supplementary info draft 12 · 2016-08-18 · supplementary information sensitivity and engineered...

Supplementary Information Sensitivity and engineered resistance of myeloid leukemia cells to BRD9 inhibition

Anja F. Hohmann1,2, Laetitia J. Martin3, Jessica Minder1, Jae-Seok Roe1, Junwei Shi1,4, Steffen

Steurer3, Gerd Bader3, Darryl McConnell3, Mark Pearson3, Thomas Gerstberger3, Teresa

Gottschamel3, Diane Thompson3, Yutaka Suzuki5, Manfred Koegl3, and Christopher R. Vakoc1,2

1 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, U.S.A.

2 Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New

York, U.S.A.

3 Boehringer Ingelheim Regional Center Vienna GmbH and Company KG, Vienna, Austria.

4 Molecular and Cellular Biology Program, Stony Brook University, Stony Brook, New York,

U.S.A.

5 Department of Medical Genome Sciences, University of Tokyo, Kashiwa, Chiba 277-8562, Japan.

Nature Chemical Biology: doi:10.1038/nchembio.2115

Supplementary Results


Supplementary Tables

Gene_Set_Name NES FWER p-valREACTOME_3_UTR_MEDIATED_TRANSLATIONAL_REGULATION 3.08 0REACTOME_PEPTIDE_CHAIN_ELONGATION 3.05 0REACTOME_TRANSLATION 3.04 0KEGG_RIBOSOME 3.04 0HALLMARK_MYC_TARGETS_V2 3.04 0REACTOME_INFLUENZA_VIRAL_RNA_TRANSCRIPTION_AND_REPLICATION 3.03 0REACTOME_SRP_DEPENDENT_COTRANSLATIONAL_PROTEIN_TARGETING_TO_MEMBRANE 2.99 0REACTOME_NONSENSE_MEDIATED_DECAY_ENHANCED_BY_THE_EXON_JUNCTION_COMPLEX 2.95 0GNF2_EIF3S6 2.95 0REACTOME_INFLUENZA_LIFE_CYCLE 2.94 0STRUCTURAL_CONSTITUENT_OF_RIBOSOME 2.92 0MORF_NPM1 2.84 0GCM_TPT1 2.82 0MORF_TPT1 2.81 0MYC_UP.V1_UP 2.76 0BILANGES_SERUM_AND_RAPAMYCIN_SENSITIVE_GENES 2.76 0MORF_ACTG1 2.75 0GNF2_FBL 2.73 0TRANSLATION 2.72 0HALLMARK_MYC_TARGETS_V1 2.71 0SCHUHMACHER_MYC_TARGETS_UP 2.71 0GNF2_ST13 2.71 0REACTOME_FORMATION_OF_THE_TERNARY_COMPLEX_AND_SUBSEQUENTLY_THE_43S_COMPLEX 2.70 0GNF2_NPM1 2.68 0REACTOME_ACTIVATION_OF_THE_MRNA_UPON_BINDING_OF_THE_CAP_BINDING_COMPLEX_AND_EIFS_AND_SUBSEQUENT_BINDING_TO_43S 2.68 0GNF2_TPT1 2.64 0REACTOME_METABOLISM_OF_PROTEINS 2.63 0DANG_MYC_TARGETS_UP 2.62 0GCM_NPM1 2.59 0MORF_NME2 2.58 0

Supplementary Table 1 | The 30 gene sets found to have the highest NES by GSEA performed on the RNA-Seq data set presented in Fig. 2d. Highlighted in red are those gene sets depicted as red dots in Fig. 2e.


Gene_Set_Name NES FWER p-valJAATINEN_HEMATOPOIETIC_STEM_CELL_DN -2.86 0GSE22886_NAIVE_CD8_TCELL_VS_MONOCYTE_DN -2.82 0HALLMARK_MITOTIC_SPINDLE -2.75 0BROWN_MYELOID_CELL_DEVELOPMENT_UP -2.72 0GSE29618_PDC_VS_MDC_DAY7_FLU_VACCINE_DN -2.68 0GNF2_CCNA2 -2.67 0TAKEDA_TARGETS_OF_NUP98_HOXA9_FUSION_8D_DN -2.67 0GSE29618_MONOCYTE_VS_PDC_UP -2.66 0GNF2_CCNB2 -2.65 0GNF2_CDC2 -2.64 0GNF2_HMMR -2.64 0MODULE_45 -2.64 0IVANOVA_HEMATOPOIESIS_MATURE_CELL -2.63 0NAKAYAMA_SOFT_TISSUE_TUMORS_PCA2_UP -2.63 0GNF2_ITGB2 -2.59 0GNF2_CDC20 -2.59 0REICHERT_MITOSIS_LIN9_TARGETS -2.58 0GSE22886_NAIVE_TCELL_VS_MONOCYTE_DN -2.58 0GSE29618_PDC_VS_MDC_DN -2.56 0GSE22886_NAIVE_CD4_TCELL_VS_MONOCYTE_DN -2.56 0GSE3982_NEUTROPHIL_VS_NKCELL_UP -2.56 0FLECHNER_BIOPSY_KIDNEY_TRANSPLANT_REJECTED_VS_OK_UP -2.55 0CHUNG_BLISTER_CYTOTOXICITY_DN -2.54 0HESS_TARGETS_OF_HOXA9_AND_MEIS1_DN -2.54 0GSE10239_KLRG1INT_VS_KLRG1HIGH_EFF_CD8_TCELL_DN -2.53 0GTTATAT,MIR-410 -2.53 0GNF2_PTPRC -2.51 0GSE11057_PBMC_VS_MEM_CD4_TCELL_UP -2.51 0CUI_TCF21_TARGETS_2_DN -2.51 0KAMIKUBO_MYELOID_CEBPA_NETWORK -2.50 0

Supplementary Table 2 | The 30 gene sets found to have the lowest NES by GSEA performed on the RNA-Seq data set presented in Fig. 2d. Highlighted in red are those gene sets depicted as blue dots in Fig. 2e.


