A ARID1A Expression in Ewing Sarcoma Patient Tumor Samples

Oncomine Khan Ohali Henderson Baird Ferreira Schaefer Postel-Vinay Data set (Nat Med (Oncogne (Genome (Cancer Res (Oncogene (Eur J Cancer (Nat Genet – (Publication) – 2001) – 2004) Biol - 2005) – 2005) – 2008) – 2008) 2012) ARID1A (21/26) (20/20) (5/5) (17/19) (14/14) (35/35) (117/117) Expression

B C D

E F G

HEK-293 A4573 TC32 +ARID1A-L +ARID1A-S WT

ARID1A-L 250 ARID1A-S

Actin

Supplementary Figure S1. Oncomine data sets for ARID1A expression in ES patients samples and cell lines. (A) ARID1A expression summary from the published Oncomine database. Publications are shown on the top, number of patients in the parenthesis and the arrow indicates up regulation of ARID1A expression. (B-D) Examples of gene expression data of ARID1A in ES patient samples from Ohali et al., 2004 (B); Schaefer et al., 2008 (C); Postel-Vinay et al., 2012 (D). (E) mRNA expression of ARID1A in a large panel of human cancer cell lines across multiple tissue types. Data was acquired from the Cancer Cell Line Encyclopedia. (F) ARID1A mRNA expression levels of 8 ES cell lines compared with that of hMSC+/- EF using qRT-PCR. Relative expression values are normalized to hMSC wild-type. (G) Western blot showing the ARID1A isoform expression in 2 ES cell lines compared with the exogenous plasmid cDNA expression of ARID1A isoforms in HEK-293 cells to establish size standards. Actin was used as a loading control. A B STA- STA-ET 7.2 SKES RDES 1.2 A4573 1.2 TC71 1.2 1.2 1.2 A4573 TC71 ET 7.2 SKES RDES 1.0 1.0 1.0 1.0 1.0 0.8 0.8 0.8 0.8 0.8 MW (bp) + - + - + - + - + - EWS-FLI1 0.6 0.6 0.6 0.6 0.6 1000 0.4 0.4 0.4 0.4 0.4 700 17 18

of EWS-FLI1 0.2 0.2 0.2 0.2 0.2 0.0 0.0 0.0 0.0 0.0 500

Relative Expression 400 300 Scrambled Scrambled Scrambled Scrambled Scrambled shEWS-FLI1 ShEWS-FLI1 ShEWS-FLI1 ShEWS-FLI1 shEWS-FLI1 200 EWS-FLI1 17 18

75 Actin PSI 69 18 92 00 86 09 91 93 93 39 C D E F G TC32 1.2 TC32 1.0 TC32 hMSC HEK 293 MW (bp) MW (bp) MW (bp) 0.8 MW (bp) + - EWSR1 - + Doxorubicin - + YK-4-279 - + YK-4-279 0.6 1000 1000 1000 1000 0.4 17 18 17 18 17 18 17 18 0.2 700 700 700 700 of EWSR1 0.0 500 500 500 500 400 400 400 400 Relative Expression 300 300 300 300 shEWSR1 Scrambled EWSR1 200 200 200 200 17 18 17 18 17 18 17 18

75 75 75 Actin 75 PSI 92 94 PSI 91 93 PSI 28 24 PSI 54 53

H I J +CHX shEWS-FLI1 e e

c TC32 c

n 20 1.2 n MW (bp) - + CHX 1.0 1000 15 17 18 bund a 0.8 700 bund a a 10 a 500 0.6 400 RNA

