CRISPR Knockout Library Research Articles

CRISPR-Mediated Library Screening of Gene-Knockout Cell Lines for Investigating Antiviral Innate Immunity.

The application of CRISPR-mediated library screening has fundamentally transformed functional genomics by revealing the complexity of virus-host interactions. This protocol describes the use of CRISPR-mediated library screening to identify key functional genes regulating the innate immune response to PEDV infection. We detail a step-by-step process, starting from the design and construction of a customized CRISPR knockout library targeting genes involved in innate immunity to the effective delivery of these constructs into cells using lentiviral vectors. Subsequently, we outline the process of identifying functional genes postviral attack, including the use of next-generation sequencing (NGS), to analyze and identify knockout cells that exhibit altered responses to infection. This integrated approach provides researchers in immunology and virology with a resource and a robust framework for uncovering the genetic basis of host-pathogen interactions and the arsenal of the innate immune system against viral invasions.

Abstract 1275: In vivo functional CRISPR screens identifies metabolic dependencies mediating triple negative breast cancer lung metastasis

Abstract Metastasis is responsible for about 90% of cancer-related mortality. Throughout the metastatic cascade, cancer cells requires substantial metabolic rewiring to adapt the nutrient micro-environmental changes enabling dissemination and seeding in distant organs. Recent insights into importance of the tumor-intrinsic metabolic reprograming has been implicated in immune escape, yet altered immune responses imposed by tumor metabolism during metastasis are poorly understood. Here, we generated genetic perturbations in triple-negative breast cancer cells using metabolism-focused CRISPR knockout library and performed in vivo screens in both immunocompetent and immunodeficient mice. By comparing enriched sgRNAs in lung metastases or primary tumors, we identify loss of one gene related to long chain fatty acid (LCFA) β-oxidation (FAO) potentiates metastatic dissemination in immunocompetent mice but no in immunodeficient mice, suggesting that impaired FAO in tumor cells promotes breast cancer lung metastasis through an lymphocytes-dependent manner. We confirmed individual sgRNAs targeting this FAO gene from the primary screen dramatically accelerate metastasis in different breast cancer orthotopic models. Specifically, we find that low expression of this gene in primary tumors of advanced breast cancer patients with high grade tumors and positive lymph nodes (LN+) is associated with reduced overall survival. In addition, deletion of this gene generates an immunosuppressive microenvironment in the lung metastatic tumors with increased neutrophils, monocytes and decreased TNFα+-IFNγ+ CD8+ T cells. Mechanistically, impaired FAO in tumor cells causes mitochondria imbalance under lipid metabolism stress, consequently triggers inflammatory response on establishing a neutrophil-rich metastatic niche via mitochondrial nucleic acid-driven innate immunity. Altogether, our data provides a new insight on how tumor cell lipid metabolism impacting immune interplay during metastatic progression, nominating potential anti-tumor immunotherapies. Citation Format: Xiaoyong Wang, Shu-ting Chou, Verra Ngwa, Yoonha Hwang, Breelyn Karno, Deanna Edwards, Jin Chen. In vivo functional CRISPR screens identifies metabolic dependencies mediating triple negative breast cancer lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1275.

Roflumilast inhibits tumor growth and migration in STK11/LKB1 deficient pancreatic cancer

Pancreatic cancer is a malignant tumor of the digestive system. It is highly aggressive, easily metastasizes, and extremely difficult to treat. This study aimed to analyze the genes that might regulate pancreatic cancer migration to provide an essential basis for the prognostic assessment of pancreatic cancer and individualized treatment. A CRISPR knockout library directed against 915 murine genes was transfected into TB 32047 cell line to screen which gene loss promoted cell migration. Next-generation sequencing and PinAPL.py- analysis was performed to identify candidate genes. We then assessed the effect of serine/threonine kinase 11 (STK11) knockout on pancreatic cancer by wound-healing assay, chick agnosia (CAM) assay, and orthotopic mouse pancreatic cancer model. We performed RNA sequence and Western blotting for mechanistic studies to identify and verify the pathways. After accelerated Transwell migration screening, STK11 was identified as one of the top candidate genes. Further experiments showed that targeted knockout of STK11 promoted the cell migration and increased liver metastasis in mice. Mechanistic analyses revealed that STK11 knockout influences blood vessel morphogenesis and is closely associated with the enhanced expression of phosphodiesterases (PDEs), especially PDE4D, PDE4B, and PDE10A. PDE4 inhibitor Roflumilast inhibited STK11-KO cell migration and tumor size, further demonstrating that PDEs are essential for STK11-deficient cell migration. Our findings support the adoption of therapeutic strategies, including Roflumilast, for patients with STK11-mutated pancreatic cancer in order to improve treatment efficacy and ultimately prolong survival.

GARP and EARP are required for efficient BoHV-1 replication as identified by a genome wide CRISPR knockout screen.

