NF-κB memory coordinates transcriptional responses to dynamic inflammatory stimuli

Many scenarios in cellular communication requires cells to interpret multiple dynamic signals. It is unclear how exposure to immune stimuli alters transcriptional responses to subsequent stimulus under inflammatory conditions. Using high-throughput microfluidic live cell analysis, we systematically profiled the NF-κB response to different signal sequences in single cells. We found that NF-κB dynamics stores the history of signals received by cells: depending on the dose and type of prior pathogenic and cytokine signal, the NF-κB response to subsequent stimuli varied widely, from no response to full activation. Using information theory, we revealed that these stimulus-dependent changes in the NF-κB response encode and reflect information about the identity and dose of the prior stimulus. Small-molecule inhibition, computational modeling, and gene expression profiling show that this encoding is driven by stimulus-dependent engagement of negative feedback modules. These results provide a model for how signal transduction networks process sequences of inflammatory stimuli to coordinate cellular responses in complex dynamic environments.

Investigating the NRAS 5' UTR as a Target for Small Molecules

Neuroblastoma RAS (NRAS) is an oncogene that is deregulated and highly mutated in cancers including melanomas and acute myeloid leukemias. Constitutively activated NRAS induces the MAPK and AKT signaling pathways and leads to uncontrolled proliferation and cell growth, making it an attractive target for small molecule inhibition. Like all RAS-family proteins, it has proven difficult to identify small molecules that directly inhibit the protein. An alternative approach would involve targeting the NRAS mRNA. The 5′ untranslated region (5′ UTR) of the NRAS mRNA is reported to contain a G-quadruplex (G4) that regulates translation of NRAS mRNA. Stabilizing the G4 structure with small molecules could reduce NRAS protein expression in cancer cells by impacting translation. Here we report a novel class of small molecule that binds to the G4 structure located in the 5′ UTR of the NRAS mRNA. We used a small molecule microarray (SMM) screen to identify molecules that selectively bind to the NRAS-G4. Biophysical studies demonstrated that compound 18 binds reversibly to the NRAS-G4 structure with submicromolar affinity. A Luciferase based reporter assay indicated that 18 inhibits the translation of NRAS via stabilizing the NRAS-G4 in vitro but showed only moderate effects on the NRAS levels in cellulo. Rapid Amplification of cDNA Ends (RACE), RT-PCR analysis on 14 different NRAS-expressing cell lines, coupled with analysis of publicly available CAGE seq experiments, revealed that predominant NRAS transcript does not possess the G4 structure. Further analysis of published rG4 and G4 sequencing data indicated the presence of G4 structure in the promoter region of NRAS gene (DNA) but not in the mRNA. Thus, although many NRAS transcripts lack a G4 in many cell lines the broader concept of targeting folded regions within 5' UTRs to control translation remains a highly attractive strategy and this work represents an intriguing example of transcript heterogeneity impacting targetability.

Gut Microbial Trimethylamine is Elevated in Alcohol-Associated Hepatitis and Contributes to Ethanol-Induced Liver Injury in Mice

Background:There is mounting evidence that microbes resident in the human intestine contribute to diverse alcohol-associated liver diseases (ALD) including the most deadly form known as alcohol-associated hepatitis (AH). However, mechanisms by which gut microbes synergize with excessive alcohol intake to promote liver injury are poorly understood. Furthermore, whether drugs that selectively target gut microbial metabolism can improve ALD has never been tested. Methods: We used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in healthy controls and AH patients, finding elevated levels of the microbial metabolite trimethylamine (TMA) in AH. In subsequent studies, we treated mice with non-lethal bacterial choline TMA lyase (CutC/D) inhibitors to blunt gut microbedependent production of TMA in the context of chronic ethanol administration. Indices of liver injury were quantified by complementary RNA sequencing, biochemical, and histological approaches. In addition, we examined the impact of ethanol consumption and TMA lyase inhibition on gut microbiome structure via 16S rRNA sequencing. Results: We show the gut microbial choline metabolite trimethylamine (TMA) is elevated in AH patients and correlates with reduced hepatic expression of the TMA oxygenase flavin-containing monooxygenase 3 (FMO3). Provocatively, we find that small molecule inhibition of gut microbial CutC/D activity protects mice from ethanol-induced liver injury. CutC/D inhibitor-driven improvement in ethanol-induced liver injury is associated with distinct reorganization of the gut microbiome and host liver transcriptome. Conclusions: The microbial metabolite TMA is elevated in patients with AH, and inhibition of TMA production from gut microbes can protect mice from ethanol-induced liver injury.

