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Background Cyclin-dependent kinase 4/6 inhibitors (CDK4/6is) are established standard-of-care therapies for the treatment of hormone receptor-positive (HR+) metastatic breast cancer (BC), yet they offer limited clinical benefit in triple-negative breast cancer (TNBC). Despite their widespread use, the immunomodulatory effects of CDK4/6is—particularly their influence on tumor antigenicity—remain poorly understood. Emerging evidence suggests that CDK4/6is such as Abemaciclib may enhance tumor immunogenicity by modulating the tumor immune microenvironment and promoting immune recognition. However, their specific impact on the repertoire of antigens presented by tumor cells remain to be defined.Methods To investigate how CDK4/6 inhibition alters tumor antigen presentation, we treated two breast cancer cell lines—CAMA1 (HR+) and MDAMB231 (TNBC)—with either Abemaciclib or DMSO, and analyzed the MHC I-bound antigen repertoire using mass spectrometry. To maximize antigen detection, mass spectrometry data were searched against customized protein databases derived from RNA sequencing (RNA-seq) of the same samples. This proteogenomic approach enabled the identification of both canonical and non-canonical antigens, including peptides originating from non-coding regions of the genome.Results Treatment with Abemaciclib significantly increased the diversity and abundance of MHC-I-associated peptides (MAPs), with the most pronounced effects observed in the HR+ cell line, consistent with its greater clinical sensitivity to CDK4/6is. Using an established pipeline developed for tumor antigen discovery, we identified a distinct subset of novel MAPs uniquely induced by Abemaciclib. These antigens—derived predominantly from non-coding genomic regions—represent promising candidates for combinatorial immunotherapies.Mechanistic investigations integrating public transcriptomic, ATAC-seq, and ChIP-seq datasets revealed that part of the Abemaciclib-induced remodeling of the immunopeptidome is driven by Retinoblastoma protein (Rb)-dependent epigenetic remodeling. Using isogenic cell lines expressing specific Rb isoforms, we demonstrate that dephosphorylation of key Rb residues facilitates interactions with BET proteins, leading to chromatin remodeling and transcriptional activation of genes encoding induced antigens.Conclusions Our study reveals a novel role for CDK4/6 inhibition in reshaping tumor antigen presentation through Rb-driven epigenetic remodeling. Abemaciclib not only upregulates MHC-I machinery but also broadens the immunopeptidome, unveiling new antigens including those from non-coding regions that may enhance immune recognition. These findings provide a strong mechanistic rationale for combining CDK4/6is with immunotherapeutic strategies, especially in HR+ subtypes traditionally considered immunologically cold. Such combinations hold promise for expanding the repertoire of targetable tumor antigens and improving BC patient responses to cancer immunotherapy.