Search

Leading researchers at the intersection of infectious disease and systems biology speak about how systems approaches have influenced modern infectious disease research and what these tools can offer for the future of the field.

Background: GATA4 (GATA-binding protein 4), a zinc finger–containing, DNA-binding transcription factor, is essential for normal cardiac development and homeostasis in mice and humans, and mutations in this gene have been reported in human heart defects. Defects in alternative splicing are associated with many heart diseases, yet relatively little is known about how cell type– or cell state–specific alternative splicing is achieved in the heart. Here, we show that GATA4 regulates cell type–specific splicing through direct interaction with RNA and the spliceosome in human induced pluripotent stem cell–derived cardiac progenitors. Methods: We leveraged a combination of unbiased approaches including affinity purification of GATA4 and mass spectrometry, enhanced cross-linking with immunoprecipitation, electrophoretic mobility shift assays, in vitro splicing assays, and unbiased transcriptomic analysis to uncover GATA4’s novel function as a splicing regulator in human induced pluripotent stem cell–derived cardiac progenitors. Results: We found that GATA4 interacts with many members of the spliceosome complex in human induced pluripotent stem cell–derived cardiac progenitors. Enhanced cross-linking with immunoprecipitation demonstrated that GATA4 also directly binds to a large number of mRNAs through defined RNA motifs in a sequence-specific manner. In vitro splicing assays indicated that GATA4 regulates alternative splicing through direct RNA binding, resulting in functionally distinct protein products. Correspondingly, knockdown of GATA4 in human induced pluripotent stem cell–derived cardiac progenitors resulted in differential alternative splicing of genes involved in cytoskeleton organization and calcium ion import, with functional consequences associated with the protein isoforms. Conclusions: This study shows that in addition to its well described transcriptional function, GATA4 interacts with members of the spliceosome complex and regulates cell type–specific alternative splicing via sequence-specific interactions with RNA. Several genes that have splicing regulated by GATA4 have functional consequences and many are associated with dilated cardiomyopathy, suggesting a novel role for GATA4 in achieving the necessary cardiac proteome in normal and stress-responsive conditions.

Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the therapeutic potential of Mac1 inhibition, we generated recombinant viruses and replicons encoding catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced activity by ∼10-fold, mutations to aspartic acid (N40D) reduced the catalytic activity by ∼100-fold relative to wildtype activity. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, N40D replicated at >1000-fold lower levels while inducing a robust interferon response, and all infected animals survived infection with the mutant, but not the wildtype virus. Our data validate SARS-CoV-2 NSP3 Mac1 domain as a critical viral pathogenesis factor and a reasonable target to develop antivirals, while emphasizing the importance of amino acid identity in viral mutagenesis studies and underscoring the limitations of solely relying on in vitro viral replication studies for target validation.

Proximity labeling (PL) coupled with mass spectrometry has emerged as a powerful technique to map proximal protein interactions in living cells. Large-scale sample processing for proximity proteomics necessitates a high-throughput workflow to reduce hands-on time and increase quantitative reproducibility. To address this issue, we developed a scalable and automated PL pipeline, including generation and characterization of monoclonal cell lines, automated enrichment of biotinylated proteins in a 96-well format, and optimization of the quantitative mass spectrometry (MS) acquisition method. Combined with data-independent acquisition (DIA) MS, our pipeline outperforms manual enrichment and data-dependent acquisition (DDA) MS regarding reproducibility of protein identification and quantification. We apply the pipeline to map subcellular proteomes for endosomes, late endosomes/lysosomes, the Golgi apparatus, and the plasma membrane. Moreover, using serotonin receptor (5HT2A) as a model, we investigated agonist-induced dynamics in protein-protein interactions. Importantly, the approach presented here is universally applicable for PL proteomics using all biotinylation-based PL enzymes, increasing both throughput and reproducibility of standard protocols.

