TScan-II: A genome-scale platform for the de novo identification of CD4+ T cell epitopes.

CD4+ T cells play fundamental roles in orchestrating immune responses and tissue homeostasis. However, our inability to associate peptide human leukocyte antigen class-II (HLA-II) complexes with their cognate T cell receptors (TCRs) in an unbiased manner has hampered our understanding of CD4+ T cell function and role in pathologies. Here, we introduce TScan-II, a highly sensitive genome-scale CD4+ antigen discovery platform. This platform seamlessly integrates the endogenous HLA-II antigen-processing machinery in synthetic antigen-presenting cells and TCR signaling in T cells, enabling the simultaneous screening of multiple HLAs and TCRs. Leveraging genome-scale human, virome, and epitope mutagenesis libraries, TScan-II facilitates de novo antigen discovery and deep exploration of TCR specificity. We demonstrate TScan-II's potential for basic and translational research by identifying a non-canonical antigen for a cancer-reactive CD4+ T cell clone. Additionally, we identified two antigens for clonally expanded CD4+ T cells in Sjögren's disease, which bind distinct HLAs and are expressed in HLA-II-positive ductal cells within affected salivary glands.

The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons.

Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.

Profound Tissue Specificity in Proliferation Control Underlies Cancer Drivers and Aneuploidy Patterns.

Genomics has provided a detailed structural description of the cancer genome. Identifying oncogenic drivers that work primarily through dosage changes is a current challenge. Unrestrained proliferation is a critical hallmark of cancer. We constructed modular, barcoded libraries of human open reading frames (ORFs) and performed screens for proliferation regulators in multiple cell types. Approximately 10% of genes regulate proliferation, with most performing in an unexpectedly highly tissue-specific manner. Proliferation drivers in a given cell type showed specific enrichment in somatic copy number changes (SCNAs) from cognate tumors and helped predict aneuploidy patterns in those tumors, implying that tissue-type-specific genetic network architectures underlie SCNA and driver selection in different cancers. In vivo screening confirmed these results. We report a substantial contribution to the catalog of SCNA-associated cancer drivers, identifying 147 amplified and 107 deleted genes as potential drivers, and derive insights about the genetic network architecture of aneuploidy in tumors.

Unbiased Screens Show CD8+ T Cells of COVID-19 Patients Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside outside the Spike Protein.

Developing effective strategies to prevent or treat coronavirus disease 2019 (COVID-19) requires understanding the natural immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used an unbiased, genome-wide screening technology to determine the precise peptide sequences in SARS-CoV-2 that are recognized by the memory CD8+ T cells of COVID-19 patients. In total, we identified 3-8 epitopes for each of the 6 most prevalent human leukocyte antigen (HLA) types. These epitopes were broadly shared across patients and located in regions of the virus that are not subject to mutational variation. Notably, only 3 of the 29 shared epitopes were located in the spike protein, whereas most epitopes were located in ORF1ab or the nucleocapsid protein. We also found that CD8+ T cells generally do not cross-react with epitopes in the four seasonal coronaviruses that cause the common cold. Overall, these findings can inform development of next-generation vaccines that better recapitulate natural CD8+ T cell immunity to SARS-CoV-2.

Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma.

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses1-3; however, the relationship between phenotypic characteristics and TCR properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8+ T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide-human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire.

Global identification of modular cullin-RING ligase substrates.

Cullin-RING ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes, and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. We demonstrate that one substrate, NUSAP1, is an SCF(Cyclin F) substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology and provides a set of proteins likely to be key indicators of cellular physiology.

A genetic interaction analysis identifies cancer drivers that modify EGFR dependency.