RNA 0.4 m 5 300 m

ve 0.2 i ve t

i 200 t a

l 0 a

l 0.0 e 17 18 e R FLI1 R FLI1 75 GAS5 APDH GAS5 APDH ANXA1G ARID1A ANXA1 SRSF2(+) G ARID1A EWS- SRSF2(+) EWS- PSI 87 89

Supplementary Figure S2. ARID1A is alternatively spliced in ES and evaluation of ARID1A transcripts for nonsense-mediated decay (NMD). (A) EWS-FLI1 levels are reduced using shRNA in ES cell lines A4573, TC71, STA-ET 7.2, SKES and RDES validations. Each of these models was confirmed by qRT-PCR showing the reduction (blue), scrambled shRNA transfected (red) of EWS-FLI1 mRNA shown on the top. Bottom panels show the immunoblots confirming the reduction of EWS-FLI1 protein. Actin was used as a loading control. (B) We measured both short and long isoforms, of ARID1A using semi-quantitative RT-PCR analysis followed by gel densitometry. The ARID1A isoform switch occurs in five ES cell lines based on the presence and absence of EWS-FLI1. Isoforms were quantified by gel densitometry to measure the percent of spliced-in (PSI) for the long exon 18 (ARID1A-L) and are reported below each lane. (C) Semi-quantitative RT-PCR (top) and immunoblot (bottom) confirm the reduction of wild-type EWSR1 in TC32 cells with shRNA. (D) ARID1A isoform levels after shEWSR1 reduction in TC32 cells. Assay and quantification as in B. (E) ARID1A isoform levels after doxorubicin treatment in TC32 cells. Assay and quantification as in B. (F, G) ARID1A isoform levels after YK-4-279 treatment in hMSC cells (F) and HEK 293 cells (G). Assay and quantification as in B. (H) RT-qPCR showing the relative expression of NMD substrates in TC32 cells were treated with 100 µg/ml cycloheximide (CHX) for 4 hrs prior to cell harvest. (I) RT-qPCR showing the relative expression of NMD substrates in TC32 cells with reduced EWS-FLI1. (J) ARID1A isoform levels after CHX treatment in TC32 cells. Assay and quantification as in B. Interpreted overlaid DIC Exon 18 L Exon 20 on DAPI

Lymph

Ovary

Thymus

Tonsil

Uterus

Supplementary Figure S3. Single Molecule FISH imaging of ARID1A isoforms in normal tissues. A frozen tissue array containing lymph node, ovary, thymus, tonsil and uterus was hybridized with exon 18 probes (Fig. 1A). The first panel is differential interference contrast (DIC), the second and third panels are a merge of z stacks from raw images from the independent probes 18L and 20, respectively, the fourth panel is an overlay of DAPI staining with merge of both channel images. A signal from the exon 18L probe is red, exon 20 is green. ARID1A-L RNA signal is identified as yellow and ARID1A-S is identified as green. The quantification is found in Figure 1H. Scale bar is 5µm. A B 1.2

alu e 1.0

0.8 ARID1A-L 0.6 ARID1A-S shScrambled shARID1A shARID1A + shARID1A +

0.4 ARID1A-L 250 ARID1A-S 0.2

Relative Expression V 0.0 50 Actin

shARID1A Sh Scramble

shARID1A+ARID1A-LshARID1A+ARID1A-S C 8

6 shScrambled 4 shARID1A * * shARID1A+ARID1A-L 2 shARID1A+ARID1A-S Cell Inde x

0 10 20 30 40 50 60 70 -2 Time (h)

