Small molecule induced STING degradation facilitated by the HECT ligase HERC4.

Stimulator of interferon genes (STING) is a central component of the cytosolic nucleic acids sensing pathway and as such master regulator of the type I interferon response. Due to its critical role in physiology and its' involvement in a variety of diseases, STING has been a focus for drug discovery. Targeted protein degradation (TPD) has emerged as a promising pharmacology for targeting previously considered undruggable proteins by hijacking the cellular ubiquitin proteasome system (UPS) with small molecules. Here, we identify AK59 as a STING degrader leveraging HERC4, a HECT-domain E3 ligase. Additionally, our data reveals that AK59 is effective on the common pathological STING mutations, suggesting a potential clinical application of this mechanism. Thus, these findings introduce HERC4 to the fields of TPD and of compound-induced degradation of STING, suggesting potential therapeutic applications.

Multiple Genes Core to ERAD, Macroautophagy and Lysosomal Degradation Pathways Participate in the Proteostasis Response in α1-Antitrypsin Deficiency.

BACKGROUND & AIMS: In the classic form of α1-antitrypsin deficiency (ATD), the misfolded α1-antitrypsin Z (ATZ) variant accumulates in the endoplasmic reticulum (ER) of liver cells. A gain-of-function proteotoxic mechanism is responsible for chronic liver disease in a subgroup of homozygotes. Proteostatic response pathways, including conventional endoplasmic reticulum-associated degradation and autophagy, have been proposed as the mechanisms that allow cellular adaptation and presumably protection from the liver disease phenotype. Recent studies have concluded that a distinct lysosomal pathway called endoplasmic reticulum-to-lysosome completely supplants the role of the conventional macroautophagy pathway in degradation of ATZ. Here, we used several state-of-the-art approaches to characterize the proteostatic responses more fully in cellular systems that model ATD. METHODS: We used clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing coupled to a cell selection step by fluorescence-activated cell sorter to perform screening for proteostasis genes that regulate ATZ accumulation and combined that with selective genome editing in 2 other model systems. RESULTS: Endoplasmic reticulum-associated degradation genes are key early regulators and multiple autophagy genes, from classic as well as from ER-to-lysosome and other newly described ER-phagy pathways, participate in degradation of ATZ in a manner that is temporally regulated and evolves as ATZ accumulation persists. Time-dependent changes in gene expression are accompanied by specific ultrastructural changes including dilation of the ER, formation of globular inclusions, budding of autophagic vesicles, and alterations in the overall shape and component parts of mitochondria. CONCLUSIONS: Macroautophagy is a critical component of the proteostasis response to cellular ATZ accumulation and it becomes more important over time as ATZ synthesis continues unabated. Multiple subtypes of macroautophagy and nonautophagic lysosomal degradative pathways are needed to respond to the high concentrations of misfolded protein that characterizes ATD and these pathways are attractive candidates for genetic variants that predispose to the hepatic phenotype.

Discovery of Ligands for TRIM58, a Novel Tissue-Selective E3 Ligase.

Redirecting E3 ligases to neo-substrates, leading to their proteasomal disassembly, known as targeted protein degradation (TPD), has emerged as a promising alternative to traditional, occupancy-driven pharmacology. Although the field has expanded tremendously over the past years, the choice of E3 ligases remains limited, with an almost exclusive focus on CRBN and VHL. Here, we report the discovery of novel ligands to the PRY-SPRY domain of TRIM58, a RING ligase that is specifically expressed in erythroid precursor cells. A DSF screen, followed by validation using additional biophysical methods, led to the identification of TRIM58 ligand TRIM-473. A basic SAR around the chemotype was established by utilizing a competitive binding assay employing a short FP peptide probe derived from an endogenous TRIM58 substrate. The X-ray co-crystal structure of TRIM58 in complex with TRIM-473 gave insights into the binding mode and potential exit vectors for bifunctional degrader design.

Expressed Barcoding Enables High-Resolution Tracking of the Evolution of Drug Tolerance.