A large number of cancer drivers have been identified through tumor sequencing efforts, but how they interact and the degree to which they can substitute for each other have not been systematically explored. To comprehensively investigate how cancer drivers genetically interact, we searched for modifiers of epidermal growth factor receptor (EGFR) dependency by performing CRISPR, shRNA, and expression screens in a non-small cell lung cancer (NSCLC) model. We elucidated a broad spectrum of tumor suppressor genes (TSGs) and oncogenes (OGs) that can genetically modify proliferation and survival of cancer cells when EGFR signaling is altered. These include genes already known to mediate EGFR inhibitor resistance as well as many TSGs not previously connected to EGFR and whose biological functions in tumorigenesis are not well understood. We show that mutation of PBRM1, a subunit of the SWI/SNF complex, attenuates the effects of EGFR inhibition in part by sustaining AKT signaling. We also show that mutation of Capicua (CIC), a transcriptional repressor, suppresses the effects of EGFR inhibition by partially restoring the EGFR-promoted gene expression program, including the sustained expression of Ets transcription factors such as ETV1 Together, our data provide strong support for the hypothesis that many cancer drivers can substitute for each other in certain contexts and broaden our understanding of EGFR regulation.

Genetic interrogation of replicative senescence uncovers a dual role for USP28 in coordinating the p53 and GATA4 branches of the senescence program.

Senescence is a terminal differentiation program that halts the growth of damaged cells and must be circumvented for cancer to arise. Here we describe a panel of genetic screens to identify genes required for replicative senescence. We uncover a role in senescence for the potent tumor suppressor and ATM substrate USP28. USP28 controls activation of both the TP53 branch and the GATA4/NFkB branch that controls the senescence-associated secretory phenotype (SASP). These results suggest a role for ubiquitination in senescence and imply a common node downstream from ATM that links the TP53 and GATA4 branches of the senescence response.

A gain-of-function senescence bypass screen identifies the homeobox transcription factor DLX2 as a regulator of ATM-p53 signaling.

Senescence stimuli activate multiple tumor suppressor pathways to initiate cycle arrest and a differentiation program characteristic of senescent cells. We performed a two-stage, gain-of-function screen to select for the genes whose enhanced expression can bypass replicative senescence. We uncovered multiple genes known to be involved in p53 and Rb regulation and ATM regulation, two components of the CST (CTC1-STN1-TEN1) complex involved in preventing telomere erosion, and genes such as REST and FOXO4 that have been implicated in aging. Among the new genes now implicated in senescence, we identified DLX2, a homeobox transcription factor that has been shown to be required for tumor growth and metastasis and is associated with poor cancer prognosis. Growth analysis showed that DLX2 expression led to increased cellular replicative life span. Our data suggest that DLX2 expression reduces the protein components of the TTI1/TTI2/TEL2 complex, a key complex required for the proper folding and stabilization of ATM and other members of the PIKK (phosphatidylinositol 3-kinase-related kinase) family kinase, leading to reduced ATM-p53 signaling and senescence bypass. We also found that the overexpression of DLX2 exhibited a mutually exclusive relationship with p53 alterations in cancer patients. Our functional screen identified novel players that may promote tumorigenesis by regulating the ATM-p53 pathway and senescence.

Functional identification of optimized RNAi triggers using a massively parallel sensor assay.

Short hairpin RNAs (shRNAs) provide powerful experimental tools by enabling stable and regulated gene silencing through programming of endogenous microRNA pathways. Since requirements for efficient shRNA biogenesis and target suppression are largely unknown, many predicted shRNAs fail to efficiently suppress their target. To overcome this barrier, we developed a "Sensor assay" that enables the biological identification of effective shRNAs at large scale. By constructing and evaluating 20,000 RNAi reporters covering every possible target site in nine mammalian transcripts, we show that our assay reliably identifies potent shRNAs that are surprisingly rare and predominantly missed by existing algorithms. Our unbiased analyses reveal that potent shRNAs share various predicted and previously unknown features associated with specific microRNA processing steps, and suggest a model for competitive strand selection. Together, our study establishes a powerful tool for large-scale identification of highly potent shRNAs and provides insights into sequence requirements of effective RNAi.

Systematic autoantigen analysis identifies a distinct subtype of scleroderma with coincident cancer.