The advances in gene editing bring unprecedented opportunities in high throughput functional genomics to animal research. Here we describe a genome wide CRISPR knockout library, btCRISPRko.v1, targeting all protein coding genes in the cattle genome. Using it, we conducted genome wide screens during Bovine Herpes Virus type 1 (BoHV-1) replication and compiled a list of pro-viral and anti-viral candidates. These candidates might influence multiple aspects of BoHV-1 biology such as viral entry, genome replication and transcription, viral protein trafficking and virion maturation in the cytoplasm. Some of the most intriguing examples are VPS51, VPS52 and VPS53 that code for subunits of two membrane tethering complexes, the endosome-associated recycling protein (EARP) complex and the Golgi-associated retrograde protein (GARP) complex. These complexes mediate endosomal recycling and retrograde trafficking to the trans Golgi Network (TGN). Simultaneous loss of both complexes in MDBKs resulted in greatly reduced production of infectious BoHV-1 virions. We also found that viruses released by these deficient cells severely lack VP8, the most abundant tegument protein of BoHV-1 that are crucial for its virulence. In combination with previous reports, our data suggest vital roles GARP and EARP play during viral protein packaging and capsid re-envelopment in the cytoplasm. It also contributes to evidence that both the TGN and the recycling endosomes are recruited in this process, mediated by these complexes. The btCRISPRko.v1 library generated here has been controlled for quality and shown to be effective in host gene discovery. We hope it will facilitate efforts in the study of other pathogens and various aspects of cell biology in cattle.

Exploring the Mechanisms of Venetoclax Resistance Via Drug Screening and Genome-Wide CRISPR Screening

Introduction: Combining Venetoclax, a selective BCL-2 inhibitor, with hypomethylating agents has revolutionized frontline acute myeloid leukemia (AML) treatment. Despite its success, the occurrence of resistance remains a significant concern, limiting the range of available treatment alternatives. Little is known about the mechanism by which the cells escape the treatment. Recent studies have observed MCL1 upregulation in AML cells to promote resistance, and the rationale to use an MCL1 inhibitor became attractive. Although MCL1 inhibitors work exceptionally well in vitro their clinical trials were unsuccessful due to their toxicity. Here, we aim to identify other genes responsible for acquiring resistance and tackle the problem through screening for clinically approved drugs to rapidly benefit the patients with our study. Methods: First, we generated a model of the venetoclax resistance from 3 AML cell lines: MOLM-13, HL-60 and MV4-11. Resistance was achieved through chronic administration of the compound, inhibiting cell viability to 80-90% in multiple rounds by gradually increasing concentrations until the cells developed resistance. We have then characterized these Venetoclax-resistant (VeR) cells using quantitative RT-PCR and RNA sequencing to reveal the mechanisms behind their resistance. VeR cell lines were subjected to drug screening with a library of 859 FDA and EMA-approved compounds with various clinical indications. Cell viability in response to the drug library was assessed after 72h by Cell-Titer Glo and evaluated against the wild-type counterparts. Top-performing compounds were validated in dose-response curves, and synergy scores were identified from combinational treatments. Simultaneously, we performed genome-wide CRISPR/Cas9 knockout screening using Brunello CRISPR knockout library on both MOLM-13 wild-type (WT) and VeR cell lines to identify genes that maintain the resistance to venetoclax and genes that may potentially synergize with targeting BCL-2. Results: Results from the CRISPR screen pointed out multiple genes whose loss of function may contribute to venetoclax resistance in MOLM-13 cells, among which BAX, NOXA, ELAVL1 and TP53 ranked as top hits. On the contrary, we also identified several genes whose loss of function sensitized the cells to venetoclax (e.g., MCL1, OPA1, CDK2, MCAT), suggesting them to be promising therapeutic targets. Among these hits, CDK2 correlates with our drug screening data indicating Flavopiridol as an effective compound in treating VeR cell lines. Moreover, we confirmed the elevated MCL1 gene expression for the MOLM-13 VeR cell line through RNAseq (and qPCR) and detected an altered expression of genes mainly in metabolic pathways and PI3K-Akt signaling pathway via differential gene expression analysis. Our drug screening results revealed overall high efficacy of DNA-damaging agents, proteasome and HDAC inhibitors for both WT and VeR cell lines. We also included an MCL1 inhibitor as a positive control in our drug library and confirmed a synergistic effect when combined with venetoclax. Conclusion: Integrating a range of screening approaches may lead to discovering previously unknown therapeutic targets for venetoclax-resistant AML. Our screens confirmed previously described involvement of the apoptotic pathway genes in venetoclax resistance as well as identified some novel promising targets. Our ongoing efforts involve thorough validations of the identified hits from CRISPR and drug screening to enhance the reliability and translatability of the results. This project was partly supported by grants MUNI/A/1330/2021, MUNI/A/1419/2021 OPRDE (No.CZ.02.2.69/0.0/0.0/19_073/0016943) and project NICR (EU program EXCELES, No. LX22NPO5102).

