Genetic Screen Research Articles

An antibiotic that mediates immune destruction of senescent cancer cells

Drugs that eliminate senescent cells, senolytics, can be powerful when combined with prosenescence cancer therapies. Using a CRISPR/Cas9-based genetic screen, we identify here SLC25A23 as a vulnerability of senescent cancer cells. Suppressing SLC25A23 disrupts cellular calcium homeostasis, impairs oxidative phosphorylation, and interferes with redox signaling, leading to death of senescent cells. These effects can be replicated by salinomycin, a cation ionophore antibiotic. Salinomycin prompts a pyroptosis-apoptosis-necroptosis (PAN)optosis-like cell death in senescent cells, including apoptosis and two forms of immunogenic cell death: necroptosis and pyroptosis. Notably, we observed that salinomycin treatment or SLC25A23 suppression elevates reactive oxygen species, upregulating death receptor 5 via Jun N-terminal protein kinase (JNK) pathway activation. We show that a combination of a death receptor 5 (DR5) agonistic antibody and salinomycin is a robust senolytic cocktail. We provide evidence that this drug combination provokes a potent natural killer (NK) and CD8+ T cell-mediated immune destruction of senescent cancer cells, mediated by the pyroptotic cytokine interleukin 18 (IL18).

Control of 3′ splice site selection by the yeast splicing factor Fyv6

Pre-mRNA splicing is catalyzed in two steps: 5ʹ splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (first and second step factors). We recently identified Fyv6 (FAM192A in humans) as a second step factor in Saccharomyces cerevisiae; however, we did not determine how widespread Fyv6’s impact is on the transcriptome. To answer this question, we have used RNA sequencing (RNA-seq) to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3ʹ SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-electron microscopy (cryo-EM) structure of a yeast product complex spliceosome containing Fyv6 at 2.3 Å. The structure reveals that Fyv6 is the only second step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the first step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv6Δ suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of consensus, BP distal 3ʹ SS.

Characterization of YdgH: a mediator of beta-lactam susceptibility in Enterobacterales.

Beta-lactam antibiotics are often the treatment of choice for serious bacterial infections. In a previous screen for novel genetic mediators affecting beta-lactam susceptibility, we discovered that deletion of ydgH, a conserved gene of unknown function, leads to increased resistance to beta-lactams, as well as increased susceptibility to detergent compounds. Here, we further characterize YdgH in Serratia marcescens, Enterobacter cloacae, and Escherichia coli using a combination of biochemical and cell biological approaches. We find that YdgH fractionates with periplasmic proteins, and this periplasmic localization is necessary for its function. Using purified recombinant protein, we demonstrate that YdgH is a relatively compact, globular monomer. The YdgH polypeptide contains three tandem DUF1471 domains. In a ΔydgH background, overexpression of polypeptides containing both the second and the third, but not the first DUF1471 domain, is necessary to rescue the deletion phenotype. To determine how YdgH function influences beta-lactam and detergent susceptibility, we tested several targeted hypotheses. We found that deletion of ydgH neither affects ompC or ompF transcript levels, nor does it alter the processing of lipopolysaccharide, nor does it activate the sigma E regulon alone or in combination with mutations in other periplasmic proteins. Finally, we delineate the results of a genetic screen for spontaneous mutants that complement the detergent susceptibility phenotype, the results of which may fuel the further studies that are necessary to determine the precise role YdgH plays in bacterial physiology.IMPORTANCEBeta-lactams such as penicillins and cephalosporins are the most commonly prescribed antibiotics for serious bacterial infections. Increasing antibiotic resistance threatens their effectiveness. We previously identified the uncharacterized gene ydgH as a modifier of beta-lactam susceptibility in Gram-negative bacteria. To begin to understand the specific role of YdgH, in this study, we perform initial characterizations of this protein. We also test hypotheses as to how the function of YdgH contributes to beta-lactam physiology.