D E 250 * 200 * ARID1A-L ARID1A-S 150 shScrambled shARID1A shARID1A + shARID1A +

100

No. of Colonie s 50

0

shARID1A shScrambled

shARID1A+ARID1A-LshARID1A+ARID1A-S

Supplementary Figure S4. ARID1A-L is necessary for ES oncogenesis in A4573 cells. Total ARID1A was reduced using shARID1A in A4573 cells followed by exogenously expressed ARID1A-L or ARID1A-S. (A) ARID1A isoform expression using specific primers to evaluate mRNA. (B) ARID1A protein levels after either shScrambled control or shARID1A followed by individual isoform exogenous expression (ARID1A-L or ARID1A-S). (C) Cell proliferation assay using xCELLigence system using electric impedence as a measure of ES cancer cell proliferation in various conditions as in (A, B). Data were collected from three independent experiments. *Indicates significantly different comparisons (p < 0.05, Two-way ANOVA). (D) Anchorage-independent growth assays showing colony formations for same conditions as in (A, B). (E) Enumeration of anchorage-independent colonies by reporting the average number of colonies per well (3 independent experiments, *p < 0.05). A B C D 1.2 A4573 1.2 TC32 alu e alu e 1.0 1.0 0.8 0.8 0.6 0.6 ARID1A-L ARID1A-L ARID1A-S ARID1A-S shScrambled shARID1A shARID1A + shARID1A + shScrambled shARID1A shARID1A + shARID1A + 0.4 0.4 0.2 0.2 ARID1A-L ARID1A-L 250 ARID1A-S 250 ARID1A-S 0.0 0.0 Relative Expression V Relative Expression V 50 50 Actin Actin shARID1A shARID1A Sh Scramble Sh Scramble A4573 TC32 shARID1A+ARID1A-LshARID1A+ARID1A-S shARID1A+ARID1A-LshARID1A+ARID1A-S E F 8 5 A4573 TC32

6 4 shScrambled 3 shScrambled 4 shARID1A * shARID1A * 2 * * 2 shARID1A+ARID1A-L shARID1A+ARID1A-L Cell Inde x Cell Inde x 1 shARID1A+ARID1A-S shARID1A+ARID1A-S 0 0 10 20 30 40 50 60 70 10 20 30 40 50 60 70 -2 Time (h) -1 Time (h) G H I J A4573 TC32 * 150 * 100 * 125 * 80 ARID1A-L ARID1A-L ARID1A-S ARID1A-S 100 shScrambled shARID1A shARID1A + shARID1A + shScrambled shARID1A shARID1A + shARID1A + 60 75 40 50

25 20 No. of Colonie s No. of Colonie s 0 0

shARID1A shARID1A shScrambled shScrambled

shARID1A+ARID1A-LshARID1A+ARID1A-S shARID1A+ARID1A-LshARID1A+ARID1A-S A4573 TC32 Supplementary Figure S5. ARID1A-L is necessary for ES oncogenesis through confirmation using alternative shRNA. To confirm the data in Figure 2 and Supplementary Figure 4, a different shARID1A targeting the 3’UTR region was used in both A4573 and TC32 cells. This was followed by exogenously expressed ARID1A-L or ARID1A-S. (A, B) ARID1A isoform expression using specific primers to evaluate mRNA in A4573 and TC32, respectively. (C, D) ARID1A protein levels after either shScrambled control or shARID1A followed by individual isoform exogenous expression (ARID1A-L or ARID1A-S) in A4573 and TC32, respectively. (E, F) Cell proliferation assay using xCELLigence system using electric impedence as a measure of ES cancer cell proliferation in various conditions as in (A, B), and data were collected from three independent experiments, in A4573 and TC32, respectively. *Indicates significantly different comparisons (p < 0.05, Two-way ANOVA). (G, H) Anchorage-independent growth assays showing colony formations for same conditions as in (A, B) in A4573 and TC32, respectively. (I, J) Enumeration of anchorage-independent colonies (3 independent experiments, *p < 0.05). A B EV +ARID1A-S EV +ARID1A-S

250 ARID1A-S 250 ARID1A-S

75 75 EWS-FLI1 EWS-FLI1

50 50 Actin Actin

A4573 TC32 C D ysate ) ysate ) A4573 TC32 2500 2500 Activit y Activit y 2000 2000

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1000 1000

500 500 Relative Caspase-3 Relative Caspase-3 0 0

EV EV (Fluorescence Units / ug Protein L (Fluorescence Units / ug Protein L

ARID1A-L ARID1A-S ARID1A-L ARID1A-S + + + + E F

6 A4573 TC32 6 EV 5 5 * +ARID1A-S x 4 x 4 3 3 ll I nd e 2 EV ll I nd e 2 e * e C 1 +ARID1A-S C 1 0 0 15 30 45 60 75 15 30 45 60 75 -1 -1 Time (In Hour) Time (In Hour)

