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Heterozygous Deletion of Chromosome 17P Renders Prostate Cancer Vulnerable to Inhibition of RNA Polymerase II

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Heterozygous Deletion of Chromosome 17P Renders Prostate Cancer Vulnerable to Inhibition of RNA Polymerase II ARTICLE DOI: 10.1038/s41467-018-06811-z OPEN Heterozygous deletion of chromosome 17p renders prostate cancer vulnerable to inhibition of RNA polymerase II Yujing Li1,2,3, Yunhua Liu 2,3,4, Hanchen Xu1,2,3, Guanglong Jiang3, Kevin Van der Jeught 2,3, Yuanzhang Fang2,3, Zhuolong Zhou3, Lu Zhang2,3, Michael Frieden3, Lifei Wang3, Zhenhua Luo5, Milan Radovich4,6, Bryan P. Schneider7, Yibin Deng8, Yunlong Liu3, Kun Huang7, Bin He9, Jin Wang 10, Xiaoming He 11,12, Xinna Zhang3,4, Guang Ji 1 & Xiongbin Lu2,3,4 1234567890():,; 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 cat- alytic 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 discovered 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 suppress the growth of CRPC in a synergistic manner, which potentiates the therapeutic effectivity of the RNAP2 inhibitor, α-amanitin-based antibody drug conjugate (ADC). Given the limited therapeutic options for CRPC, our findings identify RBX1 as a potentially therapeutic target for treating human CRPC harboring heterozygous deletion of 17p. 1 Institute of Digestive Diseases, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, 200032 Shanghai, China. 2 Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 3 Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA. 4 Indiana University Melvin and Bren Simon Cancer Center, Indiana University School of Medicine, Indianapolis, IN 46202, USA. 5 The Liver Care Center and Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA. 6 Department of Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA. 7 Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA. 8 Laboratory of Cancer Genetics, The University of Minnesota Hormel Institute, Austin, MN 55912, USA. 9 Biomarker Research Program Center, Houston Methodist Research Institute, Houston, TX 77030, USA. 10 Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, TX 77030, USA. 11 Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA. 12 Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD 21201, USA. These authors contributed equally: Yujing Li, Yunhua Liu, Hanchen Xu. Correspondence and requests for materials should be addressed to X.Z. (email: [email protected]) or to G.J. (email: [email protected]) or to X.L. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:4394 | DOI: 10.1038/s41467-018-06811-z | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06811-z rostate cancer is among the most common male malig- human 17p13.1) cooperates with Trp53 (mouse orthologue of nancies and one major leading cause of cancer mortality in TP53) deletion to produce more aggressive disease in lymphoma P 1,2 18 fi men . Since the discovery of androgen dependence in and leukemia . In this study, we nd that 63% of metastatic prostate cancer, the backbone therapy for prostate cancer has prostate cancer harbors heterozygous deletion of a region that been to lower androgen levels by surgical castration or androgen- spans up to 20 megabases of DNA at 17p. We demonstrate that deprivation therapy3. However, although many patients initially POLR2A is included in the 17p deletion region along with TP53 respond to androgen deprivation therapy, nearly all the patients in a majority of prostate cancers. POLR2A is the catalytic subunit relapse and eventually develop castration-resistant prostate can- of the RNAP2 complex that is solely in charge of mRNA synthesis cer (CRPC)4. Over the past decade, it has become clear that the in cells. POLR2A and RNAP2 activity is specifically inhibited by androgen receptor (AR) plays a pivotal role in the development of α-amanitin, a small molecule peptide produced by the death cap resistance to hormone therapies in both primary and recurrent mushroom (Amanita phalloides)19,20. Inhibition of POLR2A with prostate cancer. New therapeutic approaches in advanced pros- α-amanitin-based ADC selectively suppresses the proliferation, tate cancer have focused on the AR protein, which led to the survival and tumor growth of CRPC cells harboring this genomic development of AR-targeting agents, abiraterone acetate and event. Using a CRISPR-Cas9 screen, we uncover that hetero- enzalutamide5. zygous deletion of 17p confers a selective dependence on RBX1, Despite