Biology subjects

Tyers, M.

Publications and source records attributed to Tyers, M..

At least 19 recordsLinked to original sources

bioRxiv2025

The scaffold protein PRR14L links the PP2A-TACC3 axis to mitotic fidelity and sensitivity to MPS1 inhibition

Aneuploidy is a hallmark of cancer and is a potential vulnerability that can be selectively targeted. To systematically identify genes that affect the incidence and fitness of aneuploid cells, we conducted a genome-wide CRISPR/Cas9 screen using NMS-P715, an inhibitor of the spindle assembly checkpoint (SAC) kinase MPS1/TTK. In this study, we identified a number of genes known to regulate aneuploidy and mitosis, and subsequently focused on PRR14L, a ubiquitously expressed gene previously implicated in chronic myelomonocytic leukemia (CMML). Proximity labeling of PRR14L using TurboID revealed several cell division proteins, including the PP2A-B56 phosphatase complex and the spindle assembly factor TACC3, as PRR14L-interacting proteins. Loss of PRR14L prolongs SAC-dependent mitotic arrest in response to microtubule depolymerization but, paradoxically, leads to catastrophic mitotic errors upon SAC abrogation by MPS1 inhibitors. A model derived from our findings provides a rationale for exploiting MPS1 inhibition as a potential vulnerability in cancers containing either PRR14L loss of function mutations or FGFR-TACC3 fusions. Significance StatementO_LIAneuploidy is a hallmark of cancer. Whether aneuploidy can be selectively targeted is not known. C_LIO_LIUtilizing a CRISPR/Cas9 screen, the authors found that loss of the gene PRR14L sensitizes cells to aneuploidy induction. Taking advantage of live cell imaging and proximity labeling, they linked PRR14L to TACC3 and mitosis. C_LIO_LIThese findings suggest that spindle checkpoint inhibitors may have therapeutic potential in cancers with either loss-of-function PRR14L and/or gain of function TACC3 mutations. C_LI

cell biology↗

bioRxiv2025

A platform for the systematic interrogation of genetic interactions in human cells: 26 gene by genome-wide double knockout screens as a proof of concept

Genetic interactions are typically studied by looking at the phenotype that results from disruption of pairs of genes, as well as from higher order combinations of perturbations. Systematically interrogating all pairwise combinations provides insights into how genes are organized into pathways and complexes to sustain cellular homeostasis and how interacting genes respond to stressors and external signals. Genetic interactions have been studied extensively in yeast, due, in part, to the availability of a systematic collection of gene knockouts, and the development of Synthetic Genetic Array (SGA) technology. In contrast, such approaches are more challenging in human cells and therefore comparable data for human cells is scarce. This study introduces an innovative approach to functionally characterize genetic interactions in human cells through CRISPR/Cas9 screens using a pooled genome-wide knockout library in NALM6 cells. By combining a single guide RNA (sgRNA) targeting the gene of interest (aka the query) in cells already infected with an inducible genome-wide sgRNA pool, it is possible to achieve near saturation of genome-wide double knockouts. We conducted 26 of these screens, which we term "gene by genome-wide" knockout screens. This approach can be rapidly performed, in part, because it bypasses the need to generate genotyped isogenic knockout clones. Data from these screens identified both expected and novel synthetic lethal and synthetic rescue interactions, demonstrating that this strategy is effective for large-scale genetic research in human cells. Additionally, we show that these GBGW screens can be combined with chemical perturbation to reveal new synthetic interactions that are not apparent without drug treatment. Finally, we show that cDNA overexpression can be incorporated with genome-wide knockouts to systematically explore gain-of-function scenarios. The complete dataset is accessible on the ChemoGenix website (URL: https://chemogenix.iric.ca).

genetics↗

bioRxiv2025

A Saccharomyces boulardii synthetic biotic platform for delivery of therapeutic nanobodies to ameliorate gastrointestinal inflammation

