Published OnlineFirst January 15, 2021; DOI: 10.1158/2159-8290.CD-20-1109 RESEARCH ARTICLE A CRISPR/Cas9-Engineered ARID1A-Deficient Human Gastric Cancer Organoid Model Reveals Essential and Nonessential Modes of Oncogenic Transformation Yuan-Hung Lo1, Kevin S. Kolahi2, Yuhong Du3, Chiung-Ying Chang2, Andrey Krokhotin4, Ajay Nair5, Walter D. Sobba1, Kasper Karlsson1,6, Sunny J. Jones5, Teri A. Longacre2, Amanda T. Mah1, Bahar Tercan7, Alexandra Sockell6, Hang Xu6, Jose A. Seoane6, Jin Chen8,9, Ilya Shmulevich7, Jonathan S. Weissman8, Christina Curtis6, Andrea Califano5, Haian Fu3, Gerald R. Crabtree2,4,10, and Calvin J. Kuo1 Downloaded from cancerdiscovery.aacrjournals.org on September 23, 2021. © 2021 American Association for Cancer Research. Published OnlineFirst January 15, 2021; DOI: 10.1158/2159-8290.CD-20-1109 ABSTRACT Mutations in ARID1A rank among the most common molecular aberrations in human cancer. However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward genetic models. Here, CRISPR/Cas9-mediatedARID1A knockout (KO) in primary TP53−/− human gastric organoids induced morphologic dysplasia, tumorigenicity, and mucinous differentiation. Genetic WNT/β-catenin activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative pathways of ARID1A KO-mediated transformation. ARID1A mutation induced transcriptional regulatory modules characteristic of microsatellite instability and Epstein–Barr virus–associated subtype human gastric cancer, including FOXM1-associated mitotic genes and BIRC5/survivin. Convergently, high-throughput compound screening indicated selective vul- nerability of ARID1A-deficient organoids to inhibition of BIRC5/survivin, functionally implicating this pathway as an essential mediator of ARID1A KO-dependent early-stage gastric tumorigenesis. Overall, we define distinct pathways downstream of oncogenicARID1A mutation, with nonessential WNT-inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-stimulated prolifera- tion, illustrating the general utility of organoid-based forward genetic cancer analysis in human cells. SIGNIFicance: We establish the first human forward genetic modeling of a commonly mutated tumor suppressor gene, ARID1A. Our study integrates diverse modalities including CRISPR/Cas9 genome editing, organoid culture, systems biology, and small-molecule screening to derive novel insights into early transformation mechanisms of ARID1A-deficient gastric cancers. INTRODUCTION ARID1A, also designated BAF250a, encodes a multifunc- tional BAF complex subunit that targets BAF to AT-rich Alterations in the epigenetic landscape are a hallmark of enhancer DNA sequences, regulates transcription, and cancer (1). The epigenetic state defines the permissible tran- recruits topoisomerase II to chromatin (6, 7). ARID1A muta- scriptome as chromatin topology determines responses to tions rank among the most common molecular aberrations oncogenes and tumor suppressors. Thus, chromatin regulators in human cancer (8–11) and are frequent in multiple cancer play critical roles in tumorigenesis, and their mutation is now types such as ovarian clear-cell carcinoma (∼57%), endo- appreciated as a pervasive feature of malignancy. The mam- metrioid carcinoma (∼30%), urothelial carcinoma (∼26%), malian SWI/SNF (mSWI/SNF; BAF) chromatin remodeling cholangiocarcinoma (∼19%), pancreatic ductal adenocar- complex actively remodels chromatin in an ATP-dependent cinoma (∼8%), and colorectal carcinoma (∼8%; ref. 12). fashion and renders DNA accessible to transcription factors Mutations in ARID1A occur in ∼31% of all gastric adenocar- and other DNA binding proteins (2) to govern development, cinomas, particularly in microsatellite instability (MSI) and homeostasis, and disease (3–5). Epstein–Barr virus-associated (EBV) subtypes, but also in the chromosomal instability (CIN) subtype with lower fre- 1Department of Medicine, Division of Hematology, Stanford University quency (13–15). ARID1A mutations dysregulate BAF com- 2 School of Medicine, Stanford, California. Department of Pathology, Stan- plex–mediated chromatin remodeling because this subunit ford University School of Medicine, Stanford, California. 3Department of Pharmacology and Chemical Biology and Emory Chemical Biology Dis- directly interfaces with DNA and recruits other transcrip- covery Center, Emory University School of Medicine, Atlanta, Georgia. tional coactivators (16). ARID1A’s function as a global 4Howard Hughes Medical Institute, Stanford University School of Medi- chromatin conformation regulator underlies the pleiotropic 5 cine, Stanford, California. Department of Systems Biology, Columbia Uni- effects observed when this gene is disrupted and renders versity, New York, New York. 6Division of Oncology, Stanford University School of Medicine, Stanford, California 7Institute for Systems Biology, the study of ARID1A’s role in oncogenesis especially chal- Seattle, Washington. 