German Edition: DOI: 10.1002/ange.201611281Protein DegradersInternational Edition: DOI: 10.1002/anie.201611281

Degradation of the BAF Complex Factor BRD9 by HeterobifunctionalLigandsDavid Remillard, Dennis L. Buckley, Joshiawa Paulk, Gerard L. Brien, Matthew Sonnett,Hyuk-Soo Seo, Shiva Dastierdi, Martin W�hr, Sirano Dhe-Paganon, Scott A. Armstrong, andJames E. Bradner*

Abstract: The bromodomain-containing protein BRD9, a sub-unit of the human BAF (SWI/SNF) nucleosome remodelingcomplex, has emerged as an attractive therapeutic target incancer. Despite the development of chemical probes targetingthe BRD9 bromodomain, there is a limited understanding ofBRD9 function beyond acetyl-lysine recognition. We havetherefore created the first BRD9-directed chemical degraders,through iterative design and testing of heterobifunctionalligands that bridge the BRD9 bromodomain and the cereblonE3 ubiquitin ligase complex. Degraders of BRD9 exhibitmarkedly enhanced potency compared to parental ligands (10-to 100-fold). Parallel study of degraders with divergent BRD9-binding chemotypes in models of acute myeloid leukemiaresolves bromodomain polypharmacology in this emergingdrug class. Together, these findings reveal the tractability ofnon-BET bromodomain containing proteins to chemicaldegradation, and highlight lead compound dBRD9 as a toolfor the study of BRD9.

Small-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 andother inhibitors of the bromodomain and extra-terminaldomain (BET) subfamily of transcriptional co-activators has

contributed an enhanced mechanistic understanding of genecontrol and has availed new therapeutic opportunities incancer.[2, 3] Indeed, multiple BET inhibitor candidates fromseveral groups are now undergoing clinical trials for diverseindications in and outside of oncology.[4–6] Further, significantresearch effort has contributed a number of high qualitychemical probes for bromodomain-containing proteinsbeyond 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 knownas SWI/SNF). Meta-analyses of whole-genome sequencingefforts have recently identified a high frequency of recurrentsomatic 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 ascontext-specific dependencies, either supporting growthwithin a residual complex following loss of function mutation,or as novel oncogenes such as the SS18-SSX fusion.[10] Theseobservations have generated interest in therapeutic strategiesto target BAF.

Beyond its presence in the BAF complex, a lack offunctional annotation for BRD9 has provided incentive fordevelopment of BRD9 selective inhibitors to interrogate itsbiological role and to assess any therapeutic potential. SeveralBRD9-directed efforts in discovery chemistry have beenreported,[11–15] developing chemotypes for BRD9-specificengagement. Further, a recent study implicated BRD9 asa dependency in acute myeloid leukemia (AML), wherebromodomain inhibition prompted a cytostatic response.[16]

Beyond the bromodomain, the function of BRD9 remainsunclear, and chemical tools to study other functions of BRD9are not available. We therefore undertook to create firstchemical probes that destabilize BRD9, anticipating that thestudy of acute BRD9 loss would offer a powerful approach tointerrogate BAF complex function.

Recently, we reported a strategy to direct protein-specificdegradation using bifunctional molecules to recruit thecereblon (CRBN) ubiquitin ligase complex to non-physio-logic protein substrates,[17] providing an all-chemical solutionto prior efforts using peptides to bridge E3 ligases and ligandtargets (PROTACs).[18, 19] In our prior research, we directedthe degradation of BET family proteins by appending CRBNligands to JQ1, resulting in rapid and potent degradation ofBRD2, BRD3, and BRD4. These findings have been wellvalidated, suggesting a broader utility of this strategy.[20–22]

Toward the elaboration of BRD9-directed degraders, weinitially evaluated putative pharmacophores from reported

[*] D. Remillard, Dr. D. L. Buckley, Dr. J. L. Paulk, S. Dastierdi,Dr. J. E. BradnerDepartment of Medical Oncology, Dana-Farber Cancer InstituteBoston, MA (USA)E-mail: james_bradner@dfci.harvard.edu

james.bradner@novartis.com

Dr. G. L. Brien, Dr. S. A. ArmstrongDepartment of Pediatric Oncology, Dana-Farber Cancer InstituteBoston, MA (USA)

M. SonnettDepartment of Systems Biology, Harvard Medical SchoolBoston, MA (USA)

M. Sonnett, Dr. M. W�hrLewis-Sigler Institute for Integrative Genomics, Princeton UniversityPrinceton, NJ (USA)

Dr. H.-S. Seo, Dr. S. Dhe-PaganonDepartment of Cancer Biology, Dana-Farber Cancer InstituteBoston, MA (USA)

Dr. M. W�hrDepartment of Molecular Biology, Princeton UniversityPrinceton, NJ (USA)

Supporting information and the ORCID identification number(s) forthe author(s) of this article can be found under:https://doi.org/10.1002/anie.201611281.