Genetic Analysis of Flagellar-Mediated Surface Sensing by Pseudomonas aeruginosa PA14.

Surface sensing is a key aspect of the early stage of biofilm formation. For P. aeruginosa, the type IV pili (TFP), the TFP alignment complex and PilY1 were shown to play a key role in c-di-GMP signaling upon surface contact. The role of the flagellar machinery in surface sensing is less well understood in P. aeruginosa. Here we show, consistent with findings from other groups, that a mutation in the gene encoding the flagellar hook protein (ΔflgK) or flagellin (ΔfliC) results in a strain that overproduces the Pel exopolysaccharide (EPS) with a concomitant increase in c-di-GMP levels. We use a candidate gene approach and genetic screens, combined with phenotypic assays, to identify key roles for the MotAB and MotCD stators and the FliG protein, a component of the flagellar switch complex, in stimulating the surface-dependent, increased c-di-GMP level noted for these flagellar mutants. These findings are consistent with previous studies showing a role for the stators in surface sensing. We also show that mutations in the genes coding for the diguanylate cyclases SadC and RoeA as well as SadB, a protein involved in early surface colonization, abrogate the increased c-d-GMP-related phenotypes of the ΔflgK mutant. Together, these data indicate that bacteria monitor the status of flagellar synthesis and/or function during surface sensing as a means to trigger the biofilm program.

Straight to the heart: Protecting the patient’s heart during chemotherapy

How do we protect the heart during chemotherapy with the anthracycline drug class? Tackling this question, Liu etal. combined pluripotent stem cell models, CRISPR genetic screens, and molecular modeling to identify indisulam as a potential cardioprotective drug in this issue of Cell Stem Cell.

Functional profiling of murine glioma models highlights targetable immune evasion phenotypes

Cancer-intrinsic immune evasion mechanisms and pleiotropy are a barrier to cancer immunotherapy. This is apparent in certain highly fatal cancers, including high-grade gliomas and glioblastomas (GBM). In this study, we evaluated two murine syngeneic glioma models (GL261 and CT2A) as preclinical models for human GBM using functional genetic screens, single-cell transcriptomics and machine learning approaches. Through CRISPR genome-wide co-culture killing screens with various immune cells (cytotoxic T cells, natural killer cells, and macrophages), we identified three key cancer-intrinsic evasion mechanisms: NFκB signaling, autophagy/endosome machinery, and chromatin remodeling. Additional fitness screens identified dependencies in murine gliomas that partially recapitulated those seen in human GBM (e.g., UFMylation). Our single-cell analyses showed that different glioma models exhibited distinct immune infiltration patterns and recapitulated key immune gene programs observed in human GBM, including hypoxia, interferon, and TNF signaling. Moreover, in vivo orthotopic tumor engraftment was associated with phenotypic shifts and changes in proliferative capacity, with murine tumors recapitulating the intratumoral heterogeneity observed in human GBM, exhibiting propensities for developmental- and mesenchymal-like phenotypes. Notably, we observed common transcription factors and cofactors shared with human GBM, including developmental (Nfia and Tcf4), mesenchymal (Prrx1 and Wwtr1), as well as cycling-associated genes (Bub3, Cenpa, Bard1, Brca1, and Mis18bp1). Perturbation of these genes led to reciprocal phenotypic shifts suggesting intrinsic feedback mechanisms that balance in vivo cellular states. Finally, we used a machine-learning approach to identify two distinct immune evasion gene programs, one of which represents a clinically-relevant phenotype and delineates a subpopulation of stem-like glioma cells that predict response to immune checkpoint inhibition in human patients. This comprehensive characterization helps bridge the gap between murine glioma models and human GBM, providing valuable insights for future therapeutic development.