mouse shRNA sequences

shRenTGCTGTTGACAGTGAGCGCAGGAATTATAATGCTTATCTATAGTGAAGCCACAGATGTATAGATAAGCATTATAATTCCTATGCCTACTGCCTCGGA

shBrg1TGCTGTTGACAGTGAGCGACTCCGTCAAGGTGAAGATCAATAGTGAAGCCACAGATGTATTGATCTTCACCTTGACGGAGCTGCCTACTGCCTCGGA

shBrd9.136TGCTGTTGACAGTGAGCGAGCCACGACTCCAGCTACTACATAGTGAAGCCACAGATGTATGTAGTAGCTGGAGTCGTGGCCTGCCTACTGCCTCGGA

shBrd9.422TGCTGTTGACAGTGAGCGCCCAGAGGCTTCTGGAACACTTTAGTGAAGCCACAGATGTAAAGTGTTCCAGAAGCCTCTGGATGCCTACTGCCTCGGA

shBrd9.510TGCTGTTGACAGTGAGCGCCCTGGGTATTCAATGATAATATAGTGAAGCCACAGATGTATATTATCATTGAATACCCAGGATGCCTACTGCCTCGGA

shBrd9.512TGCTGTTGACAGTGAGCGATGGGTATTCAATGATAATAAATAGTGAAGCCACAGATGTATTTATTATCATTGAATACCCAGTGCCTACTGCCTCGGA

shBrd9.561TGCTGTTGACAGTGAGCGCGACAAGATTGTAGCTAATGAATAGTGAAGCCACAGATGTATTCATTAGCTACAATCTTGTCTTGCCTACTGCCTCGGA

shBrd9.783TGCTGTTGACAGTGAGCGACAAGTAGAAACTACCAAGAAATAGTGAAGCCACAGATGTATTTCTTGGTAGTTTCTACTTGCTGCCTACTGCCTCGGA

shBrd9.1061TGCTGTTGACAGTGAGCGCGGACCTGAGTTCACTGTCTAATAGTGAAGCCACAGATGTATTAGACAGTGAACTCAGGTCCATGCCTACTGCCTCGGA

shBrd9.1116TGCTGTTGACAGTGAGCGAAAAGATGAAAGAAGAAATAAATAGTGAAGCCACAGATGTATTTATTTCTTCTTTCATCTTTGTGCCTACTGCCTCGGA

shBrd9.1211TGCTGTTGACAGTGAGCGCTGAGATGGAGCTTCTGTATTATAGTGAAGCCACAGATGTATAATACAGAAGCTCCATCTCATTGCCTACTGCCTCGGA

shBrd9.1630TGCTGTTGACAGTGAGCGCCTCGGCCATCCTCCAACCTTATAGTGAAGCCACAGATGTATAAGGTTGGAGGATGGCCGAGATGCCTACTGCCTCGGA

shBrd7.536TGCTGTTGACAGTGAGCGATACCATGAAAGAAAAGATCAATAGTGAAGCCACAGATGTATTGATCTTTTCTTTCATGGTACTGCCTACTGCCTCGGA

shBrd7.548TGCTGTTGACAGTGAGCGCAAAGATCAAGAATAACGACTATAGTGAAGCCACAGATGTATAGTCGTTATTCTTGATCTTTTTGCCTACTGCCTCGGA

shBrd7.569TGCTGTTGACAGTGAGCGCCCAGTCCATAGAAGAACTAAATAGTGAAGCCACAGATGTATTTAGTTCTTCTATGGACTGGTTGCCTACTGCCTCGGA

shBrd7.1726TGCTGTTGACAGTGAGCGATGGGTCCTTCTTACAGAGAAATAGTGAAGCCACAGATGTATTTCTCTGTAAGAAGGACCCAGTGCCTACTGCCTCGGA

shBrd7.1803TGCTGTTGACAGTGAGCGCCCAGGTGATGTTGTAAGCATATAGTGAAGCCACAGATGTATATGCTTACAACATCACCTGGATGCCTACTGCCTCGGA

shBrd7.1894TGCTGTTGACAGTGAGCGCCAGGAGAGTGTGAAGAACCTATAGTGAAGCCACAGATGTATAGGTTCTTCACACTCTCCTGTTGCCTACTGCCTCGGA

Supplementary Table 3 | Sequences of mouse shRNAs used in this study.


human shRNA sequences

shBRD9.508TGCTGTTGACAGTGAGCGACTCCTGGATATTCAATGATAATAGTGAAGCCACAGATGTATTATCATTGAATATCCAGGAGCTGCCTACTGCCTCGGA

shBRD9.510TGCTGTTGACAGTGAGCGCCCTGGATATTCAATGATAATATAGTGAAGCCACAGATGTATATTATCATTGAATATCCAGGATGCCTACTGCCTCGGA

shBRD9.512TGCTGTTGACAGTGAGCGATGGATATTCAATGATAATAAATAGTGAAGCCACAGATGTATTTATTATCATTGAATATCCAGTGCCTACTGCCTCGGA

shBRD9.561TGCTGTTGACAGTGAGCGCGACAAAATTGTAGCTAATGAATAGTGAAGCCACAGATGTATTCATTAGCTACAATTTTGTCTTGCCTACTGCCTCGGA

Supplementary Table 4 | Sequences of human shRNAs used in this study.


Category Parameter Description Description Description Assay Type of assay Thermal Shift Assay (DSF) Surface Plasmon Resonance

Microscale Thermophoresis

Target BRD9 BRD9 BRD9 Primary

measurement Protein Stability Mass increase Protein mobility in

temperature gradient

Key reagents Fluorescent Dye His-tagged protein Fluorescent Dye Assay protocol Anal Biochem. 2004 Sep

1;332(1):153-9.

J.Biomol. Screening 2009 14:337-49

ChemMedChem. 2015 Sep;10(9):1511-21

Library Library size 1.697 1.697 1.697

Library composition

Generic fragment set Generic fragment set Generic fragment set

Source BI pool BI pool BI pool Screen Format 384 well 384 well 384 well Concentration(s)

tested 400 µM, 2% DMSO 100 µM, 1 % DMSO 500 µM, 5% DMSO

Plate controls Positive (in-house bromodomain binder) and negative (DMSO) controls

Positive (in-house bromodomain binder) and negative (buffer) controls

Positive (in-house bromodomain binder) and negative (DMSO) controls

Reagent/ compound dispensing system

Hamilton Star system CyBio System 6 Hamilton Star system

Detection instrument and software

Bio-Rad CFX384 Real-Time System (C1000Touch Thermal Cycler;) / Bio-Rad CFX Manager-Data Analysis software

Biacore T200 / BiaEvaluation software

In house implemented automation of Nanotemper NT.015 / Nanotemper Analysis software

Assay validation/QC

Z’ = 0.55 Z’ = 0.93

Correction factors DMSO solvent correction Normalization Normalization to positive control

Post-HTS analysis

Hit criteria ΔT≥1°C BR ≥20% ∆MST(DMSO, Cpd) ≥ MST(2sd DMSO)

Hit rate 2.1% Hit rate 2.7% 6.6% Additional

assay(s)

Confirmation of hit purity and structure

Entire screening library QC’ed for purity and structural identity



Additional comments

94.4% Hit confirmation by 15N HSQC

72.2% Hit confirmation by 15N HSQC

26 % Hit confirmation by 15N HSQC

Supplementary Table 5 | Overview of small molecule screening methods


Data collection and refinement statistics BI-7273 Data collection* Space group P 21 21 2 Cell dimensions a, b, c (Å) 70.80, 125.34, 29.92 α, β, γ (°) 90.00, 90.00, 90.00 Resolution (Å) 1.60 (1.79 – 1.60) ** Rmerge (%) 3.0 (67.0) CC1/2(ref. 2) 1.0 (0.924) I / σI 26.2 (3.1) Completeness (%) 99.9 (99.7) Redundancy 6.3 (6.3) Refinement Resolution (Å) 1.60 No. reflections 35816 Rwork / Rfree (%) 17.8/19.2 No. atoms Protein 1847 Ligand/ion 52 Water 352 B-factors (Å2) Protein 35.75 Ligand/ion 32.13 Water 47.24 R.m.s. deviations Bond lengths (Å) 0.009 Bond angles (°) 0.81 * Values in parentheses are for highest-resolution shell. ** Resolution cutoffs have been determined using the standard parameters of autoPROC(ref. 1)

1. Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr D Biol Crystallogr 67, 293-302 (2011). 2. Karplus, P.A., Diederichs, K. Linking crystallographic model and data quality. Linking crystallographic model and data quality. Science 336, 1030-33 (2012).

Stereo image of BI-7273 bound to BRD9 (wall-eye stereo). The refind 2Fo-Fc electron density is countoured at 1 σ.

Supplementary Table 6 | Crystal structure data refinement and statistics


Supplementary Figures

Supplementary Figure 1 | BRD9 binding to the MYC locus in RN2 and NOMO-1 cells

(a) ChIP-Seq occupancy profiles for BRD9 at the MYC locus in NOMO-1 cells derived using two different antibodies for BRD9. The y-axis reflects the number of cumulative ChIP-Seq tag counts within a 50-100 bp bin surrounding each region. Ab1: Bethyl #A303-781A; Ab2: Abcam #ab66443.

(b) Magnified ChIP-Seq occupancy profile of BRD9 at the MYC promoter and super-enhancer region in NOMO-1 cells.

(c) Ranking of Brd9-occupied sites based on tag counts obtained from ChIP-Seq analysis in RN2 cells.

