Supporting Information
Materials and Methods
Synthesis of BETi-211, BETd-246 and BETd-260
Scheme I: Synthesis of BETi-211
Ethyl cyanoacetate (2.26 g, 20 mmol) was dissolved in anhydrous DMF (50 mL) and the
solution was cooled to 0 oC. NaH (1.2 g, 60% in mineral oil, 30 mmol) was added in small
portions. The resulting reaction mixture was stirred for 0.5 h at 0 oC and an anhydrous DMF
solution of known compounds S1 (20 mmol, J. Med. Chem. 55: 449-464 (2012)) was added.
The resulting solution was stirred at 0 oC for 3 h before quenching with 1 N HCl. The aqueous
layer was extracted with ethyl acetate and combined organic layers were washed with brine and
dried over anhydrous Na2SO4. The volatile components were removed on a rotary evaporator
and the residue was purified by flash column chromatogram. The desired intermediate S2 was
isolated as colorless oil (75% yield). 1H NMR (300 MHz, CDCl3): 8.41 (s, 1H), 7.11 (s, 1H), 5.60
(s, 1H), 4.24 (q, J = 7.03 Hz, 2H), 4.01 (s, 3H), 1.25 (t, J = 7.14 Hz, 3H).
S2 (1.43 g, 4.2 mmol), 3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole
(2.34 g, 10.5 mmol), and K2CO3 (2.03 g, 14.7 mmol) were added to a round-bottom flask. DME
(30 mL) and water (15 mL) were added at room temperature. The solution was degassed, then
Pd(PPh3)4 (242 mg, 0.21 mmol) was added in one portion. The solution was again degassed,
then heated at reflux for 14 h. The aqueous layer was extracted with ethyl acetate, the
combined organic layers were washed with brine, then dried over anhydrous Na2SO4. The
volatile components were removed on a rotary evaporator and the residue was purified by flash
column chromatogram. The desired intermediate S3 was isolated in 80% yield. 1H NMR (CDCl3,
300 MHz): 8.10 (s, 1H), 7.27 (s, 1H), 5.78 (s, 1H), 4.35 (q, J = 7.12 Hz, 2H), 3.99 (s, 3H), 2.33
(s, 3H), 2.18 (s, 3H), 1.37 (t, J = 7.14 Hz, 3H).
1
To an AcOH (30 mL) solution of S3 (1.47 g) at 80 oC, 0.8 g Zn powder was added in small
portions. The mixture was stirred at 80 oC for 1 h, another 0.8 g Zn powder was added, and the
reaction was kept at the same temperature for 2 h. The reaction was cooled, filtered, and
washed with AcOH. The AcOH solution was combined and the volatile components were
removed on a rotary evaporator. Purification by flash column chromatogram furnished the
desired intermediate 4 (40% yield). 1H NMR (CDCl3, 300 MHz): 8.01 (br, s, 1H), 7.44 (s, 1H),
6.78 (s, 1H), 5.73 (br, s, 2H), 4.40 (q, J = 7.08 Hz, 2H), 3.82 (s, 3H), 2.29 (s, 3H), 2.15 (s, 3H),
1.45 (t, J = 7.08 Hz, 3H). ESI-MS calculated for C17H20N3O4 [M+H]+: 330.15, Obtained: 330.25.
To a round-bottom flask, intermediate 4 (1 g), MeOCH2CN (4 mL) and hydrogen chloride
solution, 4 M in dioxane (4 mL) were added at room temperature. The reaction mixture was
stirred overnight. The volatile components were removed on a rotary evaporator. To this crude
mixture, 10% NaOH aqueous solution (10 mL) and EtOH (20 mL) were added and the solution
was heated at reflux for 8 h. The volatile components were then removed on a rotary evaporator
and the aqueous residue was acidified with 2N HCl aqueous solution. The product was allowed
to precipitate at 0 oC. Filtration of the mixture furnished pure intermediate 5 in 0.8 g. ESI-MS
calculated for C18H19N4O4 [M+H]+ = 355.14; Observed: 355.44. 1H NMR (300 MHz, DMSO) δ
12.15 (s, 1H), 12.09 (s, 1H), 7.59 (s, 1H), 7.26 (s, 1H), 4.39 (s, 2H), 3.84 (s, 3H), 3.38 (s, 3H),
2.29 (s, 3H), 2.09 (s, 3H).