G EWS-FLI1 1.5 e u a l V 1.0 ve i on a t e l ess i

R 0.5 r x p E 0.0

EV D1A-L

+ARI +ARID1A-L shEWS-FLI1 +shEWS-FLI1 Supplementary Figure S6. ARID1A-S is toxic to ES cell growth. (A, B) Exogenous expression of ARID1A-S followed by ARID1A immunoblotting of A4573 and TC32. (C, D) Caspase-3 assay as a measure of apoptosis evaluated the effect of exogenous expression of ARID1A-L or ARID1A-S. Cells were transfected for 18 hours followed by protein extraction and ten µg were incubated with the caspase-3 substrate Ac-DEVD-AMC for 4 hrs. Relative fluorescence units of caspase activation of three independent experiments are shown in A4573 (C) and TC32 (D). (E, F) Real-time cell proliferation assay using xCELLigence system showing cell proliferation in control empty vector (EV) and ARID1A-S using the same cells as in (A, B). (G) qRT-PCR for EWS-FLI1 expression as in Figure 4C. The color code is the same as Figure 4C; red is empty vector, green is stable exogenous ARID1A-L, blue is EV + shEWS-FLI1, and brown is ARID1A-L expression followed by shEWS-FLI1. A B C TC32 TC32 Jurkat

I P

I P I P TN L BRG1 I P IgG TN L EWSR1 I P IgG TN L EWSR1 I P IgG 250 ARID1A (BAF250a) ARID1A ARID1A 250 250 250 BRG1 (SMARCA4) 250 BRG1 250 BRG1

150 BAF170 (SMARCC2) 150 BAF155 150 BAF155 150 BAF155 (SMARCC1) 50 50 BAF53 BAF53

50 BAF47 (SMARCB1) 50 50 BAF47 BAF47 75 EWS-FLI1 EWSR1 EWSR1 75 75

D TC32 (Exp. 1) TC32 (Exp. 2) DMSO YK-4-279 DMSO YK-4-279

I P I P

I P I P TN L FLI1 I P IgG TN L FLI1 I P IgG TN L FLI1 I P IgG TN L FLI1 I P IgG ARID1A ARID1A 250 250 250 BRG1 250 BRG1

150 BAF155 150 BAF155

50 BAF53 50 BAF53 50 50 BAF47 BAF47 75 75 EWS-FLI1 EWS-FLI1

E F TC32 Jurkat DMSO YK-4-279 DMSO YK-4-279

I P I P I P I P TN L TN L BRG1 I P IgG TN L BRG1 I P IgG TN L BRG1 I P IgG BRG1 I P IgG

ARID1A ARID1A 250 250

250 BRG1 250 BRG1

150 BAF155

50 BAF53 150 BAF155

50 BAF47 50 BAF53

75 50 EWS-FLI1 BAF47

Supplementary Figure S7. Evaluation of multiple BAF complexes and their dissociation from EWS-FLI1 following treatment with YK-4-279. (A) BAF complexes associated with EWS-FLI1 were demonstrated using immunoprecipitation (IP) with BRG1 (SMARCA4). Antibodies used for the immuno blots are indicated on the right. 10% of total nuclear lysate (TNL) was used as input (lane 1), BRG1 antibody pull downs showing the presence of proteins as a part of the complex with EWS-FLI1 (lane 2). Immunoglobulin control precipitations (lane 3) lack meaningful protein associations. (B, C) An EWSR1 C-terminal antibody was used for co-IP in TC32 ES cells (B) and in Jurkat leukemia cells (C). Antibodies used for the immuno blots are indicated on the right. (D) TC32 cells were treated with 3 µM YK-4-279 for 16 hrs followed by precipitation with a FLI1 antibody (two independent experiments shown). (E, F) TC32 ES cells (E) and Jurkat leukemia cells (F) were treated with 3 µM YK-4-279 for 16 hrs and followed by BRG1 precipitation. A B C

