The context-dependent epigenetic and organogenesis programs determine 3D vs. 2D cellular fitness of MYC-driven cancer.

3D cellular-specific epigenetic and transcriptomic reprogramming is critical to organogenesis and tumorigenesis. Here we dissect the distinct cell fitness in 2D (normoxia vs. chronic hypoxia) vs 3D (normoxia) culture conditions. We identify over 600 shared essential genes and additional context-specific fitness genes and pathways. Knockout of the VHL-HIF1 pathway results in incompatible fitness defects under normoxia vs. 1% oxygen or 3D culture conditions. Moreover, deletion of each of the mitochondrial respiratory electron transport chain complex has distinct fitness outcomes. Notably, multicellular organogenesis signaling pathways including TGFß-SMAD specifically constrict the uncontrolled cell proliferation in 3D while inactivation of epigenetic modifiers (Bcor, Kmt2d, Mettl3 and Mettl14) has opposite outcomes in 2D vs. 3D. We further identify a 3D-dependent synthetic lethality with partial loss of Prmt5 due to a reduction of Mtap expression resulting from 3D-specific epigenetic reprogramming. Our study highlights unique epigenetic, metabolic and organogenesis signaling dependencies under different cellular settings.

Generation of iPSC lines and isogenic gene-corrected lines from two individuals with RPS19-mutated Diamond-Blackfan anemia syndrome.

Diamond-Blackfan anemia syndrome (DBAS) is an inherited bone marrow failure disorder that typically presents in infancy as hypoplastic anemia and developmental abnormalities in approximately 50% of cases. DBAS is caused by haploinsufficiency in one of 24 ribosomal protein genes, with RPS19 mutations accounting for 25% of cases. We generated iPSC lines from two patients with different heterozygous RPS19 mutations (c.191T > C and c.184C > T) and isogenic lines in which the mutations were corrected by Cas9-mediated homology directed repair.

Melanoma antigens in pediatric medulloblastoma contribute to tumor heterogeneity and species-specificity of group 3 tumors.

Background: Medulloblastoma (MB) is the most malignant childhood brain cancer. Group 3 MB subtype accounts for about 25% of MB diagnoses and is associated with the most unfavorable outcomes. Herein, we report that more than half of group 3 MB tumors express melanoma antigens (MAGEs), which are potential prognostic and therapeutic markers. MAGEs are tumor antigens, expressed in several types of adult cancers and associated with poorer prognosis and therapy resistance; however, their expression in pediatric cancers is mostly unknown. The aim of this study was to determine whether MAGEs are activated in pediatric MB. Methods: To determine MAGE frequency in pediatric MB, we obtained formalin-fixed paraffin-embedded tissue (FFPE) samples of 34 patients, collected between 2008 - 2015, from the Children's Medical Center Dallas pathology archives and applied our validated reverse transcription quantitative PCR (RT-qPCR) assay to measure the relative expression of 23 MAGE cancer-testis antigen genes. To validate our data, we analyzed several published datasets from pediatric MB patients and patient-derived orthotopic xenografts, totaling 860 patients. We then examined how MAGE expression affects the growth and oncogenic potential of medulloblastoma cells by CRISPR-Cas9- and siRNA-mediated gene depletion. Results: Our RT-qPCR analysis suggested that MAGEs were expressed in group 3/4 medulloblastoma. Further mining of bulk and single-cell RNA-sequencing datasets confirmed that 50-75% of group 3 tumors activate a subset of MAGE genes. Depletion of MAGEAs, B2, and Cs alter MB cell survival, viability, and clonogenic growth due to decreased proliferation and increased apoptosis. Conclusions: These results indicate that targeting MAGEs in medulloblastoma may be a potential therapeutic option for group 3 medulloblastomas. Key Points: Several Type I MAGE CTAs are expressed in >60% of group 3 MBs. Type I MAGEs affect MB cell proliferation and apoptosis. MAGEs are potential biomarkers and therapeutic targets for group 3 MBs. Importance of the Study: This study is the first comprehensive analysis of all Type I MAGE CTAs ( MAGEA , -B , and -C subfamily members) in pediatric MBs. Our results show that more than 60% of group 3 MBs express MAGE genes, which are required for the viability and growth of cells in which they are expressed. Collectively, these data provide novel insights into the antigen landscape of pediatric MBs. The activation of MAGE genes in group 3 MBs presents potential stratifying and therapeutic options.

