Proteomics: A new dimension to decode small cell lung cancer.

Small cell lung cancer (SCLC) is a recalcitrant malignancy. Conquering it will require deep insight into its biology. In this issue of Cell, Liu and colleagues describe proteomic and phosphoproteomic landscapes of resected SCLC tumors and illustrate the potential of this knowledge to identify new SCLC vulnerabilities.

Safe and effective in vivo delivery of DNA and RNA using proteolipid vehicles.

Genetic medicines show promise for treating various diseases, yet clinical success has been limited by tolerability, scalability, and immunogenicity issues of current delivery platforms. To overcome these, we developed a proteolipid vehicle (PLV) by combining features from viral and non-viral approaches. PLVs incorporate fusion-associated small transmembrane (FAST) proteins isolated from fusogenic orthoreoviruses into a well-tolerated lipid formulation, using scalable microfluidic mixing. Screening a FAST protein library, we identified a chimeric FAST protein with enhanced membrane fusion activity that improved gene expression from an optimized lipid formulation. Systemically administered FAST-PLVs showed broad biodistribution and effective mRNA and DNA delivery in mouse and non-human primate models. FAST-PLVs show low immunogenicity and maintain activity upon repeat dosing. Systemic administration of follistatin DNA gene therapy with FAST-PLVs raised circulating follistatin levels and significantly increased muscle mass and grip strength. These results demonstrate the promising potential of FAST-PLVs for redosable gene therapies and genetic medicines.

CD1 lipidomes reveal lipid-binding motifs and size-based antigen-display mechanisms.

The CD1 system binds lipid antigens for display to T cells. Here, we solved lipidomes for the four human CD1 antigen-presenting molecules, providing a map of self-lipid display. Answering a basic question, the detection of >2,000 CD1-lipid complexes demonstrates broad presentation of self-sphingolipids and phospholipids. Whereas peptide antigens are chemically processed, many lipids are presented in an unaltered form. However, each type of CD1 protein differentially edits the self-lipidome to show distinct capture motifs based on lipid length and chemical composition, suggesting general antigen display mechanisms. For CD1a and CD1d, lipid size matches the CD1 cleft volume. CD1c cleft size is more variable, and CD1b is the outlier, where ligands and clefts show an extreme size mismatch that is explained by uniformly seating two small lipids in one cleft. Furthermore, the list of compounds that comprise the integrated CD1 lipidome supports the ongoing discovery of lipid blockers and antigens for T cells.

Mechanopathology of biofilm-like Mycobacterium tuberculosis cords.

Mycobacterium tuberculosis (Mtb) cultured axenically without detergent forms biofilm-like cords, a clinical identifier of virulence. In lung-on-chip (LoC) and mouse models, cords in alveolar cells contribute to suppression of innate immune signaling via nuclear compression. Thereafter, extracellular cords cause contact-dependent phagocyte death but grow intercellularly between epithelial cells. The absence of these mechanopathological mechanisms explains the greater proportion of alveolar lesions with increased immune infiltration and dissemination defects in cording-deficient Mtb infections. Compression of Mtb lipid monolayers induces a phase transition that enables mechanical energy storage. Agent-based simulations demonstrate that the increased energy storage capacity is sufficient for the formation of cords that maintain structural integrity despite mechanical perturbation. Bacteria in cords remain translationally active despite antibiotic exposure and regrow rapidly upon cessation of treatment. This study provides a conceptual framework for the biophysics and function in tuberculosis infection and therapy of cord architectures independent of mechanisms ascribed to single bacteria.

Genomic surveillance reveals dynamic shifts in the connectivity of COVID-19 epidemics.

The maturation of genomic surveillance in the past decade has enabled tracking of the emergence and spread of epidemics at an unprecedented level. During the COVID-19 pandemic, for example, genomic data revealed that local epidemics varied considerably in the frequency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage importation and persistence, likely due to a combination of COVID-19 restrictions and changing connectivity. Here, we show that local COVID-19 epidemics are driven by regional transmission, including across international boundaries, but can become increasingly connected to distant locations following the relaxation of public health interventions. By integrating genomic, mobility, and epidemiological data, we find abundant transmission occurring between both adjacent and distant locations, supported by dynamic mobility patterns. We find that changing connectivity significantly influences local COVID-19 incidence. Our findings demonstrate a complex meaning of "local" when investigating connected epidemics and emphasize the importance of collaborative interventions for pandemic prevention and mitigation.