A Functional Genomic Screen to Identify Novel Genes Involved in Multiple Myeloma Tumour Development

Multiple myeloma (MM) is an incurable malignancy characterised by uncontrolled proliferation of plasma cells (PCs) in the bone marrow (BM). MM is a genetically heterogeneous disease with each patient's PCs harbouring unique genetic mutations, however the development of MM tumours is not only dependent on the underlying genetics but also on the selective pressures applied by the BM microenvironment. Hence, we hypothesise that identifying the dependencies which promote MM cell outgrowth in the BM microenvironment will allow for the identification of new drug targets. This project aims to use an in vivo functional screen, combining CRISPR-Cas9 gene editing, using a single guide RNA (sgRNA) library targeting thousands of genes, with a murine model of MM, to identify novel genes involved in MM tumour development. The Bassik human apoptosis and cancer CRISPR knockout library (Addgene #101926) was used to transduce MM cells with 31,324 unique sgRNAs targeting 3,015 genes and 1,500 control regions. The human MM cell line OPM2 was transduced with the Cas9 transgene (FuCas9GFP), followed by the sgRNA expression vector (pMCB320) generating OPM2-Cas9-sgRNA cells. These cells were then injected (5x10 5 cells/mouse, n=8 mice + 1 tumour-naive control) into the tibiae of immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl/SzJ (NSG) mice. Four weeks post-injection, the primary tumour within the injected tibia was isolated and sgRNA frequencies were assessed via next generation sequencing and compared with that of the initial library and the injected cells using the MAGeCK algorithm. To identify genes that play roles in MM tumour formation in patients, we assessed the expression of our identified genes in CD138-selected BM PCs from newly diagnosed MM patients (n=155) when compared with normal PCs from healthy donors (n=5) in microarray dataset E-MTAB-363 (ArrayExpress). Furthermore, to identify potential pathway involvement of our identified genes we performed gene set enrichment analysis (GSEA) using the University of California San Diego and Broad Institute developed GSEA software, comparing to Hallmark Gene Sets. Comparison of the in vitro cultured cells with the initial library identified 34 genes with undetectable (p<0.05) sgRNAs post in vitro culture, suggesting these genes play key roles in the survival of OPM2 cells. This list contained a number of genes known to control MM cell survival, such as IRF4 and CCND2. Additionally, sgRNAs targeting a further 115 genes were significantly depleted (p<0.05) in vitro and were completely undetectable in vivo, suggesting that these are implicated in cell proliferation and tumour formation in vivo. This gene list contained some already known MM drivers such as MYC, KRAS and DIS3. Furthermore, we identified 28 genes where CRISPR knockout had no significant effect in vitro but were critical dependencies for tumour development, with sgRNAs being undetectable (p<0.05) in vivo. This list contained genes known to be important for MM and BM stromal cell interaction such as ALCAM and SPP1. Notably, 25% of the genes identified as critical dependencies for OPM2 cells in vitro and/or in vivo are significantly upregulated >2-fold (p<0.05; limma) in MM patient PCs when compared with normal PCs (E-MTAB-363), suggesting these may play a role in tumour development in patients. Gene set enrichment analysis revealed that the genes that were essential in vitro were predominantly involved in cell survival related pathways such as cell cycle regulation (G2M checkpoint) and DNA replication/repair; however, the uniquely in vivo genes are predominantly involved in cell metabolism pathways such as MTORC1 signalling, oxidative phosphorylation and glycolysis. Overall, this study has successfully used a functional genetic screen to identify novel gene dependencies required for in vitro MM cell growth and in vivo MM tumour development. A secondary genetic screen will be performed in vivo to validate and refine this list of candidate genes. These validated genes will be further investigated using patient derived data sets to determine if they represent common vulnerabilities in patients and in multiple MM cell lines to investigate their specific mechanistic and pathway involvement. Identification and understanding the driving dependencies which promote MM cell outgrowth in the BM microenvironment will allow for the identification and development of new drug targets to improve patient outcomes.

Decoding the Epigenetic Drivers of Menin-MLL Inhibitor Resistance in KMT2A-Rearranged Acute Myeloid Leukemia

