Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1ß processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1ß maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1ß secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

Systematic evaluation of antibody-mediated siRNA delivery using an industrial platform of THIOMAB-siRNA conjugates.

Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody-siRNA complexes provide a possible solution. However, initial reports of antibody-siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, we built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. We report that such coupling generates monomeric antibody-siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technology, we generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. We identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only observed using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. Our broad characterization of multiple parameters using therapeutic-grade conjugate technology provides a thorough assessment of this delivery technology, highlighting both examples of success as well as remaining challenges.

Quantitative evaluation of first, second, and third generation hairpin systems reveals the limit of mammalian vector-based RNAi.

Incorporating miRNA-like features into vector-based hairpin scaffolds has been shown to augment small RNA processing and RNAi efficiency. Therefore, defining an optimal, native hairpin context may obviate a need for hairpin-specific targeting design schemes, which confound the movement of functional siRNAs into shRNA/artificial miRNA backbones, or large-scale screens to identify efficacious sequences. Thus, we used quantitative cell-based assays to compare separate third generation artificial miRNA systems, miR-E (based on miR-30a) and miR-3G (based on miR-16-2 and first described in this study) to widely-adopted, first and second generation formats in both Pol-II and Pol-III expression vector contexts. Despite their unique structures and strandedness, and in contrast to first and second-generation RNAi triggers, the third generation formats operated with remarkable similarity to one another, and strong silencing was observed with a significant fraction of the evaluated target sequences within either promoter context. By pairing an established siRNA design algorithm with the third generation vectors we could readily identify targeting sequences that matched or exceeded the potency of those discovered through large-scale sensor-based assays. We find that third generation hairpin systems enable the maximal level of siRNA function, likely through enhanced processing and accumulation of precisely-defined guide RNAs. Therefore, we predict future gains in RNAi potency will come from improved hairpin expression and identification of optimal siRNA-intrinsic silencing properties rather than further modification of these scaffolds. Consequently, third generation systems should be the primary format for vector-based RNAi studies; miR-3G is advantageous due to its small expression cassette and simplified, cost-efficient cloning scheme.

Right- and left-loop short shRNAs have distinct and unusual mechanisms of gene silencing.

Small hairpin RNAs (shRNAs) having duplex lengths of 25-29 bp are normally processed by Dicer into short interfering RNAs (siRNAs) before incorporation into the RNA-induced silencing complex (RISC). However, shRNAs of ≤ 19 bp [short shRNAs (sshRNAs)] are too short for Dicer to excise their loops, raising questions about their mechanism of action. sshRNAs are designated as L-type or R-type according to whether the loop is positioned 3' or 5' to the guide sequence, respectively. Using nucleotide modifications that inhibit RNA cleavage, we show that R- but not L-sshRNAs require loop cleavage for optimum activity. Passenger-arm slicing was found to be important for optimal functioning of L-sshRNAs but much less important for R-sshRNAs that have a cleavable loop. R-sshRNAs could be immunoprecipitated by antibodies to Argonaute-1 (Ago1); complexes with Ago1 contained both intact and loop-cleaved sshRNAs. In contrast, L-sshRNAs were immunoprecipitated with either Ago1 or Ago2 and were predominantly sliced in the passenger arm of the hairpin. However, 'pre-sliced' L-sshRNAs were inactive. We conclude that active L-sshRNAs depend on slicing of the passenger arm to facilitate opening of the duplex, whereas R-sshRNAs primarily act via loop cleavage to generate a 5'-phosphate at the 5'-end of the guide strand.

CheekAge: a next-generation buccal epigenetic aging clock associated with lifestyle and health.

Epigenetic aging clocks are computational models that predict age using DNA methylation information. Initially, first-generation clocks were developed to make predictions using CpGs that change with age. Over time, next-generation clocks were created using CpGs that relate to both age and health. Since existing next-generation clocks were constructed in blood, we sought to develop a next-generation clock optimized for prediction in cheek swabs, which are non-invasive and easy to collect. To do this, we collected MethylationEPIC data as well as lifestyle and health information from 8045 diverse adults. Using a novel simulated annealing approach that allowed us to incorporate lifestyle and health factors into training as well as a combination of CpG filtering, CpG clustering, and clock ensembling, we constructed CheekAge, an epigenetic aging clock that has a strong correlation with age, displays high test-retest reproducibility across replicates, and significantly associates with a plethora of lifestyle and health factors, such as BMI, smoking status, and alcohol intake. We validated CheekAge in an internal dataset and multiple publicly available datasets, including samples from patients with progeria or meningioma. In addition to exploring the underlying biology of the data and clock, we provide a free online tool that allows users to mine our methylomic data and predict epigenetic age.