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bromodomain probes LP99 and I-BRD9, and subsequentlyexpanded our study to a third probe, BI7273, reported duringthe course of this research (Figure 1A).[12, 14,15] To explore thepotential of bifunctional derivatives to induce BRD9 degra-dation, we initially selected as our starting point a closechemical analog of I-BRD9 described by GlaxoSmithKline(GSK-39).[14] This ligand was attractive in that it offered highbinding affinity (IC50 = 7.9 nm) as well as a solvent exposedmethoxy substituent amenable to chemical derivatization. Inour initial design strategy, we adapted this ligand by installingan ether-linked acetyl moiety as a handle for E3 ligandattachment, as exemplified by compounds 1–3 (Figure 1B).Using this approach, we prepared a series of analogs thatdiffer in linker length and composition, and explored variedattachment chemistries to CRBN or von Hippel–Lindau(VHL) E3 ligase ligands (Table S1 in the Supporting Infor-mation).

To characterize these compounds biochemically, wedeveloped competitive ligand binding assays to the BRD9bromodomain and the co-purified CRBN-DDB1 complex.High BRD9 affinity was retained across all compounds of thisseries relative to the parental bromodomain ligand, asexemplified by IC50 values for compounds 1–3 that closelyapproximate the published IC50 for GSK-39 (7.9 nm ; Fig-ure 1C, Table S1). Moderate differences were observed inCRBN–DDB1 affinity among compounds with divergentlylinked phthalimides (Table S1). For example, the direct alkylether phthalimide linkage of 3 showed slightly improvedbinding over acetamide ethers 1 and 2. Interestingly, mea-sured affinities of all compounds exceeded that of unmodifiedthalidomide, perhaps reflecting positive affinity contributionsof the pendant linker substituents.

To elicit protein degradation, bifunctional molecules mustbe able to efficiently associate the E3 ligase with the target. Tomeasure this activity, we developed a homogenous lumines-cence assay to report on compound-induced proximity of

BRD9 and CRBN. All of the bifunctional compounds in ourinitial series were able to significantly induce proximity of theBRD9 bromodomain and CRBN-DDB1 relative to unmodi-fied thalidomide; an activity subsequently referred to simplyas “dimerization” (Figure 1D, Table S1). This ternary inter-action exhibited a characteristic auto-inhibitory concentra-tion dependence consistent with bimolecular interactionsdominating at saturating ligand concentrations.[23]

Across a range of concentrations, the intermediate lengthalkyl 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 poseadopted by the bromodomain warhead confirmed a conservedbinding mode relative to the free probe, with the derivatizedmethoxy position projected to solvent as envisioned (Fig-ure 1E, Figure S1). In-silico modeling of the ternary assemblyincluding CRBN–DDB1 demonstrated the steric feasibility ofternary formation, with the two ligand-binding domainsbrought into close assembly by compound 3 (Figure 1F).[24]

To evaluate the ability of these compounds to degradeBRD9 in a cellular context, we treated a human AML cell line(MOLM-13) for 4 hours at varied concentrations and assessedBRD9 protein levels by immunoblot. While 1 and theextended PEG-linked S1 (see supplement) had little effecton BRD9 protein abundance, marked BRD9 loss wasobserved with the more potent biochemical dimerizers 2and 3 (Figure 2 A, Table S1). Encouraged by this activity, weprepared an additional focused set of analogs exploringvarious molecular features. We examined the effect of likerrigidity by installing a conformationally constrained bi-piperidine linker in compound S2. This molecule showedsignificant improvement in both dimerization and cellularpotency, 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

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 �SEM).D) Compound-induced ternary complex formation of recombinant BRD9(bd) and CRBN–DDB1 (AlphaScreen quadruplicate means �SEM).E) Cocrystal structure of 3 with BRD9(bd) (PDB 5TWX). F) Docking of (E) into the published CRBN–DDB1 structure (4CI3).