Menin inhibitors that disrupt Menin-MLL interaction are candidate therapeutic agents for several acute myeloid leukemia (AML) subtypes such as KMT2A rearrangement and NPM1 mutation. Several Menin inhibitors are currently being assessed in clinical trials and have demonstrated promising initial results. However, the development of both innate and acquired resistance to Menin inhibitors could limit their therapeutic potential. While studies have found that around 40% of these resistance cases can be traced back to mutations in the Menin gene, the Menin gene remains unaltered in other majorities, suggesting the presence of other mechanisms driving resistance. Here, we developed a unique chromatin-focused CRISPR knockout library, named Advanced Catalogue of Epigenetic Regulators (ACER). The ACER library contains approximately 800 traditional and newly-predicted epigenetic regulators using epigenetic network analyses that integrated publicly available gene codependency (DepMap) and protein-protein interaction data (BioPlex) (Figure A). To understand the chromatin mechanisms responsible for Menin-MLL inhibition resistance, we employed the ACER library for CRISPR screen in the context of Menin inhibition. Notably, our screen identified non-canonical polycomb repressive complex PRC1.1 (PCGF1, BCOR, and RYBP) and PRC2.2 (EZH2, SUZ12, and EED) among the top hits that conferred resistance to MLL-Menin inhibition upon gene depletion (Figure B). Consistent with our screen results, the depletion of PRC1.1 components led to a markedly increase in IC50 values when treated with the Menin inhibitor VTP50469 in both human and murine KMT2A-rearranged cell models. While treatment with Menin inhibitors typically induces cellular differentiation in leukemia cells, depletion of PRC1.1 genes resulted in an almost complete blockage in this differentiation process. Menin inhibitors evict Menin from chromatin and repress the expression levels of Menin-MLL target genes like MEIS1, PBX3, and MEF2C. Strikingly, our ChIP-seq and RNA-seq data indicatethat the loss of PRC1.1 components doesn't impact Menin's displacement from chromatin or the repression of KMT2A target genes by the Menin inhibitor. This is consistent with recent data from phase I clinical trial of revumenib (SNDX-5613) and preclinical patient derived xenograft models, wherein the Menin inhibitor persistently inhibitedkey KMT2A targets, even in resistant samples. These findings highlightthe possibility that resistance to Menin inhibition arising from mechanisms independent of canonical KMT2A target genes. Importantly, our unbiased transcriptomic analysis revealed that MYC signatures as the most affected pathway in PRC1.1-depleted cells following Menin inhibition. Furthermore, we show that both the transcriptionally active Menin-MLL and the repressive PRC1.1 complexes coexist at the MYC promoter, and the interplay between these two complexes determines MYC transcription output in AML. Moreover, enforced expression of MYC leads to resistance against Menin inhibition, while genetic and pharmacologic targeting MYC in PRC1.1 deficient cells rescued the resistance to Menin inhibition. Finally, in our RNA-seq datasets, we observed a diminished monocyte differentiation signature after PRC1.1 depletion. Considering AML cells with monocytic signatures, typically seen in KMT2A-rearranged AML, often exhibit resistance to BCL-2 inhibitor venetoclax, we hypothesized that AML cells lacking PRC1.1 could be more susceptible to BCL2 inhibition. This was confirmed in our ACER library screen with venetoclax treatment, where the PRC1.1 components PCGF1 and BCOR stood out as top epigenetic factors rendering drug sensitivity. Our subsequent studies using murine AML models and human primary samples further validated that PRC1.1-deficient AML cells are markedly sensitive to venetoclax. Furthermore, we show that venetoclax can be employed to overcome the resistance to Menin-MLL inhibition. In summary, our study identifiesthe non-canonical PRC1.1 as a key epigenetic driver of Menin-MLL resistance through a KMT2A target gene-independent mechanism which involves aberrant activation of MYC. We have further provided evidence that AML cells with loss of PRC1.1 are hypersensitive to BCL-2 inhibitor Venetoclax, opening a new therapeutic avenue for tackling Menin-resistant AMLs driven by polycomb inactivation.

Functional Interrogation of Clinically Associated Venetoclax-Sensitive Genes Identifies an Essential Role of Transcription Factor ZNF740 in Therapeutic Response

Venetoclax, a targeted inhibitor of BCL-2, has shown therapeutic benefits in acute myeloid leukemia (AML). However, the emergence of drug resistance remains a significant clinical challenge, emphasizing the need for deeper understanding of its mechanisms. While comprehensive transcriptome analyses have identified many genes associated with venetoclax responsiveness, it remains crucial to distinguish between genes that play a direct role and those that are simply associated. In this study, we focused on genes strongly linked to a positive venetoclax response in primary AML samples using the BeatAML2.0 dataset. We identified ~1,400 top genes whose expression inversely correlated with venetoclax AUC. We theorized that if any of these genes were essential for venetoclax sensitivity, their absence would lead to resistance. To explore their significance, we developed a focused CRISPR knockout library of these ~1,400 genes, with each gene represented by 7 guide RNAs, and an additional 250 guide RNAs as negative controls. We then performed CRISPR screens on AML cell lines, using either DMSO or venetoclax as treatments. Interestingly, the majority of the gene knockouts did not lead to resistance, suggesting that these genes likely have an associative rather than a direct impact on venetoclax sensitivity. However, our screen did identify several genes that, when depleted, consistently resulted in resistance across different AML cell models. One of the top hits was the zinc finger transcription factor ZNF740, whose expression is strongly correlated with venetoclax AUC ( R = -0.52, p = 2.71e-26). For verification, we used CRISPR approach to knock out ZNF740 in Cas9-expressing OCI-AML2 and MOLM-13 cell lines. We then performed IC 50 and competitive proliferation assays under venetoclax treatment. We found that AML cells without ZNF740 exhibited a markedly higher IC 50 and demonstrated enhanced survival under venetoclax exposure. Conversely, enforced overexpression of ZNF740 rendered hypersensitivity to venetoclax. Intriguingly, our transcriptomic study showed that ZNF740 deletion upregulated monocyte differentiation signature genes, a change previously linked to venetoclax resistance. Collectively, our findings indicate that ZNF740 serves as a newly identified, clinically relevant transcriptional modulator of venetoclax responsiveness. We are now investigating the molecular targets of ZNF740, particularly its role in guiding AML lineage decisions and its impact on venetoclax response. Gaining insight into ZNF740's function and mechanism could pave the way for innovative strategies to counteract venetoclax resistance observed in clinical settings.

