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ATRX-Mutant Cancers to WEE1 Inhibition Junbo Liang1, Hong Zhao2,3, Bill H

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ATRX-Mutant Cancers to WEE1 Inhibition Junbo Liang1, Hong Zhao2,3, Bill H Published OnlineFirst September 24, 2019; DOI: 10.1158/0008-5472.CAN-18-3374 CANCER RESEARCH | TRANSLATIONAL SCIENCE Genome-Wide CRISPR-Cas9 Screen Reveals Selective Vulnerability of ATRX-Mutant Cancers to WEE1 Inhibition Junbo Liang1, Hong Zhao2,3, Bill H. Diplas4, Song Liu5, Jianmei Liu2,3, Dingding Wang1, Yan Lu1, Qing Zhu6, Jiayu Wu1, Wenjia Wang1, Hai Yan4, Yi-Xin Zeng6, Xiaoyue Wang1, and Yuchen Jiao2,7 ABSTRACT ◥ The tumor suppressor gene ATRX is frequently mutated in a induced apoptosis. AZD1775 also selectively inhibited the prolif- variety of tumors including gliomas and liver cancers, which are eration of patient-derived primary cell lines from gliomas with highly unresponsive to current therapies. Here, we performed a naturally occurring ATRX mutations, indicating that the synthetic genome-wide synthetic lethal screen, using CRISPR-Cas9 genome lethal relationship between WEE1 and ATRX could be exploited in a editing, to identify potential therapeutic targets specific for ATRX- broader spectrum of human tumors. As WEE1 inhibitors have been mutated cancers. In isogenic hepatocellular carcinoma (HCC) cell investigated in several phase II clinical trials, our discovery provides lines engineered for ATRX loss, we identified 58 genes, including the the basis for an easily clinically testable therapeutic strategy specific checkpoint kinase WEE1, uniquely required for the cell growth of for cancers deficient in ATRX. ATRX null cells. Treatment with the WEE1 inhibitor AZD1775 robustly inhibited the growth of several ATRX-deficient HCC cell Significance: ATRX-mutant cancer cells depend on WEE1, lines in vitro, as well as xenografts in vivo. The increased sensitivity which provides a basis for therapeutically targeting WEE1 in to the WEE1 inhibitor was caused by accumulated DNA damage– ATRX-deficient cancers. Introduction variety of human cancers, including pancreatic neuroendocrine tumors (PanNET), glioma, liver cancer, and neuroblastoma. Although Over 20 years ago, germline variations in ATRX, a member of the ATR-X syndrome-related germline variations are mainly missense in SWI/SNF chromatin remodeling superfamily, were found to underlie a the histone methylation recognition domain and the ATPase rare developmental disorder, X-linked mental retardation with alpha- domain (6), the cancer-associated somatic ATRX mutations are thalassemia (ATR-X syndrome; ref. 1). More recently, genomic predominantly truncating mutations, resulting in loss of ATRX studies (2–5) have revealed that ATRX is frequently mutated in a expression (7), indicating that ATRX functions as a tumor suppressor gene in these cancers. Although genomic sequencing has enabled the rapid discovery of 1 State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical the mutations driving the development of human cancer, significant Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, challenges remain regarding the effective translation of these data into Beijing, China. 2State Key Lab of Molecular Oncology, National Cancer Center/ National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy viable therapies. Most driver mutations are damaging the functions of of Medical Sciences and Peking Union Medical College, Beijing, China. 3Depart- suppressor genes and cannot serve as a direct therapeutic target. One ment of Hepatobiliary Surgery, National Cancer Center/National Clinical strategy for developing targeted therapies against such loss-of-function Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical mutations is “synthetic lethality.” Synthetic lethality refers to the 4 Sciences and Peking Union Medical College, Beijing, China. The Preston Robert relationship between 2 genes for which loss of both genes results in Tisch Brain Tumor Center at Duke, Pediatric Brain Tumor Foundation Institute at cell death but loss of either one alone does not. For example, poly Duke, and Department of Pathology, Duke University Medical Center, Durham, PARP1 BRCA1/2 North Carolina. 5Department of Central Laboratory, Peking Union Medical (ADP)-ribose polymerase 1 ( ) and are synthetic lethal College Hospital, Peking Union Medical College & Chinese Academy of Medical because loss of both genes abolishes 2 parallel DNA damage repair Sciences, Beijing, China. 