Cancer Cell, Volume 33

Supplemental Information

EWS/FLI Confers Tumor Cell Synthetic Lethality

to CDK12 Inhibition in Ewing Sarcoma

Amanda Balboni Iniguez, Björn Stolte, Emily Jue Wang, Amy Saur Conway, GabrielaAlexe, Neekesh V. Dharia, Nicholas Kwiatkowski, Tinghu Zhang, Brian J. Abraham, JaumeMora, Peter Kalev, Alan Leggett, Dipanjan Chowdhury, Cyril H. Benes, Richard A.Young, Nathanael S. Gray, and Kimberly Stegmaier

Figure S1, related to Figure 1: THZ1 represses p-RNA Pol II levels in an EWS/FLI independent manner and does not cause weight loss in mice. A. Western blot of A673 cells lentivirally infected with CTRL or two unique EWS/FLI hairpins and treated with DMSO or the indicated concentrations of THZ1 for 1 hour. Blots were probed for p-Ser2 RNA Pol II, p-Ser5 RNAPol II and EWS/F-LI. Vinculin is used as a loading control. B. Relative weight measurements for mice with TC32 Ewing sarcoma xenografts treated with 10 mg/kg THZ1 IP BID or vehicle control (10% DMSO in D5W) IP BID for 42 days. Data were plotted out to day 32 when > 50% of mice in the vehicle group were still alive. Data are plotted as mean values ±SD (n=8). (ns, not significant, t-test).

p-Ser2 RNA Pol II

THZ1 (µM)0 0.5 1 0 0.5 1 0 0.5 1

EWS/FLI

shCTRL shEF#6 shEF#7

p-Ser5 RNA Pol II

Vinculin

ns

125120115

110105

10095

900 10 20 30 40

Vehicle

THZ1

Day of Treatment

Rel

ativ

e W

eigh

t (%

)

A B

A B C

0.00.10.20.30.40.50.60.70.80.91.0

−1.5 −1.0CDK7 Dependency Score

CD

K7 S

cale

d R

ank

Other cancer cell lines

Ewing sarcoma Neuroblastoma T-ALL Small cell lung cancer

0.00.10.20.30.40.50.60.70.80.91.0

−0.75 −0.50 −0.25 0.00CDK12 Dependency Score

CD

K12

Scal

ed R

ank

Other cancer cell lines

Ewing sarcomaNeuroblastomaT-ALLSmall cell lung cancer

0.00.10.20.30.40.50.60.70.80.91.0

−0.5 0.0 0.5CDK13 Dependency Score

CD

K13

Scal

ed R

ank

Other cancer cell lines

Ewing sarcomaNeuroblastomaT-ALLSmall cell lung cancer

1600 40THZ531 (nM)

80p-Ser2 RNA Pol II

p-Ser5 RNA Pol II

p-Ser7 RNA Pol II

Total RNA Pol II

E

0 4 0 8 0 1 6 0 3 2 0 0 4 0 8 0 1 6 0 3 2 0 0 4 0 8 0 1 6 0 3 2 0

THZ531 (nM) THZ531 (nM) THZ531 (nM)A673 TC32 TC71

F

0

5

10

15

20

25

30

35

40 DMSOTHZ531 40 n MTHZ531 80 nMTHZ531 160 n MTHZ531 320 nM

0

100

200

300

400

500

600

DMSOTHZ531 40 n MTHZ531 80 nMTHZ531 160 n MTHZ531 320 nM

****

************ ***

**

**

*

Anne

xin

V+ (%

)