(d) Ranking of BRD9-occupied sites based on tag counts obtained from ChIP-Seq analysis in NOMO-1 cells.

aScalechr8:

DNase ClustersTxn Factor ChIP

RhesusMouse

DogElephantChicken

X_tropicalisZebrafishLamprey

Common SNPs(142)

1 Mb hg19129,000,000 129,500,000 130,000,000 130,500,000

BC042052MYCMYCMYC

HV975509

MYC

MYCPVT1

MIR1204

PVT1TMEM75

MIR1205

MIR1206

U4

MIR1207

MIR1208 BC009730 BC014119 LOC7287247SK

CCDC26

Nomo1_Brd9Bethyl.bigw

52 -

1 _

Nomo1_ab66443.bigwig

138 -

1 _

Layered H3K27Ac100 -

0 _

100 Vert. Cons4.88 -

-4.5 _

0 -

1 Mb

BRD9 Ab1

52-

1_

BRD9 Ab2

138-

1_MYC super-enhancer

BRD9 Ab2

Scalechr8:

CCDC26CCDC26CCDC26CCDC26

50 kb hg19130,560,000 130,570,000 130,580,000 130,590,000 130,600,000 130,610,000 130,620,000 130,630,000 130,640,000 130,650,000 130,660,000 130,670,000 130,680,000 130,690,000

Nomo1_ab66443.bigwig

138 -

1 _

Nomo1_Brd9Bethyl.bigw

52 -

1 _

//MYC

50 kb hg19128,730,000 128,740,000 128,750,000 128,760,000 128,770,000 128,780,000 128,790,000 128,800,000 128,810,000 128,820,000 128,830,000 128,840,000 128,850,000

138-

1_

MYC super-enhancer

50 kbb

MLL-AF9; KRASG13D human AML (NOMO-1)


E1 E2 E3 E4 E5

0 1000 2000 30000

500

1000

1500

Peak_name

c1500

1000

500

020000 30001000

MLL-AF9; NrasG12D murine AML (RN2)

3,305 Brd9 ChIP peaks ranked by tag counts

(5) Myc E1(6) Myc E2

(57) Myc E3(77) Myc E4Br

d9 ta

g co

unts

BRD9

tag

coun

tsd

2500

2000

1500

1000

500

00 2000 4000 6000

0

500

1000

1500

2000

2500

Peak_name2000 4000 60000


7,175 BRD9 ChIP peaks ranked by tag counts

(1) MYC E2

(8) MYC E5

(53) MYC E1(64) MYC E4.1

(198) MYC E3(325) MYC E4.2


a

Gapdh Brd9 Brd93’UTR

Brg1 Brd4

Gapdh, Brd9, Brg1, Brd4

Gapdh

Brd9

Brd9 3

' UTR

Smarca

4Brd

4 0.0

0.5

1.0DoxD2 Rluc.713DoxD2 Brd9.s1/510DoxD2 Brd9.s2/561DoxD2 Brd9.841/783DoxD2 Brd9.u5/1061DoxD2 Brd9.1174/1116

1.0

0.5

0.0

Rela

tive

mRN

A ex

pres

sion

shRen

shBrd9783

1116

510561

1061

Data 4 - no s4

Gapdh

Brd9

Brd9 3'

UTR

Myc

Myc 3'

UTR

Smarca4

Brd4

0.0

0.5


RN2

b

2 d6 d10 d14 d18 d22 d

empt

y +

RLuc

.713

empt

y +

Brd9

.561

empt

y +

Brd9

.111

6

BRD9

+ R

Luc.

713

BRD9

+ B

rd9.

561

BRD9

+ B

rd9.

1116

0.0

0.5

1.0

1.5

Day 2Day 4Day 6Day 8Day 10Day 12

Norm

alize

d %

GFP

and

mCh

erry

+ve

cel

ls

RLu

c

Rpa3

Brd

9.8

41

Brd

9.1

17

4

0

50

100

150


No

rma

lize

d %

of

GF

P +

ve c

ells

1.5

1.0

0.5

0.0

Rela

tive

% G

FP+

shBrd9

783

1116

shRen

shRpa

3

iMEF

c


Gapdh, Brd9

Gapdh

Brd9

Brd9 3' UTR

0.0

0.5

1.0TRIN Rluc.713TRIN Brd9.s1/510TRIN Brd9.841/783TRIN Brd9.u5/1061TRIN Brd9.1174/1116

1.0

0.5

0.0Rela

tive

mRN

A ex

pres

sion

shRen

shBrd9783

1116

510

1061

MEF

Gapdh

Brd9

Brd9 3'

UTR

Myc

Myc 3'

UTR

TRIN Rluc.713TRIN Brd9.s1/510TRIN Brd9.841/783TRIN Brd9.u5/1061TRIN Brd9.1174/1116

iMEF 2 d4 d6 d8 d10 d12 d

empt

y +

RLuc

.713

empt

y +

Brd9

.561

empt

y +

Brd9

.111

6

BRD9

+ R

Luc.

713

BRD9

+ B

rd9.

561

BRD9

+ B

rd9.

1116

0.0

0.5

1.0

1.5


Norm

alize

d %

GFP

and

mCh

erry

+ve

cel

ls d

2.0

1.0

0.0

3.0

shBrd718

94536

548

569

1726

1803

shRen

RLuc

Brd7

.775

Brd7

.787

Brd7

.808

Brd7

.196

5

Brd7

.204

2

Brd7

.213

3

0

1

2

3Day 2Day 4Day 6Day 8Day 10Day 12

Norm

alize

d %

GFP

+ve

cells

Rela

tive

% G

FP+

RN2

g h

NOMO-1

MOLM-13

THP-1

MV4-11

ML-2HL-60

CMKEoL-1

A549H1299

HEK293T

K-562JURKAT

HCC1954639VHeLa

MLL-AF9, KRASG13D; AML M5a

MLL-AF9, FLT3ITD; AML M5a

MLL-AF9, NRASmut; AML M5

MLL-AF4; AML M5

MLL-AF6; AML M4MYC amplification; AML M2

JAK3A572V; AML-M7 + Down’s SyndromeFIP1L1-PDGFRA, MLL-ITD; Eosinophilic AML

SMARCA4mut; Non-small cell lung cancerSMARCA4mut; Non-small cell lung cancer

Embryonic kidney cells

BCR-ABL1; CML blast crisisT-ALL

BRD9 amplification; breast cancerUreter transitional cell carcinomaCervical adenocarcinoma

Cell line Genetics or disease descriptionshRen shBRD9

17013010070

55

40

508 510 512 561

55

40

kDa

BRD9

Actin

50% 100%


1.0

0.5

0.0

e

Rela

tive

mRN

A ex

pres

sion

Gapdh, Brd7

Gapdh

Brd7

Brd7 3

' UTR

0.0

0.5

1.0RLuc.713Brd7.775/536Brd7.787/548Brd7.808/569Brd7.1965/1726Brd7.2402/1803Brd7.2133/1894

RN2 shRen536

shBrd71726

1894

548569

1803

RN2

Gapdh

Brd7

Brd7 3'

UTR

Myc

Myc 3'

UTR

0.0

0.5


Rel

ativ

e m

RN

A e

xpre

ssio

n

kDa

BRD9

actin

f

shRen shBRD9

508 510 512 56150% 100%

130

100

70

40

HeLa

OPM1


Supplementary Figure 2 | Experiments to examine shRNA knockdown efficiencies, the effect of Brd9 knockdown in iMEF cells, and the consequences of Brd7 knockdown in RN2 cells

(a) qRT-PCR analysis to test the knockdown efficiency of Brd9 shRNAs in RN2 cells. mRNA levels were examined after 48 hours of shRNA expression (TRMPV-Neo vector). Values were normalized to Gapdh expression within each sample and to shRen values across samples. n = 3

(b) Competition-based assay to measure effect of Brd9 shRNAs on growth of iMEF cells. Transduced (shRNA-expressing) cells were identified by co-expression of GFP (LMN vector). The percentage of GFP+ cells was normalized to GFP percentage on day 2. shRpa3 targets Replication Protein A3 and serves as a positive control. n = 3, except for shRpa3 for which n = 2

(c) qRT-PCR analysis to test the knockdown efficiency of Brd9 shRNAs in iMEF cells. Performed as in (a). n = 2

(d) Competition-based assay to measure the effect of Brd7 shRNAs on the growth of RN2 cells. Performed as in (b). n = 3, except of shRen for which n = 2

(e) qRT-PCR analysis to test Brd7 shRNA knockdown efficiency in RN2 cells. Performed as in (a), except that the LMN vector was used for shRNA expression. n = 1

(f,g) Western blot to test knockdown efficiency of human BRD9 shRNAs in HeLa (f) and OPM-1 (g) cells. Actin blot serves to control for gel loading.

