Degradation of the BAF Complex Factor BRD9 by Heterobifunctional Ligands

Article abstract of DOI:10.1002/anie.201611281

The bromodomain-containing protein BRD9, a subunit of the human BAF (SWI/SNF) nucleosome remodeling complex, has emerged as an attractive therapeutic target in cancer. Despite the development of chemical probes targeting the BRD9 bromodomain, there is a limited understanding of BRD9 function beyond acetyl-lysine recognition. We have therefore created the first BRD9-directed chemical degraders, through iterative design and testing of heterobifunctional ligands that bridge the BRD9 bromodomain and the cereblon E3 ubiquitin ligase complex. Degraders of BRD9 exhibit markedly enhanced potency compared to parental ligands (10- to 100-fold). Parallel study of degraders with divergent BRD9-binding chemotypes in models of acute myeloid leukemia resolves bromodomain polypharmacology in this emerging drug class. Together, these findings reveal the tractability of non-BET bromodomain containing proteins to chemical degradation, and highlight lead compound dBRD9 as a tool for the study of BRD9.

Full text of DOI:10.1002/anie.201611281

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Communications
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International Edition: DOI: 10.1002/anie.201611281
German Edition: DOI: 10.1002/ange.201611281
Protein Degraders
Degradation of the BAF Complex Factor BRD9 by Heterobifunctional
Ligands
David Remillard, Dennis L. Buckley, Joshiawa Paulk, Gerard L. Brien, Matthew Sonnett,
Hyuk-Soo Seo, Shiva Dastierdi, Martin Wꢀhr, Sirano Dhe-Paganon, Scott A. Armstrong, and
James E. Bradner*
Abstract: The bromodomain-containing protein BRD9, a sub-
unit of the human BAF (SWI/SNF) nucleosome remodeling
complex, has emerged as an attractive therapeutic target in
cancer. Despite the development of chemical probes targeting
the BRD9 bromodomain, there is a limited understanding of
BRD9 function beyond acetyl-lysine recognition. We have
therefore created the first BRD9-directed chemical degraders,
through iterative design and testing of heterobifunctional
ligands that bridge the BRD9 bromodomain and the cereblon
E3 ubiquitin ligase complex. Degraders of BRD9 exhibit
markedly enhanced potency compared to parental ligands (10-
to 100-fold). Parallel study of degraders with divergent BRD9-
binding chemotypes in models of acute myeloid leukemia
resolves bromodomain polypharmacology in this emerging
drug class. Together, these findings reveal the tractability of
non-BET bromodomain containing proteins to chemical
degradation, and highlight lead compound dBRD9 as a tool
for the study of BRD9.
contributed an enhanced mechanistic understanding of gene
control and has availed new therapeutic opportunities in
cancer.[2,3] Indeed, multiple BET inhibitor candidates from
several groups are now undergoing clinical trials for diverse
indications in and outside of oncology.[4–6] Further, significant
research effort has contributed a number of high quality
chemical probes for bromodomain-containing proteins
beyond the BET family.[7,8]
The bromodomain-containing protein BRD9 has gar-
nered particular attention as a component of the human ATP-
dependent chromatin remodeling BAF complex (also known
as SWI/SNF). Meta-analyses of whole-genome sequencing
efforts have recently identified a high frequency of recurrent
somatic mutations in BAF factors in diverse human cancers.[9]
Within several subsets of these genetically defined malignan-
cies, components of the BAF complex have emerged as
context-specific dependencies, either supporting growth
within a residual complex following loss of function mutation,
or as novel oncogenes such as the SS18-SSX fusion.[10] These
observations have generated interest in therapeutic strategies
to target BAF.
