A CRISPR/Cas9-Engineered ARID1A

Author Manuscript Published OnlineFirst on January 15, 2021; DOI: 10.1158/2159-8290.CD-20-1109 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model 2 reveals essential and non-essential modes of oncogenic transformation 3 4 Yuan-Hung Lo1, Kevin S. Kolahi2, Yuhong Du3, Chiung-Ying Chang2, Andrey Krokhotin4, Ajay 5 Nair5, Walter D. Sobba1, Kasper Karlsson1,6, Sunny J. Jones5, Teri A. Longacre2, Amanda T. 6 Mah1, Bahar Tercan7, Alexandra Sockell6, Hang Xu6, Jose A. Seoane6, Jin Chen8,9, Ilya 7 Shmulevich7, Jonathan S. Weissman8, Christina Curtis6, Andrea Califano5, Haian Fu3, Gerald R. 8 Crabtree2,4,10, Calvin J. Kuo1* 9 10 11 Department of Medicine, Divisions of Hematology1 and Oncology6, Stanford University School of 12 Medicine, Stanford, CA 94305, USA 13 14 2Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA 15 16 3Department of Pharmacology and Chemical Biology and Emory Chemical Biology Discovery 17 Center, Emory University School of Medicine, Atlanta, GA 30322, USA 18 19 4Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, 20 USA 21 22 5Department of Systems Biology, Columbia University, New York, NY 10032, USA 23 24 7Institute for Systems Biology, Seattle, WA 98109, USA 25 26 8Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, 27 University of California, San Francisco, CA 94158, USA 28 29 9Department of Pharmacology and Cecil H. and Ida Green Center for Reproductive Biology 30 Sciences, UT Southwestern Medical Center, Dallas, TX 75390, USA 31 32 10Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 33 94305, USA 34 35 36 *Corresponding author: Calvin J. Kuo 37 Email: [email protected] 38 Phone: 650-498-9047 39 Address: Stanford University School of Medicine, Lokey G2034A, 265 Campus Dr., Stanford, 40 CA 94305, USA 41 42 43 Running title 44 Modeling ARID1A-deficient tumorigenesis in human organoids 45 46 47 Key words 48 ARID1A, Organoids, CRISPR/Cas9, FOXM1, BIRC5/survivin, WNT, Gastric Cancer 49 50 51 Conflict of Interest Statement 52 The authors declare no potential conflicts of interest 53

Downloaded from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 15, 2021; DOI: 10.1158/2159-8290.CD-20-1109 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

54 Abstract

56 Mutations in ARID1A rank amongst the most common molecular aberrations in human cancer.

57 However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined

58 due to lack of forward genetic models. Here, CRISPR/Cas9-mediated ARID1A knockout in

59 primary TP53-/- human gastric organoids induced morphologic dysplasia, tumorigenicity and

60 mucinous differentiation. Genetic Wnt/-catenin activation rescued mucinous differentiation, but

61 not hyperproliferation, suggesting alternative pathways of ARID1A KO-mediated transformation.

62 ARID1A mutation induced transcriptional regulatory modules characteristic of MSI and EBV

63 subtype human gastric cancer, including FOXM1-associated mitotic genes and BIRC5/survivin.

64 Convergently, high-throughput compound screening indicated selective vulnerability of ARID1A-

65 deficient organoids to inhibition of BIRC5/survivin, functionally implicating this pathway as an

66 essential mediator of ARID1A KO-dependent early-stage gastric tumorigenesis. Overall, we

67 define distinct pathways downstream of oncogenic ARID1A mutation, with non-essential Wnt-

68 inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-

69 stimulated proliferation, illustrating the general utility of organoid-based forward genetic cancer

70 analysis in human cells.

72 Statement of significance

74 We establish the first human forward genetic modeling of a commonly mutated tumor

75 suppressor gene, ARID1A. Our study integrates diverse modalities including CRISPR/Cas9

76 genome editing, organoid culture, systems biology and small molecule screening to derive novel

77 insights into early transformation mechanisms of ARID1A-deficient gastric cancers.

79 Introduction

81 Alterations in the epigenetic landscape are a hallmark of cancer(1). The epigenetic state defines

82 the permissible transcriptome as chromatin topology determines responses to oncogenes and

83 tumor suppressors. Thus, chromatin regulators play critical roles in tumorigenesis, and their

84 mutation is now appreciated as a pervasive feature of malignancy. The mammalian SWI/SNF

85 (mSWI/SNF, BAF) chromatin remodeling complex actively remodels chromatin in an ATP-

86 dependent fashion and renders DNA accessible to transcription factors and other DNA binding

87 proteins(2) to govern development, homeostasis and disease(3–5).

88 ARID1A, also designated BAF250a, encodes a multifunctional BAF complex subunit that

89 targets BAF to AT-rich enhancer DNA sequences, regulates transcription and recruits

90 topoisomerase II to chromatin(6,7). ARID1A mutations rank amongst the most common

91 molecular aberrations in human cancer(8–11) and are frequent in multiple cancer types such as

92 ovarian clear-cell carcinoma (~57%), endometrioid carcinoma (~30%), urothelial carcinoma

93 (~26%), cholangiocarcinoma (~19%), pancreatic ductal adenocarcinoma (~8%), and colorectal

94 carcinomas (~8%)(12). Mutations in ARID1A occur in ~31% of all gastric adenocarcinomas,

95 particularly in microsatellite instability (MSI) and Epstein-Barr virus-associated (EBV) subtypes,

96 but also in the chromosomal instability (CIN) subtype with lower frequency(13–15). ARID1A

97 mutations dysregulate BAF complex-mediated chromatin remodeling since this subunit directly

98 interfaces with DNA and recruits other transcriptional co-activators(16). ARID1A’s function as a

99 global chromatin conformation regulator underlies the pleiotropic effects observed when this

100 gene is disrupted, and renders the study of ARID1A’s role in oncogenesis especially challenging.

101 Transgenic Arid1a knockout mouse models in embryo(17), ovarian(18), colon(19), small

102 intestine(20), endometrium(21), pancreas(22–25), liver(26), and hematopoietic cells(27) have

103 provided tremendous insight into ARID1A-associated tumorigenesis. However, despite these

104 extensive mouse studies, forward genetic human models are crucially needed to elaborate

105 mechanisms of ARID1A-dependent oncogenic transformation in a more clinically relevant

106 context.

107 Organoid culture is a robust in vitro culture method that recapitulates many essential

108 attributes of primary human tissue including 3-dimensional (3D) structure, multilineage

109 differentiation, signaling nodes, histology, and pathology with high fidelity and thus represents

110 an emerging approach to cancer biology(28). Bridging cell and tissue scales, organoids offer an

111 attractive hybrid between transgenic mouse models and transformed 2D human cancer cell

112 lines that enables an engineered “bottom up” approach to study temporal and sequential

113 oncogenic events and permits the functional validation of oncogenic loci. Successful multi-hit

114 oncogenic transformation of normal wild-type organoids to adenocarcinoma has been achieved

115 by introducing simultaneous oncogenic mutations into tissues such as colon, stomach and

116 pancreas(29–32).

117 Here, we utilize wild-type human gastric organoids to establish the first forward genetic

118 human ARID1A-deficient oncogenic transformation model, using CRISPR/Cas9-engineered

119 ARID1A depletion alongside mutation of TP53, a co-occurring tumor suppressor. These

120 engineered ARID1A-deficient organoids mirror several clinicopathologic features of ARID1A-

121 mutant gastric cancer. Coupled with a regulatory network-based analysis and high-throughput

122 drug screening, we have leveraged this human organoid model to discover potential

123 mechanisms underlying the role of ARID1A during oncogenic transformation of gastric

124 epithelium.

125

126 Results

127

128 Establishment of clonal TP53 and TP53/ARID1A knockout human gastric organoid lines

129 Arid1a is indispensable for stem-cell maintenance and self-renewal, as genetic deletion results

130 in lethal compromise of gastrulation at E6.5, and knockout embryonic stem cells cannot be

131 established(17,20,27,33). Consistent with these observations, using wild-type human gastric

132 corpus organoids(34) from partial gastrectomy obesity surgeries, we could not expand and

133 maintain ARID1A CRISPR/Cas9 KO derivatives in long-term culture. Four independent

134 experiments were attempted, and a total of 12 clonal ARID1A CRISPR/Cas9 KO organoid lines

135 were continuously tracked for at least two weeks. However, all of these ARID1A KO organoid

136 lines eventually failed to grow, leading us to surmise that additional bypass mutation(s) could be

137 needed. Thus, to establish an ARID1A-deficient human gastric cancer transformation model, we

138 first disrupted TP53, the most frequently mutated locus (~49%) in gastric adenocarcinoma(14),

139 by CRISPR/Cas9 into the same wild-type human gastric corpus organoids, followed by

140 secondary CRISPR/Cas9 KO of ARID1A. Transient transfection of an all-in-one construct

141 expressing both Cas9 and sgRNA targeting TP53 exon 4 followed by a recently developed

142 nutlin-3 functional selection(30,31) yielded numerous organoid colonies, whereas no growth

143 was seen in non-transfected cells. After clonal expansion, a nutilin-3-resistant organoid clone

144 harboring a 1 bp cytosine deletion (327delC; TTCCG to TTCG) within TP53 exon 4 was chosen

145 for further analysis (Fig. 1A).

146 Serial genome editing in primary human organoids to generate sequential oncogenic

147 mutations has been largely restricted by limited absolute knockout efficiency and a paucity of

148 available functional selection strategies(35). To overcome these limitations and introduce

149 inactivating mutations in ARID1A in these newly generated TP53 KO gastric organoids, we

150 utilized a two-vector, sequential lentiviral-based CRISPR/Cas9 system. First, TP53 KO

151 organoids were transfected with a Cas9 construct conferring blasticidin resistance and

152 constitutive Cas9 protein expression was verified (Fig. 1B). Cas9-expressing organoids did not

153 exhibit growth defects, suggesting low Cas9 toxicity after blasticidin selection. To quantify the

154 efficiency of CRISPR/Cas9 cleavage, we delivered a second lentivirus containing a sgRNA

155 targeting the GFP reporter in the same construct and a puromycin resistance gene (Fig. 1C). In

156 the parental TP53 KO organoids, nearly all cells showed GFP expression after puromycin

157 selection. However, in Cas9-expressing TP53 KO organoids, over 95% of cells were GFP-

158 negative as quantified by flow cytometry, indicating highly efficient CRISPR cleavage (Fig. 1C).

159 We next applied this dual lentiviral system to ARID1A genetic knockout in TP53-null

160 organoids. Of note, CRISPR can be mutagenic by introducing random insertions or deletions

161 (indels) during cleavage, resulting in heterogenous cell populations. To address this potential

162 pitfall and more precisely characterize sequelae of ARID1A loss in gastric tumorigenesis, we

163 established a spectrum of clonal TP53/ARID1A DKO organoid lines by sgRNA targeting of

164 ARID1A exon 1 or exon 11 in a lentiviral vector with BFP reporter. After lentivirus sgRNA-BFP

165 delivery of Cas9-TP53 KO organoids, single dissociated BFP positive cells were sorted into

166 single wells of a 96-well plate, clonally expanded and ARID1A indels at sgRNA-targeted regions

167 were confirmed by Sanger sequencing (Fig. 1D). The corresponding wild-type organoids

168 possessed wild-type TP53 (Fig. 1A) and ARID1A (Fig. 1D) alleles. The loss of ARID1A

169 expression, but not ARID1B, was further confirmed by Western blotting (Fig. 1E) and

170 immunohistochemical (IHC) staining (Fig. 1F). A total of five TP53/ARID1A DKO organoids

171 lines were chosen for this study. In parallel, an empty lentiviral sgRNA-BFP vector was

172 transduced into the same Cas9-TP53 KO organoids, and represented the control. The TP53 KO

173 organoids (control) and TP53/ARID1A DKO organoids (ARID1A KO) were established, grown,

174 maintained and passaged using identical culture conditions throughout this study. We

175 performed whole-genome sequencing of control TP53 KO, TP53/ARID1A DKO clone 3 (indels

176 in ARID1A exon 1), and TP53/ARID1A DKO clone 5 (indels in ARID1A exon 11) at 3 months

177 after ARID1A sgRNA delivery to outline the genetic background of these engineered organoids.

178 The genome of the parental wild-type organoids was used as the reference. As expected, TP53

179 mutation induced a moderate degree of chromosomal instability in both TP53 KO and

180 TP53/ARID1A DKO organoids (Supplementary Fig. 1A), and a few shared and clonal-specific

181 nonsynonymous mutations were detected (Supplementary Fig. 1B). Importantly, no additional

182 canonical TCGA gastric cancer driver mutations were identified in either TP53 KO or

183 TP53/ARID1A DKO organoids, thus excluding promiscuous alteration of additional oncogenes

184 or tumor suppressors (Supplementary Fig. 1B).

185

186 Loss of ARID1A promotes gastric malignancy

187 To elucidate consequences of ARID1A loss in gastric tumorigenesis, we initially examined

188 histology of TP53 KO and TP53/ARID1A DKO organoids by hematoxylin and eosin (H&E)

189 staining. TP53 KO organoids harboring control sgRNA predominantly grew as variably-sized

190 acini composed of a single layer of polarized epithelium (Fig. 2A). Cytologically, the cells in

191 TP53 KO organoids were well-organized with an apically oriented cytoplasm and basally placed

192 nucleus, indicating preservation of apicobasal polarity. In contrast, all five TP53/ARID1A DKO

193 organoid lines exhibited different degrees of architectural complexity and cytologic changes

194 characteristic of high-grade dysplasia, including but not limited to cribriform growth, stratification,

195 increased nuclear to cytoplasmic ratios, and nuclear pleomorphism with nuclear membrane

196 irregularities (Fig. 2A). These features were rarely identified in the control TP53 KO organoids.

197 The cribriform features of ARID1A KO organoids resulted in multi-cystic organoids containing

198 several lumina (Fig. 2A), and epithelia were haphazardly arranged with loss of the distinctive

199 apicobasal orientation evident on H&E stained histologic sections in TP53 KO organoids. The

200 latter observations, along epithelial stratification, raise the possibility that cell intrinsic apicobasal

201 polarity is disrupted in ARID1A-deficient organoids (Fig. 2A). Immunofluorescence staining of

202 the apical-specific marker ZO1 further confirmed inappropriate basolateral ZO1 expression

203 facing the extracellular matrix in a subset of TP53/ARID1A DKO organoid cells, suggesting

204 disrupted apicobasal polarity (Fig. 2B). Additionally, TP53/ARID1A DKO organoids exhibited

205 several high-grade dysplasia cytologic features, including nuclear pleomorphism, nuclear

206 membrane irregularities and conspicuous nucleoli (Supplementary Fig. 2)(36,37). Functionally,

207 TP53/ARID1A DKO organoid lines proliferated more rapidly than TP53 KO organoids, resulting

208 in the larger size (Fig. 2C), as well as increased EdU-positive cells (Fig. 2D), revealing a growth

209 advantage conferred by ARID1A loss. Consistent with these results, compared to TP53 KO

210 organoids, TP53/ARID1A DKO organoids exhibited higher metabolic activity (Fig. 2E).

211 Subcutaneous xenografts of TP53 KO organoids showed poor in vivo engraftment and

212 diminutive outgrowth (n=16, 18.75% success rate); however, TP53/ARID1A DKO organoids

213 engrafted at a significantly greater rate (n=17, 76.47% success rate) and formed larger masses

214 (Fig. 2F). Of note, TP53/ARID1A DKO xenografts in vivo also reflected high-grade dysplasia

215 (Fig. 2F). Taken together, these results suggested that ARID1A mutation morphologically and

216 functionally enhances tumorigenesis in primary human gastric organoids. Additionally, our

217 review of histopathology and immunohistochemical ARID1A expression in a gastric cancer

218 tissue microarray of 197 patients from Stanford Hospital indicated a significant inverse

219 association between ARID1A staining and tumor grade (Fig. 2G).