Protein-based pharmaceuticals, such as engineered antibodies, form a major drug class of steadily increasing market share. However, these biologic medicines are costly to manufacture, are subject to strict supply chain and storage constraints and often require invasive administration routes. Engineered microbes that secrete bioactive products directly within the microbiome milieu may mitigate these challenges. Here, we describe a cell microfactory platform based on the probiotic yeast Saccharomyces boulardii for the production of nanobody biologics in the gastrointestinal (GI) tract. High-level secretion of nanobodies by S. boulardii was achieved by optimizing promoters, secretion signals, and antibody formats. In mice, oral gavage of S. boulardii allowed efficient and transient colonization of the colonic compartment and in situ production of a therapeutic nanobody directed against tumor necrosis factor (TNF). In a mouse model of chemical-induced colitis, GI-delivery of anti-mTNF nanobody via live S. boulardii improved both survival and disease severity without causing overt perturbation of microbiome composition. These results position S. boulardii as a synthetic biotic platform for the in situ production and delivery of protein-based therapeutics to the GI tract.

synthetic biology↗

bioRxiv2025

Engineered spermidine-secreting Saccharomyces boulardii enhances olfactory memory in Drosophila melanogaster

The polyamines putrescine, spermidine and spermine are ubiquitous metabolites synthesized in all cells. The intracellular levels of polyamines, especially spermidine, decrease in aging. Oral spermidine supplementation has been reported to alleviate aspects of age-related disease in animal models, including decline in learning and memory. The diverse health benefits of spermidine supplementation, often at doses that do not significantly alter spermidine levels of target organs, suggests that exogenous spermidine may have a common site of action, the gastrointestinal (GI) tract. To directly deliver spermidine to the GI tract with minimum impact on the global spermidine levels, we engineered the probiotic yeast Sacchromyces boulardii (Sb) to overproduce and secrete spermidine. We tested the effects of a spermidine-producing yeast strain (Sb576) on age-associated learning and memory decline in an olfactory classical conditioning in Drosophila melanogaster. Feeding of newly emerged adult flies [w1118(isoCJ1)] for 30 days with food supplemented with live Sb576, but not live wild-type Sb (SbWT) or free spermidine, reduced age-associated short-term memory (STM) decline. Notably, Sb576 supplementation, but not SbWT or spermidine supplementation, of either young flies or old flies for only three days also enhanced STM without affecting locomotive ability. Transcriptome analysis of the gut revealed relatively few (30) differentially overexpressed genes in the Sb576 group compared to the SbWT group including the gene coding neuropeptide Dh31, which has been implicated in memory in the flies. These results demonstrate that in situ production of spermidine by a synthetic biotic yeast in the GI tract can enhance STM, and further suggest a mechanism involving the gut-brain axis.

synthetic biology↗

bioRxiv2025

Engineered spermidine-secreting Saccharomyces boulardii ameliorate colitis and colon cancer in mice

Experimental studies suggest that the probiotic yeast Saccharomyces boulardii can mitigate the symptoms of inflammatory bowel disease. However, these results are equivocal and S boulardii probiotic therapy has not gained widespread acceptance in clinical practice. To assess whether the therapeutic properties of S boulardii might be improved upon, we engineered S boulardii to overproduce and secrete spermidine, a pro-regenerative natural metabolite. We employed CRISPR gene deletion and transposon-mediated gene integration to manipulate expression of key enzymes in the polyamine synthetic and transport pathways. We tested the engineered yeast by oral gavage of mice treated with azoxymethane and dextran sulfate sodium to induce chronic colitis and colon cancer. We demonstrate that oral delivery of spermidine-secreting S boulardii in mice populates the gastrointestinal tract with viable spermidine-secreting S boulardii cells and raises free spermidine levels in the gastrointestinal tract. Strikingly, spermidine-secreting S boulardii strains were significantly more effective than wild-type S boulardii in reducing dextran sulfate sodium-induced colitis as well as colitis-associated colon cancer in mice. These results suggest that in situ spermidine secretion by engineered synthetic biotic yeast strains may be an effective and low-cost therapy to mitigate inflammatory bowel disease and colon cancer.

