CRISPR-Cas9 Screens Research Articles

Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens.

Toxoplasma gondii is an intracellular parasite that subverts host cell functions via secreted virulence factors. Up to 70% of parasite-controlled changes in the host transcriptome rely on the MYR1 protein, which is required for the translocation of secreted proteins into the host cell. Mice infected with MYR1 knock-out (KO) strains survive infection, supporting a paramount function of MYR1-dependent secreted proteins in Toxoplasma virulence and proliferation. However, we have previously shown that MYR1 mutants have no growth defect in pooled in vivo CRISPR-Cas9 screens in mice, suggesting that the presence of parasites that are wild-type at the myr1 locus in pooled screens can rescue the phenotype. Here, we demonstrate that MYR1 is not required for the survival in IFN-γ-activated murine macrophages, and that parasites lacking MYR1 are able to expand during the onset of infection. While ΔMYR1 parasites have restricted growth in single-strain murine infections, we show that the phenotype is rescued by co-infection with wild-type (WT) parasites in vivo, independent of host functional adaptive immunity or key pro-inflammatory cytokines. These data show that the major function of MYR1-dependent secreted proteins is not to protect the parasite from clearance within infected cells. Instead, MYR-dependent proteins generate a permissive niche in a paracrine manner, which rescues ΔMYR1 parasites within a pool of CRISPR mutants in mice. Our results highlight an important limitation of otherwise powerful in vivo CRISPR screens and point towards key functions for MYR1-dependent Toxoplasma-host interactions beyond the infected cell.

CRISPR-Cas9 screening identifies the role of FER as a tumor suppressor.

It is important to systematically identify tumor suppressor genes (TSGs) to improve our understanding of tumorigenesis and develop strategies for early diagnosis and mitigating disease progression. In the present study, we used an in vivo genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) screen and identified FPS/FES-related (FER) as a TSG. Single-cell RNA sequencing (scRNA-seq) revealed that normal cells with low FER expression exhibited elevated malignant transformation potential and stemness properties. FER knockout promoted the tumorigenic transformation, characterized by high colony-forming efficiency and suspension growth ability, acquired tumorigenicity in vivo, increased metabolic activity, dedifferentiation properties, and immune evasion. Moreover, analysis revealed that low FER expression tumors share molecular phenotypes with FER knockout cells, suggesting the consistent role of FER in tumor initiation and progression. Taken together, our findings not only provide insights into the essential role of FER as a tumor suppressor in tumor initiation and progression but also highlight its potential as a target for future clinical diagnosis. © 2024 The Pathological Society of Great Britain and Ireland.

CRISPR-Cas9 screening identified novel subtypes of cutaneous melanoma based on essential cancer genes.

Our primary objective was to identify genes critical for cutaneous melanoma (CM) and related typing, based on essential genes, to generate novel insights for clinical management and immunotherapy of patients with CM. We analyzed RNA sequencing (RNA-seq) data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx), and sequencing data of 29 CM cell line from Cancer Cell Line Encyclopedia (CCLE) databases. Combined with DepMap database, 406 CM essential cancer genes were finally obtained. Based on the expression of essential genes in cancer, the patients included in TCGA and Gene Expression Omnibus (GEO) databases were divided into three different molecular subtypes (C1, C2, and C3) by the NMF algorithm. Data analysis from TCGA and GEO datasets revealed that subtype C3 had the poorest prognosis, while subtype C1 exhibited the best prognosis. Combined with the CIBERSORT, ESTIMATE and ssGSEA algorithm, patients with different molecular subtypes can be divided into two immune subtypes (hot and cold). We found that subtype C1 was characterized by hot tumors, in contrast to subtypes C2 and C3, which were characterized by cold tumors. Then, we used univariate Cox regression, LASSO, and multifactor Cox regression analysis to select risk genes and constructed a prognostic model based on eight genes: RABIF, CDCA8, FOXM1, SPRR2E, AIP, CAP1, CTSW, and IFITM3. All patients were divided into two risk subtypes (high and low ) according to the median of risk scores. We found that most hot tumor subtypes were found in the low-risk subtypes and most patients with this subtype survived for longer. Ultimately, we selected RABIF, which exhibits the highest risk coefficient, for histological and cytological verification. The results showed that RABIF was overexpressed in melanoma. Inhibition of RABIF expression could suppress the proliferation and invasion of melanoma cells and promote the apoptosis of melanoma cells. In conclusion, we used CRISPR-Cas9 screening to verify the association between molecular subtypes (C1, C2, and C3), immune subtypes (hot and cold), and risk subtypes (high and low) in patients with CM, particularly in distinguishing survival and prognosis. These findings can be used to guide clinical management and immunotherapy of patients with CM.