Inhibitors directed toward PARP1 and PARP2 are approved agents for the treatment of BRCA1 and BRCA2-related cancers. Other members of the PARP family have also been implicated in cancer and are being assessed as therapeutic targets in cancer and other diseases. Recently, an inhibitor of PARP7 (RBN-2397) has reached early-stage human clinical trials. Here, we performed a genome-wide CRISPR screen for genes that modify the response of cells to RBN-2397. We identify the polycyclic aromatic hydrocarbon receptor AHR and multiple components of the cohesin complex as determinants of resistance to this agent. Activators and inhibitors of AHR modulate the cellular response to PARP7 inhibition, suggesting potential combination therapy approaches.

Supplementary Table from A Whole-Genome CRISPR Screen Identifies AHR Loss as a Mechanism of Resistance to a PARP7 Inhibitor

Previously Reported HPV-Human PPIs. Related to Figures 1 and 2.

Supplementary Data from A Whole-Genome CRISPR Screen Identifies AHR Loss as a Mechanism of Resistance to a PARP7 Inhibitor

This combined Supplementary Document contains Supplementary Notes, Methods and References, Legends to Supplementary Tables S1-S6, and Supplementary Figures S1-S5.

Cancer drugs targeting the identified virus-host protein-protein interactions of SARS-CoV-2

Supplementary Table S2. Drugs and their concentrations used in the isogenic drug screen.

Supplementary Table S3. Chemical-genetic interaction scores derived in this study

MiST Scores for all HPV-Human PPIs. Related to Figures 1 and 2.

Differentially Mutated Genes in HPV(-) vs. HPV(+) Samples. Related to Figure 3.

Supplementary Figure 1. Distribution of gene alterations in Breast TCGA and verification of expression of MCF10A cells. Supplementary Figure 2. Analysis of the MCF10A drug screen. Supplementary Figure 3. Significance of overlap between interactions found in this study and in the CGP. Supplementary Figure 4. Response of isogenic engineered cells to dasatinib. Supplementary Figure 5. Dasatinib sensitivity of CML versus AML cancer cell lines. Supplementary Figure 6. Verification of LYN knockdown via siRNA and response of LYN T319I to dasatinib. Supplementary Figure 7. Co-­�expression of MYC and LYN in breast cancer cell lines.

Gene Ontology Terms. Related to Figure 1.

Summary:The mapping of SARS-CoV-2 human protein–protein interactions by Gordon and colleagues revealed druggable targets that are hijacked by the virus. Here, we highlight several oncogenic pathways identified at the host–virus interface of SARS-CoV-2 to enable cancer biologists to apply their knowledge for rapid drug repurposing to treat COVID-19, and help inform the response to potential long-term complications of the disease.

Differentially Expressed Genes upon L2 Overexpression. Related to Figure 5.

Supplementary Table S1. List of constructs used to generate stable cell lines.

Meningiomas are the most common primary intracranial tumors. There are no effective medical therapies for meningioma patients, and new treatments have been encumbered by limited understanding of meningioma biology. Here, we use DNA methylation profiling on 565 meningiomas integrated with genetic, transcriptomic, biochemical, proteomic and single-cell approaches to show meningiomas are composed of three DNA methylation groups with distinct clinical outcomes, biological drivers and therapeutic vulnerabilities. Merlin-intact meningiomas (34%) have the best outcomes and are distinguished by NF2/Merlin regulation of susceptibility to cytotoxic therapy. Immune-enriched meningiomas (38%) have intermediate outcomes and are distinguished by immune infiltration, HLA expression and lymphatic vessels. Hypermitotic meningiomas (28%) have the worst outcomes and are distinguished by convergent genetic and epigenetic mechanisms driving the cell cycle and resistance to cytotoxic therapy. To translate these findings into clinical practice, we show cytostatic cell cycle inhibitors attenuate meningioma growth in cell culture, organoids, xenografts and patients.

