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1 mutations are synthetic lethal with stimulation of WNT signaling 2 3 4 Chue Vin Chin1,2, Jisha Antony1,2, Sarada Ketharnathan1, Gregory Gimenez1, Kate M. 5 Parsons3, Jinshu He3, Amee J. George3, Antony Braithwaite1,2, Parry Guilford4, Ross D. 6 Hannan3 and Julia A. Horsfield1,2,5 7 8 1 Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New 9 Zealand. 10 2 Maurice Wilkins Centre for Molecular Biodiscovery, Private Bag 92019, The University of 11 Auckland. 12 3 The John Curtin School of Medical Research, Australian National University, Canberra, 13 Australia. 14 4 Department of Biochemistry, University of Otago, Dunedin, New Zealand. 15 5 Correspondence: Julia Horsfield, Department of Pathology, Dunedin School of Medicine, 16 University of Otago, PO Box 913, Dunedin 9016, New Zealand. Tel.: 64 3-479-7436; E-mail: 17 [email protected].

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18 Abstract 19 Mutations in encoding subunits of the cohesin complex are common in several cancers, 20 but may also expose druggable vulnerabilities. We generated isogenic MCF10A cell lines 21 with deletion mutations of genes encoding cohesin subunits SMC3, RAD21 and STAG2 and 22 screened for synthetic lethality with 3,009 FDA-approved compounds. The screen identified 23 several compounds that interfere with transcription, DNA damage repair and the . 24 Unexpectedly, one of the top ‘hits’ was a GSK3 inhibitor, an agonist of Wnt signaling. We 25 show that sensitivity to GSK3 inhibition is likely due to stabilization of b-catenin in cohesin 26 mutant cells, and that Wnt-responsive expression is highly sensitized in STAG2-mutant 27 CMK leukemia cells. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin 28 subunit . Our results suggest that cohesin mutations could progress oncogenesis by 29 enhancing Wnt signaling, and that targeting the Wnt pathway may represent a novel 30 therapeutic strategy for cohesin mutant cancers.

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31 Introduction 32 33 The cohesin complex is essential for sister cohesion, DNA replication, DNA 34 repair, and genome organization. Three subunits, SMC1A, SMC3 and RAD21, form the core 35 ring-shaped structure of human cohesin [1, 2]. A fourth subunit of either STAG1 or STAG2 36 binds to cohesin by contacting RAD21 and SMC subunits [3], and is required for the 37 association of cohesin with DNA [1-3]. The STAG subunits of cohesin are also capable of 38 binding RNA in the nucleus [4]. Cohesin associates with DNA by interaction with loading 39 factors NIPBL and MAU2 [5], its stability on DNA is regulated by the acetyltransferases 40 ESCO1 [6] and ESCO2 [7], and its removal is facilitated by PDS5 and WAPL [8, 9]. The 41 cohesin ring itself acts as a molecular motor to extrude DNA loops, and this activity is 42 thought to underlie its ability to organize the genome [10, 11]. Cohesin works together with 43 CCCTC-binding factor (CTCF) to mediate three-dimensional genome structures, including 44 enhancer-promoter loops that instruct gene accessibility and expression [12, 13]. The 45 consequences of cohesin mutation therefore manifest as segregation errors, 46 DNA damage, and deficiencies in genome organization leading to changes. 47 48 All cohesin subunits are essential to life: homozygous mutations in genes encoding complex 49 members are embryonic lethal [14]. However, haploinsufficient germline mutations in 50 NIPBL, ESCO2, and in cohesin genes, cause human developmental syndromes known as the 51 “cohesinopathies” [14]. Somatic mutations in cohesin genes are prevalent in several different 52 types of cancer, including bladder cancer (15-40%), endometrial cancer (19%), glioblastoma 53 (7%), Ewing’s sarcoma (16-22%) and myeloid leukemias (5-53%) [15-17]. The prevalence of 54 cohesin gene mutations in myeloid malignancies [18-22] reflects cohesin’s role in 55 determining lineage identity and differentiation of hematopoietic cells [23-26]. Of the 56 cohesin genes, STAG2 is the most frequently mutated, with about half of cohesin mutations in 57 cancer involving STAG2 [17]. 58 59 While cancer-associated mutations in genes encoding RAD21, SMC3 and STAG1 are always 60 heterozygous [21, 27, 28], mutations in the -located genes SMC1A and STAG2 61 can result in complete loss of function due to hemizygosity (males), or silencing of the wild 62 type during X-inactivation (females). STAG2 and STAG1 have redundant roles in cell 63 division, therefore complete loss of STAG2 is tolerated due to partial compensation by 64 STAG1. Loss of both STAG2 and STAG1 leads to lethality [29, 30]. STAG1 inhibition in

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65 cancer cells with STAG2 mutation causes defects and subsequent 66 lethality [31]. Therefore, although partial depletion of cohesin can confer a selective 67 advantage to cancer cells, a complete block of cohesin function will cause cell death. The 68 multiple roles of cohesin provides an opportunity to inhibit the growth of cohesin mutant 69 cancer cells via chemical interference with pathways that depend on normal cohesin function. 70 For example, poly ADP-ribose polymerase (PARP) inhibitors were previously shown to 71 exhibit synthetic lethality with cohesin mutations [17, 31-34]. PARP inhibitors prevent DNA 72 double-strand break repair [35], a process that also relies on cohesin function. 73 74 To date, only a limited number of compounds have been identified as inhibitors of cohesin- 75 mutant cells [17]. Here, we sought to identify additional compounds of interest by screening 76 libraries of FDA-approved molecules against isogenic MCF10A cells with deficiencies in 77 RAD21, SMC3 or STAG2. Unexpectedly, our screen identified a novel sensitivity of 78 cohesin-deficient cells to a GSK3 inhibitor that acts as an agonist of the Wnt signaling 79 pathway. We found that b-catenin stabilization upon cohesin deficiency likely contributes to 80 an acute sensitivity of Wnt target genes. The results raise the possibility that sensitization to 81 Wnt signaling in cohesin mutant cells may participate in oncogenesis, and suggest that Wnt 82 agonism could be therapeutically useful for cohesin mutant cancers. 83 84 Results 85 86 Cohesin gene deletion in MCF10A cells results in minor cell cycle defects 87 To avoid any complications with pre-existing oncogenic mutations, we chose the relatively 88 ‘normal’ MCF10A line for creation and screening of isogenic deletion clones of cohesin 89 genes SMC3, RAD21, and STAG2 (Supplementary Fig. 1a). MCF10A is a near-diploid, 90 immortalized, breast epithelial cell line that exhibits normal epithelial characteristics [36] and 91 has been successfully used for siRNA and small molecule screening [37]. We isolated several 92 RAD21 and SMC3 deletion clones, and selected single clones for further characterization that 93 grew normally and were essentially heterozygous. In the selected RAD21 deletion clone, one 94 of three RAD21 alleles (on , triploid in MCF10A) was confirmed deleted, with 95 one wild type and one undetermined allele (Supplementary Fig. 1c, e-g). In the selected 96 SMC3 deletion clone, one of two SMC3 alleles (on chromosome 10) was deleted 97 (Supplementary Fig. 1d, e-g). In the selected STAG2 deletion clone (Supplementary Fig. 1b),

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98 homozygous loss of STAG2 was determined by the absence of STAG2 mRNA and 99 (Supplementary Fig. 1e-g). For convenience here, we named the three cohesin mutant clones 100 RAD21+/-, SMC3+/- and STAG2-/-.

a b Hours after double thymidine release 0 6 12 36 48 100 WT WT ** RAD21+/- RAD21+/-

en ce (%) 50 SMC3+/- **** STAG2-/-

Conflu SMC3+/-

0 0 24 48 72 96 120 STAG2-/- Time (h)

100 c Chromosome spreads d

Normal Partial loss Complete loss Normal

ds (%) ea ds Partial loss

50 Complete loss aph ase spr Met

Normal 0 PCS 1+/- 2-/- WT 2 G D A A R SMC3+/-ST e f g WT RAD21+/- SMC3+/- STAG2-/- 300 **** 0.25 ****

0.20 M) 2

(μ 250 * 0.15

DAPI 0.10 ea r area 200

Nucl 0.05 Micronuclei per cell (%)

150 0.00

1+/- 2-/- WT 1+/- 2-/- WT 2 G 2 G D A D A A A SMC3+/- R SMC3+/-ST R ST

Figure 1. minor deficiencies in cell cycle progression, chromosome segregation and nuclear morphology in cohesin-deficient isogenic cell lines. a Proliferation curves (Incucyte) of MCF10A parental and cohesin-deficient clones (n = 3 independent experiments, mean ± sd, two-way ANOVA test: **p ≤ 0.01; ****p ≤ 0.0001). Doubling time in hours of MCF10A parental cells are 15.5±0.1; RAD21+/- , 17.1±0.2; SMC3+/-, 21.4±0.9; STAG2-/-, 16.4±0.2 respectively. b Flow cytometry analysis of cell cycle progression. c Representative spread images of cohesin-deficient cells. Black arrow indicates a normal chromosome; Red arrow indicates premature chromatid separation (PCS). d Quantification of chromosome cohesion defects. A minimum of 20 metaphase spreads were examined per individual experiment. (n=2 independent experiments, mean ± s.d.). e Representative confocal images of nuclear morphology. Scale bar, 15 M. Yellow arrow indicates a micronucleus. f Quantification of nuclear area. g Quantification of micronuclei (MN). A minimum of 1000 cells was examined per individual experiment. (n = 3 independent experiments, mean ± s.d., *p ≤ 0.05; ****p ≤ 0.0001). 101 102 The RAD21+/-, SMC3+/- and STAG2-/- clones had essentially normal cell cycle progression 103 when compared to parental cells, although the RAD21+/- and SMC3+/- clones proliferated 104 more slowly than the others (Fig. 1a, b). Chromosome spreads revealed that only the STAG2-

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105 /- clone had noteworthy chromosome cohesion defects characterized by partial or complete 106 loss of chromosome cohesion and gain or loss of more than one chromosome (Fig. 1c-d, 107 Supplementary Fig. 2a). Remarkably, SMC3+/- cells had noticeably larger nuclei that 108 appeared to be less dense (Fig. 1e-f, Supplementary Fig. 2b), possibly owing to decompaction 109 of DNA in this clone. RAD21+/- and SMC3+/- clones exhibited occasional lagging 110 and micronuclei, while the STAG2-/- clone did not (Fig. 1g, Supplementary 111 Fig. 2c-e). Cell growth and morphology in monolayer culture was essentially normal in all 112 three cohesin mutant clones (Supplementary Fig. 3). 113 114 Overall the cohesin deletion clones appear to have infrequent but specific cell cycle 115 anomalies that are shared between some, but not all clones. Anomalies include loss of 116 chromosome cohesion, lagging chromosomes, or micronuclei, but these features do not 117 appear to majorly impact on cell cycle progression or morphology. 118 119 Cohesin-depleted cells have altered nucleolar morphology and are sensitive to DNA 120 damaging agents 121 Cohesin-deficient cells have been demonstrated to display compromised nucleolar 122 morphology and ribosome biogenesis [38, 39], as well as sensitivity to DNA damaging 123 agents [40, 41]. Analysis of our RAD21+/-, SMC3+/- and STAG2-/- clones confirmed these 124 findings. Cohesin-deleted cells in steady-state growth had abnormal nucleolar morphology, 125 as revealed by fibrillarin and nucleolin staining (Supplementary Fig. 4). Furthermore, we 126 found that treatment with DNA intercalator/transcription inhibitor Actinomycin D caused 127 marked fragmentation of nucleoli in all three cohesin-deficient cell lines as determined by 128 fibrillarin staining (Fig. 2a, b). Actinomycin D treatment increased g-H2AX and TP53 in the 129 nuclei of RAD21+/- and SMC3+/- cells, in particular, indicating that these cells are 130 compromised for DNA damage repair relative to the parental MCF10A cells (Fig. 2c-f). In 131 contrast, immunostaining for g-H2AX and TP53 levels were comparable at baseline between 132 cohesin-deficient clones and parental cells. Interestingly, the STAG2-/- deletion clone was 133 much more resistant to DNA damage caused by Actinomycin D (Fig. 2c-f), even though 134 nucleoli are abnormal in this clone (Supplementary Fig. 4). 135 136 Overall, increased susceptibility of RAD21+/- and SMC3+/- clones to DNA damage is 137 consistent with cohesin’s role in DNA double-strand break repair [42], and highlights the