To a round-bottom flask, compound 5 (0.278 g, 0.8 mmol) and POCl3 (8 mL) were added. The
mixture was heated at 90 oC for 6 h. The reaction mixture was cooled to room temperature and
the volatile components were removed on a rotary evaporator. Water (20 mL) and ethyl acetate
(20 mL) were added and the pH was adjusted to 8 using NaHCO3 saturated aqueous solution.
Filtration of the mixture furnished intermediate 6 as a brown solid in 0.22 g. ESI-MS calculated
for C18H18ClN4O3 [M+H]+ = 373.10; Observed: 373.44. 1H NMR (300 MHz, DMSO) δ 7.83 (s,
1H), 7.48 (s, 1H), 4.62 (s, 2H), 3.90 (s, 3H), 3.42 (s, 3H), 2.32 (s, 3H), 2.12 (s, 3H).
Pd2(dba)3 (18 mg) and BINAP (26 mg) were mixed in anhydrous toluene. And the mixture was
heated at reflux for 3-4 minutes. This mixture was transferred into a round-bottom flask
containing compound 6 (60 mg), 3-cyclopropyl-1-ethyl-1H-pyrazol-5-amine (84 mg), K3PO4 (130
mg), and toluene (2 mL). The mixture was heated at reflux for overnight before quenching with
methanol. The reaction mixture was filtered and the mixture was purified by HPLC to yield BETi211 as a CF3CO2H salt in 39 mg. ESI-MS calculated for C26H30N7O3 [M+H]+ = 488.24;
Observed: 488.44. 1H NMR (400 MHz, MeOD) δ 7.46 (s, 1H), 7.15 (s, 1H), 6.00 (s, 1H), 4.66 (s,
2H), 4.12 (q, J = 7.2 Hz, 2H), 3.87 (s, 3H), 3.57 (s, 3H), 2.32 (s, 3H), 2.15 (s, 3H), 2.04 – 1.88
(m, 1H), 1.44 (t, J = 7.2 Hz, 3H), 1.03 – 0.91 (m, 2H), 0.78 – 0.64 (m, 2H).13C (100 M Hz,
MeOD-d4): 166.28, 159.70, 158.95, 154.72, 154.47, 154.40, 153.57, 152.68, 136.56, 131.60,
119.55, 118.62, 144.40, 113.44, 104.01, 98.51, 96.69, 72.35, 58.26, 55.36, 42.76, 13.98, 10.06,
9.22, 8.84, 7.10.
Scheme II: Synthesis of BETd-246
2
To a round-bottom flask, compound 4 (0.37 g, 1.1 mmol) and ethyl cyanoformate (3 mL) were
added at room temperature. Hydrogen chloride solution in dioxane was added and the reaction
mixture was warmed up to reflux (82 oC) for 2.5 h. The reaction was then cooled to room
temperature and the volatile components were removed on a rotary evaporator. To this crude
mixture, 10% NaOH aqueous solution (20 mL) and EtOH (50 mL) were added and the solution
was heated at reflux for 6 h. The volatile components were then removed on a rotary
evaporator and the aqueous residue was acidified with 2N HCl aqueous solution. The product
5' was allowed to precipitate at 0 oC. Filtration of the mixture furnished pure intermediate 5' as
a solid in 0.31 g (80% yield, 2 steps). ESI-MS calculated for C17H15N4O5 [M+H]+ = 355.10,
Obtained: 355.45.
