Fig 2: DYRK1A regulated NFATC1 to facilitate glioma cell migration. A, T98G cells were transfected with DYRK1A and NFATC1 expression vectors alone or simultaneously. Transwell assay showed that co-expression of DYRK1A and NFATC1 greatly increased T98G cell migration. B, T98G cells were transfected with vectors knocking down DYRK1A and/or NFATC1. Simultaneous knockdown of DYRK1A and NFATC1 decreased T98G cell migration. C, T98G cells were transfected with DYRK1A or NFATC1 mutant expression vector alone or simultaneously. Transwell assay showed that co-expression of DYRK1A and NFATC1 mutants marginally increased T98G cell migration. D, MTT assay showed that T98G cell viability was not affected by expression levels of DYRK1A and/or NFATC1. E, MTT assay showed that T98G cell viability was not affected by knockdown of DYRK1A and/or NFATC1. F, MTT assay showed that T98G cell viability was not affected by expression of DYRK1A and/or NFATC1 mutants. Values represent means ± SD, n = 3

Fig 3: Under hypoxic conditions, Har enhances the apoptotic effect of Rego on liver cancer cells. (A and B) PI staining and flow cytometric analysis of the cell death of HepG2 and Hep3B cells treated with the indicated concentrations of Har and/or Rego for 48 h under hypoxia. One-way ANOVA followed by Tukey's post hoc test was used to examine the significant differences among multiple groups. *P<0.05 vs. control, ***P<0.001 vs. control, ###P<0.001 vs. combination. Western blot analysis of the protein expression levels of (C) Mcl-1, caspase-3, cleaved-caspase-3, and cleaved-PARP and (D) DYRK1A, STAT3, p-STAT3(Tyr705), HIF-1α, AKT, and p-AKT (ser473) in HepG2 and Hep3B cells treated with the indicated concentrations of Har and/or Rego for 48 h under hypoxic conditions. Apop, apoptosis; DYRK1A, dual-specificity tyrosine-phosphorylation-regulated kinase 1A; Har, harmine; HIF-1α, hypoxia inducible factor-1α; Mcl1, myeloid cell leukemia-1; p, phosphorylated; PARP, poly(ADP-ribose) polymerase; Rego, regorafenib.

Fig 4: Evidence for DYRK1A and DYRK2 as priming kinases for MTHFR T34 phosphorylation. (a) Western blot analysis of immunoprecipitated, N-terminal-Flag-tagged wild-type or S30A-MTHFR from MDA-MB-468 and 293FT cells, treated with 1 µM harmine for the indicated times. (b) Western blot analysis of MTHFR from MDA-MB-468 cells treated with various combinations of non-targeting control, siDYRK1A, siDYRK2 for 48 h. The endogenous MTHFR was probed using MTHFR monoclonal antibody 5D3. DYRK2 knockdown was assessed by qRT-PCR analysis. n = 4. Data are represented as mean ± SD.

Fig 5: 108600 suppresses growth of TNBC cell line and PDX tumor grafts.A 108600 inhibits phosphorylation of CK2a and DYRK1A substrates in vivo. NSG mice bearing CTG1883 PDX tumor grafts were treated with 108600 (100 mg/kg) or DMSO for 5 days (n = 5). Box plots show levels of phospho-AKT1Ser129 and phospho-CYCLIN D1Thr 286 normalized to those of their respective total proteins and GAPDH. Variances were tested using F-tests (two-sided). P values were calculated using a t-test (two-sided). Samples derive from the same experiment and the blots processed in parallel. B Nude mice bearing MDA-MB-231 xenografts were treated with vehicle or 108600 (50 mg/kg or 100 mg/kg) for 21 days. Tumor volumes were measured on the indicated days. (n = 7 for vehicle- and 108600 (100 mg/kg)-treated animals; n = 6 for 108600 (50 mg/kg)-treated animals). Statistical differences were assessed by two-way ANOVA (two-sided). Data represent mean ± SEM. C 108600 suppresses growth of paclitaxel-sensitive PDX tumors. TM00098 tumor-bearing NSG mice were randomly assigned to groups and treated with vehicle (n = 4) or 108600 (100 mg/kg q2D) (n = 5). Error bars represent mean ± SEM. Statistical differences were assessed by two-way ANOVA (two-sided). D Chemotherapy-resistant PDX growth suppression by 108600. PDX models were treated with DMSO (n = 5 for CTG1883; n = 7 for CTG2397), paclitaxel (10 mg/kg, n = 6 for CTG1883; n = 5 for CTG2397), 108600 (100 mg/kg, n = 6 for CTG1883; n = 7 for CTG2397) or a combination of paclitaxel and 108600 (n = 6 for CTG1883; n = 6 for CTG2397). Paclitaxel and 108600 were administered q4d and q2d, respectively. Statistical differences were assessed by two-way ANOVA (two-sided). Weights of tumors harvested at study endpoints are shown. Data represent mean ± SD. Statistical differences were assessed using a t-test with false discovery rate correction calculated by the Benjamini–Hochberg method. Experiments were performed twice with each model. E 108600 decreased the ALDHhigh fraction in CTG2397 organoids. ALDH activity was measured in organoids treated with 108600 for 48 h. DEAB-treated organoids served as negative controls. Mean values ± SD from three independent experiments are shown. Statistical differences between the ALDHhigh fractions were tested using one-way ANOVA (two-sided) and Dunnett’s test (two-sided). p values of less than 0.05 were considered statistically significant. Source data are provided as a source data file.