8Howard Hughes Medical Institute, Department of lenging. Transgenic Arid1a knockout (KO) mouse models in Cellular and Molecular Pharmacology, University of California, San Fran- embryo (17), ovarian (18), colon (19), small intestine (20), cisco, California. 9Department of Pharmacology and Cecil H. and Ida Green endometrium (21), pancreas (22–25), liver (26), and hemat- Center for Reproductive Biology Sciences, The University of Texas South- opoietic cells (27) have provided tremendous insight into western Medical Center, Dallas, Texas. 10Department of Developmental Biology, Stanford University School of Medicine, Stanford, California. ARID1A-associated tumorigenesis. However, despite these Note: Supplementary data for this article are available at Cancer Discovery extensive mouse studies, forward genetic human models Online (http://cancerdiscovery.aacrjournals.org/). are crucially needed to elaborate mechanisms of ARID1A- Corresponding Author: Calvin J. Kuo, Stanford University School of Medi- dependent oncogenic transformation in a more clinically cine, Lokey G2034A, 265 Campus Drive, Stanford, CA 94305. Phone: 650- relevant context. 498-9047; E-mail: [email protected] Organoid culture is a robust in vitro culture method that Cancer Discov 2021;11:1–20 recapitulates many essential attributes of primary human doi: 10.1158/2159-8290.CD-20-1109 tissue including three-dimensional (3-D) structure, multilin- ©2021 American Association for Cancer Research. eage differentiation, signaling nodes, histology, and pathology JUNE 2021 CANCER DISCOVERY | OF2 Downloaded from cancerdiscovery.aacrjournals.org on September 23, 2021. © 2021 American Association for Cancer Research. Published OnlineFirst January 15, 2021; DOI: 10.1158/2159-8290.CD-20-1109 RESEARCH ARTICLE Lo et al. with high fidelity and thus represents an emerging approach CRISPR/Cas9 system. First, TP53 KO organoids were trans- to cancer biology (28). Bridging cell and tissue scales, orga- fected with a Cas9 construct conferring blasticidin resist- noids offer an attractive hybrid between transgenic mouse ance, and constitutive Cas9 protein expression was verified models and transformed 2-D human cancer cell lines that (Fig. 1B). Cas9-expressing organoids did not exhibit growth enables an engineered “bottom-up” approach to study defects, suggesting low Cas9 toxicity after blasticidin selec- temporal and sequential oncogenic events and permits the tion. To quantify the efficiency of CRISPR/Cas9 cleavage, we functional validation of oncogenic loci. Successful multi-hit delivered a second lentivirus containing a sgRNA targeting oncogenic transformation of normal wild-type organoids to the GFP reporter in the same construct and a puromycin resist- adenocarcinoma has been achieved by introducing simulta- ance gene (Fig. 1C). In the parental TP53 KO organoids, nearly neous oncogenic mutations into tissues such as colon, stom- all cells showed GFP expression after puromycin selection. ach, and pancreas (29–32). However, in Cas9-expressing TP53 KO organoids, more than Here, we utilize wild-type human gastric organoids to estab- 95% of cells were GFP negative as quantified by flow cytometry, lish the first forward genetic humanARID1A -deficient oncogenic indicating highly efficient CRISPR cleavage (Fig. 1C). transformation model, using CRISPR/Cas9-engineered ARID1A We next applied this dual lentiviral system to ARID1A depletion alongside mutation of TP53, a co-occurring tumor genetic knockout in TP53-null organoids. Of note, CRISPR suppressor. These engineered ARID1A-deficient organoids mir- can be mutagenic by introducing random insertions or dele- ror several clinicopathologic features of ARID1A-mutant gas- tions (indels) during cleavage, resulting in heterogeneous tric cancer. Coupled with a regulatory network-based analysis cell populations. To address this potential pitfall and more and high-throughput drug screening, we have leveraged this precisely characterize sequelae of ARID1A loss in gastric tumo- human organoid model to discover potential mechanisms rigenesis, we established a spectrum of clonal TP53/ARID1A underlying the role of ARID1A during oncogenic transforma- DKO organoid lines by sgRNA targeting of ARID1A exon 1 tion of gastric epithelium. or exon 11 in a lentiviral vector with BFP reporter. After lenti- virus sgRNA-BFP delivery of Cas9-TP53 KO organoids, single RESULTS dissociated BFP-positive cells were sorted into single wells of a 96-well plate and clonally expanded, and ARID1A indels at Establishment