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a FBL b WT RAD21+/- SMC3+/- STAG2-/- 10 *** ** DMSO * ActD 8 nM

5

DMSO No. FBL spots per cell 1+/- 2-/- WT 2 G ActD D A A R SMC3+/-ST

c d γH2AX WT RAD21+/- SMC3+/- STAG2-/- 6000 ** * 4000

DMSO

2000

ActD H2AX intensity per cell (R.U.) 1+/- 2-/- γ WT 2 G D A A R SMC3+/-ST p53 e WT RAD21+/- SMC3+/- STAG2-/- f

*** 1.5 x 107

DMSO 1.0 x 107

5.0 x 106 intensity per cell (R.U.)

ActD

p53 1+/- 2-/- WT 2 G D A A R SMC3+/-ST

Figure 2. Cohesin-deficient cells have increased sensitivity to nucleolar stress and DNA damaging agents. a Representative image and b quantification of nucleolar dispersal observed in MCF10A cells after exposure to a DNA damaging agent, Actinomycin D (ActD) 8 nM. Fibrillarin (FBL) staining is used as a marker for nucleoli. c Representative image and d quantification of DNA damage foci observed in MCF10A cells after exposure to ActD. γH2AX staining is used to detect DNA double-strand breaks; arrows indicate γH2AX foci. e Representative image and f quantification of nuclear p53 in MCF10A cells after exposure to ActD. A minimum of 500 cells was examined per individual experiment. (n = 3 independent experiments, mean ± s.d., *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.0005; ****p ≤ 0.0001). Scale bar, 15 M. 138 139 different requirements for RAD21 and SMC3 versus STAG2 in this process. Cohesin 140 mutations were previously shown to sensitize cells to PARP inhibitor, Olaparib [32, 43]. We 141 confirmed a mild to moderate sensitivity to Olaparib in our cohesin-deficient MCF10A 142 clones relative to parental cells (Supplementary Fig. 5a). Collectively, characterization of our 143 cohesin deficient clones provided confidence that they represent suitable models for synthetic 144 lethal screening. 145

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146 A synthetic lethal screen of FDA-approved compounds identifies common sensitivity of 147 cohesin mutant cells to WNT activation and BET inhibition 148 To identify additional compounds that inhibit the growth of cohesin-deficient cells, we 149 screened the cohesin-deficient MCF10A cell lines with five dose concentrations (1-5,000 150 nM) of 3,009 compounds, including FDA-approved compounds (2399/3009), kinase 151 inhibitors (429/3009), and epigenetic modifiers (181/3009) (Fig. 3a). DMSO and 152 Camptothecin were included as negative and positive viability controls, respectively 153 (Supplementary Fig. 5b, c). We assayed cell viability after 48 hours of compound treatment.

Figure 3. A synthetic lethal screen identifies common sensitivity of cohesin-deficient cells to WNT activation and BET inhibition. a Schematic overview of the synthetic lethal screen. b-d Overview of the differential area over the curve (AOC) activity of all compounds tested in cohesin- deficient cell lines in the primary screen. A threshold of differential AOC ≥ 0.15 (red dashed lines) was used to filter candidate compounds of interest. e Venn diagram showing the number of common and unique compounds that inhibited RAD21+/-, SMC3+/- and STAG2-/- in the primary screen. f, g Dose-response curves of I-BET-762 and LY2090314. 154 155 Synthetic lethal candidate compounds were ranked based on the differential area over curve 156 (AOC) values that are derived from a growth rate-based metric (GR) (Fig. 3b-d). The GR 157 metric takes into account the varying growth rate of dividing cells to mitigate inconsistent 158 comparison of compound effects across cohesin-deficient cell lines [44]. Compounds that 159 caused ≥30% growth inhibition in cohesin-deficient clones compared with parental MCF10A 160 cells were selected for further analysis. The screen identified candidate 206 synthetic lethal 161 compounds, of which 18 inhibited all three cohesin-deficient cell lines ≥30% more than 162 parental MCF10A cells (Fig. 3e, Table 1, Supplementary Table 1). Most of the 206 163 compounds inhibited at least two cohesin-deficient cell lines and were classed in similar 164 categories of inhibitor (Supplementary Fig. 6a-d, Supplementary Table 1). Of the 206 165 primary screen hits, 85 (including common and unique hits identified in each cohesin-

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166 deficient clone) were subjected to secondary screening in an 11-point dose curve ranging 167 from 0.5 nM to 10 μM. Notable sensitive pathways included: DNA damage repair, the 168 PI3K/AKT/mTOR pathway, epigenetic control of transcription, and stimulation of the Wnt 169 signaling pathway (Table 1, Supplementary Table 2). 170 171 Table 1. Active compounds identified from the synthetic lethal screen. Compound Rank 1° Rank 2° Target Pathway Differential AOC activity Screen Screen RAD21+/- SMC3+/- STAG2-/- WAY-600 1 3 mTORC1/2 PI3K/AKT/mTOR 0.30 0.37 0.32 I-BET-762 2 8 BET Epigenetics 0.25 0.27 0.29 LY2090314 3 6 GSK3 WNT 0.25 0.22 0.25 Vistusertib (AZD2014) 4 1 mTORC1/2 PI3K/AKT/mTOR 0.18 0.33 0.26 P276-00 5 17 CDK1/4/9 Cell Cycle 0.17 0.29 0.28 MK-8745 6 22 Aurora A Cell Cycle 0.18 0.30 0.26 Ethacridine lactate 7 28 Anti-infection Microbiology 0.18 0.25 0.27 CUDC-101 8 36 EGFR, HDAC, Epigenetics 0.16 0.25 0.24 HER2 Dabrafenib 9 9 BRAF MAPK 0.18 0.27 0.18 (GSK2118436)* SAR131675 10 34 VEGFR3 Protein Tyrosine 0.15 0.17 0.33 Kinase ZM 447439 11 41 Aurora A/B Cell Cycle 0.16 0.22 0.24 Gitoxigenin Diacetate 12 32 NA Other 0.17 0.24 0.20 UNC669 13 62 Epigenetic Epigenetics 0.18 0.23 0.20 Reader Domain 4-Phenylbutyric Acid 14 29 Endoplasmic Other 0.20 0.17 0.20 reticulum stress Ipatasertib (GDC- 15 12 AKT PI3K/AKT/mTOR 0.21 0.19 0.16 0068) VX-702 16 43 P38 MAPK MAPK 0.15 0.19 0.21 RVX-208 17 58 BET proteins Epigenetics 0.16 0.20 0.17 Dihydroergotamine 18 49 NA Other 0.16 0.19 0.16 mesylate Olaparib 351 PARP1/2 DNA Damage 0.11 0.13 0.22 172 173 The identification of DNA damage repair inhibitors in our screen agrees with previous 174 studies showing synthetic lethality of PARP inhibition with cohesin mutation [33]. 175 Differential sensitivity of the cohesin deletion cell lines to PI3K/AKT/mTOR inhibitors is 176 consistent with the observed nucleolar deficiencies in cohesin mutant cells (Supplementary 177 Fig. 4). The PI3K/AKT/mTOR pathway stimulates ribosome biogenesis and rDNA 178 transcription; because rDNA is contained in nucleoli, it is likely that rDNA transcription and 179 ribosome production is already compromised in the cohesin gene deletion cell lines. We had 180 previously shown that BET inhibition is effective in blocking precocious gene expression in 181 the chronic myelogenous leukemia cell line K562 containing a STAG2 mutation [45]. Growth 182 inhibition of cohesin-deficient MCF10A cells by I-BET-762 (Fig. 3f, Supplementary Fig. 6e) 183 reinforces the idea that targeting BET could be therapeutically effective in cohesin-mutant 184 cancers. 185

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186 Interestingly, we found that LY2090314, a GSK3 inhibitor and stimulator of the Wnt 187 signaling pathway, inhibits all three cohesin deletion lines (Table 1, Fig. 3g, Supplementary 188 Fig. 6f). LY2090314 also inhibited the growth of K562 STAG2 R614* mutant leukemia cells 189 that we had previously characterized [45] (Supplementary Fig. 6g). Wnt signaling appears to 190 act upstream of cohesin [46, 47], and also to be primarily downregulated downstream of 191 cohesin mutation [48-50]. Therefore, we were prompted to further investigate why Wnt 192 stimulation via GSK3 inhibition might cause lethality in cohesin-deficient cells. 193 194 Stabilization of b-catenin in cohesin-deficient MCF10A cells 195 In “off” state of canonical Wnt signaling, β-catenin forms a complex including Axin, APC, 196 GSK3, and CK1 proteins. β-catenin phosphorylated by CK1 and GSK3 is recognized by the 197 E3 ubiquitin ligase β-Trcp, which targets β-catenin for proteasomal degradation. Activation 198 of Wnt signaling by ligand binding, or by GSK3 inhibition, releases β-catenin, allowing it to 199 accumulate in the nucleus where it binds TCF to activate Wnt target gene transcription [51, 200 52]. We found that there was no difference in GSK3 levels between parental MCF10A cells 201 and the cohesin-deficient clones, and upon treatment of cells with LY2090314, levels of 202 GSK3 were reduced in all cells as expected (Supplementary Fig. 7). In contrast, in untreated 203 cells we found that b-catenin is stabilized in all three cohesin-deficient clones compared with 204 parental MCF10A cells. When Wnt signaling is in the “off” state, phospho-Ser33/37/Thr41 205 marks b-catenin targeted for degradation. Membrane-associated phospho-Ser33/37/Thr41-b- 206 catenin was increased in cohesin-deficient clones, and disappeared upon treatment with 207 LY2090314 (Fig. 4a). Phospho-Ser675 b-catenin, the active form of b-catenin that is induced 208 upon Wnt signaling, was noticeably increased in the cytoplasm of cohesin-deficient cells 209 following treatment with LY2090314 (Fig. 4b, c). The results imply that inactive b-catenin 210 that would normally be targeted for degradation is instead stabilized in cohesin-deficient 211 cells, and is available to be converted into the active form following inhibition of GSK3. 212 213 Immunofluorescence labeling of cells showed that in LY2090314-treated cells, the stabilized 214 active phospho-Ser675 b-catenin (and total b-catenin, Supplementary Fig. 8a) mainly locates 215 to puncta in the cytoplasm (Fig. 4d). In contrast, no b-catenin accumulation was observed in 216 DMSO-treated cells (Supplementary Fig. 8b). b-catenin-containing puncta were also 217 observed upon WNT3A treatment of cohesin-deficient clones (Supplementary Fig. 8c). This 218 indicates that Wnt stimulation rather than a secondary effect of LY2090314 is responsible for

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Figure 4. LY2090314-mediated WNT stimulation leads to β-catenin stabilization in cohesin- deficient MCF10A cells. a Immunoblot of the membrane fraction of cohesin-deficient MCF10A cells shows increased basal level of β-catenin phosphorylation at Ser33/37/Thr41. b Immunoblot of the cytoplasmic fraction shows increased level of both total and phosphorylated β-catenin at Ser675 after MCF10A cells were treated with LY2090314 at 100 nM for 24 hours. c Quantification of protein levels for total and phosphorylated beta-catenin at Ser675. (n = 3 independent experiments, mean ± s.d., **p ≤ 0.01). d Immunofluorescence images show cytosolic accumulation of active β-catenin in cohesin-deficient MCF10A cells treated with LY2090314 100 nM for 24 hours. White arrows indicate puncta of β-catenin (Ser675). Scale bar = 25 M.