Integrative structure modeling is increasingly used for determining the architectures of biological assemblies, especially those that are structurally heterogeneous. Recently, we reported on how to convert in vivo genetic interaction measurements into spatial restraints for structural modeling: first, phenotypic profiles are generated for each point mutation and thousands of gene deletions or environmental perturbations. Following, the phenotypic profile similarities are converted into distance restraints on the pairs of mutated residues. We illustrate the approach by determining the structure of the histone H3-H4 complex. The method is implemented in our open-source IMP program, expanding the structural biology toolbox by allowing structural characterization based on in vivo data without the need to purify the target system. We compare genetic interaction measurements to other sources of structural information, such as residue coevolution and deep-learning structure prediction of complex subunits. We also suggest that determining genetic interactions could benefit from new technologies, such as CRISPR-Cas9 approaches to gene editing, especially for mammalian cells. Finally, we highlight the opportunity for using genetic interactions to determine recalcitrant biomolecular structures, such as those of disordered proteins, transient protein assemblies, and host-pathogen protein complexes.

Understanding the effects of genetic variation is a fundamental problem in biology that requires methods to analyse both physical and functional consequences of sequence changes at systems-wide and mechanistic scales. To achieve a systems view, protein interaction networks map which proteins physically interact, while genetic interaction networks inform on the phenotypic consequences of perturbing these protein interactions. Until recently, understanding the molecular mechanisms that underlie these interactions often required biophysical methods to determine the structures of the proteins involved. The past decade has seen the emergence of new approaches based on coevolution, deep mutational scanning and genome-scale genetic or chemical–genetic interaction mapping that enable modelling of the structures of individual proteins or protein complexes. Here, we review the emerging use of large-scale genetic datasets and deep learning approaches to model protein structures and their interactions, and discuss the integration of structural data from different sources., Large-scale genetic datasets and deep learning approaches are being used to model the structures of proteins or protein complexes. This Review describes approaches based on coevolution, deep mutational scanning and genome-scale genetic or chemical–genetic interaction mapping and their application and integration to inform structural modelling.

Supplementary Data from Resistance to ATR Inhibitors Is Mediated by Loss of the Nonsense-Mediated Decay Factor UPF2

Pan-phospho-RTK profiling identifies HER3 to be hyper-phosphorylated after BYL719 treatment in HPV(+) cell lines

Over one million cases of gastric cancer are diagnosed each year globally, and the metastatic disease continues to have a poor prognosis. A significant proportion of gastric tumors have defects in the DNA damage response pathway, creating therapeutic opportunities through synthetic lethal approaches. Several small-molecule inhibitors of ATR, a key regulator of the DNA damage response, are now in clinical development as targeted agents for gastric cancer. Here, we performed a large-scale CRISPR interference screen to discover genetic determinants of response and resistance to ATR inhibitors (ATRi) in gastric cancer cells. Among the top hits identified as mediators of ATRi response were UPF2 and other components of the nonsense-mediated decay (NMD) pathway. Loss of UPF2 caused ATRi resistance across multiple gastric cancer cell lines. Global proteomic, phosphoproteomic, and transcriptional profiling experiments revealed that cell-cycle progression and DNA damage responses were altered in UPF2-mutant cells. Further studies demonstrated that UPF2-depleted cells failed to accumulate in G1 following treatment with ATRi. UPF2 loss also reduced transcription–replication collisions, which has previously been associated with ATRi response, thereby suggesting a possible mechanism of resistance. Our results uncover a novel role for NMD factors in modulating response to ATRi in gastric cancer, highlighting a previously unknown mechanism of resistance that may inform the clinical use of these drugs.Significance:Loss of NMD proteins promotes resistance to ATR inhibitors in gastric cancer cells, which may provide a combination of therapeutic targets and biomarkers to improve the clinical utility of these drugs.

HPV(+) cell lines are less responsive to BKM120 and BEZ235 than HPV(-) cell lines