Abstract 1692: mTOR inhibition attenuates chemosensitivity through induction of a persister state

Abstract Effective cancer therapeutics often kill a large fraction of tumor cells, while sparing a small surviving population, which can resurface as future tumor recurrence. Mechanisms of resistance in these residual tumors may involve rare preexisting clones with mutations, as well as de novo development of resistance through various genetic and/or non-genetic processes. In the context of acute stress resulting from cancer therapeutics, the initial adaptation is essential for tumor cell survival and allows for subsequent development of resistance through additional genetic and non-genetic mechanisms. As an integrator of environmental signals and cellular response, the mTOR pathway is a critical driver of cellular adaptation to environmental changes. In particular, mTOR regulates embryonic diapause, a reversible dormancy state in mammalian development in response to unfavorable environments. Analogous to its role in regulating embryonic diapause, mTOR inhibition may induce a diapause-like state in cancer cells, leading to drug tolerance and persistence of residual tumors following therapy. In line with this notion, recent studies have reported markedly reduced mTOR activity in residual tumors. However, the causal relationship between mTOR inhibition and chemosensitivity has not been investigated. Through a genome-wide CRISPR knockout library screen in pancreatic cancer cells treated with chemotherapeutic agents, we have identified the mTOR pathway as a prominent determinant of chemosensitivity. Pharmacological suppression of mTOR activity in cancer cells from diverse tissue origins leads to the persistence of a reversibly resistant population, which is otherwise eliminated by chemotherapeutic agents. Conversely, activation of the mTOR pathway increases chemosensitivity and predicts better survival among various human cancers. Survival of the persisters is dependent on the regulation of autophagy, G2/M checkpoint, and cell death pathways, as revealed by a small-molecule chemical library screen. Thus, mTOR plays a causal yet paradoxical role in regulating chemotherapeutic response; inhibition of the mTOR pathway, while suppressing tumor expansion, facilitates the development of a reversible drug-tolerant persister state. Citation Format: Yuanhui Liu, Nancy Azizian, Delaney Sullivan, Yulin Li. mTOR inhibition attenuates chemosensitivity through induction of a persister state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1692.

Abstract 251: Identification of therapeutic vulnerabilities by genome-wide CRISPR knockout library screening of colon cancer organoids

Abstract The promise of precision medicine is based on the idea that genetic alterations present only in tumor cells create vulnerabilities that can be targeted for therapeutic intent. In some instances, somatically mutated driver genes such as KRAS, BRAF or PIK3CA can be targeted with small molecule inhibitors specific for mutant oncoproteins. In other cases therapeutic vulnerability is an indirect consequence, such as response to immune checkpoint inhibitors caused by somatic inactivation of mismatch repair genes and subsequent creation of neo-antigens in tumors. Synthetic lethal vulnerabilities dependent on common somatic alterations would be highly specific therapeutic targets applicable to a large number of cancer patients. We sought to identify potential therapeutic vulnerabilities for colon cancer by performing a genome wide CRISPR knockout screen in a colon cancer organoid in both TP53-Wild-Type and TP53-Knockout backgrounds. We identified 1784 gene knockouts with TP53-dependent effects on Darwinian fitness. Examples include MDM2 and PPM1D knockouts, both of which selectively harmed the TP53 WT organoids. 250 gene knockouts selectively harmed the TP53 KO organoids and represent novel avenues for development of TP53 synthetic lethal targeted therapeutics. We also identified ~1000 gene knockouts under significant negative Darwinian selection in both TP53 WT and TP53 KO organoids which are not known to be common essential genes. Several of these organoid model specific vulnerabilities are in pathways that are downstream of the somatic mutations present in the tumor and are therefore candidates for highly-specific targeted therapeutic intervention. Examples include Werner’s pathway dependencies (WRN, EME1, MUS81) which are known to result from mismatch repair deficiency, WNT pathway dependencies (CTNNB1, PORCN) which result from RNF43 inactivating somatic mutation, and BRAF dependency resulting from its own activating oncogenic somatic mutation. In summary, genome-wide CRISPR knockout library screening of human colon cancer organoids can provide a comprehensive overview of dependencies interacting with either natural or engineered mutations and are a promising novel platform for discovering personalized or pathway based therapeutic targets. Future work will focus on target validation and additional primary screens using our biobank of tumor-normal organoid pairs. Citation Format: Phillip J. Buckhaults, Sana Khalili, Carolyn Banister, Prashanth R. Gokare, Dave Pocalyko, Kurtis Bachman. Identification of therapeutic vulnerabilities by genome-wide CRISPR knockout library screening of colon cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 251.

MTOR inhibition attenuates chemosensitivity through the induction of chemotherapy resistant persisters

Chemotherapy can eradicate a majority of cancer cells. However, a small population of tumor cells often survives drug treatments through genetic and/or non-genetic mechanisms, leading to tumor recurrence. Here we report a reversible chemoresistance phenotype regulated by the mTOR pathway. Through a genome-wide CRISPR knockout library screen in pancreatic cancer cells treated with chemotherapeutic agents, we have identified the mTOR pathway as a prominent determinant of chemosensitivity. Pharmacological suppression of mTOR activity in cancer cells from diverse tissue origins leads to the persistence of a reversibly resistant population, which is otherwise eliminated by chemotherapeutic agents. Conversely, activation of the mTOR pathway increases chemosensitivity in vitro and in vivo and predicts better survival among various human cancers. Persister cells display a senescence phenotype. Inhibition of mTOR does not induce cellular senescence per se, but rather promotes the survival of senescent cells through regulation of autophagy and G2/M cell cycle arrest, as revealed by a small-molecule chemical library screen. Thus, mTOR plays a causal yet paradoxical role in regulating chemotherapeutic response; inhibition of the mTOR pathway, while suppressing tumor expansion, facilitates the development of a reversible drug-tolerant senescence state.

