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The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.

Targeted protein degradation is a rapidly developing therapeutic modality that promises lower dosing and enhanced selectivity as compared to traditional occupancy-driven inhibitors, and the potential to modulate historically intractable targets. While the well-characterized E3 ligases CRBN and VHL have been successfully redirected to degrade numerous proteins, there are approximately 600 predicted additional E3 family members that may offer improved activity, substrate selectivity, and/or tissue distribution; however, characterizing the potential applications of these many ligases for targeted protein degradation has proven challenging. Here, we report the development of an approach to evaluate the ability of recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for hit finding to identify specific E3 ligase binders, this approach makes use of simple chemistry for Covalent Functionalization Followed by E3 Electroporation into live cells (COFFEE). We demonstrate this method by electroporating recombinant VHL, covalently functionalized with JQ1 or dasatinib, to induce degradation of BRD4 or kinase targets, respectively. Furthermore, by applying COFFEE to SPSB2, a SOCS box and SPRY-domain E3 ligase that has not previously been redirected for targeted protein degradation, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligases against neo-substrates.

Targeted protein degradation is a rapidly developing therapeutic modality that promises lower dosing and enhanced selectivity as compared to traditional occupancy-driven inhibitors, and the potential to modulate historically intractable targets. While well-characterized E3 ligases such as CRBN and VHL have been successfully redirected to degrade numerous proteins, there are approximately 600 predicted additional E3 family members that may offer improved activity, substrate selectivity, and/or tissue distribution. Characterizing the potential applications of these many ligases for targeted protein degradation has proven challenging. Here, we report the development of an approach to evaluate the ability of recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for hit finding to identify specific E3 ligase binders, this approach makes use of simple maleimide-thiol chemistry for Covalent Functionalization Followed by E3 Electroporation into live cells (COFFEE). We demonstrate this method by electroporating recombinant VHL, covalently functionalized with JQ1 or dasatinib, to induce degradation of BRD4 or tyrosine kinase targets, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F-box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only ligase receptors but also adaptors can be directly hi-jacked for neo-substrate degradation.

Bromodomains have been pursued intensively over the past several years as emerging targets for the development of anti-cancer and anti-inflammatory agents. It has recently been shown that some kinase inhibitors are able to potently inhibit the bromodomains of BRD4. The clinical activities of PLK inhibitor BI-2536 and JAK2-FLT3 inhibitor TG101348 have been attributed to this unexpected poly-pharmacology, indicating that dual-kinase/bromodomain activity may be advantageous in a therapeutic context. However, for target validation and biological investigation, a more selective target profile is desired. Here we report that benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones, versatile ATP-site directed kinase pharmacophores utilized in the development of inhibitors of multiple kinases including a number of previously reported kinase chemical probes, are also capable of exhibiting potent BRD4-dependent pharmacology. Using a dual kinase-bromodomain inhibitor of the kinase domains of ERK5 and LRRK2, and the bromodomain of BRD4 as a case study, we define the structure-activity relationships required to achieve dual kinase/BRD4 activity as well as how to direct selectivity towards inhibition of either ERK5 or BRD4. This effort resulted in identification of one of the first reported kinase-selective chemical probes for ERK5 (JWG-071), a BET selective inhibitor with 1 μM BRD4 IC(50) (JWG-115), and additional inhibitors with rationally designed polypharmacology (JWG-047, JWG-069). Co-crystallography of seven representative inhibitors with the first bromodomain of BRD4 demonstrate that distinct atropisomeric conformers recognize the kinase ATP-site and the BRD4 acetyl lysine binding site, conformational preferences supported by rigid docking studies.

The addressable pocket of a protein is often not functionally relevant in disease. This is true for the multidomain, bromodomain-containing transcriptional regulator TRIM24. TRIM24 has been posited as a dependency in numerous cancers, yet potent and selective ligands for the TRIM24 bromodomain do not exert effective anti-proliferative responses. We therefore repositioned these probes as targeting features for heterobifunctional protein degraders. Recruitment of the VHL E3 ubiquitin ligase by dTRIM24 elicits potent and selective degradation of TRIM24. Using dTRIM24 to probe TRIM24 function, we characterize the dynamic genome-wide consequences of TRIM24 loss on chromatin localization and gene control. Further, we identify TRIM24 as a novel dependency in acute leukemia. Pairwise study of TRIM24 degradation versus bromodomain inhibition reveals enhanced anti-proliferative response from degradation. We offer dTRIM24 as a chemical probe of an emerging cancer dependency, and establish a path forward for numerous selective yet ineffectual ligands for proteins of therapeutic interest.