219 b-catenin accumulation. We could not reliably detect an increase of b-catenin in the nucleus 220 of cohesin-deficient MCF10A clones by immunofluorescence (Fig. 4d), therefore we decided 221 to use transcriptional response to determine the consequences of b-catenin stabilization for 222 Wnt signaling. 223 224 Cohesin-deficient leukemia cells are hypersensitive to Wnt signaling 225 RNA sequencing analysis of the cohesin gene deletion clones compared with parental 226 MCF10A cells revealed that gene expression changes strongly track with the identity of the 227 deleted cohesin gene (Supplementary Fig. 9a). However, expressed transcripts encoding Wnt 228 signaling pathway components did not cluster differently in cohesin-deficient clones 229 compared with parental MCF10A cells (Supplementary Fig. 9b), and cohesin mutation-based

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a Hoechst c d Total β-catenin Total β-catenin up down

400 upregulated STAG2-/- STAG2-/- 350 616 downregulated 404 171 WT 253 273 -400 214 102

Number of genes 136 151 DMSO WT WT WT STAG2-/-

STAG2-/- e WT LY2090314 STAG2-/-

b 0.4 p <0.0001 0.3 ti ve 0.2

Re la luo resce nce f 0.1

0.0 WT STAG2-/-

Figure 5. Cohesin-STAG2 mutant CMK cells show increased sensitivity to Wnt signaling. a Immunofluorescence images showing slightly increased nuclear accumulation of β-catenin in STAG2-CMK cells (STAG2-/-) compared to parental (WT) following treatment with LY2090314 at 100 nM for 24 hours. b Quantification of nuclear total β-catenin in parental (WT) and STAG2- CMK cells (STAG2-/-) cells. Fluorescence of nuclear total β-catenin was determined relative to the nuclear area. Graph depicts S.E.M. from analyses of 170-188 cell nuclei. c Histogram showing the number of genes upregulated or downregulated at FDR ≤ 0.05 upon WNT3A treatment in WT and STAG2-/- CMK cells. d Overlap of genes significantly upregulated or downregulated (FDR ≤ 0.05 ) upon WNT3A treatment between parental and STAG2 mutant cells. e Top ten enriched pathways (ranked by gene count) from the significantly downregulated and upregulated genes (FDR ≤ 0.05) following Wnt3a treatment in STAG2-/- and parental (WT) CMK cells using the ClusterProfiler R package on Biological Process dataset modelling for both cell types (WT or STAG2-/-) and regulation pattern (up- or downregulation). 230 clustering remained dominant. We reasoned that stimulation of Wnt signaling in a more 231 responsive cell type might be necessary to determine how stabilized b-catenin in cohesin- 232 deficient cells affects transcription. 233 234 Myeloid leukemias are frequently characterized by cohesin mutations, particularly in STAG2. 235 Furthermore, activation of Wnt signaling is associated with transformation in AML [53-55] 236 and AML patients were identified with mutations in AXIN1 and APC that lead to stabilization 237 of b-catenin [56]. CMK is a Down Syndrome-derived megakaryoblastic cell line that typifies 238 myeloid leukemias that are particularly prone to cohesin mutation [57]. We edited CMK to 239 contain the STAG2-AML associated mutation R614* [45] and selected single clones with

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240 complete loss of the STAG2 protein to create the cell line CMK-STAG2-/- (Supplementary 241 Fig. 10a-c). 242 243 Immunofluorescence showed that b-catenin is increased by 20% in the nuclei of CMK- 244 STAG2-/- cells relative to parental cells upon WNT3A stimulation (Fig. 5a, b). To determine 245 immediate early transcriptional responses to Wnt signaling, RNA-sequencing was performed 246 on CMK-STAG2-/- and parental CMK cells at baseline and after stimulation with WNT3A 247 for 4 hours. Around 76% more genes were upregulated in response to WNT3A in CMK- 248 STAG2-/- compared with CMK parental cells (616 in CMK-STAG2-/- vs 350 in CMK 249 parental, FDR ≤0.05, Fig. 5c), while around the same number were downregulated. About 250 one quarter of differentially expressed genes overlapped between CMK-STAG2-/- and CMK 251 parental cells. 252 253 Strikingly, 404 genes that were not Wnt-responsive in CMK parental cells became Wnt- 254 responsive upon introduction of the STAG2 R614* mutation (Fig. 5d). Genes upregulated in 255 CMK-STAG2-/- markedly increased the number of Wnt-sensitive biological pathways 256 following WNT3A treatment compared with parental CMK cells (Fig. 5e). A strongly 257 upregulated cluster of 244 transcripts in CMK-STAG2-/- included genes encoding signaling 258 molecules (JAG2, IL6ST, SEM3G) and transcription factors (SP7, KLF3, SMAD3, EP300 259 and EPHA4) (Supplementary Fig. 10d). Genes in this cluster were enriched for LEF1 and 260 TCF7 binding indicating their potential to be directly regulated by b-catenin. Enriched 261 biological pathways included Wnt signaling, cell cycle and metabolism (Supplementary Fig. 262 10e). Pathway analyses of genes significantly downregulated only in CMK-STAG2-/- also 263 showed enrichment for metabolism (Supplementary Fig. 10f, g). 264 265 Overall, our results show that CMK-STAG2-/- cells are exquisitely sensitive to Wnt 266 signaling. Introduction of the STAG2 R614* mutation amplified expression of Wnt- 267 responsive genes and sensitized genes and pathways that are not normally Wnt-responsive in 268 CMK. This sensitivity could be due at least in part to stabilized b-catenin. 269 270 Conservation of WNT sensitivity in cohesin-deficient zebrafish 271 To determine if enhanced Wnt sensitivity is conserved in a cohesin-deficient animal model, 272 we used our previously described zebrafish mutant, rad21nz171 [58], which has a nonsense

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273 point mutation in the rad21 gene that eliminates Rad21 protein. To provide a readout for Wnt 274 signaling, we used transgenic zebrafish carrying a TCF/b-catenin reporter in which 275 exogenous Wnt stimulation induces nuclear mCherry: Tg(7xTCF-Xla.Siam:nslmCherry)ia5 276 [59]. The Wnt reporter construct, which drives nuclear mCherry red fluorescence in Wnt- 277 responsive cells, was introduced into zebrafish carrying the rad21nz171 mutation by crossing. 278 Zebrafish embryos heterozygous for Tg(7xTCF-Xla.Siam:nslmCherry)ia5, and either 279 homozygous for rad21nz171 or wild type, were exposed to 0.15 M LiCl, an agonist of the Wnt 280 signaling pathway (Fig. 6). Expression of mCherry in the midbrain of embryos was detected 281 at 20 hours post-fertilization (hpf) by epifluorescence and confocal imaging. A constitutive 282 low level of mCherry is present in the developing midbrain of both untreated wild type and 283 rad21nz171 embryos (Fig. 6a-d). LiCl treatment dramatically increased the existing midbrain 284 mCherry expression in rad21nz171 mutants compared with wild type embryos (Fig. 6e-h).

Tg(7xTCF-Xla.Siam:nlsmCherry)ia5 Tg(7xTCF-Xla.Siam:nlsmCherry)ia5;rad21nz171 a a’ b c c’ d

mb mb Untreated mb mb

e e’ f g g’ h

mb mb

mb mb 0.15 M Lithium chloride

Figure 6. Zebrafish rad21 cohesin mutants show increased sensitivity to Wnt stimulation. Wnt ia5 reporter Tg(7xTCF-Xla.Siam:nlsmCherry) control embryos and Tg(7xTCF- Xla.Siam:nlsmCherry)ia5;rad21nz171 cohesin mutant embryos were treated with 0.15 M LiCl from 4 hpf to ia5 20 hpf. a, a’ Brightfield/fluorescent and b confocal images of Tg(7xTCF-Xla.Siam:nlsmCherry) transgenic control embryos. c, c’ Brightfield/fluorescent and d confocal Tg(7xTCF- Xla.Siam:nlsmCherry)ia5;rad21nz171 cohesin mutant embryos. Both control and rad21nz171 genotypes show constitutive Wnt reporter activity in the midbrain at 20 hpf. Control brightfield/fluorescent (e, e’) and confocal (f) images compared with rad21nz171 brightfield/fluorescent (g, g’) and confocal (h) images show that in the cohesin mutation background, Wnt reporter activity is enhanced in the midbrain (arrow) upon LiCl treatment, relative to controls. mb, midbrain. Scale bar, 50 µm. For Tg(7xTCF- ia5 ia5 nz171 Xla.Siam:nlsmCherry) control embryos n=15/15; and for Tg(7xTCF-Xla.Siam:nlsmCherry) ;rad21 cohesin mutant, n=4/4 with the same expression pattern. 285 286 The results show that Wnt signaling is sensitized in rad21 cohesin mutant zebrafish embryos, 287 similar to our observations in MCF10A and CMK cell lines. Our findings agree with previous