Bone morphogenetic protein (BMP) signaling determines neuroblastoma cell fate and sensitivity to retinoic acid.

Retinoic acid (RA) is a standard-of-care neuroblastoma drug thought to be effective by inducing differentiation. Curiously, RA has little effect on primary human tumors during upfront treatment but can eliminate neuroblastoma cells from the bone marrow during post-chemo consolidation therapy-a discrepancy that has never been explained. To investigate this, we treated a large cohort of neuroblastoma cell lines with RA and observed that the most RA-sensitive cells predominantly undergo apoptosis or senescence, rather than differentiation. We conducted genome-wide CRISPR knockout screens under RA treatment, which identified BMP signaling as controlling the apoptosis/senescence vs differentiation cell fate decision and determining RA's overall potency. We then discovered that BMP signaling activity is markedly higher in neuroblastoma patient samples at bone marrow metastatic sites, providing a plausible explanation for RA's ability to clear neuroblastoma cells specifically from the bone marrow, seemingly mimicking interactions between BMP and RA during normal development.

The protein phosphatase PP6 promotes RIPK1-dependent PANoptosis.

BACKGROUND: The innate immune system serves as the first line of host defense. Transforming growth factor-ß-activated kinase 1 (TAK1) is a key regulator of innate immunity, cell survival, and cellular homeostasis. Because of its importance in immunity, several pathogens have evolved to carry TAK1 inhibitors. In response, hosts have evolved to sense TAK1 inhibition and induce robust lytic cell death, PANoptosis, mediated by the RIPK1-PANoptosome. PANoptosis is a unique innate immune inflammatory lytic cell death pathway initiated by an innate immune sensor and driven by caspases and RIPKs. While PANoptosis can be beneficial to clear pathogens, excess activation is linked to pathology. Therefore, understanding the molecular mechanisms regulating TAK1 inhibitor (TAK1i)-induced PANoptosis is central to our understanding of RIPK1 in health and disease. RESULTS: In this study, by analyzing results from a cell death-based CRISPR screen, we identified protein phosphatase 6 (PP6) holoenzyme components as regulators of TAK1i-induced PANoptosis. Loss of the PP6 enzymatic component, PPP6C, significantly reduced TAK1i-induced PANoptosis. Additionally, the PP6 regulatory subunits PPP6R1, PPP6R2, and PPP6R3 had redundant roles in regulating TAK1i-induced PANoptosis, and their combined depletion was required to block TAK1i-induced cell death. Mechanistically, PPP6C and its regulatory subunits promoted the pro-death S166 auto-phosphorylation of RIPK1 and led to a reduction in the pro-survival S321 phosphorylation. CONCLUSIONS: Overall, our findings demonstrate a key requirement for the phosphatase PP6 complex in the activation of TAK1i-induced, RIPK1-dependent PANoptosis, suggesting this complex could be therapeutically targeted in inflammatory conditions.

RBM39 degrader invigorates natural killer cells to eradicate neuroblastoma despite cancer cell plasticity.

The cellular plasticity of neuroblastoma is defined by a mixture of two major cell states, adrenergic (ADRN) and mesenchymal (MES), which may contribute to therapy resistance. However, how neuroblastoma cells switch cellular states during therapy remains largely unknown and how to eradicate neuroblastoma regardless of their cell states is a clinical challenge. To better understand the lineage switch of neuroblastoma in chemoresistance, we comprehensively defined the transcriptomic and epigenetic map of ADRN and MES types of neuroblastomas using human and murine models treated with indisulam, a selective RBM39 degrader. We showed that cancer cells not only undergo a bidirectional switch between ADRN and MES states, but also acquire additional cellular states, reminiscent of the developmental pliancy of neural crest cells. The lineage alterations are coupled with epigenetic reprogramming and dependency switch of lineage-specific transcription factors, epigenetic modifiers and targetable kinases. Through targeting RNA splicing, indisulam induces an inflammatory tumor microenvironment and enhances anticancer activity of natural killer cells. The combination of indisulam with anti-GD2 immunotherapy results in a durable, complete response in high-risk transgenic neuroblastoma models, providing an innovative, rational therapeutic approach to eradicate tumor cells regardless of their potential to switch cell states.

