Fig 2: Nilotinib inhibits p38aMAPK-MK2/MK3 signaling axis. (A) MALDI-TOF MS relative quantitation of the ratio m/z 4964/4632 in THP-1 cells pre-treated with DMSO, 5 µM nilotinib, imatinib, 1 µM losmapimod, or 1 µM MRT68601 for 1 h before stimulation with live E. coli for up to 24 h. (B) Western blot analysis of the p38a MAPK pathway in THP-1 cells pre-treated with DMSO, 5 µM nilotinib, 5 µM imatinib, 1 µM losmapimod, or 1 µM MRT68601 for 1 h before stimulation with live E. coli for up to 15 min shows loss of p38 phosphorylation in response to nilotinib. Nilotinib and losmapimod, but not imatinib, block phosphorylation of the downstream MK2. a-Tubulin serves as a loading control. A representative image with two biological replicates of four replicates is shown. Relative mobilities of reference proteins (masses in kDa) are shown on the left of each blot. Relative quantification is shown in Figure S6C. (C) TNF-a, (D) IL-6, and (E) IL-1ß secretion was measured by ELISA at 24 h. The statistical significance of the comparisons with E. coli is indicated as follows: ns, not significant; **, P = 0.01; ***, P = 0.001. (F) TNF-a and (G) IL-6 expressions were determined by RT-qPCR in nontransfected or transfected HEK293 cells with p38a WT or p38a M107T for 48 h and pre-treated with DMSO, 5 µM nilotinib, or imatinib for 1 h before stimulation with LPS for up to 24 h. The results were analyzed using the 2-??Ct method and normalized using GAPDH and TBP as the reference genes and nontransfected DMSO sample as the reference sample. Statistical significance of the comparisons: LPS nontransfected is indicated as ‡; LPS transfected with p38a WT is indicated as †; LPS transfected with p38a T106M is indicated as $. Error bars represent the standard deviation of four biological replicates. The statistical significance of the comparisons between p38a WT and p38a M107T is indicated as follows: ns, not significant; **, P = 0.01. Significant differences between two groups were determined by the Mann–Whitney U-test.

Fig 3: SMYD2 monomethylates MAPKAPK3 at Lys355. (A) SMYD2 methylation on a protein array platform identified candidate substrates. A representative image (n = 2 independent experiments) shows a SMYD2 methylation assay on a ProtoArray. The right panel is a close-up of the indicated space on the array. (B) SMYD2 directly methylates MAPKAPK3. An in vitro methylation assay on full-length recombinant MAPKAP3, p53 (as a positive control), and GST (as a negative control) with recombinant wild-type (WT) SMYD3 or the indicated catalytic mutants in a representative radiolabeled methylation assay is shown. n = 5. (Top panel) Autoradiogram of a methylation assay. (Bottom panel) Coomassie stain of proteins in the reaction. (C) SMYD2 methylates MAPKAPK3 at K355. In vitro methylation assays as in B with the indicated MAPKAPK3 mutants or truncation are shown (full-length [FL], truncated [300–382], and point mutants K352R, K355R, and K364R). n = 2. An asterisk indicates SMYD2 automethylation. (D) SMYD2 monomethylates MAPKAPK3 at K355. Tandem mass spectrometry (MS/MS) spectrum identifying monomethylated K355 present on the MAPKAPK3 peptide after in vitro SMYD2 methylation of MAPKAPK3. Note that deuterated S-adenosyl-l-methionine was used as a methyl donor and that samples were chemically propionylated prior to trypsin digestion (see the Materials and Methods). n = 2. (E) MAPKAPK3 is a specific substrate of SMYD2. In vitro methylation assays as in B on MAPKAPK3 using the indicated KMTs are shown. n = 2. GST was used as a negative control. An asterisk indicates SMYD2 automethylation. (F) SMYD2 specifically methylates MAPKAPK3. In vitro SMYD2 methylation assays as in B on the indicated 12 different MAPK pathway-related proteins are shown. n = 3. An asterisk indicates SMYD2 automethylation.