220

221 Loss of ARID1A induces mucinous metaplasia

222 Precancerous transformation of gastric epithelial cells is incited by alterations in genes involved

223 in lineage differentiation and stem cell activity(38). Human gastric homeostasis is maintained, in

224 part, by a gradient of canonical Wnt/-catenin activity generated from the gland base, where the

225 chief cells reside, extending to mucin-producing populations such as neck (TFF2+, LYZ+) and

226 upper gland pit cells (TFF1+, MUC5AC+)(39,40). To determine if ARID1A loss altered organoid

227 differentiation, we assessed several lineage-specific markers (Fig. 3A). In comparison to

228 TP53 KO organoids, TP53/ARID1A DKO organoids upregulated TFF1, TFF2 and LYZ but the

229 enteroendocrine marker CHGA was unaltered (Fig. 3B). We further confirmed significantly up-

230 regulation of additional pit cell markers, GKN1 and GKN2, in TP53/ARID1A DKO organoids

231 (Supplementary Fig. 3A).

232 Next, we performed TFF1, MUC5AC, TFF2 and LYZ immunofluorescence staining of

233 engineered organoids and the original cognate donor primary gastric tissues. In primary healthy

234 tissues, TFF1 and MUC5AC were specifically expressed in the pit domain at gland tops (Fig.

235 3C). In addition, TFF2 specifically marked mucous neck cells (Supplementary Fig. 3B) and

236 LYZ labeled pit cells, with additional scattered positivity in gland bases (Supplementary Fig.

237 3C). TP53 KO organoids expressed very low levels of TFF1 and only sporadically expressed

238 MUC5AC, TFF2 and LYZ. In contrast, TP53/ARID1A DKO organoids profoundly induced TFF1,

239 MUC5AC, TFF2, and LYZ, consistent with acquisition of a mucinous phenotype (Fig. 3C and

240 Supplementary Fig. 3B-C). Chief cell mRNAs LGR5, MIST1, PGC, and CPB1 were down-

241 regulated in TP53/ARID1A DKO organoids (Supplementary Fig. 3D).

242 Gastrointestinal cell fate decisions can increase mucin production in reaction to injury, a

243 phenomenon termed mucous cell metaplasia(41,42). During metaplasia the epithelium is

244 repopulated by cell lineages non-endemic to gastric tissues. Importantly, metaplastic

245 transformation occurs in the earliest stages of progression of precancerous lesions to gastric

246 cancer. We tested if ARID1A loss induced mucous cell metaplasia by Alcian blue staining,

247 which marks acidic mucins in mucinous cancers but not normal stomach, and further does not

248 stain pH-neutral mucins in healthy gastric epithelium (Fig. 3D). Accordingly, Alcian blue-positive

249 cells were significantly increased in all five TP53/ARID1A DKO lines, versus TP53 KO

250 organoids (Fig. 3D). In addition, in vivo xenografts from TP53/ARID1A DKO organoids retained

251 the mucinous phenotype with Periodic Acid-Schiff (PAS)-positive gastric pit cell-like and Alcian

252 blue-positive intestinal goblet cell-like dysplastic cells (Fig. 3E).

253 Interestingly, some Alcian blue and PAS double-positive mucin lakes were rimmed by

254 Alcian blue-negative pit-like cells, suggesting an intermediate differentiation state between

255 gastric-type and intestinal-type mucin-producing cells (Supplementary Fig. 3E). ARID1A

256 deficient mucinous organoid cells were indeed proliferative, as 21.3% of mitotic cells exhibited

257 mucinous histology, which could be subdivided into goblet-like (1.3%) and pit-like (20%) cells

258 (Fig. 3F). Moreover, KI67-positive proliferating mucinous cells were identified (Fig. 3G). To

259 further investigate the gastric versus intestinal mucinous state in organoid xenografts, we

260 performed IHC staining of CDX2, an intestinal epithelium specific transcription factor. Both TP53

261 KO and TP53/ARID1A DKO xenografts exhibited clusters of CDX2-positive cells, indicating foci

262 of intestinal metaplasia (IM) in vivo (Fig. 3H). Interestingly, intestinal goblet cell-like MUC2+

263 cells were exclusively identified in TP53/ARID1A DKO xenografts, but not in TP53/ARID1A DKO

264 organoids (Fig. 3I), suggesting potential host tumor microenvironmental regulation of the IM

265 phenotype. Accordingly, the gastric epithelium-specific tight junction protein CLDN18, was

266 dramatically decreased in TP53/ARID1A DKO xenografts in vivo, versus TP53/ARID1A DKO

267 organoids in vitro (Fig. 3J). Of note, a small proportion of CLDN18-positive TP53/ARID1A DKO

268 xenografts (<10%) resembled spasmolytic polypeptide-expressing metaplasia (SPEM)(42), a

269 metaplastic mucous cell lineage, by co-expressing the chief cell digestive enzyme PGC and

270 mucous neck cell specific marker MUC6 (Fig. 3J).

271 We further confirmed these findings in gastric cancer patients by demonstrating a

272 significant inverse correlation between ARID1A expression and mucin production by

273 simultaneous ARID1A and Alcian blue staining of a 197-patient gastric cancer tissue microarray

274 (Fig. 3K). Of note, in a few cases of heterogeneous ARID1A tumor expression, mucin was

275 present in association with tumor areas having low, but not high ARID1A expression (Fig. 3K),

276 again reiterating the mucous cell metaplasia associated with ARID1A loss.

277

278 Loss of ARID1A inhibits canonical Wnt/-catenin activity

279 Wnt activity is inversely correlated with gastric mucinous differentiation since canonical Wnt

280 signaling is lowest in the mucinous pit cell-containing regions occupying the apical-most

281 domains of gastric glands(43) and withdrawal of Wnt and R-spondin from human gastric

282 organoids directs cell fate from gland-type to mucin-expressing pit lineages(34). We thus

283 hypothesized that organoid ARID1A KO induced the mucin-producing pit-like cell phenotype by

284 impairing Wnt/-catenin signaling (Fig. 3). This was directly tested by delivering a Wnt-

285 activated TOPflash luciferase construct containing an mCherry reporter by lentiviral-based

286 transduction into our engineered organoids. An equivalent number of mCherry-positive single

287 cells were sorted from TP53 KO and TP53/ARID1A DKO organoids followed by quantification of

288 luciferase activity. Consistent with this model, Wnt/-catenin-induced reporter activity was

289 significantly reduced in all five ARID1A-deficient lines (Fig. 4A) despite their increased

290 proliferation (Fig. 2C-D).

291 To determine if the mucinous metaplasia induced by ARID1A loss could be rescued by

292 constitutively activated Wnt signaling, we transduced an N-terminal truncated gain-of-function -

293 catenin (CTNNB1N90) lentivirus bearing neomycin resistance into TP53 KO and

294 TP53/ARID1A DKO organoids, yielding CTNNB1N90/TP53 KO and

295 CTNNB1N90/TP53/ARID1A DKO organoid lines. The gain-of-function -catenin mutant

296 strongly induced TOPflash reporter activity (Supplementary Fig. 4A) and extinguished the

297 ectopic MUC5AC, TFF1 and TFF2 expression in TP53/ARID1A DKO organoids, while LYZ was

298 relatively unaffected (Fig. 4B). Similarly, induction of MUC1, an apically-restricted, gastric

299 cancer-associated transmembrane mucin(44), in TP53/ARID1A DKO organoids was profoundly

300 reversed by CTNNB1N90, reverting these organoids to a non-mucinous phenotype with re-

301 establishment of apicobasal polarity indicated by uniformly apical MUC1 expression (Fig. 4C).

302 To delineate the inhibitory effect of extracellular Wnt and R-Spondin on the mucin-producing

303 phenotype, organoids were grown for 9 days in the fully supplemented culture medium (WENR)

304 followed by withdrawal of Wnt and R-Spondin from the medium (EN) for an additional 5 days to

305 induce mucous cell differentiation. The expression of TFF1 and TFF2, but not LYZ and CHGA

306 were increased in both TP53 KO and TP53/ARID1A DKO organoids in the absence of Wnt and

307 R-Spondin, suggesting withdrawal of Wnt stimulation is sufficient to induce mucinous

308 differentiation (Supplementary Fig. 4B). Taken together, these results suggested that the

309 mucin-producing phenotype of TP53/ARID1A DKO organoids results from inhibition of Wnt/-

310 catenin activity, indicating a redirection of gland- to pit-like cell fate determination.

311 To mechanistically investigate ARID1A mutation-repressed canonical Wnt/-catenin

312 signaling and mucous cell differentiation, we studied Wnt/-catenin-regulated transcripts upon

313 CTNNB1N90 rescue of either TP53 KO or TP53/ARID1A DKO organoids. (Fig. 4D and

314 Supplementary Table 1). Gene Ontology (GO) analysis of up-regulated genes showed

315 enrichment of biological processes that are associated with Wnt activation, such as tissue

316 development, regulation of Wnt signaling and epithelial to mesenchymal transition (EMT) (Fig.

317 4E). As expected, Wnt/-catenin target genes such as TCF1, LEF1, ASCL2, AXIN2, CTNNB1,

318 LGR5, LGR6 and RNF43 were induced along with MEX3A(45), and PROX1(46) which mark

319 injury-inducible intestinal stem cells (Fig. 4F). In contrast, down-regulated gene GO terms

320 implicated digestive tract development (Fig. 4E). Consistent with CTNNB1N90 abrogation of

321 the mucinous phenotype, markers of gastric pit cells (TFF1, LYZ), gastric mucous neck cells

322 (TFF2, LYZ, AGR2), and intestinal goblet cells (TFF3, AGR2, REG4) were significantly

323 decreased (Fig. 4F) alongside transcription factors SPDEF, SOX21, THRB and SIX2 (Fig. 4G).

324 Notably, SPDEF is a master transcription factor regulating mucin-producing cell differentiation

325 and maturation across many tissue types, such as gastric mucous neck cells(47) and intestinal

326 goblet cells(48,49). On balance, these results suggested ARID1A- and Wnt-dependent control

327 of mucous cell differentiation via SPDEF regulation.

328 In addition to the mucinous phenotype, CTNNB1N90/TP53/ARID1A DKO organoids

329 rescued many dysplastic features characteristic of TP53/ARID1A DKO organoids, with reduced

330 epithelial stratification and architectural complexity (Fig. 4H). We next examined if the

331 hyperproliferation phenotype of TP53/ARID1A DKO organoids (Fig. 2C) could be reverted by

332 activated Wnt signaling. However, CTNNB1N90 notably did not rescue the elevated cell

333 proliferation of any of the five clonal TP53/ARID1A DKO organoid lines (Fig. 4I). These results

334 thus dissociated the non-essential Wnt repression-dependent mucous metaplasia from

335 alternative undefined yet essential mechanisms governing ARID1A loss-associated

336 hyperproliferation.

337

338 ARID1A loss-associated gene regulatory modules recapitulates TCGA gastric cancers

339 To identify the critical Wnt-independent biological processes governing the hyperproliferation

340 associated with ARID1A loss, we investigated ARID1A-associated transcripts by bulk RNA-

341 sequencing (RNA-seq) in the control TP53 KO and two of the TP53/ARID1A DKO organoid

342 lines. Compared to TP53 KO organoids, the TP53/ARID1A DKO biological replicates contained

343 1,087 differentially expressed genes that were consistently up-regulated (472 genes) or down-

344 regulated (675 genes) (Fig. 5A and Supplementary Table 2). GO enrichment analysis of up-

345 regulated genes in ARID1A-deficient organoids indicated several key biological processes

346 including regulation of mitotic cell cycle, cell division, chromatin segregation, and cytoskeletal

347 organization (Fig. 5B and Supplementary Fig. 5). On the other hand, the top GO terms of the

348 down-regulated genes in ARID1A-deficient cells included cell morphogenesis, nervous system

349 development, cell differentiation, cell adhesion, cell migration, and negative regulation of cellular

350 response to growth factor stimulus (Fig. 5B and Supplementary Fig. 5). These findings were in

351 agreement with our conclusions that ARID1A loss altered cell proliferation and differentiation in

352 TP53/ARID1A DKO organoids (Figs. 2, 3). Of note, the abnormal mitotic and chromatin

353 segregation signatures suggested that ARID1A loss might be implicated in chromosome

354 instability, consistent with our prior studies(7).

355 To gain deeper insights into how ARID1A loss influences gene regulatory architecture, we

356 performed master regulator (MR) analysis using the VIPER(50) algorithm to elucidate ARID1A-

357 regulated gene hierarchies(51). Akin to highly multiplexed gene reporter assays, VIPER infers

358 the activity of 2,782 regulator proteins based on expression of their positively regulated and

359 repressed transcriptional targets. Transcriptional targets were identified by analyzing a set of

360 200 TCGA stomach adenocarcinoma (STAD) gene expression profiles(14) using the

361 ARACNe(52) algorithm. VIPER analysis identified several MRs representing candidate effector

362 proteins that were significantly associated with ARID1A loss in two independent

363 TP53/ARID1A DKO organoid lines (Fig. 5C and Supplementary Table 3). FOXM1, a classical

364 proliferation-associated transcription factor that is intimately involved in tumorigenesis(53), was

365 listed as the top-ranked MR that was differentially enriched in both TP53/ARID1A DKO organoid

366 biological replicates versus the control TP53 KO organoids. Consistent with this result, several

367 FOXM1 targets, such as BIRC5, CKS1B, CDC25C, CCNB1, CCNB2, CDK1, AURKA and

368 AURKB, were simultaneously upregulated in parallel with FOXM1 in ARID1A-deficient

369 organoids (Fig. 5C and Supplementary Table 4). The upregulation of the FOXM1 targets

370 BIRC5 and AURKB was further confirmed by Western blotting (Fig. 5D). Notably, the global MR

371 profile of ARID1A-deficient organoids revealed strong overlap with MRs independently identified

372 in TCGA STAD gastric cancers, with particularly significant similarity to STAD MSI (p<7.56E-12)

373 and EBV (p<0.03) clusters where ARID1A mutations are highly enriched(14,15) but not GS

374 (p>1), CIN (p>0.97), and HM-SNV (p>0.23) subtypes (Fig. 5E). In addition, compared to a

375 gastric cancer patient-derived organoid (PDO) data set that was established in previous

376 studies(54), the MR profile of ARID1A-deficient organoids again exhibited significant similarity to

377 the MSI subtype PDOs (Fig. 5F).

378

379 ARID1A deletion confers therapeutic vulnerability to Survivin inhibition

380 A potential advantage of the use of isogenic paired TP53 KO and TP53/ARID1A DKO organoids

381 engineered from non-neoplastic gastric tissue is a reduced likelihood of simultaneous

382 confounding co-occurring mutations that are common to transformed cancer cell lines, as

383 confirmed by lack of driver alterations upon whole genome sequencing (Supplementary Fig. 1).

