Inhibition of bromodomain-containing protein 9 for the prevention of epigenetically-defined drug resistance

Article abstract of DOI:10.1016/j.bmcl.2017.05.063

Bromodomain-containing protein 9 (BRD9), an epigenetic “reader” of acetylated lysines on post-translationally modified histone proteins, is upregulated in multiple cancer cell lines. To assess the functional role of BRD9 in cancer cell lines, we identified a small-molecule inhibitor of the BRD9 bromodomain. Starting from a pyrrolopyridone lead, we used structure-based drug design to identify a potent and highly selective in vitro tool compound 11, (GNE-375). While this compound showed minimal effects in cell viability or gene expression assays, it showed remarkable potency in preventing the emergence of a drug tolerant population in EGFR mutant PC9 cells treated with EGFR inhibitors. Such tolerance has been linked to an altered epigenetic state, and 11 decreased BRD9 binding to chromatin, and this was associated with decreased expression of ALDH1A1, a gene previously shown to be important in drug tolerance. BRD9 inhibitors may therefore show utility in preventing epigenetically-defined drug resistance.

Full text of DOI:10.1016/j.bmcl.2017.05.063

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of bromodomain-containing protein 9 for the prevention of
epigenetically-defined drug resistance
a
b
b
b
Terry D. Crawford a, , Steffan Vartanian , Alexandre Côté , Steve Bellon , Martin Duplessis ,
E. Megan Flynn a, Michael Hewitt b, Hon-Ren Huang b, James R. Kiefer a, Jeremy Murray a,
Christopher G. Nasveschuk b, Eneida Pardo b, F. Anthony Romero a, Peter Sandy b, Yong Tang b,
Alexander M. Taylor b, Vickie Tsui a, Jian Wang c, Shumei Wang a, Laura Zawadzke b, Brian K. Albrecht b,
⇑
Steven R. Magnuson a, Andrea G. Cochran a, David Stokoe a,
⇑
a Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, United States
b Constellation Pharmaceuticals, 215 First Street, Suite 200, Cambridge, MA 02142, United States
c Wuxi Apptec Co., Ltd., 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Bromodomain-containing protein 9 (BRD9), an epigenetic ‘‘reader” of acetylated lysines on post-transla-
tionally modified histone proteins, is upregulated in multiple cancer cell lines. To assess the functional
role of BRD9 in cancer cell lines, we identified a small-molecule inhibitor of the BRD9 bromodomain.
Starting from a pyrrolopyridone lead, we used structure-based drug design to identify a potent and highly
selective in vitro tool compound 11, (GNE-375). While this compound showed minimal effects in cell via-
bility or gene expression assays, it showed remarkable potency in preventing the emergence of a drug
tolerant population in EGFR mutant PC9 cells treated with EGFR inhibitors. Such tolerance has been
linked to an altered epigenetic state, and 11 decreased BRD9 binding to chromatin, and this was associ-
ated with decreased expression of ALDH1A1, a gene previously shown to be important in drug tolerance.
BRD9 inhibitors may therefore show utility in preventing epigenetically-defined drug resistance.
Ó 2017 Elsevier Ltd. All rights reserved.
Received 18 April 2017
Revised 19 May 2017
Accepted 20 May 2017
Available online xxxx
Keywords:
Epigenetics
Bromodomain
BRD9
Inhibitor
Resistance
Bromodomains are a series of 61 different ꢀ110 amino-acid
protein modules that recognize acetylated lysine residues on his-
tones as well as on other proteins.1–7 Significant progress has been
made in elucidating the biological function of the bromodomain
and extra terminal domain (BET) family consisting of dual bromod-
omains in bromodomain-containing protein 2 (BRD2), bromod-
bitors of BRD9 have been disclosed recently.10–13 Herein we report
our discovery of novel inhibitors of BRD9.
