Genome-wide Screen Research Articles

Strategic and Innovative Roles of lncRNAs Regulated by Naturally-derived Small Molecules in Cancer Therapy.

In the field of precision and personalized medicine, the next generation sequencing method has begun to take an active place as genome-wide screening applications in the diagnosis and treatment of diseases. Studies based on the determination of the therapeutic efficacy of personalized drug use in cancer treatment in the size of the transcriptome and its extension, lncRNA, have been increasing rapidly in recent years. Targeting and/or regulating noncoding RNAs (ncRNAs) consisting of long noncoding RNAs (lncRNAs) are promising strategies for cancer treatment. Within the scope of rapidly increasing studies in recent years, it has been shown that many natural agents obtained from biological organisms can potentially alter the expression of many lncRNAs associated with oncogenic functions. Natural agents include effective small molecules that provide anti-cancer effects and have been used as chemotherapy drugs or in combination with standard anti-cancer drugs used in routine treatment. In this review, it was aimed to provide detailed information about the potential of natural agents to regulate and/or target non-coding RNAs and their mechanisms of action to provide an approach for cancer therapy. The discovery of novel anti-cancer targets and subsequent development of effective drugs or combination strategies that are still needed for most cancers will be promising for cancer treatment.

GGNBP2 regulates MDA5 sensing triggered by self double-stranded RNA following loss of ADAR1 editing.

Adenosine-to-inosine (A-to-I) editing of double-stranded RNA (dsRNA) by ADAR1 is an essential modifier of the immunogenicity of cellular dsRNA. The role of MDA5 in sensing unedited cellular dsRNA and the downstream activation of type I interferon (IFN) signaling are well established. However, we have an incomplete understanding of pathways that modify the response to unedited dsRNA. We performed a genome-wide CRISPR screen and showed that GGNBP2, CNOT10, and CNOT11 interact and regulate sensing of unedited cellular dsRNA. We found that GGNBP2 acts between dsRNA transcription and its cytoplasmic sensing by MDA5. GGNBP2 loss prevented induction of type I IFN and autoinflammation after the loss of ADAR1 editing activity by modifying the subcellular distribution of endogenous A-to-I editing substrates and reducing cytoplasmic dsRNA load. These findings reveal previously undescribed pathways to modify diseases associated with ADAR mutations and may be determinants of response or resistance to small-molecule ADAR1 inhibitors.

The facilitated osteogenic differentiation by extracellular proline treatment in in vitro cell cultivation using MC3T3E1 and hPDLF.

Proline is a major substrate in collagen biosynthesis and is required for collagen molecule formations. However, detailed explanations of the molecular basis through which proline functions in collagen biosynthesis have yet to be provided. Thus, genome-wide screening was employed to elucidate these in the pre-osteoblastic MC3T3-E1 and human periodontal ligament fibroblast (hPDLF) cell lines. Indeed, both cell lines represent important sources for collagen biosynthesis and tissue regeneration in the dental region, specifically treating extracellular proline during cultivations. The altered gene expression patterns were identified, and the precise expression patterns were confirmed by microarray. Cell viability and osteogenic differentiation patterns were examined using a range of experimental methods, such as the MTS assay, ALP staining, ARS staining, and collagen (COL)-type1A ELISA. Overall, we revealed a cell line-specific function of exogenous proline in collagen biosynthesis during osteogenic differentiation conditions with the candidate signaling pathways. These putative signaling networks could represent plausible answers to understanding collagen biosynthesis for regenerating connective tissues such as skin, muscle, and bone.

CRISPR/Cas9 system: a novel approach to overcome chemotherapy and radiotherapy resistance in cancer.

Cancer presents a global health challenge with rising incidence and mortality. Despite treatment advances in cancer therapy, radiotherapy and chemotherapy remained the most common treatments for all types of cancers. However, resistance phenotype in cancer cells leads to unsatisfactory results in the efficiency of therapeutic strategies. Therefore, researchers strive to propose effective solutions to overcome treatment failure, which requires a deep knowledge of treatment-resistant mechanisms. The progression and occurrence of tumors can be attributed to gene mutation. Over the past decade, the emergence of clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) genome editing has revolutionized cancer research. This versatile technology enables cancer modeling, manipulation of specific DNA sequences, and genome-wide screening. CRISPR/Cas9 is an effective tool for identifying radio- and chemoresistance genes and offering potential adjunctive treatments to overcome tumor recurrence after chemo- and radiotherapy. This article aims to explain the potential of the CRISPR/Cas9 system in improving the effectiveness of chemo- and radiotherapy and ultimately overcoming treatment failure.