Num

ber o

f Col

onie

s

A673 TC32 TC71 A673 TC32 TC71

D

Figure S2, related to Figure 2: CDK7, and not CDK12 or CDK13, is a pan-lethal depdendency across diverse cancer cell lines. (A-C) Scatter plots showing gene dependency scores on the x-axis and gene dependency rank within each cell line on the y-axis for CDK7 (A) CDK12 (B) and CDK13 (C) across 341 cancer cell lines screened with the Avana CRISPR library. Each circle represents an individual cancer cell line. Ewing sarcoma cell lines are shown in red, neuroblastoma cell lines are shown in blue, T-ALL cell lines are shown in green, and small cell lung cancer cell lines are shown in purple. D. Western blot demonstrating effects of THZ531 on p-Ser2/5/7-RNA Pol II in A673 cells at 6 hrs. Total RNA Pol II is used as loading control. E. Western blot demonstrating the effects of THZ531 on cleaved PARP levels in Ewing sarcoma cell lines at 24 hrs. Vinculin is used as loading control. F. Effects of THZ531 on percentage of total Annexin V positive (PI positive and negative) Ewing sarcoma cells measured by flow cytometry at 24 hrs. Data shown is a representative experiment of three biological replicates. G. Colony formation in soft agar of Ewing sarcoma cell lines treated with increasing concentrations of THZ531. The experiment was performed in biological triplicate. Data are presented as mean values of a representative experiment ± SD of duplicate points (*p value <0.05, **p value<0.01, ***p value<0.001, ****p value<0.0001, t-test).

G

Vinculin

Cleaved PARPFL PARP

981982 160994

A673 TC32THZ531 100 nM vs DMSO

98141 1087

A673 TC32

9813,429 778730

A673 TC32THZ531 500 nM vs DMSO

98139 2723

A673 TC32

A B

DACOSTA_UV_RESPONSE_VIA_ERCC3_DNDAZARD_RESPONSE_TO_UV_NHEK_DNDAZARD_UV_RESPONSE_CLUSTER_G6DACOSTA_UV_RESPONSE_VIA_ERCC3_TTD_DNDACOSTA_UV_RESPONSE_VIA_ERCC3_XPCS_DNDNA_DAMAGE_RESPONSESIGNAL_TRANSDUCTIONDNA_INTEGRITY_CHECKPOINTDNA_DAMAGE_CHECKPOINTDOUBLE_STRAND_BREAK_REPAIRDNA_REPLICATIONDNA_DEPENDENT_DNA_REPLICATIONRESPONSE_TO_DNA_DAMAGE_STIMULUSDNA_REPAIRPUJANA_BRCA2_PCC_NETWORKKEGG_HOMOLOGOUS_RECOMBINATIONPUJANA_BREAST_CANCER_WITH_BRCA1_MUTATED_UP

BRCA1BLMBRCA2RAD51CATRRAD17ATRIPBRSK1XRCC4RAD50NDC80WDHD1ORC5EXO1MSH5CDC6RFC4PMS1TUBGCP3WDR37FAM179BDOCK4E2F5ZHX2CHD9APPBP2KIF14ZMIZ1ZYMND8ZZZ3IRS1PLRT2ZNF638

DAC

OST

A_U

V_R

ESPO

NSE

_VIA

_ER

CC

3_D

ND

AZAR

D_R

ESPO

NSE

_TO

_UV_

NH

EK_D

ND

AZAR

D_U

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NSE

_CLU

STER

_G6

DAC

OST

A_U

V_R

ESPO

NSE

_VIA

_ER

CC

3_TT

D_D

ND

ACO

STA_

UV_

RES

PON

SE_V

IA_E

RC

C3_

XPC

S_D

ND

NA_

DAM

AGE_

RES

PON

SESI

GN

AL_T

RAN

SDU

CTI

ON

DN

A_IN

TEG

RIT

Y_C

HEC

KPO

INT

DN

A_D

AMAG

E_C

HEC

KPO

INT

DO

UBL

E_ST

RAN

D_B

REA

K_R

EPAI

RD

NA_

REP

LIC

ATIO

ND

NA_

DEP

END

ENT_

DN

A_R

EPLI

CAT

ION

RES

PON

SE_T

O_D

NA_

DAM

AGE_

STIM

ULU

SD

NA_

REP

AIR

PUJA

NA_

BRC

A2_P

CC

_NET

WO

RK

KEG

G_H

OM

OLO

GO

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REC

OM

BIN

ATIO

NPU

JAN

A_BR

EAST

_CAN

CER

_WIT

H_B

RC

A1_M

UTA

TED

_UP

Category Name AnnotationBRCA1 7BLM 7BRCA2 5RAD51C 5ATR 10RAD17 7ATRIP 4BRSK1 4XRCC4 5RAD50 5NDC80 4WDHD1 4ORC5 4EXO1 5MSH5 4CDC6 4RFC4 4PMS1 8TUBGCP3 5WDR37 4FAM179B 5DOCK4 5E2F5 4ZHX2 4CHD9 4APPBP2 4KIF14 5ZMIZ1 4ZMYND8 5ZZZ3 4IRS1 4FLRT2 4ZNF638 4