For a majority of patients with non-small cell lung cancer with EGFR mutations, treatment with EGFR inhibitors (EGFRi) induces a clinical response. Despite this initial reduction in tumor size, residual disease persists that leads to disease relapse. Elucidating the preexisting biological differences between sensitive cells and surviving drug-tolerant persister cells and deciphering how drug-tolerant cells evolve in response to treatment could help identify strategies to improve the efficacy of EGFRi. In this study, we tracked the origins and clonal evolution of drug-tolerant cells at a high resolution by using an expressed barcoding system coupled with single-cell RNA sequencing. This platform enabled longitudinal profiling of gene expression and drug sensitivity in response to EGFRi across a large number of clones. Drug-tolerant cells had higher expression of key survival pathways such as YAP and EMT at baseline and could also differentially adapt their gene expression following EGFRi treatment compared with sensitive cells. In addition, drug combinations targeting common downstream components (MAPK) or orthogonal factors (chemotherapy) showed greater efficacy than EGFRi alone, which is attributable to broader targeting of the heterogeneous EGFRi-tolerance mechanisms present in tumors. Overall, this approach facilitates thorough examination of clonal evolution in response to therapy that could inform the development of improved diagnostic approaches and treatment strategies for targeting drug-tolerant cells. SIGNIFICANCE: The evolution and heterogeneity of EGFR inhibitor tolerance are identified in a large number of clones at enhanced cellular and temporal resolution using an expressed barcode technology coupled with single-cell RNA sequencing.

Convergence of multiple RNA-silencing pathways on GW182/TNRC6.

The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development, and cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Here, we uncover that TRIM71 represses its targets through RNA-supported interaction with TNRC6/GW182, a core component of the miRNA-induced silencing complex (miRISC). We demonstrate that AGO2, TRIM71, and UPF1 each recruit TNRC6 to specific sets of transcripts to silence them. As cellular TNRC6 levels are limiting, competition occurs among the silencing pathways, such that the loss of AGO proteins or of AGO binding to TNRC6 enhances the activities of the other pathways. We conclude that a miRNA-like silencing activity is shared among different mRNA silencing pathways and that the use of TNRC6 as a central hub provides a means to integrate their activities.

Cell adhesion molecule KIRREL1 is a feedback regulator of Hippo signaling recruiting SAV1 to cell-cell contact sites.

The Hippo/YAP pathway controls cell proliferation through sensing physical and spatial organization of cells. How cell-cell contact is sensed by Hippo signaling is poorly understood. Here, we identified the cell adhesion molecule KIRREL1 as an upstream positive regulator of the mammalian Hippo pathway. KIRREL1 physically interacts with SAV1 and recruits SAV1 to cell-cell contact sites. Consistent with the hypothesis that KIRREL1-mediated cell adhesion suppresses YAP activity, knockout of KIRREL1 increases YAP activity in neighboring cells. Analyzing pan-cancer CRISPR proliferation screen data reveals KIRREL1 as the top plasma membrane protein showing strong correlation with known Hippo regulators, highlighting a critical role of KIRREL1 in regulating Hippo signaling and cell proliferation. During liver regeneration in mice, KIRREL1 is upregulated, and its genetic ablation enhances hepatic YAP activity, hepatocyte reprogramming and biliary epithelial cell proliferation. Our data suggest that KIRREL1 functions as a feedback regulator of the mammalian Hippo pathway through sensing cell-cell interaction and recruiting SAV1 to cell-cell contact sites.

Genome-wide CRISPR screen identifies protein pathways modulating tau protein levels in neurons.

Aggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.

Identification of the HECT E3 ligase UBR5 as a regulator of MYC degradation using a CRISPR/Cas9 screen.