RAD52 and ERCC6L/PICH have a compensatory relationship for genome stability in mitosis.

Mammalian RAD52 is a DNA repair factor with strand annealing and recombination mediator activities that appear important in both interphase and mitotic cells. Nonetheless, RAD52 is dispensable for cell viability. To query RAD52 synthetic lethal relationships, we performed genome-wide CRISPR knock-out screens and identified hundreds of candidate synthetic lethal interactions. We then performed secondary screening and identified genes for which depletion causes reduced viability and elevated genome instability (increased 53BP1 nuclear foci) in RAD52-deficient cells. One such factor was ERCC6L, which marks DNA bridges during anaphase, and hence is important for genome stability in mitosis. Thus, we investigated the functional interrelationship between RAD52 and ERCC6L. We found that RAD52 deficiency increases ERCC6L-coated anaphase ultrafine bridges, and that ERCC6L depletion causes elevated RAD52 foci in prometaphase and interphase cells. These effects were enhanced with replication stress (i.e. hydroxyurea) and topoisomerase IIα inhibition (ICRF-193), where post-treatment effect timings were consistent with defects in addressing stress in mitosis. Altogether, we suggest that RAD52 and ERCC6L co-compensate to protect genome stability in mitosis.

Ablation of FAS confers allogeneic CD3- CAR T cells with resistance to rejection by T cells and natural killer cells.

Allogeneic chimaeric antigen receptor T cells (allo-CAR T cells) derived from healthy donors could provide rapid access to standardized and affordable batches of therapeutic cells if their rejection by the host's immune system is avoided. Here, by means of an in vivo genome-wide CRISPR knockout screen, we show that the deletion of Fas or B2m in allo- T cells increases their survival in immunocompetent mice. Human B2M- allo-CAR T cells become highly sensitive to rejection mediated by natural killer (NK) cells, whereas FAS- CAR T cells expressing normal levels of human leukocyte antigen I remain resistant to NK cells. CD3- FAS- CAR T cells outperformed CD3- B2M- CAR T cells in the control of leukaemia growth in mice under allogeneic pressure by T cells and NK cells. The partial protection of CD3- FAS- allo-CAR T cells from cellular rejection may improve the efficacy of allogeneic cellular therapies in patients with cancer.

Abstract PR014: DNA associated with EVs is uniquely chromatinized and prevents metastasis by enhancing anti-tumor immunity

Abstract Cells release extracellular vesicles (EVs) loaded with functional biomolecules that facilitate intercellular communication in health and disease. These EVs contain DNA (EV-DNA), mirroring the mutational profile of parental cells, yet the mechanism of DNA packaging onto EVs and its role in diseases like cancer remain unclear. Using advanced imaging and biochemical techniques, we discovered that EV-DNA predominantly resides on the vesicle surface, bound to modified histones. Through genome-wide CRISPR knockout screens in cancer cell lines, we identified immune-related pathways and genes crucial for EV-DNA loading, such as APAF1 and NCF1. In several cancer models, loss of APAF1 reduced EV-DNA packaging, and enhancing metastasis, thus implicating EV-DNA in immune surveillance against cancer spread. We demonstrated that EV-DNA uptake by liver macrophages triggers DNA damage response, altering cytokine production and promoting immune activation in pre-metastatic sites. Moreover, quantifying EV-DNA in colorectal cancer biopsies revealed its potential as a predictive metastatic risk biomarker. Our findings shed light on EV-DNA packaging mechanisms and its significance, suggesting that elevated levels of tumor-derived EV-DNA bolster anti-tumor immunity and inhibit metastasis. Citation Format: Inbal Wortzel, Han-Sang Kim, David C. Lyden. DNA associated with EVs is uniquely chromatinized and prevents metastasis by enhancing anti-tumor immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR014.

