A genome-wide enrichment screen identifies NUMA1-loss as a resistance mechanism against mitotic cell-death induced by BMI1 inhibition

AbstractBMI1 is a core protein of the polycomb repressive complex 1 (PRC1) that is overexpressed in several cancer types, making it a promising target for cancer therapies. However, the underlying mechanisms and interactions associated with BMI1-induced tumorigenesis are often context-dependent and complex. Here, we performed a drug resistance screen on mutagenized human haploid HAP1 cells treated with the BMI1 inhibitor PTC-318 to find new genetic and mechanistic features associated with BMI1-dependent cancer cell proliferation. Our screen identified NUMA1-mutations as the most significant inducer of PTC-318 cell death resistance. Independent validations on NUMA1-proficient HAP1 and non-small cell lung cancer cell lines exposed to BMI1 inhibition by PTC-318 or BMI1 knockdown resulted in cell death following mitotic arrest. Interestingly, cells with CRISPR-Cas9 derived NUMA1 knockout also showed a mitotic arrest phenotype following BMI1 inhibition but, contrary to cells with wildtype NUMA1, these cells were resistant to BMI1-dependent cell death. The current study brings new insights to BMI1 inhibition-induced mitotic lethality in cancer cells and presents a previously unknown role for NUMA1 in this process.

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Pathway-Wide Genetic Risks in Chlamydial Infections Overlap between Tissue Tropisms: A Genome-Wide Association Scan

Mediators of Inflammation ◽

10.1155/2018/3434101 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9

Author(s):

Chrissy H. Roberts ◽

Sander Ouburg ◽

Mark D. Preston ◽

Henry J. C. de Vries ◽

Martin J. Holland ◽

...

Keyword(s):

Cell Cycle ◽

Chlamydia Trachomatis ◽

Mitotic Cell ◽

Genome Wide Association ◽

Serological Response ◽

Transmitted Infection ◽

Genome Wide ◽

A Genome ◽

Tissue Tropisms ◽

Chlamydial Infections

Chlamydia trachomatis is the most commonly diagnosed bacterial sexually transmitted infection and can lead to tubal factor infertility, a disease characterised by fibrosis of the fallopian tubes. Genetic polymorphisms in molecular pathways involving G protein-coupled receptor signalling, the Akt/PI3K cascade, the mitotic cell cycle, and immune response have been identified in association with the development of trachomatous scarring, an ocular form of chlamydia-related fibrotic pathology. In this case-control study, we performed genome-wide association and pathways-based analysis in a sample of 71 Dutch women who attended an STI clinic who were seropositive for Chlamydia trachomatis antibodies and 169 high-risk Dutch women who sought similar health services but who were seronegative. We identified two regions of within-gene SNP association with Chlamydia trachomatis serological response and found that GPCR signalling and cell cycle pathways were also associated with the trait. These pathway-level associations appear to be common to immunological sequelae of chlamydial infections in both ocular and urogenital tropisms. These pathways may be central mediators of human refractoriness to chlamydial diseases.

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SEC24A identified as an essential mediator of thapsigargin-induced cell death in a genome-wide CRISPR/Cas9 screen

Cell Death Discovery ◽

10.1038/s41420-018-0135-5 ◽

2018 ◽

Vol 4 (1) ◽

Cited By ~ 6

Author(s):

Tamutenda Chidawanyika ◽

Elizabeth Sergison ◽

Michael Cole ◽

Kenneth Mark ◽

Surachai Supattapone

Keyword(s):

Cell Death ◽

Genome Wide ◽

A Genome

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Intracellular Staphylococcus aureus Perturbs the Host Cell Ca2+ Homeostasis To Promote Cell Death

mBio ◽

10.1128/mbio.02250-20 ◽

2020 ◽

Vol 11 (6) ◽

pp. e02250-20

Author(s):

Kathrin Stelzner ◽

Ann-Cathrin Winkler ◽

Chunguang Liang ◽

Aziza Boyny ◽

Carsten P. Ade ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Death ◽

Host Cell ◽

Intracellular Bacterium ◽

Tissue Destruction ◽

Content Type ◽

Host Cell Death ◽

Genome Wide ◽

A Genome ◽

Spread Of Infection

ABSTRACTThe opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca2+ increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca2+ concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca2+ rise led to an increase in mitochondrial Ca2+ concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca2+ homeostasis and induces cytoplasmic Ca2+ overload, which results in both apoptotic and necrotic cell death in parallel or succession.IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca2+ overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca2+ homeostasis.

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Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy

Science Translational Medicine ◽

10.1126/scitranslmed.aay0271 ◽

2020 ◽

Vol 12 (536) ◽

pp. eaay0271 ◽

Cited By ~ 6

Author(s):

Angela Lek ◽

Yuanfan Zhang ◽

Keryn G. Woodman ◽

Shushu Huang ◽

Alec M. DeSimone ◽

...