Caenorhabditis elegans inositol hexaphosphate pathways couple to RNA interference and pathogen defense

RNA interference (RNAi) is an evolutionarily conserved pathway that defends against viral infections in diverse organisms. Caenorhabditis elegans mutations that enhance RNAi have revealed pathways that may regulate antiviral defense. A genetic screen for C. elegans mutations that fail to up-regulate a defense response reporter transgene detected mutations that enhance RNAi to silence this reporter gene in the inositol polyphosphate multikinase impk-1, the synMuv B gene lin-15B, and the pathogen defense response gene pals-22. Using other assays for enhanced RNAi, we found that the impk-1 alleles and an ippk-1 gene inactivation of a later step in inositol hexaphosphate (IP6) synthesis, and the lin-15B and pals-22 alleles enhance RNAi. IP6 has been known for decades to bind and stabilize human adenosine deaminase that acts on RNA (ADAR) as well as the paralog tRNA editing ADAT. We show that the C. elegans IP6 pathway is also required for mRNA and tRNA editing. Thus, a deficiency in two axes of RNA editing enhances the already potent C. elegans RNAi antiviral defense, suggesting adenosine to inosine RNA editing may normally moderate this siRNA antiviral defense pathway. The C. elegans IP6-deficient mutants are synthetic lethal with a set of enhanced RNAi mutants that act in the polyploid hypodermis to regulate collagen secretion and signaling from that tissue, implicating IP6 signaling especially in this tissue. This enhanced antiviral RNAi response uses the C. elegans RIG-I-like receptor DRH-1 to activate the unfolded protein response (UPR). The production of primary siRNAs, rather than secondary siRNAs, contributes to this activation of the UPR through XBP-1 signaling. The gon-14 and pal-17 mutants that also emerged from this screen act in the mitochondrial defense pathway rather than by enhancing RNAi.

Glucose limitation protects cancer cells from apoptosis induced by pyrimidine restriction and replication inhibition.

Cancer cells often experience nutrient-limiting conditions because of their robust proliferation and inadequate tumour vasculature, which results in metabolic adaptation to sustain proliferation. Most cancer cells rapidly consume glucose, which is severely reduced in the nutrient-scarce tumour microenvironment. In CRISPR-based genetic screens to identify metabolic pathways influenced by glucose restriction, we find that tumour-relevant glucose concentrations (low glucose) protect cancer cells from inhibition of de novo pyrimidine biosynthesis, a pathway that is frequently targeted by chemotherapy. We identify two mechanisms to explain this result, which is observed broadly across cancer types. First, low glucose limits uridine-5-diphosphate-glucose synthesis, preserving pyrimidine nucleotide availability and thereby prolonging the time to replication fork stalling. Second, low glucose directly modulates apoptosis downstream of replication fork stalling by suppressing BAK activation and subsequent cytochrome c release, key events that activate caspase-9-dependent mitochondrial apoptosis. These results indicate that the low glucose levels frequently observed in tumours may limit the efficacy of specific chemotherapeutic agents, highlighting the importance of considering the effects of the tumour nutrient environment on cancer therapy.

CRISPR-based genetic screens in human pluripotent stem cells derived neurons and brain organoids.

Recent large-scale genome-wide association and single-cell RNA sequencing (scRNA-seq) studies have uncovered disease-associated genetic risk factors and cell type-specific genetic alterations. However, our understanding of how these genetic variants cause diseases and the underlying mechanisms remains largely unknown. Functional genomics screens using CRISPR-based technologies offer an effective tool for studying genes relevant to disease phenotypes. Here, we summarize recent CRISPR-based functional genomics screen approaches applied to human pluripotent stem cell (hPSC)-derived neurons and brainorganoids. These screens have identified genes crucial for neurogenesis, neuronal survival, morphological development, and migration. Combining CRISPR-based genetic screens with scRNA-seq, researchers have revealed downstream genes and cellular pathways impacted by these genetic variants in human neural cells, providing new insights into the pathogenesis of neurodevelopmental disorders, such as microcephaly and autism spectrum disorders. Finally, we discuss current challenges and future directions for using CRISPR-based screens in furthering our understanding of neurological diseases and developing potential therapeutic strategies. Despite challenges, CRISPR-based screens have enormous potential for advancing the therapeutic development of many diseases.