C

0

0.5

1

1.5

BRCA1 RAD51 FANCF XRCC2

Rel

ativ

e Ex

pres

sion

TC32

0

0.5

1

1.5

BRCA1 RAD51 FANCF XRCC2

A673DMSOTHZ531 100 nMTHZ531 500 nM

DDMSOTHZ531 100 nMTHZ531 500 nM

**

****

* **

**** **

** ** ** ** ****

***

*

100 500

Log2

Fol

d-C

hang

e in

Gen

e

Exp

ress

ion

Log2

Fol

d-C

hang

e in

Gen

e

Exp

ress

ion

100 500

THZ531 (nM) THZ531 (nM)

−4

−3

−2

−1

0

1

−4

−3

−2

−1

0

1

All

TE SE FLI

All

TE SE FLI

All

TE SE FLI

All

TE SE FLI

E

ns

ns

ns

ns

TC32A673

Rel

ativ

e Ex

pres

sion

0

50

100

0.01 0.1THZ531, µM

% L

umin

esce

nce

(Rel

ativ

e to

CTL

)

0 2 4 60

10

20

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40

Day

Rel

ativ

e Lu

min

esce

nce

0 2 4 60

10

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30

Day

Rel

ativ

e Lu

min

esce

nce

F G

H I0.1% FBS1% FBS2.5% FBS5% FBS10% FBS

Figure S3 related to Figure 3: THZ531 preferentially represses genes involved in DNA damage repair in Ewing sarcoma. A. Venn diagrams depicting the 1,976 genes downregulated by 100 nM THZ531 in the A673 cell line and the 1,142 genes down-regulated by 100 nM THZ531 in the TC32 cell line. They have a significant overlap of 982 genes (representing 8.2% of the 11,963 genes analyzed in the genome) based on the two-tailed Fisher test (Odds Ratios = 63.7, p value < 0.001). The 48 genes upreg-ulated by 100 nM THZ531 in the A673 cell line and the 149 genes upregulated by 100 nM THZ531 in the TC32 cell line have a significant overlap of 41 genes based on the two-tailed Fisher test (Odds Ratios > 100, p value < 0.001). B. Venn diagrams depicting the 4,159 genes downregulated by 500 nM THZ531 in the A673 cell line and the 4,207 genes downregulated by 500 nM THZ531 in the TC32 cell line have a significant overlap of 3,429 genes (representing 28.6% of the 11,963 genes analyzed in the genome) based on the two-tailed Fisher test (Odds Ratios = 66.3, p value < 0.001). The 62 genes upregulated by 500 nM THZ531 in the A673 cell line and the 66 genes upregulated by 500 nM THZ531 in the TC32 cell line have a significant overlap of 39 genes based on the two-tailed Fisher test (Odds Ratios > 100, p value < 0.001). C. Leading edge (consensus) genes in the DNA Damage and Repair MSigDB gene sets significantly enriched among genes downregulated by 100 nM THZ531 vs. DMSO in both the A673 and TC32 cell lines. Rows contain the 33 leading edge genes that belong to at least four of the enriched DNA damage and repair MSigDB gene sets (columns). The red dots in the heatmap depict leading edge genes that belong to each gene set. D. QPCR analysis of select DDR genes in THZ531-treated A673 (left) and TC32 (right) cells. Data are presented as mean values ± SD of triplicate points (*p value<0.05, **p value<0.01, ***p value<0.001, ****p value<0.0001, t-test). E. Box plots showing the Log2 fold change in gene expression (y-axis) for all expressed genes in THZ531-treated cells versus DMSO control cells, A673 (left) and TC32 (right). Shown are all genes (ALL), typical enhancer–associated genes (TE), super–enhancer associ-ated genes (SE), and FLI target genes. ns = not significant. Data are presented as median values with whiskers representing min and max values and the outer edges of the boxes representing the 25th percentile (lower quartile) and 75th percentile (upper quartile). (F-G) Growth rates of A673 (F) and TC32 (G) cells cultured with the indicated percentage of fetal bovine serum (FBS) over five days. Data are plotted as mean luminescence values (measured by CellTiter-Glo) relative to Day 0. The experiment was performed in biological triplicate. Results are presented as mean values of a representative experiment ± SD of eight technical replicates. (H-I) Dose-response curves of A673 (H) and TC32 (I) cells cultured under varying serum starvation condi-tions and treated with increasing concentrations of THZ531 for 72 hours. Data are plotted as the percentage of luminescence (measured by CellTiter-Glo) relative to DMSO controls. The experiment was performed in biological triplicate. Results are presented as mean values of a representative experiment ± SD of eight technical replicates.

