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.
e13011 Background: Trastuzumab is a monoclonal antibody that targets the human epidermal growth factor receptor-2 (HER2). Its use may result in cardiotoxicity. There are no formal guidelines for left ventricular ejection fraction (LVEF) monitoring in patients with metastatic disease treated with trastuzumab. Our local guideline include an echocardiogram every 3 months for these patients. Methods: We collected data from electronic records and hard copy files of patients treated with Trastuzumab as first line, between the years 2011-2014. Results: One hundred patients met the inclusion criteria. Median treatment duration of all patients was 30 months, with an average of 12 echocardiogram follow-ups per patient. Ninety-nine patients (99%) received additional chemotherapy. Thirty patients (30%) also received Pertuzumab. Ten (10%) of the eligible 100 patients showed significant decline in EF (≥10%) to a final LVEF of < 50%. The median follow-up duration of these patients was 52 months. Median EF at the beginning of treatment was 61.5% in all patients. Cardiotoxicity occurred at an average of 23 months [median 18 months] after beginning of Trastuzumab treatment (range; 3-54 months), with an average EF decline of 14% (range; 10-18%). Three patients (30%) required treatment change due to the decline in EF. Temporary cessation of treatment was necessary in 4 (40%) of the patients for a period of time between 4 weeks to 8 months. The remaining 3 patients (30%), although developing significant decline in EF, did not require treatment cessation or change. None of the 10 patients presented any other clinical sign of cardiotoxicity. In the group with cardiac events, there were no previous reports of congestive heart failure (CHF), ischemic heart failure (IHF), myocardial infraction (MI) or diabetes. Only one patient was a smoker and another received hormone replacement therapy. The average age of this group at the time of diagnosis of metastatic disease was younger (50.6 vs. 55.8, P-value 0.5). Conclusions: Monitoring heart function by trimonthly echocardiogram does not seem to be the optimal monitoring modality in metastatic breast cancer patients who receive a trastuzumab-containing regimen as first line. This study results suggest that less frequent monitoring may be sufficient, thus diminishing patients' inconvenience and allowing better allocation of health care resources.
Immunotherapies have yet to demonstrate significant efficacy in the treatment of hormone receptor positive (HR+) breast cancer. Given that endocrine therapy (ET) is the primary approach for treating HR+ breast cancer, we investigated the effects of ET on the tumor immune microenvironment (TME) in HR+ breast cancer. Spatial proteomics analysis of primary HR+ breast cancer samples obtained at baseline and after ET from patients enrolled in a neoadjuvant clinical trial (NCT02764541) indicated that ET upregulated B2-microglobulin and influenced the TME in a manner that promotes enhanced immunogenicity. To gain a deeper understanding of the underlying mechanisms, the intrinsic effects of ET on cancer cells were explored, which revealed that ET plays a crucial role in facilitating the chromatin binding of RelA, a key component of the NF-κB complex. Consequently, heightened NF-κB signaling enhanced the response to interferon-gamma, leading to the upregulation of β2-microglobulin and other antigen presentation-related genes. Further, modulation of NF-κB signaling using a SMAC-mimetic in conjunction with ET augmented T-cell migration and enhanced MHC-I specific T-cell mediated cytotoxicity. Remarkably, the combination of ET and SMAC-mimetics, which also block pro-survival effects of NF-κB signaling through the degradation of inhibitors of apoptosis (IAP) proteins, elicited tumor regression through cell-autonomous mechanisms, providing additional support for their combined use in HR+ breast cancer.
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