Beyond its presence in the BAF complex, a lack of
functional annotation for BRD9 has provided incentive for
development of BRD9 selective inhibitors to interrogate its
biological role and to assess any therapeutic potential. Several
BRD9-directed efforts in discovery chemistry have been
reported,[11–15] developing chemotypes for BRD9-specific
engagement. Further, a recent study implicated BRD9 as
a dependency in acute myeloid leukemia (AML), where
bromodomain inhibition prompted a cytostatic response.[16]
Beyond the bromodomain, the function of BRD9 remains
unclear, and chemical tools to study other functions of BRD9
are not available. We therefore undertook to create first
chemical probes that destabilize BRD9, anticipating that the
study of acute BRD9 loss would offer a powerful approach to
interrogate BAF complex function.
Recently, we reported a strategy to direct protein-specific
degradation using bifunctional molecules to recruit the
cereblon (CRBN) ubiquitin ligase complex to non-physio-
logic protein substrates,[17] providing an all-chemical solution
to prior efforts using peptides to bridge E3 ligases and ligand
targets (PROTACs).[18,19] In our prior research, we directed
the degradation of BET family proteins by appending CRBN
ligands to JQ1, resulting in rapid and potent degradation of
BRD2, BRD3, and BRD4. These findings have been well
validated, suggesting a broader utility of this strategy.[20–22]
Toward the elaboration of BRD9-directed degraders, we
initially evaluated putative pharmacophores from reported
S
mall-molecule inhibitors of BRD4 established the feasi-
bility of inhibiting acetyl-lysine recognition domains (bromo-
domains).[1] The broad use of the chemical probe JQ1 and
other inhibitors of the bromodomain and extra-terminal
domain (BET) subfamily of transcriptional co-activators has
[*] D. Remillard, Dr. D. L. Buckley, Dr. J. L. Paulk, S. Dastierdi,
Dr. J. E. Bradner
Department of Medical Oncology, Dana-Farber Cancer Institute
Boston, MA (USA)
E-mail: james_bradner@dfci.harvard.edu
james.bradner@novartis.com
Dr. G. L. Brien, Dr. S. A. Armstrong
Department of Pediatric Oncology, Dana-Farber Cancer Institute
Boston, MA (USA)
M. Sonnett
Department of Systems Biology, Harvard Medical School
Boston, MA (USA)
M. Sonnett, Dr. M. Wꢀhr
Lewis-Sigler Institute for Integrative Genomics, Princeton University
Princeton, NJ (USA)
Dr. H.-S. Seo, Dr. S. Dhe-Paganon
Department of Cancer Biology, Dana-Farber Cancer Institute
Boston, MA (USA)
Dr. M. Wꢀhr
Department of Molecular Biology, Princeton University
Princeton, NJ (USA)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201611281.
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Figure 1. Design and characterization of thienopyridinone BRD9-targeted degraders. A) Structures of select BRD9 bromodomain probes.
B) Schematic representation of degrader design. C) Vehicle-normalized BRD9(bd) displacement (AlphaScreen quadruplicate means ÆSEM).
D) Compound-induced ternary complex formation of recombinant BRD9(bd) and CRBN–DDB1 (AlphaScreen quadruplicate means ÆSEM).
E) Cocrystal structure of 3 with BRD9(bd) (PDB 5TWX). F) Docking of (E) into the published CRBN–DDB1 structure (4CI3).
bromodomain probes LP99 and I-BRD9, and subsequently
expanded our study to a third probe, BI7273, reported during
the course of this research (Figure 1A).[12,14,15] To explore the
potential of bifunctional derivatives to induce BRD9 degra-
dation, we initially selected as our starting point a close
chemical analog of I-BRD9 described by GlaxoSmithKline
(GSK-39).[14] This ligand was attractive in that it offered high
binding affinity (IC50 = 7.9 nm) as well as a solvent exposed
methoxy substituent amenable to chemical derivatization. In
our initial design strategy, we adapted this ligand by installing
an ether-linked acetyl moiety as a handle for E3 ligand
attachment, as exemplified by compounds 1–3 (Figure 1B).
Using this approach, we prepared a series of analogs that
differ in linker length and composition, and explored varied
attachment chemistries to CRBN or von Hippel–Lindau
(VHL) E3 ligase ligands (Table S1 in the Supporting Infor-
mation).