Abstract PR013: Identification, therapeutic potential, and regulatory networks of tumor suppressing miRNAs in angiosarcoma

Abstract Angiosarcoma (AS) is an aggressive cancer of endothelial cells arising in the blood and lymphatic vasculature. Among all the rare soft tissue sarcomas, AS patients have some of the worst outcomes, with an estimated 5-year survival rate of only 30%. In mouse models, we discovered that endothelial loss of miRNAs through the conditional deletion of Dicer1 leads to AS development. Since most cells, and even most cancer cells require some level of miRNA expression for survival, this demonstrates that endothelial cells are uniquely sensitive to miRNA loss mediated transformation. Consistent with this finding we have found that Dgcr8 deletion with aP2-Cre phenocopies Dicer1 loss resulting in AS with 100% penetrance. This implicates miRNAs as critical tumor suppressors in AS. Thus, we tested the repurposing of the antibiotic, enoxacin as an anticancer therapeutic based on its known potential to enhance miRNA processing. Indeed, enoxacin significantly reduces AS cell proliferation and migration. Small RNA-sequencing revealed that several known tumor suppressing miRNAs were increased in abundance following enoxacin treatment. To discover other miRNAs critical in AS, we conducted an unbiased CRISPR-Cas9 screen with a miRNA specific gRNA library and identified several potential candidates. Among these, our preliminary data indicates miR-410 as a potent tumor suppressor. To determine the miRNA and mRNA target interactome of these miRNAs in AS, we are utilizing AgoTRIBE. This method fuses the Ago2 miRNA effector protein to the RNA editing domain of ADAR2 to generate RNA edits on transcripts that are associated with Ago2-miRNAs as determined by RNA-seq. This work will further utilize in vitro and in vivo tools to dissect the underlying biology and role of these tumor suppressing miRNAs in AS. We aim to leverage these findings to develop novel strategies for therapeutic interventions, including miRNA-based approaches that may improve patient outcomes. Citation Format: Bozhi Liu, Ant Murphy, Annaleighh Benton, Lauren E. Gartenhaus, Jason A Hanna. Identification, therapeutic potential, and regulatory networks of tumor suppressing miRNAs in angiosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR013.

TRIM21-mediated ubiquitination and phosphorylation of ERK1/2 promotes cell proliferation and drug resistance in pituitary adenomas.

Pituitary adenomas (PAs) are common intracranial tumors and TRIM family plays a crucial role in cell proliferation, and therapeutic resistance of tumors. However, the role of the TRIM family in PAs is not well recognized. CRISPR screening explored the role of TRIM family in the cell proliferation and drug resistance in PAs. In vitro and in vivo experiments were performed to evaluate the effects of Tripartite Motif Containing 21 (TRIM21). RNA-sequencing, mass spectrometry, immunoprecipitation and ubiquitination experiments were performed to explore the molecular mechanism. NanoBiT assays were used to screen the drugs reducing TRIM21 expression. CRISPR-Cas9 screens identified that TRIM21 facilitated cell proliferation and drug resistance in PAs. Mechanistically, TRIM21 interacted with ERK1/2 through PRY-SPRY domain, leading to ERK1/2 K27-linked ubiquitination. The ERK1/2 ubiquitination promotes the interaction between ERK1/2 and MEK1/2, thereby facilitating the phosphorylation of ERK1/2. However, an excess presence of TRIM21 supressed the phosphorylation of ERK1/2 and cell proliferation via activating ERK1/2 negative feedback pathways. Importantly, TRIM21 was upregulated in dopamine-resistant prolactinomas and cabergoline-resistant MMQ cells. Furthermore, drug screening identified that Fimepinostat and Quisinostat, can reduce the protein levels of TRIM21, inhibit tumor progression, and increase drug sensitivity. TRIM21 may represent a therapeutic target for tumors, and inhibiting TRIM21 could be a potential strategy for tumor treatment.

