Cellular signaling is often propagated by multivalent interactions. Multivalency creates avidity, allowing stable biophysical recognition. Multivalency is an attractive strategy for achieving potent binding to protein targets, as the affinity of bivalent ligands is often greater than the sum of monovalent affinities. The bromodomain and extraterminal domain (BET) family of transcriptional coactivators features tandem bromodomains through which BET proteins bind acetylated histones and transcription factors. All reported antagonists of the BET protein BRD4 bind in a monovalent fashion. Here we describe, to our knowledge for the first time, a bivalent BET bromodomain inhibitor-MT1-which has unprecedented potency. Biophysical and biochemical studies suggest MT1 is an intramolecular bivalent BRD4 binder that is more than 100-fold more potent, in cellular assays, than the corresponding monovalent antagonist, JQ1. MT1 significantly (P < 0.05) delayed leukemia progression in mice, as compared to JQ1. These data qualify a powerful chemical probe for BET bromodomains and a rationale for further development of multidomain inhibitors of epigenetic reader proteins.

BCL-2 family proteins are high-priority cancer targets whose structures provide essential blueprints for drug design. Whereas numerous structures of anti-apoptotic BCL-2 protein complexes with α-helical BH3 peptides have been reported, the corresponding panel of apo structures remains incomplete. Here, we report the crystal structure of apo BFL-1 at 1.69-Å resolution, revealing similarities and key differences among unliganded anti-apoptotic proteins. Unlike all other BCL-2 proteins, apo BFL-1 contains a surface-accessible cysteine within its BH3-binding groove, allowing for selective covalent targeting by a NOXA BH3-based stapled peptide inhibitor. The crystal structure of this complex demonstrated the sulfhydryl bond and fortuitous interactions between the acrylamide-bearing moiety and a newly formed hydrophobic cavity. Comparison of the apo and BH3-liganded structures further revealed an induced conformational change. The two BFL-1 structures expand our understanding of the surface landscapes available for therapeutic targeting so that the apoptotic blockades of BFL-1-dependent cancers can be overcome.

p53 is a critical tumor-suppressor protein that guards the human genome against mutations by inducing cell-cycle arrest or apoptosis. Cancer cells subvert p53 by deletion, mutation, or overexpression of the negative regulators HDM2 and HDMX. For tumors that retain wild-type p53, its reactivation by pharmacologic targeting of HDM2 and/or HDMX represents a promising strategy, with a series of selective small-molecule HDM2 inhibitors and a dual HDM2/HDMX stapled-peptide inhibitor being evaluated in clinical trials. Because selective HDM2 targeting can cause hematologic toxicity, selective HDMX inhibitors could provide an alternative p53-reactivation strategy, but clinical candidates remain elusive. Here, we applied a mutation-scanning approach to uncover p53-based stapled peptides that are selective for HDMX. Crystal structures of stapled-peptide/HDMX complexes revealed a molecular mechanism for the observed specificity, which was validated by HDMX mutagenesis. Thus, we provide a blueprint for the development of HDMX-selective inhibitors to dissect and target the p53/HDMX interaction.

Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal α-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities. Further, we define changes in BAF complex architecture upon nucleosome engagement and compare the structural model of endogenous BAF to those of related SWI/SNF-family complexes. Finally, we assign and experimentally interrogate cancer-associated hot-spot mutations localizing within the endogenous human BAF complex, identifying those that disrupt BAF subunit-subunit and subunit-nucleosome interfaces in the nucleosome-bound conformation. Taken together, this integrative structural approach provides important biophysical foundations for understanding the mechanisms of BAF complex function in normal and disease states.

Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5BN642H, a frequently-occurring oncogenic driver mutation, promotes aggressive T-cell leukemia/lymphoma in patient carriers, although the molecular origins remain unclear. Herein, we emphasize the aggressive nature of STAT5BN642H in driving T-cell neoplasia upon hematopoietic expression in transgenic mice, revealing evidence of multiple T-cell subset organ infiltration. Notably, we demonstrate STAT5BN642H-driven transformation of γδ T-cells in in vivo syngeneic transplant models, comparable to STAT5BN642H patient γδ T-cell entities. Importantly, we present human STAT5B and STAT5BN642H crystal structures, which propose alternative mutation-mediated SH2 domain conformations. Our biophysical data suggests STAT5BN642H can adopt a hyper-activated and hyper-inactivated state with resistance to dephosphorylation. MD simulations support sustained interchain cross-domain interactions in STAT5BN642H, conferring kinetic stability to the mutant anti-parallel dimer. This study provides a molecular explanation for the STAT5BN642H activating potential, and insights into pre-clinical models for targeted intervention of hyper-activated STAT5B.