Human papillomavirus (HPV) type 16 is implicated in approximately 75% of head and neck squamous cell carcinomas (HNSCC) that arise in the oropharynx, where viral expression of the E6 and E7 oncoproteins promote cellular transformation, tumor growth, and maintenance. An important oncogenic signaling pathway activated by E6 and E7 is the PI3K pathway, a key driver of carcinogenesis. The PI3K pathway is also activated by mutation or amplification of PIK3CA in over half of HPV(+) HNSCC. In this study, we investigated the efficacy of PI3K-targeted therapies in HPV(+) HNSCC preclinical models and report that HPV(+) cell line- and patient-derived xenografts are resistant to PI3K inhibitors due to feedback signaling emanating from E6 and E7. Receptor tyrosine kinase profiling indicated that PI3K inhibition led to elevated expression of the HER3 receptor, which in turn increased the abundance of E6 and E7 to promote PI3K inhibitor resistance. Targeting HER3 with siRNA or the mAb CDX-3379 reduced E6 and E7 abundance and enhanced the efficacy of PI3K-targeted therapies. Together, these findings suggest that cross-talk between HER3 and HPV oncoproteins promotes resistance to PI3K inhibitors and that cotargeting HER3 and PI3K may be an effective therapeutic strategy in HPV(+) tumors.Significance: These findings suggest a new therapeutic combination that may improve outcomes in HPV(+) head and neck cancer patients. Cancer Res; 78(9); 2383–95. ©2018 AACR.

Blockade of the PI3K pathway with BKM120 or BEZ235 increases HER3/AKT signaling

Legend for supplemental figures

Supplementary Figure from Resistance to ATR Inhibitors Is Mediated by Loss of the Nonsense-Mediated Decay Factor UPF2

HER3 blockade enhances the sensitivity of HPV(+) cell lines to BKM120

Clinical characteristics of patients prior to surgery and PDX establishment

SummaryInfection by hepatitis B virus (HBV) increases risk for liver cancer by inducing inflammation, cellular stress and cell death. To elucidate the molecular pathways by which HBV promotes cancer development and progression, we used affinity purification mass spectrometry to comprehensively map a network of 145 physical interactions between HBV and human host proteins in hepatocellular carcinoma (HCC). We find that viral proteins target host factors that are preferentially mutated in non-HBV-associated HCC, implicating cancer pathways whose interaction with HBV plays a role in HCC. Focusing on proteins that directly interact with the HBV oncoprotein X (HBx), we show that HBx remodels the PP2A phosphatase complex, altering its effect on tumor signaling. HBx excludes striatin-family regulatory subunits from PP2A, causing Hippo kinase activation and unmasking a requirement for mTOR complex 2 to maintain expression of the YAP oncoprotein in HCC. Thus, HBV rewires HCC to expose potentially targetable signaling dependencies.SignificancePrecision medicine has revolutionized cancer treatment but remains elusive for HCC. We used proteomics to define HBV/host interactions and integrated them with HCC mutations. The results implicate modifiers of HCC behavior via remodeling of host complexes and illuminate new biological mechanisms in advanced disease for therapeutic investigation.

The DNA damage response (DDR) entails reorganization of proteins and protein complexes involved in DNA repair. The coordinated regulation of these proteomic changes maintains genome stability. Traditionally, regulators and mediators of DDR have been investigated individually. However, recent advances in mass spectrometry (MS)-based proteomics enable us to globally quantify changes in protein abundance, post-translational modifications (PTMs), protein localization, and protein-protein interactions (PPIs) in cells. Furthermore, structural proteomics approaches, such as crosslinking MS (XL-MS), hydrogen/deuterium exchange MS (H/DX-MS), Native MS (nMS), provide large structural information of proteins and protein complexes, complementary to the data collected from conventional methods, and promote integrated structural modeling. In this review, we will overview the current cutting-edge functional and structural proteomics techniques that are being actively utilized and developed to help interrogate proteomic changes that regulate the DDR.