384 Thus, the syngeneic and low background somatic mutational burden of these engineered

385 organoids provided a unique opportunity to study ARID1A growth dependencies in a system

386 having reduced interference from modifier loci. Thus, we tested ARID1A-specific growth

387 dependencies by high-throughput small molecule screening of an FDA-approved and bioactive

388 chemical library (2,036 compounds) in TP53/ARID1A DKO versus control TP53 organoid lines

389 (Fig. 6A). TP53/ARID1A DKO organoids were dissociated into smaller clusters, re-plated into

390 384-well plates, cultured for 5 days followed by drug treatment and cell viability was quantified

391 after 3 additional days (Fig. 6A). Notably, this screening system exhibited robust assay

392 performance, with signal-to-background (S/B) ratio > 8 and Z’ > 0.5 (Fig. 6B). To discover

393 compounds exhibiting selective synthetic lethality with ARID1A deficiency, we performed 12-

394 point concentration counter-screening in the control TP53 KO versus two additional

395 TP53/ARID1A DKO lines for the top 50 hits from the initial primary DKO organoid screening.

396 Among these, several candidates such as YM-155, BMS-526924, HS-173 and Torin-2

397 selectively inhibited proliferation of TP53/ARID1A DKO versus TP53 KO organoids, whereas

398 many hits such as AP-26113 showed no obvious differences (Supplementary Fig. 6A).

399 We then performed secondary counter-screening with repurchased compounds to repeat

400 and further confirm enhanced sensitivity in TP53/ARID1A DKO organoids. While some

401 variability in the magnitude of sensitivities were observed, the results of the secondary

402 confirmatory assay were generally consistent with our primary screen, yielding 14 candidate

403 compounds that selectively enhanced killing of ARID1A-mutant organoids (Supplementary Fig.

404 6B). Consistent with previous studies of ARID1A-mutated cancer cells, engineered ARID1A-

405 deficient gastric organoids were selectively sensitive to histone deacetylase (HDAC)

406 inhibitors(55,56) and PI3K/AKT inhibitors(57,58) (Supplementary Fig. 6B). Among these

407 compounds, ARID1A-deficient gastric organoids were also sensitive to YM-155, a small

408 molecule inhibitor of BIRC5/survivin(59), a member of the inhibitor of apoptosis (IAP) family,

409 which inhibits caspase-mediated apoptosis(60) and controls mitotic spindle dynamics and

410 chromosome segregation(61) (Supplementary Fig. 6A-B). We additionally confirmed the

411 potent YM-155 repression of BIRC5 protein in TP53 KO and TP53/ARID1A DKO organoids

412 (Supplementary Fig. 6C). Crucially, YM-155 exhibited selective lethality with ARID1A mutation

413 consistently across all five TP53/ARID1A DKO lines (average IC50=0.03 M) versus the two

414 TP53 KO lines (average IC50=0.23 M) (Fig. 6C-D).

415 We further evaluated the therapeutic effect of YM-155 in conventional 2D gastric cancer

416 cell lines, as opposed to oncogene-engineered organoids. Six isogenic pairs of ARID1A wild-

417 type and mutant cancer cell lines were generated by CRISPR/Cas9, and sensitivities to YM-155

418 were compared. In contrast to 3D engineered organoids, ARID1A KO 2D gastric cancer cell

419 lines did not exhibit selective sensitivity to YM-155 (Supplementary Fig. 7). These results

420 indicated that highly transformed gastric cancer cell lines are less dependent on BIRC5/survivin

421 after ARID1A loss than our DKO organoids, which appear to harbor only TP53 and ARID1A

422 oncogenic driver mutations and thus model early gastric cancer (Supplementary Fig. 1A-B).

423

424 Rescue and functional independence of ARID1A KO-regulated BIRC5/survivin and Wnt

425 pathways

426 To test if constitutive expression of BIRC5 was sufficient to rescue YM-155-associated ARID1A

427 synthetic lethality, we lentivirally overexpressed MYC-DDK-tagged BIRC5 in TP53 KO versus

428 TP53/ARID1A DKO organoid lines (Fig. 6E). As expected, single TP53 KO control organoids

429 exhibited YM-155 insensitivity at the IC50 of 0.03 M for TP53/ARID1A DKO organoids, which

430 was not altered by BIRC5 overexpression. However, the YM-155 hypersensitivity of multiple

431 independent TP53/ARID1A DKO organoid lines was significantly rescued by BIRC5

432 overexpression, which additionally confirmed the specificity of YM-155 for BIRC5 (Fig. 6F).

433 This unexpected convergence between the MR analysis, in which the mostly highly ranked hit

434 was a ARID1A KO-induced FOXM1BIRC5/survivin regulatory node with concurrent

435 upregulation of FOXM1 mRNA and BIRC5 mRNA and protein (Fig. 5), and the small molecule

436 screen, revealing selective sensitivity of ARID1A KO organoids to the BIRC5/survivin inhibitor

437 YM-155 (Fig. 6), functionally implicated FOXM1BIRC5/survivin as an essential pathway

438 mediating hyperproliferation following ARID1A loss. Consistent with these findings in the

439 organoids, BIRC5 expression was significantly higher in TCGA STAD patients harboring

440 ARID1A mutations (Supplementary Table 5).

441 We then probed the functional independence of the ARID1A KO-induced, YM-155-senstive,

442 FOXM1BIRC5/survivin essential proliferation pathway, as distinct from the non-essential Wnt-

443 regulated mucinous differentiation pathway. Importantly, YM-155 did not inhibit Wnt-dependent

444 mucous metaplasia in ARID1A-deficient organoids (Fig. 6G). LEF1 and TCF1 are two Wnt/-

445 catenin targets that are robustly induced by the CTNNB1N90 gain-of-function -catenin mutant

446 (Fig. 6H, lane 1 versus lane 3). As expected, LEF1 and TCF1 proteins were decreased in

447 TP53/ARID1A DKO organoids having impaired Wnt signaling, versus control TP53 organoid

448 lines (Fig. 6H, lane 1 versus lanes 4, 7 and 10). However, YM-155 did not revert the ARID1A

449 KO-associated decrease in LEF1 or TCF1 protein (Fig. 6H, lanes 1 versus 2, 4 versus 5, 7

450 versus 8, 10 versus 11), indicating that YM-155 did not affect Wnt/-catenin activity. Consistent

451 with these observations, Wnt/-catenin-induced TOPflash reporter activity was also not altered

452 by YM-155 treatment (Fig. 6I). Conversely, CTNNB1N90 Wnt pathway activation did not

453 rescue the expression of BIRC5 (Fig. 6J), or the selective YM-155 proliferation sensitivity of

454 ARID1A-deficient organoids (Fig. 6K) despite potently reversing the mucinous metaplasia

455 phenotype (Fig. 4B-C). In total, these selective perturbation results confirmed the independent

456 functionality of the ARID1A KO-induced Wnt/mucinous metaplasia versus

457 FOXM1BIRC5/survivin-mediated proliferation pathways.

458

459

460 Discussion

461

462 Primary human organoids have proven to be invaluable models of tumorigenesis(28).

463 Organoids mimic oncogenic transformation on a collective tissue scale and accurately replicate

464 the in vivo biology of their original native tissues. Coupled with contemporary experimental

465 methods, organoid systems provide enormous experimental flexibility and capacity for studying

466 molecular mechanisms of gene function in human cells. CRISPR/Cas9 gene editing of primary

467 human organoids from various tissues including colon(30,31,62), stomach(32),

468 pancreas(63,64) , breast(65) and liver(66) has contributed tremendous mechanistic insight into

469 the functional basis of diverse oncogenic loci identified from large-scale next-generation

470 sequencing studies of human cancers. Here, we leveraged primary human gastric organoids to

471 establish the first forward genetic human ARID1A transformation model, whose multi-omic

472 analysis revealed phenotypic and functional recapitulation of numerous features of ARID1A-

473 mutated gastric cancer.

474 The inability to establish ARID1A KO organoids from wild-type human gastric organoids

475 could originate in the anaphase bridge formation and G2/M cell cycle arrest upon loss of BAF

476 subunits(7). TP53 deficiency, as in the current study, may bypass this arrest, allowing

477 establishment of organoids mutated in both ARID1A and TP53. Although concomitant mutation

478 of ARID1A and TP53 occurs sporadically (~4-13%) in human gastric cancers and ~30% of MSI

479 gastric cancer(14,15,67), the engineered TP53/ARID1A DKO organoids nevertheless faithfully

480 recapitulate numerous features of ARID1A-mutated gastric cancer; similar studies could be

481 extended to model ARID1A loss in the context of other driver mutations.

482 Notably, ARID1A KO elicits global transcriptional regulatory programs significantly

483 reminiscent of MSI- and EBV-type gastric cancers, precisely those subtypes in which ARID1A

484 mutation is most prevalent(14,15). Moreover, the absence of engineered MSI mutations in

485 ARID1A-defieicient organoids suggests that ARID1A loss may be a major determinant of the

486 overall transcriptional regulatory program of MSI stomach adenocarcinoma. Crucially, our

487 multiscale analysis of ARID1A KO organoids, integrating transcriptional, small molecule and

488 computational approaches, defines a bifurcated model of ARID1A-dependent oncogenic

489 transformation where non-essential Wnt-regulated mucinous metaplasia is distinct from

490 essential YM-155-sensitive, FOXM1BIRC5-regulated proliferation (Fig. 7).

491 Mechanistically, ARID1A loss inhibits canonical Wnt/-catenin activity leading to a

492 redirection of gland- to pit-like cell fate determination. During homeostasis, gastric cell

493 determination is maintained by a gradient of canonical Wnt/-catenin activity that is established

494 and most intense at the gland base, and extends up toward the mucin-producing pit cells in the

495 upper gland where canonical Wnt/-catenin activity is virtually absent(43). Emerging evidence

496 suggests Wnt and R-Spondin agonists are critical microenvironmental cues for maintaining

497 gastric stem cells(34,68,69). Consistent with this, ARID1A-deficient organoids displayed

498 reduced canonical Wnt/-catenin signaling, accompanied by a shift to pit-like mucin-producing

499 lineage differentiation which was potently rescued by constitutive -catenin activation. Of note,

500 constitutive -catenin activation significantly downregulated several gastric mucous cell and

501 intestinal goblet cell genes, including SPDEF, encoding a transcription factor regulating

502 epithelial goblet cell differentiation(47,48). Consistent with these results, previous studies

503 identify SPDEF as a Wnt-responsive gene(48) that functions as a colorectal cancer tumor

504 suppressor by regulating Wnt signaling(70,71). Together with the inverse relationship between

505 ARID1A expression and mucinous differentiation in human gastric cancer microarrays, our

506 findings confirm prior transgenic mouse studies where Arid1a loss promotes mucinous

507 tumorigenesis in colon(19) and pancreas(22–24) but where a molecular mechanism was not

508 established. In contrast, non-mucinous differentiation associated with Arid1a mutation occurs in

509 ovarian and uterine tumors(18,21). Thus, lineage metaplasia may be a pervasive feature of

510 ARID1A-deficient cancer, which our studies reveal can be driven by Wnt pathway dysregulation.

511 Surprisingly, despite robust Wnt-dependency of ARID1A loss-induced mucous metaplasia,

512 this pathway did not regulate cell division, indicating non-essentiality. Instead, the unexpected

513 convergence of our master regulator and small molecule selective lethal screens identified a 20

514 YM-155-sensitive FOXM1BIRC5 transcriptional node as a essential regulator of ARID1A KO-

515 induced proliferation. The functional independence of the Wnt/mucin versus

516 FOXM1BIRC5/proliferation pathways is attested by the inability of -catenin rescue to alter

517 YM-155 sensitivity, while conversely YM-155 does not reverse Wnt-dependent mucinous

518 differentiation or target expression.

519 Several studies have pursued discovery of targets exhibiting synthetic lethality with

520 ARID1A deficiency in transformed cancer cell lines. Such ARID1A selective lethal compounds

521 include inhibitors of EZH2 methyltransferase, a PRC2 core subunit that opposes BAF complex

522 activity(72,73) and glutathione synthesis antagonists(74). Our results clearly indicate that

523 ARID1A mutation confers selective sensitivity to BIRC5/survivin inhibition in engineered gastric

524 organoids, reflecting early-stage gastric tumorigenesis. In contrast, multiple conventional 2D

525 cancer cell lines did not exhibit selective sensitivity to BIRC5/survivin inhibition. Thus, BIRC5

526 dependency appears more stringent during earlier stages of ARID1A-deficient gastric

527 oncogenesis, as in engineered organoids, while late-stage gastric cancers may possess

528 redundant pro-survival mechanisms. We also cannot exclude confounding effects on drug

529 sensitivity from 2D cancer cell line versus 3D engineered organoid culture, which can influence

530 oncogenic phenotypes(75), or from genetic drift and resistance mechanisms in long-passaged

531 cell lines. Thus, further work will be required to explore YM-155 efficacy in established gastric

532 cancer and define the range of ARID1A-deficient malignancies for which YM-155 may be

533 effective.

534 Mouse models have proven invaluable for study of molecular mechanisms underlying

535 gastric metaplasia and its neoplastic progression. However murine models, while recapitulating

536 early-stage gastric mucous cell hyperplasia and SPEM, are limited in modeling the later stages

537 of carcinogenesis in humans, such as progression to IM, high-grade dysplasia, and infiltrating

538 adenocarcinoma. Our engineered TP53/ARID1A DKO human organoids recapitulate high-grade

539 dysplasia in vitro and acquired intestinal goblet cell features in vivo, the latter suggesting that

540 stromal and/or inflammatory cells within the tumor microenvironment may promote development

541 of late-stage gastric tumors. Interestingly, upon in vivo implantation, ~10% of TP53/ARID1A

542 DKO organoid cells exhibit SPEM features, indicating that ARID1A loss could potentially

543 predispose to SPEM, possibly in concert with environmental cues. Thus, engineered human

544 tumor organoids together with in vivo xenotransplantation provide a valuable platform for

545 studying previously inaccessible stages of human gastric cancer development. Future studies

546 will be required to determine whether additional oncogenic drivers or microenvironmental cues

547 facilitate evolution of ARID1A-deficient cells to metastatic adenocarcinoma.

548 Overall, our forward genetic study of engineered ARID1A-deficient human gastric

549 organoids enabled a functional deconstruction of essential versus non-essential mechanisms of

550 early ARID1A-dependent tumorigenesis. These analyses were greatly facilitated by the

551 synthesis of genome-scale omics approaches, high-throughput small molecule screening and

552 computational models, affording mechanistic insights into the genesis of ARID1A-deficient

553 gastric cancer. Conceivably, analogous multimodal approaches to oncogene-engineered

554 organoids may be further generalizable to additional cancer-associated loci and malignancies,

555 yielding clinically relevant insights regarding cancer initiation and ultimately therapy.

556

557 Acknowledgements

558 We thank generous support from NIH fellowship K00CA212433 (Y-H.L.), a Swedish Research

559 Council International Postdoctoral Fellowship (K.K.), Stanford Bio-X Undergraduate Fellowship

560 (W.D.S.), the NCI Cancer Target Discovery and Development (CTD2) Network (C.J.K., H.F.,

561 A.C. and C.N.C.), NIH U01CA217875 to H.F., NIH R01CA163915 to G.R.C and grants from the

562 NIH (U01CA217851, U54CA224081, U01CA199241 and U19 AI116484), Emerson Collective

563 and Ludwig Cancer Research to C.J.K.