cancer biology↗

bioRxiv2024

Reduction in ETFDH expression optimizes cancer cell bioenergetics

Mitochondrial electron transport flavoprotein (ETF) insufficiency causes metabolic diseases known as a multiple acyl-CoA dehydrogenase deficiency (MADD). In contrast to muscle, ETFDH is a non-essential gene in acute lymphoblastic leukemia NALM-6 cells, and its expression is reduced across human cancers. ETF insufficiency caused by decreased ETFDH expression limits flexibility of OXPHOS fuel utilization but paradoxically increases cancer cell bioenergetics and accelerates neoplastic growth by activation of the mTORC1/BCL-6/4E-BP1 axis. Collectively, these findings reveal that while ETF insufficiency is rare and has detrimental effects in non-malignant tissues, it is common in neoplasia, where ETFDH downregulation leads to bioenergetic and signaling reprogramming that accelerate neoplastic growth.

cancer biology↗

bioRxiv2024

The Role of ATP Synthase Subunit e (ATP5I) in Mediating the Metabolic and Antiproliferative Effects of Biguanides

Here we identify the subunit e of F F -ATP synthase (ATP5I) as a target of metformin, a first-in-class antidiabetic biguanide. ATP5I maintains the stability of F F -ATP synthase dimers which is crucial for shaping cristae morphology. We demonstrate that ATP5I interacts with a biguanide analogue in vitro and disabling its expression by CRISPR-Cas9 in pancreatic cancer cells leads to the same phenotype as biguanide treated cells including mitochondrial morphology alterations, reduction of the NAD+/NADH ratio, inhibition of oxidative phosphorylation (OXPHOS), rescue of respiration by uncouplers and a compensatory increase in glycolysis. Notably, metformin affects the oligomerization of the F F -ATP synthase leading to accumulation of vestigial assembly intermediates also observed upon ATP5I inactivation. Moreover, ATP5I knockout (KO) cells exhibit resistance to the antiproliferative effects of biguanides, but reintroduction of ATP5I rescues the metabolic and anti-proliferative effects of metformin and phenformin. Finally, a genome-wide CRISPR screening in NALM-6 lymphoma cells revealed that metformin-treated cells exhibit genetic interaction profiles similar to those observed with the F F -ATP synthase inhibitor oligomycin, but not with the complex I inhibitor rotenone. This provides unbiased support for the relevance of the newly proposed target.

cancer biology↗

bioRxiv2024

De novo design of potent inhibitors of Clostridioides difficile toxin B

Clostridioides difficile is a major cause of secondary disease in hospitals. During infection, C. difficile toxin B drives disease pathology. Here we use deep learning and Rosetta-based approaches to de novo design small proteins that block the entry of TcdB into cells. These molecules have binding affinities and neutralization IC50s in the pM range and are compelling candidates for further clinical development. By directly targeting the toxin rather than the pathogen, these molecules have the advantage of immediate cessation of disease and lower selective pressure for escape compared to conventional antibiotics. As C. difficile infects the colon, the protease and pH resistance of the designed proteins opens the door to oral delivery of engineered biologics. Significance statementC. difficile infection (CDI) is a major public health concern with over half a million cases in the United States annually resulting in 30,000 deaths. Current therapies are inadequate and frequently result in cycles of recurrent infection (rCDI). Progress has been made in the development of anti-toxin mAb therapies that can reduce the rate of rCDI, but these remain unaffordable and out of reach for many patients. Using de novo protein design, we developed small protein inhibitors targeting two independent receptor binding sites on the toxin that drives pathology during CDI. These molecules are high affinity, potently neutralizing and stable in simulated intestinal fluid, making them strong candidates for the clinical development of new CDI therapies.

microbiology↗

bioRxiv2024

PRMT5 is required for full-length HTT expression by repressing multiple proximal intronic polyadenylation sites

