Figure 6 from Long-term Multimodal Recording Reveals Epigenetic Adaptation Routes in Dormant Breast Cancer Cells

Targeting the dormant epigenome. A, Clustered heat maps of histone posttranslational modifications of super-SILAC mass spectrometry for TRADITIOM MCF7 and T47D samples [time zero (T0), latency (time between treatment onset and dormancy entry), dormancy, awakening (early progression), and TEPs (late progression)]. Significantly enriched (dormancy 30 days vs. TEPs, two-tailed t test: *, P < 0.01; **, P < 0.001; ***, P < 0.0001) modifications are depicted in bold, and the ones found to be associated with dormancy are highlighted in yellow. B, Schematic representation of small-molecule inhibitor experiments. Inhibitors against G9a (H3K9me2), EZH2 (H3K27me3), and KMT5B/C (H4K20me3) were used either alone or in combination. Start time of the inhibition was either at the beginning of estrogen deprivation to target persister pool generation or at 30 days of estrogen deprivation (dormancy) to target established dormant cells. C, Proliferation dynamics of MCF7 cells in E2-supplemented conditions (+E2) after treatment with inhibitors against EHMT2, EZH2, KMT5B/C, dual combinations of each and vehicle. D, Proliferation dynamics of MCF7 cells in estrogen-deprived conditions (−E2) after treatment with inhibitors against G9a, EZH2, KMT5B/C, dual combinations of each and vehicle. Proliferation dynamics of T47D cells in E2-supplemented (+E2; E) and deprived (−E2) conditions (F) after treatment with inhibitors against G9a, EZH2, KMT5B/C, dual combinations of each and vehicle (one-way ANOVA with Dunnett correction: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Error bars represent standard deviation (n = 3). G, Relapse-free survival (RFS) curves for ER⁺ breast cancer patients stratified based on the expression of the epigenetic dormancy signature (high vs. low EHMT2/EZH2/KMT5C expression). Left: no adjuvant treatment; middle: adjuvant endocrine therapy (TAM/AI); right: AI adjuvant treatment. Multivariate analysis for clinically relevant prognostic biomarkers is shown in the onset table. H, Proliferation dynamics of MCF7 dormant cells (pretreated for 30 days with –E2) after treatment with inhibitors against G9a, EZH2, KMT5B/C, dual combinations of each and vehicle (one-way ANOVA with Dunnett correction: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Error bars represent standard deviation (n = 3). I, Model: endocrine therapy-induced dormancy is characterized by a consistent epigenetic reprogramming involving a global increase in histone repressive marks (H3K9me2, H3K27me3, and H4K20me3). The dormant epigenome is unstable and through a progressive loss of the histone repressive marks (erosion), cells resume proliferation in a process that mimics patient relapse (awakening). Epidrugs (G9a/EZH2/KMT5B/C inhibitors) can interfere with epigenetic reprogramming and block the formation of persister dormant clones. During adaptation, dormant cells engage in sporadic cycling (failed awakening) while under therapeutic stress possibly forcing cells into a subsequent round of epigenetic reprogramming that could also be antagonized with epidrugs.