Keyword(s):

Cell Death ◽

Muscular Dystrophy ◽

Phenotypic Selection ◽

Facioscapulohumeral Muscular Dystrophy ◽

Loss Of Function ◽

Hypoxia Response ◽

Genome Wide ◽

A Genome ◽

Structural And Functional Properties ◽

Cellular Hypoxia

The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.

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Identification of TMEM206 proteins as pore of PAORAC/ASOR acid-sensitive chloride channels

eLife ◽

10.7554/elife.49187 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 10

Author(s):

Florian Ullrich ◽

Sandy Blin ◽

Katina Lazarow ◽

Tony Daubitz ◽

Jens Peter von Kries ◽

...

Keyword(s):

Cell Death ◽

Plasma Membrane ◽

Ion Channels ◽

Gene Family ◽

Chloride Channels ◽

Anion Channel ◽

Ion Permeation ◽

Acid Sensing Ion Channels ◽

Genome Wide ◽

A Genome

Acid-sensing ion channels have important functions in physiology and pathology, but the molecular composition of acid-activated chloride channels had remained unclear. We now used a genome-wide siRNA screen to molecularly identify the widely expressed acid-sensitive outwardly-rectifying anion channel PAORAC/ASOR. ASOR is formed by TMEM206 proteins which display two transmembrane domains (TMs) and are expressed at the plasma membrane. Ion permeation-changing mutations along the length of TM2 and at the end of TM1 suggest that these segments line ASOR’s pore. While not belonging to a gene family, TMEM206 has orthologs in probably all vertebrates. Currents from evolutionarily distant orthologs share activation by protons, a feature essential for ASOR’s role in acid-induced cell death. TMEM206 defines a novel class of ion channels. Its identification will help to understand its physiological roles and the diverse ways by which anion-selective pores can be formed.

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Identification of Genes Regulating Cell Death inStaphylococcus aureus

10.1101/569053 ◽

2019 ◽

Author(s):

Rebecca Yee ◽

Jie Feng ◽

Jiou Wang ◽

Jiazhen Chen ◽

Ying Zhang

Keyword(s):

Cell Death ◽

Acetic Acid ◽

Drug Targets ◽

Opportunistic Pathogen ◽

Infection Model ◽

Chronic Infections ◽

Bacterial Cell Death ◽

Genome Wide ◽

A Genome ◽

Genetic Mechanisms

AbstractStaphylococcus aureusis an opportunistic pathogen that causes acute and chronic infections. Due toS. aureus’ s highly resistant and persistent nature, it is paramount to identify better drug targets in order to eradicateS. aureusinfections. Despite the efforts in understanding bacterial cell death, the genes and pathways ofS. aureuscell death remain elusive. Here, we performed a genome-wide screen using a transposon mutant library to study the genetic mechanisms involved inS. aureuscell death. Using a precisely controlled heat-ramp and acetic acid exposure assays, mutations in 27 core genes (hsdR1, hslO, nsaS, sspA, folD, mfd, vraF, kdpB, USA300HOU_2684, 0868, 0369, 0420, 1154, 0142, 0930, 2590, 0997, 2559, 0044, 2004, 1209, 0152, 2455, 0154, 2386, 0232, 0350 involved in transporters, transcription, metabolism, peptidases, kinases, transferases, SOS response, nucleic acid and protein synthesis) caused the bacteria to be more death-resistant. In addition, we identified mutations in core 10 genes (capA, gltT, mnhG1,USA300HOU_1780, 2496, 0200, 2029, 0336, 0329, 2386, involved in transporters, metabolism, transcription, cell wall synthesis) from heat-ramp and acetic acid that caused the bacteria to be more death-sensitive or with defect in persistence. Interestingly, death-resistant mutants were more virulent than the parental strain USA300 and caused increased mortality in aCaenorhabditis elegansinfection model. Conversely, death-sensitive mutants were less persistent and formed less persister cells upon exposure to different classes of antibiotics. These findings provide new insights into the mechanisms ofS. aureuscell death and offer new therapeutic targets for developing more effective treatments caused byS. aureus.