While the function of BRD9 is unknown, over-expression has
been observed for several disease tissues. The gene encoding
BRD9 is located on the 5p arm of chromosome 5 and is over-
expressed in cervical cancer as well as non-small cell lung
cancer.14,15 Proteomic analysis has also identified BRD9 as a dedi-
cated member of the mammalian SWI/SNF complex, which has
been postulated as being involved in tumor suppression.16 Finally,
Hohmann, et al. have disclosed that BRD9 may play a role in
hematopoietic cancers and have used a combination of protein
engineering and inhibitor studies to validate the bromodomain
as a target.17
To further understand the functional role of BRD9, we sought an
in vitro BRD9 bromodomain inhibitor tool compound. Our goal was
to achieve <100 nM cellular potency against BRD9, while maintain-
ing at least 100-fold selectivity over bromodomain family mem-
bers in our internal panel of fluorescence resonance energy
transfer (FRET) assays. We also sought BRD4 FRET activity > 10 mM
to ensure that any phenotypic readout at full BRD9 target coverage
would not be obscured by the broad cellular activities observed for
omain-containing protein
3 (BRD3), bromodomain-containing
protein (BRD4), and bromodomain testis-specific protein
4
(BRDT).8,9 However, little is known regarding the biological role
of the other bromodomain family members. Because of this, it is
important to identify potent and selective bromodomain inhibitors
for cellular phenotypic screening in order to elucidate the thera-
peutic significance of targeting bromodomains for oncology,
immunology, and inflammatory diseases. Bromodomain-contain-
ing protein 9 (BRD9) is one such non-BET bromodomain, and inhi-
⇑
Corresponding authors at: Calico Life Sciences, 1170 Veterans Blvd, South San
Francisco, CA 94080, United States (D. Stokoe).
E-mail addresses: terrydc@gene.com (T.D. Crawford), dhstokoe@calicolabs.com
(D. Stokoe).
http://dx.doi.org/10.1016/j.bmcl.2017.05.063
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Crawford T.D., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.05.063

2
T.D. Crawford et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
BET inhibitors. The BRD4 bromodomains served as a surrogate for
all BET bromodomains due to the high degree of sequence identity
among BET family members.
remaining unchanged. We also hypothesized that the methoxy
group would create a steric clash with bromodomains containing
the more rigid WPF shelf (TAF1, BRD4, CECR2) and therefore
increase the selectivity for BRD9. Indeed, we found the selectivity
window for 3 increased at least 5 to 7-fold over most of our inter-
nal bromodomain panel. BRD4(1) was an exception, showing only
a modest selectivity gain. We believe the retained potency for
BRD4(1) may be due to the methoxy group being directed away
from the BRD4(1) WPF shelf and into the ZA channel, therefore
avoiding the steric clash and subsequent potency loss.
The level of cellular engagement by these early lead compounds
was assessed by visualization of the displacement of an inducible
ZsGreen fusion protein localized to chromatin that forms aggrega-
tion ‘‘dots” when displaced.19 These dots can be quantified in a
dose-dependent manner to enable EC50 determination (Table 1).
We found that both the 2-propanol (3) and morpholine amide
(5) substituents have sub-micromolar cellular affinity, making
them attractive candidates for initial phenotypic screening.
We profiled 3 and 5 in a 3-day viability assay across a panel of
652 cancer cell lines representing 32 distinct indications. Activity
was largely restricted to a subset of lines derived from blood and
lymph node (Fig. 2A,B, Supporting Information Tables S1 and S2).
Analysis of 3 in a subset of the full panel (125 lines) in a longer
8-day assay did not reveal any additional sensitivities (Fig. 2C, Sup-
porting information Table S3).
We recently reported the identification of compound 1, a frag-
ment-derived BRD9 small-molecule lead (Fig. 1).18 Importantly,
this molecule incorporates a highly ligand-efficient pyrrolopyri-
done core that forms a two-point hydrogen bonding interaction
with the conserved asparagine N100 (PDB Code: 5I7Y, 1.45 Å).18
The N-crotyl substituent extending from the pyridone induces a
hydrophobic channel adjacent to the conserved binding-pocket
water network and conveys significant selectivity for BRD9 over
the majority of the bromodomain family.
