In vivo CRISPR screens reveal the landscape of immune evasion pathways across cancer.

The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1b/HLA-E. Surprisingly, loss of tumor interferon-γ (IFNγ) signaling sensitizes many models to immunity. The immune inhibitory effects of tumor IFN sensing are mediated through two mechanisms. First, tumor upregulation of classical MHC class I inhibits natural killer cells. Second, IFN-induced expression of Qa-1b inhibits CD8+ T cells via the NKG2A/CD94 receptor, which is induced by ICB. Finally, we show that strong IFN signatures are associated with poor response to ICB in individuals with renal cell carcinoma or melanoma. This study reveals that IFN-mediated upregulation of classical and non-classical MHC class I inhibitory checkpoints can facilitate immune escape.

A Compendium of Genetic Modifiers of Mitochondrial Dysfunction Reveals Intra-organelle Buffering.

Mitochondrial dysfunction is associated with a spectrum of human conditions, ranging from rare, inborn errors of metabolism to the aging process. To identify pathways that modify mitochondrial dysfunction, we performed genome-wide CRISPR screens in the presence of small-molecule mitochondrial inhibitors. We report a compendium of chemical-genetic interactions involving 191 distinct genetic modifiers, including 38 that are synthetic sick/lethal and 63 that are suppressors. Genes involved in glycolysis (PFKP), pentose phosphate pathway (G6PD), and defense against lipid peroxidation (GPX4) scored high as synthetic sick/lethal. A surprisingly large fraction of suppressors are pathway intrinsic and encode mitochondrial proteins. A striking example of such "intra-organelle" buffering is the alleviation of a chemical defect in complex V by simultaneous inhibition of complex I, which benefits cells by rebalancing redox cofactors, increasing reductive carboxylation, and promoting glycolysis. Perhaps paradoxically, certain forms of mitochondrial dysfunction may best be buffered with "second site" inhibitors to the organelle.

Defining a Cancer Dependency Map.

Most human epithelial tumors harbor numerous alterations, making it difficult to predict which genes are required for tumor survival. To systematically identify cancer dependencies, we analyzed 501 genome-scale loss-of-function screens performed in diverse human cancer cell lines. We developed DEMETER, an analytical framework that segregates on- from off-target effects of RNAi. 769 genes were differentially required in subsets of these cell lines at a threshold of six SDs from the mean. We found predictive models for 426 dependencies (55%) by nonlinear regression modeling considering 66,646 molecular features. Many dependencies fall into a limited number of classes, and unexpectedly, in 82% of models, the top biomarkers were expression based. We demonstrated the basis behind one such predictive model linking hypermethylation of the UBB ubiquitin gene to a dependency on UBC. Together, these observations provide a foundation for a cancer dependency map that facilitates the prioritization of therapeutic targets.

Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma.

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC-family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets.

NSD1 mediates antagonism between SWI/SNF and polycomb complexes and is required for transcriptional activation upon EZH2 inhibition.

Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.

KRAS and YAP1 converge to regulate EMT and tumor survival.

Cancer cells that express oncogenic alleles of RAS typically require sustained expression of the mutant allele for survival, but the molecular basis of this oncogene dependency remains incompletely understood. To identify genes that can functionally substitute for oncogenic RAS, we systematically expressed 15,294 open reading frames in a human KRAS-dependent colon cancer cell line engineered to express an inducible KRAS-specific shRNA. We found 147 genes that promoted survival upon KRAS suppression. In particular, the transcriptional coactivator YAP1 rescued cell viability in KRAS-dependent cells upon suppression of KRAS and was required for KRAS-induced cell transformation. Acquired resistance to Kras suppression in a Kras-driven murine lung cancer model also involved increased YAP1 signaling. KRAS and YAP1 converge on the transcription factor FOS and activate a transcriptional program involved in regulating the epithelial-mesenchymal transition (EMT). Together, these findings implicate transcriptional regulation of EMT by YAP1 as a significant component of oncogenic RAS signaling.

Structure-function analysis of the SHOC2-MRAS-PP1C holophosphatase complex.

Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling1. SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes2-5. However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development .

Three million images and morphological profiles of cells treated with matched chemical and genetic perturbations.

The identification of genetic and chemical perturbations with similar impacts on cell morphology can elucidate compounds' mechanisms of action or novel regulators of genetic pathways. Research on methods for identifying such similarities has lagged due to a lack of carefully designed and well-annotated image sets of cells treated with chemical and genetic perturbations. Here we create such a Resource dataset, CPJUMP1, in which each perturbed gene's product is a known target of at least two chemical compounds in the dataset. We systematically explore the directionality of correlations among perturbations that target the same protein encoded by a given gene, and we find that identifying matches between chemical and genetic perturbations is a challenging task. Our dataset and baseline analyses provide a benchmark for evaluating methods that measure perturbation similarities and impact, and more generally, learn effective representations of cellular state from microscopy images. Such advancements would accelerate the applications of image-based profiling of cellular states, such as uncovering drug mode of action or probing functional genomics.