(h) Genetic background and/or disease descriptions of human cell lines profiled for sensitivity to BRD9 knockdown.

shRen targets Renilla luciferase and serves as a negative control. All error bars in this figure represent SEM.


Supplementary Figure 3 | Effect of Brd9 or Brd7 knockdown on Myc expression

(a) qRT-PCR analysis to test the effect Brd9 knockdown on Myc expression in RN2 cells. mRNA levels were examined after 48 hours of shRNA expression (TRMPV-Neo vector). Values were normalized to Gapdh expression within each sample and to shRen values across samples. n = 3

(b) qRT-PCR analysis to test effect of Brd7 knockdown on Myc expression in RN2 cells. Performed as in (a), except that the LMN vector was used for shRNA expression. n = 1

(c) GSEA plots of the top 100 genes downregulated after 4 days of shBrg1 expression in RN2 cells (shBrg1_Top100Down), genes up-regulated in Burkitt’s Lymphoma cells induced to express MYC (Schuhmacher_MYC_Targets_Up), genes expressed in leukemia stem cells

Gapdh, Myc

Gapdh

Myc

Myc 3' U

TR

0.0

0.5

1.0

TRIN Rluc.713TRIN Brd9.s1/510TRIN Brd9.841/783TRIN Brd9.u5/1061TRIN Brd9.1174/1116

Gapdh Myc Myc3’UTR

1.0

0.5

0.0

b

Re

lative

mR

NA

exp

ressio

n

shRen

536

shBrd71726

1894

548

569

1803

RN2

Gapdh

Brd7

Brd7 3'

UTR

Myc

Myc 3'

UTR

0.0

0.5


Rel

ativ

e m

RN

A e

xpre

ssio

n Gapdh, Myc

Gapdh Myc

Myc 3'

UTR

0.0

0.5


a

1.0

0.5

0.0

Gapdh

Re

lative

mR

NA

exp

ressio

n

Myc Myc3’UTR

shRen

shBrd9783

1116

510

561

1061

Data 4 - no s4

Brd9

Brd9 3'

UTR

Myc

Myc 3'

UTR

Smarca4

Brd4

DoxD2 Rluc.713DoxD2 Brd9.s1/510DoxD2 Brd9.s2/561DoxD2 Brd9.841/783DoxD2 Brd9.u5/1061DoxD2 Brd9.1174/1116

Gapdh, Myc

Gapd

hMyc

Myc 3' U

TR

0.0

0.5


RN2 RN2

e

1.0

0.5

0.0

Gapdh Myc

Re

lative

mR

NA

exp

ressio

n

Myc3’UTR

shRen

shBrd9783

1116

510

1061

MEF

Gapdh

Brd9

Brd9 3'

UTR

Myc

Myc 3'

UTR

0.0

0.5

1.0

1.5 TRIN Rluc.713TRIN Brd9.s1/510TRIN Brd9.841/783TRIN Brd9.u5/1061TRIN Brd9.1174/1116

Rel

ativ

e m

RN

A e

xpre

ssio

n

iMEF

f g

Lo

g2 (

FP

KM

sh

Brd

9 /

FP

KM

sh

Re

n) 2

0

-2

d

-12

4

-1

1

-3

-6

-12-6-3

-2

-1

0

1

24

8657 genes with FPKM > 5 in shRen sample

iMEF

Myc (4066)

Brd9 (42)

-2

-1

0

2

3

0 0.5 1

NE

S

FWER p-val

71

50

ge

ne

se

ts

1

-3

Myc target

gene

signatures

Myeloid

differentiation

signatures

shBrd9

Top100Down

/Up

-3-2

-10

12

30.00.51.0

NES

c

0.0

0.6

shBrg1_Top100Down

shBrd9shRen

0.0

0.7

Schuhmacher_MYC_Targets_Up

shBrd9shRen

0.0

0.6

LSC_Signature_Somervaille

shBrd9shRen

-0.5

0.0

Ivanova_Hematopoiesis

Mature_Cell

shBrd9shRen

En

rich

me

nt

sco

re

En

rich

me

nt

sco

re

NES: 2.50

FWER p-val: 0

NES: 2.13

FWER p-val: 0.015

NES: -2.63

FWER p-val: 0

NES: 2.71

FWER p-val: 0

-0.2

0.3

shBrd9_Top100Up

shBrd9shRen

-0.1

0.2

shBrd9_Top100Down

shBrd9shRen

En

rich

me

nt

sco

re

En

rich

me

nt

sco

re

NES: 0.78

FWER p-val: 1

NES: 1.00

FWER p-val: 1

0

0


(LSC_Signature_Somervaille), and genes up-regulated in mature blood cell populations from adult bone marrow and fetal liver (Ivanova_Hematopoiesis_Mature_Cell).

(d) RNA-Seq analysis of gene expression changes in iMEF cells expressing Brd9 shRNAs for 2 days (TRMPV-Neo vector). Averaged FPKM values for two independent Brd9 shRNAs were normalized to mRNA levels in control cells expressing shRen.

(e) qRT-PCR analysis to test the effect of Brd9 knockdown on Myc expression in iMEF cells. Performed as in (a). n = 2

(f) Gene set enrichment analysis (GSEA) on the RNA-Seq data presented in (c). FWER p-val, familywise-error rate p-value.

(g) GSEA plots of the top 100 genes up- and down-regulated after 2 days of shBrd9 expression in RN2 cells.

shRen targets Renilla luciferase and serves as a negative control. All error bars in this figure represent SEM.


Supplementary Figure 4 | Effect of Brd9 knockdown on cell death and cell cycle

(a) Flow cytrometry analysis of Kit (top) and Mac1 (bottom) cell surface expression after 4 days of shRen or shBrd9 expression (TRMPV-Neo vector) in RN2 cells.

(b) qRT-PCR analysis to test Brd9 knockdown efficiency in RN2-derived shRNA-expressing clones. mRNA levels were examined after 48 hours after dox-induced shRNA expression (TRMPV-Neo vector). Values were normalized to Gapdh expression within each sample and to shRen clone 1 values across samples. n = 1

(c) Cell death assay. Flow cytometry analysis of Annexin V and DAPI stained RN2-derived clones 4 days after induction of shRen or shBrd9 expression (TRMPV-Neo vector). Annexin V-/DAPI- cells were considered live; Annexin V+/DAPI-, cells were considered pre-apoptotic; DAPI+ cells were considered dead. n = 3

(d) Cell cycle assay. Flow cytrometry analysis of BrdU labeled; BrdU and DAPI stained RN2-derived clones 4 days after induction of shRen or shBrd9 expression (TRMPV-Neo vector). BrdU-/2N cells were considered to be in G1 phase; BrdU+ cells were considered to be in S phase; BrdU-/4N cells were considered to be in G2/M phase. n = 4

clone 1 clone 3 783clone 3

1116clone 21

1116clone 29

shBrd9shRen

1.4 MNaCl

% o

f cel

ls

DeadPre-apoptoticLive

c Data 1

RLuc_cl1

RLuc_cl3

Brd9.84

1_cl3

Brd9.11

74_c

l21

Brd9.11

74_c

l29

500nM JQ1 Day2

0

50

100G1SG2

Data 1

RLuc_

cl1

RLuc_

cl3

Brd9.84

1_cl3

Brd9.11

74_c

l21

Brd9.11

74_c

l29

500n

M JQ1 D

ay2

0

50

100G1SG2

G1S

G2/M

d

100

50

0clone 1 clone 3 783

clone 31116

clone 211116

clone 29

shBrd9shRen

500 nMJQ1

% o

f cel

ls

100

50

0

b

1.0

0.5

0.0clone 1 clone 3 783

clone 31116

clone 21

Rel

ativ

e m

RN

A ex

pres

sion

1116clone 29

Data 1

Rluc

.713

Cl.1

Dox

D2

Rluc

.713

Cl.3

Dox

D2

Brd9

.841

Cl.3

Dox

D2

Brd9

.117

4 Cl.21 Do

xD2

Brd9

.117

4 Cl.29 Do

xD2

0.0

0.5

1.0GapdhBrd9Brd9 3' UTRMycMyc 3' UTR

Gapdh

Brd9 3’UTR

Myc 3’UTR

Brd9

Myc

MEF

Gapdh

Brd9

Brd9 3'