Genome-Wide Screening and Characterization of Methyl-CpG-Binding Domain (MBD) Proteins in Arabidopsis Species.

Methyl-CpG-binding domain (MBD) proteins play vital roles in epigenetic gene regulation, and they have diverse molecular, cellular, and biological functions in plants. MBD proteins have been functionally characterized in a few plant species. However, the structure and function of MBD proteins in Arabidopsis halleri and Arabidopsis lyrata remain unknown. In this study, 12 A. halleri MBD (AhMBD) and 13 A. lyrata MBD (AlMBD) genes were identified. A phylogenetic analysis of the Arabidopsis genus showed that the MBD proteins of three species (Arabidopsis thaliana, A. helleri, and A. lyrata) could be classified into eight classes. Expression patterns suggested that the AtMBD genes were expressed in different tissues. We characterized the function of AtMBD3 and found that it was constitutively localized to the nucleus and interacted with several AtMBD protein members. Our results reveal that AtMBD3 is involved in the development of A. thaliana, which may be helpful in further studies on these genes in A. helleri and A. lyrata.

CRISPR knockout genome-wide screens identify the HELQ-RAD52 axis in regulating the repair of cisplatin-induced single-stranded DNA gaps.

Treatment with genotoxic agents, such as platinum compounds, is still the mainstay therapeutical approach for the majority of cancers. Our understanding of the mechanisms of action of these drugs is, however, imperfect and continuously evolving. Recent advances highlighted single-stranded DNA (ssDNA) gap accumulation as a potential determinant underlying cisplatin chemosensitivity, at least in some genetic backgrounds, such as BRCA mutations. Cisplatin-induced ssDNA gaps form upon restart of DNA synthesis downstream of cisplatin-induced lesions through repriming catalyzed by the PRIMPOL enzyme. Here, we show that PRIMPOL overexpression in otherwise wild-type cells results in accumulation of cisplatin-induced ssDNA gaps without sensitizing cells to cisplatin, suggesting that ssDNA gap accumulation does not confer cisplatin sensitivity in BRCA-proficient cells. To understand how ssDNA gaps may cause cellular sensitivity, we employed CRISPR-mediated genome-wide genetic screening to identify factors which enable the cytotoxicity of cisplatin-induced ssDNA gaps. We found that the helicase HELQ specifically suppresses cisplatin sensitivity in PRIMPOL-overexpressing cells, and this is associated with reduced ssDNA accumulation. We moreover identify RAD52 as a mediator of this pathway. RAD52 promotes ssDNA gap accumulation through a BRCA-mediated mechanism. Our work identified the HELQ-RAD52-BRCA axis as a regulator of ssDNA gap processing and cisplatin sensitization.