564

565 Author Contributions

566 Y-H.L., K.S.K., W.D.S., K.K. and A.S. generated and analyzed organoids. Y.D. performed small

567 molecule screening. C.-Y.C. performed RNA-seq, A.K., J.A.S. B.T., H.X., I.S. and C.C. analyzed

568 genomic data. A.N., S.J.J., and A.C. performed master regulator analysis. T.A.L. provided

569 gastric cancer tissue microarrays. J.C. and J.S.W. provided reagents for CRISPR/Cas9

570 genome-editing. Y.-H.L., K.S.K. and C.J.K. wrote the manuscript. Y.-H.L., H. F., G.R.C. and

571 C.J.K. conceived and designed experiments.

572 Methods

573

574 Cell lines and maintenance. L-WRN cells (ATCC, CRL-3276) that produced Wnt-3A/R-

575 spondin/Noggin conditional media, and HEK293T cells were maintained in DMEM (Life

576 Technologies, #11995-073) supplemented with 10% FBS. Gastric cancer cell lines were

577 purchased from ATCC. SNU-16, AGS, NCI-N87 and MKN7 cells were maintained in RPMI1640

578 supplemented with 10% FBS. HGC27 cells were maintained in DMEM supplemented with 10%

579 FBS. KATO-III cells were maintained in DMEM supplemented with 20% FBS. All cells were

580 cultured at 37C with 5% CO2. All cell lines have been tested for mycoplasma at least once

581 every 6 months.

582

583 Organoid culture media.

584 The organoid culture media contained Advanced DMEM/F-12 (Thermo Fisher Scientific,

585 #12634028) with 0.5% Penicillin/Streptomycin/Glutamine (Thermo Fisher Scientific, #10378016),

586 5% FBS, 1 mM HEPES (Thermo Fisher Scientific, #15630080), 1 mM N-Acetylcysteine (Sigma,

587 A9165), 1X B-27 Supplement (Thermo Fisher Scientific, #12587001), 500 nM A83-01 (Tocris

588 Bioscience, #2939), 1X GlutaMax Supplement (Thermo Fisher Scientific, #35050061), 10 μM

589 SB-202190 (Biogems, #1523072), 10 mM Nicotinamide (Sigma, #N0636), 50 ng/mL EGF

590 (PeproTech, AF-100-15), 100 μg/mL Normocin (InvivoGen, ant-nr-1), 10 mM Gastrin (Sigma,

591 G9145), 200 ng/mL fibroblast growth factor (FGF) (Peprotech, #100-26), and 50% Wnt-3A/R-

592 spondin/Noggin conditioned media.

593

594 Establishment of normal gastric organoid cultures. Clinical samples used for gastric

595 organoid establishment were obtained from gastric corpus of patients at Stanford University

596 Hospital’s Tissue Procurement Shared Resource facility. Healthy gastric tissues were collected

597 by surgical resection. Gastric organoids were established as previously reported(34). Briefly,

598 surgical specimens were washed vigorously three times with sterile, cold phosphate-buffered

599 saline (PBS) in a 15 mL conical tube, and then were dissected into smaller pieces in cold

600 chelation buffer (5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM

601 Sucrose, 54.9 mM D-Sorbitol, 0.5 mM DTT) plus 10 mM EDTA. The tissues were incubated 4ºC

602 for 3-5 hours in a rocking chamber. After incubation, tissues were washed by fresh cold

603 chelation buffer and vigorously shaken by hands. After shaking, the supernatant was checked

604 for the presence of gastric crypts. This step was repeated 8-10 times and each wash produced

605 supernatant containing gastric crypts that were examined under bright-field microscope. Finally,

606 crypts collected from different fractions were combined and centrifuged at 600 g at 4C for 5

607 minutes. Gastric crypts were resuspended in Matrigel (R&D systems, Basement Membrane

608 Extract type 2) and plated in a 24-well plate. After Matrigel polymerization, organoid culture

609 media was added to each well (described above) plus 10 M Y-27632 (Peprotech, #1293823)

610 and 3 M CHIR-99021 (R&D Systems, #4223). After 3 days, the media was changed to

611 organoid culture media without Y-27632 and CHIR-99021, and cultures were maintained in

612 organoid culture media with routine media changes occurring every 3-4 days until subsequent

613 passage. Fibroblast growth factor (FGF) was dispensable for engineered TP53 KO and

614 TP53/ARID1A DKO organoids. Organoids were passaged to prevent overgrowth every 10-14

615 days. For passaging, organoids were washed by PBS and mechanically dissociated into smaller

616 pieces by pipetting and resuspension in TrypLETM (Invitrogen, #12604-012) at 37C for 10-20

617 minutes. After incubation, fetal bovine serum (FBS) was added to quench TrypLETM activity.

618 Organoids were then centrifuged at 600 g for 5 minutes and washed once using organoid

619 culture media before resuspension in matirgel and plating onto a new 24-well plate.

620

621 Guide RNA expression vector cloning. The lentiviral sgRNA vectors were generously

622 provided by Dr. Jonathan Weissman(76,77). The sgRNA vector was digested by BstXI (New

623 England BioLabs, R0113) and BlpI (New England BioLabs, R0585) at 37C for 6 hours. The

624 linearized vectors were separated on a 1% agarose gel. Linearized vectors were cut and then

625 purified by QIAquick Gel Extraction Kit (Qiagen, #28706). The lentiviral sgRNA expression 25

626 vectors were cloned by inserting annealed sgRNA oligos into the linearized sgRNA vectors. The

627 ligation of the linearized vectors and the annealed sgRNA oligos were completed by T4 DNA

628 ligase (New England BioLabs, M0202) at 25C for 2 hours. Ligation reactions were transformed

629 into Stellar Competent E. coli Cells (TaKaRa, #636763) following the manufacturer’s instructions.

630 Competent cells were plated on LB agar plates supplemented with 100 µg/mL carbenicillin and

631 incubated at 37C overnight. Colonies were randomly picked from each plate and inoculated

632 into 4 mL LB supplemented with 100 µg/mL carbenicillin and then grown at 37C for 14 hours.

633 The lentiviral sgRNA expression vectors were purified by QIAprep Spin Miniprep Kit (Qiagen,

634 #27106) for subsequent confirmation by Sanger-sequencing. The sgRNA sequences used in

635 this study were listed in the key resources table.

636

637 Generation of clonal organoid lines. Organoids were washed by PBS dissociated with

638 TrypLETM (Invitrogen, #12604-012) for 30 minutes at 37C. Cell clumps were removed using 35

639 mm cell strainer (BD Falcon, #352235) and the flow-through was pelleted at 600 g at 4C for 5

640 minutes. Cells pellets were resuspended in organoid culture media with 10 M Y-27632

641 (Peprotech, #1293823). Single cells were sorted in single wells of a 96-well plate. The 96-well

642 plate was pre-coated by 10 L Matrigel (R&D systems, Basement Membrane Extract type 2)

643 and covered by 100 L organoid culture media. FACS Aria II (BD Biosciences) equipped with a

644 100 mm nozzle was used for cell sorting. Wells containing a single organoid 12-14 days after

645 cell sorting were dissociated with TrypLETM and replated for clonal expansion. The clonal lines

646 were verified by Sanger-sequencing, immunoblot analysis, or immunostaining. For Sanger-

647 sequencing, genomic DNA was isolated from organoids by using DNeasy blood and tissue kit

648 (Qiagen, #69506). The targeted loci were amplified by PCR using Phusion High-Fidelity DNA

649 Polymerase (New England BioLabs, M0530) and then sequenced directly. Primers for PCR

650 amplification and Sanger-sequencing used in this study were listed in the key resources table.

651

652 Generation of lentivirus. Lentiviral plasmids were co-transfected with viral packaging plasmid

653 psPAX2 (Addgene, #12260) and viral envelope plasmid pCMV-VSV-G (Addgene, #8454) into

654 293T cells by Lipofectamine 2000 (Invitrogen, #11668-019) following the manufacturer’s

655 instructions. Lentiviral supernatants were collected at 48 hours and 72 hours post-transfection

656 and concentrated by PEG-it Virus Precipitation Solution (System Biosciences, LV825A-1).

657 Precipitated lentiviral particles were pelleted at 1500 g at 4C for 30 minutes and resuspended

658 in organoid culture media containing 10 M Y-27632 (Peprotech, #1293823). Lentiviral plasmids

659 used in this study were listed in the key resources table.

660

661 Lentiviral transduction of organoids. Organoids were washed by PBS and dissociated into

662 smaller clusters with TrypLETM (Invitrogen, #12604-012) for 15 minutes at 37C. Organoids were

663 resuspended into 500 L transduction solution containing 10 μM Y-27632 (Peprotech,

664 #1293823), 8 μg/mL polybrene (Sigma, #107689) and concentrated lentivirus in organoid

665 culture media. Spinoculation of resuspended organoids was performed at 800 g for 1 hour at

666 32C. After spinoculation, organoids were incubated for 12-14 hours at 37C and then replated

667 onto a new 24-well plate.

668

669 Immunoblotting. Western blot analyses were performed using standard method. Briefly, the

670 pellet was lysed in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1%

671 SDS, 50 mM Tris-HCl at pH 7.5) with protease inhibitor cocktail (Roche, #04-693-124-001) and

672 phosphatase inhibitor cocktail (Sigma, P5726). Protein concentration was measured by the BCA

673 kit (Thermo Scientific, #23227). Cell lysates were separated by SDS Poly-acrylamide-gel-

674 electrophoresis (Invitrogen, NP0323). PageRuler Plus Prestained Protein Ladder (Thermo

675 Scientific, #26619) was used as molecular weight marker. Proteins were transferred to a PVDF

676 membrane (Millipore, IPVH00010), blocked by 5% non-fat dry milk in 1X TBS buffer at pH 7.4

677 (Quality Biological, #351-086-151) with 0.05% Tween-20, and then probed with the indicated

678 primary antibodies at 4C overnight. Bound antibodies were visualized by chemiluminescence 27

679 (Thermo Scientific, #34580) using a horseradish peroxidase-conjugated secondary antibody

680 and exposure of AccuRay blue X-Ray films (E&K Scientific, EK5129). Antibodies used for

681 immunoblotting were listed in the key resources table.

682

683 Immunohistochemistry and immunofluorescence staining. Organoids were fixed with 2%

684 paraformaldehyde (Electron Microscopy Sciences, #15714) in PBS for 30 minutes at room

685 temperature, washed with PBS twice, embedded in HistoGelTM (Thermo Scientific, HG-4000-

686 012), and then transferred to 70% ethanol for paraffin-embedding. Organoids were sectioned at

687 5-mm thickness. Paraffin-embedded sections were deparaffinized and rehydrated before

688 staining. For immunohistochemistry staining, antigen retrieval was achieved in sodium citrate

689 buffer (10 M sodium citrate at pH 6.0). Slides were incubated in 3% H2O2 solution (Fisher

690 Scientific, H325-100) in methanol at room temperature for 10 minutes to block endogenous

691 peroxidase activity. After washing, slides were blocked in Avidin/Biotin Solution (Vector

692 Laboratories, SP2001) at room temperature for 30 minutes and then in blocking buffer (5%

693 normal goat or donkey serum in PBS) for 1 hour. After blocking, slides were incubated with

694 primary antibody in blocking buffer at 4C overnight. Slides were washed by PBST (PBS with

695 0.05% Tween-20) and incubated with secondary antibody at room temperature for 30 minutes.

696 Slides were washed by PBST and ABC reagent was applied (Vector Laboratories, PK-6101).

697 After washing with PBST, DAB staining was performed for signal detection (Vector Laboratories,

698 SK-4100). The slides were counterstained with hematoxylin (Sigma, MHS16) for 2 minutes and

699 rinsed with water for 1 minute. Subsequent treatment with 1% acid alcohol 3 times to

700 differentiate nuclear detail was performed along with sequential treatment with 0.2% ammonia

701 for bluing, each of these steps were followed by a water rinse for 1 minute. Following this, the

702 slides were rehydrated and mounted using mounting solution (Thermo Scientific, #4112).

703 For immunofluorescence staining, deparaffinization and rehydration procedures were as

704 described above. Slides were blocked in blocking buffer at room temperature for 1 hour. After

705 blocking, slides were incubated with primary antibody in blocking buffer at 4C overnight. Slides

706 were washed by PBST and incubated with secondary antibody at room temperature for 30

707 minutes. After washes with PBST, slides were mounted by mounting solution with DAPI (Vector

708 Laboratories, H-1500). Imaging was performed using fluorescence microscopy (Keyence, BZ-

709 X700 series). For Alcian blue staining, slides were stained with Alcian blue (Thermo Fisher,

710 #88043) following the manufacturer's instructions. Antibodies used for immunostaining were

711 listed in the key resources table.

712

713 Real-time quantitative PCR. Total RNA from organoids was isolated with the RNeasy kit

714 (Qiagen, #74106). The on-column DNase digestion (Qiagen, #79254) was used to eliminate

715 genomic DNA. A total of 0.5-1 g RNA was used to synthesize complementary DNA using

716 iScriptTM Reverse Transcription Supermix (Bio-Rad, #1708841). Quantitative PCR was

717 performed with Power SYBRTM Green PCR Master Mix (Thermo Scientific, #4368708). The

718 primers used for quantitative PCR were listed in the key resources table.

719

720 Cell proliferation and viability assay. Organoids were dissociated into smaller aggregates

721 and single cells were sorted by FACS Aria II (BD Biosciences) as described above. A total of

722 20,000 cells were resuspended into 40L Matrigel (R&D systems, Basement Membrane Extract

723 type 2) and plated in a well of a 24-well plate. Over a period of 14 days, organoid growth was

724 recorded daily by bright-field microscopy. YM-155 (Cayman Chemicals, #11490) was dissolved

725 in DMSO. For 12-point full titration treatment of YM-155, a total of 5,000 cells were resuspended

726 into 10 L Matrigel and cultured in a well of a 96-well plate for 5 days before drug treatment.

727 Cell viability was quantified 3 days after YM-155 treatment. For the cell viability assay,

728 AlamarBlueTM Cell Viability Reagent (Invitrogen, DAL1100) in organoid culture media was

729 added into the plate and incubated with organoids for 4 hours before being quantified using a

730 Synergy H1 Hybrid Multi-mode Plate Reader (BioTek).

731

732 Luciferase assay. A total of 20,000 TOPflash mCherry-positive single cells were sorted by

733 FACS Aria II (BD Biosciences) as described above. Cells were washed by PBS and the pellet

734 was lysed in Passive Lysis Buffer (Promega, E194A). Firefly luciferase activities were measured

735 using the Dual Luciferase Reporter Assay System (Promega, D1980).

736

737 sgRNA design. Knockout sgRNA were designed using a combination of empirical data and on-

738 target and off-target predictions. When available, empirical data from published CRISPR

739 screens were used to pick the most active sgRNAs(78–80), otherwise the sgRNAs were

740 designed as described previously(78). The sgRNA sequences used in this study are listed in the

741 key resources table.

742

743 High-throughput compound screening. Screening of the Emory Enriched Bioactive Library

744 (EEBL), which includes 2,036 U.S. Food and Drug Administration (FDA) approved and bioactive

745 compounds(81), was carried out using our miniaturized organoid culture platform in a 384-well

746 format for HTS. Briefly, organoids grown in a 50 l Matrigel droplet on a single well of a 24-well

747 plate were harvested as described and re-suspended in ice-cold Matrigel (R&D systems,

748 Basement Membrane Extract type 2) to form a cell/Matrigel mixture. 8 L/well of the

749 cells/Matrigel (~1,000 cells/well) mixture was dispensed onto a 384-well plate using a Multidrop

750 Combi dispenser (Thermo Fisher Scientific). The plates were immediately centrifuged at 500

751 rpm for 1 min and incubated for 30 min at cell culture incubator to allow Matrigel solidification.

752 35 L per well of organoid culture media was dispensed into the wells. The plates were sealed

753 using gas permeable plate sealer (Breathe-Easy Sealing Film, Diversified Biotech, #BEM-1) and

754 incubated for 5 days in cell culture incubator to allow organoid formation. Then, 0.1 L of library

755 compounds diluted in DMSO were added to each well using Pin-tool integrated with Beckman

756 NX automated liquid handling system (Beckman Coulter, Danaher Corporation). The plates

757 were centrifuged at 800 rpm for 5 min to ensure the uniform distribution of the compound into

758 the wells. The final compound concentration was 4.6 M and the final DMSO concentration was 30

759 0.2%. The plates were sealed with gas permeable plate sealer. After incubating with compound

760 for 3 days, the viability of organoids was determined by CellTiter Blue reagent (Promega).