MYC oncoprotein is a multifunctional transcription factor that regulates the expression of a large number of genes involved in cellular growth, proliferation and metabolism. Altered MYC protein level lead to cellular transformation and tumorigenesis. MYC is deregulated in > 50% of human cancers, rendering it an attractive drug target. However, direct inhibition of this class of proteins using conventional small molecules is challenging due to their intrinsically disordered state. To discover novel posttranslational regulators of MYC protein stability and turnover, we established a genetic screen in mammalian cells by combining a fluorescent protein-based MYC abundance sensor, CRISPR/Cas9-based gene knockouts and next-generation sequencing. Our screen identifies UBR5, an E3 ligase of the HECT-type family, as a novel regulator of MYC degradation. Even in the presence of the well-described and functional MYC ligase, FBXW7, UBR5 depletion leads to accumulation of MYC in cells. We demonstrate interaction of UBR5 with MYC and reduced K48-linked ubiquitination of MYC upon loss of UBR5 in cells. Interestingly, in cancer cell lines with amplified MYC expression, depletion of UBR5 resulted in reduced cell survival, as a consequence of MYC stabilization. Finally, we show that MYC and UBR5 are co-amplified in more than 40% of cancer cells and that MYC copy number amplification correlates with enhanced transcriptional output of UBR5. This suggests that UBR5 acts as a buffer in MYC amplified settings and protects these cells from apoptosis.

An iron-dependent metabolic vulnerability underlies VPS34-dependence in RKO cancer cells.

VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.

Author Correction: CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.

Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC.

EGFR-mutant NSCLCs frequently respond to EGFR tyrosine kinase inhibitors (TKIs). However, the responses are not durable, and the magnitude of tumor regression is variable, suggesting the existence of genetic modifiers of EGFR dependency. Here, we applied a genome-wide CRISPR-Cas9 screening to identify genetic determinants of EGFR TKI sensitivity and uncovered putative candidates. We show that knockout of RIC8A, essential for G-alpha protein activation, enhanced EGFR TKI-induced cell death. Mechanistically, we demonstrate that RIC8A is a positive regulator of YAP signaling, activation of which rescued the EGFR TKI sensitizing phenotype resulting from RIC8A knockout. We also show that knockout of ARIH2, or other components in the Cullin-5 E3 complex, conferred resistance to EGFR inhibition, in part by promoting nascent protein synthesis through METAP2. Together, these data uncover a spectrum of previously unidentified regulators of EGFR TKI sensitivity in EGFR-mutant human NSCLC, providing insights into the heterogeneity of EGFR TKI treatment responses.

IRF2 is a master regulator of human keratinocyte stem cell fate.

Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. The stem cell potential of human epidermal keratinocytes is retained in vitro but lost over time suggesting extrinsic and intrinsic regulation. Transcription factor-controlled regulatory circuitries govern cell identity, are sufficient to induce pluripotency and transdifferentiate cells. We investigate whether transcriptional circuitry also governs phenotypic changes within a given cell type by comparing human primary keratinocytes with intrinsically high versus low stem cell potential. Using integrated chromatin and transcriptional profiling, we implicate IRF2 as antagonistic to stemness and show that it binds and regulates active cis-regulatory elements at interferon response and antigen presentation genes. CRISPR-KD of IRF2 in keratinocytes with low stem cell potential increases self-renewal, migration and epidermis formation. These data demonstrate that transcription factor regulatory circuitries, in addition to maintaining cell identity, control plasticity within cell types and offer potential for therapeutic modulation of cell function.

USP7 inhibits Wnt/ß-catenin signaling through promoting stabilization of Axin.

Axin is a key scaffolding protein responsible for the formation of the ß-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/ß-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/ß-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/ß-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/ß-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/ß-catenin signaling.

YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury.

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/ß-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/ß-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.

Genome-Scale CRISPR Screens Identify Human Pluripotency-Specific Genes.

Human pluripotent stem cells (hPSCs) generate a variety of disease-relevant cells that can be used to improve the translation of preclinical research. Despite the potential of hPSCs, their use for genetic screening has been limited by technical challenges. We developed a scalable and renewable Cas9 and sgRNA-hPSC library in which loss-of-function mutations can be induced at will. Our inducible mutant hPSC library can be used for multiple genome-wide CRISPR screens in a variety of hPSC-induced cell types. As proof of concept, we performed three screens for regulators of properties fundamental to hPSCs: their ability to self-renew and/or survive (fitness), their inability to survive as single-cell clones, and their capacity to differentiate. We identified the majority of known genes and pathways involved in these processes, as well as a plethora of genes with unidentified roles. This resource will increase the understanding of human development and genetics. This approach will be a powerful tool to identify disease-modifying genes and pathways.