Engineering human pluripotent stem cell lines to evade xenogeneic transplantation barriers.

Successful allogeneic human pluripotent stem cell (hPSC)-derived therapies must overcome immunological rejection by the recipient. To build reagents to define these barriers, we genetically ablated ß2M, TAP1, CIITA, CD74, MICA, and MICB to limit expression of HLA-I, HLA-II, and natural killer (NK) cell activating ligands in hPSCs. Transplantation of these cells that also expressed covalent single chain trimers of Qa1 and H2-Kb to inhibit NK cells and CD55, Crry, and CD59 to inhibit complement deposition led to persistent teratomas in wild-type mice. Transplantation of HLA-deficient hPSCs into mice genetically deficient in complement and depleted of NK cells also led to persistent teratomas. Thus, T cell, NK cell, and complement evasion are necessary to prevent immunological rejection of hPSCs and their progeny. These cells and versions expressing human orthologs of immune evasion factors can be used to define cell type-specific immune barriers and conduct preclinical testing in immunocompetent mouse models.

Single-cell CRISPR screens in vivo map T cell fate regulomes in cancer.

CD8+ cytotoxic T cells (CTLs) orchestrate antitumour immunity and exhibit inherent heterogeneity1,2, with precursor exhausted T (Tpex) cells but not terminally exhausted T (Tex) cells capable of responding to existing immunotherapies3-7. The gene regulatory network that underlies CTL differentiation and whether Tex cell responses can be functionally reinvigorated are incompletely understood. Here we systematically mapped causal gene regulatory networks using single-cell CRISPR screens in vivo and discovered checkpoints for CTL differentiation. First, the exit from quiescence of Tpex cells initiated successive differentiation into intermediate Tex cells. This process is differentially regulated by IKAROS and ETS1, the deficiencies of which dampened and increased mTORC1-associated metabolic activities, respectively. IKAROS-deficient cells accumulated as a metabolically quiescent Tpex cell population with limited differentiation potential following immune checkpoint blockade (ICB). Conversely, targeting ETS1 improved antitumour immunity and ICB efficacy by boosting differentiation of Tpex to intermediate Tex cells and metabolic rewiring. Mechanistically, TCF-1 and BATF are the targets for IKAROS and ETS1, respectively. Second, the RBPJ-IRF1 axis promoted differentiation of intermediate Tex to terminal Tex cells. Accordingly, targeting RBPJ enhanced functional and epigenetic reprogramming of Tex cells towards the proliferative state and improved therapeutic effects and ICB efficacy. Collectively, our study reveals that promoting the exit from quiescence of Tpex cells and enriching the proliferative Tex cell state act as key modalities for antitumour effects and provides a systemic framework to integrate cell fate regulomes and reprogrammable functional determinants for cancer immunity.

Inflammatory cell death, PANoptosis, screen identifies host factors in coronavirus innate immune response as therapeutic targets.

The COVID-19 pandemic, caused by the ß-coronavirus (ß-CoV) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to cause significant global morbidity and mortality. While vaccines have reduced the overall number of severe infections, there remains an incomplete understanding of viral entry and innate immune activation, which can drive pathology. Innate immune responses characterized by positive feedback between cell death and cytokine release can amplify the inflammatory cytokine storm during ß-CoV-mediated infection to drive pathology. Therefore, there remains an unmet need to understand innate immune processes in response to ß-CoV infections to identify therapeutic strategies. To address this gap, here we used an MHV model and developed a whole genome CRISPR-Cas9 screening approach to elucidate host molecules required for ß-CoV infection and inflammatory cell death, PANoptosis, in macrophages, a sentinel innate immune cell. Our screen was validated through the identification of the known MHV receptor Ceacam1 as the top hit, and its deletion significantly reduced viral replication due to loss of viral entry, resulting in a downstream reduction in MHV-induced cell death. Moreover, this screen identified several other host factors required for MHV infection-induced macrophage cell death. Overall, these findings demonstrate the feasibility and power of using genome-wide PANoptosis screens in macrophage cell lines to accelerate the discovery of key host factors in innate immune processes and suggest new targets for therapeutic development to prevent ß-CoV-induced pathology.