ACK1 and BRK non-receptor tyrosine kinase deficiencies are associated with familial systemic lupus and involved in efferocytosis.

Systemic lupus erythematosus (SLE) is an autoimmune disease, the pathophysiology and genetic basis of which are incompletely understood. Using a forward genetic screen in multiplex families with SLE, we identified an association between SLE and compound heterozygous deleterious variants in the non-receptor tyrosine kinases (NRTKs) ACK1 and BRK. Experimental blockade of ACK1 or BRK increased circulating autoantibodies in vivo in mice and exacerbated glomerular IgG deposits in an SLE mouse model. Mechanistically, NRTKs regulate activation, migration, and proliferation of immune cells. We found that the patients' ACK1 and BRK variants impair efferocytosis, the MERTK-mediated anti-inflammatory response to apoptotic cells, in human induced pluripotent stem cell (hiPSC)-derived macrophages, which may contribute to SLE pathogenesis. Overall, our data suggest that ACK1 and BRK deficiencies are associated with human SLE and impair efferocytosis in macrophages.

Cotranscriptional splicing is required in the cold to produce COOLAIR isoforms that repress Arabidopsis FLC

Plants use seasonal cold to time the transition to reproductive development. Short- and long-term cold exposure is registered via parallel transcriptional shutdown and Polycomb-dependent epigenetic silencing of the Arabidopsis thaliana major flowering repressor locus FLOWERING LOCUS C (FLC). The cold-induced antisense transcripts (COOLAIR) determine the dynamics of FLC transcriptional shutdown, but the thermosensory mechanisms are still unresolved. Here, through a forward genetic screen, we identify a mutation that perturbs cold-induced COOLAIR expression and FLC repression. The mutation is a hypomorphic allele of SUPPRESSORS OF MEC-8 AND UNC-52 1 (SMU1), a conserved subunit of the spliceosomal B complex. SMU1 interacts in vivo with the proximal region of nascent COOLAIR and RNA 3' processing/cotranscriptional regulators and enhances COOLAIR proximal intron splicing to promote specific COOLAIR isoforms. SMU1 also interacts with ELF7, an RNA Polymerase II Associated Factor (Paf1) component and limits COOLAIR transcription. Cold thus changes cotranscriptional splicing/RNA Pol II functionality in an SMU1-dependent mechanism to promote two different isoforms of COOLAIR that lead to reduced FLC transcription. Such cotranscriptional mechanisms are emerging as important regulators underlying plasticity in gene expression.

Redistribution of PU.1 partner transcription factor RUNX1 binding secures cell survival during leukemogenesis

Transcription factors (TFs) orchestrating lineage-development often control genes required for cellular survival. However, it is not well understood how cells survive when such TFs are lost, for example in cancer. PU.1 is an essential TF for myeloid fate, and mice with downregulated PU.1 levels develop acute myeloid leukemia (AML). Combining a multi-omics approach with a functional genetic screen, we reveal that PU.1-downregulated cells fundamentally change their survival control from cytokine-driven pathways to overexpression of an autophagy-predominated stem cell gene program, for which we also find evidence in human AML. Control of this program involves redirected chromatin occupancy of the PU.1 partner TF Runx1 to a lineage-inappropriate binding site repertoire. Hence, genomic reallocation of TF binding upon loss of a partner TF can act as a pro-oncogenic failsafe mechanism by sustaining cell survival during leukemogenesis.

