Figure 3 from NXP800 Activates the Unfolded Protein Response, Altering AR and E2F Function to Impact Castration-Resistant Prostate Cancer Growth

NXP800 inhibits the growth of AR-dependent and AR-independent prostate cancer models with activation of the UPR and inhibition of key signaling pathways. A and B, PDX-O [CP50, CP89, CP129, and CP142 (A)], AR-positive (VCaP, LNCaP, LNCaP95, and 22Rv1), and AR-negative (PC3 and DU145) prostate cancer cell lines (B) were treated with vehicle (DMSO 0.1%) or various concentrations (5, 10, 50, 100, and 250 nmol/L) of NXP800 (active, red line), CCT365248 (inactive, blue line), and in the case of organoids, various concentrations (1 and 10 μmol/L) of enzalutamide (gray scale), and growth was determined after 5 days for cell lines and 7 days for organoids by CellTiter-Glo Cell Viability Assay as defined in “Materials and Methods.” Mean growth (compared with vehicle; defined as 1) with SD from a single experiment with three to six replicates is shown. *Abiraterone given in the castration-sensitive setting. ^CP89 and CP129 derived from two temporally separated mCRPC biopsies from the same patient. P values were calculated for NXP800 compared with CCT365248 for each concentration and for enzalutamide, compared with vehicle using the unpaired Student t test. P values ≤ 0.05 are shown (*). C–E, VCaP, LNCaP95, and 22Rv1 prostate cancer cells were treated with NXP800 (active, 100 or 250 nmol/L) or CCT365248 (inactive, 250 nmol/L) for 48 hours. RNA-seq was performed on each single experiment in triplicate (duplicate for 250 nmol/L NXP800 in LNCaP95). Analysis of RNA-seq with gene set enrichment analysis shows the enrichment and de-enrichment of Hallmark pathways in response to 100 and 250 nmol/L NXP800 (compared with 250 nmol/L CCT365248) in VCaP (C), LNCaP95 (D), and 22Rv1 (E) prostate cancer cells. NES and FDR are shown as volcano plots. Colored dots denote significantly (FDR 0.05) enriched (red dots) and de-enriched (blue dots) pathways with NXP800 (active compound) treatment. Table shows the NES associated with pathways wherein the FDR was ≤0.05 for 100 and/or 250 nmol/L (asterisk indicates those in which FDR was >0.05). F, VCaP (A), LNCaP95 (B), and 22Rv1 (C) prostate cancer cells were treated with NXP800 (active, 100 or 250 nmol/L) or CCT365248 (inactive, 250 nmol/L) for 48 hours. RNA-seq was performed on each single experiment in triplicate (duplicate for 250 nmol/L NXP800 in LNCaP95). Log₂ fold expression level changes of “activating” E2F (E2F1–3) family members treated with NXP800 (active, 100 or 250 nmol/L) were compared with CCT365248 (inactive, 250 nmol/L). P values were calculated by DESeq2 using the Wald test. P values ≤ 0.05 are shown (*). G, VCaP, LNCaP95, and 22Rv1 prostate cancer cells were treated with 250 nmol/L CCT365248 (inactive) or 250 nmol/L NXP800 (active) for 24 hours. PERK, phospho-eIF2α, and ATF4 (PERK arm); ATF6 (ATF6 arm); IRE1 (IRE1 arm); E2F1 (E2F); and GAPDH (housekeeping) protein expression was determined by Western blot from one experiment performed in triplicate. H, Association between GO cellular response to heat gene expression signature and Hallmark E2F Targets in transcriptome cohorts of patients with PCF-SU2C and ICR-RMH. Pearson r and P values are shown.