Short tandem repeat profiling via next-generation sequencing for cell line authentication.

Cell lines are indispensable models for modern biomedical research. A large part of their usefulness derives from the ability of a cell line to proliferate over multiple passages (often indefinitely), allowing multiple experiments to be performed. However, over time, cell line identity and purity can be compromised by human errors. Cross-contamination from other cell lines and complete misidentification are both possible. Routine cell line authentication is a necessary preventive measure and has become a requirement for many funding applications and publications. Short tandem repeat (STR) profiling is the most common method for cell line authentication and is usually carried out using standard polymerase chain reaction-capillary electrophoresis analysis (STR-CE). Here, we evaluated next-generation sequencing (NGS)-based STR profiling of human and mouse cell lines at 18 and 15 loci, respectively, in a high-throughput format. Using the Python program STRight, we demonstrate that NGS-based analysis (STR-NGS) is superior to standard STR-CE in terms of the ability to report the sequence context of repeat motifs, sensitivity and flexible multiplexing capability. STR-NGS is thus a valuable alternative for cell line authentication.

Engineering Human Pluripotent Stem Cell Lines to Evade Xenogeneic Transplantation Barriers.

Allogeneic human pluripotent stem cell (hPSC)-derived cells and tissues for therapeutic transplantation must necessarily overcome immunological rejection by the recipient. To define these barriers and to create cells capable of evading rejection for preclinical testing in immunocompetent mouse models, we genetically ablated ß2m, Tap1, Ciita, Cd74, Mica, and Micb to limit expression of HLA-I, HLA-II, and natural killer cell activating ligands in hPSCs. Though these and even unedited hPSCs readily formed teratomas in cord blood-humanized immunodeficient mice, grafts were rapidly rejected by immunocompetent wild-type mice. Transplantation of these cells that also expressed covalent single chain trimers of Qa1 and H2-Kb to inhibit natural killer cells and CD55, Crry, and CD59 to inhibit complement deposition led to persistent teratomas in wild-type mice. Expression of additional inhibitory factors such as CD24, CD47, and/or PD-L1 had no discernible impact on teratoma growth or persistence. Transplantation of HLA-deficient hPSCs into mice genetically deficient in complement and depleted of natural killer cells also led to persistent teratomas. Thus, T cell, NK cell, and complement evasion are necessary to prevent immunological rejection of hPSCs and their progeny. These cells and versions expressing human orthologs of immune evasion factors can be used to refine tissue- and cell type-specific immune barriers, and to conduct preclinical testing in immunocompetent mouse models.

Stromal-induced epithelial-mesenchymal transition induces targetable drug resistance in acute lymphoblastic leukemia.

The bone marrow microenvironment (BME) drives drug resistance in acute lymphoblastic leukemia (ALL) through leukemic cell interactions with bone marrow (BM) niches, but the underlying mechanisms remain unclear. Here, we show that the interaction between ALL and mesenchymal stem cells (MSCs) through integrin ß1 induces an epithelial-mesenchymal transition (EMT)-like program in MSC-adherent ALL cells, resulting in drug resistance and enhanced survival. Moreover, single-cell RNA sequencing analysis of ALL-MSC co-culture identifies a hybrid cluster of MSC-adherent ALL cells expressing both B-ALL and MSC signature genes, orchestrated by a WNT/ß-catenin-mediated EMT-like program. Blockade of interaction between ß-catenin and CREB binding protein impairs the survival and drug resistance of MSC-adherent ALL cells in vitro and results in a reduction in leukemic burden in vivo. Targeting of this WNT/ß-catenin-mediated EMT-like program is a potential therapeutic approach to overcome cell extrinsically acquired drug resistance in ALL.

Genome-wide mapping of cancer dependency genes and genetic modifiers of chemotherapy in high-risk hepatoblastoma.

