An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity.

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.

Direct Tumor Killing and Immunotherapy through Anti-SerpinB9 Therapy.

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.

Recurrent SMARCB1 Mutations Reveal a Nucleosome Acidic Patch Interaction Site That Potentiates mSWI/SNF Complex Chromatin Remodeling.

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.

Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α.

Exercise benefits the human body in many ways. Irisin is secreted by muscle, increased with exercise, and conveys physiological benefits, including improved cognition and resistance to neurodegeneration. Irisin acts via αV integrins; however, a mechanistic understanding of how small polypeptides like irisin can signal through integrins is poorly understood. Using mass spectrometry and cryo-EM, we demonstrate that the extracellular heat shock protein 90α (eHsp90α) is secreted by muscle with exercise and activates integrin αVß5. This allows for high-affinity irisin binding and signaling through an Hsp90α/αV/ß5 complex. By including hydrogen/deuterium exchange data, we generate and experimentally validate a 2.98 Å RMSD irisin/αVß5 complex docking model. Irisin binds very tightly to an alternative interface on αVß5 distinct from that used by known ligands. These data elucidate a non-canonical mechanism by which a small polypeptide hormone like irisin can function through an integrin receptor.

Regulation of GTPase function by autophosphorylation.

A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts.

Lactate regulates cell cycle by remodelling the anaphase promoting complex.

Lactate is abundant in rapidly dividing cells owing to the requirement for elevated glucose catabolism to support proliferation1-6. However, it is not known whether accumulated lactate affects the proliferative state. Here we use a systematic approach to determine lactate-dependent regulation of proteins across the human proteome. From these data, we identify a mechanism of cell cycle regulation whereby accumulated lactate remodels the anaphase promoting complex (APC/C). Remodelling of APC/C in this way is caused by direct inhibition of the SUMO protease SENP1 by lactate. We find that accumulated lactate binds and inhibits SENP1 by forming a complex with zinc in the SENP1 active site. SENP1 inhibition by lactate stabilizes SUMOylation of two residues on APC4, which drives UBE2C binding to APC/C. This direct regulation of APC/C by lactate stimulates timed degradation of cell cycle proteins, and efficient mitotic exit in proliferative human cells. This mechanism is initiated upon mitotic entry when lactate abundance reaches its apex. In this way, accumulation of lactate communicates the consequences of a nutrient-replete growth phase to stimulate timed opening of APC/C, cell division and proliferation. Conversely, persistent accumulation of lactate drives aberrant APC/C remodelling and can overcome anti-mitotic pharmacology via mitotic slippage. In sum, we define a biochemical mechanism through which lactate directly regulates protein function to control the cell cycle and proliferation.

An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme.

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.

Depletion of creatine phosphagen energetics with a covalent creatine kinase inhibitor.

Creatine kinases (CKs) provide local ATP production in periods of elevated energetic demand, such as during rapid anabolism and growth. Thus, creatine energetics has emerged as a major metabolic liability in many rapidly proliferating cancers. Whether CKs can be targeted therapeutically is unknown because no potent or selective CK inhibitors have been developed. Here we leverage an active site cysteine present in all CK isoforms to develop a selective covalent inhibitor of creatine phosphagen energetics, CKi. Using deep chemoproteomics, we discover that CKi selectively engages the active site cysteine of CKs in cells. A co-crystal structure of CKi with creatine kinase B indicates active site inhibition that prevents bidirectional phosphotransfer. In cells, CKi and its analogs rapidly and selectively deplete creatine phosphate, and drive toxicity selectively in CK-dependent acute myeloid leukemia. Finally, we use CKi to uncover an essential role for CKs in the regulation of proinflammatory cytokine production in macrophages.

Reduced SARS-CoV-2 mRNA vaccine immunogenicity and protection in mice with diet-induced obesity and insulin resistance.

