Fig 2: HT1376 xenografts with KDM6A and ARID1A mutations are sensitive to EZH2 inhibition alone and in combination with cisplatin. a 500 mg/kg EPZ011989 alone or in combination with 3 mg/ml cisplatin reduced HT1376 tumor volume in nude mice. The Y-axis represents tumor volume in mm3 and the X-axis represents number of days in the study. There is a significant difference in the tumor growth rate between the combination treatment and cisplatin alone treatment. b Mathematical modeling of tumor growth rate indicated that the vehicle, Cisplatin, EPZ011989 would grow tumors from 100 to 1000 mm3 in 58.5, 77.7, and 399.8 days, respectively. The combination treatment is predicted to not reach 1000 mm3. c End-point tumor weights showed that the combination treatment had significantly lower tumor weight as compared cisplatin alone treatment. The Y-axis represents the tumor weight in grams and the X-axis represents the different groups. Error bars represent standard error of means of all the mice in each group. One-way ANOVA model with pairwise comparisons made as appropriate were performed to test differences between single and combination treatments. *p < 0.05, **p < 0.01, ***p < 0.001, triangle indicates significant difference as compared to vehicle, hash indicates significant difference between the EPZ011989 and cisplatin alone treatments

Fig 3: The interactome of pEZH2(T367) reveals new cytoplasmic binding proteins. a Schematic of mass spectrometry experiment to identify binding partners of EZH2 in MDA-MB-231 cells. Experiment was performed in triplicate. b Venn diagram displaying interactors overlap in proteins co-immunoprecipitating with WT- or T367A-EZH2 from the three biological replicates analyzed. c DAVID functional annotation analysis of processes enriched in WT-EZH2 over T367A-EZH2. d List of differential interactors identified from actin-binding set with fold-change (FC) scores and normalized FC scores based on total EZH2 pulldown. Average WT and T367A spectral counts (SC) and SAINT probabilities (SP) are also tabulated

Fig 4: EZH2 inhibition rescues cell fate bias upon chemotherapy exposure.All the experiments were performed in MDA-MB-468 cells. a. Histogram representing the number of cells after treatment with 5-FU alone or 5-FU and EZH2i over 21 days, relative to the number of cells at D0. Cells were pre-treated with EZH2i-1, inactive EZH2i-1 or EZH2i-2 for 10 days prior to chemotherapy treatment. (n = 3, Mean ± s.d., ANOVA test). b. Clustering of samples according to lineage barcode frequencies, detected by bulk analysis, using Spearman correlation score. c. UMAP representation of scRNA-seq datasets, colored according to the sample of origin. Cells were treated with DMSO (untreated) or with 5-FU alone (persister) or with 5-FU and EZH2i-1 (EZH2i-1 persister). d. UMAP representation of scRNA-seq datasets, barcoded cells were selected and colored according to hierarchical clusters e. (Left) UMAP representation of scRNA-seq datasets, barcoded cells were selected and colored according to lineage barcode. (Right) Histogram of the lineage barcodes diversity detected in the scRNA-seq data within hierarchical clusters, and across samples. Colors correspond to sample ID as in 4c. Ratio of unique barcodes/total barcodes and P value are indicated (two-sided Fisher’s test).

Fig 5: TRIM25 blocks TRAF6-mediated ubiquitination of EZH2.A Endogenous interaction between TRIM25 and EZH2 was determined using co-immunoprecipitation with anti-TRIM25 or anti-EZH2 antibodies in SW480 cells. B Exogenous interaction between TRIM25 and EZH2 was determined using co-immunoprecipitation with anti-Flag or anti-HA antibodies in HEK293T cells co-transfected with Flag-EZH2 and HA-TRIM25. C Immunofluorescence images showing colocalization of endogenous EZH2 and TRIM25 in SW480 cells. D Flag-EZH2, siTRIM25, His-tagged ubiquitin wild-type (WT) or mutation plasmids (K48 or K63 mutants) were transfected into HEK293T cells as the indicated combinations. Immunoprecipitation with anti-Flag antibodies was performed to detect the ubiquitination level of EZH2. E Immunoprecipitation assay using IgG and anti-EZH2 antibodies in SW480 cells transfected with siNC or siTRIM25, followed by western blotting analysis with the indicated antibodies. WCE: Whole-cell extracts. F Flag-EZH2, siTRIM25, siTRAF6, His-tagged ubiquitin wild-type (WT) were transfected into HEK293T cells as the indicated combinations. Immunoprecipitation with anti-Flag antibodies and western blotting with anti-His antibodies were performed to detect the ubiquitination level of EZH2 under different conditions. G Western blotting analysis of EZH2 in SW480 cells transfected with siTRIM25 and/or siTRAF6.