Fig 2: Targeting the MAPK pathway is inadequate and compromised by HER2 activation.a Western blots showing dose-dependent increases in phosphorylated ERK1/2, p90RSK, and cleaved caspase-3 levels in PDAC cells treated with the indicated concentrations of gemcitabine for 24 h. b Western blots showing changes in p-ERK1/2, p-AKT(S473), and cleaved caspase-3 levels after treatment with the indicated agents for 24 h. c Correlation plots with Pearson coefficients (R) showing strong positive correlation between MAPK and two independent gemcitabine resistance signatures in PDAC samples from TCGA PanCancer database. d Growth kinetics of subcutaneous Pa01c and Pa02c xenograft tumours treated as indicated when the tumour volume reached ~100 mm3. Data are presented as mean ± SEM. P-values were calculated using two-way ANOVA with Tukey’s multiple comparison test. e Heatmap of RPPA data showing significantly upregulated and downregulated markers in Pa01c and HPAC cells treated with ulixertinib or trametinib for 24 h. f Venn diagram showing the shared changes in both cell lines. Only markers showing a Log2 fold change or <-1 or >1 are illustrated. Data are presented as the mean ± SEM of two biological samples. g Western blots showing changes in p-ERK1/2, DUSP4, and DUSP6 levels in 293 T cells transfected with HER1, HER2, or HER3 for 36 h and then treated with DMSO or afatinib for 16 h. h Western blots showing changes in different phosphorylated-HER2 signals, p-HER3, p-ERK1/2, p-AKT, DUSP4, and DUSP6, in Pa01c and HPAC cells treated with ulixertinib or trametinib for 24 h. a, b, g, h were conducted two times, and one set of data was presented. Source data are provided in Source Data file.

Fig 3: KRAS recruits TRIM21 to regulate DUSP6 stability.a Schematic of the immunoprecipitation followed by mass spectrometry (IP-MS) experiment employed to identify binding partners of FLAG-KRASG12V. 3 biological replicates from each condition were pooled for mass spectrometry. Linear plot shows the enriched proteins. b IP western blots showing the interaction between HA-KRASG12V and endogenous TRIM21 and DUSP6 in 293 T cells stably expressing vector or HA-KRASG12V treated with DMSO, trametinib, or ulixertinib for 16 h. The bar graph includes quantification of three independent experiments. Data was presented as mean ± SEM. P-values were calculated using one-way ANOVA followed by Dunnet’s multiple comparisons test. c Western blots, and quantification of serial endogenous DUSP6 protein levels in 293 T cells expressing empty vector or GFP-tagged TRIM21 following treatment with CHX (10 µg/mL) for the indicated durations. d IP experiment showing differences in K48-polyubiquitination of FLAG-tagged DUSP6 in 293 T cells transfected with the vector, wild-type, or enzymatically inactive (C54Y) TRIM21. e IP experiment showing differences in K48-polyubiquitination of FLAG-tagged DUSP6 with/without TRIM21 co-expression following 16 h treatment with DMSO, ulixertinib, or trametinib in 293 T cells. f Western blots showing changes in p-HER2, p-ERK1/2, and DUSP6 in Pa01c and HPAC cells stably expressing a scramble shRNA or two different shRNAs against TRIM21. b was conducted three times, (c–f) were conducted two times, and one set of data for each was shown. g 2-dimensional colony formation assay showing the colony-forming ability of the indicated Pa01c and HPAC cells co-treated with DMSO or afatinib for 2 weeks. h GSEA plots showing the enrichment of ERBB2 and KRAS signatures in TRIM21-silenced HPAC cells subjected to bulk RNA sequencing. i proposed model of the mechanism by which KRAS/MAPK inhibitors dissociate TRIM21 from DUSP6 and causes the latter to be destabilized, leading to sustained phospho-activation of HER2. Phospho-activated HER2 may in turn promotes DUSP6 degradation while simultaneously activate other survival cascades such as the PI3K-AKT pathway. Source data are provided in Source Data file.