To characterize these compounds biochemically, we
developed competitive ligand binding assays to the BRD9
bromodomain and the co-purified CRBN-DDB1 complex.
High BRD9 affinity was retained across all compounds of this
series relative to the parental bromodomain ligand, as
exemplified by IC50 values for compounds 1–3 that closely
approximate the published IC50 for GSK-39 (7.9 nm; Fig-
ure 1C, Table S1). Moderate differences were observed in
CRBN–DDB1 affinity among compounds with divergently
linked phthalimides (Table S1). For example, the direct alkyl
ether phthalimide linkage of 3 showed slightly improved
binding over acetamide ethers 1 and 2. Interestingly, mea-
sured affinities of all compounds exceeded that of unmodified
thalidomide, perhaps reflecting positive affinity contributions
of the pendant linker substituents.
BRD9 and CRBN. All of the bifunctional compounds in our
initial series were able to significantly induce proximity of the
BRD9 bromodomain and CRBN-DDB1 relative to unmodi-
fied thalidomide; an activity subsequently referred to simply
as “dimerization” (Figure 1D, Table S1). This ternary inter-
action exhibited a characteristic auto-inhibitory concentra-
tion dependence consistent with bimolecular interactions
dominating at saturating ligand concentrations.[23]
Across a range of concentrations, the intermediate length
alkyl ether analog 3 afforded robust dimerization. To under-
stand BRD9 recognition by this compound, we solved a co-
crystal structure with the BRD9 bromodomain. The pose
adopted by the bromodomain warhead confirmed a conserved
binding mode relative to the free probe, with the derivatized
methoxy position projected to solvent as envisioned (Fig-
ure 1E, Figure S1). In-silico modeling of the ternary assembly
including CRBN–DDB1 demonstrated the steric feasibility of
ternary formation, with the two ligand-binding domains
brought into close assembly by compound 3 (Figure 1F).[24]
To evaluate the ability of these compounds to degrade
BRD9 in a cellular context, we treated a human AML cell line
(MOLM-13) for 4 hours at varied concentrations and assessed
BRD9 protein levels by immunoblot. While 1 and the
extended PEG-linked S1 (see supplement) had little effect
on BRD9 protein abundance, marked BRD9 loss was
observed with the more potent biochemical dimerizers 2
and 3 (Figure 2A, Table S1). Encouraged by this activity, we
prepared an additional focused set of analogs exploring
various molecular features. We examined the effect of liker
rigidity by installing a conformationally constrained bi-
piperidine linker in compound S2. This molecule showed
significant improvement in both dimerization and cellular
potency, possibly by enforcement of an extended ternary-
competent linker conformation (Table S1). To pursue degra-
dation by alternate E3 ligases, we prepared the VHL-ligand
To elicit protein degradation, bifunctional molecules must
be able to efficiently associate the E3 ligase with the target. To
measure this activity, we developed a homogenous lumines-
cence assay to report on compound-induced proximity of
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Figure 2. Performance of thienopyrininone degraders. A) Immunoblot
for BRD9 and actin after 4 hour treatment of MOLM-13 with indicated
concentrations of 1, 2, and 3. B) Chemical structures of 4 and 5.
C) Vehicle-normalized CRBN-DDB1 displacement (AlphaScreen quad-
ruplicate means ÆSEM). D) Immunoblot for BRD9 and actin after 4
hour treatment of MOLM-13 with indicated concentrations of 4 and 5.
Figure 3. Temporal and mechanistic characterization of BRD9 degrada-
tion by 5. A) Immunoblot for BRD9 and actin after treatment of
MOLM-13 Cells with 100 nm 5 for the indicated times. B) Immunoblot
for BRD9 and actin after a 4 h pre-treatment of MM.1S cells with
vehicle, I-BRD9, Lenalidomide, Carfilzomib,[*] or MLN-4924, followed
by a 2-hour treatment with 5 (100 nm). [*] Car pretreatment 30 min.
