Journal Article

Models of Early Resistance to CDK4/6 Inhibitors Unveil Potential Therapeutic Treatment Sequencing

Pulliam, Nicholas, Ding, Yan, de Dios, Alfonso, Ortiz-Ruiz, Maria Jesus, Lallena, María José, Zapatero-Solana, Elisabet

2025

Overview

Background: CDK4/6 inhibitors (CDK4/6i) combined with hormone therapies have demonstrated clinical benefit in HR+, HER2- breast cancer patients. However, the onset of resistance remains a concern and highlights a need for therapeutic strategies to improve outcomes. The objective of this study was to develop an in vitro model to better understand the mechanisms of resistance to CDK4/6i + hormone therapies and identify therapeutic strategies with potential to overcome this resistance. Methods: The HR+, HER2− T47D breast cancer cell line genetically modified with a Geminin–Venus reporter construct was treated with CDK4/6i (abemaciclib or palbociclib) in combination with 4-hydroxytamoxifen (tamoxifen). Resistant cells were identified by cell sorting for Geminin (%GEM+), a marker of the S/G2/M phases of the cell cycle, and confirmed by treatment with tamoxifen plus the CDK4/6i used to drive resistance. In resistant cells, following treatment with CDK4/6i + ET (tamoxifen or fulvestrant), the effects on cell proliferation (%GEM+) and viability, gene expression, and protein analysis to evaluate CDK4/6–cyclin D complex composition were examined. Results: Palbociclib + tamoxifen-resistant (PTxR) cells treated with abemaciclib + ET showed decreased %GEM+, %Ki67, and colony formation ability, compared to abemaciclib + tamoxifen-resistant (ATxR) cells treated with palbociclib + ET. Additionally, PTxR cells showed increased CDK4-p21 interaction, compared to ATxR. The CDK6 levels were greater in ATxR cells compared to PTxR cells, associated with CDK4/6i resistance. Additionally, abemaciclib + fulvestrant continued to robustly decrease pRb levels in PTxR models compared to palbociclib + fulvestrant in ATxR models. Transcriptome analysis revealed a depression of the cell cycle and E2F- and Rb-related genes in PTxR cells following treatment with abemaciclib + ET, not present in ATxR cells treated with palbociclib + ET. Both resistant models showed increased EGFR-related gene expression. Conclusion: Taken together, we describe CDK4/6i-dependent mechanisms resulting in early-onset resistance to CDK4/6i + ET, using clinically relevant drug concentrations, in preclinical breast cancer cell models. The characterization of these preclinical models post progression on CDK4/6 inhibitor + ET treatment highlights the potential that the specific sequencing of CDK4/6 inhibitors could offer to overcome acquired resistance to CDK4/6i + ET. Abemaciclib + fulvestrant is currently under clinical investigation in patients with HR+, HER2− breast cancer and progression on prior CDK4/6i + ET (NCT05169567, postMONARCH).

• Publisher: MDPI AG,MDPI
• Subject: Acrylamides - pharmacology / Aminopyridines - pharmacology / Apoptosis / Benzimidazoles - pharmacology / Biomarkers / Breast cancer / Breast Neoplasms - drug therapy / Breast Neoplasms - genetics / Breast Neoplasms - metabolism / Breast Neoplasms - pathology / Cancer / Cell cycle / Cell growth / Cell Line, Tumor / Cell Proliferation - drug effects / Cyclin-Dependent Kinase 4 - antagonists & inhibitors / Cyclin-Dependent Kinase 4 - metabolism / Cyclin-Dependent Kinase 6 - antagonists & inhibitors / Cyclin-Dependent Kinase 6 - metabolism / Cyclin-dependent kinases / Drug dosages / Drug resistance / Drug Resistance, Neoplasm - drug effects / Drug Resistance, Neoplasm - genetics / Drug therapy / Female / Fulvestrant - pharmacology / Gene Expression Regulation, Neoplastic - drug effects / Humans / Kinases / Medical diagnosis / Medical prognosis / Oncology, Experimental / Phosphorylation / Piperazines - pharmacology / Protein expression / Protein Kinase Inhibitors - pharmacology / Proteins / Pyridines - pharmacology / Tamoxifen - analogs & derivatives / Tamoxifen - pharmacology