UTR

Myc

Myc 3'

UTR

0.0

0.5

1.0

1.5 TRIN Rluc.713TRIN Brd9.s1/510TRIN Brd9.841/783TRIN Brd9.u5/1061TRIN Brd9.1174/1116

Rel

ativ

e m

RN

A e

xpre

ssio

n

Data 2

RLuc_

cl1

RLuc_

cl3

Brd9.84

1_cl3

Brd9.11

74_c

l21

Brd9.11

74_c

l29

RLuc_

cl1/3_

1.4M_N

aCl

0

50

100DAPI +veAnnexinV +veNeg

a

Mac1

% o

f Max

- shRen

- shBrd9

% o

f Max - shRen

- shBrd9

Kit

shBrd9.783

103 1050

20406080

100

103 1050

20406080

100

shBrd9.1061

103 1050

20406080

100

103 1050

20406080

100

shBrd9.1116

103 1050

20406080

100

103 1050

20406080

100


Supplementary Figure 5 | The bromodomain pocket is required for BRD9 function in leukemia

(a) Western blot to test retroviral expression of wt and mutant BRD9 in RN2 cells. The antibody recognizes human BRD9 exclusively. For this reason no Brd9 band is observed in the ‘empty’ lane. An actin blot serves to control for loading.

(b) qRT-PCR analysis to test the effect of wt or mutant BRD9 expression (via retrovirus) on Myc mRNA levels in RN2 cells. mRNA expression was examined 48 hours after retroviral transduction of transgenes (MSCV-based vector). Values were normalized to Gapdh expression within each sample and to empty vector values across samples. n = 3

(c,d) Competition-based assay to measure the effect of wt or mutant BRD9 expression on the growth of RN2 (c) and iMEF cells (d). Transduced cells were identified by co-expression of GFP (MSCV-based vector). The percentage of GFP+ cells was tracked and normalized to GFP percentage on day 2. n = 2

empty BRD9 BRD9 dBD BRD9 N216A

0.0

0.5

1.0

1.5Day 2Day 4Day 6Day 8Day 10Day 12

No

rma

lize

d %

GF

P +

ve

ce

lls

Gapdh

Myc

Myc 3'UTR

0.0

0.5

1.0

1.5

Data 2

PiGBRD9dBDN216A

Re

lativ

e m

RN

A e

xp

ressio

n

1.5

1.0

0.5

0.0Gapdh Myc

empty

Rel

ativ

e m

RN

A ex

pres

sion

Myc3’UTR

wt

ΔBD

N216A

Gapdh Myc

Myc 3'

UTR

0.0

0.5

1.0

1.5

Data 2

PiGBRD9dBDN216A

Rel

ativ

e m

RN

A e

xpre

ssio

n

BRD9

a

c

Rel

ativ

e %

GFP

+

BRD9 dBD BRD9 N216A


2 d4 d6 d8 d10 d12 d

empty wt ΔBD N216A

BRD9

0.0

0.5

1.0

1.5

empty wt ΔBD N216A

BRD9

empty BRD9 BRD9 dBD BRD9 N216A

0.0

0.5

1.0

1.5Day 2Day 4Day 6Day 8Day 10Day 12

Norm

alize

d %

GFP

+ve

cel

ls

4 d8 d12 d16 d20 d24 d

PiG BRD9 BRD9 dBD BRD9 N216A

0.0

0.5

1.0

1.5


No

rma

lize

d %

of

GF

P +

ve

ce

lls

Rel

ativ

e %

GFP

+

0.0

0.5

1.0

1.5

dRN2 iMEF

empty

wt ΔBD

N216A

BRD9

kDa

BRD9

actin

70

55

100

40

130

RN2b

actinBRD9

empty

wt ΔBD

N216A

BRD9

marker

empty

wt ΔBD

N216A

BRD9

marker

kDa

7055

100130170

e


(e) Uncropped images of the Western blots shown in (a). All error bars in this figure represent SEM.


Supplementary Figure 6 | CRISPR-Screen of SWI/SNF bromodomains in RN2 cells (a) CRISPR-Cas9 targeting of SWI/SNF bromodomains to assess their relevance for the growth

of RN2 cells in a competition-based assay. Transduced (sgRNA-expressing) cells were identified by the co-expression of GFP. The percentage of GFP+ cells on day 2 was divided by the GFP percentage on ~day 14 to calculate the fold depletion. Higher fold depletion indicates greater anti-proliferative effects of sgRNAs. Each bar represents a different sgRNA. A sgRNA targeting the Rosa26 locus served as a negative control. sgRNAs targeting Brd9 are highlighted in red. Error bars represent SEM. n = 3

a

Brd7_BD

Brd9_BD

Cecr2_BD

Pbrm1_BD1

Pbrm1_BD2

Pbrm1_BD3

Pbrm1_BD4

Pbrm1_BD5

Pbrm1_BD6

0 2 4 6

Rosa26

0 2 4 6

Pbrm1_BD6_e18.6Pbrm1_BD6_e18.7Pbrm1_BD6_e18.8Pbrm1_BD6_e18.9Pbrm1_BD5_e18.1Pbrm1_BD5_e18.2Pbrm1_BD5_e18.3Pbrm1_BD5_e18.4Pbrm1_BD5_e18.5Pbrm1_BD4_e16.1Pbrm1_BD4_e16.2Pbrm1_BD4_e16.3Pbrm1_BD4_e16.4Pbrm1_BD4_e17.1Pbrm1_BD3_e13.1Pbrm1_BD3_e13.2Pbrm1_BD3_e14.1Pbrm1_BD3_e14.2Pbrm1_BD2_e7.1Pbrm1_BD2_e8.1Pbrm1_BD2_e9.1Pbrm1_BD2_e9.2Pbrm1_BD1_e4.1Pbrm1_BD1_e4.2Pbrm1_BD1_e6.1Pbrm1_BD1_e6.2Cecr2_BD_e12.1Cecr2_BD_e12.2Cecr2_BD_e13.1Cecr2_BD_e14.1

Brd9_BD_e5.1Brd9_BD_e5.2Brd9_BD_e5.3Brd9_BD_e6.1Brd9_BD_e6.2Brd7_BD_e5.1Brd7_BD_e5.2Brd7_BD_e5.3Brd7_BD_e6.1

Rosa 26

Fold depletion (% GFP+ d2 / d14)


Supplementary Figure 7 | Additional BRD9 inhibitor binding assays

(a,b) AlphaScreen assays to determine binding affinities of BI-7271 (a) and BI-7189 (b) for the bromodomains of BRD9, BRD7 and BRD4. Curves were fit by four parameter non-linear regression using the least squares fitting method. Representative graph out of 2-6 (BI-7271, a) and 7-9 (BI-7189, b) replicates is shown, respectively.

(c) Control AlphaScreen assay of JQ1 binding to the bromodomains of BRD4. Curves were fit by four parameter non-linear regression using the least squares fitting method. Representative graph out of 3 replicates is shown

(d) Summary of IC50 values derived from the AlphaScreen graphs shown in (a), (b), and Main Fig. 3 (d). IC50 values were obtained from non-linear regression curves with the bottom constrained to 0 and the top constrained to 100.

