Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has high relapse and low 5-year survival rates. Single-cell profiling in relapsed HCC may aid in the design of effective anticancer therapies, including immunotherapies. We profiled the transcriptomes of ∼17,000 cells from 18 primary or early-relapse HCC cases. Early-relapse tumors have reduced levels of regulatory T cells, increased dendritic cells (DCs), and increased infiltrated CD8+ T cells, compared with primary tumors, in two independent cohorts. Remarkably, CD8+ T cells in recurrent tumors overexpressed KLRB1 (CD161) and displayed an innate-like low cytotoxic state, with low clonal expansion, unlike the classical exhausted state observed in primary HCC. The enrichment of these cells was associated with a worse prognosis. Differential gene expression and interaction analyses revealed potential immune evasion mechanisms in recurrent tumor cells that dampen DC antigen presentation and recruit innate-like CD8+ T cells. Our comprehensive picture of the HCC ecosystem provides deeper insights into immune evasion mechanisms associated with tumor relapse.

Base-editing mutagenesis maps alleles to tune human T cell functions.

CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1-6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency7,8. Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules targeting base editors to protein-coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base-editing screens in primary immune cells thus provide biochemical insights with the potential to accelerate immunotherapy design.

Improving prime editing with an endogenous small RNA-binding protein.

Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.

Rolling back human pluripotent stem cells to an eight-cell embryo-like stage.

After fertilization, the quiescent zygote experiences a burst of genome activation that initiates a short-lived totipotent state. Understanding the process of totipotency in human cells would have broad applications. However, in contrast to in mice1,2, demonstration of the time of zygotic genome activation or the eight-cell (8C) stage in in vitro cultured human cells has not yet been reported, and the study of embryos is limited by ethical and practical considerations. Here we describe a transgene-free, rapid and controllable method for producing 8C-like cells (8CLCs) from human pluripotent stem cells. Single-cell analysis identified key molecular events and gene networks associated with this conversion. Loss-of-function experiments identified fundamental roles for DPPA3, a master regulator of DNA methylation in oocytes3, and TPRX1, a eutherian totipotent cell homeobox (ETCHbox) family transcription factor that is absent in mice4. DPPA3 induces DNA demethylation throughout the 8CLC conversion process, whereas TPRX1 is a key executor of 8CLC gene networks. We further demonstrate that 8CLCs can produce embryonic and extraembryonic lineages in vitro or in vivo in the form of blastoids5 and complex teratomas. Our approach provides a resource to uncover the molecular process of early human embryogenesis.

Cell transcriptomic atlas of the non-human primate Macaca fascicularis.

Studying tissue composition and function in non-human primates (NHPs) is crucial to understand the nature of our own species. Here we present a large-scale cell transcriptomic atlas that encompasses over 1 million cells from 45 tissues of the adult NHP Macaca fascicularis. This dataset provides a vast annotated resource to study a species phylogenetically close to humans. To demonstrate the utility of the atlas, we have reconstructed the cell-cell interaction networks that drive Wnt signalling across the body, mapped the distribution of receptors and co-receptors for viruses causing human infectious diseases, and intersected our data with human genetic disease orthologues to establish potential clinical associations. Our M. fascicularis cell atlas constitutes an essential reference for future studies in humans and NHPs.

Ectopic expression of meiotic cohesin generates chromosome instability in cancer cell line.

Many tumors express meiotic genes that could potentially drive somatic chromosome instability. While germline cohesin subunits SMC1B, STAG3, and REC8 are widely expressed in many cancers, messenger RNA and protein for RAD21L subunit are expressed at very low levels. To elucidate the potential of meiotic cohesins to contribute to genome instability, their expression was investigated in human cell lines, predominately in DLD-1. While the induction of the REC8 complex resulted in a mild mitotic phenotype, the expression of the RAD21L complex produced an arrested but viable cell pool, thus providing a source of DNA damage, mitotic chromosome missegregation, sporadic polyteny, and altered gene expression. We also found that genomic binding profiles of ectopically expressed meiotic cohesin complexes were reminiscent of their corresponding specific binding patterns in testis. Furthermore, meiotic cohesins were found to localize to the same sites as BORIS/CTCFL, rather than CTCF sites normally associated with the somatic cohesin complex. These findings highlight the existence of a germline epigenomic memory that is conserved in cells that normally do not express meiotic genes. Our results reveal a mechanism of action by unduly expressed meiotic cohesins that potentially links them to aneuploidy and chromosomal mutations in affected cells.