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.

DDDR-41. A GENOME-WIDE CRISPR/CAS9 SCREEN IN GLIOBLASTOMA STEM CELLS UNVEILS SYNTHETIC LETHAL INTERACTIONS AND RESISTANCE MECHANISMS OF JIN001, A BLOOD-BRAIN BARRIER-PENETRANT HSP90 INHIBITOR

Abstract Glioblastomas (GBMs) are aggressive brain tumors characterized by extensive genetic heterogeneity and therapeutic resistance. Heat shock protein 90 (Hsp90) is a molecular chaperone that stabilizes and facilitates the proper folding of numerous proteins, including oncogenic drivers and is overexpressed in cancers. Hsp90 inhibitors can disrupt multiple oncogenic signaling pathways by degrading various client proteins essential for tumor growth and survival, making them attractive therapeutic candidates for GBMs. We have previously reported on the efficacy of Hsp90 inhibitors against gliomas. However, cancer cells develop resistance to Hsp90 inhibitors by upregulating compensatory chaperones (e.g., Hsp70), mutating client proteins, or activating alternative survival pathways, reducing the long-term efficacy of these agents. Combination approaches overcoming such resistance mechanisms are needed. JIN001, a novel Hsp90 inhibitor, demonstrates superior blood-brain barrier (BBB) penetration, and high potential for clinical translation. we performed a genome-wide CRISPR/Cas9 knockout screen using the Toronto Knockout Library v3 (TKOv3) with screen selection pressure from JIN001 in GBM stem cells (GSC272) to identify synthetic lethal interactions and resistance mechanisms associated with JIN001 treatment. Experiments were performed in triplicate with two time points, T22 and T37. Quality control analysis of the NGS data indicated excellent quality and consistency between different replicates. DrugZ analysis revealed a set of sensitive and resistant genes, informing potential combination therapies and further elucidating JIN001’s mechanism of action. Validation studies for the top synthetic lethal targets are underway to confirm the identified interactions and will be presented. This CRISPR-Cas9 screen offers valuable insights into JIN001’s therapeutic potential, reveals new combination strategies to overcome resistance to Hsp90 inhibitors and for use of these agents in other cancers, and paves the way for the development of more effective GBM combination treatment strategies.

MED12 loss activates endogenous retroelements to sensitise immunotherapy in pancreatic cancer

ObjectivePancreatic ductal adenocarcinoma (PDAC) stands as one of the most lethal cancers, marked by its lethality and limited treatment options, including the utilisation of checkpoint blockade (ICB) immunotherapy. Epigenetic dysregulation...

IMMU-65. TARGETING AXONAL GUIDANCE DEPENDENCIES IN GLIOBLASTOMA WITH ROBO1 CAR T CELLS

Abstract Resistance to genotoxic therapies and subsequent disease recurrence are hallmarks of aggressive cancers, including glioblastoma (GBM). Here, we uncover functional drivers of post-treatment recurrent GBM using genome scale CRISPR-Cas9 screens accompanied by integrative genomic analysis. Using patient-matched pre- and post-treatment GBM models, our findings uncover large-scale reorganization of functional dependencies at tumor recurrence and implicate protein tyrosine phosphatases 4A2 (PTP4A2) as a novel driver of tumorigenicity in recurrent GBM. Small molecule inhibition and phospho-proteomic analyses reveal a novel PTP4A-ROBO1 signaling axis that modulates tumor invasion, self-renewal and proliferation in recurrent GBM. Since a pan-PTP4A targeting small molecule suffers from poor blood-brain-barrier penetrance, we engineered a novel second generation chimeric antigen receptor (CAR) targeting human ROBO1, a cell surface receptor enriched across GBM specimens. Not only do ROBO1 CAR T cells exhibit a potent and specific anti-tumor effect in vitro, a single dose of ROBO1 CAR T cells doubles median survival in a patient-derived xenograft (PDX) model of recurrent GBM. Expansion of ROBO1 CAR T cells to other invasive brain cancers leads to tumor eradication in ~50% of mice in PDX models of pediatric medulloblastoma and adult lung-to-brain metastases. Together, we provide insights into functional remodeling of GBM at recurrence and present a multi-targetable PTP4A-ROBO1 signaling axis with potential across multiple malignant brain tumors.