Discovery of a ZIP7 inhibitor from a Notch pathway screen.

The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.

Bile acid analogues are activators of pyrin inflammasome.

Bile acids are critical metabolites in the gastrointestinal tract and contribute to maintaining intestinal immune homeostasis through cross-talk with the gut microbiota. The conversion of bile acids by the gut microbiome is now recognized as a factor affecting both host metabolism and immune responses, but its physiological roles remain unclear. We conducted a screen for microbiome metabolites that would function as inflammasome activators and herein report the identification of 12-oxo-lithocholic acid (BAA485), a potential microbiome-derived bile acid metabolite. We demonstrate that the more potent analogue 11-oxo-12S-hydroxylithocholic acid methyl ester (BAA473) can induce secretion of interleukin-18 (IL-18) through activation of the inflammasome in both myeloid and intestinal epithelial cells. Using a genome-wide CRISPR screen with compound induced pyroptosis in THP-1 cells, we identified that inflammasome activation by BAA473 is pyrin-dependent (MEFV). To our knowledge, the bile acid analogues BAA485 and BAA473 are the first small molecule activators of the pyrin inflammasome. We surmise that pyrin inflammasome activation through microbiota-modified bile acid metabolites such as BAA473 and BAA485 plays a role in gut microbiota regulated intestinal immune response. The discovery of these two bioactive compounds may help to further unveil the importance of pyrin in gut homeostasis and autoimmune diseases.

mTORC1 signaling suppresses Wnt/ß-catenin signaling through DVL-dependent regulation of Wnt receptor FZD level.

Wnt/ß-catenin signaling plays pivotal roles in cell proliferation and tissue homeostasis by maintaining somatic stem cell functions. The mammalian target of rapamycin (mTOR) signaling functions as an integrative rheostat that orchestrates various cellular and metabolic activities that shape tissue homeostasis. Whether these two fundamental signaling pathways couple to exert physiological functions still remains mysterious. Using a genome-wide CRISPR-Cas9 screening, we discover that mTOR complex 1 (mTORC1) signaling suppresses canonical Wnt/ß-catenin signaling. Deficiency in tuberous sclerosis complex 1/2 (TSC1/2), core negative regulators of mTORC1 activity, represses Wnt/ß-catenin target gene expression, which can be rescued by RAD001. Mechanistically, mTORC1 signaling regulates the cell surface level of Wnt receptor Frizzled (FZD) in a Dishevelled (DVL)-dependent manner by influencing the association of DVL and clathrin AP-2 adaptor. Sustained mTORC1 activation impairs Wnt/ß-catenin signaling and causes loss of stemness in intestinal organoids ex vivo and primitive intestinal progenitors in vivo. Wnt/ß-catenin-dependent liver metabolic zonation gene expression program is also down-regulated by mTORC1 activation. Our study provides a paradigm that mTORC1 signaling cell autonomously regulates Wnt/ß-catenin pathway to influence stem cell maintenance.

Identification of ICAT as an APC Inhibitor, Revealing Wnt-Dependent Inhibition of APC-Axin Interaction.

Adenomatous polyposis coli (APC) and Axin are core components of the ß-catenin destruction complex. How APC's function is regulated and whether Wnt signaling influences the direct APC-Axin interaction to inhibit the ß-catenin destruction complex is not clear. Through a CRISPR screen of ß-catenin stability, we have identified ICAT, a polypeptide previously known to block ß-catenin-TCF interaction, as a natural inhibitor of APC. ICAT blocks ß-catenin-APC interaction and prevents ß-catenin-mediated APC-Axin interaction, enhancing stabilization of ß-catenin in cells harboring truncated APC or stimulated with Wnt, but not in cells deprived of a Wnt signal. Using ICAT as a tool to disengage ß-catenin-mediated APC-Axin interaction, we demonstrate that Wnt quickly inhibits the direct interaction between APC and Axin. Our study highlights an important scaffolding function of ß-catenin in the assembly of the destruction complex and suggests Wnt-inhibited APC-Axin interaction as a mechanism of Wnt-dependent inhibition of the destruction complex.