Fig 2: Pharmacodynamic assays for ONC201. Maximum fold induction of serum prolactin levels (a) and cleaved cytokeratin 18 (b) in patients relative to baseline levels. c Max fold induction of prolactin and cleaved cytokeratin 18 in patients treated at once every three weeks (Q3W) and on the weekly (Q1W) schedule relative to baseline levels. Median prolactin levels: 1.79 and 1.84 for Q3W and Q1W cohorts, respectively. Median cleaved cytokeratin levels: 1.25 and 1.61 for Q3W and Q1W cohorts, respectively. d IHC analyses of CHOP, DR5, and double-stranded DNA breaks (TUNEL) in baseline and ONC201-treated biopsies for an endometrial cancer patient. The biopsy was done 9.8 months after starting ONC201 treatment (7 days after the most recent ONC201 dose). Each error bar indicates SEM

Fig 3: MCL1 and JUNB mediate resistance to FasL- and TRAIL-induced cell death through the mitochondrial apoptosis pathway.a Caspase 8 activity measured using a colorimetric cleavage assay in A375 cells overexpressing candidate genes after treating with 500 ng/µL of FasL or TRAIL for 3 h. N = 3. Repeated measures ANOVA with adjustments for multiple comparisons were performed. b Dox-induction of genes in the mitochondrial apoptosis pathway in A375 cells overexpressing MCL1 or GFP. Cell survival against ESO T cell cytotoxicity (n = 8) and expression of MCL1 interaction partners (n = 4) were measured at different Dox concentrations. Two-tailed t tests with adjustments for multiple comparisons were performed. c Expression of cell surface FasL or TRAIL receptor, FAS or TNFRSF10B, measured by antibody staining and flow cytometry in A375 cells overexpressing candidate genes. Data are displayed as histograms (top), median fluorescence intensity (MFI; bottom left), and percent cells expressing receptor with gating at the gray dashed line (bottom right). N = 3. Two-tailed t tests were performed. d Cell survival against 500 ng/µL of FasL- or TRAIL-induced cell death in A375 cells overexpressing JUNB or GFP with BCL2A1 knocked down. N = 4. KD knockdown, NT non-targeting. Two-tailed t tests were performed. e Expression of the NF-?B pathway genes overlapping JUNB ChIP-seq and RNA-seq. Values represent fold change in A375 cells overexpressing JUNB relative to GFP control. N = 3. Two-tailed t tests with adjustments for multiple comparisons were performed. f Western blots of phosphorylated or total p65 (RELA) protein in A375 cells overexpressing candidate genes. Data are representative of two independent experiments. g Schematic describing the pathways MCL1 and JUNB are involved in to mediate resistance to T cell cytotoxicity. All values are mean ± s.e.m. ns not significant. Source data are provided in Source Data 3.

Fig 4: DHCP increases DR5 expression in a p53-CHOP-denpendent pathway.A and B, HCT116 cells were treated with indicated concentrations of DHCP for 24 h (A) or 200 μM of DHCP for indicated times (B), then the cells were lysed, and the proteins were tested by western blot. C, HCT116 and HCT116 p53−/− cells were treated with 200 μM of DHCP for 24 h, whole-cell extracts were prepared and analyzed by western blot. D, HCT116 and HCT116 p53−/− cells were treated with 200 μM of DHCP with or without 25 ng/ml of TRAIL for 24 h, cell death was analyzed by flow cytometry. E and F, HCT116 cells were transfected with control siRNA, p53 siRNAs (E), or CHOP siRNAs (F), then treated with DHCP for 24 h. Whole-cell extracts were prepared and analyzed by western blot.

Fig 5: DHCP induces the upregulation of DR5.A and B, HCT116 cells were treated with indicated concentrations of DHCP for 24 h. The expressions of cell survival proteins (A) and procell death proteins (B) were detected. C and D, HCT116 cells were treated with indicated concentrations of DHCP for 24 h. The whole-cell extracts were prepared and subjected to western blot for the analysis of DR4/DR5 proteins (C), the surface DR4 and DR5 expression was tested by flow cytometry (D). E and F, HCT116 cells were transfected with control siRNA or DR5 siRNAs, then treated with 200 μM of DHCP for 24 h. The total expression of DR5 (E) and the cell surface DR5 expression (F) were tested. G, HCT116 cells were transfected with control siRNA or DR5 siRNAs, then treated with 200 μM of DHCP with or without 25 ng/ml of TRAIL for 24 h. Cell death was tested by flow cytometry after stained by PI.