SummaryMycobacterium tuberculosisis currently the leading cause of death by any bacterial infection1. The mycolic acid layer of the cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are therefore front line targets for antimycobacterial drug development2,3. Polyketide synthase 13 (Pks13) is a module comprised of a closely symmetric parallel dimer of chains, each encoding several enzymatic and transport functions, that carries out the condensation of two different very long chain fatty acids to produce mycolic acids that are essential components of the mycobacterial cell wall. Consequently individual enzymatic domains of Pks13 are targets for antimycobacterial drug development4. To understand this machinery, we sought to determine the structure and domain trajectories of the dimeric multi-enzyme Pks13, a 2×198,426 Dalton complex, from protein purified endogenously from mycobacteria under normal growth conditions, to capture it with normal substrates bound trapped ‘in action’.Structures of the multi-domain assembly revealed by cryogenic electron microscopy (cryoEM) define the ketosynthase (KS), linker, and acyltransferase (AT) domains, each at atomic resolution (1.8Å), with bound substrates defined at 2.4Å and 2.9Å resolution. Image classification reveals two distinct structures with alternate locations of the N-terminal acyl carrier protein (termed ACP1a, ACP1b) seen at 3.6Å and 4.6Å resolution respectively. These two structures suggest plausible intermediate states, related by a ~60Å movement of ACP1, on the pathway for substrate delivery from the fatty acyl-ACP ligase (FadD32) to the ketosynthase domain. The linking sequence between ACP1 and the KS includes an 11 amino acid sequence with 6 negatively charged side chains that lies in different positively charged grooves on the KS in ACP1a versus ACP1b structures. This charge complementarity between the extended chain and the grooves suggests some stabilization of these two distinct orientations. Other domains are visible at lower resolution and indicate flexibility relative to the KS-AT core. The chemical structures of three bound endogenous long chain fatty acid substrates with their proximal regions defined in the structures were determined by electrospray ionization mass spectrometry.The domain proximities were probed by chemical cross-linking and identified by mass spectrometry. These were incorporated into integrative structure modeling to define multiple domain configurations that transport the very long fatty acid chains throughout the multistep Pks13 mediated synthetic pathway.

Genetic alterations that activate protein kinase A (PKA) are found in many tumor types. Yet, their downstream oncogenic signaling mechanisms are poorly understood. We used global phosphoproteomics and kinase activity profiling to map conserved signaling outputs driven by a range of genetic changes that activate PKA in human cancer. Two signaling networks were identified downstream of PKA: RAS/MAPK components and an Aurora Kinase A (AURKA)/glycogen synthase kinase (GSK3) sub-network with activity toward MYC oncoproteins. Findings were validated in two PKA-dependent cancer models: a novel, patient-derived fibrolamellar carcinoma (FLC) line that expresses a DNAJ-PKAc fusion and a PKA-addicted melanoma model with a mutant type I PKA regulatory subunit. We identify PKA signals that can influence both de novo translation and stability of the proto-oncogene c-MYC. However, the primary mechanism of PKA effects on MYC in our cell models was translation and could be blocked with the eIF4A inhibitor zotatifin. This compound dramatically reduced c-MYC expression and inhibited FLC cell line growth in vitro. Thus, targeting PKA effects on translation is a potential treatment strategy for FLC and other PKA-driven cancers.

Host-pathogen interactions (HPIs) are pivotal in regulating establishment, progression, and outcome of an infection. Affinity-purification mass spectrometry has become instrumental for the characterization of HPIs, however the targeted nature of exogenously expressing individual viral proteins has limited its utility to the analysis of relatively small pathogens. Here we present the use of co-fractionation mass spectrometry (SEC-MS) for the high-throughput analysis of HPIs from native viral infections of two jumbophages (ϕKZ andϕPA3) inPseudomonas aeruginosa. This enabled the detection>6000 unique host-pathogen and>200 pathogen-pathogen interactions for each phage, encompassing>50% of the phage proteome. Interactome-wide comparison across phages showed similar perturbed protein interactions suggesting fundamentally conserved mechanisms of phage predation within the KZ-like phage family. Prediction of novel ORFs revealed aϕPA3 complex showing strong structural and sequence similarity toϕKZ nvRNAp, suggestingϕPA3 also possesses two RNA polymerases acting at different stages of the infection cycle. We further expanded our understanding on the molecular organization of the virion packaged and injected proteome by identifying 23 novel virion components and 5 novel injected proteins, as well as providing the first evidence for interactions between KZ-like phage proteins and the host ribosome. To enable accessibility to this data, we developed PhageMAP, an online resource for network query, visualization, and interaction prediction (https://phagemap.ucsf.edu/). We anticipate this study will lay the foundation for the application of co-fractionation mass spectrometry for the scalable profiling of hostpathogen interactomes and protein complex dynamics upon infection.