Fig 4: SMYD2 inhibition enhances PDAC chemosensitivity in vitro and in vivo. (A) BAY-598 inhibits SMYD2. An immunoblot analysis of methylation assays (of MAP3K2 by SMYD3 and MAPKAPK3 by SMYD2) with the SMYD2 inhibitor BAY-598 is shown. n = 3. A Coomassie stain of the proteins in the reaction is shown in the bottom panel. (B) Cotreatment with BAY-598 and gemcitabine inhibits the expansion of PDAC cells from low density. Representative images of the response of primary cancer cell lines established form Kras;p53 and Kras;p53;Smyd2 mutant mice to treatment with DMSO (control), 10 µM BAY-598, and 0.1 µM gemcitabine for 9 d are shown. n = 2 independent experiments performed in triplicates. One example of Sapphire 700 staining is shown, and the average value of the triplicate for that experiment is shown. (C) BAY-598 enhances the toxicity of doxorubicin. Changes in cell viability (MTT assay) in response to treatment with 10 µM BAY-598, 1 µM doxorubicin, and cotreatment for 48 h are shown. Combined results of two independent experiments performed in triplicates are shown. (D) Schematic protocol for acute gemcitabine treatment protocol in Kras;p53 mutant mice with advanced PDAC. (E) Representative images for HE and cleaved Caspase 3 immunohistochemistry (arrowheads) in controls and gemcitabine-treated PDAC tumors. n = 5 for each experimental group. Bars, 100 µm. (F) Quantification of cleaved Caspase 3-positive cells in PDAC tumors from the indicated genotypes of mice treated with gemcitabine. n = 5 for each experimental group. (G,H) Tumor volume quantification of human SW1990 PDAC cells growing subcutaneously in immunocompromised mice following SMYD2 inactivation and chemotherapy. (G) SMYD2 knockdown (control cells and empty vector). (H) SMYD2 inhibition by 50 mg/kg BAY-598 once daily. Mice were treated with 100 mg/kg gemcitabine every third day (G,H) or vehicle control (G,H). n = 5 mice for each treatment group in one experiment. (*) P < 0.05; (**) P < 0.01; (***): P < 0.001, P-value calculated by two-tailed unpaired Student's t-test. Data are represented as mean ± SEM.

Fig 5: MAPKAPK3 is methylated in PDAC cells and regulates PDAC-associated phenotypes. (A) SMYD2 induces MAPKAPK3-K355me1 in cells. High-performance liquid chromatography (HPLC) elution profiles show a K355-containing peptide that is unmethylated (left panel) and methylated (right panel) from HEK293T cells overexpressing either MAPKAPK3 alone (blue line) or MAPKAPK3 and SMYD2 (red line) (n = 2). See Supplemental Figure S4C for the MS/MS spectrum of MAPKAPK3-K355me1. (B) Characterization of the MAPKAPK3 K355me1 antibody. Immunoblot analysis with the anti-MAPKAPK3-K355me1 antibody on the product of in vitro methylation assays using combinations of either wild-type or mutant forms of recombinant SMYD2 and MAPKAPK3, as indicated. The Coomassie gel is shown as loading control. n = 3. (C) SMYD2 is required for methylation of MAPKAP3 in cells. An immunoblot analysis with the indicated antibodies after MAPKAPK3 immunoprecipitation using extracts from SW1990 cells stably expressing control, SMYD2, or MAPKPAK3 shRNAs is shown. Inputs are shown as loading and shRNA efficiency controls. n = 2. An asterisk indicates nonspecific bands. (D) MAPKAPK3 promotes pancreatic cancer cell growth in a xenograft model. Xenografts tumor volume analysis for SW1990 human PDAC cells growing subcutaneously in immunocompromised mice with a control knockdown or MAPKAPK3 knockdown. n = 6 tumors for each experimental group. (E) Inhibition of MAPKAPK2/3/5 attenuates the growth of human PDAC cells. SW1990 cells were treated with DMSO (control) or 1 µM MAPKAPK2/3/5 inhibitor PF-3644022 for 9 d. After drug treatment, cells were stained with Sapphire 700 (left panel), and the fluorescence signal was quantified (right panel). (F) Schematic of the pancreatitis-induced precancerous lesion formation protocol in Kras mutant mice and treatment protocol with the PF-3644022 inhibitor. (G) Representative pancreata images in control mice and mice treated with the PF-3644022 inhibitor 7 d after pancreatitis induction. n = 5 for each experimental group. Bars, 1 cm. (H) Representative images of HE staining and immunohistochemistry for MUC5AC, Ki67, and cleaved Caspase 3 in Kras mutant mice treated with vehicle (control) or the PF-3644022 inhibitor 7 d after pancreatitis induction. Bars, 100 µm. (I–K) Quantification of MUC5AC-, Ki67-, and cleaved Caspase 3-positive cells in caerulein-treated pancreata from Kras mutant mice treated with vehicle control (n = 5) or the PF-3644022 inhibitor (n = 5) 7 d after pancreatitis induction. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, P-value calculated by two-tailed unpaired Student's t-test. Data are represented as mean ± SEM.