Abstract 6605: Spatially-resolved single-cell transcriptome landscape of response and resistance to CDK4/6 inhibitors in patients with hormone receptor-positive metastatic breast cancer

Abstract Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) have significantly improved the treatment of hormone receptor (HR+) positive breast cancer. Clinical trials have demonstrated that addition of CDK4/6i to endocrine therapy (ET) improves both progression-free and overall survival in patients with HR+ metastatic breast cancer (MBC). Nevertheless, their impact remains limited by the eventual development of resistance. While a number of resistance mechanisms have been identified using both preclinical model systems and translational interrogation of patient specimens, these are not apparent in all cases. As part of the Human Tumor Atlas Network, we integrated genomic (whole-exome sequencing; n=47), single-nucleus transcriptomic (snRNA-seq; n=56), and spatial expression profiling (MERFISH and multiplex immunofluorescence; n=22 and n=17, respectively) from 58 clinically annotated MBC biopsy specimens collected before or after treatment with CDK4/6i plus ET to dissect the roles of tumor-intrinsic programs and the tumor microenvironment (TME) in CDK4/6i resistance. Transcriptomic analysis of the tumor cell compartment with snRNA-seq not only extended prior findings including an association between expression of cell-cycle programs and CDK4/6i resistance, but importantly identified additional programs associated with response and resistance including ciliogenesis, immune evasion, and inflammation. Additionally, tumor-intrinsic gene programs related to epithelial-to-mesenchymal transition had a decreased activity in ESR1 mutant biopsies, a mutational feature of CDK4/6i-resistant MBC associated with upregulated estrogen receptor pathway activity. In the TME, CD8+ T cells were depleted in the CDK4/6i-resistant biopsies, both before and after treatment. In contrast, the CDK4/6i-sensitive biopsies displayed pro-inflammatory features including an increased number of CXCL9+/CXCL10+ macrophages and clustering of CD8+ T cells in the vicinity of the malignant cells spatially. Notably, MERFISH also demonstrated an association between spatial architecture and CDK4/6i response, with the malignant cells in the CDK4/6i-resistant biopsies dispersed spatially compared to the sensitive biopsies. In summary, to the best of our knowledge, this study represents the largest spatially-resolved single-cell MBC cohort with curated CDK4/6i response status established to date. Our findings offer insights into potential biomarkers for CDK4/6i response based on tumor-intrinsic and tumor-microenvironment mechanisms, as well as therapeutic opportunities. Citation Format: Junko Tsuji, Jorge Gómez Tejeda Zañudo, Timothy R. Blosser, Ha T. Vu, Danielle Firer, Nick Maus, Kathleen L. Pfaff, Billie A. Porter, Jason L. Weirather, Anne Carlisle, Allison M. Frangieh, Elliot Boblitt, Aaron R. Thorner, Karla E. Helvie, Isabella E. Kallassy, Laura K. DelloStritto, Ragnhild Laursen, François Aguet, Mendy Miller, Melissa E. Hughes, Nancy U. Lin, Sara M. Tolaney, Eliezer M. Van Allen, Samouil L. Farhi, Xiaowei Zhuang, Barbara Engelhardt, Scott J. Rodig, Alex K. Shalek, Aviv Regev, Bruce E. Johnson, Nikhil Wagle, Gad Getz, Daniel L. Abravanel. Spatially-resolved single-cell transcriptome landscape of response and resistance to CDK4/6 inhibitors in patients with hormone receptor-positive metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6605.