Fig 2: RBX1 depletion sensitized 17ploss prostate cancer cells to POLR2A inhibition. a Protein levels of POLR2A, p53, Rbx1, and β-Actin in human prostate cancer cell lines. b, c Cell proliferation of 17pneutral (22Rv1 and DU145) and 17ploss cells (PC3 and VCaP) treated with α-amanitin (b) or actinomycin D (c). d, e RBX1 depletion sensitizes the 17ploss DU145 cells to the treatment of the POLR2A inhibitor, α-amanitin. Representative images (d) and quantitative results (e) of cell survival are shown. f Cell proliferation of human prostate cancer cell lines under α-amanitin treatment. 17pneutral (22Rv1 and DU145) and 17ploss (PC3 and VCaP) cells, with or without Dox-induced RBX1 knockdown, were treated with increasing doses of α-amanitin. Data are representative of three independent experiments

Fig 3: Prostate cancer cells with 17p deletion are highly sensitive to RBX1 depletion. a, b Effect of RBX1 knockdown on the proliferation of the parental and isogenic 17ploss DU145 cells, determined by direct competition assay. Cells expressing RFP and control nonspecific shRNA (shNT) or shRBX1 were sorted and mixed with control RFP-negative cells (1:1) and the RFP-positive cells were quantified at passages 2, 4, and 6 (a). Representative cell survival measured by staining with crystal violet was shown in b. c–e Cell growth curves, based on crystal violet staining, of human prostate cancer cell lines expressing Dox-inducible shNT or RBX1-specific shRNA (shRBX1 #1, shRBX1 #2) (c). RBX1 knockdown efficiency and representative image were shown in d and e, respectively. f Fraction of apoptotic cells in the 17pneutral (22Rv1 and DU145) and 17ploss (PC3 and VCaP) cells expressing Dox-induced RBX1 shRNA at 4 days post Dox treatment. g, h Cell survival measured by crystal violet staining (g) and protein expression levels (h) of RBX1 in PC3 and VCaP cells expressing shRBX1, control or ectopic RBX1. Data are representative of three independent experiments and analyze by unpaired two-tailed t-test. Error bars denote SD. **, p < 0.01; ***, p < 0.001

Fig 4: CRISPR-Cas9 screen identifies RBX1 as an essential gene for 17ploss prostate cancer cells. a Schematic illustration of CRISPR screening procedure in the isogenic pair of DU145 cells. b Box plots showing the distribution of sgRNA frequencies at different time points. c Overlapping of essential genes from this screen and from the previous reports. d Frequency histograms of enriched or depleted sgRNAs. POLR2A and EIF6 are two representatives of common essential genes. RBX1 and GTF2H1 are representatives of selective essential genes in the context of 17p loss

Fig 5: Inhibition of RBX1 sensitizes 17ploss CRPC to the treatment of α-amanitin-based ADC. a Schematic illustration of orthotopic injection of 17ploss DU145 cells (1 × 106 cells) followed by twice i.p. injection of ADC and Dox food treatment. b, c Representative bioluminescent tumor images and individual tumor growth curves of xenograft tumors derived from orthotopically implanted 17ploss DU145 cells without (b) or with (c) Dox treatment. Once tumor was established, mice were randomly divided to 4 groups (n = 8) and treated with either free anti-EpCAM antibody or different doses of anti-EpCAM-amanitin conjugates (ADC). d Quantification of RBX1 knockdown efficiency, cell proliferation (Ki-67 staining) and apoptosis (cleaved caspase-3 staining) in the xenografted tumor tissues described above. Data are analyze by unpaired two-tailed t-test and are presented as the mean ± SD. **, p < 0.01; ***, p < 0.001