The receptor TREML4 amplifies TLR7-mediated signaling during antiviral responses and autoimmunity.

The molecules and pathways that fine-tune innate inflammatory responses mediated by Toll-like receptor 7 (TLR7) remain to be fully elucidated. Using an unbiased genome-scale screen with short hairpin RNA (shRNA), we identified the receptor TREML4 as an essential positive regulator of TLR7 signaling. Macrophages from Treml4(-/-) mice were hyporesponsive to TLR7 agonists and failed to produce type I interferons due to impaired phosphorylation of the transcription factor STAT1 by the mitogen-activated protein kinase p38 and decreased recruitment of the adaptor MyD88 to TLR7. TREML4 deficiency reduced the production of inflammatory cytokines and autoantibodies in MRL/lpr mice, which are prone to systemic lupus erythematosus (SLE), and inhibited the antiviral immune response to influenza virus. Our data identify TREML4 as a positive regulator of TLR7 signaling and provide insight into the molecular mechanisms that control antiviral immunity and the development of autoimmunity.

ß-Catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis.

Wnt/ß-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic ß-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of ß-catenin in transformation, we classified ß-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that ß-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with ß-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of ß-catenin-dependent cancers in both cell lines and animal models. These observations define a ß-catenin-YAP1-TBX5 complex essential to the transformation and survival of ß-catenin-driven cancers.

Cancer vulnerabilities unveiled by genomic loss.

Due to genome instability, most cancers exhibit loss of regions containing tumor suppressor genes and collateral loss of other genes. To identify cancer-specific vulnerabilities that are the result of copy number losses, we performed integrated analyses of genome-wide copy number and RNAi profiles and identified 56 genes for which gene suppression specifically inhibited the proliferation of cells harboring partial copy number loss of that gene. These CYCLOPS (copy number alterations yielding cancer liabilities owing to partial loss) genes are enriched for spliceosome, proteasome, and ribosome components. One CYCLOPS gene, PSMC2, encodes an essential member of the 19S proteasome. Normal cells express excess PSMC2, which resides in a complex with PSMC1, PSMD2, and PSMD5 and acts as a reservoir protecting cells from PSMC2 suppression. Cells harboring partial PSMC2 copy number loss lack this complex and die after PSMC2 suppression. These observations define a distinct class of cancer-specific liabilities resulting from genome instability.

Identification of regulators of polyploidization presents therapeutic targets for treatment of AMKL.

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.

Total loss of VHL gene function impairs neuroendocrine cancer cell fitness due to excessive HIF2α activity.

Loss-of-function germline von Hippel-Lindau (VHL) tumor suppressor mutations cause VHL disease, which predisposes individuals to kidney cancer, hemangioblastomas, and paragangliomas. The risk that a given VHL disease family will manifest some or all these tumor types is profoundly influenced by the VHL allele it carries. For example, almost all VHL disease families that develop paraganglioma have missense VHL mutations. VHL families with null VHL alleles develop kidney cancer and hemangioblastomas without a high risk of paraganglioma. The latter is surprising because the VHL gene product, pVHL, suppresses the HIF2 transcription factor and gain-of-function HIF2 mutations are also linked to paraganglioma. Paragangliomas arise from the sympathetic or parasympathetic nervous system. Given the lack of human paraganglioma cell lines, we studied the effects of inactivating VHL in neuroblastoma cell lines, which also arise from the sympathetic nervous system. We found that total loss of pVHL function profoundly impairs the fitness of neuroblastoma cell lines in a HIF2-dependent manner both ex vivo and in vivo. This fitness defect can be rescued by pVHL variants linked to paraganglioma, but not by pVHL variants associated with a low risk of paraganglioma. These findings suggest that HIF2 activity above a critical threshold prevents the development of paraganglioma.

Systematic discovery of TLR signaling components delineates viral-sensing circuits.

Deciphering the signaling networks that underlie normal and disease processes remains a major challenge. Here, we report the discovery of signaling components involved in the Toll-like receptor (TLR) response of immune dendritic cells (DCs), including a previously unkown pathway shared across mammalian antiviral responses. By combining transcriptional profiling, genetic and small-molecule perturbations, and phosphoproteomics, we uncover 35 signaling regulators, including 16 known regulators, involved in TLR signaling. In particular, we find that Polo-like kinases (Plk) 2 and 4 are essential components of antiviral pathways in vitro and in vivo and activate a signaling branch involving a dozen proteins, among which is Tnfaip2, a gene associated with autoimmune diseases but whose role was unknown. Our study illustrates the power of combining systematic measurements and perturbations to elucidate complex signaling circuits and discover potential therapeutic targets.

Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5.

Activation-induced cytidine deaminase (AID) initiates antibody gene diversification by creating U:G mismatches. However, AID is not specific for antibody genes; Off-target lesions can activate oncogenes or cause chromosome translocations. Despite its importance in these transactions little is known about how AID finds its targets. We performed an shRNA screen to identify factors required for class switch recombination (CSR) of antibody loci. We found that Spt5, a factor associated with stalled RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for CSR. Spt5 interacts with AID, it facilitates association between AID and Pol II, and AID recruitment to its Ig and non-Ig targets. ChIP-seq experiments reveal that Spt5 colocalizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II stalling is predictive of AID-induced mutation. We propose that AID is targeted to sites of Pol II stalling in part via its association with Spt5.

