Abstract PO3-23-09: ESR1 mutations drive resistance to CDK4/6 inhibitors in ER+ Breast Cancer
Authors: Chica-Parrado M Rosario , Lin Chang-Ching , Jaeger Ellen , Harris Michelle , Guo Lei , Bikorimana Emmanuel , Napolitano Fabiana , Chao Calvin , Hanker Ariella , Arteaga Carlos 
Published: May 2024

Abstract
Mutations in ESR1 (the gene encoding the estrogen receptor, ERα) are major drivers of resistance to antiestrogen therapy in ER+ breast cancer. However, it remains unclear whether ESR1 mutations also drive resistance to CDK4/6 inhibitors (CDK4/6i). We analyzed de-identified next generation sequencing (NGS) data of tumor tissue DNA (n=1,820) and circulating tumor (ct) DNA (n=2,138) from patients with ER+/HER2- metastatic breast cancer. NGS of tumor tissue DNA and ctDNA previously had been performed using the Tempus xT tumor assay (595-648 genes) and Tempus xF liquid biopsy (105-523 genes), respectively. Patients were stratified into treated (xT:1,070; xF:1,885) or untreated (xT:750; xF:253) with CDK4/6i prior to biopsy, and untreated patients were restricted to those biopsied within 30 days of metastatic diagnosis. ESR1 was the only gene for which mutations were significantly enriched in patients who received CDK4/6i vs. those who did not in both xT (32% vs. 4.8%, q&lt; 0.001) and xF (29% vs. 9.5%, q&lt; 0.001) cohorts.
To determine whether ESR1 mutations drive resistance to CDK4/6i, we used MCF-7 cells carrying ESR1 wild-type (WT), Y537S, or D538G ‘hotspot’ knock-in mutations to assess cell proliferation upon treatment with the CDK4/6i palbociclib. Cells harboring the Y537S and D538G mutations displayed a 3-fold increase in the IC50 of palbociclib single agent compared to ESR1WT cells. When treated with palbociclib + fulvestrant or palbociclib + estrogen-free medium (mimicking estrogen suppression in patients treated with aromatase inhibitors), cells harboring the Y537S and D538G mutations displayed an 11.2 to 46.9-fold increase in the IC50 of palbociclib. Treatment with palbociclib arrested growth of MCF-7 ESR1WT xenografts established in ovariectomized nude mice whereas MCF-7 ESR1Y537S and MCF-7 ESR1D538G tumors continued to grow. These results suggest that ESR1 mutations enable cell proliferation and tumor growth in the presence of CDK4/6i and the combination of CDK4/6i + antiestrogens.
Next, we performed RNA-seq to investigate the mechanisms underlying ESR1 mutation-mediated resistance to CDK4/6i. Gene set enrichment analysis (GSEA) of the RNA-seq data showed significant upregulation of cell cycle-related Hallmark gene signatures such as E2F targets, G2/M checkpoints, and mitotic spindle in ESR1Y537S vs. ESR1WT and ESR1D538G vs. ESR1WT MCF-7 cells upon treatment with palbociclib. Upregulation of these gene signatures in ESR1-mutant vs. WT cells was also observed upon treatment with palbociclib + fulvestrant and palbociclib in estrogen-free media, suggesting that ESR1 mutations hamper the inhibitory effects of CDK4/6i alone or in combination with antiestrogens on cell cycle progression. Additionally, RNA-seq analysis showed that cells expressing either the Y537S or D538G mutation had elevated basal gene signatures (METABRIC_basal, TCGA_basal, and Huper_basal) compared to ESR1WT cells, consistent with previous studies suggesting that ESR1 mutations promote a luminal-to-basal subtype switch in ER+ breast cancers. We are currently investigating whether this lineage reprogramming underlies resistance to CDK4/6i in ESR1-mutant cells. In addition, we will validate our findings by analyzing the tumor tissue transcriptome from the patients included in this study.
Conclusions: ESR1 mutations are enriched following treatment with CDK4/6i in a cohort of 3,958 patients with ER+/HER2- metastatic breast cancer. Mutations in ESR1 in ER+ breast cancer cells directly promote resistance to CDK4/6i alone or CDK4/6i + antiestrogens in vitro and in vivo.
Citation Format: M Rosario Chica-Parrado, Chang-Ching Lin, Ellen Jaeger, Michelle Harris, Lei Guo, Emmanuel Bikorimana, Fabiana Napolitano, Calvin Chao, Ariella Hanker, Carlos Arteaga. ESR1 mutations drive resistance to CDK4/6 inhibitors in ER+ Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-23-09.