Epigenetic reader SP140 loss of function drives Crohn's disease due to uncontrolled macrophage topoisomerases.

How mis-regulated chromatin directly impacts human immune disorders is poorly understood. Speckled Protein 140 (SP140) is an immune-restricted PHD and bromodomain-containing epigenetic "reader," and SP140 loss-of-function mutations associate with Crohn's disease (CD), multiple sclerosis (MS), and chronic lymphocytic leukemia (CLL). However, the relevance of these mutations and mechanisms underlying SP140-driven pathogenicity remains unexplored. Using a global proteomic strategy, we identified SP140 as a repressor of topoisomerases (TOPs) that maintains heterochromatin and macrophage fate. In humans and mice, SP140 loss resulted in unleashed TOP activity, de-repression of developmentally silenced genes, and ultimately defective microbe-inducible macrophage transcriptional programs and bacterial killing that drive intestinal pathology. Pharmacological inhibition of TOP1/2 rescued these defects. Furthermore, exacerbated colitis was restored with TOP1/2 inhibitors in Sp140-/- mice, but not wild-type mice, in vivo. Collectively, we identify SP140 as a TOP repressor and reveal repurposing of TOP inhibition to reverse immune diseases driven by SP140 loss.

Molecular Epigenetics: Chemical Biology Tools Come of Age.

The field of epigenetics has exploded over the last two decades, revealing an astonishing level of complexity in the way genetic information is stored and accessed in eukaryotes. This expansion of knowledge, which is very much ongoing, has been made possible by the availability of evermore sensitive and precise molecular tools. This review focuses on the increasingly important role that chemistry plays in this burgeoning field. In an effort to make these contributions more accessible to the nonspecialist, we group available chemical approaches into those that allow the covalent structure of the protein and DNA components of chromatin to be manipulated, those that allow the activity of myriad factors that act on chromatin to be controlled, and those that allow the covalent structure and folding of chromatin to be characterized. The application of these tools is illustrated through a series of case studies that highlight how the molecular precision afforded by chemistry is being used to establish causal biochemical relationships at the heart of epigenetic regulation.

Multimodal single-cell datasets characterize antigen-specific CD8+ T cells across SARS-CoV-2 vaccination and infection.

The immune response to SARS-CoV-2 antigen after infection or vaccination is defined by the durable production of antibodies and T cells. Population-based monitoring typically focuses on antibody titer, but there is a need for improved characterization and quantification of T cell responses. Here, we used multimodal sequencing technologies to perform a longitudinal analysis of circulating human leukocytes collected before and after immunization with the mRNA vaccine BNT162b2. Our data indicated distinct subpopulations of CD8+ T cells, which reliably appeared 28 days after prime vaccination. Using a suite of cross-modality integration tools, we defined their transcriptome, accessible chromatin landscape and immunophenotype, and we identified unique biomarkers within each modality. We further showed that this vaccine-induced population was SARS-CoV-2 antigen-specific and capable of rapid clonal expansion. Moreover, we identified these CD8+ T cell populations in scRNA-seq datasets from COVID-19 patients and found that their relative frequency and differentiation outcomes were predictive of subsequent clinical outcomes.

G-CSF drives autoinflammation in APLAID.

Missense mutations in PLCG2 can cause autoinflammation with phospholipase C gamma 2-associated antibody deficiency and immune dysregulation (APLAID). Here, we generated a mouse model carrying an APLAID mutation (p.Ser707Tyr) and found that inflammatory infiltrates in the skin and lungs were only partially ameliorated by removing inflammasome function via the deletion of caspase-1. Also, deleting interleukin-6 or tumor necrosis factor did not fully prevent APLAID mutant mice from autoinflammation. Overall, these findings are in accordance with the poor response individuals with APLAID have to treatments that block interleukin-1, JAK1/2 or tumor necrosis factor. Cytokine analysis revealed increased granulocyte colony-stimulating factor (G-CSF) levels as the most distinct feature in mice and individuals with APLAID. Remarkably, treatment with a G-CSF antibody completely reversed established disease in APLAID mice. Furthermore, excessive myelopoiesis was normalized and lymphocyte numbers rebounded. APLAID mice were also fully rescued by bone marrow transplantation from healthy donors, associated with reduced G-CSF production, predominantly from non-hematopoietic cells. In summary, we identify APLAID as a G-CSF-driven autoinflammatory disease, for which targeted therapy is feasible.

