Fig. 2

CDX models as drug testing platforms to study responses and overcoming drug resistance by blocking MYC/MAX dimerization: A Mice (n = 5) bearing MU150 and MU197 CDX tumors were intraperitoneally injected with standard-of-care doublet carboplatin/paclitaxel versus vehicle control. Tumor growth was monitored. Upper panel: Treatment schedules. Middle panel: Tumor growth curves demonstrate that MU150 CDX is resistant towards chemotherapy. Lower panel: Tumor growth curves demonstrate that MU197 CDX is sensitive towards chemotherapy. n = 5, error bars represent mean ± standard error of the mean (SEM) (ns-not significant, **p < 0.01; Student’s t-test). B Differential expression of Hallmark MYC target genes and MYC protein between chemoresistant MU150 CDX versus chemosensitive MU197 CDX. Upper panel: Differential expression between MU150 and MU197 CDX models were obtained by integrating snRNA-seq data sets by MAST algorithm. Violin plots depicting differential log fold change expression of Hallmark MYC targets that were significantly higher in chemoresistant MU150 CDX tumors. Middle panel: Western blots for MYC protein (and β-actin control) in CDX tumor lysates (biological triplicates) show higher MYC expression in chemoresistant MU150. Lower panel: MYC immunostaining shows higher expression in chemoresistant MU150 versus chemosensitive MU197 CDX tumor tissues (human aorta served as negative control tissue, IgG as isotype control). Scale bar, 20 μm. C Experimental design of MYC/MAX dimerization blockade in chemotherapy resistant MU150 CDX tumor-derived cells in vitro. D CDX tumor-derived cells were cultured and treated with carboplatin/paclitaxel with or without MYC blocker 10058-F4. Live/dead cell staining demonstrates cell death on day 4 in the Carbo/Pacli/MYC blocker (C + P + M) group versus Carbo/Pacli (C + P), MYC blocker alone (M) and vehicle (V) groups, indicating that MYC blockade reverses drug resistance. (4X magnification, scale bar 200 μm). Images represent biological triplicates. E Quantification of live/dead cell percentage by hybrid cell count method, and F Cell proliferation assay of MU150 showing significant reduction in proliferation in the Carbo/Pacli/MYC blocker group (***p < 0.001, ns-not significant; Multiple t-test and significance was determined by Holm-Sidak method; error bars represent mean ± SEM; biological triplicates). G Western blots showing direct MYC/MAX dimerization target TERT protein expression inhibition in MYC blocker-treated groups (loading control: β-actin) (biological triplicates). H Blocking MYC/MAX dimerization overcomes chemotherapy resistance in MU150 CDX model in vivo. Upper panel: Treatment outline. Mice (n = 4) bearing MU150 CDX tumors were treated with doublet carboplatin/paclitaxel (Carbo/Pacli) with or without MYC blocker and MYC blocker alone (vs. vehicle control). Lower panel: Tumor growth graphs and representative tumor images demonstrate that MYC blockade overcomes drug resistance (error bars: ±SEM; ns-not significant, *** p < 0.001; two-way ANOVA). I H&E staining of representative tumor images for all the groups, with highest degree of necrosis (arrows) in carboplatin/paclitaxel/MYC blocker-treated groups (scale bar, 50 μm). J Immunohistochemistry of TERT demonstrate lower expression with MYC/MAX dimerization inhibition, and higher expression of apoptotic markers cPARP and cCASP3 in the group treated with Carbo/Pacli/MYC blocker (human aorta: negative control tissue; IgG: isotype control; Scale bar, 20 μm) Images are representatives from biological triplicates per model