Expansion of the CAG trinucleotide repeat tract in exon 1 of the Huntingtin (HTT) gene above a threshold of [~]36 repeats causes Huntingtons disease (HD) through the expression of a polyglutamine-expanded form of the HTT protein. This mutation triggers wide-ranging cellular and biochemical pathologies leading to cognitive, motor, and psychiatric symptoms in HD patients. As accurate splicing is required to produce the full-length HTT protein of [~]348 kDa, targeting HTT splicing with small molecule drugs is a compelling approach to lower HTT protein levels to treat HD, and splice modulators are being tested in the clinic. Here, we identify PRMT5 as a novel regulator of HTT mRNA splicing and alternative polyadenylation. PRMT5 inhibition disrupts the splicing of HTT introns 9 and 10, leading to activation of multiple proximal intronic polyadenylation sites within these introns and promoting premature termination, cleavage and polyadenylation (PCPA) of the HTT mRNA, thus lowering total HTT protein levels. We also detected increasing levels of these truncated, intron-containing HTT transcripts across a series of neuronal differentiation samples which correlated with lower PRMT5 expression. Notably, PRMT5 inhibition in glioblastoma (GBM) stem cells potently induced neuronal differentiation. We posit that PRMT5-mediated regulation of intronic polyadenylation, premature termination and cleavage of the HTT mRNA modulates HTT expression and plays an important role during embryonic development and neuronal differentiation.

neuroscience↗

bioRxiv2023

Swi4-dependent SWI4 transcription couples cell size to cell cycle commitment

Growth-dependent accumulation of the limiting SBF transcription factor, composed of Swi4 and Swi6, occurs in G1 phase in budding yeast and is limiting for commitment to division, termed Start. Here we measure size-dependence of Swi4 protein copy number under different genetic contexts using the scanning number and brightness technique. Mutation of SBF binding sites in the SWI4 promoter or disruption of SBF activation resulted in [~]33-50% decrease in Swi4 accumulation rate and concordantly increased cell size at Start. Ectopic inducible expression of Swi4 in G1 phase cells increased production of Swi4 from the endogenous promoter, upregulated transcription of the G1/S regulon, and accelerated Start. Despite the potential for Swi4 positive feedback, G1 phase Swi4 accumulation was linear unless the Whi5 transcriptional repressor was inactivated. A threshold model in which Swi4 titrates SBF binding sites in G1/S promoters predicted the effects of nutrients, ploidy, and G1/S regulatory mutations on cell size. These results exemplify how transcription factor auto-production can contribute to a cell state transition.

cell biology↗

bioRxiv2021

Active growth signalling promotes cancer cell sensitivity to the CDK7 inhibitor ICEC0942

CDK7 has a central role in promoting cellular proliferation, through the activation of the mitotic CDKs, and by driving global gene expression, through targeting RNA polymerase II. Several recently developed CDK7 inhibitors (CDK7i) have been shown to be non-toxic and to limit tumour growth for a number of cancer cell types and are now in Phase I/II clinical trials. However, the mechanisms underlying the sensitivity of particular cancer cells to CDK7 inhibition remain largely unknown. To improve the outcome of individual patients and increase the chances of successful CDK7i approval, we assessed which fundamental cellular processes determine sensitivity to CDK7 inhibition, using the highly specific CDK7 inhibitor ICEC0942. Our data shows that selective CDK7 inhibition acutely arrests cells in the G1 phase of the cell cycle, which over time leads to senescence. Through a genome-wide CRISPR knock-out chemogenetic screen we identified active mTOR (mammalian target of rapamycin) signalling, as an important determinant of ICEC0942-induced senescence and show that a cancer-associated mutation that promotes cell growth can increase sensitivity to ICEC0942. Our work indicates that cellular growth is an important predictive marker for sensitivity to CDK7i.