761 Briefly, 5 L of CellTiter Blue reagent was added to each well in 384-well plates using a

762 MultiDrop Combi. After incubating at 37°C for 4 hours, the fluorescence intensity (FI), which is

763 correlated with the number of viable cells, was measured using PHERAstar FSX multi-label

764 plate reader (BMG LABTECH) with excitation at 540/20 nm and Emission at 590/20 nm.

765

766 Data analysis for high throughput drug screening. Screening data were analyzed using

767 CambridgeSoft Bioassay software. The performance of the organoids HTS viability assay in

768 384-well format was evaluated by Z’ factor and Signal-to-background (S/B) ratio and were

769 calculated as the following equations:

770 Z’=1 - (3SDDMSO control + 3SDblank) / (FIDMSO control – FI blank)

771 S/B = FIDMSO control / FIblank

772 Where SD DMSO and SD blank are the standard deviations, and FI DMSO control and FI blank are the

773 corresponding average FI signal for the wells with DMSO control or blank with medium only

774 without cells, respectively. A Z’ factor between 0.5 and 1.0 indicating that the assay is robust for

775 HTS(82). The effect of compound on the growth of organoids was expressed as % of control

776 and calculated based on per plate as the following equations:

777 % of control = (FI compound – FI blank)/( FI DMSO control – FI blank ) X 100

778 The dose-response effect of selected hit compounds from HTS on the growth of organoids was

779 analyzed using GraphPad Prism 7 (GraphPad Software, Inc.).

780

781 Master regulator analysis. The context-specific regulatory network used in this analysis was

782 reverse-engineered from a collection of 200 gene expression profiles from STAD patients in

783 TCGA(14) using the ARACNe algorithm(52). Specifically, ARACNe was used to infer regulatory

784 targets of 1,813 transcription factors—including genes annotated in Gene Ontology molecular

785 function database (GO) as ‘transcription factor activity’, ‘DNA binding’, ‘transcription regulator

786 activity’, or ‘regulation of transcription’ (GO:0003700, GO:0004677, GO:0030528, GO:0004677,

787 GO: 0045449)—and a manually curated list of 969 transcriptional cofactors—including genes

788 annotated as ‘transcription cofactor activity’, (GO:0003712, GO:0030528, GO:0045449). For

789 each of these regulators, its protein activity was computed by VIPER(50) analysis of genes

790 differentially expressed in TP53/ARID1A DKO compared to TP53 KO samples, using the STAD-

791 specific ARACNe regulatory network. The list of regulators and of their inferred differential

792 activity in TP53/ARID1A DKO samples were then compared to the VIPER inferred protein

793 activity profiles of all TCGA-STES patient samples, using the ‘viperSimilarity’ method of the

794 VIPER package. This method computes the similarity between two samples based on the

795 conservation of their differentially active proteins. This is accomplished by performing a gene set

796 enrichment analysis of statistically significantly differentially active proteins in one context (e.g.,

797 TP53/ARID1A DKO) to protein differentially active in the other context (e.g. STES patients) and

798 vice versa, using the aREA algorithm, an analytic extension of GSEA(83). The similarity scores

799 obtained from viperSimilarity method are the z-scores of enrichment analysis. TCGA-STES

800 samples and their subtype annotations were obtained from literature(84). The context-specific

801 regulatory network of the TCGA-STES samples was reverse-engineered using ARACNe

802 algorithm, and protein activity profiles of all samples were computed by VIPER analysis of

803 genes differentially expressed in each TCGA-STES sample compared to the average gene

804 expression in all samples, using STES specific ARACNe regulatory network.

805

806 RNA-seq and data analysis. For the RNA-seq, two technical duplicates were included for each

807 sample. RNA-seq libraries were generated by using NEBNext Ultra II Directional RNA Library

808 Prep Kit coupled with Poly(A) mRNA Magnetic Isolation Module and NEBNext multiplex oligos

809 for Illumina (New England Biolabs). The deep sequencing was performed on the NextSeq

810 500 sequencing system (Illumina) with 75-cycle, paired-end sequencing. RNA-seq data were

811 aligned to hg38 human genome assembly using kallisto (v 0.44.0) with default parameters.

812 Differential gene expression analysis was performed using DESeq2(85). Change in gene

813 expression between two conditions was defined as significant if |log2FC|>0.5 and adjusted p-

814 value <0.05. ComplexHeatmap was used to produce heat maps(86).

815

816 Somatic variant calling. Short reads produced by WGS on the Illumina platform were aligned

817 to hg38 using BWA (v0.7.17). Following GATK (v4.1.4.1) best practice workflow(87), the raw

818 alignment files (BAMs) were then pre-processed through marking duplicated reads and base

819 recalibration. SNV and INDEL calls were made using MuTect2 in GATK package. The calls

820 were then filtered and annotated using FilterMutectCalls and Funcotator in GATK. Somatic copy

821 number aberrations (SCNAs) were estimated using CNVkit (v0.9.6)(88).

822

823 Subcutaneous xenografts. NOD-scid IL2Rgammanull (NSG) immunodeficient mice were

824 obtained from the Jackson Laboratory (#005557). For xenograft studies, male adult NSG mice

825 (~8-10 weeks old) were randomly divided into experimental groups. Mice were subcutaneously

826 injected with organoids (1.5 x 106 cells in 150 L 100% Matrigel per injection). Mice were

827 sacrificed 3 months after inoculation of organoids. All mouse studies were approved by the

828 Stanford Institutional Animal Care and Use Committee (IACUC).

829

830 Data availability. The datasets generated in this study are available from the corresponding

831 author on reasonable request. Raw and processed sequencing data were deposited into the

832 Gene Expression Omnibus (GEO) under accession code GSE164179.

833

834 References 835 836 1. Flavahan WA, Gaskell E, Bernstein BE. Epigenetic plasticity and the hallmarks of cancer. 837 Science. 2017;357:eaal2380. 838 2. Hodges C, Kirkland JG, Crabtree GR. The many roles of BAF (mSWI/SNF) and PBAF 839 complexes in cancer. Cold Spring Harb Perspect Med. 2016;6:a026930. 840 3. Mashtalir N, D’Avino AR, Michel BC, Luo J, Pan J, Otto JE, et al. Modular Organization 841 and Assembly of SWI/SNF Family Chromatin Remodeling Complexes. Cell. 842 2018;175:1272–88. 843 4. Wu JI, Lessard J, Crabtree GR. Understanding the Words of Chromatin Regulation. Cell. 844 2009;136:200–6. 845 5. Ho L, Crabtree GR. Chromatin remodelling during development. Nature. 2010;463:474– 846 84. 847 6. Pulice JL, Kadoch C. Composition and function of mammalian SWI/SNF chromatin 848 remodeling complexes in human disease. Cold Spring Harb Symp Quant Biol. 849 2016;81:53–60. 850 7. Dykhuizen EC, Hargreaves DC, Miller EL, Cui K, Korshunov A, Kool M, et al. BAF 851 complexes facilitate decatenation of DNA by topoisomerase IIα. Nature. 2013;497:624–7. 852 8. Kadoch C, Hargreaves DC, Hodges C, Elias L, Ho L, Ranish J, et al. Proteomic and 853 bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in 854 human malignancy. Nat Genet. 2013;45:592–601. 855 9. Kadoch C, Crabtree GR. Reversible disruption of mSWI/SNF (BAF) complexes by the 856 SS18-SSX oncogenic fusion in synovial sarcoma. Cell. 2013;153:71–85. 857 10. Wilson BG, Wang X, Shen X, McKenna ES, Lemieux ME, Cho YJ, et al. Epigenetic 858 antagonism between polycomb and SWI/SNF complexes during oncogenic 859 transformation. Cancer Cell. 2010;18:316–28. 860 11. McBride MJ, Pulice JL, Beird HC, Ingram DR, D’Avino AR, Shern JF, et al. The SS18- 861 SSX Fusion Oncoprotein Hijacks BAF Complex Targeting and Function to Drive Synovial 862 Sarcoma. Cancer Cell. 2018;33:1128–41. 863 12. Wu JN, Roberts CWM. ARID1A mutations in cancer: Another epigenetic tumor 864 suppressor? Cancer Discov. 2013;3:35–43. 865 13. Zang ZJ, Cutcutache I, Poon SL, Zhang SL, Mcpherson JR, Tao J, et al. Exome 866 sequencing of gastric adenocarcinoma identifies recurrent somatic mutations in cell 867 adhesion and chromatin remodeling genes. Nat Genet. 2012;44:570–4. 868 14. Bass AJ, Thorsson V, Shmulevich I, Reynolds SM, Miller M, Bernard B, et al. 869 Comprehensive molecular characterization of gastric adenocarcinoma. Nature. 870 2014;513:202–9. 871 15. Cristescu R, Lee J, Nebozhyn M, Kim KM, Ting JC, Wong SS, et al. Molecular analysis of 872 gastric cancer identifies subtypes associated with distinct clinical outcomes. Nat Med. 873 2015;21:449–56. 874 16. Chandler RL, Brennan J, Schisler JC, Serber D, Patterson C, Magnuson T. ARID1a-DNA 875 Interactions Are Required for Promoter Occupancy by SWI/SNF. Mol Cell Biol. 876 2013;33:265–80. 877 17. Gao X, Tate P, Hu P, Tjian R, Skarnes WC, Wang Z. ES cell pluripotency and germ-layer 878 formation require the SWI/SNF chromatin remodeling component BAF250a. Proc Natl 879 Acad Sci U S A. 2008;105:6656–61. 880 18. Chandler RL, Damrauer JS, Raab JR, Schisler JC, Wilkerson MD, Didion JP, et al. 881 Coexistent ARID1A-PIK3CA mutations promote ovarian clear-cell tumorigenesis through 882 pro-tumorigenic inflammatory cytokine signalling. Nat Commun. 2015;27:6118. 883 19. Mathur R, Alver BH, San Roman AK, Wilson BG, Wang X, Agoston AT, et al. ARID1A 884 loss impairs enhancer-mediated gene regulation and drives colon cancer in mice. Nat 885 Genet. 2017;49:296–302. 886 20. Hiramatsu Y, Fukuda A, Ogawa S, Goto N, Ikuta K, Tsuda M, et al. Arid1a is essential for

887 intestinal stem cells through Sox9 regulation. Proc Natl Acad Sci U S A. 2019;116:1704– 888 13. 889 21. Wilson MR, Reske JJ, Holladay J, Wilber GE, Rhodes M, Koeman J, et al. ARID1A and 890 PI3-kinase pathway mutations in the endometrium drive epithelial transdifferentiation and 891 collective invasion. Nat Commun. 2019;10:3554. 892 22. Kimura Y, Fukuda A, Ogawa S, Maruno T, Takada Y, Tsuda M, et al. ARID1A Maintains 893 Differentiation of Pancreatic Ductal Cells and Inhibits Development of Pancreatic Ductal 894 Adenocarcinoma in Mice. Gastroenterology. 2018;155:194–209. 895 23. Wang W, Friedland SC, Guo B, O’Dell MR, Alexander WB, Whitney-Miller CL, et al. 896 ARID1A, a SWI/SNF subunit, is critical to acinar cell homeostasis and regeneration and is 897 a barrier to transformation and epithelial-mesenchymal transition in the pancreas. Gut. 898 2019;68:1245–58. 899 24. Wang SC, Nassour I, Xiao S, Zhang S, Luo X, Lee J, et al. SWI/SNF component ARID1A 900 restrains pancreatic neoplasia formation. Gut. 2019;68:1259–70. 901 25. Livshits G, Alonso-Curbelo D, Morris JP, Koche R, Saborowski M, Wilkinson JE, et al. 902 Arid1a restrains Kras-dependent changes in acinar cell identity. Elife. 2018;7:e35216. 903 26. Sun X, Wang SC, Wei Y, Luo X, Jia Y, Li L, et al. Arid1a Has Context-Dependent 904 Oncogenic and Tumor Suppressor Functions in Liver Cancer. Cancer Cell. 2017;32:574– 905 89. 906 27. Han L, Madan V, Mayakonda A, Dakle P, Woon TW, Shyamsunder P, et al. Chromatin 907 remodeling mediated by ARID1A is indispensable for normal hematopoiesis in mice. 908 Leukemia. 2019;33:2291–305. 909 28. Lo Y-H, Karlsson K, Kuo CJ. Applications of organoids for cancer biology and precision 910 medicine. Nat Cancer. 2020;1:761–773. 911 29. Li X, Nadauld L, Ootani A, Corney DC, Pai RK, Gevaert O, et al. Oncogenic 912 transformation of diverse gastrointestinal tissues in primary organoid culture. Nat Med. 913 2014;20:769–77. 914 30. Matano M, Date S, Shimokawa M, Takano A, Fujii M, Ohta Y, et al. Modeling colorectal 915 cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat 916 Med. 2015;21:256–62. 917 31. Drost J, Van Jaarsveld RH, Ponsioen B, Zimberlin C, Van Boxtel R, Buijs A, et al. 918 Sequential cancer mutations in cultured human intestinal stem cells. Nature. 919 2015;521:43–7. 920 32. Nanki K, Toshimitsu K, Takano A, Fujii M, Shimokawa M, Ohta Y, et al. Divergent Routes 921 toward Wnt and R-spondin Niche Independency during Human Gastric Carcinogenesis. 922 Cell. 2018;174:856–69. 923 33. Ho L, Ronan JL, Wu J, Staahl BT, Chen L, Kuo A, et al. An embryonic stem cell 924 chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal 925 and pluripotency. Proc Natl Acad Sci U S A. 2009;106:5181–6. 926 34. Bartfeld S, Bayram T, Van De Wetering M, Huch M, Begthel H, Kujala P, et al. In vitro 927 expansion of human gastric epithelial stem cells and their responses to bacterial infection. 928 Gastroenterology. 2015;148:126–36. 929 35. Fujii M, Clevers H, Sato T. Modeling Human Digestive Diseases With CRISPR-Cas9– 930 Modified Organoids. Gastroenterology. 2019;156:562–76. 931 36. Uhler C, Shivashankar G V. Nuclear Mechanopathology and Cancer Diagnosis. Trends in 932 Cancer. 2018;4:320–31. 933 37. Webster M, Wikin KL, Cohen-Fix O. Sizing up the nucleus: Nuclear shape, size and 934 nuclear-envelope assembly. J Cell Sci. 2009;122:1477–86. 935 38. Tan P, Yeoh KG. Genetics and Molecular Pathogenesis of Gastric Adenocarcinoma. 936 Gastroenterology. 2015;149:1153–62. 937 39. Mills JC, Shivdasani RA. Gastric epithelial stem cells. Gastroenterology. 2011;140:412– 938 24. 939 40. Hayakawa Y, Fox JG, Wang TC. The Origins of Gastric Cancer From Gastric Stem Cells:

