Activation Of Stress Response Research Articles

Abstract PR006: DNA repair inhibition via targeting ATR activates cGAS/STING signaling, promotes anti-tumor immunity, and is a synthetic lethal strategy to augment immunotherapy response in preclinical models and clinical samples of small cell lung cancer

Abstract Introduction: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer. Clinical trials of immune checkpoint blockade (ICB) combined with chemotherapy delivered only very modest benefits. In this study, we investigated the effect of targeting ataxia telangiectasia and rad3-related (ATR), the primary activator of the replication stress response, in SCLC. Clinical samples from treatment naïve and treated SCLC patients were analyzed by single cell and bulk RNA sequencing to ascertain the intratumoral transcriptional heterogeneity and the surrounding tumor microenvironment (TME). Methods: In this study, we investigated the effect of ATR inhibition either alone or in combination with PD-L1 blockade in multiple immunocompetent mouse models of SCLC. The downstream effects of ATR inhibition were assessed by bulk RNA sequencing, and multicolor flow cytometry. The SCLC clinical samples from treatment naïve and patients treated were analyzed by single cell and bulk RNA sequencing. Results: In multiple immunocompetent SCLC mouse models, ATR inhibition (ATRi) remarkably enhanced the anti-tumor effect of PD-L1 blockade. We next tested the ATR inhibition either alone or in combination with PD-L1 in the second-line regimen for SCLC. We observed that ATR inhibition in combination with PD-L1 blockade significantly reduced tumor volume and prolonged survival of aggressive mice models when compared to PD-L1 alone. Targeting ATR enhanced the expression of PD-L1, activated the cGAS/STING pathway, induced the expression of Type I and II interferon pathways, and caused significant infiltration of cytotoxic and memory/effector T-cells into tumors. Interestingly, ATRi also led to significant induction of MHC class I in SCLC in vitro and in vivo models. Single-cell RNA sequencing data provided a deep annotation of SCLC phenotypes and the surrounding immune TME. Treatment and subtype are associated with substantial phenotypic changes in the SCLC immune microenvironment, with greater T-cell dysfunction in SCLC-N than in SCLC-A. Moreover, a recurrent highly metastatic SCLC subset is associated with exhausted CD8+ T-cells and a pro-fibrotic, immunosuppressive monocyte/macrophage population, suggesting possible tumor-immune coordination to promote metastasis. Specifically, analysis of pre-and post-treatment clinical samples from a proof-of-concept study of a first-in-class ATR inhibitor, VX970 (berzosertib), and topotecan, in patients with relapsed SCLCs, validated the induction of MHC class I and interferon pathway genes, for the first time in this disease. Conclusion: We provide insight into tumor and immune biology in SCLC at single-cell resolution. Further, we show ATRi as a potentially transformative vulnerability of SCLC. Given the increasing importance of immunotherapy for the management of SCLC and that ATR inhibitors are already in clinical trials, combining ATR inhibitors with PD-L1-blockade offers an attractive strategy for the treatment of SCLC and contributes to the rapid translation of this combination into the clinic. Citation Format: Triparna Sen. DNA repair inhibition via targeting ATR activates cGAS/STING signaling, promotes anti-tumor immunity, and is a synthetic lethal strategy to augment immunotherapy response in preclinical models and clinical samples of small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR006.

Acute stress promotes effort mobilization for safety-related goals

Although the acute stress response is a highly adaptive survival mechanism, much remains unknown about how its activation impacts our decisions and actions. Based on its resource-mobilizing function, here we hypothesize that this intricate psychophysiological process may increase the willingness (motivation) to engage in effortful, energy-consuming, actions. Across two experiments (n = 80, n = 84), participants exposed to a validated stress-induction protocol, compared to a no-stress control condition, exhibited an increased willingness to exert physical effort (grip force) in the service of avoiding the possibility of experiencing aversive electrical stimulation (threat-of-shock), but not for the acquisition of rewards (money). Use of computational cognitive models linked this observation to subjective value computations that prioritize safety over the minimization of effort expenditure; especially when facing unlikely threats that can only be neutralized via high levels of grip force. Taken together, these results suggest that activation of the acute stress response can selectively alter the willingness to exert effort for safety-related goals. These findings are relevant for understanding how, under stress, we become motivated to engage in effortful actions aimed at avoiding aversive outcomes.

The integrated stress response promotes neural stem cell survival under conditions of mitochondrial dysfunction in neurodegeneration.

Impaired mitochondrial function is a hallmark of aging and a major contributor to neurodegenerative diseases. We have shown that disrupted mitochondrial dynamics typically found in aging alters the fate of neural stem cells (NSCs) leading to impairments in learning and memory. At present, little is known regarding the mechanisms by which neural stem and progenitor cells survive and adapt to mitochondrial dysfunction. Using Opa1-inducible knockout as a model of aging and neurodegeneration, we identify a decline in neurogenesis due to impaired stem cell activation and progenitor proliferation, which can be rescued by the mitigation of oxidative stress through hypoxia. Through sc-RNA-seq, we identify the ATF4 pathway as a critical mechanism underlying cellular adaptation to metabolic stress. ATF4 knockdown in Opa1-deficient NSCs accelerates cell death, while the increased expression of ATF4 enhances proliferation and survival. Using a Slc7a11 mutant, an ATF4 target, we show that ATF4-mediated glutathione production plays a critical role in maintaining NSC survival and function under stress conditions. Together, we show that the activation of the integrated stress response (ISR) pathway enables NSCs to adapt to metabolic stress due to mitochondrial dysfunction and metabolic stress and may serve as a therapeutic target to enhance NSC survival and function in aging and neurodegeneration.

