Fig 2: Combined targeting of EZH2 and p38 enzymatic activities reduces primary breast cancer growth and metastasis(A) Primary tumor growth curves of NOD/SCID mice orthotopically implanted with MDA-MB-231 cells. When primary tumors reached 100 mm3, mice were treated intraperitoneally with EPZ-6438 (10 mg/kg/day), SB202190 (p38i, 1 mg/kg/day), combination, or control (4% DMSO-30% PEG 300-5% Tween 80), 5 days/week for 56 days (n = 10/group). Primary tumor growth as assessed by caliper measurements, shown as mean ± SEM.(B) Quantification of tumor volume at day 56 shown as mean ± SEM.(C) MDA-MB-231 cells were injected intracardially in nude mice (n = 10/group) and treated as in (A), for 3 weeks. Bars show the number of metastases per mouse in each group on day 21 after heart inoculation ±SEM.(D) Representative H&E-stained sections of lung metastases. Magnification 600x. Scale bar 50 µm.(E) Co-immunoprecipitation and immunoblots of methylated p38a in whole-cell lysates of primary MDA-MB-231 xenograft tumors derived from (A).(F) Immunoblots for the indicated proteins in lysates obtained from the MDA-MB-231 primary orthotopic xenografts in (A). For A-C, *p = 0.05; **p = 0.01; ***p = 0.005; ****p = 0.0001.

Fig 3: Combined pharmacological blockade of EZH2 and p38 enzymatic activities reduces neoplastic functions(A) Immunoblots of MDA-MB-231 cells treated with GSK-343 (3 µM for 48 h), SB202190 (p38i, 20 µM for 48 h), or the combination.(B) Cells in A were subjected to growth assays.(C) Synergistic effect of EZH2 inhibitor and p38 inhibitor in MDA-MB-231 cells incubated with various doses of GSK-343 and SB202190 for 4 d. A matrix for synergy score was calculated (Ianevski et al., 2017).(D and E) Wound healing assay to quantify cell migration (D) and reconstituted Boyden basement membrane-invasive chamber assay of MDA-MB-231 cells treated as in (A). Representative chambers after crystal violet staining are shown above bars. Data for B-E are from at least three independent experiments carried out in at least triplicate. Data for B, (D and E) are presented as mean ± SEM. *p = 0.05; **p = 0.01; ***p = 0.005; ****p = 0.0001.

Fig 4: Combined inhibition of EZH2 methyltransferase and p38 kinase activities reduce AKT signaling in vivo(A) RNA sequencing studies of mammary tumors treated with EPZ-6438 (10 mg/kg/day), SB202190 (p38i, 1 mg/kg/day), combination, or control (4% DMSO-30% PEG 300-5% Tween 80) and excised at day 56. The graph shows significantly deregulated pathways by the combination EPZ/p38i vs. control.(B) Immunoblot of primary xenografts treated as indicated in (A). Combined EPZ/p38i reduces p-AKT compared to single inhibitors.(C) Immunoblots for pAKT and total AKT in MDA-MB-231 cells transduced with HA-tagged WT-p38a, K139A-p38a, K165A-p38a, and K139A/K165A-p38a. P38a mutants display reduced pAKT levels compared to WT-p38a.(D) Immunoblots of MDA-MB-231 EZH2 KD rescued with Myc-tagged WT-EZH2, T367A-EZH2, or vector (pBabe) show that T367 phosphorylation is necessary to upregulate pAKT1 without changes in total AKT1.(E) Representative images of human primary invasive carcinomas. Case 1 shows an invasive high-grade ductal carcinoma with concordant high cyto-pEZH2-T367 and high pAKT, while case 2 shows an intermediate-grade invasive carcinoma with low expression of both proteins. Bars, 50 µm.(F) Schematic illustrating our working model of EZH2 function in breast cancer through H3K27me3-dependent and independent activities. P and Me represent phosphorylation and methylation, respectively.

Fig 5: EZH2 methylates p38a, and EZH2 phosphorylation at T367 is critical for p38a methylation and phosphorylation in TNBC(A) Co-immunoprecipitation (co-IP) and immunoblots of methylated p38a in a panel of breast cancer cells. EZH2 shRNA knockdown decreased methylated p38a.(B) Lysine N-methyltransferase (KMTase) activity assay in whole-cell lysates of MDA-MB-231 cells at the indicated conditions. KMTase activity was assessed using 0.1 mg/mL of human recombinant p38a and measuring S-adenosyl homocysteine (SAH) production detected by bioluminescence. shEZH2 and EPZ significantly reduced methylated p38a compared to control (lanes 1–3), which was rescued by WT-EZH2 (lane 4). EPZ reduced methylated p38a compared to WT-EZH2 (lanes 4–5). Bars depict mean ± SEM, *p = 0.05.(C) KMTase activity assay in whole-cell lysates of T4 and Vari068 patient-derived TNBC cells control and treated with EPZ as in (B). Bars show mean ± SEM, *p = 0.05.(D) IP and immunoblots of methylated p38a in MDA-MB-231 cells transduced with scrambled shRNA (control) or 3' UTR EZH2-targeting shRNA (shEZH2) rescued with Myc-tagged WT-EZH2, T367A-EZH2, or vector (pBabe).(E) Immunoblot of cells in (D).