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 6 (dBRD9) as a tool for the study of BRD9.

Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo. To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.

The discovery that thalidomide derivatives recruit the E3 ligase CRBN to induce neomorphic degradation of proteins critical for multiple myeloma (MM) cells stimulated the research into proteolysis-targeting chimeric compounds (PROTACs), led to development of several CRBN- or VHL-based PROTACs against various oncoproteins and put a new spotlight on the biology and therapeutic targeting of E3 ligases in human neoplasias. However, so far only a few of the ~600 known/presumed E3 ligases have been leveraged for generation of PROTACs. The mechanisms regulating the function of most E3 ligases have not been systematically examined. Because the function of an E3 ligase is considered essential for anti-tumor activity of its respective PROTACs, we applied CRISPR knock-out (KO) systems to identify candidate regulators of E3 ligase function, via characterizing the the network of genes which modulate MM cell responses to PROTACs. We thus performed genome-scale CRISPR-based gene editing (for loss-of-function, LOF) studies in MM.1S cells treated with PROTACs targeting BET bromodomain proteins through MDM2 (A1874), CRBN (dBET6) or VHL (ARV-771 or MZ-1) or targeting CDK9 through CRBN (Thal-SNS-032); and validated key hits with individual sgRNAs in different MM cell lines. The top individual LOF events conferring resistance to PROTACs did not involve a compensatory mechanism or "work-around" the loss of the respective oncoprotein, but were predominantly associated with LOF of the respective E3 ligase; or with LOF for genes with known or plausible role in regulating the respective E3 ligases. For instance, sgRNAs against members of the COP9 signalosome complex decreased MM cell responses to CRBN- and (to a lesser extent) VHL-, but not MDM2-based PROTACs. PROTACs leveraging different E3 ligases were regulated by different cullin ring ligase (CRL) complex members (e.g. CUL2, RBX1, TCEB1, TCEB2 for VHL- vs. DDB1 for CRBN- vs. no CRL member for MDM2-based PROTACs) or E2 conjugating enzymes (UBE2R2 vs. UBE2G1 for VHL- vs. CRBN-based PROTACs). Collectively, these results suggest that MDM2 regulation is largely CRL- and COP9-signalosome independent; while VHL regulation is less COP9 signalosome-dependent compared to CRBN. These mechanistic differences suggest that PROTACs targeting the same oncoprotein through different E3 ligases should not be associated with cross-resistance, a result which we validated in experiments involving sequential administration of different PROTACs against BRD4/3/2. In turn, this observation implied that developing PROTACs that leverage a more extended spectrum of E3 ligases may facilitate sequential uses of existing and these new PROTACs to delay or prevent treatment resistance. Building on results of our genome-scale CRISPR essentiality screens, we examined the dependency landscape of known E3 ligases of MM (n=20 cell lines) and 500+ non-MM cell lines. CRBN is redundant for nearly all MM or non-MM cell lines tested, while most other E3 ligases leveraged for PROTACs (e.g. MDM2, BIRC2, DCAF15, DCAF16, RNF114) are essential for only modest or small subsets of human cancer cell lines, suggesting that resistance to respective PROTACs may readily emerge through LOF of these E3 ligases without major fitness cost to tumor cells. We thus sought to identify E3 ligases which are highly expressed in subsets of human tumor cell lines (at levels well above the large majority of normal tissues) and are major dependencies for these "high expressor" cell lines: we identified MDM2 as a major dependency for p53-wild-type cell lines (consistent with MDM2 role as E3 ligase for p53) and we validated this result by documenting the preferential activity of a MDM2-based PROTAC for BRD4/3/2 against p53 wild-type cells. We also identified other E3 ligases genes with well-known roles in tumor cell biology (e.g. members of anaphase promoting complex/cyclosome); as well as E3 ligases (e.g. KCMF1, RNF4) which, to our knowledge, have not been leveraged for design of PROTACs, but warrant consideration given their patterns of essentiality in "high expressor" tumor cells. Our study provides insights on differential regulation and distinct patterns of essentiality for different E3 ligases and informs the design of new PROTACs which leverage different E3 ligases to help delay/overcome treatment resistance in MM and beyond. Disclosures Schlossman: Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Richardson:Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Ebert:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.; Celgene: Research Funding; Deerfield: Research Funding. Tsherniak:Tango Therapeutics: Consultancy. Boise:Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