Network modeling suggests the inflammatory factors secreted due to HIV infection, even in the presence of antiretroviral therapy, activate PI3K-AKT signaling in cervical cells and accelerate cervical cancer progression in HPV/HIV coinfected women.

Molecular switch proteins whose cycling between states is controlled by opposing regulators1,2 are central to biological signal transduction. As switch proteins function within highly connected interaction networks3, the fundamental question arises of how functional specificity is achieved when different processes share common regulators. Here we show that functional specificity of the small GTPase switch protein Gsp1 in Saccharomyces cerevisiae (the homologue of the human protein RAN)4 is linked to differential sensitivity of biological processes to different kinetics of the Gsp1 (RAN) switch cycle. We make 55 targeted point mutations to individual protein interaction interfaces of Gsp1 (RAN) and show through quantitative genetic5 and physical interaction mapping that Gsp1 (RAN) interface perturbations have widespread cellular consequences. Contrary to expectation, the cellular effects of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle and not by the targeted interfaces. Instead, we show that interface mutations allosterically tune the GTPase cycle kinetics. These results suggest a model in which protein partner binding, or post-translational modifications at distal sites, could act as allosteric regulators of GTPase switching. Similar mechanisms may underlie regulation by other GTPases, and other biological switches. Furthermore, our integrative platform to determine the quantitative consequences of molecular perturbations may help to explain the effects of disease mutations that target central molecular switches. Interface mutations in the GTPase switch protein Gsp1 (the yeast homologue of human RAN) allosterically affect the kinetics of the switch cycle, revealing a systems-level mechanism of multi-specificity.

Macrophages employ an array of pattern recognition receptors to detect and eliminate intracellular pathogens that access the cytosol. The cytosolic carbohydrate sensors Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) recognize damaged pathogen-containing phagosomes, and Gal-3 and Gal-8 are reported to restrict bacterial growth via autophagy in cultured cells. However, the contribution of these galectins to host resistance during bacterial infection remains unclear. We found that Gal-9 binds directly toMycobacterium tuberculosis(Mtb) andSalmonella entericaserovar Typhimurium (Stm) and localizes toMtbin macrophages. To determine the combined contribution of membrane damage-sensing galectins to immunity in vivo, we generated Gal-3, -8, and - 9 triple knockout (TKO) mice.Mtbinfection of primary macrophages from TKO mice resulted in defective lysosomal trafficking but normal bacterial replication. Surprisingly, these mice had no discernable defect in resistance to acute infection withMtb,Stm or Listeria monocytogenes, and had only modest impairments in bacterial growth restriction and CD4 T cell activation during chronicMtbinfection. Collectively, these findings indicate that while Gal-3, -8, and -9 respond to an array of intracellular pathogens, together these membrane damage-sensing galectins play a limited role in host resistance to bacterial infection.Author SummaryIntracellular bacterial pathogens cause many of the world’s most deadly infectious diseases. A common requirement for nearly all intracellular pathogens is the ability to damage the endomembrane compartments in which they reside, which allows pathogens access to the nutrient-rich cytosol of the host. However, membrane damage also creates a “pattern of pathogenesis” that triggers antimicrobial immune responses. Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) act as a surveillance system for membrane damage and Gal-3 and Gal-8 inhibit bacterial growth by activating autophagy, a cellular pathway that can capture cytosolic bacteria and degrade them in lysosomes. Membrane damage-sensing galectins were hypothesized to promote bacterial killing during acute infection yet their role in the immune response of an infected animal remains unclear. Here, we show that mice deficient for Gal-3, -8, and -9 had no defects in resistance to acute infection with the pathogensListeria monocytogenes, Salmonella entericaserovar Typhimurium, andMycobacterium tuberculosis(Mtb), and were only modestly susceptible to chronicMtbinfection. Our data suggest that Gal-3, -8 and -9 are not critical for innate immune responses during acute infection and may play a more prominent role in the adaptive immune response. These results broaden our understanding of the role of membrane damage-sensing pathways in host defense against bacterial infection.

