Fig 2: Detection of tyrosine autophosphorylation in DYRK1A, DYRK1B and MNB. (A) Domain structure of selected class 1 DYRKs and design of expression constructs. Conserved sequence regions include the catalytic domain (cat), the DH box and the N-terminal region (NT), which harbors a binding site for the adaptor protein DCAF7 and a bipartite nuclear localization signal (NLS). Expression constructs employed in the present study (designated ?C) were fused to a Strep-tag (S), glutathione S-transferase (GST), or the FLAG (F) and HiBiT (H) tags. The shorter constructs (?N?C) have been described previously11. The exact boundaries of the different subdomains are listed in the supplementary information. (B) Sequence surrounding the autophosphorylated tyrosine in the activation loop. The alignment illustrates the structural basis for the cross-reaction of the HIPK2(pY361) antibody with class 1 DYRKs. (C) Detection of tyrosine autophosphorylation in E. coli. The indicated deletion constructs of rat DYRK1A, human DYRK1B and Drosophila MNB were expressed in E. coli for 3 h at 37 °C. Total cell lysates were subjected to immunoblot analysis with different commercial antibodies that were directed against the tyrosine in the activation loop of HIPK2 (pY361) or DYRK1A/B (pY321/273), or with an antibody that detects phosphotyrosine (pTyr) independent of the sequence context (PY99). Total recombinant protein was visualized by detection of the N-terminal Strep-tag. Point mutants used as controls include a catalytically inactive point mutant of DYRK1A (DN, D287N) and mutants of the autophosphorylation site in the activation loop (YF, Y321F in DYRK1A and Y326F in MNB). (D) Comparison of DYRK1A and DYRK1B. DYRK1-?C constructs were expressed in E. coli for 3 h at 37 °C or for 20 h at room temperature (20–25 °C). Tyrosine autophosphorylation was detected by immunoblot analysis of total bacterial lysates with the pY361(HIPK2) antibody. Relative phosphorylation was densitometrically quantitated (ratio of pTyr to Strep-tag signal). The graphs illustrate the results of n = 4 experiments (means and SD; One sample t test).

Fig 3: 108600 suppresses the growth of TNBC stem cells.A Chemical structure of 108600. B IC50 values for inhibition of 108600 target kinases. C 108600 inhibits phosphorylation of CK2a, DYRK1A and TNIK substrates. Lysates derived from MDA-MB-231 and Hs578T cells treated for 24 h with 108600 at the indicated concentrations were probed for expression of phospho-AKT1 (Ser129), phospho-CYCLIN D1 (Thr286), and AXIN2. The blots are representative of three independent experiments. Samples derive from the same experiment and the blots processed in parallel. D GI50 values for growth inhibition of breast cancer and normal cells treated with 108600. E Simultaneous siRNA-mediated knockdown of CK2a, DYRK1A, and TNIK suppresses proliferation of MDA-MB-231 and Hs578T cells. siRNA-transfected cells were counted on the indicated days. All data represent mean values ± standard deviation obtained from three independent biological replicates. The statistical difference between the target and nontarget siRNA treated groups was tested using two-way ANOVA (two-sided). Knockdown was confirmed by western blot analysis, which was performed three times. Samples derive from the same experiment and the blots processed in parallel. F 108600 treatment suppresses colony and G mammosphere formation of CD44high/CD24low stem cell fraction of MDA-MB-231 and Hs578T cells. For colony assays, CD44high/CD24low cells were purified by FACS, treated with the indicated concentrations of 108600 for 72 h, washed and allowed to grow for 7–14 days in drug-free medium. Cells were fixed with 4% paraformaldehyde in PBS, stained with crystal violet solution and colonies quantified microscopically. All data points represent mean value ± standard deviation. Scale bars = 50 µm. Values in F and G represent the mean percentage of colony and sphere formation ±SD, respectively, relative to that of DMSO, which was set to 100%, in three independent experiments. Source data are provided as a source data file.

Fig 4: 108600 suppresses growth of TNBC cell line and PDX tumor grafts.A 108600 inhibits phosphorylation of CK2α 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.

Fig 5: Effect of ganetespib on the maturation of the DYRK1A-DYRK1B chimera in mammalian cells. HEK293 cells transiently expressing DYRK1A, DYRK1B or chimeric constructs with an N-terminal FLAG tag and a C-terminal HiBiT tag were treated with 100 nM ganetespib (Gan) for 24 h or were not treated. Cells were extracted using a non-denaturing lysis buffer, and detergent-soluble fractions and insoluble fractions were separated by centrifugation. (A,B) Tyrosine autophosphorylation. The relative phosphotyrosine content of DYRK constructs in the soluble fraction was assayed by anti-FLAG immunoprecipitation and immunoblot analysis. HiBiT-containing proteins were detected using a fragment complementation assay (HiBiT blotting detection system). The graph shows means and SD of 3 experiments (B). (C,D) Accumulation in the insoluble fraction. Igepal-insoluble proteins in the pellets were solubilized in SDS buffer and DYRK1 constructs were quantitatively using the HiBiT blotting detection system (C). The concentration of HiBiT-tagged proteins in the soluble fraction was measured using the HiBiT lytic detection system. For each construct, the relative proportion of insoluble protein was calculated by dividing the HiBiT signals in the pellet by the HiBiT lytic values for the soluble protein as obtained in the respective supernatants (panel D; means and SD, n = 3). Statistical significance is indicated only for the treatment effects (*p < 0.05, **p < 0.01, ***p < 0.001).