C) Immunoblot for BRD9 and actin after 4 hour treatment with 5 at
the indicated doses in MM.1Swt or MM.1SCRBNÀ/À cells.
conjugates S3 and S4; however, these were found to be
ineffective (Table S1).
Additional analogs explored substitution of the phenolic
attachment as found in 3 for the amine type linkages found in
compounds S5, 4, and 5 (Figure 2B, Table S1). These com-
pounds tightly bound CRBN, and effectively induced degra-
dation of BRD9 (Figure 2C,D). The lenalidomide-based
analog 5 showed the best overall performance, effectively
downregulating BRD9 protein over a broad range of
concentrations. We therefore selected this molecule for
further characterization.
To evaluate the kinetics of BRD9 degradation, we
exposed MOLM-13 cells to 5 at a fixed concentration
(100 nm) and assessed BRD9 abundance over time by
immunoblot. Near complete BRD9 loss was observed
within 1 hour, with no detectable return observed for the
duration of the 24-hour treatment period (Figure 3A). This
profile is appropriate to enable study of primary consequen-
ces of acute BRD9 loss, as well as viability defects manifested
over longer periods.
To interrogate the mechanism of degradation by com-
pound 5 in a cellular context, we assessed the requirement for
target binding, proteasome activity, and activated cullin E3
ligases via chemical and genetic perturbations (Figure 3B,C).
Pretreatment with excess I-BRD9 or lenalidomide competed
with 5 for binding to BRD9 or CRBN, respectively, and
prevented degradation, consistent with a requirement for
intracellular engagement of both targets (Figure 3B).
Degradation was abolished by the co-treatment with the
proteasome inhibitor carfilzomib, confirming a requirement
for proteasome function. Pretreatment using a mechanism-
based inhibitor of neddylation also rescued BRD9 levels, as
expected given the requirement for neddylation of CRL E3
ligases for activity.[25,26] We further established a requirement
for CRBN by examining the effects of compound 5 treatment
in cells rendered CRBN deficient by CRISPR/Cas9
(CRBNÀ/À). While treatment of wild type MM.1S cells
resulted in marked dose-dependent BRD9 loss, treatment of
the paired MM.1S CRBNÀ/À line failed to induce BRD9
degradation (Figure 3C).[27] These data support CRBN- and
proteasome-dependent degradation of BRD9 by 5.
We aimed to further characterize 5 by establishing the
biochemical selectivity profile among 32 representative
members of the human bromodomain family. While the
results of this analysis confirmed potent engagement of
BRD9, we also observed substantial off-target binding
activity, notably including BET bromodomains (Figure 4A).
Because of the confounding transcriptional and anti-prolifer-
ative effects associated with BET inhibition or loss, we felt
selectivity over this family to be an important concern. At this
stage of research, the concurrent publication of BI-7273,
a highly selective BRD9 probe from Boehringer Ingel-
heim,[14,15] inspired exploration of a novel chemical series of
bifunctional degraders (Table S2).
Compound 6 (dBRD9), a PEG-linked pomalidomide
conjugate, was found to prompt rapid BRD9 degradation
over a broad range of concentrations (Figure 4B,C). Gratify-
ingly, dBRD9 also showed an improved bromodomain
engagement profile, with reduced binding activity across the
BET family (Figure 4D). A comparison of biochemical
affinity of 5 and dBRD9 for the BET bromodomain, BRD4-
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Figure 4. Naphthiridinone degrader 6 (dBRD9) offers improved biochemical and cellular selectivity. A) Selectivity of phage-displayed bromodomain
displacement by 5 (Bromoscan). B) Chemical structure of dBRD9. C) Immunoblot of BRD9 and actin after 4 h treatment of MOLM-13 cells with
indicated concentrations of dBRD9. D) Selectivity of phage-displayed bromodomain displacement by dBRD9 (Bromoscan). E) Compound-induced
ternary complex formation of recombinant BRD9(bd) and CRBN–DDB1 (AlphaScreen quadruplicate means ÆSEM). F) Compound-induced ternary
complex formation of recombinant BRD4(1) and CRBN–DDB1 as in (E). G) Immunoblot for BRD4 and actin after 4 h treatment of MOLM-13
cells with indicated concentrations of 5 or dBRD9. H) Immunoblot for BRD7 following treatment as in (G).