Lead-optimization efforts targeted substitution along multiple
vectors from compound 1. Structural analysis of 1 bound to
BRD9 indicated significant accessible volume extending from the
para position of the benzamide deeper into the ZA channel. Also,
we believed there was an opportunity to occupy a small pocket
formed by the Gly43-Phe44-Phe45 lipophilic shelf in BRD9 by
introduction of small substituents at either the meta or ortho posi-
tion of the benzamide. While potentially beneficial to BRD9
potency, we believed substitution would also result in an unfavor-
able interaction with the corresponding WPF shelves found in
CECR2, TAF1(2), and BET bromodomains, and thereby improve
the selectivity profile. It also appeared possible to introduce func-
tionality at the pyrrolopyridone 2-position, extending toward the
BC loop region with a small aliphatic substituent.
To extend assay conditions beyond conventional 2-dimensional
growth, we applied 3 to cells plated at low density and allowed to
grow for 9 days into single colonies (clonogenic assays). We ana-
lyzed 18 cell lines from skin, lung, breast and pancreatic cancers,
and noted that two cell lines (PC9 and HCC1954) showed modest
sensitivity to 3, albeit only at high concentrations (Fig. 3A). This
is in contrast to sensitivity to the Brd4 inhibitor JQ1,20 which is
extremely potent in this assay (Fig. 3B).
As we probed SAR on the 4-phenyl ring, we found that polar
substituents at the para position such as 2-propanol 2 or morpho-
line amide 5 both improved BRD9 potency, while also providing
increased selectivity over all bromodomains screened (Table 1).
We believe that this may be the result of the ability of the polar
functional groups to interact with solvent in the relatively large
binding pocket found in BRD9.
We next incorporated a methoxy group at the ortho position of
compound 2, as this would rest in a low energy conformation pla-
nar to the phenyl ring and effectively fill the available volume adja-
cent to the BRD9 lipophilic shelf region. While the terminal methyl
may be beneficial for potency, desolvation of the ether oxygen in
this lipophilic region may account for the overall BRD9 potency
To further extend phenotypic assays to those with known
dependence on epigenetic alterations, we tested the ability of
BRD9 inhibitors to affect the drug tolerant state of cells treated
with growth inhibitory compounds. This drug tolerant population
(DTP) is dynamic, transient, and mediated by differences in
chromatin state relative to the untreated parental population.21
Fig. 1. A) Profile of 1 in biochemical bromodomain binding assays (TR-FRET). IC50 values are the average from at least 2 independent experiments B) Compound 1 (cyan)
bound to BRD9 (residues in yellow). PDB Code: 5I7Y, 1.45 Å.
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T.D. Crawford et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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Table 1
SAR of pyrrolopyridone BRD9 inhibitors.a
Bromodomain TR-FRET (
l
M)
BRD9 Dot EC50 (lM)
Compound
R1
H
R2
BRD9
0.053
BRD4(1)
3.3 (62x)
BRD4(2)
CECR2
TAF1(2)
2
6.1 (115x)
4.7 (89x)
5.5 (104x)
0.24
R2
O
7
H 1
N
3
4
H
0.048
0.008
5.1
>20
>20
>20
4.3
>20
>20
0.19
N
6
5
O
O
R1
2
3
4
Me
>20
0.034
R2
O
HO
5
6
7
H
0.031
0.012
0.007
3.4
1.6
3.5
8.3
2.9
1.5
0.38
Me
Me
6.7
0.81
0.46
0.75
1.4
0.045
0.079
12.3
Cl
O
8
9
Me
Me
0.009
0.008
6
14.4
13.5
1.0
2.8
0.035
0.031
H
N
N
N
R1
4.2
0.37
0.52
R2
N
O
10
Me
Me
0.006
0.005
3.3
17.4
1.0
1.7
0.010
0.013
O
O
11 (GNE-375)
>20 (>4000x)
>20 (>4000x)
2.4 (>480x)
>20 (>4000x)
O
O
a
All bromodomain assays were run in TR-FRET format. IC50 data are an average of values from at least 2 independent experiments.