cancer biology↗

bioRxiv2020

A synthetic lethal interaction with a viral-induced hypomorph of GBF1

Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal partners of cancer-specific mutations. Synthetic lethal interactions of viral-induced hypomorphs have the potential to be similarly targeted for the development of host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for synthetic lethal partners of GBF1 revealed ARF1 as the top hit, disruption of which, selectively killed cells that synthesize poliovirus 3A. Thus, viral protein interactions can induce hypomorphs that render host cells vulnerable to perturbations that leave uninfected cells intact. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2. SummaryUsing a viral-induced hypomorph of GBF1, Navare et al., demonstrate that the principle of synthetic lethality is a mechanism to selectively kill virus-infected cells.

molecular biology↗

bioRxiv2020

Nsr1, a nitrogen source-regulated microprotein, confers an alternative mechanism of G1/S transcriptional activation in budding yeast

Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae, is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c, renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon mTORCl inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6, which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogeny conditions. Author SummarUnicellular microorganisms must adapt to ever-changing nutrient conditions and hence must adjust cell growth and proliferation to maximize fitness. In the budding yeast Saccharomyces cerevisiae, commitment to cell division, termed Start, is heavily influenced by nutrient availability. Our understanding of how Start is activated is based mainly on experiments carried out under rich nutrient conditions. To identify potential new Start regulators specific to poor nutrient environments, we screened for genes able to bypass a genetic Start arrest caused by loss of the G1 cyclin Cln3 and the transcriptional activator Bck2. This screen uncovered YLR053c, which we renamed NRS1 for Nitrogen-Responsive Start regulator. Sequence analysis across yeast species indicated that Nrs1 is a recently-evolved microprotein. We showed that NRS1 is nutrient- and cell cycle-regulated, and directly binds the main G1/S transcription factor complex SBF. We demonstrated that Nrs1 has an intrinsic trans-activation activity and provided genetic evidence to suggest that Nrs1 can bypass the requirement for normal Cln3-dependent activation of G1/S transcription. These results uncover a new mechanism of Start activation and illustrate how microproteins can rapidly emerge to rewire fundamental cellular processes.

cell biology↗

bioRxiv2020

Chemical-Genetic Interrogation of Nuclear Size Control Reveals Cancer-Specific Effects on Cell Migration and Invasion

Lower survival rates for many cancer types correlate with increases or decreases in nuclear size/scaling in a tumor-type/tissue-specific manner. Postulating that nuclear size changes confer a fitness advantage on tumor cells, we screened for FDA/EMA-approved compounds that reverse tumor nuclear size changes in cell lines from three such tumor types: prostate adenocarcinoma, colonic adenocarcinoma, and small-cell squamous lung cancer. We found distinct, largely non-overlapping sets of compounds that either rectify or exacerbate nuclear size changes for each tumor type. Nuclear size phenotypes across cell lines clustered particular classes of compounds including serotonin uptake inhibitors, cyclo-oxygenase inhibitors, beta-adrenergic receptor agonists, monoamine oxidase inhibitors, and Na+/K+ ATPase inhibitors. Nearly all compounds selected for further investigation inhibited cell migration and/or invasion, suggesting that targeting nuclear size control pathways in chemotherapy regimens could improve patient survival.

cancer biology↗

bioRxiv2019

G1/S transcription factors assemble in discrete clusters that increase in number as cells grow

The spatio-temporal organization of transcription factor (TF)-promoter interactions is critical for the coordination of transcriptional programs. In budding yeast, the main G1/S transcription factors, SBF and MBF, are limiting with respect to target promoters in small G1 phase cells and accumulate as cells grow, raising the question of how SBF/MBF are dynamically distributed across the G1/S regulon. Super-resolution Photo-Activatable Localization Microscopy (PALM) mapping of the static positions of SBF/MBF subunits revealed that 85% were organized into discrete clusters containing [~]8 copies regardless of cell size, while the number of clusters increased with growth. Stochastic simulations with a mathematical model based on co-localization of promoters in clusters recapitulated observed cluster behavior. A prediction of the model that SBF/MBF should exhibit both fast and slow dynamics was confirmed in PALM experiments on live cells. This spatio-temporal organization of the TFs that activate the G1/S regulon may help coordinate commitment to division.