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conjugates S3 and S4 ; however, these were found to beineffective (Table S1).

Additional analogs explored substitution of the phenolicattachment as found in 3 for the amine type linkages found incompounds 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-basedanalog 5 showed the best overall performance, effectivelydownregulating BRD9 protein over a broad range ofconcentrations. We therefore selected this molecule forfurther characterization.

To evaluate the kinetics of BRD9 degradation, weexposed MOLM-13 cells to 5 at a fixed concentration(100 nm) and assessed BRD9 abundance over time byimmunoblot. Near complete BRD9 loss was observedwithin 1 hour, with no detectable return observed for theduration of the 24-hour treatment period (Figure 3A). Thisprofile is appropriate to enable study of primary consequen-ces of acute BRD9 loss, as well as viability defects manifestedover longer periods.

To interrogate the mechanism of degradation by com-pound 5 in a cellular context, we assessed the requirement fortarget binding, proteasome activity, and activated cullin E3ligases via chemical and genetic perturbations (Figure 3B,C).Pretreatment with excess I-BRD9 or lenalidomide competedwith 5 for binding to BRD9 or CRBN, respectively, andprevented degradation, consistent with a requirement forintracellular engagement of both targets (Figure 3B).

Degradation was abolished by the co-treatment with theproteasome inhibitor carfilzomib, confirming a requirementfor proteasome function. Pretreatment using a mechanism-based inhibitor of neddylation also rescued BRD9 levels, as

expected given the requirement for neddylation of CRL E3ligases for activity.[25, 26] We further established a requirementfor CRBN by examining the effects of compound 5 treatmentin cells rendered CRBN deficient by CRISPR/Cas9(CRBN�/�). While treatment of wild type MM.1S cellsresulted in marked dose-dependent BRD9 loss, treatment ofthe paired MM.1S CRBN�/� line failed to induce BRD9degradation (Figure 3C).[27] These data support CRBN- andproteasome-dependent degradation of BRD9 by 5.

We aimed to further characterize 5 by establishing thebiochemical selectivity profile among 32 representativemembers of the human bromodomain family. While theresults of this analysis confirmed potent engagement ofBRD9, we also observed substantial off-target bindingactivity, notably including BET bromodomains (Figure 4A).Because of the confounding transcriptional and anti-prolifer-ative effects associated with BET inhibition or loss, we feltselectivity over this family to be an important concern. At thisstage 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 ofbifunctional degraders (Table S2).

Compound 6 (dBRD9), a PEG-linked pomalidomideconjugate, was found to prompt rapid BRD9 degradationover a broad range of concentrations (Figure 4B,C). Gratify-ingly, dBRD9 also showed an improved bromodomainengagement profile, with reduced binding activity across theBET family (Figure 4D). A comparison of biochemicalaffinity of 5 and dBRD9 for the BET bromodomain, BRD4-

Figure 2. Performance of thienopyrininone degraders. A) Immunoblotfor BRD9 and actin after 4 hour treatment of MOLM-13 with indicatedconcentrations of 1, 2, and 3. B) Chemical structures of 4 and 5.C) Vehicle-normalized CRBN-DDB1 displacement (AlphaScreen quad-ruplicate means �SEM). D) Immunoblot for BRD9 and actin after 4hour 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 ofMOLM-13 Cells with 100 nm 5 for the indicated times. B) Immunoblotfor BRD9 and actin after a 4 h pre-treatment of MM.1S cells withvehicle, I-BRD9, Lenalidomide, Carfilzomib,[*] or MLN-4924, followedby a 2-hour treatment with 5 (100 nm). [*] Car pretreatment 30 min.C) Immunoblot for BRD9 and actin after 4 hour treatment with 5 atthe indicated doses in MM.1Swt or MM.1SCRBN�/� cells.

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(1), by competitive ligand displacement confirmed this result(Figure S2). Moreover, while 5 was able to effectively inducebiochemical association of CRBN-DDB1 with either BRD9or BRD4, dBRD9 lost all ability to dimerize BRD4 withCRBN-DDB1 above background levels, but retained robustdimerization of BRD9 (Figure 4E,F). Consistent with thisresult, dBRD9 showed improved cellular selectivity. Off-target degradation activity on BRD4 and BRD7, observed athigh concentrations of 5, was not detectable by western blotfollowing dBRD9 treatment (Figure 4G,H).