Treatment of PC9 cells with 10 mM 3 or 5 alone for eight days had
minimal effects on cell viability (Fig. 4A). Erlotinib alone decreased
the number of cells by >98%, as expected for PC9 cells (which
ning with the more potent morpholine amide 5, we focused on
substitutions at the 2-position of the pyrrolopyridone, where we
predicted that small substituents would be tolerated. Introducing
a methyl substituent (6) yielded nearly 3-fold biochemical and 8-
fold cellular assay potency gains in BRD9; however we also found
this potency boost to be a common trend with all bromodomains
in our panel (Table 1).
We next explored substitution at the 3-position of the benza-
mide ring, which we hypothesized would direct away from the
lipophilic shelf and further into the ZA channel. Chlorine 7, methyl
8, nitrile 9, and methoxy 10 all provided single-digit nanomolar
potencies against BRD9. Methoxy 10 provided the best cellular
potency (EC50 = 10 nM) as well as the best overall selectivity across
our bromodomain panel, with the window now 550-fold over
BRD4(1), 283-fold over TAF1(2), and 167-fold over CECR2. Finally,
we incorporated a 2,5-dimethoxybenzene with both the 2-propa-
nol (4) and morpholine amide (11) at the 4-position. We hypothe-
sized that this substitution would be well tolerated in BRD9 with
the 2-methoxy group occupying the accessible pocket adjacent to
the lipophilic shelf, while inducing an unfavorable interaction with
BRD4 by forcing one of the two methoxy groups to be directed
towards the BRD4 WPF shelf. Gratifyingly, we found that the 2,5-
dimethoxy substitution maintained single-digit BRD9 potency for
both 4 and 11, with compound 4 now 540-fold selective over
express the activating EGFR mutant
DE746-A750 and are depen-
dent upon EGFR activity); the remaining 2% are the DTPs. The his-
tone deacetylase inhibitor Trichostatin A (TSA) does not affect cell
growth when used alone; however, in combination with erlotinib,
it depletes the drug tolerant persistor (DTP) population, leaving no
residual surviving cells. When BRD9 inhibitor 3 or 5 was combined
with erlotinib, the DTP population was depleted in a dose-depen-
dent manner (Fig. 4B). The BRD4 inhibitor JQ1 was also able to
inhibit DTP formation, but, in contrast to BRD9 inhibitors, the high
concentration of JQ1 required to deplete the DTPs (Fig. 4B) also
decreased viability of PC9 cells in the absence of erlotinib (Fig 4A).
While the initial biology results were intriguing and suggested a
direct role for BRD9 in promoting the appearance of DTP’s, we were
concerned that these results might not be due to BRD9 inhibition
but instead to weak BET family inhibition, as the effects of BRD4
inhibition are highly pleiotropic.22 While both compounds 3 and
5 were >100-fold selective over BRD4(1), the effects of BET bro-
modomain inhibition might still be observable at the higher con-
centrations used in the biological assays (>10 lM). We therefore
continued chemistry efforts to limit BRD4 (1 & 2) biochemical
potencies to >20 mM while also increasing BRD9 potency. Begin-
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T.D. Crawford et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 2. 652 cell lines comprising multiple indications were profiled against 3 (A) and 5 (B) in a 9-point dilution curve using 20 lM as the top concentration. Viability was
measured 3 days (A and B) or 8 days (C) following drug treatment using a CellTiter-Glo luminescent assay. IC50 values on the y-axis are plotted against indication on the x-
axis.
CECR2 and >2500-fold selective over the remaining enzymes in the
panel, while compound 11 was 480-fold selective over CECR2 and
>4000-fold selective against all the other targets we tested. Com-
pounds 4 and 11 also maintained low nanomolar activity in the
BRD9 cellular target engagement assay, with EC50s of 34 and
13 nM respectively.
be selective against the panel, with only BRD7 and CECR2
(KD = 3600 nM, 1800-fold selectivity) producing measurable KDs
under 10 mM, while the selectivity over the remaining bromod-
omains was at least 5000-fold.