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SEC24A facilitates colocalization and Ca2+ flux between the endoplasmic reticulum and mitochondria

Journal of Cell Science ◽

10.1242/jcs.249276 ◽

2021 ◽

Vol 134 (6) ◽

Author(s):

Tamutenda Chidawanyika ◽

Rajarshi Chakrabarti ◽

Kathryn S. Beauchemin ◽

Henry N. Higgs ◽

Surachai Supattapone

Keyword(s):

Endoplasmic Reticulum ◽

Cell Death ◽

Fold Increase ◽

Cellular Mechanism ◽

Autophagic Flux ◽

Wild Type ◽

Genome Wide ◽

Fold Reduction ◽

A Genome ◽

Indicator Dyes

ABSTRACT A genome-wide screen recently identified SEC24A as a novel mediator of thapsigargin-induced cell death in HAP1 cells. Here, we determined the cellular mechanism and specificity of SEC24A-mediated cytotoxicity. Measurement of Ca2+ levels using organelle-specific fluorescent indicator dyes showed that Ca2+ efflux from endoplasmic reticulum (ER) and influx into mitochondria were significantly impaired in SEC24A-knockout cells. Furthermore, SEC24A-knockout cells also showed ∼44% less colocalization of mitochondria and peripheral tubular ER. Knockout of SEC24A, but not its paralogs SEC24B, SEC24C or SEC24D, rescued HAP1 cells from cell death induced by three different inhibitors of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases (SERCA) but not from cell death induced by a topoisomerase inhibitor. Thapsigargin-treated SEC24A-knockout cells showed a ∼2.5-fold increase in autophagic flux and ∼10-fold reduction in apoptosis compared to wild-type cells. Taken together, our findings indicate that SEC24A plays a previously unrecognized role in regulating association and Ca2+ flux between the ER and mitochondria, thereby impacting processes dependent on mitochondrial Ca2+ levels, including autophagy and apoptosis.

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The E3 ligase UBR2 regulates cell death under caspase deficiency via Erk/MAPK pathway

Cell Death and Disease ◽

10.1038/s41419-020-03258-3 ◽

2020 ◽

Vol 11 (12) ◽

Author(s):

Elodie Villa ◽

Rachel Paul ◽

Ophélie Meynet ◽

Sophie Volturo ◽

Guillaume Pinna ◽

...

Keyword(s):

Cell Death ◽

Mapk Pathway ◽

Underlying Mechanism ◽

Erk Pathway ◽

Breast Cancers ◽

Cell Death Pathway ◽

Main Cell ◽

Caspase Inhibition ◽

Genome Wide ◽

A Genome

AbstractEscape from cell death is a key event in cancer establishment/progression. While apoptosis is often considered as the main cell death pathway, upon caspase inhibition, cell death is rather delayed than blocked leading to caspase-independent cell death (CICD). Although described for years, CICD’s underlying mechanism remains to be identified. Here, we performed a genome-wide siRNA lethality screening and identified the RING-Type E3 Ubiquitin Transferase (UBR2) as a specific regulator of CICD. Strikingly, UBR2 downregulation sensitized cells towards CICD while its overexpression was protective. We established that UBR2-dependent protection from CICD was mediated by the MAPK/Erk pathway. We then observed that UBR2 is overexpressed in several cancers, especially in breast cancers and contributes to CICD resistance. Therefore, our work defines UBR2 as a novel regulator of CICD, found overexpressed in cancer cells, suggesting that its targeting may represent an innovative way to kill tumor cells.

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A genome-wide RNAi screen reveals multiple regulators of caspase activation

The Journal of Cell Biology ◽

10.1083/jcb.200708090 ◽

2007 ◽

Vol 179 (4) ◽

pp. 619-626 ◽

Cited By ~ 54

Author(s):

Caroline H. Yi ◽

Dodzie K. Sogah ◽

Michael Boyce ◽

Alexei Degterev ◽

Dana E. Christofferson ◽

...

Keyword(s):

Cell Death ◽

Mammalian Cells ◽

Caspase Activation ◽

Functional Conservation ◽

Cellular Processes ◽

Regulatory Circuits ◽

Genome Wide ◽

A Genome ◽

Dependent Cell ◽

Functionally Diverse

Apoptosis is an evolutionally conserved cellular suicide mechanism that can be activated in response to a variety of stressful stimuli. Increasing evidence suggests that apoptotic regulation relies on specialized cell death signaling pathways and also integrates diverse signals from additional regulatory circuits, including those of cellular homeostasis. We present a genome-wide RNA interference screen to systematically identify regulators of apoptosis induced by DNA damage in Drosophila melanogaster cells. We identify 47 double- stranded RNA that target a functionally diverse set of genes, including several with a known function in promoting cell death. Further characterization uncovers 10 genes that influence caspase activation upon the removal of Drosophila inhibitor of apoptosis 1. This set includes the Drosophila initiator caspase Dronc and, surprisingly, several metabolic regulators, a candidate tumor suppressor, Charlatan, and an N-acetyltransferase, ARD1. Importantly, several of these genes show functional conservation in regulating apoptosis in mammalian cells. Our data suggest a previously unappreciated fundamental connection between various cellular processes and caspase-dependent cell death.

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