were different in the presence of the Y537S mutation versus WT-ER. In contrast, the clones enriched in the ABR cells were comparable between WT and mutant ER. Furthermore, the ER mutations led to decreased diversity of the enriched clones in the PDR but not in the ABR cells. Interestingly, the barcodes enriched in the PDR and ABR models did not overlap. Unsupervised analyses showed that the samples clustering based on the barcodes fractions and the mutations were similar, suggesting that the clonal selection was driven by cellular populations with specific mutational landscapes. All the ER-WT and ER-Mut resistant models had different transcriptional profiles and by single-cell RNAseq showed various degrees of intra-sample heterogeneity. At the protein level, the PDR and the ABR cells displayed downregulation of ER, Rb and p27 and upregulation of p21. In the ER-Mut conditions Cyclin D1 was upregulated in the PDR cells, while Cyclin E was upregulated in the ABR cells. Finally, the barcode sequencing of the mice metastases revealed that the clonal selection in ER-Mut metastases and in ER-Mut CDK4/6i-resistant cells is different. Conclusion Our study suggests that the development of resistance to CDK4/6i is due to the selection of pre-existing resistant clones. We also demonstrate that the expression of the Y537S ER mutation impacts the clonal evolution and the mechanisms of acquired resistance to Palbo but not to Abema. Finally, we show that the clonal evolution and mechanisms are disparate in Palbo and Abema resistance. These results support the addition of a third drug to CDK4/6i and ET, early in treatment, to delay the selection of pre-existing resistant clones and prolong the response to treatment and highlight differences between Palbo and Abema. Citation Format: Cristina Guarducci, Simona Cristea, Avery Feit, Sergey Naumenko, Agostina Nardone, Wen Ma, Douglas Russo, Gabriella Cohen Feit, Ariel Feiglin, Francisco Hermida-Prado, Shira Sherman, Myles Brown, Franziska Michor, Rinath Jeselsohn. GS3-07 Clonal evolution and mechanisms of acquired resistance to CDK4/6 inhibitors in ER-wild type and ER-mutant breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS3-07. |