Compounds 3, 5, and 11 were screened in an Invitrogen panel of
40 kinases at 1
porting information Tables S5 and S6). Only 5 was found to show
kinase inhibition of >20% at 1 mM (JNK1_ 1 = 25%, TrkA = 21%),
while compounds 5 and 11 had >20% inhibition at 10 mM in the
Cerep panel (PPAR agonist = 49%, 5-HT2B agonist = 22% for 5, ben-
lM as well as a panel of 37 assays at Cerep (Sup-
To further evaluate the selectivity of this series across bromod-
omain and non-bromodomain proteins, compounds 3–5 and 11
were profiled in a BROMOscan panel of 40 bromodomains (Sup-
porting Info Fig. S1, Table S4). Surprisingly, we observed nanomo-
lar potency toward GCN5L2 when incorporating the 2-propanol
functionality (KD = 410 nM for 3, 160 nM for 5), while no activity
was observed with the morpholine amide. Additionally, it is note-
worthy that while potent against GCN5L2, compounds 3 and 5
were inactive against homolog PCAF.23 Compound 4 unexpectedly
retained <10 mM binding to several BET family members. Intrigu-
ingly, while the mono-methoxy compounds 3 and 5 were a modest
10-fold selective for BRD9 over homolog BRD7, the di-methoxy
group yielded additional selectivity over BRD7 with both subseries
(104-fold for 4, 55-fold for 11). Compound 11 not only had exqui-
site BRD9 potency in the panel (KD = 2 nM), but was also found to
a
c
zodiazepine agonist = 86% for 11). Based on the low nanomolar cel-
lular activity, high degree of selectivity, excellent molecular
properties (LogD = 2.4
weight = 452) and high kinetic solubility (135
@
pH 7.4, TPSA = 85, molecular
M), compound 11
l
was identified as our BRD9 in vitro probe compound, GNE-375.
To confirm that 11 binds to endogenous BRD9 in cell lysates we
used the cellular thermal shift (CETSA) assay, which measures pro-
tein denaturation at increasing temperatures in lysates from com-
pound-treated cells.24 Compound 11 (2 mM) significantly increased
the thermal stability of BRD9 (e.g. at 44–50 °C; Fig. 5), but had no
effect on BRD4, consistent with in vitro bromodomain profiling.
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T.D. Crawford et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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Fig. 3. (A) 18 cell lines representing 4 indications were profiled against 3 in a 5-point dilution curve with 20 lM as the top concentration. Cells were stained nine days
following drug treatment with MTT and imaged. Activity is indicated qualitatively by + (strong activity at the highest dose), -/+ (weak inhibition at the highest doses) and –
(no inhibition at any dose). (B) Number of colonies were quantitated and plotted against inhibitor concentrations for PC9 cells (top graph) and HCC1954 cells (bottom graph).
The left-most value for each compound is the number of colonies observed in DMSO.
Fig. 4. PC9 cells were incubated with the indicated compounds for 3 days, and then cell numbers were counted. Erlotinib was added at 1 mM and TSA was added at 50 nM. The
right-hand graph shows a dose response of bromodomain inhibitors added at the same time as 1 mM erlotinib, and cells counted nine days later. The dashed line indicates the
expansion of the y axis between the two graphs, and the dotted line represents the number of cells remaining nine days after erlotinib and 50 nM TSA. Error bars represent
standard deviations, N = 3.
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T.D. Crawford et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 5. 236 cell lines (A) or 254 cell lines (B) comprising multiple indications were profiled against 11 in a 9-point dilution curve using 5 lM (A) or 2 lM (B) as the top
concentration. Viability was measured 3 days (A) or 8 days (B) following drug treatment using a CellTiter-Glo luminescent assay. IC50 values on the y-axis are plotted against
indication on the x-axis.