Cannabidivarin and cannabigerol induce unfolded protein response and angiogenesis dysregulation in placental trophoblast HTR-8/SVneo cells.

Cannabidivarin (CBDV) and cannabigerol (CBG) are minor phytocannabinoids from Cannabis sativa, whose health benefits have been reported. However, studies about the impact of these cannabinoids on fundamental cellular processes in placentation are scarce. Placental development involves physiological endoplasmic reticulum (ER) stress, however when exacerbated it can lead to altered angiogenesis and pregnancy disorders, such as intrauterine growth restriction and preeclampsia. In this work, the effects of CBDV and CBG (1-10µM) on placental extravillous trophoblasts were studied, using the in vitro model HTR-8/SVneo cells. Both cannabinoids induced anti-proliferative effects and reactive oxygen/nitrogen species generation, which was dependent on transient receptor potential vanilloid 1 (TRPV1) activation. Moreover, CBDV and CBG significantly upregulated, in a TRPV-1 dependent manner, the gene expression of HSPA5/Glucose-regulated protein 78 (GRP78/BiP), a critical chaperone involved in ER stress and unfolded protein response (UPR) activation. Nevertheless, the UPR pathways were differentially activated. Both cannabinoids were able to recruit the IRE branch, while only CBDV enhanced the expression of downstream effectors of the PERK pathway, namely p-eIF2α, ATF4 and CHOP. It also augmented the activity of the apoptotic initiator caspases-8 and -9, though the effector caspases-3/-7 were not activated. TRB3 expression was increased by CBDV, which may hinder apoptosis termination. Moreover, both compounds upregulated the mRNA levels of the angiogenic factors VEGFA, PGF and sFLT1, and disrupted the endothelial-like behavior of HTR-8/SVneo cells, by reducing tube formation. Thus, CBDV and CBG treatment interferes with EVTs functions and may have a negative impact in placentation and in pregnancy outcome.

Cadmium binding by the F-box domain induces p97-mediated SCF complex disassembly to activate stress response programs

The F-box domain is a highly conserved structural motif that defines the largest class of ubiquitin ligases, Skp1/Cullin1/F-box protein (SCF) complexes. The only known function of the F-box motif is to form the protein interaction surface with Skp1. Here we show that the F-box domain can function as an environmental sensor. We demonstrate that the F-box domain of Met30 is a cadmium sensor that blocks the activity of the SCFMet30 ubiquitin ligase during cadmium stress. Several highly conserved cysteine residues within the Met30 F-box contribute to binding of cadmium with a KD of 8 µM. Binding induces a conformational change that allows for Met30 autoubiquitylation, which in turn leads to recruitment of the segregase Cdc48/p97/VCP followed by active SCFMet30 disassembly. The resulting inactivation of SCFMet30 protects cells from cadmium stress. Our results show that F-box domains participate in regulation of SCF ligases beyond formation of the Skp1 binding interface.

Sensitivity and activation of endoplasmic reticulum stress response and apoptosis in the perinatal sheep heart.

We determined in vitro sensitivity to thapsigargin in sheep cardiomyocytes from four perinatal ages. In utero cardiac activation of endoplasmic reticulum (ER) stress and apoptotic pathways was determined at these same ages. Thapsigargin-induced phosphorylation of EIF2A decreased 72% between 135 and 143 dGA (P=0.0096) and remained low at 1 dPN (P=0.0080). Conversely, thapsigargin-induced caspase cleavage was highest around the time of birth: cleaved caspase 3 was highest at 1 dPN (3.8-fold vs. 135 dGA, P=0.0380; 7.8-fold vs. 5 dPN, P=0.0118), cleaved caspase 7 and cleaved caspase 12 both increased between 135 and 143 dGA (25-fold and 6.9-fold respectively, both P<0.0001), and remained elevated at 1 dPN. Induced apoptosis, measured by TUNEL assay, was highest around the time of birth (P<0.0001). There were changes in myocardial ER stress pathway components in utero. GRP78 protein levels were high in the fetus and declined after birth (P<0.0001). EIF2A phosphorylation was profoundly depressed at 1 dPN (vs. 143 dGA, P=0.0113). There is dynamic regulation of ER proteostasis, ER stress, and the apoptosis cascade in the perinatal heart. Apoptotic signaling is more readily activated in fetal cardiomyocytes near birth, leading to widespread caspase cleavage in the newborn heart. These pathways are important for regulation of normal maturation in the healthy perinatal heart.