Small molecule inhibition of ATM kinase increases CRISPR-Cas9 1-bp insertion frequency

Mutational outcomes following CRISPR-Cas9-nuclease cutting in mammalian cells have recently been shown to be predictable and, in certain cases, skewed toward single genotypes. However, the ability to control these outcomes remains limited, especially for 1-bp insertions, a common and therapeutically relevant class of repair outcomes. Here, through a small molecule screen, we identify the ATM kinase inhibitor KU-60019 as a compound capable of reproducibly increasing the fraction of 1-bp insertions relative to other Cas9 repair outcomes. Small molecule or genetic ATM inhibition increases 1-bp insertion outcome fraction across three human and mouse cell lines, two Cas9 species, and dozens of target sites, although concomitantly reducing the fraction of edited alleles. Notably, KU-60019 increases the relative frequency of 1-bp insertions to over 80% of edited alleles at several native human genomic loci and improves the efficiency of correction for pathogenic 1-bp deletion variants. The ability to increase 1-bp insertion frequency adds another dimension to precise template-free Cas9-nuclease genome editing.

Single-Molecule Dynamics of SARS-CoV-2 5′ Cap Recognition by Human eIF4F

Coronaviruses initiate translation through recognition of the viral RNA 5′ m7GpppAm cap by translation factor eIF4F. eIF4F is a heterotrimeric protein complex with cap-binding, RNA-binding, and RNA helicase activities. Modulating eIF4F function through cellular regulation or small-molecule inhibition impacts coronavirus replication, including for SARS-CoV-2. Translation initiation involves highly coordinated dynamics of translation factors with messenger or viral RNA. However, how the eIF4F subunits coordinate on the initiation timescale to define cap-binding efficiency remains incompletely understood. Here we report that translation supported by the SARS-CoV-2 5′ UTR is highly sensitive to eIF4A inhibition by rocaglamide. Through a single-molecule fluorescence approach that reports on eIF4E-cap interaction, we dissect how eIF4F subunits contribute to cap-recognition efficiency on the SARS-CoV-2 5′ UTR. We find that free eIF4A enhances cap accessibility for eIF4E binding, but eIF4G alone does not change the kinetics of eIF4E-RNA interaction. Conversely, formation of the full eIF4F complex significantly alters eIF4E-cap interaction, suggesting that coordinated eIF4E and eIF4A activities establish the net eIF4F-cap recognition efficiency. Moreover, the eIF4F complex formed with phosphomimetic eIF4E(S209D) binds the viral UTR more efficiently than with wild-type eIF4E. These results highlight a dynamic interplay of eIF4F subunits and mRNA that determines cap-recognition efficiency.

Small-molecule inhibition of Lats kinases may promote Yap-dependent proliferation in postmitotic mammalian tissues

Hippo signaling is an evolutionarily conserved pathway that restricts growth and regeneration predominantly by suppressing the activity of the transcriptional coactivator Yap. Using a high-throughput phenotypic screen, we identified a potent and non-toxic activator of Yap. In vitro kinase assays show that the compound acts as an ATP-competitive inhibitor of Lats kinases—the core enzymes in Hippo signaling. The substance prevents Yap phosphorylation and induces proliferation of supporting cells in the murine inner ear, murine cardiomyocytes, and human Müller glia in retinal organoids. RNA sequencing indicates that the inhibitor reversibly activates the expression of transcriptional Yap targets: upon withdrawal, a subset of supporting-cell progeny exits the cell cycle and upregulates genes characteristic of sensory hair cells. Our results suggest that the pharmacological inhibition of Lats kinases may promote initial stages of the proliferative regeneration of hair cells, a process thought to be permanently suppressed in the adult mammalian inner ear.