Cell-autonomous effects of APOE4 in restricting microglial response in brain homeostasis and Alzheimer's disease.

Microglial involvement in Alzheimer's disease (AD) pathology has emerged as a risk-determining pathogenic event. While apolipoprotein E (APOE) is known to modify AD risk, it remains unclear how microglial apoE impacts brain cognition and AD pathology. Here, using conditional mouse models expressing apoE isoforms in microglia and central nervous system-associated macrophages (CAMs), we demonstrate a cell-autonomous effect of apoE3-mediated microglial activation and function, which are negated by apoE4. Expression of apoE3 in microglia/CAMs improves cognitive function, increases microglia surrounding amyloid plaque and reduces amyloid pathology and associated toxicity, whereas apoE4 expression either compromises or has no effects on these outcomes by impairing lipid metabolism. Single-cell transcriptomic profiling reveals increased antigen presentation and interferon pathways upon apoE3 expression. In contrast, apoE4 expression downregulates complement and lysosomal pathways, and promotes stress-related responses. Moreover, in the presence of mouse endogenous apoE, microglial apoE4 exacerbates amyloid pathology. Finally, we observed a reduction in Lgals3-positive responsive microglia surrounding amyloid plaque and an increased accumulation of lipid droplets in APOE4 human brains and induced pluripotent stem cell-derived microglia. Our findings establish critical isoform-dependent effects of microglia/CAM-expressed apoE in brain function and the development of amyloid pathology, providing new insight into how apoE4 vastly increases AD risk.

Soluble Methane Monooxygenase.

Aerobic life is possible because the molecular structure of oxygen (O2) makes direct reaction with most organic materials at ambient temperatures an exceptionally slow process. Of course, these reactions are inherently very favorable, and they occur rapidly with the release of a great deal of energy at high temperature. Nature has been able to tap this sequestered reservoir of energy with great spatial and temporal selectivity at ambient temperatures through the evolution of oxidase and oxygenase enzymes. One mechanism used by these enzymes for O2 activation has been studied in detail for the soluble form of the enzyme methane monooxygenase. These studies have revealed the step-by-step process of O2 activation and insertion into the ultimately stable C-H bond of methane. Additionally, an elegant regulatory mechanism has been defined that enlists size selection and quantum tunneling to allow methane oxidation to occur specifically in the presence of more easily oxidized substrates.

Structure of the Nanobody-Stabilized Active State of the Kappa Opioid Receptor.

The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics.

5-HT2C Receptor Structures Reveal the Structural Basis of GPCR Polypharmacology.

Drugs frequently require interactions with multiple targets-via a process known as polypharmacology-to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here, we solved two structures of the 5-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C receptor-selective inverse agonist ritanserin at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs.

Dysregulated Microbial Fermentation of Soluble Fiber Induces Cholestatic Liver Cancer.

Dietary soluble fibers are fermented by gut bacteria into short-chain fatty acids (SCFA), which are considered broadly health-promoting. Accordingly, consumption of such fibers ameliorates metabolic syndrome. However, incorporating soluble fiber inulin, but not insoluble fiber, into a compositionally defined diet, induced icteric hepatocellular carcinoma (HCC). Such HCC was microbiota-dependent and observed in multiple strains of dysbiotic mice but not in germ-free nor antibiotics-treated mice. Furthermore, consumption of an inulin-enriched high-fat diet induced both dysbiosis and HCC in wild-type (WT) mice. Inulin-induced HCC progressed via early onset of cholestasis, hepatocyte death, followed by neutrophilic inflammation in liver. Pharmacologic inhibition of fermentation or depletion of fermenting bacteria markedly reduced intestinal SCFA and prevented HCC. Intervening with cholestyramine to prevent reabsorption of bile acids also conferred protection against such HCC. Thus, its benefits notwithstanding, enrichment of foods with fermentable fiber should be approached with great caution as it may increase risk of HCC.

Chemistry-First Approach for Nomination of Personalized Treatment in Lung Cancer.

Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.

Guanylate-binding proteins convert cytosolic bacteria into caspase-4 signaling platforms.