Glycine and aging: Evidence and mechanisms.

The restriction of calories, branched-chain amino acids, and methionine have all been shown to extend lifespan in model organisms. Recently, glycine was found to boost longevity in genetically heterogenous mice. This simple amino acid similarly extends lifespan in rats and improves health in mammalian models of age-related disease. While compelling data indicate that glycine is a pro-longevity molecule, divergent mechanisms may underlie its effects on aging. Glycine is abundant in collagen, a building block for glutathione, a precursor to creatine, and an acceptor for the enzyme glycine N-methyltransferase (GNMT). A review of the literature strongly implicates GNMT, which clears methionine from the body by taking a methyl group from S-adenosyl-L-methionine and methylating glycine to form sarcosine. In flies, Gnmt is required for reduced insulin/insulin-like growth factor 1 signaling and dietary restriction to fully extend lifespan. The geroprotector spermidine requires Gnmt to upregulate autophagy genes and boost longevity. Moreover, the overexpression of Gnmt is sufficient to extend lifespan and reduce methionine levels. Sarcosine, or methylglycine, declines with age in multiple species and is capable of inducing autophagy both in vitro and in vivo. Taken all together, existing evidence suggests that glycine prolongs life by mimicking methionine restriction and activating autophagy.

Real-time quantification of antibody-short interfering RNA conjugate in serum by antigen capture reverse transcription-polymerase chain reaction.

Short interfering RNA (siRNA) has therapeutic potential. However, efficient delivery is a formidable task. To facilitate delivery of siRNA into cells, we covalently conjugated siRNA to antibodies that bind to cell surface proteins and internalize. Understanding how these antibody-siRNA conjugates function in vivo requires pharmacokinetic analysis. Thus, we developed a simple real-time antigen capture reverse transcription-polymerase chain reaction (RT-PCR) assay to detect intact antibody-siRNA conjugates. Biotinylated antigen bound to streptavidin-coated PCR tubes was used to capture antibody-siRNA conjugate. The captured antibody-siRNA conjugate was then reverse-transcribed in the same tube, avoiding a sample transfer step. This reproducible assay had a wide standard curve range of 0.029 to 480ng/ml and could detect as low as 0.58ng/ml antibody-siRNA conjugates in mouse serum. The presence of unconjugated antibody that could be generated from siRNA degradation in vivo did not affect the assay as long as the total antibody concentration in the antigen capture step did not exceed 480ng/ml. Using this assay, we observed a more rapid decrease in serum antibody-siRNA conjugate concentrations than the total antibody concentrations in mice dosed with antibody-siRNA conjugates, suggesting loss of siRNA from the antibody. This assay is useful for optimizing antibody-siRNA and likely aptamer-siRNA conjugates to improve pharmacokinetics and aid siRNA delivery.

A set of common buccal CpGs that predict epigenetic age and associate with lifespan-regulating genes.

Epigenetic aging clocks are computational models that use DNA methylation sites to predict age. Since cheek swabs are non-invasive and painless, collecting DNA from buccal tissue is highly desirable. Here, we review 11 existing clocks that have been applied to buccal tissue. Two of these were exclusively trained on adults and, while moderately accurate, have not been used to capture health-relevant differences in epigenetic age. Using 130 common CpGs utilized by two or more existing buccal clocks, we generate a proof-of-concept predictor in an adult methylomic dataset. In addition to accurately estimating age (r = 0.95 and mean absolute error = 3.88 years), this clock predicted that Down syndrome subjects were significantly older relative to controls. A literature and database review of CpG-associated genes identified numerous genes (e.g., CLOCK, ELOVL2, and VGF) and molecules (e.g., alpha-linolenic acid, glycine, and spermidine) reported to influence lifespan and/or age-related disease in model organisms.

Human age reversal: Fact or fiction?