To a round-bottom flask, conc. H2SO4 (0.5 mL) were added to a solution of compound 5' (0.2 g)
in MeOH (30 mL) at room temperature. The mixture was stirred for 10 hour at reflux and the
volatile components were removed on a rotary evaporator. Then ethyl acetate (5 mL) was
added. The product 6' was allowed to precipitate. Filtration of the mixture furnished pure 6' as a
solid in 0.16 g. 1H NMR (300 MHz, MeOD-d4) δ 7.86 (s, 1H), 7.39 (s, 1H), 4.07 (s, 3H), 3.93 (s,
3H), 2.34 (s, 3H), 2.17 (s, 3H).
3
To a round-bottom flask, 6' (0.278 g) and POCl3 (8 mL) were added. The mixture was heated
at 90 oC for 6 h. The reaction mixture was cooled to room temperature and the volatile
components were removed on a rotary evaporator. Water (20 mL) and ethyl acetate (20 mL)
were added and the pH was adjusted to 8 using NaHCO3 saturated aqueous solution. Filtration
of the mixture furnished intermediate 7 as a brown solid in 0.208 g. 1H NMR (300 MHz, MeODd4) δ 8.02 (s, 1H), 7.55 (s, 1H), 4.07 (s, 3H), 3.99 (s, 3H), 2.37 (s, 3H), 2.20 (s, 3H).
Pd2(dba)3 (18 mg) and BINAP (26 mg) were mixed in anhydrous toluene. And the mixture was
heated at reflux for 3-4 minutes. This mixture was transferred into a round-bottom flask
containing 7 (60 mg), 3-cyclopropyl-1-ethyl-1H-pyrazol-5-amine (84 mg), K3PO4 (130 mg), and
toluene (2 mL). The mixture was heated at reflux for overnight before quenching with methanol.
Then MeOH(4 mL), H2O (4 mL) and LiOH (10 mg) was added and the reaction mixture was
stirred at room temperature for 2 hours. Then the reaction mixture was acidified with 2N HCl
aqueous solution and was purified by HPLC to yield intermediate 8 as a CF3CO2H salt in
10 mg. ESI-MS calculated for C25H26N7O4 [M+H]+ = 488.20; Observed: 488.4.
To a round-bottom flask, the commercially available 9 (276 mg, 1.0 mmol) was dissolved in 3.0
mL of anhydrous DMF. Amine 10 (320 mg, 1.0 mmol) and DIPEA (259 mg, 2.0 mmol) were
added. The reaction mixture was stirred at 90 oC for 12 h. The mixture was cooled to room
temperature, poured into water and extracted with ethyl acetate for two times. The combined
organic layer was washed with brine, dried over anhydrous Na2SO4. After filtration and
evaporation, the crude residue was purified by HPLC with H2O/ MeCN to give compound 11 as
colorless oil (172 mg, 30% yield). ESI-MS calculated for C28H41N4O9 [M+H]+ = 577.2; Observed:
577.3.
To a round-bottom flask, 11 (15 mg) was dissolved in 3 mL of DCM and TFA (2:1). After stirring
for 1 h, the solvent was evaporated to give the crude product 12, which was used in the next
step without further purification.
To a round-bottom flask, N,N-diisopropylethylamine (50 mg) were added to a solution of 8 (20
mg), HATU (20 mg), and 12 (40 mg) in DMF (1 mL) at room temperature. The mixture was
stirred for 30 min and purified by HPLC to yield BETd-246 as a CF3CO2H salt in 14 mg. ESIMS calculated for C48H56N11O10 [M+H]+ = 946.4; Observed: 946.5. 1H NMR (400 MHz, MeOD) δ
7.55 (s, 1H), 7.39 – 7.30 (m, 2H), 6.86 (d, J = 8.6 Hz, 1H), 6.80 (d, J = 7.0 Hz, 1H), 6.20 (s,
1H), 4.97 (dd, J = 11.7, 4.4 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 3.88 (s, 3H), 3.71 – 3.48 (m,
16H), 2.86 – 2.57 (m, 3H), 2.36 (s, 3H), 2.19 (s, 3H), 2.08 – 1.99 (m, 2H), 1.95 – 1.86 (m, 2H),
1.83 – 1.74 (m, 2H), 1.48 (t, J = 7.2 Hz, 3H), 1.12 – 1.04 (m, 2H), 0.87 – 0.80 (m, 2H).