Abstract B011: Identifying druggable lipid synthesis targets as novel therapeutics for pancreatic cancer

Abstract Lipid metabolic reprogramming is a hallmark of cancer that contributes to tumorigenesis by supporting energy metabolism and the synthesis of signaling molecules, cellular membranes, and protein modifications. Cells modulate lipid metabolic genes through the regulation of sterol regulatory element-binding proteins (SREBPs), which serve as transcriptional activators of genes responsible for fatty acid and cholesterol synthesis and uptake. Despite efforts demonstrating that the SREBP pathway is an attractive chemotherapeutic target, a potent bioavailable inhibitor of the pathway has not yet been discovered. Here, we identify druggable SREBP target genes as potential targets in pancreatic ductal adenocarcinoma (PDAC). Results: To discover candidate genes, we constructed a CRISPR knockout library containing 148 known SREBP target genes. We used a patient-derived PDAC cell line to conduct the knockout screens in vivo (n = 7 tumors) by injecting library-infected cells into the pancreas of nude mice. After 4 weeks of growth, mouse PDAC tumors showed a statistically significant depletion of guide RNAs targeting 8 genes within the mevalonate pathway, which is responsible for synthesizing cholesterol and isoprenoids such as farnesyl-diphosphate and geranyl-diphosphate. One of the most significant hits was HMG-CoA reductase (HMGCR), which catalyzes the formation of mevalonate. We chose to investigate statins, which are a well-characterized class of FDA-approved drugs that inhibit HMGCR. Using flow cytometry, we stained for annexin-V as a marker for apoptosis and found minimal cell death induced by Fluvastatin alone. Given that inhibiting HMGCR would disrupt lipid homeostasis and in turn activate the SREBP pathway, we hypothesized that a combination therapy with an SREBP inhibitor, TMDP, would potentiate statin-induced cell death. We observed minimal cell death with TMDP alone, but striking synergy between Fluvastatin and TMDP, showing a five-fold increase in annexin-V staining after 48 hours. Mevalonate and geranylgeranyl-diphosphate both fully rescued this cell death to basal levels. Conclusions: In vivo CRISPR screening is a powerful tool for the identification of novel therapeutic targets for cancer. We found that the mevalonate pathway plays a significant role in pancreatic tumor growth in vivo and that existing FDA-approved drugs can be used in combination with an SREBP inhibitor to dramatically enhance cell killing in vitro. Citation Format: Casie S. Kubota, Chiaki T. Ishida, Stephanie Myers, Peter J. Espenshade. Identifying druggable lipid synthesis targets as novel therapeutics for pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B011.

PICKLES v3: the updated database of pooled in vitro CRISPR knockout library essentiality screens.

PICKLES (https://pickles.hart-lab.org) is an updated web interface to a freely available database of genome-scale CRISPR knockout fitness screens in human cell lines. Using a completely rewritten interface, researchers can explore gene knockout fitness phenotypes across cell lines and tissue types and compare fitness profiles with fitness, expression, or mutation profiles of other genes. The database has been updated to include data from three CRISPR libraries (Avana, Score, and TKOv3), and includes information from 1162 whole-genome screens probing the knockout fitness phenotype of 18 959 genes. Source code for the interface and the integrated database are available for download.

A Genome-Wide CRISPR Screen Identifies Factors Regulating Pluripotency Exit in Mouse Embryonic Stem Cells

Pluripotency maintenance and exit in embryonic stem cells is a focal topic in stem cell biology. However, the effects of screening under very stringent culture conditions (e.g., differentiation medium, no leukemia inhibitory factor, no chemical inhibitors such as PD0325901 and CHIR99021, and no feeder cells) and of prolonging culture for key factors that regulate pluripotency exit, have not yet been reported. Here, we used a genome-wide CRISPR library to perform such a screen in mouse embryonic stem cells. Naïve NANOG-GFP mESCs were first transfected with a mouse genome-wide CRISPR knockout library to obtain a mutant mESCs library, followed by screening for two months in a strict N2B27 differentiation medium. The clones that survived our stringent screening were analyzed to identify the inserted sgRNAs. In addition to identifying the enriched genes that were reported in previous studies (Socs3, Tsc1, Trp53, Nf2, Tcf7l1, Csnk1a1, and Dhx30), we found 17 unreported genes, among which Zfp771 and Olfr769 appeared to be involved in pluripotency exit. Furthermore, Zfp771 knockout ESCs showed a differentiation delay in embryonic chimera experiments, indicating Zfp771 played an important role in pluripotency exit. Our results show that stringent screening with the CRISPR library can reveal key regulators of pluripotency exit.

