Fig 2: The MYC accumulation induced by activated FGFR3 is dependent on the activation of p38 and AKT MGH‐U3 and RT112 human bladder tumor cells were transfected with control siRNA (Ctr) or with siRNAs targeting FGFR3. Lysates were obtained and the levels of p38, phospho‐p38 [P‐p38 Thr180/Tyr182), AKT, phospho‐AKT (P‐AKT Ser473)], and FGFR3 were assessed by Western blotting. Different siRNAs were used in the two cell lines (see Materials and Methods).MGH‐U3 and RT112 cells were transfected for 72 h with a siRNA targeting either FGFR3 or MAPK14 (p38α). Lysates were obtained and MYC and p38 protein levels were analyzed by Western blotting.MGH‐U3 and RT112 cells were transfected with MAPK14 (p38α) siRNA for 72 h. The level of MYC mRNA level was determined by RT–qPCR (left panel). The results presented are the means and standard errors of two independent experiments carried out in triplicate. Unpaired Student's t‐tests were used for comparison with appropriate siRNA control (Ctr), *P < 0.05; **P < 0.005.MGH‐U3 and RT112 cells were treated for 2 h with DMSO or a FGFR inhibitor (0.5 μM PD173074). Lysates were obtained and analyzed by Western blotting with antibodies against MYC, phospho‐AKT (Ser473) and phospho‐GSK3β (Ser9). Tubulin was used as a loading control.Western blot comparing MYC, phospho‐AKT (Ser473) and phospho‐GSK3β (Ser9) levels in MGH‐U3 and RT112 cells treated for 3 h with a PI3 kinase inhibitor (20 μM LY294002) or control DMSO. Tubulin was used as a loading control.The level of phosphorylation of p38 (left panel) and AKT (right panel) was assessed by reverse‐phase protein array (RPPA) in 129 human bladder tumors, as described in the Materials and Methods. FGFR3 mutations were present in 38 tumors. No tumor harbored an FGFR3‐TACC3 or FGFR3‐BAIAP2L1 fusion gene. Mann–Whitney test was used for comparisons between mutated and non‐mutated tumors. Means and standard errors are represented.

Fig 3: The inhibition of p38 or AKT reduces the growth and transformation of bladder cancer cells expressing aberrantly activated FGFR3 MGH‐U3 and RT112 cells were treated with control DMSO, PD [PD173074 (FGFR inhibitor)], SB [SB203580 (p38 inhibitor)] or LY [LY294002 (PI3 kinase inhibitor)] for 72 h and cell viability was then assessed by measuring MTT incorporation.Impact of PD (PD173074), SB (SB203580), or LY (LY294002) treatment on the cell anchorage‐independent growth of MGH‐U3 cells. Colonies in soft agar with diameters greater than 50 μm were counted 14 days after seeding in the presence of inhibitors.Comparison of the effects of MAPK14 (p38α isoform) and FGFR3 knockdown on the viability of MGH‐U3 and RT112 cells, as measured by MTT incorporation.Soft agar colony formation assay for MGH‐U3 cells transfected with siRNA against FGFR3 or MAPK14 (p38α isoform). Cells were grown for 14 days before counting.MGH‐U3 bladder cancer cells were injected into nude mice (n = 5 animals/group), two xenografts per animal (one in each flank). Nine days later, the mice received an injection of vehicle or SB203580 (100 μl of 20 μM SB203580) into the tumor, once daily, 6 days per week. Tumor size was measured at the indicated time point, and tumor volume was calculated.Data information: (A–D) The results presented are the means of two independent experiments carried out in triplicate; the standard errors are indicated. Unpaired Student's t‐tests were used to assess the significance of differences, *P < 0.05; **0.001 < P < 0.005; ***0.0001 < P < 0.001; ****P < 0.0001. (E) Data are presented as means ± SEM. Results were compared in Mann–Whitney test.