A lack of relevant genetic models and cell lines hampers our understanding of hepatoblastoma pathogenesis and the development of new therapies for this neoplasm. Here, we report an improved MYC-driven hepatoblastoma-like murine model that recapitulates the pathological features of embryonal type of hepatoblastoma, with transcriptomics resembling the high-risk gene signatures of the human disease. Single-cell RNA-sequencing and spatial transcriptomics identify distinct subpopulations of hepatoblastoma cells. After deriving cell lines from the mouse model, we map cancer dependency genes using CRISPR-Cas9 screening and identify druggable targets shared with human hepatoblastoma (e.g., CDK7, CDK9, PRMT1, PRMT5). Our screen also reveals oncogenes and tumor suppressor genes in hepatoblastoma that engage multiple, druggable cancer signaling pathways. Chemotherapy is critical for human hepatoblastoma treatment. A genetic mapping of doxorubicin response by CRISPR-Cas9 screening identifies modifiers whose loss-of-function synergizes with (e.g., PRKDC) or antagonizes (e.g., apoptosis genes) the effect of chemotherapy. The combination of PRKDC inhibition and doxorubicin-based chemotherapy greatly enhances therapeutic efficacy. These studies provide a set of resources including disease models suitable for identifying and validating potential therapeutic targets in human high-risk hepatoblastoma.

Whole-genome CRISPR screen identifies RAVER1 as a key regulator of RIPK1-mediated inflammatory cell death, PANoptosis.

Transforming growth factor-ß-activated kinase 1 (TAK1) is a central regulator of innate immunity, cell death, inflammation, and cellular homeostasis. Therefore, many pathogens carry TAK1 inhibitors (TAK1i). As a host strategy to counteract this, inhibition or deletion of TAK1 induces spontaneous inflammatory cell death, PANoptosis, through the RIPK1-PANoptosome complex, containing the NLRP3 inflammasome and caspase-8/FADD/RIPK3 as integral components; however, PANoptosis also promotes pathological inflammation. Therefore, understanding molecular mechanisms that regulate TAK1i-induced cell death is essential. Here, we report a genome-wide CRISPR screen in macrophages that identified TAK1i-induced cell death regulators, including polypyrimidine tract-binding (PTB) protein 1 (PTBP1), a known regulator of RIPK1, and a previously unknown regulator RAVER1. RAVER1 blocked alternative splicing of Ripk1, and its genetic depletion inhibited TAK1i-induced, RIPK1-mediated inflammasome activation and PANoptosis. Overall, our CRISPR screen identified several positive regulators of PANoptosis. Moreover, our study highlights the utility of genome-wide CRISPR-Cas9 screens in myeloid cells for comprehensive characterization of complex cell death pathways to discover therapeutic targets.

High-Throughput Analysis of CRISPR-Cas9 Editing Outcomes in Cell and Animal Models Using CRIS.py.

Genome editing using the CRISPR-Cas9 platform creates precise modifications in cells and whole organisms. Although knockout (KO) mutations can occur at high frequencies, determining the editing rates in a pool of cells or selecting clones that contain only KO alleles can be a challenge. User-defined knock-in (KI) modifications are achieved at much lower rates, making the identification of correctly modified clones even more challenging. The high-throughput format of targeted next-generation sequencing (NGS) provides a platform allowing sequence information to be gathered from a one to thousands of samples. However, it also poses a challenge in terms of analyzing the large amount of data that is generated. In this chapter, we present and discuss CRIS.py, a simple and highly versatile Python-based program for analyzing NGS data for genome-editing outcomes. CRIS.py can be used to analyze sequencing results for any kind of modification or multiplex modifications specified by the user. Moreover, CRIS.py runs on all fastq files found in a directory, thereby concurrently analyzing all uniquely indexed samples. CRIS.py results are consolidated into two summary files, which allows users to sort and filter results and quickly identify the clones (or animals) of greatest interest.

cBAF complex components and MYC cooperate early in CD8+ T cell fate.