Conversely, JQ1 (10 mM) showed strong thermostabilization on
BRD4 but had no effect on BRD9 (full temperature range shown
in Fig. S2A).
parental PC9 cells (Fig. 6A), but caused >60% reduction in the num-
ber of DTPs when co-dosed with erlotinib, showing increased
potency relative to 5, consistent with its in vitro properties
(Fig. 6B). We examined the effects of inhibitor treatment on the
expression and localization of BRD9 in parental and drug-tolerant
cells. The overall levels of BRD9 do not change in the drug-resistant
population. However, BRD9 shows redistribution to the nuclear
and chromatin fractions in the DTP population, which is partially
prevented if the cells are pre-treated with 11 (Fig. S2B).
Finally, to understand the consequences of BRD9 inhibition that
might contribute to its effects on DTP formation, we performed
RNAseq analysis of gene expression following 24 h treatment with
the BRD9 inhibitors 3, 4, 5, and 11. Overall the gene expression
changes in response to BRD9 inhibitors were modest (Table S9
and Fig. S4). Compounds 3 and 5 inhibited the expression by >2-
fold of a relatively small number of genes in the drug-tolerant
Because of the enhanced selectivity properties of 11, we revis-
ited the phenotypic effects of BRD9 inhibition in cells. Using a
smaller panel of 236 cell lines, 11 showed no activity in any cell
line tested, even in the 78 blood and lymph node lines tested in
the experiments shown in Fig. 2 (Fig. 5A, Table S7). This suggests
that the activities of 3 and 5 in this assay were off-target (likely
due to BRD4 inhibition). In an extended 8-day viability assay, activ-
ity was restricted to a very small number of cell lines (Fig. 5B,
Table S8). Consistent with reduced cellular activity, 11 was also
inactive in the clonogenic assay in PC9 and HCC1954 cells (Fig. S3).
Next we tested whether the more selective BRD9 inhibitor
retained the ability to suppress DTP numbers following erlotinib
treatment in PC9 cells. 11 showed no growth inhibition of the
Fig. 6. (A) PC9 cells were incubated with the indicated compounds for 3 days, and then cell numbers were counted. (B) Compounds 5 and 11 were added to PC9 cells together
with 1 M erlotinib, and cells counted nine days later. The dotted line represents the number of cells remaining nine days after erlotinib and 50 nM TSA. Error bars represent
l
standard deviations, N = 3.
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T.D. Crawford et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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Fig. 7. (A) Venn diagram showing the number of genes down-regulated >2-fold in DTPs treated with the indicated compounds. (B) mRNA expression of ALDH1A1 in parental
cells and DTPs in untreated (DMSO) and BRD9 inhibitor treated cells. Error bars represent standard deviations, N = 3.
population (59 and 48 respectively), whereas 4 and 11 only inhib-
ited 4 and 7 genes respectively, confirming the increased selectiv-
ity of the later compounds. Interestingly, ALDH1A1 was one of only
three genes commonly down-regulated by all four BRD9 inhibitors
in the drug-tolerant population (Fig. 7A). ALDH genes in general,
and ALDH1A1 in particular, have previously been shown to be
important for the drug-tolerant phenotype.25 Fig. 7B shows that
this gene is down-regulated by BRD9 inhibitors in both parental
and drug-resistant population.
gastric cancer cell lines.25 Moreover, inhibition of total ALDH activ-
ity using disulfiram showed no effect on parental cells, but reduced
the drug-tolerant population following treatment with crizotinib,
similar to the effects seen with BRD9 inhibition. The consequences
of disulfiram were also seen using PC9 cells and erlotinib treat-
ment. These observations beg the question: How might BRD9 be
regulating ALDH1A1 expression? Both ALDH1A1 itself,29 as well
as its promoter30 are regulated by acetylation, suggesting that
BRD9 could be involved in either or both of these mechanisms.