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288 work showing that canonical Wnt signaling is hyperactivated in cohesin loader nipblb-loss- 289 of-function zebrafish embryos [60]. Altogether, the results suggest that hyperactivation of 290 Wnt signaling is a conserved feature of cohesin deficient cells, and that enhanced sensitivity 291 to Wnt is at least in part due to stabilization of b-catenin. 292 293 Discussion 294 295 The cohesin complex and its regulators are encoded by several different loci, and genetic 296 alterations in any one of them may occur in up to 26% of patients included in The Cancer 297 Genome Atlas (TCGA) studies [61]. Therefore, we were motivated to identify compounds 298 that interfere with cell viability in more than just one type of cohesin mutant. The generation 299 of RAD21, SMC3 and STAG2 cohesin mutations in the breast epithelial cell line MCF10A 300 resulted in mild cell cycle and nucleolar phenotypes that are consistent with the many cellular 301 roles of cohesin. Differences between RAD21 and SMC3 heterozygotes vs STAG2 302 homozygotes could be explained by the fact that RAD21 and SMC3 are obligate members of 303 the cohesin ring, whereas STAG2 can be compensated by STAG1. 304 305 Synthetic lethal sensitivities of cohesin mutant cells that emerged from our screen mostly 306 related to the phenotypes of cohesin mutant cells, and/or their previously identified 307 vulnerabilities. For example, cohesin mutant cells were sensitive to inhibitors of the 308 PI3K/Akt/mTOR pathway that feeds into ribosome biogenesis, epigenetic inhibitors that 309 could interfere with cohesin’s genome organization and gene expression roles, and a limited 310 sensitivity to PARP inhibitors. The observed sensitivity to PI3K/AKT/mTOR inhibitors is 311 consistent with the nucleolar disruption present in cohesin-deficient cell lines, and with 312 cohesin’s known involvement in rDNA transcription and ribosome biogenesis [38]. We have 313 also previously described evidence for sensitivity of cohesin-deficient cell lines to 314 bromodomain (BET) inhibition [45]. However, the Wnt agonist LY2090314, which mimics 315 Wnt activation by inhibiting GSK3-β [62], emerged as a novel class of compound that 316 inhibited the growth of all three cohesin mutants tested. Because there is pathway 317 convergence between Wnt signaling and the PI3K/AKT/mTOR pathway [63] it is possible 318 that these pathways represent a common avenue of sensitivity. The identification of 319 PI3K/AKT/mTOR inhibitors in the screen supports this notion. 320

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321 Wnt signals are important for stem cell maintenance and renewal in multiple mammalian 322 tissues [52]. Canonical Wnt signaling is required for self-renewal of leukemia stem cells 323 (LSCs) [55], and is reactivated in more differentiated granulocyte-macrophage progenitors 324 (GMPs) when they give rise to LSCs [53]. Wnt signaling is an important regulator of 325 hematopoietic stem cell (HSC) self-renewal as well [64]. Our results suggest that cohesin 326 deficient cells are sensitive to Wnt agonism because they already have stabilization of β- 327 catenin. Interestingly, multiple experimental systems showed that dose-dependent reduction 328 in cohesin function causes HSC expansion accompanied by a block in differentiation [65, 329 66]. It is possible that the effects of cohesin deficiency on HSC development could be in part 330 due to enhanced Wnt signaling. 331 332 Cohesin mutations are particularly frequent (53%) in Down Syndrome-associated Acute 333 Megakaryoblastic Leukemia (DS-AMKL) patients [57]. In the edited DS-AMKL cell line 334 STAG2-CMK, there was a dramatically enhanced immediate early transcriptional response to 335 WNT3A, including induction of hundreds of genes that are not normally responsive to 336 WNT3A in this cell line. It is possible that STAG2 deficiency in CMK leads to an altered 337 structure that sensitizes genes to Wnt signaling. However, we observed increased 338 nuclear localization of β-catenin in STAG2-CMK, increasing the likelihood that stabilization 339 of β-catenin in STAG2-CMK cells accounts for amplified gene expression in response to 340 WNT3A. Interestingly, DS-AMKL leukemias are often associated with amplified Wnt 341 signaling caused by Trisomy 21 [67] suggesting a possible synergy between cohesin 342 mutations and Wnt signaling dysregulation in DS-AMKL. 343 344 We observed an enhanced Wnt reporter response to Wnt activation in rad21 mutant 345 zebrafish, arguing that Wnt sensitivity upon cohesin mutation is a conserved phenomenon. 346 Previous research shows that cohesin genes are at once both targets [46, 68] and upstream 347 regulators [47, 49, 50, 60] of Wnt signaling. For example, depletion of cohesin loader 348 in zebrafish embryos was reported to downregulate Wnt signaling at 24 hpf [69], but to 349 upregulate it at 48 hpf [60]. What determines the directionality of cohesin’s effect on Wnt 350 signaling is unclear. It is possible that feedback loops operate differently in cell- and signal- 351 dependent contexts [70] in cohesin-deficient backgrounds. 352

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353 A remaining question is exactly what causes b-catenin stabilization and Wnt hyperactivation 354 in cohesin mutant cells. One possibility is that stabilization of b-catenin upon cohesin 355 deficiency is linked to energy metabolism. For example, in developing amniote embryos, 356 glycolysis in actively growing cells of the embryonic tail bud raises the intracellular pH, 357 which in turn causes b-catenin stabilization [71, 72]. This mode of glycolysis is similar to 358 that observed in cancer cells and is known as the Warburg effect [73, 74]. Metabolic and 359 oxidative stress disturbances in cohesin mutant cells observed in this study and others [38, 360 75] might similarly influence b-catenin stability and in turn, sensitivity to incoming Wnt 361 signals. Further research will be needed to determine how b-catenin is stabilized in cohesin 362 deficient cells, whether b-catenin stabilization is part of a transition to malignancy, and if the 363 associated Wnt sensitivity represents a therapeutic opportunity in cohesin mutant cancers. 364 365 366 Material and Methods 367 368 Cell culture 369 MCF10A and its cohesin-deficient derivatives were maintained in Dulbecco’s modified 370 Eagle medium (DMEM) (Life Technologies) supplemented with 5% horse serum (Life 371 Technologies), 20 ng/mL of epidermal growth factor (EGF) (Peprotech), 0.5 mg/mL of 372 hydrocortisone, 100 ng/mL of cholera toxin and 10 μg/mL of insulin (local pharmacy). All 373 supplements were purchased from Sigma-Aldrich unless otherwise stated. K562 cells were 374 maintained in Isocove’s Modified Dulbecco’s Media (IMDM) (Life Technologies) containing 375 10% fetal bovine. CMK cells were maintained in RPMI 1640 media containing 10% fetal 376 bovine serum. CMK cells and adherent K562-STAG2 null line were detached for subculture 377 using trypsin-EDTA (0.005% final concentration, Life Technologies). All cells were cultured 378 at 37 °C at 5% CO2. 379 380 Generation of isogenic cell lines using CRISPR-CAS9 editing 381 We used CRISPR-CAS9 sgRNAs targeting the 5' and 3' UTR regions of RAD21, SMC3 and 382 STAG2 gene to create MCF10A RAD21+/-, MCF10A SMC3+/-, and MCF10A STAG2-/-. 383 Specific sgRNAs were cloned into the px458 plasmid [76] and transfected into MCF10A 384 cells. Single GFP-positive cells were isolated into 96-well plates using a FACSAriaII (Becton 385 Dickinson) and clonally expanded. PCR screening and Sanger sequencing identified cells

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386 with heterozygous deletion of RAD21 or SMC3 and homozygous deletion of STAG2. Primer 387 and guide sequences are provided in Supplementary Tables 3 and 4. Editing of K562 to 388 create STAG2 R614* mutation has been described previously [45]. The CMK line with the 389 STAG2 R614* mutation was generated using the same sgRNA and method. 390 391 Quantitative PCR (RT-qPCR) 392 Total RNA was extracted using NucleoSpin RNA kit (Machery-Nagel). cDNA was 393 synthesized using qScriptTM cDNA SuperMix (Quanta Biosciences). RT-qPCR was 394 performed on a LightCycler® 480 II (Roche Life Science) using SYBR® Premix Ex Taq™ 395 (Takara). Expression values relative to reference genes cyclophilin and glyceraldehyde 3- 396 phosphate dehydrogenase (GAPDH) were derived using qBase Plus (Biogazelle). Primer 397 sequences are provided in Supplementary Table 5. 398 399 Antibodies 400 Primary antibodies used are as follows: anti-RAD21 (ab992), anti-SMC3 (#5696, CST), anti- 401 STAG2 (ab4463), anti-γ-Tubulin (T5326; Sigma-Aldrich) in 1:5000, anti-fibrillarin (ab5821), 402 anti-nucleolin (ab13541), anti-gamma H2AX (ab26350), anti-TP53 (ab131442), anti-β- 403 catenin (#9562, CST), anti-phospho-β-catenin (Ser675) (#9567, CST), anti-phospho- 404 phospho-β-catenin (Ser33/37/Thr41) (#9561, CST). Secondary antibodies used for 405 immunofluorescence were anti-mouse Alexa Fluor 488 (1:2000, Life Technologies), anti- 406 rabbit Alexa Fluor 568 (1:2000, Life Technologies). All antibodies were used in 1:1000 for 407 immunoblotting or immunofluorescence unless otherwise stated. 408 409 Immunoblotting 410 Immunoblotting was carried out as described previously [77, 78]. Primary antibodies were 411 detected using IRDye 800CW Donkey anti-Goat IgG and IRDye 680CW Goat anti-mouse 412 IgG, IRDye 800CW Goat anti-rabbit IgG (LICOR). LI-COR Odyssey® and LI-COR Image 413 Studio software was used to image and quantify blots. 414 415 Proliferation assays 416 MCF10A parental and isogenic cohesin deficient cell lines were seeded in 96-well plates at 417 2,000 cells per well. Cell confluence was monitored by time-lapse microscopy using 418 IncuCyteTM FLR with a 10X objective for 5 days. 419

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420 Cell cycle analysis 421 Cells were synchronized using double thymidine block as described previously [78]. Samples 422 were harvested, fixed and stained with 10 μg/ml propidium iodide (PI) and 250 μg/ml RNase 423 A, 37 °C for 30 min. Cells were then analyzed using a Beckman Coulter Gallios Flow 424 Cytometer. 425 426 Immunofluorescence and imaging 427 Cells stained for FBL, gH2AX, and TP53 were imaged using an Opera Phenix high content 428 screening system, with 63x water objectives in confocal mode. Spot enumeration and signal 429 intensities were analyzed using Harmony software (PerkinElmer). CMK cells were fixed with 430 4% (v/v) paraformaldehyde in PBS for 10 minutes, washed, stained with Hoechst 33342 (1 431 μg/mL) for 10 min. CMK cells were spun onto slides using SHANDON cytospin prior to 432 fixation. Cells were blocked with 2% (w/v) bovine serum albumin in PBS, then incubated 433 with primary antibody overnight at 4 °C. The next day, cells were washed with PBS and 434 incubated with secondary antibody at room temperature for 1 hour. Cells were then washed 435 and mounted with microscope slides using ProLong Gold Anti-fade (Life Technology) or 436 DAKO mounting medium. Imaging was done using a Nikon C2 confocal microscope with 437 NIS Elements and images were processed and quantified using Image J or FIJI software. 438 439 High-throughput compound screen 440 3,009 compounds from the FDA-approved, kinase inhibitors and epigenetic libraries 441 (Compounds Australia, Griffith University, Australia) were screened against MCF10A 442 parental and MCF10A cohesin-deficient clones. MCF10A parental cells were seeded at 600 443 cells/well, RAD21+/- at 800 cells/well, SMC3+/- at 1,200 cells/well and STAG2-/- at 700 444 cells/well, into 384-well CellCarrier-384 Ultra microplates (PerkinElmer) with EL406 445 microplate washer dispenser (BioTek). 24 hours later, growth media was aspirated from the 446 plates and 35uL of fresh MCF10A complete growth media was added into each well using 447 BioTek EL406TM Microplate washer dispenser. 5uL of 8x concentrated compound solution 448 (diluted in MCF10A complete growth media) was added into each well with a JANUS 449 automated liquid handling system (PerkinElmer). At the time of drug treatment, one 450 untreated plate was retained to determine the cell number at t=0. For the rest of the assay 451 plate, compounds were added in 40 µL of complete medium per well using an Echo 550 452 Liquid Handler (Labcyte). Positive (Campthothecin, 40 nM) and negative controls (DMSO 453 0.1%) were added to each assay plate for quality control. Duplicated control plates of each