Mammalian switch/sucrose non-fermentable (mSWI/SNF) complexes are multi-component machines that remodel chromatin architecture. Dissection of the subunit- and domain-specific contributions to complex activities is needed to advance mechanistic understanding. Here, we examine the molecular, structural, and genome-wide regulatory consequences of recurrent, single-residue mutations in the putative coiled-coil C-terminal domain (CTD) of the SMARCB1 (BAF47) subunit, which cause the intellectual disability disorder Coffin-Siris syndrome (CSS), and are recurrently found in cancers. We find that the SMARCB1 CTD contains a basic α helix that binds directly to the nucleosome acidic patch and that all CSS-associated mutations disrupt this binding. Furthermore, these mutations abrogate mSWI/SNF-mediated nucleosome remodeling activity and enhancer DNA accessibility without changes in genome-wide complex localization. Finally, heterozygous CSS-associated SMARCB1 mutations result in dominant gene regulatory and morphologic changes during iPSC-neuronal differentiation. These studies unmask an evolutionarily conserved structural role for the SMARCB1 CTD that is perturbed in human disease.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

T cell exhaustion limits antitumor immunity, but the molecular determinants of this process remain poorly understood. Using a chronic stimulation assay, we performed genome-wide CRISPR-Cas9 screens to systematically discover regulators of T cell exhaustion, which identified an enrichment of epigenetic factors. In vivo CRISPR screens in murine and human tumor models demonstrated that perturbation of the INO80 and BAF chromatin remodeling complexes improved T cell persistence in tumors. In vivo Perturb-seq revealed distinct transcriptional roles of each complex and that depletion of canonical BAF complex members, including Arid1a, resulted in the maintenance of an effector program and downregulation of exhaustion-related genes in tumor-infiltrating T cells. Finally, Arid1a depletion limited the acquisition of exhaustion-associated chromatin accessibility and led to improved antitumor immunity. In summary, we provide an atlas of the genetic regulators of T cell exhaustion and demonstrate that modulation of epigenetic state can improve T cell responses in cancer immunotherapy.

Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally-uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function study, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, computational modeling, and biochemical analyses, to characterize a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted α-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, our data support a structural basis for VLCAD deficiency in patients with discrete mutations in an α-helical membrane-binding motif, resulting in pathologic enzyme mislocalization.

Development of SARS-CoV-2 vaccines that protect vulnerable populations is a public health priority. Here, we took a systematic and iterative approach by testing several adjuvants and SARS-CoV-2 antigens to identify a combination that elicits antibodies and protection in young and aged mice. While demonstrating superior immunogenicity to soluble receptor-binding domain (RBD), RBD displayed as a protein nanoparticle (RBD-NP) generated limited antibody responses. Comparison of multiple adjuvants including AddaVax, AddaS03, and AS01B in young and aged mice demonstrated that an oil-in-water emulsion containing carbohydrate fatty acid monosulphate derivative (CMS:O/W) most effectively enhanced RBD-NP-induced cross-neutralizing antibodies and protection across age groups. CMS:O/W enhanced antigen retention in the draining lymph node, induced injection site, and lymph node cytokines, with CMS inducing MyD88-dependent Th1 cytokine polarization. Furthermore, CMS and O/W synergistically induced chemokine production from human PBMCs. Overall, CMS:O/W adjuvant may enhance immunogenicity and protection of vulnerable populations against SARS-CoV-2 and other infectious pathogens.

Deubiquitinating enzymes (DUBs) are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors. Despite growing interest in DUB biological function and potential as therapeutic targets, few selective small-molecule inhibitors and no approved drugs currently exist. To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family, we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs, spanning three well-characterized DUB families. Promising hit compounds were validated in a series of counter-screens and orthogonal assays, as well as further assessed for selectivity across expanded panels of DUBs. Through these efforts, we have identified multiple highly selective DUB inhibitors and developed a roadmap for rapidly identifying and validating selective inhibitors of related enzymes.

The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here, we present the discovery and characterization of a pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/β/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.

Mammalian SWI/SNF chromatin remodelling complexes exist in three distinct, final-form assemblies: canonical BAF (cBAF), PBAF and a newly characterized non-canonical complex (ncBAF). However, their complex-specific targeting on chromatin, functions and roles in disease remain largely undefined. Here, we comprehensively mapped complex assemblies on chromatin and found that ncBAF complexes uniquely localize to CTCF sites and promoters. We identified ncBAF subunits as synthetic lethal targets specific to synovial sarcoma and malignant rhabdoid tumours, which both exhibit cBAF complex (SMARCB1 subunit) perturbation. Chemical and biological depletion of the ncBAF subunit, BRD9, rapidly attenuates synovial sarcoma and malignant rhabdoid tumour cell proliferation. Importantly, in cBAF-perturbed cancers, ncBAF complexes maintain gene expression at retained CTCF-promoter sites and function in a manner distinct from fusion oncoprotein-bound complexes. Together, these findings unmask the unique targeting and functional roles of ncBAF complexes and present new cancer-specific therapeutic targets.