TMED inhibition suppresses cell surface PD-1 expression and overcomes T cell dysfunction

BackgroundBlockade of the programmed cell death protein 1 (PD-1) immune checkpoint (ICB) is revolutionizing cancer therapy, but little is known about the mechanisms governing its expression on CD8 T cells....

Transcriptional Regulation of Protein Synthesis by Mediator Kinase Represents a Therapeutic Vulnerability in MYC-driven Medulloblastoma.

MYC-driven medulloblastoma (MB) is a highly aggressive cancer type with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening of MB cell lines, we identified the Mediator-associated kinase CDK8 as a critical regulator of MYC-driven MB. Loss of CDK8 substantially reduces MYC expression and induces pronounced transcriptional changes, consequently inhibiting MB growth and suppressing monosome assembly, resulting in decreased ribosome biogenesis and protein synthesis. Mechanistically, CDK8 regulates the occupancy of RNA polymerase II at specific chromatin loci, facilitating an epigenetic alteration that promotes the transcriptional regulation of ribosomal genes. Targeting CDK8 effectively diminishes the stem-like neoplastic cells characterized by hyperactive ribosome biogenesis. Furthermore, we demonstrated that the combined inhibition of CDK8 and mTOR synergizes to optimize therapeutic outcomes in vivo and in vivo. Overall, our findings establish a connection between CDK8-mediated transcriptional regulation and mRNA translation, suggesting a promising new therapeutic approach that targets the protein synthesis for MYC-driven MB. .

High TNF and NF-κB Pathway Dependency Are Associated with AZD5582 Sensitivity in OSCC via CASP8-Dependent Apoptosis.

Mechanistically guided drug repurposing has been made possible by systematically integrating pharmacologic and CRISPR-Cas9 screen data. Our study discovers the biomarker and cell death mechanisms underpinning sensitivity toward AZD5582, an antagonist of the inhibitor of apoptosis family protein. Our findings have important implications for improving future trial design for patients with OSCC using this emerging drug class.

Reciprocal inhibition of NOTCH and SOX2 shapes tumor cell plasticity and therapeutic escape in triple-negative breast cancer.

Cancer cell plasticity contributes significantly to the failure of chemo- and targeted therapies in triple-negative breast cancer (TNBC). Molecular mechanisms of therapy-induced tumor cell plasticity and associated resistance are largely unknown. Using a genome-wide CRISPR-Cas9 screen, we investigated escape mechanisms of NOTCH-driven TNBC treated with a gamma-secretase inhibitor (GSI) and identified SOX2 as a target of resistance to Notch inhibition. We describe a novel reciprocal inhibitory feedback mechanism between Notch signaling and SOX2. Specifically, Notch signaling inhibits SOX2 expression through its target genes of the HEY family, and SOX2 inhibits Notch signaling through direct interaction with RBPJ. This mechanism shapes divergent cell states with NOTCH positive TNBC being more epithelial-like, while SOX2 expression correlates with epithelial-mesenchymal transition, induces cancer stem cell features and GSI resistance. To counteract monotherapy-induced tumor relapse, we assessed GSI-paclitaxel and dasatinib-paclitaxel combination treatments in NOTCH inhibitor-sensitive and -resistant TNBC xenotransplants, respectively. These distinct preventive combinations and second-line treatment option dependent on NOTCH1 and SOX2 expression in TNBC are able to induce tumor growth control and reduce metastatic burden.