Scheme III: Synthesis of BETd-260
4
To a round-bottomed flask were added 13 (1.6 g, 4.96 mmol, 1.0 eq), 14 (1 g, 5.46 mmol, 1.1
eq), Pd(PPh3)Cl2 (348 mg, 0.50 mmol, 0.1 eq) and CuI (190 mg, 0.992 mmol, 0.2 eq). 15 mL of
DMF was added. The solution was vacuumed by sonication and refilled with nitrogen for three
times. Then 15 mL of trimethylamine was injected and the solution was vacuumed by sonication
and refilled with nitrogen again for three times. The resulted solution was stirred at 80 oC for 412 h. After cooling to ambient temperature, the solution was diluted in methanol and filtered
through celite. Workup with DCM and saturated brine afforded the crude product, which was
purified by anhydrous Na2SO4. After filtration and concentration, the residue was purified by
flash column chromatography with DCM: MeOH to afford compound 15.
The above obtained compound 15 was dissolved in 20 mL of DCM and 5 mL of TFA. The
reaction mixture was stirred at room temperature for 1 h. After concentration, the residue was
purified by reverse semi-preparative HPLC to afford compound 16.
The above obtained compound 16 was dissolved in methanol. 10% Pd/C was added under
nitrogen. The solution was vacuumed and refilled by hydrogen for three times. The solution was
stirred at room temperature for 5 h. Filtration and concentration afforded the desired compound
s5 as a white solid (after frozen-drying on lyophilizer), which was used in next step without
further column purification (1.18 g, 54 % yield for three steps).
To a round bottomed flask were added compound 8 (315 mg, 0.647 mmol, 1.0 eq), 17 (0.906
mmol, 1.4 eq), HATU (319 mg, 0.841 mmol, 1.3 eq) and 5.0 mL of DMF. DIPEA (0.56 mL, 3.235
mmol, 5.0 eq) was added. The solution was stirred at room temperature for 1-2 h. The solution
5
was purified by reverse semi-preparative HPLC to afford the desired product BETd-260 as a
yellow solid (388 mg, 75.2 % yield).
H NMR (400 MHz, DMSO-d6) δ (ppm) 12.21 (s, 1H), 10.98 (s, 1H), 9.29 (s, 1H), 8.18 (t, J = 5.2
Hz, 1H), 7.57 (d, J = 6.8 Hz, 2H), 7.49-7.43 (m, 2H), 7.35 (s, 1H), 5.93 (s, 1H), 5.13 (dd, J =
13.2 Hz, J = 5.2 Hz, 1H), 4.49 (d, J = 17.2 Hz, 1H), 4.33 (d, J = 17.2 Hz, 1H), 3.95 (q, J = 7.2
Hz, 2H), 3.83 (s, 3H), 3.29-3.24 (m, 2H), 2.96-2.87 (m, 1H), 2.70-2.44 (m, 4H), 2.30 (s, 3H),
2.10 (s, 3H), 2.03-2.00 (m, 1H), 1.88-1.85 (m, 1H), 1.67-1.65 (m, 2H), 1.58-1.54 (m, 2H), 1.391.24 (m, 5H), 0.84-0.82 (m, 2H), 0.63-0.59 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ (ppm)
172.87, 171.04, 168.37, 165.48, 162.96, 159.17, 156.48, 155.10, 154.07, 152.42, 152.20,
140.49, 137.46, 136.47, 131.88, 131.56, 131.43, 128.26, 120.60, 119.04, 117.43, 113.85,
113.41, 104.51, 98.35, 97.22, 56.01, 51.55, 46.24, 42.41, 31.16, 29.00, 28.80, 26.30, 22.51,
14.86, 11.28, 10.34, 9.58, 7.59; ESI+-MS calculated for C43H47N10O6 [M+1]+: 799.37, found
799.19.