Deubiquitinase-targeting chimeras for targeted protein stabilization.

Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48-ubiquitin-specific deubiquitinase OTUB1. We showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.

Ligandability of E3 Ligases for Targeted Protein Degradation Applications.

Targeted protein degradation (TPD) using proteolysis targeting chimeras (PROTACs) and molecular glue degraders has arisen as a powerful therapeutic modality for eliminating disease-causing proteins from cells. PROTACs and molecular glue degraders employ heterobifunctional or monovalent small molecules, respectively, to chemically induce the proximity of target proteins with E3 ubiquitin ligases to ubiquitinate and degrade specific proteins via the proteasome. Whereas TPD is an attractive therapeutic strategy for expanding the druggable proteome, only a relatively small number of E3 ligases out of the >600 E3 ligases encoded by the human genome have been exploited by small molecules for TPD applications. Here we review the existing E3 ligases that have thus far been successfully exploited for TPD and discuss chemoproteomics-enabled covalent screening strategies for discovering new E3 ligase recruiters. We also provide a chemoproteomic map of reactive cysteines within hundreds of E3 ligases that may represent potential ligandable sites that can be pharmacologically interrogated to uncover additional E3 ligase recruiters.

Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors.

While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly "druggable" or relatively easy-to-drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.

MYBL2 and ATM suppress replication stress in pluripotent stem cells.

Replication stress, a major cause of genome instability in cycling cells, is mainly prevented by the ATR-dependent replication stress response pathway in somatic cells. However, the replication stress response pathway in embryonic stem cells (ESCs) may be different due to alterations in cell cycle phase length. The transcription factor MYBL2, which is implicated in cell cycle regulation, is expressed a hundred to a thousand-fold more in ESCs compared with somatic cells. Here we show that MYBL2 activates ATM and suppresses replication stress in ESCs. Consequently, loss of MYBL2 or inhibition of ATM or Mre11 in ESCs results in replication fork slowing, increased fork stalling and elevated origin firing. Additionally, we demonstrate that inhibition of CDC7 activity rescues replication stress induced by MYBL2 loss and ATM inhibition, suggesting that uncontrolled new origin firing may underlie the replication stress phenotype resulting from loss/inhibition of MYBL2 and ATM. Overall, our study proposes that in addition to ATR, a MYBL2-MRN-ATM replication stress response pathway functions in ESCs to control DNA replication initiation and prevent genome instability.

Transposable element sequence fragments incorporated into coding and noncoding transcripts modulate the transcriptome of human pluripotent stem cells.

Transposable elements (TEs) occupy nearly 40% of mammalian genomes and, whilst most are fragmentary and no longer capable of transposition, they can nevertheless contribute to cell function. TEs within genes transcribed by RNA polymerase II can be copied as parts of primary transcripts; however, their full contribution to mature transcript sequences remains unresolved. Here, using long and short read (LR and SR) RNA sequencing data, we show that 26% of coding and 65% of noncoding transcripts in human pluripotent stem cells (hPSCs) contain TE-derived sequences. Different TE families are incorporated into RNAs in unique patterns, with consequences to transcript structure and function. The presence of TE sequences within a transcript is correlated with TE-type specific changes in its subcellular distribution, alterations in steady-state levels and half-life, and differential association with RNA Binding Proteins (RBPs). We identify hPSC-specific incorporation of endogenous retroviruses (ERVs) and LINE:L1 into protein-coding mRNAs, which generate TE sequence-derived peptides. Finally, single cell RNA-seq reveals that hPSCs express ERV-containing transcripts, whilst differentiating subpopulations lack ERVs and express SINE and LINE-containing transcripts. Overall, our comprehensive analysis demonstrates that the incorporation of TE sequences into the RNAs of hPSCs is more widespread and has a greater impact than previously appreciated.

The regulation of glucose and lipid homeostasis via PLTP as a mediator of BAT-liver communication.

While brown adipose tissue (BAT) is well-recognized for its ability to dissipate energy in the form of heat, recent studies suggest multifaced roles of BAT in the regulation of glucose and lipid homeostasis beyond stimulating thermogenesis. One of the functions involves interorgan communication with metabolic organs, such as the liver, through BAT-derived secretory factors, a.k.a., batokine. However, the identity and the roles of such mediators remain insufficiently understood. Here, we employed proteomics and transcriptomics in human thermogenic adipocytes and identified previously unappreciated batokines, including phospholipid transfer protein (PLTP). We found that increased circulating levels of PLTP, via systemic or BAT-specific overexpression, significantly improve glucose tolerance and insulin sensitivity, increased energy expenditure, and decrease the circulating levels of cholesterol, phospholipids, and sphingolipids. Such changes were accompanied by increased bile acids in the circulation, which in turn enhances glucose uptake and thermogenesis in BAT. Our data suggest that PLTP is a batokine that contributes to the regulation of systemic glucose and lipid homeostasis as a mediator of BAT-liver interorgan communication.