BACKGROUND: Obesity and type 2 diabetes mellitus (T2DM) are associated with an increased risk of severe outcomes from infectious diseases, including coronavirus disease 2019. These conditions are also associated with distinct responses to immunization, including an impaired response to widely used severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines. OBJECTIVE: We sought to establish a connection between reduced immunization efficacy via modeling the effects of metabolic diseases on vaccine immunogenicity that is essential for the development of more effective vaccines for this distinct vulnerable population. METHODS: A murine model of diet-induced obesity and insulin resistance was used to model the effects of comorbid T2DM and obesity on vaccine immunogenicity and protection. RESULTS: Mice fed a high-fat diet (HFD) developed obesity, hyperinsulinemia, and glucose intolerance. Relative to mice fed a normal diet, HFD mice vaccinated with a SARS-CoV-2 mRNA vaccine exhibited significantly lower anti-spike IgG titers, predominantly in the IgG2c subclass, associated with a lower type 1 response, along with a 3.83-fold decrease in neutralizing titers. Furthermore, enhanced vaccine-induced spike-specific CD8+ T-cell activation and protection from lung infection against SARS-CoV-2 challenge were seen only in mice fed a normal diet but not in HFD mice. CONCLUSIONS: The study demonstrated impaired immunity following SARS-CoV-2 mRNA immunization in a murine model of comorbid T2DM and obesity, supporting the need for further research into the basis for impaired anti-SARS-CoV-2 immunity in T2DM and investigation of novel approaches to enhance vaccine immunogenicity among those with metabolic diseases.

Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo.

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

Transcription control by the ENL YEATS domain in acute leukaemia.

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.

Identification of a potent and selective covalent Pin1 inhibitor.

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.

Functional TRIM24 degrader via conjugation of ineffectual bromodomain and VHL ligands.

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.

p53-related protein kinase confers poor prognosis and represents a novel therapeutic target in multiple myeloma.

p53-related protein kinase (TP53RK, also known as PRPK) is an upstream kinase that phosphorylates (serine residue Ser15) and mediates p53 activity. Here we show that TP53RK confers poor prognosis in multiple myeloma (MM) patients, and, conversely, that TP53RK knockdown inhibits p53 phosphorylation and triggers MM cell apoptosis, associated with downregulation of c-Myc and E2F-1-mediated upregulation of pro-apoptotic Bim. We further demonstrate that TP53RK downregulation also triggers growth inhibition in p53-deficient and p53-mutant MM cell lines and identify novel downstream targets of TP53RK including ribonucleotide reductase-1, telomerase reverse transcriptase, and cyclin-dependent kinase inhibitor 2C. Our previous studies showed that immunomodulatory drugs (IMiDs) downregulate p21 and trigger apoptosis in wild-type-p53 MM.1S cells, Importantly, we demonstrate by pull-down, nuclear magnetic resonance spectroscopy, differential scanning fluorimetry, and isothermal titration calorimetry that IMiDs bind and inhibit TP53RK, with biologic sequelae similar to TP53RK knockdown. Our studies therefore demonstrate that either genetic or pharmacological inhibition of TP53RK triggers MM cell apoptosis via both p53-Myc axis-dependent and axis-independent pathways, validating TP53RK as a novel therapeutic target in patients with poor-prognosis MM.

Inhibition of USP10 induces degradation of oncogenic FLT3.

Oncogenic forms of the kinase FLT3 are important therapeutic targets in acute myeloid leukemia (AML); however, clinical responses to small-molecule kinase inhibitors are short-lived as a result of the rapid emergence of resistance due to point mutations or compensatory increases in FLT3 expression. We sought to develop a complementary pharmacological approach whereby proteasome-mediated FLT3 degradation could be promoted by inhibitors of the deubiquitinating enzymes (DUBs) responsible for cleaving ubiquitin from FLT3. Because the relevant DUBs for FLT3 are not known, we assembled a focused library of most reported small-molecule DUB inhibitors and carried out a cellular phenotypic screen to identify compounds that could induce the degradation of oncogenic FLT3. Subsequent target deconvolution efforts allowed us to identify USP10 as the critical DUB required to stabilize FLT3. Targeting of USP10 showed efficacy in preclinical models of mutant-FLT3 AML, including cell lines, primary patient specimens and mouse models of oncogenic-FLT3-driven leukemia.