RBIO-07. IMMUNOMODULATORY PATHWAYS MEDIATE RADIOTHERAPY RESPONSE IN MALIGNANT PERIPHERAL NERVE SHEATH TUMORS

Abstract PURPOSE Malignant peripheral nerve sheath tumors (MPNSTs) are the most common cause of death in neurofibromatosis type 1 (NF-1) and are resistant to radiation therapy (RT), yet the mechanisms underlying RT response are poorly understood. Here, we elucidate mechanisms of RT response in NF-1 associated peripheral nerve sheath tumors through genome-wide CRISPRi screens, genomic analysis of mouse models, and molecular analysis of patient-derived tumor specimens. METHODS Patient derived NF1 mutant plexiform neurofibroma (pNF) cells (NF9511b, NF95.6), and MPNST cells (ST88-14; JH2-002) were used to measure RT responses by cell counts and flow cytometry. Genome-wide CRISPRi screens were used to identify functional modifiers of RT response in ST88-14 and JH2-002 MPNST cells. Nf1-/-; Tp53-/- mouse MPNSTs were subcutaneously implanted in immunocompetent C57/B6 mice, irradiated (2Gyx5), and dissociated for single-cell RNA sequencing (scRNA-seq) using 10x Genomics. Human MPNST tumor specimens (n=45) were analyzed with targeted DNA mutation sequencing and methylation arrays for cell type deconvolution. RESULTS Radiation single-dose response curves revealed MPNST cells (IC50 6.85Gy) were radioresistant compared to pNF cells (IC50 3.13Gy). Genome wide CRISPRi screens in ST88-14 and JH2-002 MPNST cells converged on cell cycle, DNA repair, and immunomodulatory gene sets mediating RT sensitivity. Irradiation of subcutaneous mouse Nf1-/-; Tp53-/- MPNSTs significantly decreased tumor growth (p=0.01, t-test). scRNA-seq of 32,763 cells from 4 irradiated and 3 unirradiated mouse Nf1-/-; Tp53-/- MPNSTs identified 8 tumor clusters and 7 non-tumor clusters. Irradiation demonstrated a pro-inflammatory effect with increased T-cell presence (5.8% versus 3.3%, p=0.04, t-test) and greater activation of immunostimulatory genes (Cxcl10, Stat1, Irf7) in T cells. In human MPNSTs, CDKN2A/B deletion (p=0.04, log-rank test) and decreased immune cell composition (p=0.03, log-rank test) were associated with significantly worse overall survival in response to RT. CONCLUSION MPNST RT responses may depend on immunomodulatory mechanisms that could be leveraged to improve clinical RT response.

DDDR-42. LEVERAGING GENOME-WIDE CRISPR/CAS9 KNOCKOUT DRUG SCREENS TO IDENTIFY SENSITIZERS FOR PROTEOSOME INHIBITORS IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common primary brain tumor with a poor prognosis despite aggressive treatment. Patient outcomes remain abysmal with 95% of patients relapsing and a median overall survival of 15 months. This necessitates the rapid search for personalized therapeutics and agents to enhance current treatments. One pathway that demonstrates dysregulated functioning is the ubiquitin-proteasome pathway (UPP). Literature has shown that UPP dependent proteolysis remains constitutively upregulated in cancer cells leading to the rapid degradation of proteins that regulate oncogenic pathways. Despite robust preclinical evaluations for the usage of proteasome inhibitors against GBM, most proteasome inhibitors failed in clinical trials, indicating resistance. We conducted an unbiased genome wide CRISPR-Cas9 screen in HAP1 cells treated with the proteasome inhibitor, Bortezomib (BTZ), to identify genes leading to a BTZ-resistant phenotype. We identified several genes that when perturbed, sensitized cells to BTZ including N-glycanase-1 (NGLY-1), Nuclear factor Erythroid 2-Like-1 (NFE2L1), and DNA damage inducible 1 homolog 2 (DDI2). Herein, we sought to evaluate the effects of perturbing these identified genes in our patient derived GBM cell lines by utilizing CRISPR/Cas9 knockout technology in vitro and in vivo. The generation of NGLY-1, DDI2, and NFE2L1 KO cell lines demonstrated functional sensitivity to the proteasome inhibitor Marizomib (MZB) as observed through a significant reduction of the IC50 in the KO cell lines compared to an adeno-associated virus integration site 1 (AAVS1) control. Functional evaluation also demonstrated reduced proliferation capacity and sphere formation in our KO GBM lines. Ongoing in vivo work will aim to evaluate the mitigation of this resistant pathway in our NSG mouse models orthotopically transplanted with our patient derived KO cell lines. In an ongoing collaborative effort, we aim to functionally assess a novel small molecule NGLY-1 inhibitor for preclinical sensitivity to MZB in our in vitro and in vivo models.

