Chromosomal instability (CIN) and epigenetic reprogramming are central drivers of breast cancer progression, yet the mechanisms connecting them remain elusive. Here, we uncover a direct role for EZH2 histone methyltransferase in promoting CIN in triple-negative breast cancer. Across breast cancers, EZH2 expression correlates with copy-number alterations, and its catalytic activity is associated with increased CIN in metastasis-initiating cells. Pharmacologic EZH2 inhibition suppresses CIN, revealing an unexpected vulnerability. Integrated chromatin and transcriptome profiling identified tankyrase (TNKS), a PARP, as a direct transcriptional target of EZH2. Mechanistically, EZH2-mediated TNKS suppression disrupts centrosomal P4.1–associated protein (CPAP), driving centrosome overduplication, multipolar mitosis, and exacerbated CIN. In vivo, CIN suppression is a critical mechanism underlying the antimetastatic effects of EZH2 inhibition. These findings delineate a previously unrecognized epigenetic mechanism governing CIN and establish EZH2 inhibitors as the first therapeutic agents capable of directly suppressing CIN, underscoring the need for trials with metastasis-focused endpoints. Significance: We elucidate epigenetic regulation of CIN through EZH2–TNKS–CPAP-axis and show that CIN suppression is important for the efficacy of EZH2 inhibition on metastasis. These mechanistic insights are informative for developing CIN-suppressing therapies.

Background: The majority of antigen-based SARS-CoV-2 (SCV2) vaccines utilized in the clinic have had the Spike protein or domains thereof as the immunogen. While the Spike protein is highly immunogenic, it is also subject to genetic drift over time, which has led to a series of variants of concern that continue to evolve, requiring yearly updates to the vaccine formulations. In this study, we investigate the potential of the N-terminal ectodomain of the ORF3a protein encoded by the orf3a gene of SCV2 to be an evolution-resistant vaccine antigen. This domain is highly conserved over time, and, unlike many other SCV2 conserved proteins, it is present on the exterior of the virion, making it accessible to antibodies. ORF3a is also important for eliciting robust anti-SARS-CoV-2 T-cell responses. Methods: We designed a DNA vaccine by fusing the N-terminal ectodomain of orf3a to macrophage-inflammatory protein 3α (MIP3α), which is a chemokine utilized in our laboratory that enhances vaccine immunogenicity by targeting an antigen to its receptor CCR6 present on immature dendritic cells. The DNA vaccine was tested in mouse immunogenicity studies, vaccinating by intramuscular (IM) electroporation and by intranasal (IN) with CpG adjuvant administrations. We also tested a peptide vaccine fusing amino acids 15–28 of the ectodomain to immunogenic carrier protein KLH, adjuvanted with Addavax. Results: The DNA IM route was able to induce 3a-specific splenic T-cell responses, showing proof of principle that the region can be immunogenic. The DNA IN route further showed that we could induce ORF3a-specific T-cell responses in the lung, which are critical for potential disease mitigation. The peptide vaccine elicited a robust anti-ORF3a antibody response systemically, as well as in the mucosa of the lungs and sinus cavity. Conclusions: These studies collectively show that this evolutionarily stable region can be targeted by vaccination strategies, and future work will test if these vaccines, alone or in combination, can result in reduced disease burden in animal challenge models.

Background: The majority of antigen-based SARS-CoV-2 (SCV2) vaccines utilized in the clinic have had the Spike protein or domains thereof as the immunogen. While the Spike protein is highly immunogenic, it is also subject to genetic drift over time that has led to a series of variants of concern that continue to evolve, requiring yearly updates to the vaccine formulations. In this study, we investigate the potential of the N-terminal ectodomain of the ORF3a protein encoded by the orf3a gene of SCV2 to be an evolution-resistant vaccine antigen. This domain is highly conserved over time, and, unlike many other SCV2 conserved proteins, it is present on the exterior of the virion, making it accessible to antibodies. ORF3a is also important for eliciting robust anti-SARS-CoV-2 T-cell responses. Methods: We designed a DNA vaccine fusing the N-terminal ectodomain of orf3a to macrophage-inflammatory protein 3α (MIP3α), which is a chemokine utilized in our laboratory that enhances vaccine immunogenicity by targeting antigen to its receptor CCR6 present on im-mature dendritic cells. The DNA vaccine was tested in mouse immunogenicity studies, vaccinating by intramuscular (IM) electroporation and by intranasal (IN) with CpG adjuvant administrations. We also tested a peptide vaccine fusing amino acids 15-28 of the ectodomain to immunogenic carrier protein KLH, adjuvanted with Addavax. Results: The DNA IM route was able to induce 3a-specific splenic T-cell responses, showing proof of principle that the region can be immunogenic. The DNA IN route further showed that we could induce ORF3a-specific T-cell responses in the lung, critical for potential disease mitigation. The peptide vaccine elicited a robust anti-ORF3a antibody response systemically as well as in the mucosa of the lungs and sinus cavity. Conclusions: These studies collectively show that this evolutionarily stable region can be targeted by vaccination strategies, and future work will test if these vaccines alone or in combination can result in reduced disease burden in animal challenge models.

Table S1 shows Overlap of differential regulated genes in EZH2high vs EZH2low and EZH2i vs vehicle of EZH2high cells comparisons identifies 12 common genes.

Supplementary materials include materials and methods, supplementary Figure S1 to S10, figure legends and references.