0.20 1.4 0.5 1.2

m 0.4 m n

1.0 n m 0.15 n 0.8 0.3 450 450 . . 450 0.10 0.6

. 0.2 O. D

0.4 O. D O. D 0.05 0.2 0.1 0.0 0.0 0.00 Block + + - - - - - ARID1A #FL - + - - ARID1A #FL - - + - + - - + EWS-FLI1 - - + + ARID1A #FS - - - + - + - DMSO - + - - - ARID1A #FL + - + - EWSR1 - + - - + + + YK-4-279 - - 3 10 30 ARID1A #FS - + - + -DDK + + + + + + + EWS-FLI1 + + + + +

Supplementary Figure S8. EWS-FLI1 directly binds to ARID1A-L. ARID1A fragments were synthesized using a coupled in vitro transcription/translation (IVTT) system (as shown in Figure 6B). (A) For direct protein-protein interaction, purified His-tagged recombinant EWS-FLI1 was coated in ELISA wells followed by ARID1A fragments. Detection with ARID1A antibody that recognizes both ARID1A-#FL and ARID1A-#FS. (B) ELISA plates were coated with ARID1A fragments and followed by DDK-tagged recombinant EWSR1. Detection with DDK antibody that recognizes recombinant DDK tagged EWSR1 protein. As a positive control, EWSR1 alone was detected with DDK antibody. Several negative controls were used for the background normalization. (C) ELISA assay of ARID1A-#FL binding to EWS-FLI1 showing a dose responsive inhibition using YK-4-279 treatment. A B 2 2 shScrambled

21 shEWS-FLI1 +ARID1A-L 20 +shEWS-FLI1 +ARID1A-L 2-1

-2 TGFBR2 2 6 * Fold Differenc e * lu e a -3 V 4 2 ve i t a

A l R2 ID2 e ess ion 2 R LOX GLI1 r

EZH2UPP1TERT p

PTPL1 x IGFBP3 VEGF NROB1 TGF E 0

C D E IGFBP3 VEGFA EZH2

lu e 6 * 1.2 * 1.2 * a * V ve i 0.8 0.8 * t 4 a l e ess ion

R * r 2 0.4 0.4 p x E 0 0.0 0.0 F G H UPP1 TERT NROB1 1.5 * 1.2 1.2 lu e * * a

V * ve

i 1.0 0.8 * 0.8 t a l e ess ion R 0.5 0.4 r 0.4 * p x E 0.0 0.0 0.0 I J K GLI1 PTPL1 ID2 1.2 1.2 lu e 1.2 * a * * * V

ve * i 0.8 0.8 0.8 t a l e ess ion

R NS r 0.4 0.4 0.4 p x E 0.0 0.0 0.0

Supplementary Figure S9. Effect of ARID1A on canonical EWS-FLI1 transcriptional targets. (A) RT-qPCR showing the relative expression of EWS-FLI1 canonical target genes in shRNA reduced ARID1A in A4573 cells. (B-K) RT-qPCR showing the relative expression of EWS-FLI1 canonical target genes: TGFBR2, IGFBP3, VEGFA, EZH2, UPP1, TERT, NROB1, GLI1, PTPL1 and ID2 in either (blue) shRNA reduced EWS-FLI1, (brown) stably expressing ARID1A-L cells followed by shEWS-FLI1, or (green) stable expression of ARID1A-L in A4573 cells. *Indicates significantly different comparisons (P < 0.05, t-test); NS - non-significant. Supplementary Table S1. Primers used for qRT-PCR

