Abstract 5827: Identification of potential novel drivers and biomarkers of CDK4/6 inhibitor resistance in metastatic breast cancer

Abstract Background: Inhibitors of the cyclin-dependent kinases 4 and 6 (CDK4/6i) have become part of the standard of care for patients with hormone receptor-positive metastatic breast cancer. Despite their initial benefit for most patients, the development of resistance to this treatment is almost inevitable. However, there is a lack of predictive or clinical markers that can be utilized to monitor resistance development, and their definition remains an unmet challenge. Methods: We aimed to identify novel drivers and potential biomarkers of CDK4/6i resistance by creating and characterizing two ribociclib-resistant breast cancer cell lines. Ribociclib-resistant derivatives of MCF7 cells and the CTC (circulating tumor cell)-derived cell line CTC-ITB-01 were created by long-term exposure to the inhibitor for a minimum of 6 months and potential novel resistance drivers were identified by transcriptome analysis using RNA-sequencing. In addition to mRNAs encoding proteins that may contribute to resistance, we also investigated microRNAs as they are poorly characterized in the context of CDK4/6i resistance. Additionally, changes in the kinome activity were assessed by using PamGene technology. Results: The RNA-sequencing analysis identified ≥ 2000 genes with differential expression in both resistant cell lines compared to their respective parental counterparts. Over Representation Analysis revealed that a high number of these genes encode proteins associated with the cell cycle, proliferation, epithelial-to-mesenchymal transition, various signaling pathways like Wnt, and the complement system. Evaluation of kinase activity using functional kinome profiling in the resistant versus parental CTC-ITB-01 cell line showed increased activity of the CDK and MAPK kinase subgroups, indicating a multifactorial resistance mechanism. Furthermore, the analysis of deregulated ncRNAs demonstrated increased expression of miR-146a-5p in both resistant cell lines and interestingly, a significant decrease of miR-205-5p levels in the resistant CTC-ITB-01 cell line. MiR-205-5p is described as a negative regulator of EMT and progression in breast cancer but has not been linked to CDK4/6i resistance yet. This result was validated by qPCR and miRNA in situ-hybridization. Conclusion: Through our comprehensive transcriptome analysis of two ribociclib-resistant cell lines, we were able to identify several novel potential resistance drivers. Their utility as predictive or monitoring biomarkers requires further investigation. Furthermore, discovering new resistance mechanisms could also help find new therapeutic strategies after CDK4/6i resistance development. Acknowledgment: This project received funding from the Deutsche Krebshilfe DETECT high project (Nr.: 70114705) and the Horizon 2020 program RNADiagon (Nr.: 824036). Citation Format: Leonie F. Ott, Malik Alawi, Malgorzata Stoupiec, Elena Laakmann, Malte Kriegs, Volkmar Müller, Klaus Pantel, Sabine Riethdorf. Identification of potential novel drivers and biomarkers of CDK4/6 inhibitor resistance in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5827.