0.1% FBS1% FBS2.5% FBS5% FBS10% FBS

0.1% FBS1% FBS2.5% FBS5% FBS10% FBS

0.1% FBS1% FBS2.5% FBS5% FBS10% FBS

0

50

100

0.01 0.1THZ531, µM

% L

umin

esce

nce

(Rel

ativ

e to

CTL

)

1.00.80.60.40.20.0

Exce

ss o

ver B

liss

3

0

0

10THZ531 (µM)

Cis

plat

in (µ

M)

5

1.00.80.60.40.20.0

Exce

ss o

ver B

liss

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0

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0.5THZ531 (µM)

Mito

myc

in C

(µM

)

1.00.80.60.40.20.0

Exce

ss o

ver B

liss

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0

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20THZ531 (µM)

KU55

93 (µ

M)

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1.00.80.60.40.20.0

Exce

ss o

ver B

liss

3

0

0

10THZ531 (µM)

VE82

21 (µ

M)

5

1.00.80.60.40.20.0

Exce

ss o

ver B

liss

3

0

0

10THZ531 (µM)

Vinc

ristin

e (µ

M)

5

Synergy (very strong)

Near additivity Synergy (weak)SynergySynergy (strong)

0.8 to 1.00.6 to 0.80.4 to 0.60.2 to 0.4

Excess over Bliss

-0.2 to 0.2

A

B

C

D

E

Figure S4, related to Figure 4: THZ531 synergizes with DNA damaging agents and DNA repair inhibitors and not microtubule inhibitors. (A-E) Excess over Bliss synergy plots for THZ531 and Cisplatin (A), Mitomycin C (B) KU5593 (C) VE821 (D) and Vincristine (E) in A673 cells.

Synergy (very strong)

Near additivity Synergy (weak)SynergySynergy (strong)

0.8 to 1.00.6 to 0.80.4 to 0.60.2 to 0.4

Excess over Bliss

-0.2 to 0.2

Synergy (very strong)

Near additivity Synergy (weak)SynergySynergy (strong)

0.8 to 1.00.6 to 0.80.4 to 0.60.2 to 0.4

Excess over Bliss

-0.2 to 0.2

Synergy (very strong)

Near additivity Synergy (weak)SynergySynergy (strong)

0.8 to 1.00.6 to 0.80.4 to 0.60.2 to 0.4

Excess over Bliss

-0.2 to 0.2

Synergy (very strong)

Near additivity Synergy (weak)SynergySynergy (strong)

0.8 to 1.00.6 to 0.80.4 to 0.60.2 to 0.4

Excess over Bliss

-0.2 to 0.2

1.00.80.60.40.20.0

Exce

ss o

ver B

liss

1.5

0

0

15THZ1 (µM)

Ola

parib

(µM

)

7.5

A673

Synergy (very strong)

Near additivity Synergy (weak)SynergySynergy (strong)

0.8 to 1.00.6 to 0.80.4 to 0.60.2 to 0.4

Excess over Bliss

-0.2 to 0.2

Figure S5, related to Figure 6: THZ1 and olaparib combinations are highly synergistic in vitro and are well tolerated across mouse models of Ewing sarcoma. A. Excess over Bliss synergy plots of THZ1 and olaparib across the indicated concen-trations in A673 cells. B-D. Relative weight measurements of all mice across studies shown in Figure 6 for A673 xenografts (B) TC71 xenografts (C) and PDXs (D). Data for a given group at a given time point are plotted if >50% of mice in the group are still alive. Data are plotted as mean values ± SD (n=10 for A673 and TC71 xenografts and n=8 for PDXs). (ns, not significant, t-test).