940 Lessons From Mouse Models. CMGH. 2017;3:331–8. 941 41. Giroux V, Rustgi AK. Metaplasia: Tissue injury adaptation and a precursor to the 942 dysplasia-cancer sequence. Nat Rev Cancer. 2017;17:594–604. 943 42. Petersen CP, Mills JC, Goldenring JR. Murine Models of Gastric Corpus Preneoplasia. 944 CMGH. 2017;3:11–26. 945 43. Stange DE, Koo BK, Huch M, Sibbel G, Basak O, Lyubimova A, et al. Differentiated 946 Troy+ chief cells act as reserve stem cells to generate all lineages of the stomach 947 epithelium. Cell. 2013;155:357–68. 948 44. Wang XT, Kong FB, Mai W, Li L, Pang LM. MUC1 immunohistochemical expression as a 949 prognostic factor in gastric cancer: Meta-analysis. Dis Markers. 2016;9421571:1–8. 950 45. Barriga FM, Montagni E, Mana M, Mendez-Lago M, Hernando-Momblona X, Sevillano M, 951 et al. Mex3a Marks a Slowly Dividing Subpopulation of Lgr5+ Intestinal Stem Cells. Cell 952 Stem Cell. 2017;20:801–16. 953 46. Yan KS, Gevaert O, Zheng GXY, Anchang B, Probert CS, Larkin KA, et al. Intestinal 954 Enteroendocrine Lineage Cells Possess Homeostatic and Injury-Inducible Stem Cell 955 Activity. Cell Stem Cell. 2017;21:78–90. 956 47. Horst D, Gu X, Bhasin M, Yang Q, Verzi M, Lin D, et al. Requirement of the epithelium- 957 specific Ets transcription factor Spdef for mucous gland cell function in the gastric antrum. 958 J Biol Chem. 2010;285:35047–55. 959 48. Gregorieff A, Stange DE, Kujala P, Begthel H, van den Born M, Korving J, et al. The Ets- 960 Domain Transcription Factor Spdef Promotes Maturation of Goblet and Paneth Cells in 961 the Intestinal Epithelium. Gastroenterology. 2009;137:1333–45. 962 49. Noah TK, Kazanjian A, Whitsett J, Shroyer NF. SAM pointed domain ETS factor (SPDEF) 963 regulates terminal differentiation and maturation of intestinal goblet cells. Exp Cell Res. 964 2010;316:452–65. 965 50. Alvarez MJ, Shen Y, Giorgi FM, Lachmann A, Ding BB, Hilda Ye B, et al. Functional 966 characterization of somatic mutations in cancer using network-based inference of protein 967 activity. Nat Genet. 2016;48:838–47. 968 51. Califano A, Alvarez MJ. The recurrent architecture of tumour initiation, progression and 969 drug sensitivity. Nat Rev Cancer. 2017;17:116–30. 970 52. Basso K, Margolin AA, Stolovitzky G, Klein U, Dalla-Favera R, Califano A. Reverse 971 engineering of regulatory networks in human B cells. Nat Genet. 2005;37:382–90. 972 53. Wierstra I. FOXM1 (Forkhead box M1) in tumorigenesis. Overexpression in human 973 cancer, implication in tumorigenesis, oncogenic functions, tumor-suppressive properties, 974 and target of anticancer therapy. Adv Cancer Res. 2013;119:191–419. 975 54. Yan HHN, Siu HC, Law S, Ho SL, Yue SSK, Tsui WY, et al. A Comprehensive Human 976 Gastric Cancer Organoid Biobank Captures Tumor Subtype Heterogeneity and Enables 977 Therapeutic Screening. Cell Stem Cell. 2018;23:882–97. 978 55. Bitler BG, Wu S, Park PH, Hai Y, Aird KM, Wang Y, et al. ARID1A-mutated ovarian 979 cancers depend on HDAC6 activity. Nat Cell Biol. 2017;19:962–73. 980 56. Fukumoto T, Park PH, Wu S, Fatkhutdinov N, Karakashev S, Nacarelli T, et al. 981 Repurposing Pan-HDAC Inhibitors for ARID1A-Mutated Ovarian Cancer. Cell Rep. 982 2018;22:3393–400. 983 57. Lee D, Yu EJ, Ham IH, Hur H, Kim YS. AKT inhibition is an effective treatment strategy in 984 ARID1A-deficient gastric cancer cells. Onco Targets Ther. 2017;10:4153–9. 985 58. Samartzis EP, Gutsche K, Dedes KJ, Fink D, Stucki M, Imesch P. Loss of ARID1A 986 expression sensitizes cancer cells to PI3K- and AKT-inhibition. Oncotarget. 2014;5:5295– 987 303. 988 59. Nakahara T, Takeuchi M, Kinoyama I, Minematsu T, Shirasuna K, Matsuhisa A, et al. 989 YM155, a novel small-molecule survivin suppressant, induces regression of established 990 human hormone-refractory prostate tumor xenografts. Cancer Res. 2007;67:8014–21. 991 60. Altieri DC. Validating survivin as a cancer therapeutic target. Nat Rev Cancer. 2003;3:46– 992 54.

993 61. Wheatley SP, Altieri DC. Survivin at a glance. J Cell Sci. 2019;132:jcs223826. 994 62. Drost J, Van Boxtel R, Blokzijl F, Mizutani T, Sasaki N, Sasselli V, et al. Use of CRISPR- 995 modified human stem cell organoids to study the origin of mutational signatures in cancer. 996 Science. 2017;358:234–8. 997 63. Lee J, Snyder ER, Liu Y, Gu X, Wang J, Flowers BM, et al. Reconstituting development 998 of pancreatic intraepithelial neoplasia from primary human pancreas duct cells. Nat 999 Commun. 2017;8:14686. 1000 64. Seino T, Kawasaki S, Shimokawa M, Tamagawa H, Toshimitsu K, Fujii M, et al. Human 1001 Pancreatic Tumor Organoids Reveal Loss of Stem Cell Niche Factor Dependence during 1002 Disease Progression. Cell Stem Cell. 2018;22:454–67. 1003 65. Dekkers JF, Whittle JR, Vaillant F, Chen H-R, Dawson C, Liu K, et al. Modeling Breast 1004 Cancer Using CRISPR-Cas9–Mediated Engineering of Human Breast Organoids. JNCI J 1005 Natl Cancer Inst. 2019;112:540–4. 1006 66. Artegiani B, van Voorthuijsen L, Lindeboom RGH, Seinstra D, Heo I, Tapia P, et al. 1007 Probing the Tumor Suppressor Function of BAP1 in CRISPR-Engineered Human Liver 1008 Organoids. Cell Stem Cell. 2019;24:927–43. 1009 67. Chen K, Yang D, Li X, Sun B, Song F, Cao W, et al. Mutational landscape of gastric 1010 adenocarcinoma in Chinese: Implications for prognosis and therapy. Proc Natl Acad Sci 1011 U S A. 2015;112:1107–12. 1012 68. Sigal M, Logan CY, Kapalczynska M, Mollenkopf HJ, Berger H, Wiedenmann B, et al. 1013 Stromal R-spondin orchestrates gastric epithelial stem cells and gland homeostasis. 1014 Nature. 2017;548:451–5. 1015 69. Barker N, Huch M, Kujala P, van de Wetering M, Snippert HJ, van Es JH, et al. Lgr5+ve 1016 Stem Cells Drive Self-Renewal in the Stomach and Build Long-Lived Gastric Units In 1017 Vitro. Cell Stem Cell. 2010;6:25–36. 1018 70. Lo YH, Noah TK, Chen MS, Zou W, Borras E, Vilar E, et al. SPDEF Induces Quiescence 1019 of Colorectal Cancer Cells by Changing the Transcriptional Targets of β-catenin. 1020 Gastroenterology. 2017;153:205–18. 1021 71. Noah TK, Lo YH, Price A, Chen G, King E, Washington MK, et al. SPDEF functions as a 1022 colorectal tumor suppressor by inhibiting β-catenin activity. Gastroenterology. 1023 2013;144:1012–23. 1024 72. Kim KH, Kim W, Howard TP, Vazquez F, Tsherniak A, Wu JN, et al. SWI/SNF-mutant 1025 cancers depend on catalytic and non-catalytic activity of EZH2. Nat Med. 2015;21:1491–6. 1026 73. Bitler BG, Aird KM, Garipov A, Li H, Amatangelo M, Kossenkov A V., et al. Synthetic 1027 lethality by targeting EZH2 methyltransferase activity in ARID1A-mutated cancers. Nat 1028 Med. 2015;21:231–8. 1029 74. Ogiwara H, Takahashi K, Sasaki M, Kuroda T, Yoshida H, Watanabe R, et al. Targeting 1030 the Vulnerability of Glutathione Metabolism in ARID1A-Deficient Cancers. Cancer Cell. 1031 2019;35:177–90. 1032 75. Han K, Pierce SE, Li A, Spees K, Anderson GR, Seoane JA, et al. CRISPR screens in 1033 cancer spheroids identify 3D growth-specific vulnerabilities. Nature. 2020;580:136–41. 1034 76. Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W, Li GW, et al. Dynamic imaging 1035 of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell. 1036 2013;155:1479–91. 1037 77. Horlbeck MA, Gilbert LA, Villalta JE, Adamson B, Pak RA, Chen Y, et al. Compact and 1038 highly active next-generation libraries for CRISPR-mediated gene repression and 1039 activation. Elife. 2016;5:e19760. 1040 78. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized 1041 sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat 1042 Biotechnol. 2016;34:184–91. 1043 79. Sanson KR, Hanna RE, Hegde M, Donovan KF, Strand C, Sullender ME, et al. Optimized 1044 libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat Commun. 1045 2018;9:5416.

1046 80. Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the 1047 CRISPR-Cas9 system. Science. 2014;343:80–4. 1048 81. Mo X, Tang C, Niu Q, Ma T, Du Y, Fu H. HTiP: High-Throughput Immunomodulator 1049 Phenotypic Screening Platform to Reveal IAP Antagonists as Anti-cancer Immune 1050 Enhancers. Cell Chem Biol. 2019;26:331–9. 1051 82. Zhang JH, Chung TDY, Oldenburg KR. A simple statistical parameter for use in 1052 evaluation and validation of high throughput screening assays. J Biomol Screen. 1053 1999;4:67–73. 1054 83. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene 1055 set enrichment analysis: A knowledge-based approach for interpreting genome-wide 1056 expression profiles. Proc Natl Acad Sci U S A. 2005;102:15545–50. 1057 84. Liu Y, Sethi NS, Hinoue T, Schneider BG, Cherniack AD, Sanchez-Vega F, et al. 1058 Comparative Molecular Analysis of Gastrointestinal Adenocarcinomas. Cancer Cell. 1059 2018;33:721–35. 1060 85. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for 1061 RNA-seq data with DESeq2. Genome Biol. 2014;15:550. 1062 86. Gu Z, Eils R, Schlesner M. Complex heatmaps reveal patterns and correlations in 1063 multidimensional genomic data. Bioinformatics. 2016;32:2847–9. 1064 87. Depristo MA, Banks E, Poplin R, Garimella K V., Maguire JR, Hartl C, et al. A framework 1065 for variation discovery and genotyping using next-generation DNA sequencing data. Nat 1066 Genet. 2011;43:491–8. 1067 88. Talevich E, Shain AH, Botton T, Bastian BC. CNVkit: Genome-Wide Copy Number 1068 Detection and Visualization from Targeted DNA Sequencing. PLoS Comput Biol. 1069 2016;12:e1004873. 1070

1071 Figure Legends

1072

1073 Figure 1. Establishment of clonal TP53/ARID1A knockout human gastric organoid lines.

1074 A, The TP53 indel created by CRISPR/Cas9 cleavage was determined by Sanger sequencing.

1075 B, Establishment of a stable Cas9-expressing engineered TP53 KO human gastric organoid line.

1076 After antibiotic (Blasticidin) selection, Cas9 expression was confirmed by immunoblot analysis.

1077 C, Highly efficient CRISPR/Cas9 cleavage in Cas9-expressing TP53 KO organoids. A lentiviral

1078 construct containing a GFP guide RNA targeting the GFP reporter in the same construct was

1079 delivered into control TP53 KO and the Cas9-expressing TP53 KO organoids. After antibiotic

1080 (Puromycin) selection, GFP-positive cells were quantified by flow cytometry. D, Five different

1081 TP53/ARID1A DKO clones were established. ARID1A indels were determined by Sanger

1082 sequencing. E, Immunoblot analysis of ARID1A and ARID1B expression. F, IHC staining of

1083 ARID1A in TP53 KO versus TP53/ARID1A DKO organoids.

1084

1085 Figure 2. CRISPR KO of ARID1A promotes gastric malignancy. A, TP53 KO (control)

1086 organoids were typically well-organized morphologically; however, TP53/ARID1A DKO

1087 organoids exhibited different degrees of architectural complexity. H&E staining. Quantitation

1088 revealed increased epithelial stratification (green bar), structural complexity (blue bar), and loss

1089 of polarity (red bar) in all five TP53/ARID1A DKO clones. B, Immunofluorescence staining of the

1090 apical-specific marker ZO1 (red) showed disruptions in apicobasal polarity in a subset of

1091 TP53/ARID1A DKO organoid cells. The arrow (orange) indicates loss of polarity with

1092 inappropriate basolateral ZO1 expression. Cell membrane was stained with CTNNB1 (green).

1093 Nuclei were stained with DAPI (blue). C, ARID1A-deficient organoids exhibit hyperproliferation.

1094 TP53 KO and TP53/ARID1A DKO organoids were grown from 20,000 single FACS-sorted

1095 BFP+ cells. Brightfield images were taken after cell sorting. Quantification of organoid size is

1096 shown (n=400 per group). D, Quantification of EdU-positive proliferating cells in TP53 KO and

1097 TP53/ARID1A DKO organoids from independent experiments (N=3) at day 6 after passage. E,

1098 Quantification of metabolic activity from independent experiments (N=6) was determined by

1099 Alamar blue assay at day 12 after passage. Relative metabolic activity was normalized to TP53

1100 KO organoids (Control). Dots indicate independent experiments. The horizontal bar indicates

1101 mean. The error bar represents SEM. *P<0.05, ***P<0.005. ns, not significant. F, ARID1A-

1102 deficient organoids exhibited efficient in vivo tumor formation upon subcutaneous xenografting

1103 into NSG mice. TP53/ARID1A DKO xenografts formed larger tumors compare with TP53 KO

1104 xenografts. H&E staining. G, A significant negative correlation between ARID1A expression

1105 and tumor grade was identified in a human gastric cancer tissue microarray (total 197 patients).

1106 ARID1A expression was assessed by IHC.

1107

1108 Figure 3. ARID1A knockout induces mucinous metaplasia. A, Schematic illustration of

1109 gastric epithelium. Different cell lineages and specific lineage markers are indicated. B,

1110 Western blot of mucin-producing pit cell and mucous neck cell markers, TFF1, TFF2 and LYZ,

1111 reveals upregulation in TP53/ARID1A DKO organoids. Quantification of expression from

1112 independent experiments (N>3) was shown. Dots indicate independent experiments. C,

1113 Immunofluorescence staining of MUC5AC (green) and TFF1 (red) in engineered organoids and

1114 the donor primary gastric tissues. Nuclei were stained with DAPI (blue). Quantification of

1115 MUC5AC-positive organoids is shown. D, Mucin production in engineered organoids and donor

1116 primary gastric tissues detected by Alcian blue staining. Nuclei were counterstained by nuclear

1117 fast red. Quantification of Alcian blue-positive organoids indicate increased mucin in all five

1118 TP53/ARID1A DKO organoids lines. E, TP53/ARID1A DKO xenografts in a subcutaneously

1119 xenografted NSG mice retain their mucin-secreting phenotype in vivo. Alcian blue and PAS

1120 staining. Goblet-like (Alcian blue -positive) and pit-like (PAS positive) cells were indicated. F,

1121 Quantification of mitotic cells. Goblet-like and pit-like cells with mitotic figures were shown. H&E

1122 staining. G, Immunofluorescence staining of TP53/ARID1A DKO organoids showing LYZ-

1123 positive (red) or MUC5-positive (red) proliferating cells (KI67+, green). H, IHC staining of CDX2

1124 in xenografts and the donor primary gastric tissues. Colon tissues were used as positive control.