WRN helicase is a synthetic lethal target in microsatellite unstable cancers.

Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins2. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach3. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.

Acquired Resistance to KRASG12C Inhibition in Cancer.

BACKGROUND: Clinical trials of the KRAS inhibitors adagrasib and sotorasib have shown promising activity in cancers harboring KRAS glycine-to-cysteine amino acid substitutions at codon 12 (KRASG12C). The mechanisms of acquired resistance to these therapies are currently unknown. METHODS: Among patients with KRASG12C -mutant cancers treated with adagrasib monotherapy, we performed genomic and histologic analyses that compared pretreatment samples with those obtained after the development of resistance. Cell-based experiments were conducted to study mutations that confer resistance to KRASG12C inhibitors. RESULTS: A total of 38 patients were included in this study: 27 with non-small-cell lung cancer, 10 with colorectal cancer, and 1 with appendiceal cancer. Putative mechanisms of resistance to adagrasib were detected in 17 patients (45% of the cohort), of whom 7 (18% of the cohort) had multiple coincident mechanisms. Acquired KRAS alterations included G12D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, and high-level amplification of the KRASG12C allele. Acquired bypass mechanisms of resistance included MET amplification; activating mutations in NRAS, BRAF, MAP2K1, and RET; oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3; and loss-of-function mutations in NF1 and PTEN. In two of nine patients with lung adenocarcinoma for whom paired tissue-biopsy samples were available, histologic transformation to squamous-cell carcinoma was observed without identification of any other resistance mechanisms. Using an in vitro deep mutational scanning screen, we systematically defined the landscape of KRAS mutations that confer resistance to KRASG12C inhibitors. CONCLUSIONS: Diverse genomic and histologic mechanisms impart resistance to covalent KRASG12C inhibitors, and new therapeutic strategies are required to delay and overcome this drug resistance in patients with cancer. (Funded by Mirati Therapeutics and others; ClinicalTrials.gov number, NCT03785249.).

Systematic identification of biomarker-driven drug combinations to overcome resistance.

The ability to understand and predict variable responses to therapeutic agents may improve outcomes in patients with cancer. We hypothesized that the basal gene-transcription state of cancer cell lines, coupled with cell viability profiles of small molecules, might be leveraged to nominate specific mechanisms of intrinsic resistance and to predict drug combinations that overcome resistance. We analyzed 564,424 sensitivity profiles to identify candidate gene-compound pairs, and validated nine such relationships. We determined the mechanism of a novel relationship, in which expression of the serine hydrolase enzymes monoacylglycerol lipase (MGLL) or carboxylesterase 1 (CES1) confers resistance to the histone lysine demethylase inhibitor GSK-J4 by direct enzymatic modification. Insensitive cell lines could be sensitized to GSK-J4 by inhibition or gene knockout. These analytical and mechanistic studies highlight the potential of integrating gene-expression features with small-molecule response to identify patient populations that are likely to benefit from treatment, to nominate rational candidates for combinations and to provide insights into mechanisms of action.

A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection.

During the course of a viral infection, viral proteins interact with an array of host proteins and pathways. Here, we present a systematic strategy to elucidate the dynamic interactions between H1N1 influenza and its human host. A combination of yeast two-hybrid analysis and genome-wide expression profiling implicated hundreds of human factors in mediating viral-host interactions. These factors were then examined functionally through depletion analyses in primary lung cells. The resulting data point to potential roles for some unanticipated host and viral proteins in viral infection and the host response, including a network of RNA-binding proteins, components of WNT signaling, and viral polymerase subunits. This multilayered approach provides a comprehensive and unbiased physical and regulatory model of influenza-host interactions and demonstrates a general strategy for uncovering complex host-pathogen relationships.

Synthetic lethal interaction between oncogenic KRAS dependency and STK33 suppression in human cancer cells.

An alternative to therapeutic targeting of oncogenes is to perform "synthetic lethality" screens for genes that are essential only in the context of specific cancer-causing mutations. We used high-throughput RNA interference (RNAi) to identify synthetic lethal interactions in cancer cells harboring mutant KRAS, the most commonly mutated human oncogene. We find that cells that are dependent on mutant KRAS exhibit sensitivity to suppression of the serine/threonine kinase STK33 irrespective of tissue origin, whereas STK33 is not required by KRAS-independent cells. STK33 promotes cancer cell viability in a kinase activity-dependent manner by regulating the suppression of mitochondrial apoptosis mediated through S6K1-induced inactivation of the death agonist BAD selectively in mutant KRAS-dependent cells. These observations identify STK33 as a target for treatment of mutant KRAS-driven cancers and demonstrate the potential of RNAi screens for discovering functional dependencies created by oncogenic mutations that may enable therapeutic intervention for cancers with "undruggable" genetic alterations.