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Heterozygous Deletion of Chromosome 17P Renders Prostate Cancer Vulnerable to the Inhibition of RNA Polymerase II IMPROVING HEALTH THROUGH RESEARCH

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Heterozygous Deletion of Chromosome 17P Renders Prostate Cancer Vulnerable to the Inhibition of RNA Polymerase II IMPROVING HEALTH THROUGH RESEARCH Heterozygous Deletion of Chromosome 17p Renders Prostate Cancer Vulnerable to the Inhibition of RNA Polymerase II IMPROVING HEALTH THROUGH RESEARCH Yujing Li1, Xiongbin Lu1,2 indianactsi.org 1 Indiana University School of Medicine, Indianapolis Indiana; 2 Corresponding authors: XL ([email protected]) ABSTRACT RESULT 3 RESULT 6 Heterozygous deletion of chromosome 17p (17p) is one of the most frequent genomic events in human cancers. Beyond the tumor suppressor TP53, the POLR2A gene encoding the catalytic subunit of RNA polymerase II (RNAP2) is also included in a ~20-megabase deletion region of 17p in 63% of metastatic castration-resistant prostate cancer (CRPC). Using a focused CRISPR-Cas9 screen, we discover that heterozygous loss of 17p confers a selective dependence of CRPC cells on the ubiquitin E3 ligase Ring-Box 1 (RBX1). RBX1 activates POLR2A by the K63-linked ubiquitination and thus elevates the RNAP2- mediated mRNA synthesis. Combined inhibition of RNAP2 and RBX1 profoundly suppresses the growth of CRPC in a synergistic manner, which potentiates the therapeutic effectivity of the RNAP2 inhibitor, α-amanitin-based antibody drug loss conjugate (ADC). Given the limited therapeutic options for RBX1 depletion sensitized 17p prostate cancer cells to POLR2A inhibition. (a) RBX1 promotes RNA transcription. Global RNA synthesis was CRPC, our findings identify RBX1 as a potentially therapeutic CRISPR/Cas9-based screen identifies RBX1 as an essential gene evaluated by measurement of 5-EU incorporation and the intensity was loss target for treating human CRPC harboring heterozygous deletion selectively for the 17p prostate cancer cells. (a) Schematic illustration of quantified with the CellProfiler Software. (b) Effect of RBX1 knockdown on the of 17p. CRISPR screening procedure in the isogenic pair of DU145 cells. (b) Box levels of lysine-63 (K63) ubiquitination of POLR2A in the parental and isogenic plots showing the distribution of sgRNA frequencies at different time points. (c) 17ploss DU145 cells. (c) Protein levels of POLR2A, p53, Rbx1 and β-Actin in Overlapping of essential genes from this screen and from the previous reports. human prostate cancer cell lines. (d) Cell proliferation of 17pneutral and 17ploss (d) Frequency histograms of enriched or depleted sgRNAs. POLR2A and EIF6 cells treated with α-amanitin or actinomycin D. (e, f) RBX1 depletion sensitizes are two representatives of common essential genes. RBX1 and GTF2H1 are the 17ploss DU145 cells to the treatment of the POLR2A inhibitor, α-amanitin. (g) representatives of selective essential genes in the context of 17p loss. Cell proliferation of human prostate cancer cell lines under α-amanitin. RESULT 1 RESULT 4 RESULT 7 Chromosome 17p loss is a frequent genomic event in prostate cancer. Prostate cancer cells with heterozygous 17p deletion are highly sensitive Inhibition of RBX1 sensitizes 17ploss CRPC to the treatment of α-amanitin- (a) Genomic alterations of TP53 (point mutation, shallow deletion and deep to RBX1 depletion. (a) Effect of RBX1 knockdown on the proliferation of the based ADC. (a) Schematic illustration of orthotopic injection of 17ploss DU145 deletion) in a TCGA prostate cancer dataset (TCGA provisional, n=499) parental and isogenic 17ploss DU145 cells, determined by direct competition cells followed by twice i.p. injection of ADC and Dox food treatment. (b, c) determined by cBioportal. (b) Integrated analysis of 17p deletion in 155 assay. (b) Representative cell survival measured by staining with crystal violet. Representative bioluminescent tumor images and individual tumor growth prostate cancer patient samples. Frequency plots