The other two genes regulated by BRD9 in this system are ROS1
and DIRC3. ROS1 levels have not previously been linked to EGFR
inhibitor resistance, but recurrent activating fusions of ROS1 are
found in lung cancer, and resistance to ROS1 inhibitors in these set-
tings can occur via a switch to EGFR dependency.31 Therefore,
ROS1 and EGFR show important cross-talk in lung cancer progres-
sion and therapy. Much less is known about DIRC3, a lncRNA that
was identified as a translocation gene in renal cancer.32 However,
polymorphisms near this gene are associated with breast and thy-
roid cancer.33,34
In conclusion, starting from lead compound 1 we utilized struc-
ture-based drug design to target a potent and selective BRD9 inhi-
bitor. Targeted interactions with the lipophilic shelf as well as
substitution further into the ZA channel provided optimal potency
and greatly enhanced selectivity over the bromodomain family,
ultimately resulting in the identification of an in vitro tool com-
pound (Compound 11, GNE-375) suitable for probing the cellular
consequences of BRD9 bromodomain engagement. In addition,
we have uncovered a previously unknown role of BRD9 in mediat-
ing drug tolerance, which could be a useful avenue to pursue for
future therapeutic intervention strategies.
Selective engagement of the BRD9 bromodomain appears to
have minimal consequences on gross cell morphology or viability
in most cell lines. Only a small subset of leukemia cell lines showed
any effect at concentrations <2 lM, a concentration >100-fold
higher than needed for cellular target engagement. Viability conse-
quences were also only seen in longer-term (8-day) assays, and
were not apparent after 3-day treatments, an observation in accord
with one made previously.17 While the effects on cell viability and
colony growth using our earlier BRD9 inhibitors were likely due to
residual BRD4 inhibition, the effects on drug tolerance remained
potent using highly selective BRD9 inhibitors. Therefore, exquisite
selectivity over BRD4 is needed to dissect out BRD9-specific func-
tions, as previously suggested.26
Genetic alterations such as EGFR T790M are present in a minor
fraction (1 in ꢀ50,000 cells) of the parental population of PC9 cells,
and underlie a rapid resistance mechanism to 1st generation EGFR
inhibitors such as erlotinib. In addition, this mutation in EGFR, as
well as mutations in additional genes, can also emerge de novo fol-
lowing drug treatment, which account for a slower resistance to
EGFR inhibitors.27 However, the nine day assay used here captures
short-term drug tolerant cells which are independent of these
genetic alterations, and instead are predominantly due to epige-
netic changes in H3K4, H3K9 and H3K27 methylation.21 Therefore,
it was not unexpected that compounds regulating epigenetic tar-
gets might show a response in this model. It will be interesting
to see if and how compounds inhibiting epigenetic mechanisms
interact with the long term genetic resistance mechanisms such
as EGFR mutation, KRas, NRas, BRaf and Ret mutations, as well as
met amplification or small cell differentiation.28
Acknowledgements
We would like to thank the Genentech DNA/RNA sequencing,
gCell (cell culture) and gCSI (drug screening) core facilities. Men-
gling Wong, Michael Hayes, and Amber Guillen are acknowledged
for compound purification, and Baiwei Lin, Yanzhou Liu, Deven
Wang, and Yutao Jiang for analytical support. Grady Howes, Jan
Seerveld, Hao Zheng, Ted Peters, Gigi Yuen, Jeff Blaney, Phil Berg-
eron, and Thomas Pillow for help with compound management,
logistics and technical support are also recognized. We would also
like to thank Shiva Malek and Jeff Settleman for helpful advice
throughout this study.
RNAseq experiments showed only very small changes in gene
expression following treatment with these inhibitors. No genes
were altered >2-fold in parental PC9 cells, and only 3 genes were
commonly downregulated in the erlotinib-resistant population.
Of these, ALDH1A1 is of particular interest, as its levels and activity
were previously shown to be increased in crizotinib resistant
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Please cite this article in press as: Crawford T.D., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.05.063