(e) NanoBRET assay to test binding of BI-7273 to the BRD9 bromodomain expressed in HEK293T cells. As a control the same assay was performed using a compound inactive in BRD9 binding. n = 3

rela

tive lum

inescence

BI-7271 (μM)

a

10010.010.0001

100

50

0

Data 1

-4 -2 0 20

50

100 BRD9BRD7BRD4-BD1BRD4-BD2BRD9BRD7BRD4-BD1BRD4-BD2

Log10[7271], uM

2

BRD9BRD7BRD4-BD1BRD4-BD2

BRD9

BRD7

BRD4-BD1

BRD4-BD2

b

rela

tive lum

inescence

BI-7189 (μM)10010.010.0001

100

50

0

Data 1

-4 -2 0 20

50

100BRD9BRD7BRD4-BD1BRD4-BD2BRD4-BD2

Log10[7189], uM

Data 1

-4 -2 0 20

50

100 BRD9BRD7BRD4-BD1BRD4-BD2

Log10[7271], uM

BRD9

BRD7

BRD4-BD1

BRD4-BD2

e

Compound concentration (μM)1010.10.010.001

NanoB

RE

T r

atio

1.5

2.0

2.5

3.0

-3 -2 -1 0 10.00.51.01.5

2.0

2.5

3.0

Log10[Compound], uM

Nan

oBR

ET ra

tio

0.00.51.0

2.0

2.5

3.0BI-7273Inactive compound

Nan

oBR

ET ra

tio

BI-7273

Inactive compound

log-dose vs response

-4 -2 0 20

50

100

Log10[JQ1]uM

c

rela

tive lum

inescence

JQ1 (μM)10010.010.0001

100

50

0

Data 1

-4 -2 0 20

50

100 BRD9BRD7BRD4-BD1BRD4-BD2

Log10[7271], uM

BRD9

BRD7

BRD4-BD1

BRD4-BD2

BI-727113 nM42 nM

22,240 nM27,330 nM

BI-727321 nM71 nM

>100,000 nM>100,000 nM

BI-718967 nM

644 nM77,990 nM

>100,000 nM

d

H3 K9,14,18,23 Ac

H3 K9,14,18,23 Ac

H4 K5,8,12,16 Ac

H4 K5,8,12,16 Ac

substrateBRD9

BRD7

BRD4-BD1

BRD4-BD2

BD

IC50


Supplementary Figure 8 | Effect of BI-7273 on FLAG-BRD9 and Brg1 chromatin binding

(a,b) ChIP-qPCR analysis of FLAG-BRD9 (a) and Brg1 (b) binding to the Myc locus in RN2 cells in the presence and absence of BI-7273. Each experiment included a parallel precipitation using an IgG antibody as a negative control. neg = a ‘negative’ region not bound by Brd9 in RN2 cells. P values were determined using the paired, two-tailed Student’s T-Test. n = 7 (a), n = 6 (b)

(c) Ranking of FLAG-BRD9 occupied sites based on fold change of tag counts obtained from ChIP-Seq analysis of BI-7273 vs. DMSO treated FLAG-BRD9 expressing RN2 cells.

(d) Ranking of Brg1 occupied sites based on fold change of tag counts obtained from ChIP-Seq analysis of BI-7273 vs. DMSO treated RN2 cells.

b

Brg2 ChIP

left 2

TSS -1.8

kb

ME1M

ME2M

ME3M

ME4M

ME5M

0.00

0.05

0.10

DMSO; IgG

BI2; IgG

DMSO; Brg1

BI2; Brg1

DMSO; IgG

10uM BI-7273; IgG

DMSO; Brg1

10uM BI-7273; Brg1

* < 0.05

** < 0.005

p-valueneg. new

TSS -1.8

kb

ME1M

ME2M

ME3M

ME4M

ME5M

0.00

0.05

0.10**

***

*

5

2.5

0

Brg1 ChIP

neg Myc-1.8 kb

1 2 3 4 5

Myc enhancer elements

-4

-2

0

2

d

2

0

-4

14,636 high confidence Brg1 ChIP peaks

Myc E5 (1577)

Myc E4 (5)

Myc E1 (187)

Myc E3 (193)

Myc E2 (442)

-2

Brg1 ChIP-Seq

-4

-2

0

2

c

2

0

-2

-4

1,403 high confidence FLAG-BRD9 ChIP peaks Log2

(tag

cou

nts

10 μ

M B

I-727

3/ ta

g co

unts

DM

SO)

Log2

(tag

cou

nts

10 μ

M B

I-727

3/ ta

g co

unts

DM

SO)

Myc E1 (79)

Myc E4 (16)

Myc E3 (21)

Myc E5 (534)

FLAG-BRD9 ChIP-Seq

a

FLAG-Brd9 ChIP

ME3M

ME4M

ME5M

DMSO; IgG

BI2; IgG

DMSO; FLAG

BI2; FLAG

DMSO; IgG

10uM BI-7273; IgG

DMSO; FLAG-BRD9

10uM BI-7273; FLAG-BRD9

* < 0.05

** < 0.005

*** < 0.0005

p-value

5

2.5

0

neg Myc-1.8 kb

1 2 3 4 5

neg. new

TSS -1.8

kb

ME1M

ME2M

ME3M

ME4M

ME5M

0.00

0.05

0.10

DMSO; IgG

BI2; IgG

DMSO; FLAG

BI2; FLAG

Myc enhancer elements

*****

****

*

FLAG-BRD9 ChIP

Fra

ctio

n o

f in

pu

t (x

10

-3)

Fra

ctio

n o

f in

pu

t (x

10

-3)


Supplementary Figure 9 | GI50 measurements of BI-7273, LP99, and I-BRD9 in human cell lines

(a) GI50 measurements for BI-7273 across human cancer cell lines. Cells were cultured in the presence of increasing BI-7273 concentrations for 7 days, then cell proliferation was assessed by CellTiter-Glo and normalized to a DMSO control. n = 2

(b,c,d) Cell counts to measure the effect of BRD9 bromodomain inhibitors on the growth of human cancer cell lines. Cells were cultured in the presence of increasing BI-7273 (b), LP99 (c), or I-BRD9 (d) concentrations for 7 days before cell numbers were determined and normalized to DMSO control. Curves were fit by four parameter non-linear regression using the least squares fitting method with the bottom constrained to 0 and the top constrained to 1. n = 3

Extra

.col

umn

EC50

.um

ol.l

Hs 852.TMHH−ES−1G−401A−673OV−90MFE−280RH−41A704QGP−1Capan−1LS411NSK−BR−3MDA−MB−415MCF7HCC1954CAMA−1CAL−85−1SBC−5NCI−H2342NCI−H2196NCI−H2172NCI−H2066NCI−H1793NCI−H1703NCI−H1623NCI−H1563NCI−H1437NCI−H1385EBC−1CAL−12TABC1A549SK−N−DZKM−H2U−2932ToledoTMD8SU−DHL−6SU−DHL−2RI1OCI−Ly3Oci−Ly1 Karpas−422HBL−1FarageBJABK−562MM.1SKMS−11SKM−1OCI−AML3OCI−AML2MV−4−11MOLM−16KG−1Kasumi−1GF−D8GDM−1F−36PEOL−1CMKCESSCESS

CMKEoL-1F-36PGDM-1GF-D8Kasumi-1KG-1MOLM-16MV-4-11OCI-AML2OCI-AML3SKM-1KMS-11MM.1SK-562BJABFarageHBL-1Karpas-422Oci-Ly1 OCI-Ly3RI1SU-DHL-2SU-DHL-6TMD8ToledoU-2932KM-H2SK-N-DZA549ABC1CAL-12TEBC-1NCI-H1385NCI-H1437NCI-H1563NCI-H1623NCI-H1703NCI-H1793NCI-H2066NCI-H2172NCI-H2196NCI-H2342SBC-5CAL-85-1CAMA-1HCC1954MCF7MDA-MB-415SK-BR-3LS411NCapan-1QGP-1A704RH-41MFE-280OV-90A-673G-401MHH-ES-1Hs 852.T