[Image: see text] Recent reports have highlighted the dual bromodomains of TAF1 (TAF1(1,2)) as synergistic with BET inhibition in cellular cancer models, engendering interest in TAF/BET polypharmacology. Here, we examine structure activity relationships within the BI-2536 PLK1 kinase inhibitor scaffold, previously reported to bind BRD4. We examine binding by this ligand to TAF1(2) and apply structure guided design strategies to discriminate binding to both the PLK1 kinase and BRD4(1) bromodomain while retaining activity on TAF1(2). Through this effort we discover potent dual inhibitors of TAF1(2)/BRD4(1), as well as biased derivatives showing marked TAF1 selectivity. We resolve X-ray crystallographic data sets to examine the mechanisms of the observed TAF1 selectivity and to provide a resource for further development of this scaffold.

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological “degraders” of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

Evaluating the engagement of a small molecule ligand with a protein target in cells provides useful information for chemical probe optimization and pharmaceutical development. While several techniques exist that can be performed in a low-throughput manner, systematic evaluation of large compound libraries remains a challenge. In-cell engagement measurements are especially useful when evaluating compound classes suspected to target multiple cellular factors. In this study we used a bioluminescent resonant energy transfer assay to assess bromodomain engagement by a compound series containing bromodomain- and kinase-biasing polypharmacophores based on the known dual BRD4 bromodomain/PLK1 kinase inhibitor BI2536. With this assay, we discovered several novel agents with bromodomain-selective specificity profiles and cellular activity. Thus, this platform aids in distinguishing molecules whose cellular activity is difficult to assess due to polypharmacologic effects.

The histone acetyl-reader BRD4 is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1. We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression, and pharmacologic inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin-associated suggests a direct role for nuclear metabolism in the control of gene expression.

NPM1 is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (AML). In AML cells, NPM1 mutations result in abnormal cytoplasmic localization of the mutant protein (NPM1c); however, it is unknown whether NPM1c is required to maintain the leukemic state. Here, we show that loss of NPM1c from the cytoplasm, either through nuclear relocalization or targeted degradation, results in immediate downregulation of homeobox (HOX) genes followed by differentiation. Finally, we show that XPO1 inhibition relocalizes NPM1c to the nucleus, promotes differentiation of AML cells, and prolongs survival of Npm1-mutated leukemic mice. We describe an exquisite dependency of NPM1-mutant AML cells on NPM1c, providing the rationale for the use of nuclear export inhibitors in AML with mutated NPM1.

The simultaneous inhibition of polo-like kinase 1 (PLK1) and BRD4 bromodomain by a single molecule could lead to the development of an effective therapeutic strategy for a variety of diseases in which PLK1 and BRD4 are implicated. Compound 23 has been found to be a potent dual kinase-bromodomain inhibitor (BRD4-BD1 IC(50) = 28 nM, PLK1 IC(50) = 40 nM). Compound 6 was found to be the most selective PLK1 inhibitor over BRD4 in our series (BRD4-BD1 IC(50) = 2,579 nM, PLK1 IC(50) = 9.9 nM). Molecular docking studies with 23 and BRD4-BD1/PLK1 as well as with 6 corroborates the biochemical assay results.

Competitive BET bromodomain inhibitors (BBIs) targeting BET proteins (BRD2, BRD3, BRD4, and BRDT) show promising preclinical activities against brain cancers. However, the BET protein-dependent glioblastoma (GBM)-promoting transcriptional network remains elusive. Here, with mechanistic exploration of a next-generation chemical degrader of BET proteins (dBET6), we reveal a profound and consistent impact of BET proteins on E2F1- dependent transcriptional program in both differentiated GBM cells and brain tumor-initiating cells. dBET6 treatment drastically reduces BET protein genomic occupancy, RNA-Pol2 activity, and permissive chromatin marks. Subsequently, dBET6 represses the proliferation, self-renewal, and tumorigenic ability of GBM cells. Moreover, dBET6-induced degradation of BET proteins exerts superior antiproliferation effects compared to conventional BBIs and overcomes both intrinsic and acquired resistance to BBIs in GBM cells. Our study reveals crucial functions of BET proteins and provides the rationale and therapeutic merits of targeted degradation of BET proteins in GBM.

Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12F36V with expression of FKBP12F36V in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRASG12V loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.

Bifunctional degraders, also referred to as proteolysis-targeting chimeras (PROTACs), are a recently developed class of small molecules. They were designed to specifically target endogenous proteins for ubiquitin/proteasome-dependent degradation and to thereby interfere with pathological mechanisms of diseases, including cancer. In this study, we hypothesized that this process of acute pharmacologic protein degradation might increase the direct MHC class I presentation of degraded targets. By studying this question, we contribute to an ongoing discussion about the origin of peptides feeding the MHC class I presentation pathway. Two scenarios have been postulated: peptides can either be derived from homeostatic turnover of mature proteins and/or from short-lived defective ribosomal products (DRiPs), but currently, it is still unclear to what ratio and efficiency both pathways contribute to the overall MHC class I presentation. We therefore generated the intrinsically stable model antigen GFP-S8L-F12 that was susceptible to acute pharmacologic degradation via the previously described degradation tag (dTAG) system. Using different murine cell lines, we show here that the bifunctional molecule dTAG-7 induced rapid proteasome-dependent degradation of GFP-S8L-F12 and simultaneously increased its direct presentation on MHC class I molecules. Using the same model in a doxycycline-inducible setting, we could further show that stable, mature antigen was the major source of peptides presented, thereby excluding a dominant role of DRiPs in our system. This study is, to our knowledge, the first to investigate targeted pharmacologic protein degradation in the context of antigen presentation and our data point toward future applications by strategically combining therapies using bifunctional degraders with their stimulating effect on direct MHC class I presentation.

A degrading game plan for cancer therapy Certain classes of proteins that contribute to cancer development are challenging to target therapeutically. Winter et al. devised a chemical strategy that, in principle, permits the selective degradation of any protein of interest. The strategy involves chemically attaching a ligand known to bind the desired protein to another molecule that hijacks an enzyme whose function is to direct proteins to the cell's protein degradation machinery. In a proof-of-concept study, they demonstrated selective degradation of a transcriptional coactivator called bromodomain-containing protein 4 and delayed the progression of leukemia in mice. Science , this issue p. 1376

Androgen Receptor (AR)-dependent transcription is a major driver of prostate tumor cell proliferation. Consequently, it is the target of several antitumor chemotherapeutic agents, including the AR antagonist MDV3100/enzalutamide. Recent studies have shown that a single AR mutation (F876L) converts MDV3100 action from an antagonist to an agonist. Here we describe the generation of a novel class of Selective Androgen Receptor Degraders (SARDs) to address this resistance mechanism. Molecules containing hydrophobic degrons linked to small molecule Androgen Receptor (AR) ligands induce AR degradation, reduce expression of AR target genes and inhibit proliferation in androgen-dependent prostate cancer cell lines. These results suggest that selective AR degradation may be an effective therapeutic prostate tumor strategy in the context of AR mutations that confer resistance to third generation AR antagonists.

The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.

Summary Heterobifunctional molecules that recruit E3 ubiquitin ligases, such as cereblon, for targeted protein degradation represent an emerging pharmacological strategy. A major unanswered question is how generally applicable this strategy is to all protein targets. In this study, we designed a multi-kinase degrader by conjugating a highly promiscuous kinase inhibitor with a cereblon-binding ligand, and used quantitative proteomics to discover 28 kinases, including BTK, PTK2, PTK2B, FLT3, AURKA, AURKB, TEC, ULK1, ITK, and nine members of the CDK family, as degradable. This set of kinases is only a fraction of the intracellular targets bound by the degrader, demonstrating that successful degradation requires more than target engagement. The results guided us to develop selective degraders for FLT3 and BTK, with potentials to improve disease treatment. Together, this study demonstrates an efficient approach to triage a gene family of interest to identify readily degradable targets for further studies and pre-clinical developments.