Chronic inflammation is linked to diverse disease processes, but the intrinsic mechanisms that determine cellular sensitivity to inflammation are incompletely understood. Here, we show the contribution of glucose metabolism to inflammation-induced changes in the survival of pancreatic islet β-cells. Using metabolomic, biochemical and functional analyses, we investigate the protective versus non-protective effects of glucose in the presence of pro-inflammatory cytokines. When protective, glucose metabolism augments anaplerotic input into the TCA cycle via pyruvate carboxylase (PC) activity, leading to increased aspartate levels. This metabolic mechanism supports the argininosuccinate shunt, which fuels ureagenesis from arginine and conversely diminishes arginine utilization for production of nitric oxide (NO), a chief mediator of inflammatory cytotoxicity. Activation of the PC-urea cycle axis is sufficient to suppress NO synthesis and shield cells from death in the context of inflammation and other stress paradigms. Overall, these studies uncover a previously unappreciated link between glucose metabolism and arginine-utilizing pathways via PC-directed ureagenesis as a protective mechanism.

Interactions between chromatin-associated proteins and the histone landscape play major roles in dictating genome topology and gene expression. Cancer-specific fusion oncoproteins, which display unique chromatin localization patterns, often lack classical DNA-binding domains, presenting challenges in identifying mechanisms governing their site-specific chromatin targeting and function. Here we identify a minimal region of the human SS18-SSX fusion oncoprotein (the hallmark driver of synovial sarcoma) that mediates a direct interaction between the mSWI/SNF complex and the nucleosome acidic patch. This binding results in altered mSWI/SNF composition and nucleosome engagement, driving cancer-specific mSWI/SNF complex targeting and gene expression. Furthermore, the C-terminal region of SSX confers preferential affinity to repressed, H2AK119Ub-marked nucleosomes, underlying the selective targeting to polycomb-marked genomic regions and synovial sarcoma-specific dependency on PRC1 function. Together, our results describe a functional interplay between a key nucleosome binding hub and a histone modification that underlies the disease-specific recruitment of a major chromatin remodeling complex.

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.

Defects in interneuron migration can disrupt the assembly of cortical circuits and lead to neuropsychiatric disease. Using forebrain assembloids derived by integration of cortical and ventral forebrain organoids, we have previously discovered a cortical interneuron migration defect in Timothy syndrome (TS), a severe neurodevelopmental disease caused by a mutation in the L-type calcium channel (LTCC) Cav1.2. Here, we find that acute pharmacological modulation of Cav1.2 can regulate the saltation length, but not the frequency, of interneuron migration in TS. Interestingly, the defect in saltation length is related to aberrant actomyosin and myosin light chain (MLC) phosphorylation, while the defect in saltation frequency is driven by enhanced γ-aminobutyric acid (GABA) sensitivity and can be restored by GABA-A receptor antagonism. Finally, we describe hypersynchronous hCS network activity in TS that is exacerbated by interneuron migration. Taken together, these studies reveal a complex role of LTCC function in human cortical interneuron migration and strategies to restore deficits in the context of disease.

Mammalian SWI/SNF (mSWI/SNF or BAF) ATP-dependent chromatin remodeling complexes play critical roles in governing genomic architecture and gene expression and are frequently perturbed in human cancers. Transcription factors (TFs), including fusion oncoproteins, can bind to BAF complex surfaces to direct chromatin targeting and accessibility, often activating oncogenic gene loci. Here, we demonstrate that the FUS::DDIT3 fusion oncoprotein hallmark to myxoid liposarcoma (MLPS) inhibits BAF complex-mediated remodeling of adipogenic enhancer sites via sequestration of the adipogenic TF, CEBPB, from the genome. In mesenchymal stem cells, small-molecule inhibition of BAF complex ATPase activity attenuates adipogenesis via failure of BAF-mediated DNA accessibility and gene activation at CEBPB target sites. BAF chromatin occupancy and gene expression profiles of FUS::DDIT3-expressing cell lines and primary tumors exhibit similarity to SMARCB1-deficient tumor types. These data present a mechanism by which a fusion oncoprotein generates a BAF complex loss-of-function phenotype, independent of deleterious subunit mutations.

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.

The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.