cell biology↗

bioRxiv2019

Genetic Landscape of Electron Transport Chain Complex I Dependency in Acute Myeloid Leukemia

Inhibition of oxidative phosphorylation (OXPHOS) is a promising therapeutic strategy in Acute Myeloid Leukemia (AML), but patients respond heterogeneously. Through chemically interrogation of 200 sequenced specimens, we identified Mubritinib as a strong in vitro and in vivo anti-leukemic compound, acting through ubiquinone-dependent inhibition of Electron Transport Chain complex I (ETC1). ETC1 targeting showed selective toxicity against a subgroup of chemotherapy-resistant leukemias exhibiting OXPHOS hyperactivity, high expression of mitochondrial activity-related genes, and mutations affecting NPM1, FLT3 and DNMT3A. Altogether, our work thus identifies a novel ETC1 inhibitor with high clinical potential and reveals the landscape of OXPHOS dependency in AML.

cancer biology↗

bioRxiv2018

Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay

The ubiquitin-proteasome system (UPS) controls the stability, localization and/or activity of the proteome. However, the identification and characterization of complex individual ubiquitination cascades and their modulators remains a challenge. Here, we report a broadly applicable, multiplexed, miniaturized on-bead technique for real-time monitoring of various ubiquitination-related enzymatic activities. The assay, termed UPS-confocal fluorescence nanoscanning (UPS-CONA), employs a substrate of interest immobilized on a micro-bead and a fluorescently labelled ubiquitin which, upon enzymatic conjugation to the substrate, is quantitatively detected on the bead periphery by confocal imaging. UPS-CONA is suitable for studying individual enzymatic activities, including various E1, E2 and HECT-type E3 enzymes, and for monitoring multi-step reactions within ubiquitination cascades in a single experimental compartment. We demonstrate the power of the UPS-CONA technique by simultaneously following ubiquitin transfer from Ube1 through Ube2L3 to E6AP. We applied this multi-step setup to investigate the selectivity of five ubiquitination inhibitors reportedly targeting different classes of ubiquitination enzymes. Using UPS-CONA, we have identified a new activity of a small molecule E2 inhibitor, BAY 11-7082, and of a HECT E3 inhibitor, heclin, towards the Ube1 enzyme. As a sensitive, quantitative, flexible and reagent-efficient method with a straightforward protocol, UPS-CONA constitutes a powerful tool for interrogation of ubiquitination-related enzymatic pathways and their chemical modulators, and is readily scalable for large experiments.

biochemistry↗

bioRxiv2017

Contextual diversity of the human cell-essential proteome

Essential genes define central biological functions required for cell growth, proliferation and survival, but the nature of gene essentiality across human cell types is not well understood. We assessed essential gene function in a Cas9-inducible human B-cell lymphoma cell line using an extended knockout (EKO) library of 278,754 sgRNAs that targeted 19,084 RefSeq genes, 20,852 alternatively-spliced exons and 3,872 hypothetical genes. A new statistical analysis tool called RANKS identified 2,280 essential genes, 234 of which had not been reported previously. Essential genes exhibited a bimodal distribution across 10 cell lines screened in different studies, consistent with a continuous variation in essentiality as a function of cell type. Genes essential in more lines were associated with more severe fitness defects and encoded the evolutionarily conserved structural cores of protein complexes. Genes essential in fewer lines tended to form context-specific modules and encode subunits at the periphery of essential complexes. The essentiality of individual protein residues across the proteome correlated with evolutionary conservation, structural burial, modular domains, and protein interaction interfaces. Many alternatively-spliced exons in essential genes were dispensable and tended to encode disordered regions. We also detected a significant fitness defect for 44 newly evolved hypothetical reading frames. These results illuminate the nature and evolution of essential gene functions in human cells.

genomics↗