CDK4/6 Inhibition reprograms the Breast Cancer immunopeptidome via Rb-Dependent chromatin and transcriptomic remodeling

The Cyclin-dependent kinase 4/6 inhibitors (CDK4/6is) are standard-of-care therapies for metastatic hormone receptor-positive (HR+) breast cancer, yet their immunomodulatory effects remain underexplored. Here, we demonstrate that CDK4/6 inhibition with Abemaciclib reprograms the tumor antigen landscape by increasing MHC-I presentation and reshaping the immunopeptidome in HR+ and triple-negative breast cancer (TNBC) cells. Through multiomics integration, we reveal that this remodeling is driven by retinoblastoma protein (Rb)-dependent transcriptional and chromatin reprogramming, reflected by increased H3K27ac deposition and enhanced chromatin accessibility revealed by ATAC-seq. CDK4/6 inhibition dephosphorylates Rb, enhancing its interaction with BET family proteins and inducing chromatin remodeling that upregulates antigen-source transcripts. We identify immunogenic Abemaciclib-induced MHC-I peptides, including antigens derived from non-coding genomic regions, which can enhance tumor immunogenicity. These findings reveal a previously unrecognized role of CDK4/6is in shaping tumor antigenicity and suggest that combining CDK4/6is with immunotherapy could broaden antigenic targets in breast cancer. Overall design: Abemaciclib (Cat#A115153) was procured from Toronto Research Chemicals Inc. The powder was dissolved in DMSO to prepare a 5 mM stock solution, filtered through a 0.22 µm filter for sterility, aliquoted, and stored at -20°C for no more than 6 months. For Abemaciclib treatment, the 5 mM stock was diluted in complete culture media to the indicated final concentrations. Cells were treated with the indicated final concentration of Abemaciclib or DMSO for 96 h before collection. Total RNA was isolated using TRIzol reagent (Thermo Scientific, cat#15596018) followed by RNeasy purification (Qiagen, Cat. #74104). RNA was quantified using Qubit (Thermo Scientific), and quality was assessed with the 2100 Bioanalyzer (Agilent Technologies). Transcriptome libraries were generated using the KAPA RNA HyperPrep (Roche) using a poly-A selection (Thermo Scientific). Sequencing was performed on the Illumina Novaseq 2000.