0 5 10 15 20 25 30

80.0

85.0

90.0

95.0

100.0

105.0

110.0

115.0

120.0

0 10 20 30 40

B

(%)

Day of TreatmentR

elat

ive

Wei

ght

90.0

95.0

100.0

105.0

110.0

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Day of Treatment

(%)

Rel

ativ

e W

eigh

t

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(%)

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eigh

t

ns

ns

A

90.0

95.0

100.0

105.0

110.0

115.0

120.0

Day of Treatment

72 hour IC50 concentrations of THZ1 and THZ531 in a panel of 10 Ewing sarcoma cell lines (black) and 2 osteosarcoma cell lines (red) from Figure 2 A-C.

THZ1 (nM) THZ531 (nM)A673 28.5 108.14EW8 39.23 139.77TC32 27.02 81.69TC71 35.05 121.59

EWS502 44.73 92.64RDES 31.82 60.85

SKNEP1 47.13 64.32EWS834 33.43 81.68

SKES1 23.95 72.77SKNMC 31.03 80.5SJSA1 112.7 395.5U2OS 74.94 1146

IC50

Table S2, related to Figure 2: IC50 values of CDK inhibitors in Ewing sarcoma cell lines.

# Gene Set Database NES -log10(p value) p value SIZE1 DAZARD_RESPONSE_TO_UV_SCC_DN MSigDB.v5 c2 -2.135 4.000 0.000 1172 DAZARD_RESPONSE_TO_UV_NHEK_DN MSigDB.v5 c2 -1.967 4.000 0.000 2933 GENTILE_UV_RESPONSE_CLUSTER_D2 MSigDB.v5 c2 -1.847 4.000 0.000 374 DAZARD_UV_RESPONSE_CLUSTER_G6 MSigDB.v5 c2 -1.826 4.000 0.000 1415 GENTILE_RESPONSE_CLUSTER_D3 MSigDB.v5 c2 -1.825 4.000 0.000 606 BRUINS_UVC_RESPONSE_MIDDLE MSigDB.v5 c2 -1.797 4.000 0.000 807 GENTILE_UV_RESPONSE_CLUSTER_D5 MSigDB.v5 c2 -1.762 4.000 0.000 368 GENTILE_UV_HIGH_DOSE_DN MSigDB.v5 c2 -1.757 4.000 0.000 2819 GENTILE_UV_RESPONSE_CLUSTER_D6 MSigDB.v5 c2 -1.650 4.000 0.000 32

10 BRUINS_UVC_RESPONSE_EARLY_LATE MSigDB.v5 c2 -1.635 4.000 0.000 25011 DNA_REPLICATION_INITIATION MSigDB.v5 c5 -1.593 1.794 0.016 16

12 DACOSTA_UV_RESPONSE_VIA_ERCC3_COMMON_DN MSigDB.v5 c2 -1.569 4.000 0.000 44913 GENTILE_UV_RESPONSE_CLUSTER_D8 MSigDB.v5 c2 -1.512 2.039 0.009 3914 DACOSTA_UV_RESPONSE_VIA_ERCC3_DN MSigDB.v5 c2 -1.505 4.000 0.000 79915 REACTOME_FANCONI_ANEMIA_PATHWAY MSigDB.v5 c2 -1.496 1.508 0.031 1816 CHROMATIN_ASSEMBLY_OR_DISASSEMBLY MSigDB.v5 c2 -1.494 1.628 0.023 2217 GENTILE_UV_RESPONSE_CLUSTER_D4 MSigDB.v5 c2 -1.493 2.215 0.006 5218 BIOCARTA_ATRBRCA_PATHWAY MSigDB.v5 c2 -1.488 1.669 0.021 2119 KEGG_HOMOLOGOUS_RECOMBINATION MSigDB.v5 c2 -1.485 1.645 0.023 2320 KEGG_RNA_POLYMERASE MSigDB.v5 c2 -1.473 1.543 0.029 2321 DNA_DEPENDENT_DNA_REPLICATION MSigDB.v5 c5 -1.469 1.917 0.012 4822 DNA_REPLICATION MSigDB.v5 c5 -1.422 2.387 0.004 7923 DACOSTA_UV_RESPONSE_VIA_ERCC3_XPCS_DN MSigDB.v5 c2 -1.392 1.793 0.016 8224 DACOSTA_UV_RESPONSE_VIA_ERCC3_TTD_DN MSigDB.v5 c2 -1.379 1.955 0.011 77