STEM-08. LIPID SATURATION HOMEOSTASIS PRESENTS A TARGETABLE VULNERABILITY IN MYC-AMPLIFIED GROUP 3 MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain cancer, originating from the cerebellum. Despite advancements in standard of care (SoC), MB remains fatal for 30% of patients. Tumor relapse, spinal metastasis and treatment resistance are the most prevalent in MYC-driven Group 3 MB (G3-MB). Patients surviving SoC are faced with life-long neurocognitive and neurodevelopmental deficits. These issues highlight the urgent need for improved treatment modalities. Reprogramming of cellular lipid metabolism is an emerging hallmark of cancer and may yield novel cancer-specific therapeutic options. Here we explore the lipidome of both G3-MB and its proposed cell of origin, human neural stem cells (hNSCs) by comparing untargeted lipidomics using Liquid-Chromatography-Mass Spectrometry (LC-MS). Comparative analyses revealed a differential abundance of distinct lipid species in G3-MB, with an overall reduced saturation level of fatty acids (FAs). These findings implicate the de novo lipid synthesis (DNL) pathway. We identified the enzymes involved in DNL to be essential for MB survival in our genome-wide CRISPR KO screen. Additionally, mRNA expression of the DNL enzymes increases at relapse in our SoC-adapted murine patient-derived xenograft (PDX) model. Pharmacological and genetic targeting of the DNL enzyme Stearoyl-CoA Desaturase 1 (SCD1) selectively targets G3-MB. Furthermore, small molecule treatment of SCD1 demonstrates efficacy against G3-MB PDX models in vivo. We identified SCD expression as a prognostic marker in Group 3 and 4 MB patients and identified possible pathways for MB treatment. Overall, these findings indicate that SCD1 is a potent target for the treatment of G3-MB.

CNSC-70. THE ROLE OF MAP4K4 IN TUMOR CELL INVASION OBSERVED IN A 3D IN VITRO MODEL OF PEDIATRIC GLIOBLASTOMA

Abstract Several CNS tumors such as pediatric glioblastoma (pGBM), diffuse midline glioma (DMG), and medulloblastoma (MB) are characterized by local invasive spread and dissemination of tumor cells into surrounding brain tissue. As identified by our group in a genome-wide CRISPR screen and several others, MAP4K4 is a mediator of cell invasion in several cancers, including glioblastoma and medulloblastoma. Recently, we validated the pro-invasive role of MAP4K4 in a panel of pGBM cell lines. To further validate this finding in the context of pGBM cellular invasion, we performed 3D Matrigel timelapse invasion assays. We performed single- and multi-spheroid encapsulation to track spheroid invasion and cell-to-cell interactions between multiple spheroids. Tumor spheroids were cultured in a round-bottom, non-adherent well plate encapsulated in a Matrigel matrix and treated with a MAP4K4 small molecule inhibitor (PF06260933 dihydrochloride) or had a MAP4K4 knockout performed with Cas9 editing. Cell invasion was recorded over 72 hours and three-dimensional structures were stained for actin and imaged 10 days post-initial seeding. We show that MAP4K4 KO (p ~ 0.00033), small-molecule inhibition in the presence of a TGFB receptor agonist (SRI-011381), (p ~ 0.001) and with drug alone (p ~ 0.013) decreased spheroid invasion and prevented single-cell invasion of pGBM cells (SF188) into the matrix. More surprisingly, abrogation of MAP4K4 prevented individual spheroids in a multi-spheroid co-culture, from forming integrated glioma networks through the formation of tube-like structures in the matrix. Based on our initial findings, it appears that MAP4K4 is necessary for single-cell invasion into a three-dimensional matrix. It also appears that MAP4K4 is required for cell-to-cell connections to form between spheroids in the 3D environment to allow for the formation of higher-ordered and spatially organized structures. Future directions include studying how MAP4K4 allows for the formation of glioma tumor networks in pediatric GBM.

Predicting tumour resistance to paclitaxel and carboplatin utilising genome-wide screening in haploid human embryonic stem cells.

Taxanes and platinum molecules, specifically paclitaxel and carboplatin, are widely used anticancer drugs that induce cell death and serve as first-line chemotherapy for various cancer types. Despite the efficient effect of both drugs on cancer cell proliferation, many tumours have innate resistance against paclitaxel and carboplatin, which leads to inefficient treatment and poor survival rates. Haploid human embryonic stem cells (hESCs) are a novel and robust platform for genetic screening. To gain a comprehensive view of genes that affect or regulate paclitaxel and carboplatin resistance, genome-wide loss-of-function screens in haploid hESCs were performed. Both paclitaxel and carboplatin screens have yielded selected plausible gene lists and pathways relevant to resistance prediction. The effects of mutations in selected genes on the resistance to the drugs were demonstrated. Based on the results, an algorithm that can predict resistance to paclitaxel or carboplatin was developed. Applying the algorithm to the DNA mutation profile of patients' tumours enabled the separation of sensitive versus resistant patients, thus, providing a prediction tool. As the anticancer drugs arsenal can offer alternatives in case of resistance to either paclitaxel or carboplatin, an early prediction can provide a significant advantage and should improve treatment. The algorithm assists this unmet need and helps predict whether a patient will respond to the treatment and may have an immediate clinically actionable application.