1
Molecular modeling
The co-crystal structure of BRD4 BD2/RX37 (PDB entry: 4Z93)(1) was used to model the
binding poses of BETi-211 with BRD4 BD2. Chain A of BRD4 BD2 from the crystal structure
was extracted and the “protonate 3D” module in MOE(2) was used to add protons to BRD4 BD2
according to the pH 7.0 condition. All water molecules from the crystal structure were saved.
The structure of BETi-211 was drawn and optimized using the MOE program. Twenty binding
poses of BETi-211 to BRD4 BD2 was generated by the GOLD program (version 4.0.1)(3, 4) in
which the highest ranked pose was selected as the binding model. In the docking simulation,
the center of the binding site as set at V439 in BRD4 BD2 and the radius of the binding site was
defined as 11 Å to cover the entire binding pocket. At the binding site, water molecules W2, W3,
W7, W9, W13 and W15 of BRD4 BD2 (PDB entry: 4Z93) were included during the docking
simulations with the flags on. GoldScore implemented in Gold 4.0.1 was used as the fitness
function to evaluate the docked poses. The parameters for the genetic algorithm (GA) run are a
maximum number of 200,000 operations for a population of 5 islands with 100 individuals each
and 95, 95,10 for the operator weights of crossover, mutation and migration.
The crystal structure of DDB1-cereblon bound to thalidomide (PDB entry: 4CI1)(5) was used to
generated the structure between cereblon and thalidomide in Fig. 1B. Fig. 1B is prepared using
the PyMOL program (www.pymol.org).
Antibodies and Reagents
Antibodies. Rabbit mAbs for Mcl-1 (#5453 and 94296), Bcl-2 (#4223), Bcl-xL (#2764), c-Myc
(#5605 and 13987), PARP (#9532), Caspase-3 (#9665) and p21Waf1/Cip1, and mouse mAbs
for Caspase-9 (C9) (#9508), Caspase-8 (1C12) (#9746), Caspase-2 (C2) (#2224), and HA-Tag
(#2367) were obtained from Cell Signaling Technology. Rabbit polyclonal antibodies for BRD2
(A302-583A), BRD3 (A302-368A) and BRD4 (A301-985A100) were from Bethyl Laboratories.
ChIP Grade Anti-Histone H3 (acetyl K27) antibody (ab4729) was from Abcam. Rabbit antiCRBN antibodies (HPA045910 and SAB2106014) were from Sigma. Rabbit polyclonal
6
antibodies for Bcl-2 (sc-492) and GAPDH (sc-25778 HRP), and mouse monoclonal antibodies
for c-Myc (9E10, sc-40), Bcl-2 (sc-509), Bcl-xL (sc-8392) and actin (sc-8432 HRP) were
obtained from Santa Cruz Technology.
Reagents. CellTiter Glo and Caspase-Glo 2/3/7/8/9 Assay Systems were purchased from
Promega. Annexin-V-FLUOS Staining Kit were from Roche Life Science.
Transcriptomic Profiling
RNA-seq. Total RNA was purified using RNeasy Mini Kit (Qiagen) following the manufacturer's
instructions. RNA was assessed for quality using the TapeStation (Agilent, Santa Clara, CA).