To assess the cellular selectivity for BRD9 degradation inan unbiased, quantitative manner, we measured effects ofdBRD9 (100 nm for 2 hours) versus vehicle (DMSO) on allcellular proteins in MOLM-13 cells detected by isobarictagging and mass spectrometry.[28] Strikingly, of the 7326proteins quantified in this experiment, BRD9 was the singularprotein showing a marked and statistically significant differ-ence in abundance, showing a median 5.5 fold lowerabundance in dBRD9 treated samples (FDR corrected q-value < 0.01) (Figure 5). Levels of other proteins wereremarkably static between treatments with 99 % of proteinsdiffering less than 0.30-fold. Consistent with quantification bywestern blot, no significant change in BRD4 or BRD7 levelswas observed.

Having characterized two potent and pharmacologicallydistinct degraders of BRD9, we next sought to evaluate theanti-proliferative activity of these molecules in comparison tothe parental bromodomain inhibitors. In the context ofhuman AML lines (EOL-1, MOLM-13, MV4;11), compound

Figure 4. Naphthiridinone degrader 6 (dBRD9) offers improved biochemical and cellular selectivity. A) Selectivity of phage-displayed bromodomaindisplacement by 5 (Bromoscan). B) Chemical structure of dBRD9. C) Immunoblot of BRD9 and actin after 4 h treatment of MOLM-13 cells withindicated concentrations of dBRD9. D) Selectivity of phage-displayed bromodomain displacement by dBRD9 (Bromoscan). E) Compound-inducedternary complex formation of recombinant BRD9(bd) and CRBN–DDB1 (AlphaScreen quadruplicate means �SEM). F) Compound-induced ternarycomplex formation of recombinant BRD4(1) and CRBN–DDB1 as in (E). G) Immunoblot for BRD4 and actin after 4 h treatment of MOLM-13cells with indicated concentrations of 5 or dBRD9. H) Immunoblot for BRD7 following treatment as in (G).

Figure 5. dBRD9 selectivity established by whole-cell lysate proteomics.Fold change in relative abundance of 7326 proteins quantified fromMOML-13 cells treated for two hours with dBRD9 (100 nm) or vehicle(DMSO), versus q-value for quintuplicate replicates.

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5 and dBRD9 both exerted a potent anti-proliferative effect,exceeding non-degrading probe potencies in excesses of 10- to100-fold (Figure 6A, Figure S3). Interestingly, although thesetwo compounds exhibited comparable low nanomolar half-maximal anti-proliferative concentrations, the maximal effect(Emax) of 5 exceeded that of dBRD9, likely owing to thepolympharmacology associated with 5, particularly activity onBRD4, a well described AML dependency.[17] These dataargue that polypharmacologic degraders are a viable chemicalstrategy, and further demonstrate the ability of cereblonligand conjugation to reveal relevant cellular off-targetactivities of chemical probes.

To further scrutinize the conclusion that the observedgrowth defect results from on-target activity of dBRD9, weextended the bromodomain-swap strategy of the Vakoclaboratory, who found that substitution of BRD4 or BRD7bromodomains for the endogenous BRD9 domain produced

recombinant BRD9 alleles that lost affinity to BI-7273, butcould functionally substitute for the wild type protein. Wetherefore stably transduced MOLM-13 cells with wild typeBRD9, bromodomain subsituted BRD9 alleles, or a GFPvector control, and re-evaluated sensitivity to dBRD9 in thepresence of each transgene. In both lines expressing domain-swap alleles, the antiproliferative affect of dBRD9 wasdramatically rescued relative to vector control. Viral expres-sion of exogenous wild type BRD9 also shifted sensitivity, butto an intermediate degree, consistent with retained succept-ibility to dBRD9-induced degradation. These responses arefully congruent with the observed activity of dBRD9 in eachline by western blot (Figure S4 A). Consistent with itspolypharmacology, the activity of compound 5 was onlypartially shifted by domain-swapped alleles, while the BRD9independent activity of BET inhibitor JQ1 was whollyunaffected (Figure S4 B). Together with expression proteo-mics, these data provide extensive support that the observedsensitivity in MOLM-13 is a BRD9 specific effect.