Elucidating the role of CYFIP2 in conferring cisplatin resistance in esophageal squamous cell carcinoma

Cisplatin (CDDP) serves as a vital component in the chemotherapeutic approach to treat esophageal squamous cell carcinoma (ESCC). However, prolonged CDDP application frequently culminates in resistance, compromising therapeutic outcomes. Through genome-wide CRISPR library screening, our study elucidates the mechanisms underlying this resistance, pinpointing CYFIP2 as a pivotal mediator. Notably, the involvement CYFIP2 is characterized by pronounced autophagic activity and the modulation of multiple cellular pathways. Empirical validation was achieved by treating ESCC cell lines with CDDP, which resulted in an upsurge of CYFIP2 expression. The functional impact of CYFIP2 was further delineated through knockdown experiments, where a marked suppression in cell proliferation was observed, alongside a discernible decline in reactive oxygen species levels. This was complemented by a suite of assays and microscopic techniques, including GFP-LC3, mRFP-GFP-LC3, electron microscopy and western blot, which collectively affirmed the inhibitory effect of CYFIP2 knockdown on autophagic processes, particularly impeding autophagosome formation and their subsequent fusion with lysosomes. In vivo studies have also confirmed that CYFIP2 knockdown limits tumor progression and increases CDDP efficacy. Conclusively, our findings introduce CYFIP2 as a novel contributor to CDDP resistance in ESCC, underscoring its potential as a therapeutic target. This revelation not only deepens our understanding of resistance mechanisms but also paves the way for novel oncotherapeutic strategies, promising enhanced treatment efficacy against ESCC.

Genome-wide selection of potential target candidates for RNAi against Nilaparvata lugens

BackgroundNilaparvata lugens is one of the most destructive pests of rice. RNAi-based N. lugens control offers one alternative strategy to traditional chemical insecticides. However, selection of potential target for RNAi against N. lugens remains a major challenge. Only two target genes for nuclear transgenic N. lugens-resistant plants have been screened. Importantly, only one or few potential target genes against N. lugens were screened every time by knowledge of essential genes from model organisms in previous study.ResultsHere, in silico genome-wide selection of potential target genes against N. lugens through homology comparison was performed. Through genome synteny comparisons, about 3.5% of Drosophila melanogaster genome was found to have conserved genomic synteny with N. lugens genome. By using N. lugens proteins to search D. melanogaster homologs defining lethal or sterile phenotype, 358 N. lugens genes were first screened as putative target genes. Transgenic rice lines expressing dsRNA of randomly selected gene (NlRan or NlSRP54) from 358 putative target genes enhanced resistance to N. lugens. After expression check and safety check, 115 N. lugens genes were screened as potential target candidates.ConclusionThe combined efforts in this study firstly provide one in silico genome-wide homology-based screening approach for RNAi-based target genes against N. lugens, which not only offer one new opportunity to batch select potential target candidates in pests of interest, but also will facilitate the selection of RNAi target in many pest species by providing more than one hundred potential target candidates.

Inhibition of GPX4 enhances CDK4/6 inhibitor and endocrine therapy activity in breast cancer.