Capturing the interactome of newly transcribed RNA.

We combine the labeling of newly transcribed RNAs with 5-ethynyluridine with the characterization of bound proteins. This approach, named capture of the newly transcribed RNA interactome using click chemistry (RICK), systematically captures proteins bound to a wide range of RNAs, including nascent RNAs and traditionally neglected nonpolyadenylated RNAs. RICK has identified mitotic regulators amongst other novel RNA-binding proteins with preferential affinity for nonpolyadenylated RNAs, revealed a link between metabolic enzymes/factors and nascent RNAs, and expanded the known RNA-bound proteome of mouse embryonic stem cells. RICK will facilitate an in-depth interrogation of the total RNA-bound proteome in different cells and systems.

Covalent targeting of the vacuolar H+-ATPase activates autophagy via mTORC1 inhibition.

Autophagy is a lysosomal degradation pathway that eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling.

Harnessing the anti-cancer natural product nimbolide for targeted protein degradation.

Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114-substrate recognition, leading to inhibition of ubiquitination and degradation of tumor suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the use of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications.

Origin-Specific Adhesive Interactions of Mesenchymal Stem Cells with Platelets Influence Their Behavior After Infusion.

We investigated the adhesive behavior of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow MSC (BMMSC) or umbilical cord MSC (UCMSC) adhered efficiently from flow to the matrix proteins, collagen, or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to predeposited platelets. The differences in behavior in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC-2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC-2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC-2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin-induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. Stem Cells 2018;36:1062-1074.

Autophagy, lipophagy and lysosomal lipid storage disorders.

Autophagy is a catabolic process with an essential function in the maintenance of cellular and tissue homeostasis. It is primarily recognised for its role in the degradation of dysfunctional proteins and unwanted organelles, however in recent years the range of autophagy substrates has also been extended to lipids. Degradation of lipids via autophagy is termed lipophagy. The ability of autophagy to contribute to the maintenance of lipo-homeostasis becomes particularly relevant in the context of genetic lysosomal storage disorders where perturbations of autophagic flux have been suggested to contribute to the disease aetiology. Here we review recent discoveries of the molecular mechanisms mediating lipid turnover by the autophagy pathways. We further focus on the relevance of autophagy, and specifically lipophagy, to the disease mechanisms. Moreover, autophagy is also discussed as a potential therapeutic target in several key lysosomal storage disorders.

Discovery of human sORF-encoded polypeptides (SEPs) in cell lines and tissue.

The existence of nonannotated protein-coding human short open reading frames (sORFs) has been revealed through the direct detection of their sORF-encoded polypeptide (SEP) products. The discovery of novel SEPs increases the size of the genome and the proteome and provides insights into the molecular biology of mammalian cells, such as the prevalent usage of non-AUG start codons. Through modifications of the existing SEP-discovery workflow, we discover an additional 195 SEPs in K562 cells and extend this methodology to identify novel human SEPs in additional cell lines and human tissue for a final tally of 237 new SEPs. These results continue to expand the human genome and proteome and demonstrate that SEPs are a ubiquitous class of nonannotated polypeptides that require further investigation.

Formaldehyde regulates S-adenosylmethionine biosynthesis and one-carbon metabolism.

One-carbon metabolism is an essential branch of cellular metabolism that intersects with epigenetic regulation. In this work, we show how formaldehyde (FA), a one-carbon unit derived from both endogenous sources and environmental exposure, regulates one-carbon metabolism by inhibiting the biosynthesis of S-adenosylmethionine (SAM), the major methyl donor in cells. FA reacts with privileged, hyperreactive cysteine sites in the proteome, including Cys120 in S-adenosylmethionine synthase isoform type-1 (MAT1A). FA exposure inhibited MAT1A activity and decreased SAM production with MAT-isoform specificity. A genetic mouse model of chronic FA overload showed a decrease n SAM and in methylation on selected histones and genes. Epigenetic and transcriptional regulation of Mat1a and related genes function as compensatory mechanisms for FA-dependent SAM depletion, revealing a biochemical feedback cycle between FA and SAM one-carbon units.

NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.

Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.