Structure of LIMP-2 provides functional insights with implications for SR-BI and CD36.

Members of the CD36 superfamily of scavenger receptor proteins are important regulators of lipid metabolism and innate immunity. They recognize normal and modified lipoproteins, as well as pathogen-associated molecular patterns. The family consists of three members: SR-BI (which delivers cholesterol to the liver and steroidogenic organs and is a co-receptor for hepatitis C virus), LIMP-2/LGP85 (which mediates lysosomal delivery of ß-glucocerebrosidase and serves as a receptor for enterovirus 71 and coxsackieviruses) and CD36 (a fatty-acid transporter and receptor for phagocytosis of effete cells and Plasmodium-infected erythrocytes). Notably, CD36 is also a receptor for modified lipoproteins and ß-amyloid, and has been implicated in the pathogenesis of atherosclerosis and of Alzheimer's disease. Despite their prominent roles in health and disease, understanding the function and abnormalities of the CD36 family members has been hampered by the paucity of information about their structure. Here we determine the crystal structure of LIMP-2 and infer, by homology modelling, the structure of SR-BI and CD36. LIMP-2 shows a helical bundle where ß-glucocerebrosidase binds, and where ligands are most likely to bind to SR-BI and CD36. Remarkably, the crystal structure also shows the existence of a large cavity that traverses the entire length of the molecule. Mutagenesis of SR-BI indicates that the cavity serves as a tunnel through which cholesterol(esters) are delivered from the bound lipoprotein to the outer leaflet of the plasma membrane. We provide evidence supporting a model whereby lipidic constituents of the ligands attached to the receptor surface are handed off to the membrane through the tunnel, accounting for the selective lipid transfer characteristic of SR-BI and CD36.

Design and characterization of bivalent BET inhibitors.

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.

Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling.

Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteins retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. In more general terms, we suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.

Degradation of the BAF Complex Factor BRD9 by Heterobifunctional Ligands.

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

Structural Characterization of Interaction between Human Ubiquitin-specific Protease 7 and Immediate-Early Protein ICP0 of Herpes Simplex Virus-1.

Human ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that prevents protein degradation by removing polyubiquitin chains from its substrates. It regulates the stability of a number of human transcription factors and tumor suppressors and plays a critical role in the development of several types of cancer, including prostate and small cell lung cancer. In addition, human USP7 is targeted by several viruses of the Herpesviridae family and is required for effective herpesvirus infection. The USP7 C-terminal region (C-USP7) contains five ubiquitin-like domains (UBL1-5) that interact with several USP7 substrates. Although structures of the USP7 C terminus bound to its substrates could provide vital information for understanding USP7 substrate specificity, no such data has been available to date. In this work we have demonstrated that the USP7 ubiquitin-like domains can be studied in isolation by solution NMR spectroscopy, and we have determined the structure of the UBL1 domain. Furthermore, we have employed NMR and viral plaque assays to probe the interaction between the C-USP7 and HSV-1 immediate-early protein ICP0 (infected cell protein 0), which is essential for efficient lytic infection and virus reactivation from latency. We have shown that depletion of the USP7 in HFF-1 cells negatively affects the efficiency of HSV-1 lytic infection. We have also found that USP7 directly binds ICP0 via its C-terminal UBL1-2 domains and mapped the USP7-binding site for ICP0. Therefore, this study represents a first step toward understanding the molecular mechanism of C-USP7 specificity toward its substrates and may provide the basis for future development of novel antiviral and anticancer therapies.