(1), by competitive ligand displacement confirmed this result
(Figure S2). Moreover, while 5 was able to effectively induce
biochemical association of CRBN-DDB1 with either BRD9
or BRD4, dBRD9 lost all ability to dimerize BRD4 with
CRBN-DDB1 above background levels, but retained robust
dimerization of BRD9 (Figure 4E,F). Consistent with this
result, dBRD9 showed improved cellular selectivity. Off-
target degradation activity on BRD4 and BRD7, observed at
high concentrations of 5, was not detectable by western blot
following dBRD9 treatment (Figure 4G,H).
To assess the cellular selectivity for BRD9 degradation in
an unbiased, quantitative manner, we measured effects of
dBRD9 (100 nm for 2 hours) versus vehicle (DMSO) on all
cellular proteins in MOLM-13 cells detected by isobaric
tagging and mass spectrometry.[28] Strikingly, of the 7326
Figure 5. dBRD9 selectivity established by whole-cell lysate proteomics.
Fold change in relative abundance of 7326 proteins quantified from
MOML-13 cells treated for two hours with dBRD9 (100 nm) or vehicle
(DMSO), versus q-value for quintuplicate replicates.
proteins quantified in this experiment, BRD9 was the singular
protein showing a marked and statistically significant differ-
ence in abundance, showing a median 5.5 fold lower
abundance in dBRD9 treated samples (FDR corrected q-
value < 0.01) (Figure 5). Levels of other proteins were
remarkably static between treatments with 99% of proteins
differing less than 0.30-fold. Consistent with quantification by
western blot, no significant change in BRD4 or BRD7 levels
was observed.
Having characterized two potent and pharmacologically
distinct degraders of BRD9, we next sought to evaluate the
anti-proliferative activity of these molecules in comparison to
the parental bromodomain inhibitors. In the context of
human AML lines (EOL-1, MOLM-13, MV4;11), compound
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recombinant BRD9 alleles that lost affinity to BI-7273, but
could functionally substitute for the wild type protein. We
therefore stably transduced MOLM-13 cells with wild type
BRD9, bromodomain subsituted BRD9 alleles, or a GFP
vector control, and re-evaluated sensitivity to dBRD9 in the
presence of each transgene. In both lines expressing domain-
swap alleles, the antiproliferative affect of dBRD9 was
dramatically rescued relative to vector control. Viral expres-
sion of exogenous wild type BRD9 also shifted sensitivity, but
to an intermediate degree, consistent with retained succept-
ibility to dBRD9-induced degradation. These responses are
fully congruent with the observed activity of dBRD9 in each
line by western blot (Figure S4A). Consistent with its
polypharmacology, the activity of compound 5 was only
partially shifted by domain-swapped alleles, while the BRD9
independent activity of BET inhibitor JQ1 was wholly
unaffected (Figure S4B). Together with expression proteo-
mics, these data provide extensive support that the observed
sensitivity in MOLM-13 is a BRD9 specific effect.
In the course of our on-target validation work, we
prepared a non-targeting control analog (compound S10),
which lacks a key hydrogen bonding moiety while being
otherwise structurally identical to dBRD9 (Figure S5A). As
expected, this molecule lost biochemical BRD9 bromdomain
affinity, the ability to degrade BRD9, and accordingly, lost
antiproliferative activity in MOLM-13 (MOLM-13 IC50
> 10 mm) (Figure S5B–D). Unexpectedly however, S10
retained considerable activity in the multiple myeloma
derived MM.1S line, which we had identified as dBRD9
sensitive in studies of activity outside AML (Figure S5E).