SUMMARY Heterobifunctional small molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality. However, we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity, which is critically required to enable rational design of degraders. Here we utilize comprehensive characterization of the ligand dependent CRBN/BRD4 interaction to demonstrate that binding between proteins that have not evolved to interact is plastic. Multiple X-ray crystal structures show that plasticity results in several distinct low energy binding conformations, which are selectively bound by ligands. We demonstrate that computational protein-protein docking can reveal the underlying inter-protein contacts and inform the design of a BRD4 selective degrader that can discriminate between highly homologous BET bromodomains. Our findings that plastic inter-protein contacts confer selectivity for ligand-induced protein dimerization provide a conceptual framework for the development of heterobifunctional ligands.

Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.

The bromodomain and extraterminal (BET) family of proteins comprises four members-BRD2, BRD3, BRD4 and the testis-specific isoform BRDT-that largely function as transcriptional coactivators and play critical roles in various cellular processes, including the cell cycle, apoptosis, migration and invasion. BET proteins enhance the oncogenic functions of major cancer drivers by elevating the expression of these drivers, such as c-Myc in leukemia, or by promoting the transcriptional activities of oncogenic factors, such as AR and ERG in prostate cancer. Pathologically, BET proteins are frequently overexpressed and are clinically linked to various types of human cancer; they are therefore being pursued as attractive therapeutic targets for selective inhibition in patients with cancer. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed and have shown promising outcomes in early clinical trials. Although resistance to BET inhibitors has been documented in preclinical models, the molecular mechanisms underlying acquired resistance are largely unknown. Here we report that cullin-3SPOP earmarks BET proteins, including BRD2, BRD3 and BRD4, for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the degradation of BET proteins, leading to their elevated abundance in SPOP-mutant prostate cancer. As a result, prostate cancer cell lines and organoids derived from individuals harboring SPOP mutations are more resistant to BET-inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor-suppressor role of SPOP in prostate cancer in which it acts as a negative regulator of BET protein stability and also provide a molecular mechanism for resistance to BET inhibitors in individuals with prostate cancer bearing SPOP mutations.

BET bromodomain inhibition has contributed new insights into gene regulation and emerged as a promising therapeutic strategy in cancer. Structural analogy of early methyl-triazolo BET inhibitors has prompted a need for structurally dissimilar ligands as probes of bromodomain function. Using fluorous-tagged multicomponent reactions, we developed a focused chemical library of bromodomain inhibitors around a 3,5-dimethylisoxazole biasing element with micromolar biochemical IC50. Iterative synthesis and biochemical assessment allowed optimization of novel BET bromodomain inhibitors based on an imidazo[1,2-a]pyrazine scaffold. Lead compound 32 (UMB-32) binds BRD4 with a Kd of 550 nM and 724 nM cellular potency in BRD4-dependent lines. Additionally, compound 32 shows potency against TAF1, a bromodomain-containing transcription factor previously unapproached by discovery chemistry. Compound 32 was cocrystallized with BRD4, yielding a 1.56 Å resolution crystal structure. This research showcases new applications of fluorous and multicomponent chemical synthesis for the development of novel epigenetic inhibitors.

Im Bereich der chemischen Biologie werden biologische Sonden- und Wirkstoffmolekule klassischerweise genutzt, um die Aktivitat von Proteinen (wie Enzymen und Rezeptoren) zu modulieren, die sich verhaltnismasig leicht durch niedermolekulare Verbindungen beeinflussen lassen. Der verbleibende Rest, der die Mehrheit des Proteoms stellt, galt lange Zeit als fur Wirkstoffe unzuganglich (“undruggable”). Mithilfe niedermolekularer Modulatoren des Ubiquitin-Proteasomsystems (UPS) ist es moglich, statt der Proteinaktivitat die Proteinmenge zu modulieren, was die Anzahl der zuganglichen Zielmolekule erhoht. Wahrend ein Angriff auf das Proteasom selbst zu einer globalen Erhohung der Proteinmenge fuhren kann, lasst sich durch das Ansteuern anderer Komponenten des UPS (z. B. der E3-Ubiquitinligasen) in gezielter Weise eine Erhohung von Proteinmengen erreichen. Als Alternative dazu beginnen sich verschiedene Strategien zur Induktion des Proteinabbaus mittels niedermolekularer “Sonden” abzuzeichnen. Durch die Fahigkeit, den Abbau bestimmter Proteine zu induzieren und/oder zu inhibieren, besitzen niedermolekulare Modulatoren des UPS das Potenzial, den fur Wirkstoffe zuganglichen Anteil des Proteoms uber klassische Zielstrukturen wie Enzyme und Rezeptoren hinaus in signifikanter Weise zu erweitern.