AML

Multiple myelomaCML

DLBCL

Hodgkin’s lymphomaBrain

Lung

Breast

ColonPancreasKidneyLiverUterusOvaryEwing’s sarcomaRhabdoidBone sarcomaMelanoma

CESS

10 30 50Value

Color Key

0 50BI-7273 GI50 (uM)

a

0.1 1 100.01

0.25

0.50

0.75

1.00

0.00

[BI-7273] (uM)

Accu

mul

ated

cel

l num

ber

b

0.1 1 100.01

0.25

0.50

0.75

1.00

0.00

[LP99] (uM)

Accu

mul

ated

cel

l num

ber

c

Jurkat

-2 -1 0 1

0.00

0.25

0.50

0.75

1.00JurkatEOL-1HL60

log10[BI-7273] uM

Jurkat HL60

10.51 μM5.028 μM

GI50

0.1 1 100.01

0.25

0.50

0.75

1.00

0.00

[I-BRD9] (uM)

Accu

mul

ated

cel

l num

ber

d

LP99

-2 -1 0 1

0.00

0.25

0.50

0.75

1.00

JurkatHL60

Log10[LP99] uM

Jurkat

0.75

1.00JurkatEOL-1HL60

Jurkat HL60

3.643 μM0.433 μM

GI50

I-BRD9

-2 -1 0 1

0.00

0.25

0.50

0.75

1.00

JurkatHL60

Log10[I-BRD9] uM

BI2

-2 -1 0 1

0.00

0.25

0.50

0.75

1.00JurkatHL60

Log10[BI-7273], uM

Jurkat

-2 -1 0 1

0.00

0.25

0.50

0.75

1.00JurkatEOL-1HL60

log10[BI-7273] uM

Jurkat HL60

>10 μM2.483 μM

GI50

LP99

BI-7273

I-BRD9


a

BRD9 BD

BRD7 BD

BRD1 BD

BRD4 BD2

CECR2 BD

BRD4 BD1

BRD9 BD

BRD7 BD

BRD1 BD

BRD4 BD2

CECR2 BD

BRD4 BD1

140 160 180

200 220 237

216

Formatted Alignments

hBRD9 isoform 1 Bromodomain

hBRD7 long isoform Bromodomain

hBRD1 Bromodomain

20 40 60

- - - - P I Q Q L L E H F L R Q L Q R K D P H - - G F F A F P V T D A I A - - P G Y S M I I K H P M D F G T M K D K I V A N

- - - - P L Q E A L N Q L M R Q L Q R K D P S - - A F F S F P V T D F I A - - P G Y S M I I K H P M D F S T M K E K I K N N

- - - - P L T V L L R S V L D Q L Q D K D P A - - R I F A Q P V S L K E V - - P D Y L D H I K H P M D F A T M R K R L E A Q

- - - - Q L K C C S G I L K E M F A K K H A A Y A W P F Y K P V D V E A L G L H D Y C D I I K H P M D M S T I K S K L E A R

R Q T N Q L Q Y L L R V V L K T L W K H Q F A - - W P F Q Q P V D A V K L N L P D Y Y K I I K T P M D M G T I K K R L E N N

- - - - D D F T A M Y K V L D V V K A H K D S - - W P F L E P V D E S Y A - - P N Y Y Q I I K A P M D I S S M E K K L N G G

hBRD9 isoform 1 Bromodomain

hBRD7 long isoform Bromodomain

hBRD1 Bromodomain

80 100 120

E Y K S V T E F K A D F K L M C D N A M T Y N R P D - - - T V Y Y K L A K K I L H A G F K M M

D Y Q S I E E L K D N F K L M C T N A M I Y N K P E - - - T I Y Y K A A K K L L H S G M K I L

G Y K N L H E F E E D F D L I I D N C M K Y N A R D - - - T V F Y R A A V R L R D Q G G V V L

E Y R D A Q E F G A D V R L M F S N C Y K Y N P P D H E V V A M A R K L Q D V F E M R F - - -

Y Y W N A Q E C I Q D F N T M F T N C Y I Y N K P G D D I V L M A E A L E K L F L Q K I N E L

L Y C T K E E F V N D M K T M F R N C R K Y N G E S S E Y T K M S D N L E R C F H R A M M K H

66 % 16 %

44 % 15 %

29 % 11 %

Identity Similarity

28 % 11 %

25 % 13 %

b c

f

0.1 1 100.01

0.25

0.50

0.75

1.00

0.00

0.001

BI-7271 (μM)

empty vector 323 nM

BRD9 160 nM

BRD9-BET 4,477 nM

GI50

-3 -2 -1 0 1

0.00

0.25

0.50

0.75

1.00

Accu

mu

late

d c

ell n

um

be

r

g

0.1 1 100.01

0.25

0.50

0.75

1.00

0.00

0.001

BI-7189 (μM)

empty vector 1,709 nM

BRD9 1,029 nM

BRD9-BET >10,000 uM

GI50

-3 -2 -1 0 1

0.00

0.25

0.50

0.75

1.00

Accu

mu

late

d c

ell n

um

be

r

d

5.0

2.5

0.0

Fra

ctio

n o

f in

pu

t (x

10

-4)

neg Myc-1.8 kb

1 2 3 4 5

Myc enhancer elementsneg

TSS -1.8kb

ME1

M

ME2

M

ME3

M

ME4

M

ME5

M

0.000

0.005

0.010

BRD9 IgGBRD9 FLAGB9BD4 IgGB9BD4 FLAG

Fra

ctio

n o

f In

pu

t

IgG

FLAG-BRD9

ME5M


IgG

FLAG-BRD9-BET

ME3MME4M

ME5M


Re

lative

% G

FP

+ &

mC

he

rry+

0.0

0.5

1.0

empty

shRen

shBrd

9

PIG

BRD9

B9BRG1BD

0.0

0.5

1.0

Day 2

Day 4

Day 6

Day 8

Day 10

Day 12

% o

f GF

P &

mC

herr

y +

ve c

ellls

BRD9

BRG1 BD

shRen

shBrd

9

wt

shRen

shBrd

9

e

kDa

70

55

40

100

40

em

pty

wt BRD7 B

D

BRD1 B

D

BRD9

BRD4 B

D1

BRG1 B

D1

BRD9

BRD9-BET

kDa

70

55

40

100

35

130

170

25

BRD9

em

pty

wt BRD7 B

D

BRD1 B

D

BRD9

BRD4 B

D1

mark

er

BRG1 B

D

kDa

70

55

40

100

35

130

170

25

actin

em

pty

wt BRD7 B

D

BRD1 B

D

BRD9

BRD4 B

D1

mark

er

BRG1 B

D


Supplementary Figure 10 | Additional experiments using the bromodomain-swap allele (a) Protein sequence alignment of the BRD9, BRD7, BRD1, BRD4 and CECR2

bromodomains. Residues in BRD7, BRD1, BRD4, CECR2 that are identical to the corresponding amino acid in BRD9 are highlighted in green boxes. The numbering on top of the alignment corresponds to the residue number in full-length BRD9. Sequence identity and similarity were calculated using the gonnet similarity matrix in MacVector.

(b) Western blot to test expression of BRD9 wt and bromodomain-swap alleles in RN2 cells. The antibody recognizes human BRD9 exclusively. For this reason no Brd9 band is observed in the ‘empty’ lane. The actin blot serves to control for loading.