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454 cell line were also prepared for cell count quantification prior to the addition of drugs, to be 455 used in the growth rate inhibition (GR) metrics. After 48 hours, control and treated cells in 456 were fixed and stained simultaneously using 4% PFA/ 0.5ug/mL DAPI/ 0.1% Triton X-100 457 solution. Cells were imaged using an Opera Phenix high content screening system and 458 analyzed using Columbus software (PerkinElmer) using 20x water objectives. To determine 459 compound activity, dose-response curves for each compound was plotted and using an R 460 package, GRmetrics [79]. Synthetic lethal candidate compounds were selected and ranked 461 based on the differential area over curve (AOC) metrics [44] derived from dose-response 462 curves of MCF10A parental and cohesin deficient cell lines. Compounds that caused ≥30% 463 growth inhibition (AOC ≥ 0.15) in cohesin deficient clones compared with parental MCF10A 464 cells were selected as hits. A secondary screen was performed with 85 candidate hits 465 identified from primary screen. Activity of the selected compounds were tested in eleven 466 concentration (0.5 nM to 10 µM). Secondary screen hit compounds were selected based on 467 the same threshold used in primary screen. 468 469 Cell viability assays to validate compounds identified from the screen 470 Individual compounds were purchased from Sigma-Aldrich, Selleck Chem or 471 MedChemExpress and dissolved in DMSO at recommended concentrations. MCF10A 472 parental cells were seeded in 96-well plates at 3,000 cells/well, RAD21+/- at 4,500 cells/well, 473 SMC3+/- at 5,000 cells/well and STAG2-/- at 3,500 cells/well. Cells were incubated for 24 474 hours then treated with compounds for 48 hours. DAPI-stained cells were counted using a 475 Lionheart FX automated microscope (BioTek). K562 parental and STAG2-/- cells were 476 seeded at 2,000 cells per well in 96 well plates, incubated with the compound for 48 hours 477 after which viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5- 478 diphenyltetrazolium bromide or MTT. 479 480 RNA sequencing and analyses 481 Total RNA was extracted using NucleoSpin RNA kit (MACHEREY-NAGEL). MCF10A 482 libraries from three biological replicates of each cell type were prepared using NEBNext® 483 Ultra™ RNA Library Prep Kit (Illumina®) and sequenced on HiSeq X by Annoroad Gene 484 Technology Ltd. (Beijing, China), contracted through Custom Science (NZ). CMK lines were 485 treated with 200 ng/ml of recombinant human WNT3A (R&D systems) for 4 hours. Libraries 486 were prepared from baseline (non-treated) and WNT3A treated CMK cell lines using 487 Illumina TruSeq stranded mRNA library and sequenced on the HiSeq 2500 V4 at the Otago

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488 Genomics Facility (Dunedin, New Zealand). RNA sequencing reads were first trimmed for 489 sequencing adapters and low quality (q<20). Cleaned reads were then aligned to the human 490 genome GRCh37 (hg19) using HISAT2 version 2.0.5 with gene annotation from Ensembl 491 version 75. Read counts were retrieved by exon and summarized by gene using featureCount 492 [80] version v1.5.3. Differentially expressed genes in the STAG2-/- mutants versus wild type 493 were identified using DESeq2 [81]. P-values were adjusted for multi-test following 494 Benjamini-Hochberg methology. Pathways analyses were performed using Molecular 495 Signature Databases ReactomePA [82] and clusterProfiler [83] on differentially expressed 496 genes. 497 498 Zebrafish methods and imaging 499 Wild type (WIK), Tg(7xTCF-Xla.Siam:nslmCherry)ia5 [59] and rad21nz171 [58] mutant fish 500 lines were maintained according to established husbandry methods [84]. Zebrafish embryos 501 were incubated with 0.15 M lithium chloride (LiCl) from 4 hpf for 16 hours. LiCl salt was 502 directly dissolved in embryo water and added to embryos in 6 well plates. 50 embryos were 503 used per treatment group. At 20 hpf, embryos were washed and mounted in 3% methyl 504 cellulose for imaging. Z-stacks were acquired using a Nikon C2 confocal microscope. 505 506 Statistical analyses 507 Unless otherwise stated, statistical analyses were carried out by a two-tailed Student’s t test. 508 A p value ≤ 0.05 is considered statistically significant. All statistical analyses were carried 509 out using R and Prism version 7 software (GraphPad). 510 511 Data availability 512 RNA sequencing data has been deposited at the GEO database under GSE154086. 513 514 Acknowledgements 515 This work was funded by Health Research Council of NZ awards 15/229 and 19/415 to 516 J.A.H., and a Maurice Wilkins Center for Biodiscovery award to J.A. and J.A.H. 517 518 Competing interests 519 The authors have no competing interests to declare. 520 521

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522 Author contributions 523 CVC, JA, SK, GG, PG, AJG, RDH, JAH designed experiments. CVC, JA, SK, GG, KMP, JH 524 performed experiments. CVC, JA, SK, GG, KMP, JH, AJG, AB, PG, RDH, JAH analyzed 525 data. CVC, JA and JAH wrote the paper with input from the other authors. All authors read 526 and approved the final manuscript. 527 528 References 529 530 1 Dorsett D, Strom L. The ancient and evolving roles of cohesin in gene expression and 531 DNA repair. Current biology : CB 2012; 22. 532 533 2 Horsfield JA, Print CG, Monnich M. Diverse developmental disorders from the one 534 ring: distinct molecular pathways underlie the cohesinopathies. Frontiers in genetics 535 2012; 3. 536 537 3 Shi Z, Gao H, Bai XC, Yu H. Cryo-EM structure of the human cohesin-NIPBL-DNA 538 complex. Science 2020; 368: 1454-1459. 539 540 4 Pan H, Jin M, Ghadiyaram A, Kaur P, Miller HE, Ta HM et al. Cohesin SA1 and SA2 are 541 RNA binding proteins that localize to RNA containing regions on DNA. Nucleic Acids 542 Res 2020; 48: 5639-5655. 543 544 5 Wendt KS. Resolving the Genomic Localization of the Kollerin Cohesin-Loader 545 Complex. Methods Mol Biol 2017; 1515: 115-123. 546 547 6 Wutz G, Ladurner R, St Hilaire BG, Stocsits RR, Nagasaka K, Pignard B et al. ESCO1 548 and CTCF enable formation of long chromatin loops by protecting cohesin(STAG1) 549 from WAPL. Elife 2020; 9. 550 551 7 van der Lelij P, Godthelp BC, van Zon W, van Gosliga D, Oostra AB, Steltenpool J et al. 552 The cellular phenotype of Roberts syndrome fibroblasts as revealed by ectopic 553 expression of ESCO2. PLoS One (Research Support, Non-U.S. Gov't) 2009; 4: e6936. 554 555 8 Wutz G, Varnai C, Nagasaka K, Cisneros DA, Stocsits RR, Tang W et al. Topologically 556 associating domains and chromatin loops depend on cohesin and are regulated by 557 CTCF, WAPL, and PDS5 proteins. EMBO J 2017; 36: 3573-3599. 558 559 9 Shintomi K, Hirano T. Releasing cohesin from chromosome arms in early : 560 opposing actions of Wapl-Pds5 and Sgo1. Genes Dev 2009; 23: 2224-2236. 561 562 10 Vian L, Pekowska A, Rao SSP, Kieffer-Kwon KR, Jung S, Baranello L et al. The 563 Energetics and Physiological Impact of Cohesin Extrusion. Cell 2018; 173: 1165-1178 564 e1120. 565

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751 61 Romero-Perez L, Surdez D, Brunet E, Delattre O, Grunewald TGP. STAG Mutations in 752 Cancer. Trends Cancer 2019; 5: 506-520. 753 754 62 Atkinson JM, Rank KB, Zeng Y, Capen A, Yadav V, Manro JR et al. Activating the 755 Wnt/beta-Catenin Pathway for the Treatment of Melanoma--Application of 756 LY2090314, a Novel Selective Inhibitor of Glycogen Synthase Kinase-3. PLoS One 757 2015; 10: e0125028. 758 759 63 Prossomariti A, Piazzi G, Alquati C, Ricciardiello L. Are Wnt/beta-Catenin and 760 PI3K/AKT/mTORC1 Distinct Pathways in Colorectal Cancer? Cell Mol Gastroenterol 761 Hepatol 2020; 10: 491-506. 762 763 64 Rattis FM, Voermans C, Reya T. Wnt signaling in the stem cell niche. Curr Opin 764 Hematol 2004; 11: 88-94. 765 766 65 Viny AD, Levine RL. Cohesin mutations in myeloid malignancies made simple. Curr 767 Opin Hematol 2018; 25: 61-66. 768 769 66 Mazumdar C, Majeti R. The role of mutations in the cohesin complex in acute 770 myeloid leukemia. Int J Hematol 2017; 105: 31-36. 771 772 67 Emmrich S, Rasche M, Schoning J, Reimer C, Keihani S, Maroz A et al. miR- 773 99a/100~125b tricistrons regulate hematopoietic stem and progenitor cell 774 homeostasis by shifting the balance between TGFbeta and Wnt signaling. Genes Dev 775 2014; 28: 858-874. 776 777 68 Ghiselli G, Coffee N, Munnery CE, Koratkar R, Siracusa LD. The cohesin SMC3 is a 778 target the for beta-catenin/TCF4 transactivation pathway. J Biol Chem 2003; 278: 779 20259-20267. 780 781 69 Pistocchi A, Fazio G, Cereda A, Ferrari L, Bettini LR, Messina G et al. Cornelia de 782 Lange Syndrome: NIPBL haploinsufficiency downregulates canonical Wnt pathway in 783 zebrafish embryos and patients fibroblasts. Cell Death Dis 2013; 4: e866. 784 785 70 MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, 786 mechanisms, and diseases. Dev Cell 2009; 17: 9-26. 787 788 71 Oginuma M, Harima Y, Tarazona OA, Diaz-Cuadros M, Michaut A, Ishitani T et al. 789 Intracellular pH controls WNT downstream of glycolysis in amniote embryos. Nature 790 2020. 791 792 72 Oginuma M, Moncuquet P, Xiong F, Karoly E, Chal J, Guevorkian K et al. A Gradient of 793 Glycolytic Activity Coordinates FGF and Wnt Signaling during Elongation of the Body 794 Axis in Amniote Embryos. Dev Cell 2017; 40: 342-353 e310. 795 796 73 Parks SK, Chiche J, Pouyssegur J. Disrupting proton dynamics and energy metabolism 797 for cancer therapy. Nat Rev Cancer 2013; 13: 611-623.