CRISPR screens unveil nutrient-dependent lysosomal and mitochondrial nodes impacting intestinal tissue-resident memory CD8+ T cell formation

Nutrient availability and organelle biology direct tissue homeostasis and cell fate, but how these processes orchestrate tissue immunity remains poorly defined. Here, using invivo CRISPR-Cas9 screens, we uncovered organelle signaling and metabolic processes shaping CD8+ tissue-resident memory T (TRM) cell development. TRM cells depended on mitochondrial translation and respiration. Conversely, three nutrient-dependent lysosomal signaling nodes-Flcn, Ragulator, and Rag GTPases-inhibited intestinal TRM cell formation. Depleting these molecules or amino acids activated the transcription factor Tfeb, thereby linking nutrient stress to TRM programming. Further, Flcn deficiency promoted protective TRM cell responses in the small intestine. Mechanistically, the Flcn-Tfeb axis restrained retinoic acid-induced CCR9 expression for migration and transforming growth factor β (TGF-β)-mediated programming for lineage differentiation. Genetic interaction screening revealed that the mitochondrial protein Mrpl52 enabled early TRM cell formation, while Acss1 controlled TRM cell development under Flcn deficiency-associated lysosomal dysregulation. Thus, the interplay between nutrients, organelle signaling, and metabolic adaptation dictates tissue immunity.

Host cell glycosylation selects for infection with CCR5- versus CXCR4-tropic HIV-1.

Human immunodeficiency virus type 1 (HIV-1) infection involves a selection bottleneck that leads to transmission of one or a few variants. C-C motif chemokine receptor 5 (CCR5) or C-X-C motif chemokine receptor 4 (CXCR4) can act as coreceptors for HIV-1 viral entry. However, initial infection mostly occurs via CCR5, despite abundant expression of CXCR4 on target cells. The host factors that influence HIV-1 susceptibility and selection during transmission are unclear. Here we conduct CRISPR-Cas9 screens and identify SLC35A2 (a transporter of UDP-galactose expressed in target cells in blood and mucosa) as a potent and specific CXCR4-tropic restriction factor in primary target CD4+ T cells. SLC35A2 inactivation, which resulted in truncated glycans, not only increased CXCR4-tropic infection levels but also decreased those of CCR5-tropic strains consistently. Single-cycle infections demonstrated that the effect is cell-intrinsic. These data support a role for a host protein that influences glycan structure in regulating HIV-1 infection. Host cell glycosylation may, therefore, affect HIV-1 selection during transmission in vivo.

CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells

Ageing impairs the ability of neural stem cells (NSCs) to transition from quiescence to proliferation in the adult mammalian brain. Functional decline of NSCs results in the decreased production of new neurons and defective regeneration following injury during ageing1–4. Several genetic interventions have been found to ameliorate old brain function5–8, but systematic functional testing of genes in old NSCs—and more generally in old cells—has not been done. Here we develop in vitro and in vivo high-throughput CRISPR–Cas9 screening platforms to systematically uncover gene knockouts that boost NSC activation in old mice. Our genome-wide screens in primary cultures of young and old NSCs uncovered more than 300 gene knockouts that specifically restore the activation of old NSCs. The top gene knockouts are involved in cilium organization and glucose import. We also establish a scalable CRISPR–Cas9 screening platform in vivo, which identified 24 gene knockouts that boost NSC activation and the production of new neurons in old brains. Notably, the knockout of Slc2a4, which encodes the GLUT4 glucose transporter, is a top intervention that improves the function of old NSCs. Glucose uptake increases in NSCs during ageing, and transient glucose starvation restores the ability of old NSCs to activate. Thus, an increase in glucose uptake may contribute to the decline in NSC activation with age. Our work provides scalable platforms to systematically identify genetic interventions that boost the function of old NSCs, including in vivo, with important implications for countering regenerative decline during ageing.

Multi-species genome-wide CRISPR screens identify conserved suppressors of cold-induced cell death.