Samples with RINs (RNA Integrity Numbers) of 8 or greater were ribosomal RNA depleted using
Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) (cat# MRZH11124) (Illumina, San Diego,
CA). The rRNA depleted samples were then prepared using the TruSeq mRNA Sample Prep v2
kit (Catalog #s RS-122-2001, RS-122-2002) (Illumina, San Diego, CA). Where the entire fraction
of 0.1-3ug of rRNA depleted total RNA was fragmented and copied into first strand cDNA using
reverse transcriptase and random primers. The 3 prime ends of the cDNA were then adenylated
and adapters were ligated. One of the adapters that was ligated had a 6 nucleotide barcode
that was unique for each sample which allowed us to sequence more than one sample in each
lane of a HiSeq flow cell (Illumina). The products are purified and enriched by PCR to create the
final cDNA library. Final libraries were checked for quality and quantity by TapeStation (Agilent)
and qPCR using Kapa’s library quantification kit for Illumina Sequencing platforms (catalog #
KK4835) (Kapa Biosystems,Wilmington MA). They were clustered on the cBot (illumina) and
sequenced 16 samples per lane on a 50 cycle single end High Output mode using version 4
reagents and HiSeq Control Software version 2.2.68 according to manufacturer’s protocols.
Demultiplexing and Fastq file generation was done using bcl2fastq version 2.17.1.14. RNA-seq
data are deposited at GEO as xx.
Data analyses. Raw data were normalized into reads per kilobase per million (RPKM) values.
Genes with the average RPKM values less than 1.00 were removed from the analysis. All the
RPKM values were then added with one before the analysis to avoid singularity in the fold
change calculations. The MultiplotPreprocess module from the GenePattern(6) website were
used to calculate the overall fold changes and the corresponding p-values in paired comparison.
They include thalidomide versus DMSO, BETi-211 versus DMSO, BETd-246 versus DMSO at 3
h treatment using three cell lines with duplicates. The data were plotted using the R program.
The GSEA program(7) obtained from Broad Institute was used in the gene set enrichment
analysis. The recently curated 50 hallmark gene sets(8) were used to identify signature genes
enriched in BETi-211 versus DMSO and BETd-246 versus DMSO treatment. In the analysis,
3000 phenotype permutations were applied to determine the significance of the enrichment for
the gene sets with FDR q value < 0.05. A threshold of the enriched gene sets was set at the
Spermatogenesis gene set which deems less relevant in the female breast cells.
QPCR primers
7
TaqMan assays:
Gene Symbol
Cat#
AJUBA
Hs01036974_m1
AURKA
Hs01582072_m1
BRD2
Hs01121986_g1
BRD3
Hs00201284_m1
BRD4
Hs04188087_m1
BTG1
Hs00982890_m1
CCNF
Hs00171049_m1
CCNT1
Hs01059085_m1
CDC25A
Hs00947994_m1
CDK9
Hs00977896_g1
CDKN1A
Hs00355782_m1
DYRK3
Hs00534092_m1
EGR1
Hs00152928_m1
FOS
Hs04194186_s1
GADD45B