Primer Gene Forward (5’-3’) Reverse (5’-3’) # Name L ARID1A AATCAGTTCTCCACCCAAGG GTACATCTCCCCTTCGTGC OTE ARID1A GCGGCTCACAATGAAAGAC AGGAGACCAGACTTGAGGG S+L ARID1A AGCAACGACATGATTCCTATGG AGGTGCGGTTCTCCATTG EWS-FLI1 CAGCCTCCCACTAGTTACCC GTTGAGGCCAGAATTCATG TGFβR2 GGAAACTTGACTGCACCGTT CTGCACATCGTCCTGTGG IGFBP3 GACGGGCTCTCCACACTG AACGCTAGTGCCGTCAGC VEGFA AGCTGCGCTGATAGACATCC CTACCTCCACCATGCCAAGT EZH2 CTGATTTTACACGCTTCCGC GGAACAACGCGAGTCGG UPP1 TTAAAAGTCTGACGGGGCAA CTCTCCTCTGACGGGTCCT TERT CAGGATCTCCTCACGCAGAC GAGCTGACGTGGAAGATGAG NROB1 CTGAGTTCCCCACTGGAGTC AGTACGCCTACCTCAAGGGG GLI1 GGCTCGCCATAGCTACTGAT CCAGCGCCCAGACAGAG PTPL1 GCCCATATTTCTTCCTCCTGA GCGTCCAGTAGCAGGAC ID2 TCAGCACTTAAAAGATTCCGTG GACAGCAAAGCACTGTGTGG SRSF2(+) CGGTGTCCTCTTAAGAAAATGATGTA CTGCTACACAACTGCGCCTTTT GAS5 TCACCCAAGCTAGAGTGCAG TCAGGCAGTCTACAAAGACCAC ANXA1 GCAGGCCTGGTTTATTGAAA GCTGTGCATTGTTTCGCTTA GAPDH AACATCATCCCTGCCTCTACTGG GTTTTTCTAGACGGCAGGTCAGG

L : Primer sets amplify only the ARID1A-L transcript. OTE : Primer sets amplify all different transcript of ARID1A S+L : Primer sets amplify both Short and Long transcript variants of ARID1A with different product size to differentiates both variants. Supplementary Table S2. Mutation analysis in BAF complex

Characteristics N=61 Median age (range), years 18 (8-60) Sex, n (%) Male 36 (59%) Female 25 (41%) Patients with Mutations in BAF Complex Genes, n (%) 2 (3.3%) ARID2 (Exon 10 – P1073S)#, SMARCA4 Mutations Found in BAF Complex Genes* (Exon 7 – Y372H) SMARCB1 (Exon 5 – A203P)# Patients with Synonymous Mutations in BAF Complex 24 (39.3%) Genes, n (%) ACTL6A - 20 patients (Exon 11 – D339D) BCL7A - 20 patients (Exon 6 – L210L) BRD7 - 6 patients (Exon 7 – A282A) DPF3 - 15 patients (Exon 3 – L85L) PBRM1 - 9 patients (Exon 17 – T737T) Synonymous Mutations$ Found in BAF Complex Genes* SMARCA2 - 4 patients (Exon 32-S1530S) SMARCA4 - 11 patients (Exon 9 -H508H) SMARCB1 - 7 patients (Exon 7 – S299S) SMARCC1 - 11 patients (Exon 19-K615K) SMARCD2 - 22 patients (Exon 13-K576K)

*Genes Assessed – ACTL6A, ACTL6B, ARID1A, ARID1B, ARID2, BCL11A, BCL11B, BCL7A, BCL7B, BCL7C, BRD7, BRD9, DPF1, DPF2, DPF3, PBRM1, PHF10, SMARCA2, SMARCA4, SMARCB1, SMARCC1, SMARCC2, SMARCD1, SMARCD2, SMARCD3, SMARCE1, SS18, SS18L1

#Both mutations were found in one patient’s tumor.

$Synonymous mutations were analyzed from 24 ES patient samples RNA-Seq data.