SummaryCell state evolution underlies tumor development and response to therapy1, but mechanisms specifying cancer cell states and intratumor heterogeneity are incompletely understood. Schwannomas are the most common tumors of the peripheral nervous system and are treated with surgery and ionizing radiation2–5. Schwannomas can oscillate in size for many years after radiotherapy6,7, suggesting treatment may reprogram schwannoma cells or the tumor microenvironment. Here we show epigenetic reprogramming shapes the cellular landscape of schwannomas. We find schwannomas are comprised of 2 molecular groups distinguished by reactivation of neural crest development pathways or misactivation of nerve injury mechanisms that specify cancer cell states and the architecture of the tumor immune microenvironment. Schwannoma molecular groups can arise independently, but ionizing radiation is sufficient for epigenetic reprogramming of neural crest to immune-enriched schwannoma by remodeling chromatin accessibility, gene expression, and metabolism to drive schwannoma cell state evolution and immune cell infiltration. To define functional genomic mechanisms underlying epigenetic reprograming of schwannomas, we develop a technique for simultaneous interrogation of chromatin accessibility and gene expression coupled with genetic and therapeutic perturbations in single-nuclei. Our results elucidate a framework for understanding epigenetic drivers of cancer evolution and establish a paradigm of epigenetic reprograming of cancer in response to radiotherapy.

Over one million cases of gastric cancer are diagnosed each year globally, and the metastatic disease continues to have a poor prognosis. A significant proportion of gastric tumors have defects in the DNA damage response pathway, creating therapeutic opportunities through synthetic lethal approaches. Several small-molecule inhibitors of ATR, a key regulator of the DNA damage response, are now in clinical development as targeted agents for gastric cancer. Here, we performed a large-scale CRISPR interference screen to discover genetic determinants of response and resistance to ATR inhibitors (ATRi) in gastric cancer cells. Among the top hits identified as mediators of ATRi response were UPF2 and other components of the nonsense-mediated decay (NMD) pathway. Loss of UPF2 caused ATRi resistance across multiple gastric cancer cell lines. Global proteomic, phosphoproteomic, and transcriptional profiling experiments revealed that cell-cycle progression and DNA damage responses were altered in UPF2-mutant cells. Further studies demonstrated that UPF2-depleted cells failed to accumulate in G1 following treatment with ATRi. UPF2 loss also reduced transcription–replication collisions, which has previously been associated with ATRi response, thereby suggesting a possible mechanism of resistance. Our results uncover a novel role for NMD factors in modulating response to ATRi in gastric cancer, highlighting a previously unknown mechanism of resistance that may inform the clinical use of these drugs. Significance: Loss of NMD proteins promotes resistance to ATR inhibitors in gastric cancer cells, which may provide a combination of therapeutic targets and biomarkers to improve the clinical utility of these drugs.

A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic—Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans.One sentence summarySystematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.

We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection bothin vitro, and in the Syrian golden hamster modelin vivo. In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants.ONE SENTENCE SUMMARYSARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.

The fate of influenza A virus (IAV) infection in the host cell depends on the balance between cellular defence mechanisms and viral evasion strategies. To illuminate the landscape of IAV cellular restriction, we generated and integrated global genetic loss-of-function screens with transcriptomics and proteomics data. Our multi-omics analysis revealed a subset of both IFN-dependent and independent cellular defence mechanisms that inhibit IAV replication. Amongst these, the autophagy regulator TBC1 domain family member 5 (TBC1D5), which binds Rab7 to enable fusion of autophagosomes and lysosomes, was found to control IAV replication in vitro and in vivo and to promote lysosomal targeting of IAV M2 protein. Notably, IAV M2 was observed to abrogate TBC1D5-Rab7 binding through a physical interaction with TBC1D5 via its cytoplasmic tail. Our results provide evidence for the molecular mechanism utilised by IAV M2 protein to escape lysosomal degradation and traffic to the cell membrane, where it supports IAV budding and growth.

International audience; Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drawn much attention. Beginning with sigma receptor ligands and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs we tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs and does not reflect specific target-based activities—rather, it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.