Hs04188837_g1
GAPDH
Hs02758991_g1
GTF2B
Hs00976255_m1
HEXIM1
Hs00538918_s1
IL6
Hs00985639_m1
ING2
Hs00357543_m1
IRF1
Hs00971960_m1
JUND
Hs04187679_s1
KLF4
Hs00358836_m1
8
MCL1
Hs01050896_m1
MED18
Hs00214189_m1
MED9
Hs00215993_m1
MP3K1
Hs00394890_m1
MYC
Hs00153408_m1
MYCL
Hs00420495_m1
PIM2
Hs00179139_m1
PLK2
Hs01573405_g1
RIPK4
Hs00221005_m1
SGK1
Hs00985033_g1
TRAF6
Hs00371512_g1
WEE1
Hs01119384_g1
9
SYBR green primers:
Oligo Name
Sequence (5' to 3')
FOS-F
CACTCCAAGCGGAGACAGAC
FOS-R
AGGTCATCAGGGATCTTGCAG
MED9-F
AACGCCCTCAAAAGCAAGTTC
MED9-R
GCAGAAGCTCATTCTTGGTCCT
MED18-F
ACAGCCAGAAATGGGAGACAA
MED18-R
TGATGCCCTTACGGAACAAATG
HEXIM1-F
CCGAGGCCAGTAAGTTGGG
HEXIM1-R
GACGGGCGTCTCCTATGTTT
PCGF1-F
CTTTGACCACTCTAAAGCCCAC
PCGF1-R
CAGCTCTAACAGAACATCGGAC
AJUBA-F
ATGGGGAAGTCCTATCATCCAG
AJUBA-R
TGGTAGTCGGTGACACAGTAT
AUNIP-F
TTGGGACTTAGGGCCGTTTC
AUNIP-R
CCTTCCTCACCGCAGATGTT
BTG1-F
AGCGGATTGGACTGAGCAG
BTG1-R
GGTGCTGTTTTGAGTGCTACC
CCNF-F
GGAAAGCGACAGGAGGACAG
CCNF-R
TGGCAGACGATCTCACTGGAA
CHAMP1-R
GCCCTGGTTTCCAAGAGCC
CHAMP1-F
CCATCCCCTTCAGAGTCTCCT
DYRK3-F
TGGTGGTCCCAATAATGGAGG
DYRK3-R
CACGTACTGTCGAAGTTTGTGAT
10
EIF2AK3-R
ACTATGTCCATTATGGCAGCTTC
EIF2AK3-F
GGAAACGAGAGCCGGATTTATT
ELL-R
TACTCGGCATTGAAGTCGTTC
ELL-F
GTCGGAGACGCCTGACTACT
EPC1-R
GAGGCGTATTCGTGCAGGTC
EPC1-F
ATGAGTAAACTGTCGTTTCGGG
FEM1C-R
CAGAAGCGGCCCATAAAGGG
FEM1C-F
GTGCCTCCATAGAAGTTGGGG
FRAT2-F
AGCTCGTGCTCTCGGGAA
FRAT2-R
ACTGGACGCTGTTGGGTCTA
GAPDH-R
GGCATGGACTGTGGTCATGAG
GAPDH-F
TGCACCACCAACTGCTTAGC
GTF2B-F
TTCTGTTCCAACCTTTGTCTTCC
GTF2B-R
GCAACACCAGCAATATCTCCA
ING2-R
GACTCCACGCACTCAAGGTA
ING2-F
GCAGCAACTGTACTCGTCG
IRF1-R
ATCCCCACATGACTTCCTCTT
IRF1-F
CTGTGCGAGTGTACCGGATG
JUND-F
TCATCATCCAGTCCAACGGG
JUND-R
TTCTGCTTGTGTAAATCCTCCAG
MAD2L1BP-R
CCGAAGCGTTGAGAGGTTCC
MAD2L1BP-F
GAGAAGTCCGAAGAAACTCACG
MCPH1-F
GTAGTCACCCCTGACCAAAAG
MCPH1-R
GCAGCCTCGGCATGATAGA
PLK2-F
CCGTCGGTGTCCTTTTCAACA
11
PLK2-R
CTCCACCATCCATGAGGTTCT
RIPK4-F
CAGAAGAAGCCGTTTGCAGAT
RIPK4-R
GAGGCGTATCAGGTGGCTG
SUV39H1-F
CAAGTTTGCCTACAATGACCAGG
SUV39H1-R
GTACCACACGATTTGGGCAGT
TIFA-F
ATACAGGTGCATGGTCAGATTCG
TIFA-R
TCTGTCGGAGAACTGCTTTGG
TRAF6-F
TTTGCTCTTATGGATTGTCCCC
TRAF6-R
CATTGATGCAGCACAGTTGTC
WEE1-F
AACAAGGATCTCCAGTCCACA
WEE1-R
GGGCAAGCGCAAAAATATCTG
mMYC-F
ATGCCCCTCAACGTGAACTTC
mMYC-R
GTCGCAGATGAAATAGGGCTG
mGAPDH-F
AGGTCGGTGTGAACGGATTTG
mGAPDH-R
GGGGTCGTTGATGGCAACA
mMCL1-F
GACGACCTATACCGCCAGTC
mMCL1-R
AGAGGCTTCGAGTCCTTGGA
Proteomic Profiling
Protein Digestion and TMT labeling: Cells were treated with DMSO, BETi-211 (1000 nM) and BETd246 (100 nM) for 2 h then lysed in RIPA buffer. Cell lysis samples (75 µg/condition) were proteolysed
and labeled with TMT 10-plex essentially by following manufacturer’s protocol (Thermo Fisher). Briefly,
upon reduction and alkylation of cysteines, the proteins were precipitated by adding 6 volumes of ice
cold acetone followed by overnight incubation at -20 C. The precipitate was spun down, and the pellet