SummaryFragment screening is widely used to identify attractive starting points for drug design. However, its potential and limitations to assess the tractability of often challenging protein:protein interfaces have been underexplored. Here, we address this question by means of a systematic deconstruction of lead-like inhibitors of the pVHL:HIF-1α interaction into their component fragments. Using biophysical techniques commonly employed for screening, we could only detect binding of fragments that violate the Rule of Three, are more complex than those typically screened against classical druggable targets, and occupy two adjacent binding subsites at the interface rather than just one. Analyses based on ligand and group lipophilicity efficiency of anchored fragments were applied to dissect the individual subsites and probe for binding hot spots. The implications of our findings for targeting protein interfaces by fragment-based approaches are discussed.

There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control. YY1 is a structural regulator of enhancer-promoter interactions and facilitates gene expression., National Institutes of Health (U.S.) (Grant HG002668/GM123511), National Institutes of Health (U.S.) (Grant R37HD045022/R01-NS088538/R01-MH104610), Virginia and Daniel K. Ludwig Graduate Fellowship, National Science Foundation (U.S.). Graduate Research Fellowship Program

Small molecule-induced protein degradation is an attractive strategy for the development of chemical probes. One method for inducing targeted protein degradation involves the use of PROTACs, heterobifunctional molecules that can recruit specific E3 ligases to a desired protein of interest. PROTACs have been successfully used to degrade numerous proteins in cells, but the peptidic E3 ligase ligands used in previous PROTACs have hindered their development into more mature chemical probes or therapeutics. We report the design of a novel class of PROTACs that incorporate small molecule VHL ligands to successfully degrade HaloTag7 fusion proteins. These HaloPROTACs will inspire the development of future PROTACs with more drug-like properties. Additionally, these HaloPROTACs are useful chemical genetic tools, due to their ability to chemically knock down widely used HaloTag7 fusion proteins in a general fashion.

A fundamental biological understanding of the individual contributions of functional domains within multidomain proteins is critical to inform therapeutic approaches to targeting mechanisms driving human disease. TRIM24 is a multi-domain protein that has been broadly characterized as a co-regulator of transcription. It is therapeutically relevant as it is implicated as a dependency in many human cancers, however, the potent and selective inhibitors of the TRIM24 bromodomain do not have well-characterized phenotypic consequences. Where inhibition of one activity of a protein does not appear to be efficacious, chemical knockdown tools allow for the acute depletion of the entire protein, therefore eliminating all protein activities. In this study, we have used chemical degradation as one strategy to target the entire TRIM24 protein, where we have shown that a bifunctional degrader molecule hijacks the ubiquitin ligase machinery for targeted TRIM24 degradation. We have shown that TRIM24 degradation is required to perturb the oncogenic state in leukemia. In this context, TRIM24 degradation rather than bromodomain inhibition alone is required to displace TRIM24 from chromatin. Using this probe, a further understanding of the contribution of TRIM24 domains to its transcriptional activation function will provide mechanistic insight as to how TRIM24 promotes a gene expression program permissive of the oncogenic state, as well as inform a therapeutic approach to target multidomain proteins, such as TRIM24, that are tightly linked to disease. Citation Format: Lara N. Gechijian, Dennis Buckley, Matthew Lawlor, Thomas Scott, Joshiawa Paulk, Jaime Reyes, Georg Winter, Michael Erb, Chris Ott, Sirano Dhe-Paganon, James Bradner. The design and characterization of a selective TRIM24 degrader [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 981. doi:10.1158/1538-7445.AM2017-981