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798 799 74 Wang CW, Purkayastha A, Jones KT, Thaker SK, Banerjee U. In vivo genetic dissection 800 of tumor growth and the Warburg effect. Elife 2016; 5. 801 802 75 Cukrov D, Newman TAC, Leask M, Leeke B, Sarogni P, Patimo A et al. Antioxidant 803 treatment ameliorates phenotypic features of SMC1A-mutated Cornelia de Lange 804 syndrome in vitro and in vivo. Hum Mol Genet 2018; 27: 3002-3011. 805 806 76 Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using 807 the CRISPR-Cas9 system. Nat Protoc 2013; 8: 2281-2308. 808 809 77 Antony J, Dasgupta T, Rhodes JM, McEwan MV, Print CG, O’Sullivan JM et al. Cohesin 810 modulates transcription of estrogen-responsive genes. Biochimica et Biophysica Acta 811 (BBA) - Gene Regulatory Mechanisms 2015; 1849: 257-269. 812 813 78 Dasgupta T, Antony J, Braithwaite AW, Horsfield JA. HDAC8 Inhibition Blocks SMC3 814 Deacetylation and Delays Cell Cycle Progression without Affecting Cohesin- 815 dependent Transcription in MCF7 Cancer Cells. J Biol Chem 2016; 291: 12761-12770. 816 817 79 Clark NA, Hafner M, Kouril M, Williams EH, Muhlich JL, Pilarczyk M et al. 818 GRcalculator: an online tool for calculating and mining dose-response data. BMC 819 Cancer 2017; 17: 698. 820 821 80 Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for 822 assigning sequence reads to genomic features. Bioinformatics 2014; 30: 923-930. 823 824 81 Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion 825 for RNA-seq data with DESeq2. Genome Biology (journal article) 2014; 15: 550. 826 827 82 Yu G, He QY. ReactomePA: an R/Bioconductor package for reactome pathway 828 analysis and visualization. Mol Biosyst 2016; 12: 477-479. 829 830 83 Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological 831 themes among gene clusters. OMICS 2012; 16: 284-287. 832 833 84 Westerfield M. The Zebrafish Book. A guide for the laboratory use of zebrafish 834 (Brachydanio rerio). University of Oregon Press: Eugene, Oregon, 1995. 835 836 837 838 Figure legends 839 840 Figure 1. minor deficiencies in cell cycle progression, chromosome segregation and 841 nuclear morphology in cohesin-deficient isogenic cell lines. a Proliferation curves 842 (Incucyte) of MCF10A parental and cohesin-deficient clones (n = 3 independent experiments,

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843 mean ± sd, two-way ANOVA test: **p ≤ 0.01; ****p ≤ 0.0001). Doubling time in hours of 844 MCF10A parental cells are 15.5±0.1; RAD21+/-, 17.1±0.2; SMC3+/-, 21.4±0.9; STAG2-/-, 845 16.4±0.2 respectively. b Flow cytometry analysis of cell cycle progression. c Representative 846 metaphase spread images of cohesin-deficient cells. Black arrow indicates a normal 847 chromosome; Red arrow indicates premature chromatid separation (PCS). d Quantification of 848 chromosome cohesion defects. A minimum of 20 metaphase spreads were examined per 849 individual experiment. (n=2 independent experiments, mean ± s.d.). e Representative 850 confocal images of nuclear morphology. Scale bar, 15 μM. Yellow arrow indicates a 851 micronucleus. f Quantification of nuclear area. g Quantification of micronuclei (MN). A 852 minimum of 1000 cells was examined per individual experiment. (n = 3 independent 853 experiments, mean ± s.d., *p ≤ 0.05; ****p ≤ 0.0001). 854 855 Figure 2. Increased sensitivity to nucleolar stress and DNA damaging agents in cohesin- 856 deficient cells. a Representative image and b quantification of nucleolar dispersal observed 857 in MCF10A cells after exposure to a DNA damaging agent, Actinomycin D (ActD) 8 nM. 858 Fibrillarin (FBL) staining is used as a marker for nucleoli. c Representative image and d 859 quantification of DNA damage foci observed in MCF10A cells after exposure to ActD. 860 γH2AX staining is used to detect DNA double-strand breaks; arrows indicate γH2AX foci. e 861 Representative image and f quantification of nuclear p53 in MCF10A cells after exposure to 862 ActD. A minimum of 500 cells was examined per individual experiment. (n = 3 independent 863 experiments, mean ± s.d., *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.0005; ****p ≤ 0.0001). Scale bar, 864 15 μM 865 866 Figure 3. A synthetic lethal screen identifies common sensitivity of cohesin-deficient 867 cells to WNT activation and BET inhibition. a Schematic overview of the synthetic lethal 868 screen. b-d Overview of the differential area over the curve (AOC) activity of all compounds 869 tested in cohesin-deficient cell lines in the primary screen. A threshold of differential AOC ≥ 870 0.15 (red dashed lines) was used to filter candidate compounds of interest. e Venn diagram 871 showing the number of common and unique compounds that inhibited RAD21+/-, SMC3+/- 872 and STAG2-/- in the primary screen. f, g Dose-response curves of I-BET-762 and 873 LY2090314. 874 875 Figure 4. LY2090314-mediated WNT stimulation leads to β-catenin stabilization in 876 cohesin-deficient MCF10A cells. a Immunoblot of the membrane fraction of cohesin-

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877 deficient MCF10A cells shows increased basal level of β-catenin phosphorylation at 878 Ser33/37/Thr41. b Immunoblot of the cytoplasmic fraction shows increased level of both 879 total and phosphorylated β-catenin at Ser675 after MCF10A cells were treated with 880 LY2090314 at 100 nM for 24 hours. c Quantification of protein levels for total and 881 phosphorylated beta-catenin at Ser675. (n = 3 independent experiments, mean ± s.d., **p ≤ 882 0.01). d Immunofluorescence images show cytosolic accumulation of active β-catenin in 883 cohesin-deficient MCF10A cells treated with LY2090314 100 nM for 24 hours. White arrows 884 indicate puncta of β-catenin (Ser675). Scale bar = 25 μM. 885 886 Figure 5. Cohesin-STAG2 mutant CMK cells show increased sensitivity to Wnt 887 signaling. a Immunofluorescence images showing slightly increased nuclear accumulation of 888 β-catenin in STAG2-CMK cells (STAG2-/-) compared to parental (WT) following treatment 889 with LY2090314 at 100 nM for 24 hours. b Quantification of nuclear total β-catenin in 890 parental (WT) and STAG2-CMK cells (STAG2-/-) cells. Fluorescence of nuclear total β- 891 catenin was determined relative to the nuclear area. Graph depicts S.E.M. from analyses of 892 170-188 cell nuclei. c Histogram showing the number of genes upregulated or downregulated 893 at FDR ≤ 0.05 upon WNT3A treatment in WT and STAG2-/- CMK cells. d Overlap of genes 894 significantly upregulated or downregulated (FDR ≤ 0.05 ) upon WNT3A treatment between 895 parental and STAG2 mutant cells. e Top ten enriched pathways (ranked by gene count) from 896 the significantly downregulated and upregulated genes (FDR ≤ 0.05) following Wnt3a 897 treatment in STAG2-/- and parental (WT) CMK cells using the ClusterProfiler R package on 898 Gene Ontology Biological Process dataset modelling for both cell types (WT or STAG2-/-) 899 and regulation pattern (up- or downregulation). 900 901 Figure 6. Zebrafish rad21 cohesin mutants show increased sensitivity to Wnt ia5 902 stimulation. Wnt reporter Tg(7xTCF-Xla.Siam:nlsmCherry) control embryos and 903 Tg(7xTCF-Xla.Siam:nlsmCherry)ia5;rad21nz171 cohesin mutant embryos were treated with 904 0.15 M LiCl from 4 hpf to 20 hpf. a, a’ Brightfield/fluorescent and b confocal images of ia5 905 Tg(7xTCF-Xla.Siam:nlsmCherry) transgenic control embryos. c, c’ Brightfield/fluorescent 906 and d confocal Tg(7xTCF-Xla.Siam:nlsmCherry)ia5;rad21nz171 cohesin mutant embryos. Both 907 control and rad21nz171 genotypes show constitutive Wnt reporter activity in the midbrain at 908 20 hpf. Control brightfield/fluorescent (e, e’) and confocal (f) images compared with 909 rad21nz171 brightfield/fluorescent (g, g’) and confocal (h) images show that in the cohesin

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910 mutation background, Wnt reporter activity is enhanced in the midbrain (arrow) upon LiCl 911 treatment, relative to controls. mb, midbrain. Scale bar, 50 µm. For Tg(7xTCF- ia5 912 Xla.Siam:nlsmCherry) control embryos n=15/15; and for Tg(7xTCF- 913 Xla.Siam:nlsmCherry)ia5;rad21nz171 cohesin mutant, n=4/4 with the same expression pattern. 914 915 Supplementary Figure Legends 916 917 Supplementary Figure 1. Creation of MCF10A isogenic cell lines with cohesin gene 918 deletions. a Top, schematic diagram shows the deletion strategy for genes encoding cohesin 919 subunits RAD21, SMC3 and STAG2 using two sgRNAs targeting the 5’UTR and the 3’UTR 920 of each gene. Bottom, heterozygous clones were identified by PCR using specific primer 921 pairs flanking the deletion region. Representative DNA gel shows the PCR products yielded 922 using specific primer pairs for MCF10A parental and RAD21+/- deletion clone. M, ladder 923 marker. b-d Schematic deletion strategy and summary of the allele sequences for the STAG2 924 homozygous clone, and the RAD21 and SMC3 heterozygous clones. e RNA levels of the 925 targeted genes in MCF10A cohesin-deficient clones. f Representative immunoblot and g 926 quantification of cohesin protein levels. γ-tubulin was used as loading control (n = 3 927 independent experiments, mean ± s.d., **p ≤ 0.01; ****p ≤ 0.0001). 928 929 Supplementary Figure 2. Cohesin deficiency causes a modest increase in chromosome 930 mis-segregation events in MCF10A cells. a Frequency of aneuploidy in cohesin-deficient 931 MCF10A clones compared to wild type (WT). b Threshold compactness in the nuclei of 932 cohesin-deficient clones compared to wild type MCF10A (R.U. = relative units). c 933 Representative images of abnormal interphase nuclei showing chromosome bridges, 934 micronuclei, and lagging chromosomes at . d Quantification of chromosome bridges 935 and micronuclei events in MCF10A cohesin deficient cells. e Quantification of chromosome 936 mis-segregation observed in MCF10A cohesin deficient clones. At least 100 mitotic cells 937 were examined. (n = 3 independent experiments, mean ± s.d., **p ≤ 0.01; ***p ≤ 0.0005; ns, 938 not significant). 939 940 Supplementary Figure 3. Cell morphology of MCF10A parental and cohesin-deficient 941 cells grown at low and high confluence. MCF10A cells were grown in low (1000 942 cells/well) and high density (4000 cells/well) in 96-well plates. Cohesin-deficient cells