of the copy number (c-e) Cell growth curves of human prostate cancer cell lines expressing Dox- curves of xenograft tumors derived from orthotopically implanted 17ploss DU145 abnormalities indicate degree of copy number loss (red) or gain (blue). inducible shNT or RBX1-specific shRNA. RBX1 knockdown efficiency (d) and cells with (b) or without (c) Dox treatment. Once tumor was established, mice r Representative genes in 17p deletion region are shown. (c, d) Distribution of representative image (e) were shown. (f) Fraction of apoptotic cells in the were randomly divided to 4 groups and treated with either free anti-EpCAM heterozygous deletion of 17p among tumor clinical (c) and pathological (d) 17pneutral (22Rv1 and DU145) and 17ploss (PC3 and VCaP) cells with RBX1 antibody or different doses of anti-EpCAM-amanitin conjugates (ADC). (d) stages in the TCGA prostate cancer dataset. knockdown. (g) Cell survival in PC3 and VCaP cells expressing shRBX1, Quantification of RBX1 knockdown efficiency, cell proliferation and apoptosis in control or ectopic RBX1. **, p < 0.01; ***, p < 0.001. the xenografted tumor tissues described above. RESULT 2 RESULT 5 CONCLUSIONS 1. 63% of CRPC harbors heterozygous deletion of a region that spans up to 20 megabases of DNA at 17p. 2. A CRISPR-based screen identified RBX1 as a selective essential gene in 17ploss CRPC cells. 3. RBX1 K63-polyubiquitinates POLR2A, a modification which can increase RNA polymerase II activity. 4. Combination of RBX1 loss and RNA polymerase II inhibition can selectively block 17 heterozygous loss tumor cell growth. ACKNOWLEDGEMENT Generation of isogenic 17ploss DU145 cell line with CRISPR-Cas9. (a) Schematic diagram of genes on chromosome 17p in human genome. (b) We thank E. Lander and D. Sabatini for providing the Cas9 expressing vector Validation of 17ploss cell colonies with PCR. To assess whether the 20 and CRISPR knockout pooled library. We thank L. Huang and M. Bar-Eli for megabases region had been excised following Cas9 cleavage, we performed a lentivirus production. We also thank J. Wang for technical support in prostate deletion PCR using a forward primer that binds to WDR81 and a reverse primer Depletion of RBX1 inhibits the growth of 17ploss CRPC tumors in vivo. (a- orthotopic tumor experiments. The following cores were used: Indiana that binds to MAP2K3. Total genomic DNA was extracted and amplified with c) Tumor growth curves (a), gross tumor images and weights (b) of xenograft University Mass Spectrometry Facility, Indiana University Flow Cytometry specific primers to validate its copy number variation. Positive cell colonies with tumors derived from subcutaneously implanted parental and isogenic 17ploss Facility, MD Anderson Flow Cytometry and Cellular Imaging Core, MD 17p loss will produce a PCR product (DNA) of small size. (c) Determination of DU145 cells expressing Dox-inducible RBX1 shRNA. (c) RBX1 expression, Anderson Clinical Pathology, Veterinary Medicine and Surgery Core, MD copy numbers of the genes in the proposed deletion region by real-time PCR cell proliferation and apoptosis in the above xenograft tumors were quantified. Anderson Sequencing and Microarray Core. This work was supported in part using gene-specific primers. Gene copy numbers are reduced by ~2-fold in the (d, e) Representative bioluminescent images (d) and gross tumor weights (e) by US National Institutes of Health grants R01CA203737 (X.L.), cell colonies with heterozygous loss of 17p. (d) Sanger sequencing of of xenograft tumors derived from orthotopically implanted 22Rv1 and PC3 R01CA206366 (X.H. & X.L.), and R01CA211861 (B.H.), Indiana University breakpoint junction (from ~1 Mb to ~21 Mb) in two single-cell-derived DU145 cells expressing Dox-inducible RBX1 shRNA. ns, not significant; **, p<0.01. Strategic Research Initiative fund (X.L.), and by Biomedical Research Grant of colonies. (e) Cell proliferation of the parental and isogenic 17ploss DU145 cells. Indiana Clinical and Translational Sciences..
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