Fig 4: MYC and FGFR3 are involved in a positive feedback loop in bladder cancer cell lines expressing an activated form of FGFR3 The expression of MYC and FGFR3 was analyzed by Western blotting in lysates from MGH‐U3 and RT112 cells transfected for 72 h with MYC siRNAs. Tubulin was used as a loading control.Relative FGFR3 mRNA levels in MGH‐U3 and RT112 cells transfected for 72 h with siRNAs targeting MYC or a control siRNA (Ctr). The results presented are the means of two independent experiments carried out in triplicate; the standard errors are indicated. Unpaired Student's t‐tests were used for comparison with the control, ****P < 0.0001.ChIP–qPCR for MYC at the FGFR3 locus in MGH‐U3 and RT112 cells (lower panel). The qPCR target loci of FGFR3 are schematized (upper panel). Data presented are representative of two replicate experiments. Error bars show standard deviation of three replicate qPCR reactions.RT112, MGH‐U3, UM‐UC‐14, RT4, and UM‐UC‐5 cells were treated for 48 h with a pan‐FGFR inhibitor (500 nM PD173074). Lysates were obtained, and levels of FGFR3 and MYC were analyzed by Western blotting with appropriate antibodies. An anti‐tubulin antibody was used as a loading control.MGH‐U3 and RT112‐derived xenograft tumors from mice treated for 9 days with vehicle or PD173074 (25 mg/kg/day) were lysed and immunoblotted with anti‐FGFR3 and anti‐MYC antibodies. Actin was used as a loading control. Black and white arrowheads indicate WT FGFR3 and FGFR3‐TACC3 bands, respectively.PDX tumors bearing the FGFR3‐S249C mutation from mice treated for 4 days with vehicle or BGJ398 (30 mg/kg/day) were lysed and immunoblotted with anti‐FGFR3 and anti‐MYC antibodies. Actin was used as a loading control. Source data are available online for this figure.

Fig 5: MYC is a key upstream regulator activated by FGFR3 that is required for FGFR3‐induced bladder cancer cell growth Venn diagram showing the number of upstream regulators (transcription factors) significantly predicted by Ingenuity Pathway Analysis to be involved in the regulation of gene expression observed after FGFR3 knockdown in RT112 and MGH‐U3 cells (left panel). List of the top 10 upstream regulators modulated by FGFR3 expression in both cell lines. The Log2FC of the transcription factor itself is also indicated. NA indicates that the FC was beyond the threshold defining genes as differentially expressed after FGFR3 depletion (see Materials and Methods).Relative MYC mRNA levels in MGH‐U3 and RT112 cells transfected for 72 h with siRNAs targeting FGFR3 or a control siRNA (Ctr). The results presented are the means of two independent experiments carried out in triplicate; the standard errors are indicated. The significance of differences was assessed in unpaired Student's t‐tests, *P < 0.05; **0.001 < P < 0.005. MYC mRNA levels in normal human urothelium (n = 4) and in the CIT cohort of human bladder tumors bearing FGFR3 mutations (n = 63) or wild‐type FGFR3 (n = 122). The significance of differences was assessed in Mann–Whitney tests, and means and standard errors are represented. MYC and FGFR3 mRNA levels in human bladder tumors harboring either mutated FGFR3 (upper panel) or wild‐type FGFR3 (lower panel). Spearman's coefficient and P‐values are indicated for the correlations between MYC and FGFR3 mRNA levels in each group. MYC mRNA levels in a PDX model bearing a FGFR3‐S249C mutation and treated daily, for 4 days, with 30 mg/kg BGJ398, a pan‐FGFR inhibitor, or with vehicle (n = 4 mice per group). Means and standard errors are represented. The significance of differences was assessed in Mann–Whitney tests.Western blot (72 h after transfection) comparing FGFR3 and MYC levels in MGH‐U3 and RT112 cells transfected with a control siRNA (Ctr) or with siRNAs targeting FGFR3.Western blot comparing MYC levels in MGH‐U3 and RT112 cells, treated for 2 h with DMSO or the pan‐FGFR inhibitor, PD173074 (500 nM).Western blot comparing MYC levels in MGH‐U3 and RT112 cells treated for 3 h with FGFR inhibitor (0.5 μM PD173074) or proteasome inhibitor (10 μM MG132), alone or in combination.Cell viability assay comparing the impact of MYC and/or FGFR3 downregulation on RT112 (left panel, CellTiter‐Glo) and MGH‐U3 (right panel, MTT assay) cell viability (72 h post‐transfection). The results presented are the means of three independent experiments carried out in triplicate, error bars represent standard deviations. Tukey's multiple comparisons tests were performed to evaluate the significance of differences. The results of the statistical analysis are summarized in Dataset EV2. Source data are available online for this figure.