Fig 2: In vivo synergistic antitumor effect of domatinostat in combination with Gemcitabine plus Abraxane in PANC28 and PANC1 xenograft models. A-B. PANC28 and PANC1 cells were s.c. injected into athymic mice as described in the Materials and Methods. When established tumors were palpable, mice were treated with vehicles or domatinostat (20 mg/Kg 5 days/week, per os) alone and in combination with gemcitabine (weekly 25 mg/Kg, i.p.) and abraxane (weekly 20 mg/Kg, i.p.) (GT) for two weeks. Relative tumor volume curves are reported as mean ± SEM tumor volume measured at the indicated timing. C-D. Percent change in tumor volume average from first day of treatment (day 0) to the end of the study (day 32 for PANC28 xenograft and day 25 for PANC1 xenograft) for each treatment group compared to vehicles group (middle panels). E-F. Tumor growth delay (TGD), determined, in both PANC28 and PANC1 xenografts, as %TGD = [(T - C) /C] × 100, where T and C are the mean times expressed in days for the treated or control groups, respectively, to reach a defined tumor volume (see Materials and Methods). G. Expression of FOXM1 and ?H2AX protein levels in lysates from three PANC1 xenograft tumor samples from each treatment group evaluated by WB. ß-actin serves as control for equal protein loading. On the right, proteins quantification reported as mean ± SD for each treatment groups. H. Paraffin-embedded tissues were generated for each group for IHC analysis for ß-Catenin as described in the Materials and Methods. Images were captured with a 20X (white scale bar: 200 µm) and 40X (white scale bar: 50 µm) objectives on a light microscope. Statistically significant results, obtained by 2-way ANOVA test, are reported (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05)

Fig 3: High activation of FoxM1–STMN1 signal axis is a risk of poor prognosis in cancers. a Overall survival and disease-free survival rate of 31 solid tumors (including ACC, BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, KICH, KIRC, KIRP, LGG, LIHC, LUAD, LUSC, MESO, OV, PAAD, PCPG, PRAD, READ, SARC, SKCM, STAD, TGCT, THCA, THYM, UCEC, UCS and UVM) from TCGA database were analyzed using the GEPIA platform; n = 9350. b Overall survival and disease-free survival rate of LIHC from TCGA database were analyzed using the GEPIA platform; n = 364. c Overall survival rate of LIHC from the TCGA database was analyzed according to the mRNA levels of FoxM1 and STMN1. HH, FoxM1High/STMN1High (n = 136). HL, FoxM1High/STMN1Low (n = 42). LH, FoxM1Low/STMN1High (n = 42). LL, FoxM1Low/STMN1High (n = 136). d A working model showing that aberrantly high activation of FoxM1–STMN1 axis contributes to tumorigenesis in cancer cells by promoting cell mitosis, while interruption of FoxM1–STMN1 axis can result in mitotic catastrophe-mediated cell death

Fig 4: Coordinated expression of FoxM1 and STMN1 in cancers. a Analysis of the mRNA levels of FoxM1 and STMN1 (cancer vs. normal) in multiple solid cancers from Oncomine Database. b The expression of FoxM1 and STMN1 in hepatocellular carcinoma cohorts (GSE14520, GSE14323), gastric cancer cohorts (GSE13911, GSE19826, GSE27342) and colon cancer cohorts (GSE8671). The data were presented as the mean ± SD of different samples. The significance was analyzed by the Student’s t-test. **P < 0.01, ***P < 0.001, ****P < 0.0001. c The correlation of FoxM1 and STMN1 expression in 31 solid tumors (including ACC, BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, KICH, KIRC, KIRP, LGG, LIHC, LUAD, LUSC, MESO, OV, PAAD, PCPG, PRAD, READ, SARC, SKCM, STAD, TGCT, THCA, THYM, UCEC, UCS and UVM) from the TCGA database was analyzed using the GEPIA platform, and the correlation of FoxM1 and STMN1 expression in LIHC (n = 373), GC (n = 415) and CRC (n = 328) TCGA data. d Protein levels of FoxM1 and STMN1 in LIHC (n = 79), GC (n = 61) and CRC (n = 68) were analyzed by IHC. The correlation of FoxM1 and STMN1 was analyzed by linear regression analysis

Fig 5: SMYD2-mediated ß-catenin methylation is required for expression of Wnt downstream genes and ß-catenin nuclear translocation by FOXM1A. SMYD2 knockdown attenuated transcriptional levels of two pivotal Wnt pathway downstream genes, CCND1 and cMYC in two HCC cell lines, SNU449 (upper left panel ) and SNU475 (upper right panel ), and two colon cancer cell lines, HCT116 (lower left panel) and SW480 cells (lower right panel). Cells were transfected with siNC or siSMYD2 (siSMYD2#1). After 48h-incubation, RNAs were prepared from these cells and transcriptional levels of SMYD2, CCND1 and cMYC were measured by quantitative RT-PCR. Statistical analyses were performed using unpaired Student's t-test (two groups). The asterisks indicate statistical significance; *, **, and *** indicate p-value of < 0.05, < 0.01 and < 0.005, respectively, compared to the corresponding value of the siNC (control) group. Error bars indicate values of one standard deviation (n = 3). B. SMYD2-mediated methylation at K133 of ß-catenin is required for its interaction with FOXM1, BCL9 and a -catenin. 293T cells were co-transfected with FLAG-WT-ß-catenin or FLAG-K133A-substituted ß-catenin, and HA-SMYD2. After 48-h incubation, cell lysates were immunoblotted with anti-FLAG (WT-ß-catenin or K133A-substituted ß-catenin), anti-FOXM1, anti-BCL9, anti-a -catenin, and anti-HA (SMYD2) antibodies before immunoprecipitation (input), and after immunoprecipitation with anti-FLAG M2 agarose. FOXM1 and BCL9 were co-immunoprecipitated with FLAG-WT-ß-catenin, but not with FLAG-K133A-substituted ß-catenin. a -catenin was co-immunoprecipitated more preferentially with FLAG-K133A-substituted ß-catenin than FLAG-WT-ß-catenin. However, HA-SMYD2 was co-immunoprecipitated almost equally with both WT- and K133A-substituted ß-catenin proteins. C. Structural analysis of an a -catenin-binding region of ß-catenin including lysine 133. Without methylation of lysine 133 of ß-catenin, hydrogen bonds are made between lysine 133 (K133) and methionine 98 (M98), and between lysine 133 and glutamic acid 101 (Glu 101) in an a -catenin-binding region of ß-catenin. When K133 is methylated, these hydrogen bonds might be affected and the affinity of ß-catenin to a -catenin could be reduced. The crystal structure of residues 84-682 of ß-catenin is obtained from PDB: 4ONS and 2Z6G.