25 REACTOME_PPARA_ACTIVATES_GENE_EXPRESSION MSigDB.v5 c2 -1.349 1.536 0.029 8626 PUJANA_BRCA2_PCC_NETWORK MSigDB.v5 c2 -1.345 4.000 0.000 35527 RESPONSE_TO_DNA_DAMAGE_STIMULUS MSigDB.v5 c2 -1.237 1.309 0.049 14028 DAZARD_UV_RESPONSE_CLUSTER_G2 MSigDB.v5 c2 4.004 4.000 0.000 2129 SESTO_RESPONSE_TO_UV_C3 MSigDB.v5 c2 2.546 4.000 0.000 1630 DAZARD_RESPONSE_TO_UV_SCC_UP MSigDB.v5 c2 2.159 4.000 0.000 7231 SESTO_RESPONSE_TO_UV_C6 MSigDB.v5 c2 1.806 4.000 0.000 2332 DAZARD_UV_RESPONSE_CLUSTER_G24 MSigDB.v5 c2 1.796 4.000 0.000 1533 REACTOME_MRNA_SPLICING_MINOR_PATHWAY MSigDB.v5 c2 1.671 4.000 0.000 3034 SIMBULAN_UV_RESPONSE_IMMORTALIZED_DN MSigDB.v5 c2 1.656 4.000 0.000 2335 GENTILE_UV_LOW_DOSE_UP MSigDB.v5 c2 1.652 4.000 0.000 2336 SESTO_RESPONSE_TO_UV_C7 MSigDB.v5 c2 1.253 4.000 0.000 37

# Gene set Database NES -log10(p- value) p value SIZE

1 DNA_DAMAGE_CHECKPOINT MSigDB.v5 c5 -1.568 2.190 0.006 192 KEGG_HOMOLOGOUS_RECOMBINATION MSigDB.v5 c2 -1.556 1.864 0.014 233 DNA_INTEGRITY_CHECKPOINT MSigDB.v5 c5 -1.502 1.660 0.022 234 BRUINS_UVC_RESPONSE_MIDDLE MSigDB.v5 c2 -1.476 2.677 0.002 805 DNA_DEPENDENT_DNA_REPLICATION MSigDB.v5 c5 -1.453 2.036 0.009 486 DOUBLE_STRAND_BREAK_REPAIR MSigDB.v5 c5 -1.429 1.337 0.046 197 DNA_REPLICATION MSigDB.v5 c5 -1.38 2.040 0.009 798 DAZARD_UV_RESPONSE_CLUSTER_G6 MSigDB.v5 c2 -1.373 2.959 0.001 1419 BRUINS_UVC_RESPONSE_EARLY_LATE MSigDB.v5 c2 -1.367 4.000 0.000 250