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943 display a cobblestone-like morphology which is similar to MCF10A parental cells. Scale bar, 944 250 μM. 945 946 Supplementary Figure 4. Cohesin deficient cells show altered nucleolar morphology. a 947 Representative images of nucleolar morphology as determined using fibrillarin (FBL) as a 948 marker of nucleoli. b, c Quantification of FBL intensity and number of FBL spots per cell in 949 each genotype. A minimum of 1000 cells was examined per individual experiment. (n = 3 950 independent experiments, mean ± s.d., *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.0005). d Types of 951 nucleolar morphology observed in MCF10A parental cells. e Representative images of 952 nucleolar morphology determined by using nucleolin (NCL) as a marker. f Quantification of 953 NCL intensity per cell in each genotype. At least 500 cells were examined per individual 954 experiment. n = 3 independent experiments, mean ± s.d., **p ≤ 0.01; ***p ≤ 0.0005). g 955 Quantification of NCL morphology classes present in cohesin deficient cells. A.U., Arbitrary 956 unit. 957 958 Supplementary Figure 5. Synthetic lethal screen controls. a Cohesin-deficient MCF10A 959 cells show differential sensitivity to the PARP inhibitor, Olaparib, after 48 hours of 960 treatment. b DMSO (negative control) and c Camptothecin (positive control) titrations were 961 used to establish a cytotoxicity range in MCF10A cells and cohesin-deleted derivatives (n = 2 962 independent experiments, mean ± s.d.). 963 964 Supplementary Figure 6. Categories of compounds that differentially inhibit cohesin 965 deficient cells. a Common and b, c, d unique categories of compounds identified in the 966 primary screen. e, f Dose-response curves were generated for validation of I-BET-762 and 967 LY2090314 in MCF10A cells. g Dose-response curve of LY2090314 in the K562 968 STAG2R614* mutant cell line. 969 970 Supplementary Figure 7. GSK3 levels are unaffected in cohesin-deficient MCF10A cells. 971 Anti-GSK3 immunoblot of membrane fraction from cohesin-deficient cells treated with 972 DMSO or 100 nM LY2090314. a Membrane fraction with DMSO and after 6 h LY2090314 973 treatment. b Cytoplasmic fraction with DMSO and after 6h LY2090314 treatment. 974 975 Supplementary Figure 8. WNT3A phenocopies LY2090314-mediated β-catenin 976 accumulation in the cytoplasm. Immunofluorescence images show that while cell in 0.5%

32 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218875; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

977 DMSO (b) have no β-catenin puncta, treatment with both a LY2090314 and c WNT3A leads 978 to formation of β-catenin puncta in cytoplasm (arrows). Scale bar = 25 µm. 979 980 Supplementary Figure 9. RNA sequencing profiling of cohesin-deficient MCF10A cells. 981 a Whole genome correlation matrix of MCF10A cells. The dendrogram shows a common 982 clustering of the biological replicates. There are two main clusters. The STAG2-/- (MT10_1- 983 3) transcriptomes are more similar to the WT (MWT_1-3). SMC3+/- (MS_1-3) and 984 RAD21+/- (MR50_1-3) populate the second cluster. b MCF10A gene expression profile of 985 genes involved in WNT signaling pathways according to Gene Ontology annotation 986 (GO:0016055). The heatmap shows the clustering of the scaled (z-score) normalized count 987 (log2RPKM) for each replicate. 988 989 Supplementary Figure 10. Enhanced sensitivity of cohesin-mutant CMK cells to Wnt 990 stimulation. a Sanger sequencing shows successful CRISPR-CAS9 editing introducing the 991 STAG2 R614* C>T mutation site into CMK cells. Reduced STAG2 mRNA (b) and protein 992 (c) in STAG2 mutant CMK cells. d Heatmap showing log2 RPKM values of 404 genes 993 upregulated only in STAG2 mutant cells (FDR ≤ 0.05) following 4 hours of WNT3A 994 treatment. e GSEA analyses of the 404 upregulated genes. f Heatmap showing log2 RPKM 995 values of 171 genes downregulated only in STAG2 mutant cells (FDR ≤ 0.05) following 4 996 hours of WNT3A treatment. g GSEA analyses of the 171 genes significantly downregulated 997 only in the STAG2 mutant line. 998 999 1000

33 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218875; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

5’UTR a 3’UTR b sgRNA STAG2 Allele 1 5’UTR 3’UTR WT sgRNA Allele 2

P1 P4

5’UTR TTTCCCCAATGGATCTATGAAGG TCCCGCGGTTCTAGAGCGGTGGG 5’UTR sgRNA PAM Heterozygous Allele 1 sgRNA PAM deletion Allele 2 P1 P2 P3 P4 460 kbp deletion

bp M Heterozygous WT M 5’UTR 3’UTR P3/P4 P1/P2 P1/P4 P3/P4 P1/P2 P1/P4 5’UTR

650 500 5’UTR 400 300 200 TTTCCCCAATGGATCTATCCCGCGGTTCTAGAGC

RAD21 SMC3 c d 5’UTR 3’UTR 5’UTR 3’UTR

CGTAAACATCCTGCCGATTTGAACCGAGGATTTGG GGTCTCCTTCCAGGCCAGGGGCCCGGAATC sgRNA PAM sgRNA PAM TGTAAACCAAGGAGTTTTCCCCGTTTGTAAAAACAG ATATAGTAATATGATTCTCATACCCAGGAACTG PAM sgRNA sgRNA PAM

29 kbp deletion 37 kbp deletion

5’UTR5’UTR 3’UTR 5’UTR5’UTR 3’UTR

P2R

5’UTR 5’UTR

ATCCTGCCGATTTGAAGTTTTCCCCGTTTGTA TTCCAGGCCAGGGCCAGGAACTG

Clone 1 Clone 1 Primer P1 and P2 Primer P1 and P2 Allele 1 Allele 1

Clone 1 Clone 1

Primer P1 and P4 +/- +/- -/- Primer P1 and P4 WT WT WT SMC3 STAG2 Allele 3 Allele 2 RAD21

1.5 Primers Clone 1 reference TGCCGATTTGAAGCCGAG Primers 1.0 Clone 1 P1F/P1R Allele 1 TGCCGATTTGAAG - - GAG reference AGGGGCCCGGAATCATG P1F/P1R 0.5 Allele 2 ? Allele 1 AGGGGCCCGGAATCATG Normalized signal reference ATCCTGCCGATTTGAA ***************GTTTTCCCCGTTTGTA reference TTCCAGGCCAGGG******************************CCAGGAACTG P1F/P2R P1F/P2R 0.0 +/- Allele 3 ATCCTGCCGATTTGAAGTTTTCCCCGTTTGTA Allele 2 TTCCAGGCCAGGGCCAGGAACTG -/- WT +/- WT WT RAD21 RNA SMC3 STAG2 e RAD21 SMC3 STAG2 f 1.5 1.5 1.5

1.0 1.0 1.0 WT RAD21+/-SMC3+/-STAG2-/- ** **** 0.5 0.5 0.5 anti-RAD21 ** Relative expression Relative expression Relative expression 0.0 0.0 0.0 anti-SMC3

WT WT WT

RAD21+/- SMC3+/- STAG2-/- anti-STAG2 Protein g 1.5 RAD21 SMC3 STAG2 anti-γ-Tubulin 1.5

1.0 1.0 1.0

0.5 0.5 0.5 Normalized signal Normalized signal Normalized signal

0.0 0.0 0.0

WT WT WT RAD21+/- SMC3+/- STAG2-/-

Supplementary Figure 1. Creation of MCF10A isogenic cell lines with cohesin gene deletions. a Top, schematic diagram shows the deletion strategy for genes encoding cohesin subunits RAD21, SMC3 and STAG2 using two sgRNAs targeting the 5’UTR and the 3’UTR of each gene. Bottom, heterozygous clones were identified by PCR using specific primer pairs flanking the deletion region. Representative DNA gel shows the PCR products yielded using specific primer pairs for MCF10A parental and RAD21+/- deletion clone. M, ladder marker. b-d Schematic deletion strategy and summary of the allele sequences for the STAG2 homozygous clone, and the RAD21 and SMC3 heterozygous clones. e RNA levels of the targeted genes in MCF10A cohesin-deficient clones. f Representative immunoblot and g quantification of cohesin protein levels. γ-tubulin was used as loading control (n = 3 independent experiments, mean ± s.d., **p ≤ 0.01; ****p ≤ 0.0001). bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218875; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a b 100 0.85 *** n < 46 ss (R.U) ne ss 0.80 n < 47 50 n = 47 n = 48 0.75 cy (%) equen cy n > 48 Fr 0.70

0 Threshold compact 0.65

WT WT

RAD21+/-SMC3+/-STAG2-/- RAD21+/-SMC3+/-STAG2-/-

c Chromosome Lagging bridge Micronucleus chromosomes

d e

50 25 *** *** 40 20 ** ** 30 15

ns 20 10 ns 10 5

0 0 Abnormal anaphase nuclei (%) Chromosome mis-segregation (%) mis-segregation Chromosome WT WT

SMC3+/-STAG2-/- RAD21+/- RAD21+/-SMC3+/-STAG2-/-

Supplementary Figure 2. Cohesin deficiency causes a modest increase in chromosome mis-segregation events in MCF10A cells. a Frequency of aneuploidy in cohesin-deficient MCF10A clones compared to wild type (WT). b Threshold compactness in the nuclei of cohesin-deficient clones compared to wild type MCF10A (R.U. = relative units). c Representative images of abnormal interphase nuclei showing chromo- some bridges, micronuclei, and lagging chromosomes at anaphase. d Quantification of chromosome bridges and micronuclei events in MCF10A cohesin deficient cells. e Quantification of chromosome mis-segregation observed in MCF10A cohesin deficient clones. At least 100 mitotic cells were examined. (n = 3 independent experiments, mean ± s.d., **p ≤ 0.01; ***p ≤ 0.0005; ns, not significant). bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218875; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

WT RAD21+/- SMC3+/- STAG2-/- Low

Confluence High

Supplementary Figure 3. Cell morphology of MCF10A parental and cohesin-deficient cells grown at low and high confluence. MCF10A cells were grown in low (1000 cells/well) and high density (4000 cells/well) in 96-well plates. Cohesin-deficient cells display a cobblestone-like morphology which is similar to MCF10A parental cells. Scale bar, 250 μM. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218875; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a b c WT RAD21+/- SMC3+/- STAG2-/- 10000 4

** **

*** FBL 5000 2 No. FBL spots per cell 0 0 FBL intensity per cell (A.U.)

WT 1+/- 2-/- 1+/- 2-/- 2 G WT 2 G D A D A

A A

DAPI / FBL SMC3+/- R ST R SMC3+/-ST

d e WT RAD21+/- SMC3+/- STAG2-/-

Normal Ring NCL

Dispersed No or Dim

DAPI / NCL

f g

2500 1.0 Dissolved 2000 ** Ring ll (A.U.) 1500 *** opu lation Normal ub p

pe r ce 0.5 No or Dim 1000

500 Fraction of s 0.0 NCL intensity

1+/- 2-/- WT 1+/- 2-/- WT 2 G 2 G D A D A A A SMC3+/- R SMC3+/-ST R ST

Supplementary Figure 4. Cohesin deficient cells show altered nucleolar morphology. a Representative images of nucleolar morphology as determined using fibrillarin (FBL) as a marker of nucleoli. b, c Quantifica- tion of FBL intensity and number of FBL spots per cell in each genotype. A minimum of 1000 cells was exam- ined per individual experiment. (n = 3 independent experiments, mean ± s.d., *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.0005). d Types of nucleolar morphology observed in MCF10A parental cells. e Representative images of nucleolar morphology determined by using nucleolin (NCL) as a marker. f Quantification of NCL intensity per cell in each genotype. At least 500 cells were examined per individual experiment. n = 3 independent experi- ments, mean ± s.d., **p ≤ 0.01; ***p ≤ 0.0005). g Quantification of NCL morphology classes present in cohesin deficient cells. A.U., Arbitrary unit. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218875; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a b c Olaparib 1 μM 1.5 * 1.5 1.0 *