Abstract I004: Systematic Discovery and Annotation of Cancer Emergent Orphan non-coding RNAs in human Cancers

Abstract We recently described orphan non-coding RNAs (oncRNAs) as a class of cancer-specific small RNAs with the potential to play functional roles in breast cancer progression1. Here, we report a systematic search to annotate and characterize cancer-emergent oncRNAs across 32 tumor types. We also leverage large-scale in vivo genetic screens in xenografted mice to functionally identify driver oncRNAs in multiple tumor types. We have not only discovered a large repertoire of oncRNAs, but also found that their presence and absence represent a digital molecular barcode that faithfully captures the types and subtypes of cancer. Importantly, we discovered that this molecular barcode is partially accessible from the cell-free space as some oncRNAs are secreted by cancer cells. In a large retrospective study across 192 breast cancer patients, we showed that oncRNAs can be reliably detected in the blood and that changes in the cell-free oncRNA burden captures both short-term and long-term clinical outcomes upon completion of a neoadjuvant chemotherapy regimen. Citation Format: Hani Goodarzi. Systematic Discovery and Annotation of Cancer Emergent Orphan non-coding RNAs in human Cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I004.

Identification and characterization of the Bacillus subtilis spore germination protein GerY.

Pathogens in the orders Bacillales and Clostridiales resist sterilization by differentiating into stress-resistant spores. Spores are metabolically inactive and can remain dormant for decades, yet upon exposure to nutrients, they rapidly resume growth, causing food spoilage, food-borne illness, or life-threatening disease. The exit from dormancy, called germination, is a key target in combating these important pathogens. Here, we report a high-throughput genetic screen using transposon sequencing to identify novel germination factors that ensure the efficient exit from dormancy. We identify several new factors and characterize one in greater detail. This factor, renamed GerY, is part of the proteinaceous coat that encapsulates the dormant spore. Our data suggest that GerY enables efficient transit of nutrients through the coat to trigger germination.

A comprehensive two-hybrid analysis to explore the Legionella pneumophila effector-effector interactome.

Secreted bacterial effector proteins are typically viewed as modulators of host activity, entering the host cytosol to physically interact with and modify the activity of one or more host proteins in support of infection. A growing body of evidence suggests that a subset of effectors primarily function to modify the activities of other effectors inside the host. These "effectors of effectors" or metaeffectors are often identified through experimental serendipity during the study of canonical effector function against the host. We previously performed the first global effector-wide genetic interaction screen for metaeffectors within the arsenal of Legionella pneumophila, an intracellular bacterial pathogen with over 300 effectors. Here, using a high-throughput, scalable methodology, we present the first global interaction network of physical interactions between L. pneumophila effectors. This data set serves as a complementary resource to identify and understand both the scope and nature of non-canonical effector activity within this important human pathogen.

TYK2 regulates tau levels, phosphorylation and aggregation in a tauopathy mouse model

Alzheimer’s disease is one of at least 26 diseases characterized by tau-positive accumulation in neurons, glia or both. However, it is still unclear what modifications cause soluble tau to transform into insoluble aggregates. We previously performed genetic screens that identified tyrosine kinase 2 (TYK2) as a candidate regulator of tau levels. Here we verified this finding and found that TYK2 phosphorylates tau at tyrosine 29 (Tyr29) leading to its stabilization and promoting its aggregation in human cells. We discovered that TYK2-mediated Tyr29 phosphorylation interferes with autophagic clearance of tau. We also show that TYK2-mediated phosphorylation of Tyr29 facilitates pathological tau accumulation in P301S tau-transgenic mice. Furthermore, knockdown of Tyk2 reduced total tau and pathogenic tau levels and rescued gliosis in a tauopathy mouse model. Collectively, these data suggest that partial inhibition of TYK2 could thus be a strategy to reduce tau levels and toxicity.

Biophysical Analysis of Vip3Aa Toxin Mutants Before and After Activation.