Scleroderma is a chronic autoimmune rheumatic disease associated with widespread tissue fibrosis and vasculopathy. Approximately two-thirds of all patients with scleroderma present with three dominant autoantibody subsets. Here, we used a pair of complementary high-throughput methods for antibody epitope discovery to examine patients with scleroderma with or without known autoantibody specificities. We identified a specificity for the minor spliceosome complex containing RNA Binding Region (RNP1, RNA recognition motif) Containing 3 (RNPC3) that is found in patients with scleroderma without known specificities and is absent in unrelated autoimmune diseases. We found strong evidence for both intra- and intermolecular epitope spreading in patients with RNA polymerase III (POLR3) and the minor spliceosome specificities. Our results demonstrate the utility of these technologies in rapidly identifying antibodies that can serve as biomarkers of disease subsets in the evolving precision medicine era.

Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks.

The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast.

A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal.

We performed a genome-wide siRNA screen in mouse embryonic stem (ES) cells to identify genes essential for self-renewal, and found 148 genes whose down-regulation caused differentiation. Many of the identified genes function in gene regulation and/or development, and are highly expressed in ES cells and embryonic tissues. We further identified target genes of two transcription regulators Cnot3 and Trim28. We discovered that Cnot3 and Trim28 co-occupy many putative gene promoters with c-Myc and Zfx, but not other pluripotency-associated transcription factors. They form a unique module in the self-renewal transcription network, separate from the core module formed by Nanog, Oct4, and Sox2. The transcriptional targets of this module are enriched for genes involved in cell cycle, cell death, and cancer. This supports the idea that regulatory networks controlling self-renewal in stem cells may also be active in certain cancers and may represent novel anti-cancer targets. Our screen has implicated over 100 new genes in ES cell self-renewal, and illustrates the power of RNAi and forward genetics for the systematic study of self-renewal.

STOP gene Phactr4 is a tumor suppressor.

Cancer develops through genetic and epigenetic alterations that allow unrestrained proliferation and increased survival. Using a genetic RNAi screen, we previously identified hundreds of suppressors of tumorigenesis and/or proliferation (STOP) genes that restrain normal cell proliferation. Our STOP gene set was significantly enriched for known and putative tumor suppressor genes. Here, we report a tumor-suppressive role for one STOP gene, phosphatase and actin regulator 4 (PHACTR4). Phactr4 is one of four members of the largely uncharacterized Phactr family of protein phosphatase 1 (PP1)-and actin-binding proteins. Our work suggests that Phactr4 restrains normal cell proliferation and transformation. Depletion of Phactr4 with multiple shRNAs leads to increased proliferation and soft agar colony formation. Phactr4 acts, in part, through an Rb-dependent pathway, because Rb phosphorylation is maintained upon growth factor withdrawal in Phactr4-depleted cells. Examination of tumor copy number analysis and sequencing revealed that PHACTR4 is significantly deleted and mutant in many tumor subtypes. Furthermore,cancer cell lines with reduced Phactr4 expression exhibit tumor suppressor hypersensitivity upon Phactr4 complementation,leading to reduced proliferation, transformation, and tumor formation. Thus, Phactr4 acts as a tumor suppressor that is deleted and mutant in several cancers.

The spliceosome factor SART1 exerts its anti-HCV action through mRNA splicing.

BACKGROUND &/AIMS: The broadly used antiviral cytokine interferon-α (IFNα)'s mechanisms of action against HCV infection are not well understood. We previously identified SART1, a host protein involved in RNA splicing and pre-mRNA processing, as a regulator of IFN's antiviral effects. We hypothesized that SART1 regulates antiviral IFN effector genes (IEGs) through mRNA processing and splicing. METHODS: We performed siRNA knockdown in HuH7.5.1 cells and mRNA-sequencing with or without IFN treatment. Selected gene mRNA variants and their proteins, together with HCV replication, were monitored by qRT-PCR and Western blot in HCV OR6 replicon cells and the JFH1 HCV infectious model. RESULTS: We identified 419 genes with a greater than 2-fold expression difference between Neg siRNA and SART1 siRNA treated cells in the presence or absence of IFN. Bioinformatic analysis identified at least 10 functional pathways. SART1 knockdown reduced classical IFN stimulating genes (ISG) mRNA transcription including MX1 and OAS3. However, SART1 did not affect JAK-STAT pathway gene mRNA expression and IFN stimulated response element (ISRE) signaling. We identified alternative mRNA splicing events for several genes, including EIF4G3, GORASP2, ZFAND6, and RAB6A that contribute to their antiviral effects. EIF4G3 and GORASP2 were also confirmed to have anti-HCV effect. CONCLUSIONS: The spliceosome factor SART1 is not IFN-inducible but is an IEG. SART1 exerts its anti-HCV action through direct transcriptional regulation for some ISGs and alternative splicing for others, including EIF4G3, GORASP2. SART1 does not have an effect on IFN receptor or canonical signal transduction components. Thus, SART1 regulates ISGs using a novel, non-classical mechanism.