Contemplating the known activity of IMiDs in MM.1S,[27] we
were lead to discover that S10 and dBRD9 retain activity
against the IKZF family of linage specific transcription
factors; an activity not previously observed with dBET-
1 (Figure S5F).[17] We therefore caution that in select cell lines
of lymphoid origin that express and depend on IKZF family
proteins, researchers should control for this feature. We
suspect that published molecules featuring similar CRBN
targeting chemistry may also retain activity.
Figure 6. Impact of BRD9 degradation on cultured human leukemia
lines. A) Viability of EOL-1 and MOML-13 cell lines treated for 7 days
with the indicated compounds (ATP-Lite quadruplicate means ÆSEM).
B) Viability of MOLM-13 AML treated with dBRD9 and measured as in
(A) following transduction with recombinant BRD9 alleles or vector
control.
In summary, the present work describes the design and
characterization of first-in-class chemical degraders of BRD9.
These studies demonstrate the utility of the targeted degra-
dation strategy to bromodomain-containing proteins beyond
the BET family. This is particularly relevant, as competitive
bromodomain inhibition has previously failed to phenocopy
the effects of protein knock-down (shRNA) or knock-out
(CRISPR-Cas9) for non-BET bromodomain proteins.[29]
Thus, using comparative biochemical and biological assays,
we have qualified a lead BRD9 chemical degrader, dBRD9,
as a selective probe useful for the study of BAF complex
biology. The rapid and potent activity of this compound
render it ideally suited to the study of fast biological responses
such as transcriptional effects and nucleosome positioning.
Finally, potent activity of dBRD9 in cellular models of human
AML is confirmed to be on target through expression
proteomics, alongside chemical and genetic controls within
the exemplar MOLM-13 line.
5 and dBRD9 both exerted a potent anti-proliferative effect,
exceeding non-degrading probe potencies in excesses of 10- to
100-fold (Figure 6A, Figure S3). Interestingly, although these
two compounds exhibited comparable low nanomolar half-
maximal anti-proliferative concentrations, the maximal effect
(Emax) of 5 exceeded that of dBRD9, likely owing to the
polympharmacology associated with 5, particularly activity on
BRD4, a well described AML dependency.[17] These data
argue that polypharmacologic degraders are a viable chemical
strategy, and further demonstrate the ability of cereblon
ligand conjugation to reveal relevant cellular off-target
activities of chemical probes.
To further scrutinize the conclusion that the observed
growth defect results from on-target activity of dBRD9, we
extended the bromodomain-swap strategy of the Vakoc
laboratory, who found that substitution of BRD4 or BRD7
bromodomains for the endogenous BRD9 domain produced
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manuscript, Eben McCue for assistance with images, N. Gray
for technical discussions, Mette Isohey for her work on
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technical contributions to protein production. D.L.B. is
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Keywords: bromodomain · cancer · drug design · epigenetics ·
protein degradation
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Manuscript received: November 17, 2016
Revised: February 24, 2017
Final Article published: && &&, &&&&
6
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 7
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Protein Degraders
D. Remillard, D. L. Buckley, J. L. Paulk,
G. L. Brien, M. Sonnett, H.-S. Seo,
S. Dastierdi, M. Wꢁhr, S. Dhe-Paganon,
S. A. Armstrong,
J. E. Bradner*
&&&& — &&&&
Degradation of the BAF Complex Factor
BRD9 by Heterobifunctional Ligands
With structural guidance alongside com-
parative biochemical and biological
assays, an iterative design strategy
resulted in the development of small-
molecule protein degraders that rapidly,
potently, and selectively eliminate
bromodomain-containing protein BRD9
from the BAF complex. These first in class
non-BET bromodomain degraders show
significant potency improvements over
existing BRD9 probes in models of acute
myeloid leukemia.
Angew. Chem. Int. Ed. 2017, 56, 1 – 7
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!