Cry toxins from Bacillus thuringiensis are effective biopesticides that kill lepidopteran pests, replacing chemical pesticides that indiscriminately attack both target and non-target organisms. However, resistance in susceptible pests is an emerging problem. B. thuringiensis also produces vegetative insecticidal protein (Vip3A), which can kill insect targets in the same group as Cry toxins but using different host receptors, making the combined application of Cry and Vip3A an exciting possibility. Vip3A toxicity requires the formation of a homotetramer. Hence, screening of Vip3A mutants for increased stability requires orthogonal biophysical assays that can test both tetrameric integrity and monomeric robustness. For this purpose, we have used herein for the first time a combination of analytical ultracentrifugation (AUC), mass photometry (MP), differential static light scattering (DSLS) and differential scanning fluorimetry (DSF) to test five mutants at domains I and II. Although all mutants appeared more stable than the wild type (WT) in DSLS, mutants that showed more dissociation into dimers in MP and AUC experiments also showed earlier thermal unfolding by DSF at domains IV-V. All of the mutants were less toxic than the WT, but toxicity was highest for domain II mutations N242C and F229Y. Activation of the protoxin was complete and resulted in a form with a lower sedimentation coefficient. Future high-resolution structural data may lead to a deeper understanding of the increased stability that will help with rational design while retaining native toxicity.

Screens for mutants defective in UapA trafficking highlight the importance of ER-exit as a primary control point in transporter biogenesis

Most transmembrane membrane proteins are thought to traffic to the plasma membrane (PM) via the conventional secretory pathway through sorting from the Golgi. However, our recent work has shown that in the filamentous fungus Aspergillus nidulans several nutrient transporters and other major membrane proteins traffic to the PM via Golgi-bypass and independently of known post-Golgi secretory mechanisms. Here in an effort to dissect the molecular mechanism underlying membrane cargo trafficking via Golgi-bypass we design and use unbiased genetic screens, based on the UapA uric acid-xanthine transporter, which allowed the isolation of mutants defective in UapA translocation to the plasma membrane. Analyses of these mutants highlight the importance of ER-exit as the primary control point in transporter trafficking via Golgi-bypass. Most mutants isolated concerned mutations within the uapA gene, albeit we also obtained uapA extragenetic mutants affecting secretion and growth pleiotropically or leading on apparent activation of an efflux transporter related to purine-detoxification. Our work paves the way to use genetic approaches targeting specifically trafficking mutations affecting Golgi-bypass.

The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

The functions of integrins are tightly regulated via multiple mechanisms including trafficking and degradation. Integrins are repeatedly internalized, routed into the endosomal system and either degraded by the lysosome or recycled back to the plasma membrane. The ubiquitin system dictates whether internalized proteins are degraded or recycled. Here, we use a genetic screen and proximity-dependent biotin identification to identify deubiquitinase(s) that control integrin surface levels. We find that a ternary deubiquitinating complex, comprised of USP12 (or the homologous USP46), WDR48 and WDR20, stabilizes β1 integrin (Itgb1) by preventing ESCRT-mediated lysosomal degradation. Mechanistically, the USP12/46-WDR48-WDR20 complex removes ubiquitin from the cytoplasmic tail of internalized Itgb1 in early endosomes, which in turn prevents ESCRT-mediated sorting and Itgb1 degradation.

A defining member of the new cysteine-cradle family is an aECM protein signalling skin damage in C. elegans.

Apical extracellular matrices (aECMs) act as crucial barriers, and communicate with the epidermis to trigger protective responses following injury or infection. In Caenorhabditis elegans, the skin aECM, the cuticle, is produced by the epidermis and decorated with circumferential furrows. We previously showed that mutants lacking cuticle furrows exhibit persistent immune activation (PIA). In a genetic suppressor screen, we identified spia-1 as a key gene downstream of furrow collagens and upstream of immune signalling. spia-1 expression oscillates during larval development, peaking between each moult together with patterning cuticular components. It encodes a secreted protein that localises to furrows. SPIA-1 shares a novel cysteine-cradle domain with other aECM proteins. SPIA-1 mediates immune activation in response to furrow loss and is proposed to act as a sensor of cuticle damage. This research provides the first molecular insight into an intricate interplay between cuticle integrity and epidermal immune activation in C. elegans.