CDK4/6 inhibition in combination with endocrine therapy is the standard of care for estrogen receptor (ER+) breast cancer, and although cytostasis is frequently observed, new treatment strategies that enhance efficacy are required. Here, we perform two independent genome-wide CRISPR screens to identify genetic determinants of CDK4/6 and endocrine therapy sensitivity. Genes involved in oxidative stress and ferroptosis modulate sensitivity, with GPX4 as the top sensitiser in both screens. Depletion or inhibition of GPX4 increases sensitivity to palbociclib and giredestrant, and their combination, in ER+ breast cancer models, with GPX4 null xenografts being highly sensitive to palbociclib. GPX4 perturbation additionally sensitises triple negative breast cancer (TNBC) models to palbociclib. Palbociclib and giredestrant induced oxidative stress and disordered lipid metabolism, leading to a ferroptosis-sensitive state. Lipid peroxidation is promoted by a peroxisome AGPAT3-dependent pathway in ER+ breast cancer models, rather than the classical ACSL4 pathway. Our data demonstrate that CDK4/6 and ER inhibition creates vulnerability to ferroptosis induction, that could be exploited through combination with GPX4 inhibitors, to enhance sensitivity to the current therapies in breast cancer.

Superior target genes and pathways for RNAi-mediated pest control revealed by genome-wide analysis in the beetle Tribolium castaneum.

An increasing human population, the emergence of resistances against pesticides and their potential impact on the environment call for the development of new eco-friendly pest control strategies. RNA interference (RNAi)-based pesticides have emerged as a new option with the first products entering the market. Essentially, double-stranded RNAs targeting essential genes of pests are either expressed in the plants or sprayed on their surface. Upon feeding, pests mount an RNAi response and die. However, it has remained unclear whether RNAi-based insecticides should target the same pathways as classic pesticides or whether the different mode-of-action would favor other processes. Moreover, there is no consensus on the best genes to be targeted. We performed a genome-wide screen in the red flour beetle to identify 905 RNAi target genes. Based on a validation screen and clustering, we identified the 192 most effective target genes in that species. The transfer to oral application in other beetle pests revealed a list of 34 superior target genes, which are an excellent starting point for application in other pests. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses of our genome-wide dataset revealed that genes with high efficacy belonged mainly to basic cellular processes such as gene expression and protein homeostasis - processes not targeted by classic insecticides. Our work revealed the best target genes and target processes for RNAi-based pest control and we propose a procedure to transfer our short list of superior target genes to other pests. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis.

Aggregation of the protein tau defines tauopathies, which include Alzheimer's disease and frontotemporal dementia. Specific neuronal subtypes are selectively vulnerable to tau aggregation and subsequent dysfunction and death, but the underlying mechanisms are unknown. To systematically uncover the cellular factors controlling the accumulation of tau aggregates in human neurons, we conducted a genome-wide CRISPRi-based modifier screen in iPSC-derived neurons. The screen uncovered expected pathways, including autophagy, but also unexpected pathways, including UFMylation and GPI anchor synthesis. We discover that the E3 ubiquitin ligase CUL5 SOCS4 is a potent modifier of tau levels in human neurons, ubiquitinates tau, and is a correlated with vulnerability to tauopathies in mouse and human. Disruption of mitochondrial function promotes proteasomal misprocessing of tau, which generates tau proteolytic fragments like those in disease and changes tau aggregation in vitro . These results reveal new principles of tau proteostasis in human neurons and pinpoint potential therapeutic targets for tauopathies.

Design, performance, processing, and validation of a pooled CRISPR perturbation screen for bacterial toxins.

Unbiased forward genetic screens have been extensively employed in biological research to elucidate functional genomics. In pooled clustered regularly interspaced short palindromic repeats (CRISPR) perturbation screens, various genetically encoded gain-of-function or loss-of-function mutations are introduced into a heterogeneous population of cells. Subsequently, these cells are screened for phenotypes, perturbation-associated genotypes are analyzed and a connection between genotype and phenotype is determined. CRISPR screening techniques enable the investigation of important biological questions, such as how bacterial toxins kill cells and cause disease. However, the broad spectrum of effects caused by diverse toxins presents a challenge when selecting appropriate screening strategies. Here, we provide a step-by-step protocol for a genome-wide pooled CRISPR perturbation screen to study bacterial toxins. We describe technical considerations, pilot experiments, library construction, screen execution, result analysis and validation of the top enriched hits. These screens are applicable for many different types of toxins and are anticipated to reveal a repertoire of host factors crucial in the intoxication pathway, such as receptors, trafficking/translocation factors and substrates. The entire protocol takes 21-27 weeks and does not require specialized knowledge beyond basic biology.