The identification of mechanisms to promote memory T (Tmem) cells has important implications for vaccination and anti-cancer immunotherapy1-4. Using a CRISPR-based screen for negative regulators of Tmem cell generation in vivo5, here we identify multiple components of the mammalian canonical BRG1/BRM-associated factor (cBAF)6,7. Several components of the cBAF complex are essential for the differentiation of activated CD8+ T cells into T effector (Teff) cells, and their loss promotes Tmem cell formation in vivo. During the first division of activated CD8+ T cells, cBAF and MYC8 frequently co-assort asymmetrically to the two daughter cells. Daughter cells with high MYC and high cBAF display a cell fate trajectory towards Teff cells, whereas those with low MYC and low cBAF preferentially differentiate towards Tmem cells. The cBAF complex and MYC physically interact to establish the chromatin landscape in activated CD8+ T cells. Treatment of naive CD8+ T cells with a putative cBAF inhibitor during the first 48 h of activation, before the generation of chimeric antigen receptor T (CAR-T) cells, markedly improves efficacy in a mouse solid tumour model. Our results establish cBAF as a negative determinant of Tmem cell fate and suggest that manipulation of cBAF early in T cell differentiation can improve cancer immunotherapy.

ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), continues to cause substantial morbidity and mortality in the ongoing global pandemic. Understanding the fundamental mechanisms that govern innate immune and inflammatory responses during SARS-CoV-2 infection is critical for developing effective therapeutic strategies. Whereas interferon (IFN)-based therapies are generally expected to be beneficial during viral infection, clinical trials in COVID-19 have shown limited efficacy and potential detrimental effects of IFN treatment during SARS-CoV-2 infection. However, the underlying mechanisms responsible for this failure remain unknown. In this study, we found that IFN induced Z-DNA-binding protein 1 (ZBP1)-mediated inflammatory cell death, PANoptosis, in human and murine macrophages and in the lungs of mice infected with ß-coronaviruses, including SARS-CoV-2 and mouse hepatitis virus (MHV). In patients with COVID-19, expression of the innate immune sensor ZBP1 was increased in immune cells from those who succumbed to the disease compared with those who recovered, further suggesting a link between ZBP1 and pathology. In mice, IFN-ß treatment after ß-coronavirus infection increased lethality, and genetic deletion of Zbp1 or its Zα domain suppressed cell death and protected the mice from IFN-mediated lethality during ß-coronavirus infection. Overall, our results identify that ZBP1 induced during coronavirus infection limits the efficacy of IFN therapy by driving inflammatory cell death and lethality. Therefore, inhibiting ZBP1 activity may improve the efficacy of IFN therapy, paving the way for the development of new and critically needed therapeutics for COVID-19 as well as other infections and inflammatory conditions where IFN-mediated cell death and pathology occur.

CRISPR screens unveil signal hubs for nutrient licensing of T cell immunity.

Nutrients are emerging regulators of adaptive immunity1. Selective nutrients interplay with immunological signals to activate mechanistic target of rapamycin complex 1 (mTORC1), a key driver of cell metabolism2-4, but how these environmental signals are integrated for immune regulation remains unclear. Here we use genome-wide CRISPR screening combined with protein-protein interaction networks to identify regulatory modules that mediate immune receptor- and nutrient-dependent signalling to mTORC1 in mouse regulatory T (Treg) cells. SEC31A is identified to promote mTORC1 activation by interacting with the GATOR2 component SEC13 to protect it from SKP1-dependent proteasomal degradation. Accordingly, loss of SEC31A impairs T cell priming and Treg suppressive function in mice. In addition, the SWI/SNF complex restricts expression of the amino acid sensor CASTOR1, thereby enhancing mTORC1 activation. Moreover, we reveal that the CCDC101-associated SAGA complex is a potent inhibitor of mTORC1, which limits the expression of glucose and amino acid transporters and maintains T cell quiescence in vivo. Specific deletion of Ccdc101 in mouse Treg cells results in uncontrolled inflammation but improved antitumour immunity. Collectively, our results establish epigenetic and post-translational mechanisms that underpin how nutrient transporters, sensors and transducers interplay with immune signals for three-tiered regulation of mTORC1 activity and identify their pivotal roles in licensing T cell immunity and immune tolerance.