Although chronological age correlates with various age-related diseases and conditions, it does not adequately reflect an individual's functional capacity, well-being, or mortality risk. In contrast, biological age provides information about overall health and indicates how rapidly or slowly a person is aging. Estimates of biological age are thought to be provided by aging clocks, which are computational models (e.g., elastic net) that use a set of inputs (e.g., DNA methylation sites) to make a prediction. In the past decade, aging clock studies have shown that several age-related diseases, social variables, and mental health conditions associate with an increase in predicted biological age relative to chronological age. This phenomenon of age acceleration is linked to a higher risk of premature mortality. More recent research has demonstrated that predicted biological age is sensitive to specific interventions. Human trials have reported that caloric restriction, a plant-based diet, lifestyle changes involving exercise, a drug regime including metformin, and vitamin D3 supplementation are all capable of slowing down or reversing an aging clock. Non-interventional studies have connected high-quality sleep, physical activity, a healthy diet, and other factors to age deceleration. Specific molecules have been associated with the reduction or reversal of predicted biological age, such as the antihypertensive drug doxazosin or the metabolite alpha-ketoglutarate. Although rigorous clinical trials are needed to validate these initial findings, existing data suggest that aging clocks are malleable in humans. Additional research is warranted to better understand these computational models and the clinical significance of lowering or reversing their outputs.

Expanded RNA-binding activities of mammalian Argonaute 2.

Mammalian Argonaute 2 (Ago2) protein associates with microRNAs (miRNAs) or small interfering RNAs (siRNAs) forming RNA-induced silencing complexes (RISCs/miRNPs). In the present work, we characterize the RNA-binding and nucleolytic activity of recombinant mouse Ago2. Our studies show that recombinant mouse Ago2 binds efficiently to miRNAs forming active RISC. Surprisingly, we find that recombinant mouse Ago2 forms active RISC using pre-miRNAs or long unstructured single stranded RNAs as guides. Furthermore, we demonstrate that, in vivo, endogenous human Ago2 binds directly to pre-miRNAs independently of Dicer, and that Ago2:pre-miRNA complexes are found both in the cytoplasm and in the nucleus of human cells.

Dicer loss in striatal neurons produces behavioral and neuroanatomical phenotypes in the absence of neurodegeneration.

MicroRNAs (miRNAs) are small noncoding RNAs that can act to repress target mRNAs by suppressing translation and/or reducing mRNA stability. Although it is clear that miRNAs and Dicer, an RNase III enzyme that is central to the production of mature miRNAs, have a role in the early development of neurons, their roles in the postmitotic neuron in vivo are largely unknown. To determine the roles of Dicer in neurons, we ablated Dicer in dopaminoceptive neurons. Mice that have lost Dicer in these cells display a range of phenotypes including ataxia, front and hind limb clasping, reduced brain size, and smaller neurons. Surprisingly, dopaminoceptive neurons without Dicer survive over the life of the animal. The lack of profound cell death contrasts with other mouse models in which Dicer has been ablated. These studies highlight the complicated nature of Dicer ablation in the brain and provide a useful mouse model for studying dopaminoceptive neuron function.

ADAR and hnRNPC deficiency synergize in activating endogenous dsRNA-induced type I IFN responses.

Cytosolic double-stranded RNA (dsRNA) initiates type I IFN responses. Endogenous retroelements, notably Alu elements, constitute a source of dsRNA. Adenosine-to-inosine (A-to-I) editing by ADAR induces mismatches in dsRNA and prevents recognition by MDA5 and autoinflammation. To identify additional endogenous dsRNA checkpoints, we conducted a candidate screen in THP-1 monocytes and found that hnRNPC and ADAR deficiency resulted in synergistic induction of MDA5-dependent IFN responses. RNA-seq analysis demonstrated dysregulation of Alu-containing introns in hnRNPC-deficient cells via utilization of unmasked cryptic splice sites, including introns containing ADAR-dependent A-to-I editing clusters. These putative MDA5 ligands showed reduced editing in the absence of ADAR, providing a plausible mechanism for the combined effects of hnRNPC and ADAR. This study contributes to our understanding of the control of repetitive element-induced autoinflammation and suggests that patients with hnRNPC-mutated tumors might maximally benefit from ADAR inhibition-based immunotherapy.