Identification of novel γ-globin inducers among all potential erythroid druggable targets

AbstractHuman γ-globin is predominantly expressed in fetal liver erythroid cells during gestation from 2 nearly identical genes, HBG1 and HBG2, that are both perinatally silenced. Reactivation of these fetal genes in adult red blood cells can ameliorate many symptoms associated with the inherited β-globinopathies, sickle cell disease, and Cooley anemia. Although promising genetic strategies to reactivate the γ-globin genes to treat these diseases have been explored, there are significant barriers to their effective implementation worldwide; alternatively, pharmacological induction of γ-globin synthesis could readily reach the majority of affected individuals. In this study, we generated a CRISPR knockout library that targeted all erythroid genes for which prospective or actual therapeutic compounds already exist. By probing this library for genes that repress fetal hemoglobin (HbF), we identified several novel, potentially druggable, γ-globin repressors, including VHL and PTEN. We demonstrate that deletion of VHL induces HbF through activation of the HIF1α pathway and that deletion of PTEN induces HbF through AKT pathway stimulation. Finally, we show that small-molecule inhibitors of PTEN and EZH induce HbF in both healthy and β-thalassemic human primary erythroid cells.

Abstract P025: Uncovering molecular actors of IDO-mediated T cell dysfunction with genome-wide CRISPR/Cas9 knockout screens

Abstract Introduction: Despite tremendous progress in cancer immunotherapy, most patients fail to benefit because of poorly characterized immune resistance mechanisms. Among these, expression of the tryptophan-catabolizing enzyme Indoleamine 2,3-dioxygenase (IDO) has been found in several tumors and associated with local immune suppression, notably by inhibiting T cell functions. However, the exact molecular pathways making T cells sensitive to IDO are still unclear. We propose to exploit the power of whole-genome single-guide RNA (sgRNA) CRISPR screens to uncover novel mechanisms of IDO-mediated T cell dysfunction. Methods: Cas9-expressing transgenic mice were crossed with mice expressing a transgenic T cell receptor (TCR) specifically recognizing a tumor antigen called P1A. Primary Cas9xTCRP1A CD8+ T cells were isolated from these mice and stimulated with P1A-expressing tumor cells. Cells were then transduced with the Teichmann retroviral genome wide CRISPR knockout library. Seven days after the first stimulation, CD8+ T cells were re-stimulated in a control (Tryptophanhigh, kynureninelow) or selective (Tryptophanlow, kynureninehigh) medium, mimicking the function of IDO in vitro. After four days of screening selection, genomic DNA was extracted from remaining living cells and sequencing libraries were prepared. sgRNA representation was then assessed by next-generation sequencing. Results: In vitro CRISPR knockout screening pipeline was successfully set up and validated. This includes: (1.) Successful transduction of the CRISPR knockout library, as well as efficient gene knockout in Cas9xTCRP1A primary CD8+ T cells. (2.) Confirming the strong inhibition of T cell proliferation and survival in the selective medium as compared to the control medium, thereby validating the screening selection strategy. (3.) Optimizing genomic DNA extraction procedure, as well as sequencing library preparation. Finally, the proposed in vitro CRISPR knockout screening was successfully launched, and analysis of potential candidate genes is ongoing. Conclusions and perspectives: Our study proposes to perform genome wide CRISPR knockout screens in T cells exposed to conditions mimicking IDO activity in vitro. Enriched sgRNA at the end of the selection should reveal genes whose inhibition improved T cell survival in Tryptophanlow/kynureninehigh medium. Resulting potential candidate targets will then be validated by knocking them out in T cells and assessing T cell functions under conditions mimicking IDO-mediated immune suppression in vitro. Similar validation of top candidate genes will also be performed in vivo in a model of adoptive cell transfer of target-knockout T cells in mice bearing IDO-expressing tumors. The identified genes/pathways involved in T cell sensitivity to Tryptophan shortage/Kynurenine enrichment should reveal new relevant mechanisms of IDO-mediated immune suppression in the tumor microenvironment. Citation Format: Raphaële Bombart, Jingjing Zhu, Benoit J. Van den Eynde. Uncovering molecular actors of IDO-mediated T cell dysfunction with genome-wide CRISPR/Cas9 knockout screens [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P025.

Abstract P096: Using CRISPR-Cas9 screens to identify microRNA involved in aggressive prostate cancer phenotypes

Abstract Background: Prostate cancer (PCa) is the most common cancer diagnosis in Canadian men. For many, the tumour can remain “dormant” negating a need for treatment, but if the tumour progresses and becomes metastatic, prognosis is poor. Despite the introduction of new treatments, metastatic PCa remains a lethal and incurable disease. This is invariably due to the onset of castration-resistance in advanced cases producing a more aggressive tumour with increased metastatic potential. We hypothesize that microRNA can promote these aggressive PCa phenotypes due to their ability to regulate diverse gene networks simultaneously. Methods: We have generated miRKOv2, the second version of our microRNA CRISPR Knockout library using the latest on- and off-target scoring algorithms and other microRNA-specific features for CRISPR-Cas9 guide RNA (gRNA) design. miRKOv2 will be used in three separate screens to identify microRNA that are essential for PCa growth, microRNA that are involved in PCa metastasis, and microRNA that can confer castration-resistance, respectively. Results: Next generation sequencing of the plasmid miRKOv2 library showed a median gRNA coverage of ~1100X. In silico comparisons to other microRNA libraries demonstrate that miRKOv2 is a superior sgRNA library. DU145 cells stably expressing Cas9 are highly active and proof of principle growth assays demonstrate that essential gene knockout and subsequent decrease in cell viability is detectable. Generation of a Cas9+ PCa cell line panel and screens are ongoing. Conclusion: miRKOv2 is an improved microRNA-focused CRISPR library with high gRNA coverage. Proof of principle assays demonstrate that knockout of essential genes results in a reduction in cell viability, indicating that a CRISPR dropout screen is appropriate for our in vitro model. This project aims to identify microRNA dependencies in metastatic and castration-resistant PCa that can be leveraged into new biomarkers and therapies. Citation Format: Jonathan Tak-Sum Chow, Daniel K. C. Lee, Martino Marco Gabra, Norman Fu, Keyue Chen, Leonardo Salmena. Using CRISPR-Cas9 screens to identify microRNA involved in aggressive prostate cancer phenotypes [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P096.