10 DACOSTA_UV_RESPONSE_VIA_ERCC3_TTD_DN MSigDB.v5 c2 -1.36 1.917 0.012 7711 DAZARD_RESPONSE_TO_UV_NHEK_DN MSigDB.v5 c2 -1.345 4.000 0.000 29312 PUJANA_BREAST_CANCER_WITH_BRCA1_MUTATED_UP MSigDB.v5 c2 -1.325 1.363 0.043 5213 DACOSTA_UV_RESPONSE_VIA_ERCC3_COMMON_DN MSigDB.v5 c2 -1.324 4.000 0.000 44914 DNA_REPAIR MSigDB.v5 c5 -1.277 1.408 0.039 10815 RESPONSE_TO_DNA_DAMAGE_STIMULUS MSigDB.v5 c2 -1.263 1.656 0.022 14016 DACOSTA_UV_RESPONSE_VIA_ERCC3_DN MSigDB.v5 c2 -1.248 4.000 0.000 79917 DAZARD_UV_RESPONSE_CLUSTER_G2 MSigDB.v5 c2 3.796 4.000 0.000 2118 GENTILE_UV_LOW_DOSE_UP MSigDB.v5 c2 2.462 4.000 0.000 2319 DAZARD_UV_RESPONSE_CLUSTER_G24 MSigDB.v5 c2 2.151 4.000 0.000 1520 DACOSTA_UV_RESPONSE_VIA_ERCC3_COMMON_UP MSigDB.v5 c2 1.360 4.000 0.000 5721 REACTOME_MRNA_SPLICING_MINOR_PATHWAY MSigDB.v5 c2 1.319 1.379 0.042

Table S3, related to Figure 3: DDR gene sets downregulated by THZ531 treatment in Ewing sarcoma cell lines.

A list of all DDR gene sets with their accompanying normalized enrichment scores (NES) and p values from GSEA of the MSigDB c2 and c5 collections among genes significantly downregulated by 100 nM (top) and 500 nM (bottom) concentrations of THZ531 in both A673 and TC32 cells.

45

# Gene Set Database NES -log10(p value) p value SIZE

1 ZHANG_TARGETS_OF_EWSR1_FLI1_FUSION MSigDB.v5 c2 -1.144 0.641 0.228 632 STAEGE_EWING_FAMILY_TUMOR MSigDB.v5 c2 -1.161 0.606 0.247 263 RIGGI_EWING_SARCOMA_PROGENITOR_UP MSigDB.v5 c2 -1.036 0.455 0.351 3134 SILIGAN_BOUND_BY_EWS_FLT1_FUSION MSigDB.v5 c2 -1.067 0.403 0.395 275 MIYAGAWA_TARGETS_OF_EWSR1_ETS_FUSIONS_UP MSigDB.v5 c2 -1.000 0.292 0.510 2016 TORCHIA_TARGETS_OF_EWSR1_FLI1_FUSION_DN MSigDB.v5 c2 -0.929 0.130 0.741 2177 MIYAGAWA_TARGETS_OF_EWSR1_ETS_FUSIONS_DNMSigDB.v5 c2 -0.849 0.055 0.880 1598 RIGGI_EWING_SARCOMA_PROGENITOR_DN MSigDB.v5 c2 -0.798 0.047 0.898 1069 TORCHIA_TARGETS_OF_EWSR1_FLI1_FUSION_UP MSigDB.v5 c2 -0.832 0.039 0.915 188

10 KINSEY_TARGETS_OF_EWSR1_FLII_FUSION_DN MSigDB.v5 c2 -0.758 0.005 0.989 248

# Gene Set Database NES -log10(p value) p value SIZE

1 RORIE_TARGETS_OF_EWSR1_FLI1_FUSION_UP MSigDB c2 -1.360 0.996 0.101 172 TSSCS_EF.KD_VS_WT_DN Ref.SweetCordero -1.164 0.935 0.116 192

3 MIYAGAWA_TARGETS_OF_EWSR1_ETS_FUSIONS_UP MSigDB c2 -1.094 0.669 0.214 201

4 RIGGI_EWING_SARCOMA_PROGENITOR_UP MSigDB c2 -1.067 0.577 0.265 3135 SCHAEFFER_EWS_CHEMORESISTANT MSigDB c2 -1.062 0.384 0.413 216 SCS_EF.KD_VS_WT_DN Ref.SweetCordero -1.031 0.451 0.354 5087 KINSEY_TARGETS_OF_EWSR1_FLII_FUSION_DN MSigDB c2 -0.881 0.064 0.862 248

8 MIYAGAWA_TARGETS_OF_EWSR1_ETS_FUSIONS_DN MSigDB c2 -0.811 0.036 0.921 159

9 STAEGE_EWING_FAMILY_TUMOR MSigDB c2 -0.807 0.117 0.764 2610 SILIGAN_BOUND_BY_EWS_FLT1_FUSION MSigDB c2 -0.673 0.051 0.888 2711 RIGGI_EWING_SARCOMA_PROGENITOR_DN MSigDB c2 -0.673 0.010 0.977 106

Table S4, related to Figure 3: Non-significant enrichment of EWS/FLI gene sets among genes downregulated by THZ531 treatment in Ewing sarcoma cell lines.