Conditional loss of Dicer disrupts cellular and tissue morphogenesis in the cortex and hippocampus.

To investigate the role of Dicer and microRNAs in the mammalian CNS, we used mice in which the second RNase III domain of Dicer was conditionally floxed. Conditional Dicer mice were bred with mice expressing an alpha-calmodulin kinase II Cre to selectively inactivate Dicer in excitatory forebrain neurons in vivo. Inactivation of Dicer results in an array of phenotypes including microcephaly, reduced dendritic branch elaboration, and large increases in dendritic spine length with no concomitant change in spine density. Microcephaly is likely caused by a 5.5-fold increase in early postnatal apoptosis in these animals as determined by active caspase-3 and TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling) staining in the cortex. Loss of Dicer function had no measurable effect on cortical lamination as determined by in situ hybridization, suggesting that microcephaly is not caused by defects in neuronal migration. Together, these results illustrate the in vivo significance of Dicer and miRNAs in the mammalian CNS and provide additional support for previous in vitro studies indicating that misregulation of this pathway may result in gross abnormalities in cell number and function that may contribute to a variety of neurological disorders.

ERBB3 and IGF1R Signaling Are Required for Nrf2-Dependent Growth in KEAP1-Mutant Lung Cancer.

Mutations in KEAP1 and NFE2L2 (encoding the protein Nrf2) are prevalent in both adeno and squamous subtypes of non-small cell lung cancer, as well as additional tumor indications. The consequence of these mutations is stabilized Nrf2 and chronic induction of a battery of Nrf2 target genes. We show that knockdown of Nrf2 caused modest growth inhibition of cells growing in two-dimension, which was more pronounced in cell lines expressing mutant KEAP1. In contrast, Nrf2 knockdown caused almost complete regression of established KEAP1-mutant tumors in mice, with little effect on wild-type (WT) KEAP1 tumors. The strong dependency on Nrf2 could be recapitulated in certain anchorage-independent growth environments and was not prevented by excess extracellular glutathione. A CRISPR screen was used to investigate the mechanism(s) underlying this dependence. We identified alternative pathways critical for Nrf2-dependent growth in KEAP1-mutant cell lines, including the redox proteins thioredoxin and peroxiredoxin, as well as the growth factor receptors IGF1R and ERBB3. IGF1R inhibition was effective in KEAP1-mutant cells compared with WT, especially under conditions of anchorage-independent growth. These results point to addiction of KEAP1-mutant tumor cells to Nrf2 and suggest that inhibition of Nrf2 or discrete druggable Nrf2 target genes such as IGF1R could be an effective therapeutic strategy for disabling these tumors. SIGNIFICANCE: This study identifies pathways activated by Nrf2 that are important for the proliferation and tumorigenicity of KEAP1-mutant non-small cell lung cancer.

A CRISPR screen identifies MAPK7 as a target for combination with MEK inhibition in KRAS mutant NSCLC.

Mutant KRAS represents one of the most frequently observed oncogenes in NSCLC, yet no therapies are approved for tumors that express activated KRAS variants. While there is strong rationale for the use of MEK inhibitors to treat tumors with activated RAS/MAPK signaling, these have proven ineffective clinically. We therefore implemented a CRISPR screening approach to identify novel agents to sensitize KRAS mutant NSCLC cells to MEK inhibitor treatment. This approach identified multiple components of the canonical RAS/MAPK pathway consistent with previous studies. In addition, we identified MAPK7 as a novel, strong hit and validated this finding using multiple orthogonal approaches including knockdown and pharmacological inhibition. We show that MAPK7 inhibition attenuates the re-activation of MAPK signaling occurring following long-term MEK inhibition, thereby illustrating that MAPK7 mediates pathway reactivation in the face of MEK inhibition. Finally, genetic knockdown of MAPK7 combined with the MEK inhibitor cobimetinib in a mutant KRAS NSCLC xenograft model to mediate improved tumor growth inhibition. These data highlight that MAPK7 represents a promising target for combination treatment with MEK inhibition in KRAS mutant NSCLC.

ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages.