Multiple myeloma (MM) remains an incurable malignancy with a clear need for novel therapeutic modalities. Moreover, acquired or de novo resistance to established or novel therapeutics remains a major challenge in this, and other, neoplasias. BET Bromodomain inhibitors (BBIs), including JQ1, have potent anti-MM activity in vitro and in in vivo, but do not provide curative outcome and do not induce apoptosis in many tumor cell types. Recently, a "next-generation" BBI, dBET, that causes degradation of BET Bromodomains (BRDs) through CRBN-mediated ubiquitination has been demonstrated to have potent activity in leukemia and myeloma. Here we sought to compare the mechanistic differences between BRD inhibition with BRD degradation in treatment-naive and drug-resistant MM. Additionally, we posited that resistance to dBET treatment could emerge through genetic perturbations and wished to uncover potential mechanisms prior to its clinical utilization. To address this, we compared effects of JQ1 with lead optimized compound dBET6, in a panel of human MM cell lines (± stromal cells), including clones resistant to JQ1, bortezomib and IMIDs, and assessed viability using CS-BLI/CTG assay. RNAseq and reverse phase protein arrays (RPPA) were employed to compare the transcriptional and translational effects of BRD degradation vs. inhibition. Using an open-ended unbiased genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)-associated Cas9 approach, we examined whether we could uncover genes associated with resistance to dBET6. MM1.S cells were transduced with Cas9 and pooled lentiviral particles of the GeCKO library, consisting of 2 pooled sgRNA sub-libraries (~120,000 sgRNAs; targeting ~19,000 genes and ~1800 miRNAs). Using this CRISPR/Cas9-based approach we sought to expedite the isolation of MM cells resistant to dBET6. We treated the pool of cells thrice with dBET (250nM), allowing regrowth between treatments and maintaining a coverage of 1000 cells/sgRNA. dBET6-resistant cells were processed to quantify sgRNA enrichment or depletion, using deep sequencing. We observed dBET6 to have significantly greater potency against MM cells than JQ1, or its combination with lenalidomide, and that MM1S.CRBN-/- cells were resistant to dBET6. Resistance to neither JQ1 nor bortezomib conferred resistance to dBET6. We observed dBET6 to induce rapid and robust ( Disclosures Bradner: Novartis Institutes for BioMedical Research: Employment.

Strategies for selectively killing HIV-infected cells present an appealing alternative to traditional antiretroviral drugs. We show here the first example of an inactive “Trojan horse” molecule that releases a cytotoxic, small-molecule proteasome inhibitor upon cleavage by HIV-1 protease. As a proof-of-concept strategy, the protein avidin was used to block entry of the compound into the proteasome in the absence of HIV-1 protease. We demonstrate that this strategy is also feasible without requiring an exogenous protein; a polylysine dendrimer-containing molecule is unable to enter the proteasome until cleaved by HIV-1 protease. These results demonstrate that conditional proteasome inhibitors could prove useful in the development of new tools for chemical biology and future therapeutics.

Traditionally, biological probes and drugs have targeted the activities of proteins (such as enzymes and receptors) that can be readily controlled by small molecules. The remaining majority of the proteome has been deemed "undruggable". By using small-molecule modulators of the ubiquitin proteasome, protein levels, rather than protein activity, can be targeted instead, thus increasing the number of druggable targets. Whereas targeting of the proteasome itself can lead to a global increase in protein levels, the targeting of other components of the UPS (e.g., the E3 ubiquitin ligases) can lead to an increase in protein levels in a more targeted fashion. Alternatively, multiple strategies for inducing protein degradation with small-molecule probes are emerging. With the ability to induce and inhibit the degradation of targeted proteins, small-molecule modulators of the UPS have the potential to significantly expand the druggable portion of the proteome beyond traditional targets, such as enzymes and receptors.