1125 I, IHC staining of MUC2 in organoids, xenografts and the donor primary gastric tissues. Colon

1126 tissues were used as positive control. J, Immunofluorescence staining of CLDN18 (white),

1127 MUC6 (red) and PGC (green) in TP53/ARID1A DKO organoids, xenografts and the donor

1128 primary gastric tissues. Cells within SPEM features (MUC6 and PGC double positive) are

1129 marked by arrows. K, A significant negative correlation between ARID1A (brown) IHC

1130 expression and mucin (blue, Alcian blue) production was identified in a human gastric cancer

1131 tissue microarray (total 197 patients).

1132

1133 Figure 4. Loss of ARID1A inhibits canonical Wnt/-catenin activity. A, Wnt/-catenin-

1134 induced activity was decreased in TP53/ARID1A DKO organoids infected by lentivirus

1135 containing TOPflash Wnt reporter and mCherry followed by luciferase assay on 20,000 sorted

1136 mCherry-positive cells. Quantification of luciferase activity from independent experiments (N=5)

1137 is shown. Luciferase activity was normalized to TP53 KO organoids (Control). B, The mucin-

1138 producing phenotype was genetically rescued by lentiviral expression of an N-terminal truncated

1139 gain-of-function -catenin mutant (CTNNB1N90). After virus transduction and antibiotic

1140 (Neomycin) selection, protein expression in the engineered organoids was analyzed by Western

1141 blot as indicated. C, Immunofluorescence staining of apically-restricted transmembrane MUC1

1142 (green) and membrane protein CDH1 (red) demonstrates that CTNNB1N90 reduces mucin

1143 production and architectural complexity of TP53/ARID1A DKO organoids. D, Venn diagram

1144 indicates overlap of genes that are significantly increased (101 genes) or decreased (143 genes)

1145 at least 2-fold in organoids with CTNNB1N90 alleles. E, Gene ontology analysis identified top

1146 key terms significantly associated with transcriptional profiles in CTNNB1N90 organoids. F,

1147 Wnt/-catenin target genes were upregulated in CTNNB1N90 organoids. G, Gastric mucous

1148 cell and intestinal goblet cell markers were significantly downregulated in CTNNB1N90

1149 organoids. The expression of transcription factors SPDEF, SOX21, THRB, SIX2 was shown. H,

1150 Phenotypic changes induced by ARID1A loss were partially restored by lentivirus CTNNB1N90.

1151 H&E staining and brightfield images. Relative stratification was quantified by counting the

1152 number of cells per length of perimeter of individual organoids. I, Constitutive Wnt signaling

1153 activation by CTNNB1N90 did not rescue ARID1A KO-mediated proliferation. Single cells

1154 (20,000/40 L Matrigel) from TP53 KO and TP53/ARID1A DKO organoids with and without

1155 lentivirus CTNNB1N90 underwent Alamar blue quantification of cell viability at day 12. Relative

1156 cell viability was normalized to control TP53 KO organoids (Control). Three independent

1157 experiments (N=3) were performed. In A, H and I, dots indicate independent experiments,

1158 horizontal bars indicate mean and error bars represent SEM. *P<0.05, ***P<0.005. ns, not

1159 significant.

1160

1161 Figure 5. ARID1A loss-associated gene master regulatory modules identify a

1162 FOXM1/BIRC5 node and recapitulate TCGA MSI and EBV human gastric cancers. A,

1163 Heatmap of significant differentially expressed genes with at least 2-fold change in each

1164 TP53/ARID1A DKO lines, compared with TP53 KO control line. A total of 412 up-regulated

1165 genes and 675 down-regulated genes were identified. Selected genes and signaling pathways

1166 are listed. B, Gene ontology analysis identified top key terms significantly associated with

1167 transcriptional profiles in TP53/ARID1A DKO organoids. C, Top 10 master regulators from

1168 ARACNe and VIPER prediction that were activated in TP53/ARID1A DKO organoids versus

1169 control TP53 KO are reported. Several FOXM1 targets, including BIRC5, CKS1B, CDC25C,

1170 CCNB1, CCNB2, CDK1, AURKA and AURKB were significantly upregulated in ARID1A-

1171 deficient cells. D, Western immunoblotting analysis demonstrated that FOXM1 targets, BIRC5

1172 and AURKB, were upregulated in TP53/ARID1A DKO organoids. Quantification of BIRC5 and

1173 AURKB expression from independent experiments (N>3) was shown. Dots indicate independent

1174 experiments. The horizontal bar indicates mean. The error bar represents SEM. E, Comparison

1175 of master transcriptional regulators in ARID1A KO organoids to TCGA STAD gastric cancer

1176 patient cases indicated significant similarities between organoids and TCGA MSI and EBV

1177 subtypes. The p-value computed by t-test (one sample) with the alternative hypothesis of true

1178 mean of the similarity score is greater than zero. Red and blue colors indicate high and low

1179 similarity concurrence, respectively. F, Comparison of master transcriptional regulators in

1180 ARID1A-deficient organoids to gastric cancer patient-derived organoids (PDOs) indicated

1181 significant similarities between engineered TP53/ARID1A DKO organoids and MSI subtype

1182 PDOs.

1183

1184 Figure 6. ARID1A deletion confers therapeutic vulnerability to BIRC5/survivin inhibition.

1185 A, High-throughput small molecule and bioactive screening in engineered organoids. B,

1186 Histogram of high-throughput screening of an FDA-approved small molecule compound library

1187 (2,036 compounds) in TP53/ARID1A DKO organoids. Organoids were dissociated into smaller

1188 clusters, re-plated into 384-well plates, and cultured for 5 days before drug treatment. Cell

1189 viability was quantified 3 days after compound treatment. The signal-to-background (S/B) ratio

1190 and Z’ indicated robust assay performance. The top 50 primary hits are indicated below the

1191 dashed red line and were selected for counter screening. C, YM-155, a BIRC5/survivin inhibitor,

1192 exhibited ARID1A-specific synthetic lethality. Fully-titrated counter screening for YM-155 was

1193 performed in two TP53 KO lines versus five additional TP53/ARID1A DKO clones. D, Brightfield

1194 images after organoid treatment with YM-155 (IC50, 0.03 M) for 3 days. YM-155 selectively

1195 inhibited growth of TP53/ARID1A DKO but not TP53 KO organoids. E, Establishment of stable

1196 BIRC5 over-expressing BIRC5/TP53 KO and BIRC5/TP53/ARID1A DKO organoid lines. After

1197 antibiotic (Neomycin) selection, BIRC5 expression was confirmed by immunoblot analysis. F,

1198 Constitutive expression of BIRC5 rescued the YM-155-associated sensitivity in

1199 TP53/ARID1A DKO organoids. Organoids were treated with YM-155 (IC50, 0.03 M) for 3 days.

1200 Three independent experiments (N=3) were performed. G, YM-155 treatment did not alter

1201 mucin production in TP53/ARID1A DKO organoids. Alcian blue staining. Nuclei were

1202 counterstained by nuclear fast red. H, Western immunoblotting analysis indicated that a gain-of-

1203 function -catenin mutant (CTNNB1N90) was sufficient to induce Wnt/-catenin targets, LEF1

1204 and TCF1; however, YM-155 treatment did not affect Wnt/-catenin activity. I, YM-155 IC50

1205 treatment (0.03 M) did not affect Wnt/-catenin-induced TOPflash reporter activity.

1206 Quantification of luciferase activity from independent experiments (N=4) is shown. Luciferase

1207 activity was normalized to DMSO treatment. A gain-of-function -catenin mutant (CTNNB1N90)

1208 organoid line was used as the positive control. J, Lentiviral expression of CTNNB1N90 did not

1209 rescue the BIRC5 expression, Western blot. K, Lentiviral expression of CTNNB1N90 did not

1210 rescue the selective YM-155 sensitivity of ARID1A-deficient cells. Fully-titrated YM-155

1211 treatment was performed in TP53 KO versus TP53/ARID1A DKO and TP53/ARID1A DKO plus

1212 CTNNB1N90 organoid clones. Alamar blue, three independent experiments (N=3).

1213

1214 Figure 7. Model of ARID1A loss-mediated oncogenic transformation in early human

1215 gastric cancer. ARID1A loss induces functionally independent transformation pathways during

1216 early gastric tumorigenesis in which non-essential Wnt-regulated mucinous differentiation

1217 operates in parallel with versus essential YM-155-sensitive FOXM1/BIRC5-regulated cell

1218 proliferation.

Author Manuscript Published OnlineFirst on January 15, 2021; DOI: 10.1158/2159-8290.CD-20-1109 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Figure 1

A C Lenti-sgGFP-PuroR-GFP TP53 WT TP53 KO mU6 EF1α T2A sgTP53 PAM LTR sgGFP PuroR GFP LTR

Parental Lenti-Cas9

G S Y G F R L 1bp deletion TP53 exon 4 GFP - B PuroR - GFP GFP Lenti-Cas9 Flag P2A EF1α sgGFP -

LTR SpCas9 BlastR LTR

Lenti Cas9 Parental Lenti-Cas9 -

Lenti kDa Parental 95.7% 130 Cas9 Parental Parental

Gastric TP53 KO Counts +Lenti-sgGFP-PuroR-GFP

Blasticidin Lenti-Cas9 + 50 µm 50 µm +Lenti-sgGFP-PuroR-GFP

GFP

D TP53 KO TP53/ARID1A DKO

sgARID1A#1 PAM E

Clone 1 Clone ARID1A KO 1 bp insertion 3 Clone 4 bp deletion 1 bp insertion

Control Q Q N S H D H G Clone 4 Clone ARID1A exon 1 2 Clone Control Clone 1 Clone 4 Clone Clone 3 Clone Clone 5 Clone 32 bp upstream deletion 297 bp downstream deletion 2 Clone ARID1A TP53 KO TP53/ARID1A DKO ARID1B PAM sgARID1A#2 β-ACTIN

Clone 5 Clone Control D L Y R L Y V S 1 bp insertion ARID1A exon 11

F TP53 KO TP53/ARID1A DKO Control Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 ARID1A ARID1A ARID1A ARID1A ARID1A ARID1A

100 µm 100 µm 100 µm 100 µm 100 µm 100 µm

A B TP53 KO TP53/ARID1A DKO TP53 KO TP53/ARID1A DKO H&E staining H&E staining Stratified CTNNB1/ZO1/DAPI CTNNB1/ZO1/DAPI (ii) (i) (ii)

lumen lumen

(iv) (iii) 50 µm 50 µm 100 µm 100 µm 50 µm

Well-organized Cribriform features Loss of polarity (i) (iii) (iv) C * TP53 TP53/ARID1A KO DKO * * Day 1

50 µm * 50 µm 50 µm Day 10 350

p < 0.0001 300

ARID1A KO ) ARID1A KO ARID1A KO m) Day 6 250

µ m

(

r 200 e Stratified High Complexity Loss of polarity t

e 150

a i Clone 2 Clone 3 Clone 1 Clone 4 Clone 5 Control Clone 2 Clone 3 Clone 2 Clone 3

Control Clone 1 Clone 4 Clone 5 Control Clone 1 Clone 4 Clone 5 100 D

100 100 100 100 100 Diameter (

)

) )

80 % 80 80 % 80 % 80 80

( 0

( ( n=93 n=77 n=54 n=67 n=84 n=117 n=54

n=93 n=77 n=93 n=77

n=67 n=54 n=67

n=84 n=84

n=117 n=117

e e

e Day 10 KO DKO

g g

60 g 60 60 KO DKO

n n

n 40

e e

40 40 e 40

e e

20 20 e 20

P P Percentage (%) Percentage

Percentage (%) Percentage 00 (%) Percentage 0 0

1 2 3 4 5

e e e e e

e e e e

Stratified Cribriform features Losse of polarity

n n n n n

o o o o o

l l l l l

C C C C C C

D E F TP53 KO TP53/ARID1A DKO TP53/ARID1A DKO

ARID1A KO ARID1A KO

Clone 2 Clone 3 Control Clone 1 Clone 4 Clone 5 Clone 2 Clone 3 Control Clone 1 Clone 4 Clone 5

5050 y 2.72.7

H&E staining 100 µm

t 100 µm i

l *

i 2.42.4 )

4040 *** b * *

% 2.1

a 2.1 i ( ns * ***

V 1.8

s ** 1.8

* * l l

3030 l 1.5

e 1.5

* e

C 1.21.2

+ cells + cells (%)

+ 2020

e 0.90.9 100 µm U

Relative Relative

i d

1010 t 0.60.6

E a EdU l 0.3

e 0.3 metabolic activity metabolic

0 R 0.00

l Xenografts # attempts # success

1 2 3 4 5

e e e e e

t ControlCloneCloneClone 1Clone 2Clone 3 4 5 TP53 KO n=16 n=3

n n n n n

o o o o o

l l l l

l TP53/ARID1A DKO n=17 n=13

C C C C C C High-grade dysplasia

G ARID1A high ARID1A low Low grade High grade

Case 1 Case 2 Human Gastric Cancer Tissue Microarray ARID1A high ARID1A low High grade 75 30 Low grade 81 3 Chi square test P = 0.0001 *** Fisher’s exact test P = 0.0001 *** 100 µm 100 µm

TFF1 TFF2 LYZ

n n

o o

25 o 25 5

s s

Pit s

e e

20 e 20 4

r r

Pit cell (MUC5AC, TFF1, LYZ) r

e e

Clone 1 Clone 3 Clone 4 Clone 3 Clone 5 Clone

Control 2 Clone 15 15

Isthmus cell

Y Y

Isthmus Y 10 10 2

Parietal cell (VEGFB, GIF) TFF1

i i

t 1

t 5

Mucous neck cell (MUC6, TFF2, LYZ) t 5

l l

TFF2 l

e e Relative fold changefold Relative Relative fold changefold Relative Relative fold changefold Relative 0

Neck 0

R R

Enteroendocrine cell (CHGA)

1 2 3 4 5

e e e e e

LYZ t

n n n n n

Chief cell (MIST1, LGR5, PGC) n

o o o o o

l l l l l

C C C C C

C C C C C Clone 2 Clone 3 Control Clone 1 Clone 4 Clone 5 Clone 2 Clone 3

CHGA Control Clone 1 Clone 4 Clone 5 Clone 2 Clone 3 Control Clone 1 Clone 4 Clone 5

Wnt Base β-ACTIN ARID1A KO ARID1A KO ARID1A KO

C TP53 KO TP53/ARID1A DKO Stomach H TP53 KO TP53/ARID1A DKO Stomach Colon

lumen CDX2 CDX2 CDX2 CDX2 DAPI / TFF1 /

100 µm 100 µm 50 µm 50 µm Xenografts Xenografts

100 µm MUC5AC MUC5AC

100 I TP53 20 40 60 80 KO TP53/ARID1A DKO (%) 0 20 40 60 80 100 MUC2 MUC2 Score 0 Score 1 Score 2 ControlK O n=42 CloneD K O 1 n=58 Stomach Colon KO

CloneD K O 2 n=43 MUC2 MUC2 CloneD K O 3 n=73 CloneD K O 4 n=56

ARID1A CloneD K O 5 n=55

Xenografts Xenografts

D So Sco Sco

TP53 KO Stomachcr

TP53/ARID1A DKO r r

e e e MUC2 MUC2

2 1 0 100 µm 100 µm Alcian Blue lumen Nuclear Red

50 µm 50 µm Organoids Organoids

J TP53/ARID1A DKO 50 µm 50 µm 100 µm Organoids Xenografts Stomach

100 20 40 60 80

(%) 0 0 20 40 60 80 100 > 99% > 90% < 10% Score 0 Score 1 Score 2 CControlo n t r o l n=462