A list of all EWS/FLI gene sets with their accompanying normalized enrichment scores (NES) and p values from GSEA of the MSigDB c2 and c5 collections as well as published EWS/FLI gene sets among genes significantly downregulated by 100 nM (top) and 500 nM (bottom) concentrations of THZ531 in both A673 and TC32 cells.

shRNA Target gene Clone ID Vector backbone Source Sequence

shLuc Luciferase TRCN0000231737 pLKO.1TRCN0000231737 was a gift from William Hahn (Addgene plasmid # 78162), Hong et al NATURE COMMUNICATIONS 7:11987

ATGTTTACTACACTCGGATAT

shCyc K #83 Cyclin K TRCN0000045183 pLKO.1 Dharmacon AAATCAAACTTGATGGTCTGC

shCyc K #84 Cyclin K TRCN0000045184 pLKO.1 Dharmacon TTATCCCAGTACCAACATGGC

shCyc K #85 Cyclin K TRCN0000045185 pLKO.1 Dharmacon AATTTGCACAAACGTCCTGCG

shCDK12 #95 CDK12 TRCN0000001795 pLKO.1 Dharmacon TTCAGAGTTATAGAGCCGAGC

shCDK12 #98 CDK12 TRCN0000001798 pLKO.1 Dharmacon AACAATCTCCTCTTTCAGTGC

CDK13 #01 CDK13 TRCN0000000701 pLKO.1 Dharmacon ATCAGCTTCTTTATCTTCAGG

CDK13 #04 CDK13 TRCN0000000704 pLKO.1 Dharmacon TAAATCAGCAAGAAGACATCG

shCTRL non-targeting RHZ4743 TRIPZ Dharmacon Minimal homology to known genes

shEWS/FLI #6 EWS/FLI V2THS227524 TRIPZ Dharmacon AATTTATACAGTAAGTTGC

shEWS/FLI #7 EWS/FLI V3THS414176 TRIPZ Dharmacon TAAACACACAAACCTTCTT

sgRNA Target gene Clone ID Vector backbone Source Sequence

NT CTRL non-targeting cloned in Stegmaier lab lentiCRISPR v2

lentiCRISPR v2 was a gift from Feng Zhang (Addgene plasmid # 52961),Sanjana et al Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047.

GTAGCGAACGTGTCCGGCGT

sgCDK12 #1 CDK12 cloned in Stegmaier lab lentiCRISPR v2 GCTTGTGCTTCGATACCAAG

sgCDK12 #2 CDK12 cloned in Stegmaier lab lentiCRISPR v2 GCTCCCAGACTGGAATTAAG

sgCDK12 #3 CDK12 cloned in Stegmaier lab lentiCRISPR v2 GTAGGAGTCATAATTGCTCG

sgCDK12 #4 CDK12 cloned in Stegmaier lab lentiCRISPR v2 ACGAACGTCGTGGATCAGAT

sgRAD51 #1 RAD51 cloned in Stegmaier lab lentiCRISPR v2 CTATAGCTTCCCATTGACCG

sgRAD51 #2 RAD51 cloned in Stegmaier lab lentiCRISPR v2 GTAGTCTGTTCTGTAAAGGG

sgRAD51 #3 RAD51 cloned in Stegmaier lab lentiCRISPR v2 GTATCTGAGGAAAGGAAGAG

sgRAD51 #4 RAD51 cloned in Stegmaier lab lentiCRISPR v2 GTTGCAGTGGTGAAACCCAT

Table S6, related to STAR methods: A list of all shRNA and sgRNA plasmids used..