Yang Bai

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 Figure S1 shows correlation of EZH2 gene expression with FGA in METABRIC and TCGA breast cancer cohort suggests a link between EZH2 and CIN. Supplementary Figure S2 shows correlation between fraction genome altered (FGA) and EZH2 gene expression in TCGA LUAD cohort. Supplementary Figure S3 shows increased CIN in various TNBC, non-TNBC and and RPE1 cells and impact of EZH2 inhibition. Supplementary Figure S4 shows EZH2 inhibition and cell cycle analysis. Supplementary Figure S5 shows CUT & RUN seq and RNA-seq analysis. Supplementary Figure S6 shows visualization and experimental validation of EZH2 target genes. Supplementary Figure S7 shows EZH2 mediated TNKS regulates CIN. Supplementary Figure S8 shows multipolar mitosis and supernumerary centrosome analysis of the TNKS KO cells. Supplementary Figure S9 shows correlation of EZH2, CPAP and aneuploidy and effect of EZH2 inhibition on CPAP expression and cell cycle analysis of TNKSWT and TNKSKO LM2 cells. Supplementary Figure S10 shows impact of CIN modulation on proliferation and metastatic progression in EZH2 WT and EZH2 KO cells.

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

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

Supplementary Figure S1 shows correlation of EZH2 gene expression with FGA in METABRIC and TCGA breast cancer cohort suggests a link between EZH2 and CIN. Supplementary Figure S2 shows correlation between fraction genome altered (FGA) and EZH2 gene expression in TCGA LUAD cohort. Supplementary Figure S3 shows increased CIN in various TNBC, non-TNBC and and RPE1 cells and impact of EZH2 inhibition. Supplementary Figure S4 shows EZH2 inhibition and cell cycle analysis. Supplementary Figure S5 shows CUT & RUN seq and RNA-seq analysis. Supplementary Figure S6 shows visualization and experimental validation of EZH2 target genes. Supplementary Figure S7 shows EZH2 mediated TNKS regulates CIN. Supplementary Figure S8 shows multipolar mitosis and supernumerary centrosome analysis of the TNKS KO cells. Supplementary Figure S9 shows correlation of EZH2, CPAP and aneuploidy and effect of EZH2 inhibition on CPAP expression and cell cycle analysis of TNKSWT and TNKSKO LM2 cells. Supplementary Figure S10 shows impact of CIN modulation on proliferation and metastatic progression in EZH2 WT and EZH2 KO cells.

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

List of antibodies and reagents used in this study

Supplementary Figures 1-13 with associated legends

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.

List of antibodies and reagents used in this study