In the course of our on-target validation work, weprepared a non-targeting control analog (compound S10),which lacks a key hydrogen bonding moiety while beingotherwise structurally identical to dBRD9 (Figure S5 A). Asexpected, this molecule lost biochemical BRD9 bromdomainaffinity, the ability to degrade BRD9, and accordingly, lostantiproliferative activity in MOLM-13 (MOLM-13 IC50> 10 mm) (Figure S5B–D). Unexpectedly however, S10retained considerable activity in the multiple myelomaderived MM.1S line, which we had identified as dBRD9sensitive in studies of activity outside AML (Figure S5 E).Contemplating the known activity of IMiDs in MM.1S,[27] wewere lead to discover that S10 and dBRD9 retain activityagainst the IKZF family of linage specific transcriptionfactors; an activity not previously observed with dBET-1 (Figure S5 F).[17] We therefore caution that in select cell linesof lymphoid origin that express and depend on IKZF familyproteins, researchers should control for this feature. Wesuspect that published molecules featuring similar CRBNtargeting chemistry may also retain activity.

In summary, the present work describes the design andcharacterization of first-in-class chemical degraders of BRD9.These studies demonstrate the utility of the targeted degra-dation strategy to bromodomain-containing proteins beyondthe BET family. This is particularly relevant, as competitivebromodomain inhibition has previously failed to phenocopythe 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 complexbiology. The rapid and potent activity of this compoundrender it ideally suited to the study of fast biological responsessuch as transcriptional effects and nucleosome positioning.Finally, potent activity of dBRD9 in cellular models of humanAML is confirmed to be on target through expressionproteomics, alongside chemical and genetic controls withinthe exemplar MOLM-13 line.

Figure 6. Impact of BRD9 degradation on cultured human leukemialines. A) Viability of EOL-1 and MOML-13 cell lines treated for 7 dayswith the indicated compounds (ATP-Lite quadruplicate means �SEM).B) Viability of MOLM-13 AML treated with dBRD9 and measured as in(A) following transduction with recombinant BRD9 alleles or vectorcontrol.

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Acknowledgements

We thank Dr. Jennifer Perry for critical reading of themanuscript, Eben McCue for assistance with images, N. Grayfor technical discussions, Mette Isohey for her work onconditions for linker synthesis, and Stephen DeAngelo fortechnical contributions to protein production. D.L.B. isa Merck Fellow of the Damon Runyon Cancer ResearchFoundation (DRG-2196-14). G.L.B. is supported by anEMBO Long-Term Fellowship (ALTF 1235–2015) with addi-tional support from Marie Curie Actions. M.S. was supportedby NIH grant F31 GM095450. This work was supported by theClaudia Adams Barr Program for Innovative CancerResearch (to D.L.B.), and by NIH/NCI grant P01-CA066996, to J.E.B. and S.A.A. J.E.B. is now an executiveand shareholder in Novartis AG.

Conflict of interest

J.E.B. is a Scientific Founder of Syros Pharmaceuticals,SHAPE Pharmaceuticals, Acetylon Pharmaceuticals, TenshaTherapeutics (now Roche) and C4 Therapeutics and is theinventor on IP licensed to these entities. J.E.B. is now anexecutive and shareholder in Novartis AG.

Keywords: bromodomain · cancer · drug design · epigenetics ·protein degradation

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Manuscript received: November 17, 2016Revised: February 24, 2017Final Article published: && &&, &&&&

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Communications

Protein Degraders

D. Remillard, D. L. Buckley, J. L. Paulk,G. L. Brien, M. Sonnett, H.-S. Seo,S. Dastierdi, M. W�hr, S. Dhe-Paganon,S. A. Armstrong,J. E. Bradner* &&&&—&&&&

Degradation of the BAF Complex FactorBRD9 by Heterobifunctional Ligands

With structural guidance alongside com-parative biochemical and biologicalassays, an iterative design strategyresulted in the development of small-molecule protein degraders that rapidly,potently, and selectively eliminate

bromodomain-containing protein BRD9from the BAF complex. These first in classnon-BET bromodomain degraders showsignificant potency improvements overexisting BRD9 probes in models of acutemyeloid leukemia.

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