The NLRC4 inflammasome recognizes bacterial flagellin and components of the type III secretion apparatus. NLRC4 stimulation leads to caspase-1 activation followed by a rapid lytic cell death known as pyroptosis. NLRC4 is linked to pathogen-free auto-inflammatory diseases, suggesting a role for NLRC4 in sterile inflammation. Here, we show that NLRC4 activates an alternative cell death program morphologically similar to apoptosis in caspase-1-deficient BMDMs. By performing an unbiased genome-wide CRISPR/Cas9 screen with subsequent validation studies in gene-targeted mice, we highlight a critical role for caspase-8 and ASC adaptor in an alternative apoptotic pathway downstream of NLRC4. Furthermore, caspase-1 catalytically dead knock-in (Casp1 C284A KI) BMDMs genetically segregate pyroptosis and apoptosis, and confirm that caspase-1 does not functionally compete with ASC for NLRC4 interactions. We show that NLRC4/caspase-8-mediated apoptotic cells eventually undergo plasma cell membrane damage in vitro, suggesting that this pathway can lead to secondary necrosis. Unexpectedly, we found that DFNA5/GSDME, a member of the pore-forming gasdermin family, is dispensable for the secondary necrosis that follows NLRC4-mediated apoptosis in macrophages. Together, our data confirm the existence of an alternative caspase-8 activation pathway diverging from the NLRC4 inflammasome in primary macrophages.

CRISPR whole-genome screening identifies new necroptosis regulators and RIPK1 alternative splicing.

The necroptotic cell death pathway is a key component of human pathogen defense that can become aberrantly derepressed during tissue homeostasis to contribute to multiple types of tissue damage and disease. While formation of the necrosome kinase signaling complex containing RIPK1, RIPK3, and MLKL has been extensively characterized, additional mechanisms of its regulation and effector functions likely remain to be discovered. We screened 19,883 mouse protein-coding genes by CRISPR/Cas9-mediated gene knockout for resistance to cytokine-induced necroptosis and identified 112 regulators and mediators of necroptosis, including 59 new candidate pathway components with minimal or no effect on cell growth in the absence of necroptosis induction. Among these, we further characterized the function of PTBP1, an RNA binding protein whose activity is required to maintain RIPK1 protein abundance by regulating alternative splice-site selection.

Silencing of retrotransposons by SETDB1 inhibits the interferon response in acute myeloid leukemia.

A propensity for rewiring genetic and epigenetic regulatory networks, thus enabling sustained cell proliferation, suppression of apoptosis, and the ability to evade the immune system, is vital to cancer cell propagation. An increased understanding of how this is achieved is critical for identifying or improving therapeutic interventions. In this study, using acute myeloid leukemia (AML) human cell lines and a custom CRISPR/Cas9 screening platform, we identify the H3K9 methyltransferase SETDB1 as a novel, negative regulator of innate immunity. SETDB1 is overexpressed in many cancers, and loss of this gene in AML cells triggers desilencing of retrotransposable elements that leads to the production of double-stranded RNAs (dsRNAs). This is coincident with induction of a type I interferon response and apoptosis through the dsRNA-sensing pathway. Collectively, our findings establish a unique gene regulatory axis that cancer cells can exploit to circumvent the immune system.

Repression of Stress-Induced LINE-1 Expression Protects Cancer Cell Subpopulations from Lethal Drug Exposure.

Maintenance of phenotypic heterogeneity within cell populations is an evolutionarily conserved mechanism that underlies population survival upon stressful exposures. We show that the genomes of a cancer cell subpopulation that survives treatment with otherwise lethal drugs, the drug-tolerant persisters (DTPs), exhibit a repressed chromatin state characterized by increased methylation of histone H3 lysines 9 and 27 (H3K9 and H3K27). We also show that survival of DTPs is, in part, maintained by regulators of H3K9me3-mediated heterochromatin formation and that the observed increase in H3K9me3 in DTPs is most prominent over long interspersed repeat element 1 (LINE-1). Disruption of the repressive chromatin over LINE-1 elements in DTPs results in DTP ablation, which is partially rescued by reducing LINE-1 expression or function.

MicroRNAs and endocrine biology.

microRNAs (miRNAs) are highly conserved, non-coding RNAs that powerfully regulate gene expression at the post-transcriptional level. These fascinating molecules play essential roles in many biological processes in mammals, including insulin secretion, B-cell development, and adipocyte differentiation. This review provides a general background regarding current knowledge about miRNA biogenesis and the potential contributions of these RNAs to endocrine function.