Cells must adapt to environmental changes to maintain homeostasis. One of the most striking environmental adaptations is entry into hibernation during which core body temperature can decrease from 37°C to as low at 4°C. How mammalian cells, which evolved to optimally function within a narrow range of temperatures, adapt to this profound decrease in temperature remains poorly understood. In this study, we conducted the first genome-scale CRISPR-Cas9 screen in cells derived from Syrian hamster, a facultative hibernator, as well as human cells to investigate the genetic basis of cold tolerance in a hibernator and a non-hibernator in an unbiased manner. Both screens independently revealed glutathione peroxidase 4 (GPX4), a selenium-containing enzyme, and associated proteins as critical for cold tolerance. We utilized genetic and pharmacological approaches to demonstrate that GPX4 is active in the cold and its catalytic activity is required for cold tolerance. Furthermore, we show that the role of GPX4 as a suppressor of cold-induced cell death extends across hibernating species, including 13-lined ground squirrels and greater horseshoe bats, highlighting the evolutionary conservation of this mechanism of cold tolerance. This study identifies GPX4 as a central modulator of mammalian cold tolerance and advances our understanding of the evolved mechanisms by which cells mitigate cold-associated damage-one of the most common challenges faced by cells and organisms in nature.

Self-sustaining long-term 3D epithelioid cultures reveal drivers of clonal expansion in esophageal epithelium

Aging epithelia are colonized by somatic mutations, which are subjected to selection influenced by intrinsic and extrinsic factors. The lack of suitable culture systems has slowed the study of this and other long-term biological processes. Here, we describe epithelioids, a facile, cost-effective method of culturing multiple mouse and human epithelia. Esophageal epithelioids self-maintain without passaging for at least 1 year, maintaining a three-dimensional structure with proliferative basal cells that differentiate into suprabasal cells, which eventually shed and retain genomic stability. Live imaging over 5 months showed that epithelioids replicate in vivo cell dynamics. Epithelioids support genetic manipulation and enable the study of mutant cell competition and selection in three-dimensional epithelia, and show how anti-cancer treatments modulate competition between transformed and wild-type cells. Finally, a targeted CRISPR–Cas9 screen shows that epithelioids recapitulate mutant gene selection in aging human esophagus and identifies additional drivers of clonal expansion, resolving the genetic networks underpinning competitive fitness.

Transcripts of repetitive DNA elements signal to block phagocytosis of hematopoietic stem cells.

Macrophages maintain hematopoietic stem cell (HSC) quality by assessing cell surface Calreticulin (Calr), an "eat-me" signal induced by reactive oxygen species (ROS). Using zebrafish genetics, we identified Beta-2-microglobulin (B2m) as a crucial "don't eat-me" signal on blood stem cells. A chemical screen revealed inducers of surface Calr that promoted HSC proliferation without triggering ROS or macrophage clearance. Whole-genome CRISPR-Cas9 screening showed that Toll-like receptor 3 (Tlr3) signaling regulated b2m expression. Targeting b2m or tlr3 reduced the HSC clonality. Elevated B2m levels correlated with high expression of repetitive element (RE) transcripts. Overall, our data suggest that RE-associated double-stranded RNA could interact with TLR3 to stimulate surface expression of B2m on hematopoietic stem and progenitor cells. These findings suggest that the balance of Calr and B2m regulates macrophage-HSC interactions and defines hematopoietic clonality.

In vivo CRISPR screens identify a dual function of MEN1 in regulating tumor–microenvironment interactions

Functional genomic screens in two-dimensional cell culture models are limited in identifying therapeutic targets that influence the tumor microenvironment. By comparing targeted CRISPR–Cas9 screens in a two-dimensional culture with xenografts derived from the same cell line, we identified MEN1 as the top hit that confers differential dropout effects in vitro and in vivo. MEN1 knockout in multiple solid cancer types does not impact cell proliferation in vitro but significantly promotes or inhibits tumor growth in immunodeficient or immunocompetent mice, respectively. Mechanistically, MEN1 knockout redistributes MLL1 chromatin occupancy, increasing H3K4me3 at repetitive genomic regions, activating double-stranded RNA expression and increasing neutrophil and CD8+ T cell infiltration in immunodeficient and immunocompetent mice, respectively. Pharmacological inhibition of the menin–MLL interaction reduces tumor growth in a CD8+ T cell-dependent manner. These findings reveal tumor microenvironment-dependent oncogenic and tumor-suppressive functions of MEN1 and provide a rationale for targeting MEN1 in solid cancers.