Tiling genomes of pathogenic viruses identifies potent antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy.

shRNAs can trigger effective silencing of gene expression in mammalian cells, thereby providing powerful tools for genetic studies, as well as potential therapeutic strategies. Specific shRNAs can interfere with the replication of pathogenic viruses and are currently being tested as antiviral therapies in clinical trials. However, this effort is hindered by our inability to systematically and accurately identify potent shRNAs for viral genomes. Here we apply a recently developed highly parallel sensor assay to identify potent shRNAs for HIV, hepatitis C virus (HCV), and influenza. We observe known and previously unknown sequence features that dictate shRNAs efficiency. Validation using HIV and HCV cell culture models demonstrates very high potency of the top-scoring shRNAs. Comparing our data with the secondary structure of HIV shows that shRNA efficacy is strongly affected by the secondary structure at the target RNA site. Artificially introducing secondary structure to the target site markedly reduces shRNA silencing. In addition, we observe that HCV has distinct sequence features that bias HCV-targeting shRNAs toward lower efficacy. Our results facilitate further development of shRNA based antiviral therapies and improve our understanding and ability to predict efficient shRNAs.

Proteasomal control of anti-CRISPRs for the regulation of CRISPR/Cas9 activity using Cas9-ACROBAT.

Small molecule-mediated proteasomal degradation of proteins is a powerful tool for synthetic regulation of biological activity. To control Cas9 activity in cells, we engineered an anti-CRISPR protein, AcrIIA4, fused to a degradation (dTAG) or small molecule assisted shutoff (SMASh) tag. Co-expression of the tagged AcrIIA4 along with Cas9 and riboswitch-regulated sgRNAs enables precise tunable control of CRISPR activity by small molecule addition.

Design of 240,000 orthogonal 25mer DNA barcode probes.

DNA barcodes linked to genetic features greatly facilitate screening these features in pooled formats using microarray hybridization, and new tools are needed to design large sets of barcodes to allow construction of large barcoded mammalian libraries such as shRNA libraries. Here we report a framework for designing large sets of orthogonal barcode probes. We demonstrate the utility of this framework by designing 240,000 barcode probes and testing their performance by hybridization. From the test hybridizations, we also discovered new probe design rules that significantly reduce cross-hybridization after their introduction into the framework of the algorithm. These rules should improve the performance of DNA microarray probe designs for many applications.

Case report: A persistently expanded T cell response in an exceptional responder to radiation and atezolizumab for metastatic non-small cell lung cancer.

Most patients with advanced non-small cell lung cancer (NSCLC) do not achieve a durable remission after treatment with immune checkpoint inhibitors. Here we report the clinical history of an exceptional responder to radiation and anti-program death-ligand 1 (PD-L1) monoclonal antibody, atezolizumab, for metastatic NSCLC who remains in a complete remission more than 8 years after treatment. Sequencing of the patient's T cell repertoire from a metastatic lesion and the blood before and after anti-PD-L1 treatment revealed oligoclonal T cell expansion. Characterization of the dominant T cell clone, which comprised 10% of all clones and increased 10-fold in the blood post-treatment, revealed an activated CD8+ phenotype and reactivity against 4 HLA-A2 restricted neopeptides but not viral or wild-type human peptides, suggesting tumor reactivity. We hypothesize that the patient's exceptional response to anti-PD-L1 therapy may have been achieved by increased tumor immunogenicity promoted by pre-treatment radiation therapy as well as long-term persistence of oligoclonal expanded circulating T cells.