Bacterial lipopolysaccharide triggers human caspase-4 (murine caspase-11) to cleave gasdermin-D and induce pyroptotic cell death. How lipopolysaccharide sequestered in the membranes of cytosol-invading bacteria activates caspases remains unknown. Here we show that in interferon-γ-stimulated cells guanylate-binding proteins (GBPs) assemble on the surface of Gram-negative bacteria into polyvalent signaling platforms required for activation of caspase-4. Caspase-4 activation is hierarchically controlled by GBPs; GBP1 initiates platform assembly, GBP2 and GBP4 control caspase-4 recruitment, and GBP3 governs caspase-4 activation. In response to cytosol-invading bacteria, activation of caspase-4 through the GBP platform is essential to induce gasdermin-D-dependent pyroptosis and processing of interleukin-18, thereby destroying the replicative niche for intracellular bacteria and alerting neighboring cells, respectively. Caspase-11 and GBPs epistatically protect mice against lethal bacterial challenge. Multiple antagonists of the pathway encoded by Shigella flexneri, a cytosol-adapted bacterium, provide compelling evolutionary evidence for the importance of the GBP-caspase-4 pathway in antibacterial defense.

Author Correction: Genome-wide CRISPR-Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genome-wide CRISPR-Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1.

Human leukocyte antigen (HLA)-independent, T cell-mediated targeting of cancer cells would allow immune destruction of malignancies in all individuals. Here, we use genome-wide CRISPR-Cas9 screening to establish that a T cell receptor (TCR) recognized and killed most human cancer types via the monomorphic MHC class I-related protein, MR1, while remaining inert to noncancerous cells. Unlike mucosal-associated invariant T cells, recognition of target cells by the TCR was independent of bacterial loading. Furthermore, concentration-dependent addition of vitamin B-related metabolite ligands of MR1 reduced TCR recognition of cancer cells, suggesting that recognition occurred via sensing of the cancer metabolome. An MR1-restricted T cell clone mediated in vivo regression of leukemia and conferred enhanced survival of NSG mice. TCR transfer to T cells of patients enabled killing of autologous and nonautologous melanoma. These findings offer opportunities for HLA-independent, pan-cancer, pan-population immunotherapies.

Spheromers reveal robust T cell responses to the Pfizer/BioNTech vaccine and attenuated peripheral CD8+ T cell responses post SARS-CoV-2 infection.

T cells are a critical component of the response to SARS-CoV-2, but their kinetics after infection and vaccination are insufficiently understood. Using "spheromer" peptide-MHC multimer reagents, we analyzed healthy subjects receiving two doses of the Pfizer/BioNTech BNT162b2 vaccine. Vaccination resulted in robust spike-specific T cell responses for the dominant CD4+ (HLA-DRB1∗15:01/S191) and CD8+ (HLA-A∗02/S691) T cell epitopes. Antigen-specific CD4+ and CD8+ T cell responses were asynchronous, with the peak CD4+ T cell responses occurring 1 week post the second vaccination (boost), whereas CD8+ T cells peaked 2 weeks later. These peripheral T cell responses were elevated compared with COVID-19 patients. We also found that previous SARS-CoV-2 infection resulted in decreased CD8+ T cell activation and expansion, suggesting that previous infection can influence the T cell response to vaccination.

Crystal Structure of an LSD-Bound Human Serotonin Receptor.

The prototypical hallucinogen LSD acts via serotonin receptors, and here we describe the crystal structure of LSD in complex with the human serotonin receptor 5-HT2B. The complex reveals conformational rearrangements to accommodate LSD, providing a structural explanation for the conformational selectivity of LSD's key diethylamide moiety. LSD dissociates exceptionally slow from both 5-HT2BR and 5-HT2AR-a major target for its psychoactivity. Molecular dynamics (MD) simulations suggest that LSD's slow binding kinetics may be due to a "lid" formed by extracellular loop 2 (EL2) at the entrance to the binding pocket. A mutation predicted to increase the mobility of this lid greatly accelerates LSD's binding kinetics and selectively dampens LSD-mediated ß-arrestin2 recruitment. This study thus reveals an unexpected binding mode of LSD; illuminates key features of its kinetics, stereochemistry, and signaling; and provides a molecular explanation for LSD's actions at human serotonin receptors. PAPERCLIP.