Fig 4: The TEY motif of HER2 promotes binding to DUSP6.a Alignment of amino acid sequences of the C-termini of ERK1, ERK2, HER1, HER2, and HER3, indicating the presence of the TEY motif in ERK1/2 and HER2. Schematic and results of IP experiments using HA-DUSP6 produced in human Pa01c cells and ERK2/HER2 variants using BL21 bacteria. IP was performed using HA beads (b) or His beads (c) to delineate the in vitro interaction between DUSP6 and HER2. d In vitro assay showing decrease phospho-HER2(Y877) of purified bead-bound FLAG-HER2 incubated with recombinant DUSP6 for 1 h. e In vitro pNPP phosphatase assay using recombinant DUSP6 and purified bead-bound FLAG-HER2 done in quadruplicates for 30 min at 37 °C in cell-free condition. The liberated p-nitrophenol which reflects DUSP6 phosphatase activity was measured at 405 nm using Biotek Synergy 2 spectrophotometer. f Representative immunofluorescence images and quantification of Duolink® proximity ligase assay identifying the interaction of endogenous HER2 and DUSP6 in Pa01c and HPAC cells treated with DMSO, ulixertinib 2 μM, or trametinib 1 μM for 4 h prior to degradation of DUSP6. Scale bar = 50 μm. For (e, f) data presented as mean ± SEM, P values were calculated by one-way ANOVA with Dunnet’s multiple comparisons test or (e) two-tailed unpaired t-test (f). g IP experiment in 293 T cells showing polyubiquitination of ectopically expressed HA-tagged DUSP6 in the absence or presence of co-transfected FLAG-tagged HER2 as well as trametinib or ulixertinib treatment for 16 h. All cells were co-treated with bortezomib for at least 4 h prior to treatment with trametinib or ulixertinib to prevent the marked downregulation of DUSP6. Experiments in (b–d, g) were conducted two times, and one set of data for each was presented. Source data are provided in Source Data file.

Fig 5: KRAS regulates DUSP6 stability.a Western blots showing changes in p-ERK1/2, DUSP4, and DUSP6 levels in 293 T cells stably expressing HA-KRASG12V treated with trametinib or ulixertinib for 16 h. The bar graph includes quantification of three independent experiments. Data was presented as mean ± SEM. P-values were calculated using one-way ANOVA followed by Dunnet’s multiple comparisons test, HA-KRAS vs control (p = 0.0031), HA-KRAS vs 2 μM ulixertinib (p = 0.0474), HA-KRAS vs 0.5 μM trametinib (p = 0.0468), HA-KRAS vs 1 μM trametinib (p = 0.0045). Western blots showing serial changes in endogenous DUSP6 protein levels in 293 T cells stably expressing an empty vector or HA-KRASG12V (b) or in Pa01c cells stably expressing a scramble sequence or an shRNA against KRAS (c), after treatment with 10 μg/mL cycloheximide (CHX) for the indicated durations. d Western blots, and quantification of serial endogenous DUSP6 protein levels in Pa01c cells stably expressing a scramble sequence or an shRNA against KRAS (c), after treatment with 2 μM of ulixertinib for the indicated durations. e Western blots, and quantification of serial endogenous DUSP6 in Pa01c cells pre-treated with DMSO or MRTX1133 (0.5 μM) for 1 h followed by 2 μM of ulixertinib at the indicated durations. f Western blots, and quantification of serial endogenous DUSP6 in MIA Paca-2 cells pre-treated with DMSO or AMG-510 (0.5 μM) for 1 h, followed by 2 μM of ulixertinib for the indicated durations. For (b–f), half-lives (t1/2) of DUSP6 were calculated by measuring the DUSP6 band intensities, normalizing to t0 and performing one-phase exponential decay analysis, as shown in the graph below. a was conducted three times, and (b–f) were conducted two times, and one set of data for each was shown. Source data are provided in Source Data file.