Fig 2: Loss of CCNC alleviates PRCC-TFE3 induced oncogene-induced senescence (OIS).(A) Growth curves of PRCC-TFE3 doxycycline (Dox)-inducible HK-2 cell lines stably established by lentiviral transduction with an empty vector (Control) or with CCNC targeting sgRNAs (CCNC-g1 and CCNC-g2), resulting in CCNC knockout (KO), and cultured in the presence or absence of Dox. Cell numbers were measured at the indicated time points (n = 3). Notably, CCNC KO alleviated the growth suppression induced by PRCC-TFE3 expression.(B) Senescence-associated β-galactosidase (SA-β-gal) staining of control and CCNC KO PRCC-TFE3 doxycycline (Dox) inducible HK-2 cells after 5 days of PRCC-TFE3 induction. Robust SA-β-gal-positive cells were observed upon PRCC-TFE3 induction in control cells, whereas CCNC KO markedly attenuated SA-β-gal staining. Black arrows indicate SA-β-gal positive cells. Representative images from three independent experiments are shown. Scale bar, 200 μm. (C) RT-qPCR analysis of lamin B1 mRNA levels in non-induced and PRCC-TFE3 induced control and CCNC KO HK-2 cells, showing restoration of lamin B1 expression upon CCNC deletion (n = 3). (D) RT-qPCR analysis of senescence associated secretory phenotype (SASP) factor mRNAs (IL1A, IL6, and VEGF) in non-induced and PRCC-TFE3 induced control and CCNC KO HK-2 cells (n = 3). Induction of PRCC-TFE3 triggered a senescence associated increase in SASP factor expression in control cells, whereas this response was markedly attenuated upon CCNC KO. (E, F) Cell-cycle analysis of PRCC-TFE3 doxycycline (Dox)-inducible control and CCNC KO HK-2 cells. Cells were cultured in the presence or absence of Dox for 3 days, labeled with BrdU for 90 min, fixed, stained with phycoerythrin (PE)-conjugated anti-BrdU antibodies, and counterstained with propidium iodide (PI). Representative flow cytometry plots are shown in (E), and quantification of the G0/G1, S, and G2/M populations is summarized in (F) (n = 3). PRCC-TFE3 induction increased the G0/G1 fraction and reduced S-phase entry in control cells, whereas CCNC KO markedly attenuated these PRCC-TFE3-induced cell cycle changes. Data are presented as means ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t-test for two-group comparisons, or two-way ANOVA followed by Sidak’s post hoc test for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Fig 3: The Mediator kinase module is required for the transcriptional and oncogenic functions of chimeric TFE3.(A) RT-qPCR analysis of representative TFE3 target genes (GPNMB, FNIP2, and RRAGD) in patient derived TFE3-RCC cell line harboring endogenous PRCC-TFE3 (UOK124), treated with or without the CDK8/19 inhibitor MSC2530818 (100 nM) (n = 3). CDK8/19 inhibition attenuated the expression of TFE3 target genes in both TFE3-RCC cell lines.(B) Flow cytometric analysis of lysosomal content in UOK124 cells treated with the CDK8/19 inhibitor MSC2530818 (100 nM) or transduced with shRNA targeting TFE3 (shTFE3). Cells were stained with LysoPrime Green to assess lysosomal volume. Representative flow cytometry histograms (left) and quantitative analysis of mean fluorescence intensity (right) are shown (n = 3). Both MSC2530818 treatment and TFE3 knockdown significantly reduced lysosomal content, indicating suppression of PRCC-TFE3 dependent lysosomal activation.(C) Colony formation assay of UOK124 cells treated with DMSO (Control) or MSC2530818 (100 nM) (n=3). Representative images of colonies are shown (left). Quantification demonstrates that MSC2530818 significantly suppresses anchorage independent colony formation by reducing colony number, without a significant effect on colony size (right).(D) In vitro cell proliferation analysis following genetic ablation of CCNC in UOK124 cells. Successful knockout of CCNC was confirmed by western blotting, with β-actin shown as a loading control (top). Cell proliferation was assessed by cell counting over time (n=8), and growth curves are shown for control and CCNC KO cells (bottom). Genetic ablation of CCNC significantly suppressed cell proliferation compared with control cells.(E) In vivo tumorigenicity of CCNC KO UOK124 cells in nude mice. Control or CCNC KO UOK124 cells (1 × 106 cells per injection) were subcutaneously injected into the left and right flanks of the same nude mice, respectively (n = 6 mice). Tumor volumes were measured at the indicated time points and are shown in the upper left panel. Representative images of tumors excised at day 39 after injection are shown in the upper right panel.Histological and immunohistochemical analyses of tumors derived from control and CCNC KO UOK124 cells. Representative hematoxylin and eosin (H&E) staining is shown in the left panels, and Cyclin C immunohistochemical staining is shown in the right panels. Scale bars, 50 μm. Tumor weights at the endpoint are summarized in the lower right panel. Genetic ablation of CCNC dramatically suppressed tumor growth and reduced final tumor weight compared with control tumors.(F) Tumor growth in an orthotopic syngeneic TFE3 RCC model derived from PRCC-TFE3 knock in mice treated with vehicle or the CDK8 and CDK19 inhibitor SEL120.Tumors derived from PRCC-TFE3 knock-in mice were orthotopically implanted into syngeneic recipient mice, which were subsequently treated with vehicle or SEL120 (60 mg/kg, oral administration, 5 days per week, n=10/arm) for 8 weeks. Tumor growth was monitored longitudinally by ultrasound imaging, and tumor volumes were quantified at the indicated time points. SEL120 treatment significantly suppressed tumor growth compared with vehicle control. Data shown are representative of two independent experiments.(G) Proposed schematic model summarizing the central findings of this study.Proposed model illustrating the interaction between TFE3 fusion proteins and the Mediator complex via the Cyclin C-CDK8/19 kinase module. While enhancer-promoter communication and chromatin organization are not directly assessed in this study, the model reflects established functions of Mediator in transcriptional regulation. Disruption of this axis, through genetic ablation or pharmacological inhibition of CDK8/19, impairs TFE3 fusion dependent transcription and suppresses tumor growth in cellular and in vivo models, identifying the Mediator kinase module as a potential therapeutic target in TFE3 RCC.Statistical significance was determined using an unpaired two-tailed Student’s t-test for two-group comparisons, one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons against a control group, or two-way ANOVA followed by Sidak’s post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.