(c) Uncropped images of the Western blots shown in (b). (d) ChIP-qPCR analysis of FLAG-BRD9 and FLAG-BRD9-BET binding to the Myc locus in

RN2 cells. Each experiment included a parallel precipitation using an IgG antibody as a negative control. neg = a ‘negative’ region not bound by Brd9 in RN2 cells. n = 3

(e) cDNA complementation assay to test functionality of bromodomain-swap alleles. wt or mutant BRD9 (linked to GFP, MSCV-based vector) was expressed in RN2 cells prior to expression of shRNAs (linked to mCherry, LMN vector). The percentage of double positive cells was tracked and normalized to day 2 values. shRen targets Renilla luciferase and serves as a negative control. n = 3

(f, g) Cell countss to measure effect of BRD9 bromodomain inhibitors on growth of RN2 cells transduced with and selected for empty vector, BRD9 or BRD9-BET (MSCV-based vector). Cells were cultured in the presence of increasing inhibitor concentrations for 5 days before cell numbers were determined and normalized to DMSO control. Curves were fit by four parameter non-linear regression using the least squares fitting method. GI50 values were derived from non-linear regression curves with the bottom constrained to 0 and the top constrained to 1. n = 3


Supplementary Figure 11 | Evaluation of published BRD9 inhibitors in the bromodomain-swap assay (a,b) Cell counts to measure effect of BRD9 bromodomain inhibitors LP99 (a) and I-BRD9 (b) on

growth of RN2 cells transduced with and selected for empty vector, BRD9 or BRD9-BET (MSCV-based vector). Cells were cultured in the presence of increasing inhibitor concentrations for 7 days (LP99, a) or 5 days (I-BRD9, b) before cell numbers were determined and normalized to DMSO control. Curves were fit by four parameter non-linear regression using the least squares fitting method. GI50 values were derived from non-linear regression curves with the bottom constrained to 0 and the top constrained to 1. n = 4 (a), n = 3 (b)

LP99

-2 -1 0 1

0.00

0.25

0.50

0.75

1.00 PiGBRD9BRD9BD4

log10[LP99], uM0.1 1 100.01

0.25

0.50

0.75

1.00

0.00

[LP99] (μM)

Accu

mul

ated

cel

l num

ber

I-BRD9

-2 -1 0 1

0.00

0.25

0.50

0.75

1.00

log10[I-BRD9] uM0.1 1 100.01

0.25

0.50

0.75

1.00

0.00

[I-BRD9] (μM)

Accu

mul

ated

cel

l num

ber

a b

LP99 I-BRD9

6.144 μM6.866 μM

empty vectorBRD9BRD9-BET 9.382 μM

GI500.757 μM1.252 μM

empty vectorBRD9BRD9-BET 2.687 μM

GI50


Supplementary Figure 12 | GSEA plots accompanying Figure 5

(a) GSEA plot of genes expressed in leukemia stem cells (LSC_Signature_Somervaille) based on RNA-Seq data presented in Fig. 5 (a) and (b) (BI-7273 vs DMSO treated RN2 cells).

(b,c,d) GSEA plots of the top 100 genes up- and down-regulated after 2 days of shBrd9 expression in RN2 cells (shBrd9_Top100Up and shBrd9_Top100Down) and genes up-regulated in Burkitt’s Lymphoma cells induced to express MYC (Schuhmacher_MYC_Targets_Up) based on BI-7273 vs DMSO treated HL60 (b), MV4-11 (c) and HeLa (d) RNA-Seq data sets presented in Fig. 5 (e), (f) and (g), respectively.

FWER p-val, familywise-error rate p-value.

b c d

shBrd9_Top100Up

Enric

hmen

tsc

ore

BI-7273DMSO

-0.7

0 NES: -2.60FWER p-val: 0

shBrd9_Top100Up

Enric

hmen

tsc

ore

BI-7273DMSO

-0.8

0 NES: -2.21FWER p-val: 0.001

shBrd9_Top100Up

Enric

hmen

tsc

ore

BI-7273DMSO

-0.4

0 NES: -1.28FWER p-val: 1

shBrd9_Top100Down

BI-7273DMSO

0.6 NES: 2.40FWER p-val: 0

0

shBrd9_Top100Down

BI-7273DMSO

0.6NES: 2.23

FWER p-val: 0.021

0

shBrd9_Top100Down

BI-7273DMSO

0.1 NES: -0.76FWER p-val: 1

0

-0.2Enric

hmen

tsc

ore

Enric

hmen

tsc

ore

Enric

hmen

tsc

ore

Schuhmacher_Myc_Targets_Up

BI-7273DMSO

0.6NES: 2.38

FWER p-val: 0

0


BI-7273DMSO

0.6 NES: 2.59FWER p-val: 0

0


BI-7273DMSO

0NES: -2.17

FWER p-val: 0.019

-0.5Enric

hmen

tsc

ore

Enric

hmen

tsc

ore

Enric

hmen

tsc

ore

HL60 - BI-7273 MV4-11 - BI-7273 HeLa - BI-7273

Enric

hmen

tsc

ore

LSC_Signature_(Somervaille)

BI-7273DMSO

0.0

0.6NES: 2.63

FWER p-val: 0

aRN2 - BI-7273


Supplementary Figure 13 | Gene expression changes with LP99 and I-BRD9 in RN2 cells

(a,d) RNA-Seq analysis of gene expression changes in RN2 cells treated with LP99 (a) or I-BRD9 (d) at 1 µM for 24 hours. FPKM values from treated cells were normalized to FPKM values recorded in cells cultured in the presence of DMSO. n = 2

(b,e) Gene set enrichment analysis (GSEA) of the RNA-Seq data presented in (a) and (d), respectively. FWER p-val, familywise-error rate p-value.

(c,f) GSEA plots of the top 100 genes up- and down-regulated after 2 days of shBrd9 expression in RN2 cells (shBrd9_Top100Up and shBrd9_Top100Down) based on the RNA-Seq data presented in (a) and (d), respectively.


Supplementary Figure 14 | Gene expression changes with BI-7273 in iMEF cells

(a) RNA-Seq analysis of gene expression changes in iMEF cells treated with after 24 hours of 1 µM BI-7273 treatment. FPKM values from treated cells were normalized to FPKM values recorded in cells cultured in the presence of DMSO. n = 2

(b) Gene set enrichment analysis (GSEA) on the RNA-Seq data presented in (a). FWER p-val, familywise-error rate p-value.

(c) GSEA plots of the top 100 genes up- and down-regulated after 2 days of shBrd9 expression in RN2 cells (shBrd9_Top100Up and shBrd9_Top100Down).


Supplementary Figure 15 | Alignment of EZH2 and EZH1 SET domains

(a) Protein sequence alignment of the EZH2 and EZH1 SET domains. Residues in EZH1 that are identical to the corresponding amino acid in EZH2 are highlighted in green boxes. The numbering on top of the alignment corresponds to the residue number in EZH2. Sequence identity and similarity were calculated using the gonnet similarity matrix in MacVector. EZH2 sequence corresponds to isoform A (NM_001203249.1/NP_001190178.1).

(b) Uncropped images of the Western blots shown in Main Fig. 6 (b).

a640 660

700 720 738

EZH2 SET

EZH1 SET

EZH2 SET

EZH1 SET 94 % 4 %

Identity Similarity

620 680

Formatted Alignments

EZH2 SET domain.prot


20 40 60

K H L L L A P S D V A GW G I F I K D P V Q K N E F I S E Y C G E I I S Q D E A D R R G K V Y D K Y M C S F L F N L N N D F V V D A T

K H L L L A P S D V A GW G T F I K E S V Q K N E F I S E Y C G E L I S Q D E A D R R G K V Y D K Y M S S F L F N L N N D F V V D A T



80 100 120

R K G N K I R F A N H S V N P N C Y A K V M M V N G D H R I G I F A K R A I Q T G E E L F F D Y R Y S Q A D A

R K G N K I R F A N H S V N P N C Y A K V V M V N G D H R I G I F A K R A I Q A G E E L F F D Y R Y S Q A D A

b

actin

kDa

130

100EZH2 40

55

35

170

70

kDa

em

pty

EZH2

EZH2EZH1-SET

mark

er

em

pty

EZH2

EZH2EZH1-SET

mark

er


Supplementary Note 1 | Diagram summarizing the small molecule screening efforts. Supplementary Note 2 | Synthetic route of small-molecules BRD9 inhibitors. Supplementary Data Set 1 | Custom gene sets used in GSEA.


supplementary info draft 12 · 2016-08-18 · supplementary information sensitivity and engineered...

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