Identification of ZDHHC17 as a Potential Drug Target for Swine Acute Diarrhea Syndrome Coronavirus Infection.

ABSTRACTThe recent emergence and spread of zoonotic viruses highlights that animal-sourced viruses are the biggest threat to global public health. Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an HKU2-related bat coronavirus that was spilled over from Rhinolophus bats to swine, causing large-scale outbreaks of severe diarrhea disease in piglets in China. Unlike other porcine coronaviruses, SADS-CoV possesses broad species tissue tropism, including primary human cells, implying a significant risk of cross-species spillover. To explore host dependency factors for SADS-CoV as therapeutic targets, we employed genome-wide CRISPR knockout library screening in HeLa cells. Consistent with two independent screens, we identified the zinc finger DHHC-type palmitoyltransferase 17 (ZDHHC17 or ZD17) as an important host factor for SADS-CoV infection. Through truncation mutagenesis, we demonstrated that the DHHC domain of ZD17 that is involved in palmitoylation is important for SADS-CoV infection. Mechanistic studies revealed that ZD17 is required for SADS-CoV genomic RNA replication. Treatment of infected cells with the palmitoylation inhibitor 2-bromopalmitate (2-BP) significantly suppressed SADS-CoV infection. Our findings provide insight on SADS-CoV-host interactions and a potential therapeutic application.

Abstract 1600: Cancer driver screens identifyregulators of immune checkpoint blockade therapy response

Abstract Cancer mutations play essential roles in tumor development by promote cell proliferation or immune evasion. However, some recent researches indicate that cancer cells with these mutation burden also expose their vulnerability to immune-based therapies such as checkpoint blockade. Here we performed comprehensive CRISPR knockout library screens in syngeneic mouse models on cancer driver mutation genes. We identified a set of genes including Kmt2d, Arid2, Rhob, Eloc, Ptpn11 and Xpo1, which have high frequency mutation in several human cancer types and their deficiency made tumor cells better respond to anti-PD-1 treatment. Furthermore, some of these deletions were validated to sensitize the tumor cells to cytotoxic T cells by in vitro co-culture with CD8+ T cells. The findings of Kmt2d was in accordance with a recent report for its role as a sensitizing modulator of immune checkpoint blockade from mouse model and patient prediction. These findings provide a new insight for understanding the interaction between the tumor heterogeneity and microenvironment. These results revealed CRISPR in vivo screen as a robust tool for both context and target discovery for tumor immunotherapy. Citation Format: Wenrong Zhou, Zhengang Peng, Dawei Huang, Min Long, Tianyu Song, Siyuan Ni, Yong Cang. Cancer driver screens identifyregulators of immune checkpoint blockade therapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1600.

Identification of SYS1 as a Host Factor Required for Shiga Toxin-Mediated Cytotoxicity in Vero Cells.

Shiga toxin (STx) or Vero toxin is a virulence factor produced by enterohemorrhagic Escherichia coli. The toxin binds to the glycosphingolipid globotriaosylceramide (Gb3) for its entry, and causes cell death by inhibiting ribosome function. Previously, we performed a loss-of-function screen in HeLa cells using a human CRISPR knockout (KO) library and identified various host genes required for STx-induced cell death. To determine whether this library targeted to the human genome is applicable to non-human primate cells and to identify previously unrecognized factors crucial for STx-induced cell death, we herein performed a similar screen in the African green monkey kidney-derived Vero C1008 subline. Many genes relevant to metabolic enzymes and membrane trafficking were enriched, although the number of enriched genes was less than that obtained in the screening for HeLa cells. Of note, several genes that had not been enriched in the previous screening were enriched: one of these genes was SYS1, which encodes a multi-spanning membrane protein in the Golgi apparatus. In SYS1 KO Vero cells, expression of Gb3 and sphingomyelin was decreased, while that of glucosylceramide and lactosylceramide was increased. In addition, loss of SYS1 inhibited the biosynthesis of protein glycans, deformed the Golgi apparatus, and perturbed the localization of trans-Golgi network protein (TGN) 46. These results indicate that the human CRISPR KO library is applicable to Vero cell lines, and SYS1 has a widespread effect on glycan biosynthesis via regulation of intra-Golgi and endosome–TGN retrograde transports.