Multiple myeloma (MM) is an incurable malignancy with an unmet need for novel therapeutic modalities. Moreover, acquired or de novo resistance to established or novel therapeutics remains a major challenge in this, and other, neoplasias. BET Bromodomain inhibitors (BBIs), including JQ1, have potent anti-MM activity in vitro and in in vivo, but do not provide curative outcome and do not induce apoptosis in most cell types. We sought to investigate dBET, a class of BBIs that induce degradation of BET Bromodomains (BRDs) through CRBN-mediated ubiquitination and proteasomal degradation, in drug-resistant MM. Additionally, we posited that resistance to dBET treatment would emerge through genetic perturbations and wished to uncover potential mechanisms prior to its clinical utilization. To address this, we compared effects of optimized lead compound, dBET6, with JQ1 on a panel of MM cell lines, including clones resistant to JQ1 or bortezomib and assessed viability using CS-BLI/CTG assay and BRD/c-MYC expression by western blot. Using an open-ended unbiased genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)-associated Cas9 approach, we examined whether we could uncover genes associated with resistance to dBET6. MM1.S cells were transduced with Cas9 and pooled lentiviral particles of the GeCKO library, consisting of 2 pooled sgRNA sub-libraries (∼120,000 sgRNAs; targeting ∼19,000 genes and ∼1800 miRNAs). Using this CRISPR/Cas9-based approach we sought to expedite the isolation of MM cells resistant to dBET6. We treated the pool of cells thrice with dBET (≥IC80), allowing regrowth between treatments and maintaining a coverage of 1000 cells/sgRNA. dBET6-resistant cells were processed to quantify sgRNA enrichment or depletion, using deep sequencing. We observed dBET6 to have significantly greater potency against MM cells than JQ1, or its combination with lenalidomide, and that MM1S.CRBN-/- cells were resistant to dBET6. Resistance to neither JQ1 nor Bortezomib conferred resistance to dBET6. We observed dBET6 to induce rapid ( Citation Format: Geoffrey M. Matthews, Yiguo Hu, Michal Sheffer, Paul J. Hengeveld, Dennis L. Buckley, Megan A. Bariteau, Haley Poarch, James E. Bradner, Constantine S. Mitsiades. BET bromodomain degradation as a therapeutic strategy in drug-resistant multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4713.

E3 ubiquitin ligases, such as the therapeutically relevant VHL, are challenging targets for traditional medicinal chemistry, as their modulation requires targeting protein-protein interactions. We report novel small-molecule inhibitors of the interaction between VHL and its molecular target HIF1α, a transcription factor involved in oxygen sensing.

E3 ubiquitin ligases, which bind protein targets, leading to their ubiquitination and subsequent degradation, are attractive drug targets due to their exquisite substrate specificity. However, the development of small-molecule inhibitors has proven extraordinarily challenging as modulation of E3 ligase activities requires the targeting of protein-protein interactions. Using rational design, we have generated the first small molecule targeting the von Hippel-Lindau protein (VHL), the substrate recognition subunit of an E3 ligase, and an important target in cancer, chronic anemia, and ischemia. We have also obtained the crystal structure of VHL bound to our most potent inhibitor, confirming that the compound mimics the binding mode of the transcription factor HIF-1 alpha, a substrate of VHL. These results have the potential to guide future development of improved lead compounds as therapeutics for the treatment of chronic anemia and ischemia.

The theme of the Third World Congress of Comparative Education Societies, "Diversity and Unity Through Education," seems especially appropriate to the present thrust in research at the UNESCO Institute for Education in Hamburg, Germany. This is because the principles of lifelong education provide a framework for unity of educational efforts, yet allow for national diversity as well.

Despite evidence that kinesin family member 14 (KIF14) can serve as a prognostic biomarker in various solid tumors, how it contributes to tumorigenesis remains unclear. We observed that experimental decrease in KIF14 expression increases docetaxel chemosensitivity in estrogen receptor–negative/progesterone receptor–negative/human epidermal growth factor receptor 2-negative, "triple-negative" breast cancers (TNBC). To investigate the oncogenic role of KIF14 , we used noncancerous human mammary epithelial cells and ectopically expressed KIF14 and found increased proliferative capacity, increased anchorage-independent grown in vitro , and increased resistance to docetaxel but not to doxorubicin, carboplatin, or gemcitabine. Seventeen benign breast biopsies of BRCA1 or BRCA2 mutation carriers showed increased KIF14 mRNA expression by fluorescence in situ hybridization compared to controls with no known mutations in BRCA1 or BRCA2 , suggesting increased KIF14 expression as a biomarker of high-risk breast tissue. Evaluation of 34 cases of locally advanced TNBC showed that KIF14 expression significantly correlates with chemotherapy-resistant breast cancer. KIF14 knockdown also correlates with decreased AKT phosphorylation and activity. Live-cell imaging confirmed an insulin-induced temporal colocalization of KIF14 and AKT at the plasma membrane, suggesting a potential role of KIF14 in promoting activation of AKT. An experimental small-molecule inhibitor of KIF14 was then used to evaluate the potential anticancer benefits of downregulating KIF14 activity. Inhibition of KIF14 shows a chemosensitizing effect and correlates with decreasing activation of AKT. Together, these findings show an early and critical role for KIF14 in the tumorigenic potential of TNBC, and therapeutic targeting of KIF14 is feasible and effective for TNBC.