Abstract
Combining cyclin-dependent kinase (CDK) inhibitors with endocrine therapy improves outcomes for metastatic estrogen receptor positive (ER+), HER2 negative, breast cancer patients. However, the value of this combination in potentially curable earlier stage patients is not clear. Using single cell transcriptomic profiling, we examined the evolutionary trajectories of early stage breast cancer tumors using serial tumor biopsies from a clinical trial of preoperative endocrine therapy (letrozole) alone or in combination with the cell cycle inhibitor ribociclib. Applying hierarchical regression and Gaussian process mathematical modelling, we classified each tumor by whether it shrinks or persists with therapy and determined cancer phenotypes related to evolution of resistance and cell cycle transcriptional rewiring. We found that all patients’ tumors undergo subclonal evolution during therapy, irrespective of the clinical response. However, tumors subjected to endocrine therapy alone showed reduced diversity over time, while those facing combination therapy exhibited increased diversity. Despite different subclonal diversity, single nuclei RNA sequencing uncovered common phenotypic changes in tumor cells that persist following treatment. In these tumors, cancer cells with accelerated loss of estrogen signaling have convergent up-regulation of the JNK pathway, while cells that maintain estrogen signaling during therapy show potentiation of CDK4/6 activation consistent with ERBB4 and ERK signaling up-regulation. These convergent phenotypes were associated with growing tumors resistant to combination therapy. Cell cycle reconstruction identified that these tumors can rebound during combination therapy treatment, indicating stronger selection and promotion of a proliferative state. These results indicate that combination therapy in early stage ER+ breast cancers with ER and CDK inhibition drives rapid evolution of resistance via a shift from estrogen signaling to alternative growth factor receptor mediated proliferation and JNK signaling activation, concordant with a bypass in the G1 checkpoint.
Competing Interest Statement
Ruth O'Regan participates on the advisory board for Cyclacel, PUMA, Biotheranostics, Lilly, Pfizer, Genentech, Novartis; declares research funding from Pfizer, Novartis, Seattle Genetics, PUMA. Priyanka Sharma declares research funding from Novartis, Merck, Bristol Myers Squibb. Consulting: Seattle Genetics, Merck, Novartis, AstraZeneca, Immunomedics, Exact Biosciences. Laura Spring participates on the advisory board for Novartis, Lumicell, Puma Biotechnology and Avrobio. Cynthia Ma declares research funding from Pfizer, Puma; Consulting: Eisai, Athenex, OncoSignal, Agendia, Biovica, AstraZeneca, Seattle Genetics. Kari Wisinski declares research funding and clinical trial involvement with Novartis, Eli Lilly, Astra Zeneca, Sanofi and Pfizer. He participated on an advisory board for Eisai, Pfizer and Astra Zeneca. Kevin Kalinsky is a medical advisor to Immunomedics, Pfizer, Novartis, Eisai, Eli-Lilly, Amgen, Merck, Seattle Genetics and Astra Zeneca; receives institutional support from Immunomedics, Novartis, Incyte, Genentech/Roche, Eli-Lilly, Pfizer, Calithera Biosciences, Acetylon, Seattle Genetics, Amgen, Zentalis Pharmaceuticals and CytomX Therapeutics; and his spouse is employed by Grail and previously by Array Biopharma and Pfizer. Anne O'Dea Consults for the Pfizer, PUMA Biotechnology, Astra Zeneca, and Daiichi Sankyo. Qamar Khan declares research funding from Novartis. All other authors have no conflicts of interest to disclose.