6Department of Experimental Research, Sun Yat-sen pathways and causes cell apoptosis (8). University Cancer Center, State Key Laboratory of Oncology in Southern China, Motivated by the clinical success of PARP inhibitors in treatment of Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong BRCA1/2-deficient tumors, scientists have performed genome-wide 7 Province, China. Department of Clinical Laboratory, National Cancer Center/ screens in human cancer cells to identify synthetic lethal partners National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. relevant to other frequently mutated tumor suppressor genes. In such screens, genes essential for each cell line are first identified by high- Note: Supplementary data for this article are available at Cancer Research throughput perturbation of genes using RNA interference (RNAi) or Online (http://cancerres.aacrjournals.org/). CRISPR-Cas9 genome editing. Synthetic lethal interactions are then J. Liang, H. Zhao, B.H. Diplas, and S. Liu contributed equally to this article. inferred by comparing the gene dependency profiles across a panel of Corresponding Authors: Xiaoyue Wang, Institute of Basic Medical Sciences, either independently derived cell lines with or without mutations at a Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing specific locus, or paired isogenic lines with engineered mutations. Such 100005, China. E-mail: [email protected]; and Yuchen Jiao, National screens have been used to identify potential pharmacologic targets that Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and enable selective killing of cancer cells with deficiencies in tumor Peking Union Medical College, Beijing 100021, China. Phone: 86-010- TP53 ARID1A 87788045; E-mail: [email protected] suppressors or (9, 10), or even with activation of “undruggable” oncogenes including RAS or MYC (11, 12). Cancer Res 2020;XX:XX–XX Among ATRX-mutant tumor types, hepatocellular carcinoma doi: 10.1158/0008-5472.CAN-18-3374 (HCC) is the 5th most common cancer in the world (13) and glioma Ó2019 American Association for Cancer Research. is the most common primary malignant brain tumor in adults. There AACRJournals.org | OF1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst September 24, 2019; DOI: 10.1158/0008-5472.CAN-18-3374 Liang et al. are currently no effective targeted therapies available for HCC or with DNAMAN multiple sequence alignment tools. The primers used glioma, making them among the most lethal tumors in the world. Here, for amplifying the flanked region of the target site were as follows: we performed a genome-wide pooled CRISPR-Cas9–based screen in 0 0 paired isogenic HCC cells with or without engineered mutations in ATRX_forward: 5 -CCGTGACTCAGATGGAATGGA-3 ATRX – WEE1 ATRX 0 0 . The G2 M checkpoint control kinase, , was one of the _reverse: 5 -GGTTACAGAGCCAGAACAGG-3 several candidate genes identified as potentially lethal in the context of ATRX deficiency. The lethality of WEE1 loss was further investigated Lentivirus production 7 2 with small molecule inhibitors of WEE1 through the treatment of Low passage HEK293T cells (4  10 ) were seeded onto 500 cm engineered ATRX-mutant HCC cell lines and xenografts, as well as culture plates 1 day before transfection. When cells reached 80% to primary cell lines from naturally occurring gliomas. The results 90% confluence, a mixture containing the following library and support inhibition of WEE1 as the basis for the development of new packaging plasmids was transfected into HEK293T cells with Neofect: therapeutic strategies to treat multiple tumor types harboring ATRX GeCKO v2 library (63 mg; Addgene plasmid 1000000048), psPAX2 (45 mutations. mg; Addgene plasmid #12260), and pMD2.G (18 mg; Addgene plasmid #12259). Culture medium was changed 12 hours after transfection. The supernatant containing lentivirus was collected 60 hours post- Materials and Methods transfection, filtered through a PVDF filter membrane (0.45 mm; Ethics statement Millipore SteriCup 250 mL; Millipore) to remove cells, aliquoted, and All animal procedures were approved by and performed according stored at À80 C. to the animal ethical committee at the Cancer Hospital, Chinese Synthetic lethal screens Academy of Medical Sciences (Beijing, China). PLC/PRF/5 parental cells or PLC/PRF/5 ATRX KO cells (200  106) Cell culture were transduced with lentivirus from the human GeCKO v2 library at PLC/PRF/5, HuH-7, and HeLa cell lines were obtained from the an MOI of 0.3 and an average of 500-fold coverage of the library. As the human GeCKO v2 library contains 123,411 sgRNAs, at least 6  National Infrastructure of Cell Line Resource (Beijing, China) and are 7 routinely authenticated via their short tandem repeat profile (latest 10 cells need to be infected to achieve 500-fold coverage. At 48 hours verification for PLC/PRF/5 and HeLa: April 2019; latest verification for postinfection, cells were selected in puromycin (2 mg/mL; Thermo fi  7 HuH-7: November 2018). PLC/PRF/5, HuH-7, HeLa, and HEK293T Fisher
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