the success of androgen deprivation and AR-blocking inhibition of which had a synergistic and robust suppression in therapies, the newly developed therapies also suffer a short the growth of CRPC along with the treatment of α-amanitin- therapeutic durability due to acquired resistance4,6. Thus, conjugated anti-EpCAM antibodies. researchers are now searching for more therapeutic targets, one of fi which is prostate-speci c membrane antigen (PSMA). PSMA is Results highly expressed on the surface of nearly all prostate cancer cells Heterozygous deletion of 17p is frequent in prostate cancer. but is present on only a few normal tissues, making it an excellent TP53, a well-documented tumor suppressor gene, is frequently target for drugs that selectively attack tumors7. Radioactive ele- inactivated by mutation or deletion in a majority of human ment lutetium-177-labeled PSMA antibody has shown promise in – tumors21. However, the mutation and homozygous deletion of treating patients who are resistant to other drug therapies8 10. TP53 are relatively infrequent events in prostate cancer, While it is hard to directly target variant forms of AR or altera- accounting for only 12% (59 out of 492) and 8% (37 out of 492) tions in the AR gene that promote castration resistance, a small of total cases, respectively. In contrast, approximately 26% (126 molecular inhibitor against ROR-γ, an upstream regulator of AR, out of 492) of all prostate tumor samples harbor heterozygous was proven to effectively shut down AR signaling11. In mouse loss of TP53 (Fig. 1a). Our recent studies have shown that het- CRPC models, treatment with ROR-γ inhibitors led to substantial erozygous deletion of TP53 in human colorectal cancer fre- and prolonged regression of tumors, and restored their sensitivity quently encompasses a neighboring essential gene POLR2A, to the treatment of enzalutamide. In-depth understanding of rendering cancer cells with heterozygous loss of TP53 susceptible prostate cancer invasion, metastasis and drug resistance will help to further inhibition of POLR2A22,23. The comprehensive ana- identify more therapeutic targets and lead to new treatment lysis of prostate cancer genomes revealed that the TP53 deletion options. is often included in a large fragment deletion that spans over The whole-genome sequencing of cancer genomes and other almost the whole short arm of chromosome 17 (17p) (Fig. 1b). associated omics efforts have empowered our knowledge of The loss of 17p is frequently detected at relatively late stages of human cancer biology and pathogenesis. These efforts have colorectal cancer development24,25, which may contribute to or facilitated personalized medicine to find new genetic alterations even determine the transition from the early-stage to advanced- in the context of a specific cancer. Comprehensive analyses of key stage cancer. Consistent with the findings in colorectal cancer, genomic changes in prostate cancer will accelerate our progress in analysis of The Cancer Genome Atlas (TCGA) prostate dataset developing more effective ways to diagnose, treat and prevent this revealed that 17p loss is readily detectable in low-grade T1 disease. Recent studies have identified recurrent somatic muta- tumors, but was significantly increased in the advanced T2-T4 tions, copy number variations, and chromosomal rearrangements tumors (Fig. 1c, d). Moreover, the loss of 17p rises up to 63% (95 in prostate cancer12,13. A number of frequent genomic changes out of 150) in metastatic prostate cancer, whereas TP53 mutation are shared by primary and metastatic prostate cancer, including and homozygous deletion account for only 39 and 6% respec- E26 transformation-specific (ETS) fusions, point mutations in tively (Fig. 1e, f). Collectively, these results indicate the 17p loss SPOP, FOXA1, and TP53, and copy number alterations involving may play an important role in prostate cancer progression and MYC, RB1, and PTEN, although these alterations are differentially metastasis. enriched at different stages of prostate cancer12,13. Interestingly, aberrations of BRCA2, BRCA1, and ATM were observed at a much higher frequency in CRPC, indicating the potential appli- RBX1 is an essential gene for 17ploss CRPC cells. In the 17p cation of PARP inhibitors in treating this subset of cancers. In a deletion region, there are as many as 200 protein-coding genes phase II study of olaparib14, a PARP inhibitor, it was proven to and noncoding RNA genes,
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