WT 1.0 1.0 RAD21+/- 0.5 SMC3+/- 0.5 0.5 STAG2-/- Relative viability Relative viability Relative viability

0 0 0

5 5 1 5 5 5 WT 0 50 0. 2. 500 000 0. 5000 50

RAD21+/-SMC3+/-STAG2-/- DMSO concentration (%) Campthothecin concentration (nM)

Supplementary Figure 5. Synthetic lethal screen controls. a Cohesin-deficient MCF10A cells show differen- tial sensitivity to the PARP inhibitor, Olaparib, after 48 hours of treatment. b DMSO (negative control) and c Camptothecin (positive control) titrations were used to establish a cytotoxicity range in MCF10A cells and cohesin-deleted derivatives (n = 2 independent experiments, mean ± s.d.). bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218875; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a Common hits b RAD21+/- c SMC3+/- d STAG2-/- 4 4 4 10

3 3 3

2 2 2 5 equen cy equen cy equen cy equen cy Fr Fr Fr 1 Fr 1 1

0 0 0 0 s le s g in is rs s le ge le gy ge sis ng ge n gy s PK -kB e PK gy a yc o a nes PK lism ing yc i asea TAT neste lismlo tionA yc A lo ase l o A o TAT in m o o o o a o in m o m M b /S Others/mTOR eneticK a /S Pr b M NF mTOR M a enetic a Others g D ta enetic/ K D ig D ta ogene T i e G e ogene ig T e Cell C crobi e i Cell C K n A JAK icrobii K Cell C n A i A M g JAK Ep si al Signali M g flamm icrobi Ep M al Signall al Signallo t M Ep M si DN DN An n K/An DN le An In K/A o y & Horm o 3 ro Tyr gy & Horm 3 g r PI y & Tyr in GPCR & g ane Transport PI e toske o in nolo Neu Neu inolo l e Cy Prot ocr ocri d Prot d En En Immuno ansmembr Tr

e f g WT WT 1.0 1.0 RAD21+/- RAD21+/- 1.0 SMC3+/- SMC3+/- WT

GR value 0.5 GR value 0.5 STAG2-/- STAG2-/- 0.5 STAG2-/- **** Normalized viability

0.0 0.0 0.0 01234 01234 0.0 0.5 1.0 1.5 2.0 log[I-BET-762],M log[LY2090314],M log[LY2090314],M

Supplementary Figure 6. Categories of compounds that differentially inhibit cohesin deficient cells. a Common and b, c, d unique categories of compounds identified in the primary screen. e, f Dose-response curves were generated for validation of I-BET-762 and LY2090314 in MCF10A cells. g Dose-response curve of LY2090314 in the K562 STAG2R614* mutant cell line. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218875; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a Membrane fraction

DMSO LY2090314 100 nM 6 hr

WT RAD21+/- SMC3+/- STAG2-/- WT RAD21+/- SMC3+/- STAG2-/-

GSK3 α/β

b Cytoplasmic fraction DMSO LY2090314 100 nM 24 hr

WT RAD21+/- SMC3+/- STAG2-/- WT RAD21+/- SMC3+/- STAG2-/-

GSK3 α/β

Supplementary Figure 7. GSK3 levels are unaffected in cohesin-deficient MCF10A cells. Anti-GSK3 immunoblot of membrane fraction from cohesin-deficient cells treated with DMSO or 100 nM LY2090314. a Membrane fraction with DMSO and after 6 h LY2090314 treatment. b Cytoplasmic fraction with DMSO and after 6h LY2090314 treatment. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218875; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a LY2090314 100 nM

WT RAD21+/- SMC3+/- STAG2-/- Total β-catenin

DAPI

Merge

DMSO b WT RAD21+/- SMC3+/- STAG2-/- β-catenin pSer675

DAPI

Merge

c WNT3A 200 ng/mL

WT RAD21+/- SMC3+/- STAG2-/- Total β-catenin

DAPI

Merge

Supplementary Figure 8. WNT3A phenocopies LY2090314-mediated β-catenin accu- mulation in the cytoplasm. Immunofluorescence images show that while cell in 0.5% DMSO (b) have no β-catenin puncta, treatment with both a LY2090314 and c WNT3A leads to formation of β-catenin puncta in cytoplasm (arrows). Scale bar = 25 µm. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218875; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a All transcripts

60 RAD21+/- 50 SMC3+/- STAG2-/- 40 WT 30

20

10

0 MS1_3 MS1_1 MS1_2 MR50_2 MR50_1 MR50_3 MT10_3 MT10_1 MT10_2 MWT_3 MWT_1 MWT_2

b Wnt signaling pathway

RAD21+/- SMC3+/- STAG2-/- WT MS1_3 MS1_1 MS1_2 MR50_2 MR50_1 MR50_3 MWT_3 MWT_1 MWT_2 MT10_3 MT10_1 MT10_2

Supplementary Figure 9. RNA sequencing profiling of cohesin-deficient MCF10A cells. a Whole genome correlation matrix of MCF10A cells. The dendrogram shows a common clustering of the biological replicates. There are two main clusters. The STAG2-/- (MT10_1-3) transcriptomes are more similar to the WT (MWT_1-3). SMC3+/- (MS_1-3) and RAD21+/- (MR50_1-3) populate the second cluster. b MCF10A gene expression profile of genes involved in WNT signaling pathways according to Gene Ontology annotation (GO:0016055). The heatmap shows the clustering of the scaled (z-score) normalized count (log2RPKM) for each replicate. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218875; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a d e WT STAG2-/- WT Genes in Key 0 hr 4 hr 0 hr 4 hr Pathway FDR the overlap 2 CTTTGT LEF1 Q2 0.0000 42 1 1 TCF7 TARGET GENES 0.0000 39 0 REACTOME RNA POLYMERASE II TRANSCRIPTION 0.0003 26 −1 KEGG WNT SIGNALING PATHWAY 0.0003 9 GGATGCCTTATTGCGACAGATCCGGAATATT −2 REACTOME METABOLISM OF LIPIDS 0.0003 18 REACTOME CELL CYCLE 0.0003 17 JAG2 C>T R614* SEM3G REACTOME MEMBRANE TRAFFICKING 0.0017 15 STAG2-/- STYK1 REACTOME DOWNREGULATION OF SMAD2 3 SMAD4 EPHA4 2 SRMS TRANSCRIPTIONAL ACTIVITY 0.0020 4 EP300 REACTOME TRANSCRIPTIONAL REGULATION OF WHITE SMAD3 KLF2 ADIPOCYTE DIFFERENTIATION 0.0021 6 SP2 SP7 REACTOME ORGANELLE BIOGENESIS AND MAINTENANCE 0.0021 10 PID NOTCH PATHWAY 0.0034 5 REACTOME FOXO MEDIATED TRANSCRIPTION 0.0049 5 GGATGCCTTATTGTGACAGATCCGGAATATT

b 3 f g STAG2-/- WT Key 0 hr 4 hr 0 hr 4 hr Genes 2 2 Pathway FDR in the A RN A 1 overlap m 0 REACTOME METABOLISM OF LIPIDS 0.0018 15 −1 1 1 REACTOME PHOSPHOLIPID METABOLISM 0.0290 7 −2 KEGG AMINO SUGAR AND NUCLEOTIDE SUGAR

STAG2 METABOLISM 0.0290 4 0 KEGG FC GAMMA R MEDIATED PHAGOCYTOSIS 0.0320 5 REACTOME REGULATION OF TP53 ACTIVITY THROUGH WT STAG2 -/- METHYLATION 0.0320 3

c WT STAG2-/- M 2

STAG2

Υ tubulin

Supplementary Figure 10. Enhanced sensitivity of cohesin-mutant CMK cells to Wnt stimulation. a Sanger sequencing shows successful CRISPR-CAS9 editing introducing the STAG2 R614* C>T mutation site into CMK cells. Reduced STAG2 mRNA (b) and protein (c) in STAG2 mutant CMK cells. d Heatmap showing log2 RPKM values of 404 genes upregulated only in STAG2 mutant cells (FDR ≤ 0.05) following 4 hours of WNT3A treatment. e GSEA analyses of the 404 upregulated genes. f Heatmap showing log2 RPKM values of 171 genes downregulated only in STAG2 mutant cells (FDR ≤ 0.05) following 4 hours of WNT3A treatment. g GSEA analyses of the 171 genes significantly downregulated only in the STAG2 mutant line. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218875; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Target gene Position sgRNA sequence (5’-3’) On-target activity (%) RAD21 5’UTR CATCCTGCCGATTTGAACCGAGG 92 RAD21 3’UTR TACAAACGGGGAAAACTCCTTGG 82 SMC3 5’UTR TGTACATGATTCCGGGCCCCTGG 94 SMC3 3’UTR GTAATATGATTCTCATACCCAGG 85 STAG2 5’UTR TTTCCCCAATGGATCTATGAAGG 87 STAG2 3’UTR TCCCGCGGTTCTAGAGCGGTGGG 96

Supplementary Table 3. sgRNA sequences targeting each of the cohesin subunit genes. The PAM sequences are highlighted in red.

Target Target sites Primer Primer sequence Expected gene product (bp) RAD21 5’UTR Forward ACAGCAGAGATGGCCAAAGAC Reverse TCTCACTGGAAGAGACTATGACAA 432

3’UTR Forward TTTTGTTTGGCTGAGGGGAGC Reverse CACACCTCTCGGAGATCCTG 401 5’UTR/3’UTR Forward TTTTGTTTGGCTGAGGGGAGC Reverse TCTCACTGGAAGAGACTATGACAA 680

SMC3 5’UTR Forward TGGCTAACACCGCAACTCTC Reverse CCTTGCGAGGTGTAGCTTCC 327

3’UTR Forward GCCCTGCCTACTACAAGGAC Reverse GGGACCACCACCAACTGAAT 650 5’UTR/3’UTR Forward TGGCTAACACCGCAACTCTC Reverse GGGACCACCACCAACTGAAT 450

STAG2 5’UTR Forward GGTGCCCCTCCAAACTTCT Reverse CCCAAACAATCCCGAAGC 465

3’UTR Forward CTTTGCAAGCAGTCTTCAGGT Reverse TTTTTGGCTACCACAGCCCA 423 5’UTR/3’UTR Forward GGTGCCCCTCCAAACTTCT Reverse TTTTTGGCTACCACAGCCCA 850

Supplementary Table 4. Primer sequences used in PCR assay to screen for cells with heterozygous deletion of RAD21 or SMC3 and homozygous deletion of STAG2.

bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218875; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Target gene Primer (Forward) Primer (Reverse) Cyclophilin ACGGCGAGCCCTTGG TTTCTGCTGTCTTTGGGACCT GAPDH TGCACCACCAACTGCTTAGC GGCATGGACTGTGGTCATGAG RAD21 CAATGCCAACCATGACTCAT CGGTGTAAGACAGCGTGTAAA SMC3 GTTTCAACCCAGCTGGCCCGTG CGATGGCTGACTTGGTCACATTCCA STAG2 ACGGAAAGTGGTTGAGGG GTGGAGGTGAGTTGTGGTGT

Supplementary Table 5. Primer sequences used in RT-qPCR.