was allowed to air dry. The pellet was resuspended in 0.1M TEAB and overnight digestion with trypsin
(1:50; enzyme:protein) at 37 C was performed with constant mixing using a thermomixer. The TMT
10-plex reagents were dissolved in 41 µl of anhydrous acetonitrile and labeling was performed by
transferring the entire digest to TMT reagent vial and incubating at room temperature for 1 h. Reaction
was quenched by adding 8 µl of 5% hydroxyl amine and further 15 min incubation. Labeled samples
12
were mixed together, and dried using a vacufuge. An offline fractionation of the combined sample
(~200 µg) into 10 fractions was performed using high pH reversed-phase peptide fractionation kit
according to the manufacturer’s protocol (Pierce; Cat #84868). Fractions were dried and reconstituted
in 12 µl of 0.1% formic acid/2% acetonitrile in preparation for LC-MS/MS analysis.
Liquid chromatography-mass spectrometry analysis (LC-multinotch MS3): In order to obtain superior
quantitation accuracy, we employed multinotch-MS3 (9) which minimizes the reporter ion ratio distortion
resulting from fragmentation of co-isolated peptides during MS analysis. Orbitrap Fusion (Thermo
Fisher Scientific) and RSLC Ultimate 3000 nano-UPLC (Dionex) was used to acquire the data. Two µl
of the sample was resolved on a PepMap RSLC C18 column (75 µm i.d. x 50 cm; Thermo Scientific) at
the flow-rate of 300 nl/min using 0.1% formic acid/acetonitrile gradient system (2-22% acetonitrile in
150 min;22-32% acetonitrile in 40 min; 20 min wash at 90% followed by 50 min re-equilibration) and
directly spray onto the mass spectrometer using EasySpray source (Thermo Fisher Scientific). Mass
spectrometer was set to collect one MS1 scan (Orbitrap; 60K resolution; AGC target 2x10 5; max IT 100
ms) followed by data-dependent, “Top Speed” (3 seconds) MS2 scans (collision induced dissociation;
ion trap; NCD 35; AGC 5x103; max IT 100 ms). For multinotch-MS3, top 10 precursors from each MS2
were fragmented by HCD followed by Orbitrap analysis (NCE 55; 60K resolution; AGC 5x104; max IT
120 ms, 100-500 m/z scan range).
Data analysis: Proteome Discoverer (v2.1; Thermo Fisher) was used for data analysis. MS2 spectra
were searched against SwissProt human protein database (release 2015-11-11; 42084 sequences)
using the following search parameters: MS1 and MS2 tolerance were set to 10 ppm and 0.6 Da,
respectively; carbamidomethylation of cysteines (57.02146 Da) and TMT labeling of lysine and Ntermini of peptides (229.16293 Da) were considered static modifications; oxidation of methionine
(15.9949 Da) and deamidation of asparagine and glutamine (0.98401 Da) were considered variable.
Identified proteins and peptides were filtered to retain only those that passed ≤1% FDR threshold.
Quantitation was performed using high-quality MS3 spectra (Average signal-to-noise ratio of 20 and
<30% isolation interference).
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