CClonel o n e 1 n=190 KO CClonel o n e 2 CLDN18 n=217 / CClonel o n e 3 n=358 CClonel o n e 4 n=261

ARID1A CClonel o n e 5 n=413 MUC6 MUC6 /

E TP53/ARID1A DKO Stomach Colon 50 µm 50 µm 50 µm 100 µm PGC Alcian Blue PAS Goblet-like K ARID1A high ARID1A low Hybrid Pit-like Mucin low Mucin high Case 1 Case 2 ARID1A high / Mucin low 100 µm Xenografts 100 µm 100 µm

F Mitotic cells Goblet-like G KI67 / LYZ (n=150) DAPI 100 µm 100µm ARID1A low / Mucin high Human Gastric Cancer Tissue Microarray Pit-like KI67 / MUC5AC ARID1A high ARID1A low Normal epithelium 100 µm DAPI Mucin high 46 60 Mucin low 61 30 1.33% Goblet-like Chi square test P = 0.0001 *** 20.00% Pit-like Fisher’s exact test P = 0.0001 *** 78.67% OtherDownloadeds from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2021 American Association for Cancer Research. D F A H Figure 4 TP53 TP53

Lenti

LTR +CTNNB1ΔN90 Parental RelativeRelativ Folde Fold C hChangeange KO KO Down 10 20 30 40 50 60 7TCF 0 2 4 6 8

TOPflash activity (%) - Parental TOPflash TCF1TCF1 100 40 60 80 20 Luciferase - Control 0

regulated in CTNNB1ΔN90 100 80 60 40 20 0

TP53

LEF1 LEF1 - KO regulated in CTNNB1ΔN90 Clone 1 Clone ASCL2ASCL2 Control *** TP53 Clone 2 Clone ARID1A

Downloaded from AXIN2AXIN2 Clone 5 +C Pa 100 µm

genes 100 µm genes 200 µm 200 µm Clone 1 ***

SV40

Clone 3 Clone - r 101 143 T

e CTNNB1 LUC CTNNB1 N n Clone 4 Clone 2 t N *** a Clone 4 Clone CTNNB1ΔN90 B l KO

1 LGR5LGR5 mCherry

Clone 3

Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited.

Δ Clone 3 *** - Clone 5 Clone N

mCherry

TP53/ARID1A LGR6 9 Author ManuscriptPublishedOnlineFirstonJanuary15,2021;DOI:10.1158/2159-8290.CD-20-1109

LGR6 KO Clone 2 0 Clone 4 RNF43RNF43 *** Clone 1

Clone 5

DKO MEX3A

MEX3A 0 20 40 60 80 100 LTR Control PROX1 PROX1 TP53/ARID1A TP53/ARID1A

cancerdiscovery.aacrjournals.org

100 µm 200 µm 100 µm 200 µm B RelativeRelati vFolde Fold C Changehange DKO DKO 10 15 20 25 30 2 3 4 5 0 1 −

Cells / 10 mm Length

TCF1 + TCF1 3 4 5 6 7 0 1 2 Control

C TP53/ARID1A

LTR o LEF1 − n Relative dysplastic feature LEF1 Lenti t Cells / 10 µm Length 1 2 3 4 5 6 7 0 r ASCL2 EF1α ASCL2 +

o C 0 1 2 3 4 5 6 7

Clone 1 o l n E tr − - C ns o AXIN2 ARID1A Parental CTNNB1ΔN90 AXIN2 l +C Pa l C CTNNB1ΔN90 o lo +C Pa

Control

r +C Pa n T ARID1A e e *** n CTNNB1 + CTNNB1 N

CTNNB1ΔN90 T r 1 n Clone 2 e N t e C N Down-regulated Up-regulated n Myc a N

lo t Clone 1 B l a B n T r −

1 e l 1 1 LGR5LGR5 2 e

143 genes 101 genes N Δ

C C Δ

N lo n

Clone 2 * N 9 l n N + Regulation of Epithelial to Mesenchymal Transition Mesenchymal to Epithelial of Regulation

e 0 LGR6 9 o t LGR6 Clone 3 3 DKO 0 a

n C B

Clonelo 3 * − KO e n l *** e 1 RNF43 RNF43

KO 4 2

Clonelo 4 Δ C n + *** MEX3AMEX3A Epithelium Gastrointestinal of Maintenance Clone 4

e l 5 N o

Clone 5 −

Embryonic Digestive Tract Development Tract Digestive Embryonic 9 n NeoR PROX1PROX1 - IRES NeoR e 0

Pathway Signaling Wnt of Regulation 3 + Clone 5

C Process Biosynthetic Aminoglycan

Regulation of Cell Differentiation Cell of Regulation

o Research. ΔN90 CTNNB1 ΔN90 CTNNB1 β CHGA TFF2 TFF1 ARID1A MUC5AC LYZ LTR Digestive System Development System Digestive n +CTNNB1ΔN90 Parental Proliferation Cell Mesenchymal

- e Relative Fold Change ACTIN Relative Fold Change Development Tract Digestive 4 0.0 0.3 0.6 0.9 1.2 C

Epithelium Development l o TFF1TFF1 Parental n

e Development Tissue

TP53 TFF2 TFF2

KO LYZLYZ C TP53

TFF3TFF3 +C Pa

Parental

+CTNNB1ΔN90 300 µm 300 µm r T AGR2AGR2 e N n t N a KO CTNNB1ΔN90 B REG4REG4 l MUC1 / CDH1 / DAPI MUC1 / CDH1 / DAPI 1

Δ N

TP53/ARID1A SPDEF SPDEF 0.0 9 0 SOX21SOX21 TP53

DKO THRBTHRB 0.5 SIX2SIX2 GO GO BiologicalProcess

1.0 GO 300 µm 300 µm RelativeRelativ Folde Fold C Changehange 1.2 0.0 0.3 0.6 0.9

B 50 µm 50 µm

Cells / 10 mm Length o -lo TFF1 1.5

TFF1 l o 0 1 2 3 4 5 6 7 g TP53/ARID1A

C g 1 TP53/ARID1A i

TFF2 0 o TFF2 c (F n Relative metabolic activity a 0.6 0.9 1.2 1.5 1.8 2.4 D l t Cells / 10 µm Length 2.1 2.0 r LYZLYZ P R 0.9 1.2 1.5 1.8 2.1 2.4 o 0.6 C r )

l o n o C t TFF3TFF3 c r +C Pa ns Parental

o CTNNB1ΔN90 l l o C e

l +C Pa o T e +C Pa Control AGR2 s n n AGR2 2.5 N ARID1A e n ns s

T r t e 1 N e a C N

n B

l N lo T r Clone 1 t REG4

1 a REG4 B n 1 e l

e ns 1 N C 2 Δ

Δ C n

N N l Clonelo 2 N SPDEFSPDEF 9 t

o 9 3.0

n DKO ns

e 0 a 0 n B

3 C

l e lo SOX21 Clone 3 1 SOX21

n DKO 2 e ns

4 50 µm 50 µm Δ

C C Clonel 4 THRBTHRB o

n ns 3.5 l e N

o 5

n SIX2

SIX2 Clone 5

e 0 3 C lo n e 4 C lo n e 5 Author Manuscript Published OnlineFirst on January 15, 2021; DOI: 10.1158/2159-8290.CD-20-1109 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Figure 5 A TP53 TP53/ARID1A B KO DKO KEYWORDS (Down-regulated Transcripts) GO Term - Keywords

Control DKO1 DKO2

Mitosis

Cell cycle Mitotic spindle DNA Signal G2/M phase organization replication Cell Cycle CCNB1 BIRC5 ORC6 EGFR Cell Division CCNB2 AURKA MCM7 MET Centromere regulated - regulated CDK1 AURKB CDC6 HDAC9 - ATP-binding MYCL 412 genes 412 412 genes CDC25C CENPN POLD1 Up

Up Microtubule MKI67 CENPU POLG2 HHEX Glycoprotein CENPP TERT …etc.

Signal

Membrane Cell adhesion / junction Signal

regulated Transmembrane TGFB2 LIN7A TGFBR2 - CLDN2 MPP7 IGFBP2 Cell Junction 675 675 genes regulated

- CDH7 AJAP1 NKX2-1 EGF-like Domain Down CDH11 ALCAM …etc. 0 2 4 6 8 10 1212 1414 1616 675 675 genes -log10(P value)

Down -log10(P value)

RNA-seq log2 signal over mean signal per peak

C e D

ARID1A KO g BIRC5 AURKB

n 55

TOP 10 MRs 66

a B 5 44

TP53 KO K h *** 44 C ** ** * * *

R * c

R * I

** U

TP53/ARID1A DKO B 33 ns 33

A d

FOXM1

v g Control Clone 1 Clone 3 Clone 4 Clone Clone 5 Clone

Clone 2 Clone

v t o 22

44 i 22

f a

n E2F7

6 l

e a

BIRC5 11 e 11

R M

TOP2A R

h K

c 0

AURKB

Relative fold change

Relative fold change l

RACGAP1 l

1 2 3 4 5

e e e e e

e e e e e 2 t

F 2

n n n n n

ZNF367 β-ACTIN n

o o o o o

l l l l l

o e

4 C

C C C C C

v Clone 2 Clone 3 ZWINT Control Clone 1 Clone 4 Clone 5 Clone 2 Clone 3

Control Clone 1 Clone 4 Clone 5

i t

Relative fold change Relative M E2F8 a l ARID1A KO ARID1A KO

K 00 e

WHSC1

5 1

1 2

B A B C

F 2

B B

K K

TRIP13 M

N N

R R X

CDK1 I

BIRC5

K C

v MCM4

C C

CKS1B

U U CCNB2 CCNB1

AURKB

O i AURKA

FOXM1

C D

CDC25C

C C A A

a C

l 0

5 1

1 2

B A B

C E F

K B

B MSI C

5 GS CIN HM-SNV EBV

K K

D N N Intestinal Mixed MSI

R Diffuse R R EBV

X (n=51) (n=297) (n=9) (n=30) (n=75)

C C

U U

O (n=17)

B (n=6) (n=5) (n=9) (n=7)

C C A A F

C TCGA ARID1A TP53 PDOs Subtypes Mutation

Clone 1 Clone Clone 1 Clone

TCGA Similarity

Organoids Organoids Clone 2 Clone

Clone 2 Clone Similarity p-value (Z-score) Diffuse EBV Intestinal Mixed MSI DKO Clone 1 0.066 1 0.593 1 2.06E-4 p-value (t-test) GS CIN HM-SNV EBV MSI DKO Clone 2 4.31E-4 0.390 1 1 1.83E-3 DKO Clone 1 1 0.1858 0.2855 0.0034 1.59E-12 DKO Clone 2 1 0.0976 0.2359 0.0025 7.56E-12

Downloaded from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 15, 2021; DOI: 10.1158/2159-8290.CD-20-1109 Figure 6 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A High throughput small molecule screening Independent validation

Organoids 384-well plates 2,036 FDA-approved drugs Identify top hits ARID1A synthetic lethal hits

ARID1A WT

5 days ARID1A KO

3 days Cell viability Cell viability

Compound No. [full titration]

B 125 C YM-155 TP53/ARID1A DKO organoids Quality control 125

100 TP53 KO 1 160 TP53 KO 2

100 TP53 KO 1 0.5 75 TP53/ARID1A DKO 1 Z’ TP53 KO 2 120 0 TP53/ARID1A DKO 2 0 1 2 3 4 5 6 7 75 TP53/ARID1A DKO 1 50 TP53/ARID1A DKO 3 Plate No. TP53/ARID1A DKO 2 80 TP53/ARID1A DKO 4 50 TP53/ARID1A DKO 3 25 TP53/ARID1A DKO 5 16 TP53/ARID1A DKO 4 40 25 TP53/ARID1A DKO 5

% of Control of % 8 0 (%) Viability Cell

Top 50 primary hits S/B -4 -3 -2 -1 0 1 2 0 10 10 10 10 10 10 10 0 0 1 2 3 4 5 6 7 0 0 500 1000 1500 2000 Plate No. 10-4 10-3 10-2 10-1 100 101 102 Compound No. Concentration (µM) Parental +BIRC5 D E F + YM-155 + YM-155 140 ns Parental

DMSO YM-155 120 *** *** *** Lenti-BIRC5-NeoR ) 100

CMV Myc-DDK IRES %

DKO (

LTR BIRC5 NeoR LTR i 8080

a i KO

V 60

TP53 60

l BIRC5 OE l

KO DKO e

C 4040

− + − + BIRC5 (%) viability Cell 2020 ParentalParent al MYC-DDK-BIRC5 +CTNNB1 ΔN90 BIRC5 OE TP53/ARID1A

Endogenous BIRC5 00

1 2 3

DKO 7

e e

1e 2 3

n n

GAPDH n

o o o

l l l

C C cloneC number TP53/ARID1A 6 TP53/ARID1A DKO

5 h TP53 t

G DMSO YM-155 H TP53/ARID1A DKO g I TP53/ARID1A DKO n

KO e

700 L

Control Clone 1 Clone 2 Clone 3 4 m

600

)

− + − − + − − + − − + − YM-155 %

1 (

3 500

KO y

− − + − − + − − + − − + s

CTNNB1ΔN90 t

TP53

i l

v 400

e i

t activity (%) activity

CTNNB1

2 v 300 A

CTNNB1ΔN90

P ns

200 LEF11 O T 100 TCF1 DKO TOPflash

5 1

β-ACTIN

l 1 2 3 4 O 5 5

o B TP53/ARID1A

r e e e e e S 1 t n n n n n 155 1 2 3 4 5 6 7 8 9 10 11 12 N n -

o o o o o M

o l l l l l M

C C C C C C N

D Y DMSO

ΔN90 T

YM C J K Average of 3 independent experiments CTNNB1 125 YM-155 Average of 3 independent experiments TP53 KO

100 TP53/ARID1A DKO KO DKO 125 TP53/ARID1A DKO +CTNNB1ΔN90 − + − + CTNNB1ΔN90 75 TP53 KO 100 TP53/ARID1A DKO BIRC5 50 TP53/ARID1A DKO +CTNNB1ΔN90 75 TCF1 25 50 (%) Viability Cell GAPDH 0 25 10-4 10-3 10-2 10-1 100 101 102 Concentration (µM) Downloaded from cancerdiscovery.aacrjournals.org0 on September 30, 2021. © 2021 American Association for Cancer 10-4 10-3 10-2 10Research.-1 100 101 102 Author Manuscript Published OnlineFirst on January 15, 2021; DOI: 10.1158/2159-8290.CD-20-1109 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Figure 7

ARID1A KO in organoids

DNA accessibility change

BAF ⇧ Wnt activity Gene expression change

MR network change Mucinous differentiation ⇧ FOXM1

YM155 ⇧ BIRC5 ⇧ Other targets

Cell proliferation

A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and non-essential modes of oncogenic transformation

Yuan-Hung Lo, Kevin S. Kolahi, Yuhong Du, et al.

Cancer Discov Published OnlineFirst January 15, 2021.

Updated version Access the most recent version of this article at: doi:10.1158/2159-8290.CD-20-1109

Supplementary Access the most recent supplemental material at: Material http://cancerdiscovery.aacrjournals.org/content/suppl/2021/01/15/2159-8290.CD-20-1109.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerdiscovery.aacrjournals.org/content/early/2021/01/14/2159-8290.CD-20-1109. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.