TabHLH27 orchestrates root growth and drought tolerance to enhance water use efficiency in wheat.

Cultivating high-yield wheat under limited water resources is crucial for sustainable agriculture in semiarid regions. Amid water scarcity, plants activate drought response signaling, yet the delicate balance between drought tolerance and development remains unclear. Through genome-wide association studies and transcriptome profiling, we identified a wheat atypical basic helix-loop-helix (bHLH) transcription factor (TF), TabHLH27-A1, as a promising quantitative trait locus candidate for both relative root dry weight and spikelet number per spike in wheat. TabHLH27-A1/B1/D1 knock-out reduced wheat drought tolerance, yield, and water use efficiency (WUE). TabHLH27-A1 exhibited rapid induction with polyethylene glycol (PEG) treatment, gradually declining over days. It activated stress response genes such as TaCBL8-B1 and TaCPI2-A1 while inhibiting root growth genes like TaSH15-B1 and TaWRKY70-B1 under short-term PEG stimulus. The distinct transcriptional regulation of TabHLH27-A1 involved diverse interacting factors such as TaABI3-D1 and TabZIP62-D1. Natural variations of TabHLH27-A1 influence its transcriptional responses to drought stress, with TabHLH27-A1Hap-II associated with stronger drought tolerance, larger root system, more spikelets, and higher WUE in wheat. Significantly, the excellent TabHLH27-A1Hap-II was selected during the breeding process in China, and introgression of TabHLH27-A1Hap-II allele improved drought tolerance and grain yield, especially under water-limited conditions. Our study highlights TabHLH27-A1's role in balancing root growth and drought tolerance, providing a genetic manipulation locus for enhancing WUE in wheat.

The E3 ubiquitin-protein ligase synoviolin (Syvn1/Hrd1) promotes adaptive decreases in cardiac myocyte protein synthesis via eIF2α/ATF4 pathway activation

As the heart undergoes pathologic hypertrophy in response to many disease conditions, an imbalance between protein synthesis and degradation in cardiac myocytes, due to the increases in protein synthesis relative to protein degradation, happens, which challenges protein folding and endoplasmic reticulum (ER) proteostasis. This leads to ER stress and the activation of the ER stress response. Synoviolin (Syvn1), also known as HMG-CoA reductase degradation protein 1 (Hrd1), is an ER-localized protein that is upregulated in cardiac myocytes during ER stress. We previously found that Syvn1 mediates the ubiquitylation and consequent degradation of toxic misfolded proteins and preserved cardiac function in a model of pressure overload-induced cardiac pathology. The objective of this study was to examine whether Syvn1 is involved in regulating protein synthesis, and if so, what its function is during hypertrophic growth of cardiac myocytes. Given that cardiac hypertrophy is caused by increases in protein synthesis, we hypothesized that Syvn1 provides an adaptive response in part through regulation of protein synthesis and consequent decrease in cardiac hypertrophic response. We examined the effects of Syvn1 on ventricular myocyte growth in vitro and found that overexpression of Syvn1 decreased cardiac myocyte surface area as well as protein synthesis when treated with phenylephrine (PE), a treatment which normally leads to increased protein synthesis and increased cardiac myocyte size, mimicking pathological cardiac hypertrophy. We next focused on identifying the mechanism of Syvn1-mediated growth inhibition and found that increases in Syvn1 lead to activation of the PERK (protein kinase RNA (PKR)-like ER kinase) branch of the ER stress response, which leads to increased phosphorylation of the α-subunit of translation initiation factor 2 (eIF2α) at Ser51, thereby inhibiting global translational initiation. PERK activation also results in selective translation of a subset of mRNAs, including activating transcription factor 4 (ATF4), which activates the transcription of a wide range of genes involved in adaptation to stress conditions. We found increased ATF4 levels and increased ATF4 target gene expression with Syvn1 overexpression during PE treatment, compared to control PE treatment. Inhibition of the PERK pathway blocked Syvn1-mediated decrease in cardiac myocyte size and protein synthesis as well as PERK activation and ATF4 target gene induction. Additionally, we found that an adeno-associated virus encoding Syvn1, administered in a therapeutically-relevant manner after the onset of pathological cardiac hypertrophy induced by transverse-aortic constriction in mice, blunted pathological remodeling and preserved cardiac function. We posit that the Syvn1/eIF2α/ATF4 pathway constitutes a newly-discovered mechanism for balancing the proteome in cardiac myocytes. Here we found that Syvn1 upregulation blunts maladaptive increase in cardiac myocyte size during pathologic hypertrophy. In conclusion, these findings suggest that Syvn1 is a critical regulator of cardiac myocyte protein synthesis and subsequent growth and that increases in Syvn1 lead to the activation of the PERK branch of the ER stress response, which results in inhibition of global protein synthesis, and activation of ATF4. This work was supported by the National Institutes of Health (R01HL157027) and the University of Arizona Health Sciences Career Development Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Integrated Stress Response Regulates Diurnal Rhythms in Liver Metabolism

Disruptions in biological rhythms are associated with the development of metabolic diseases. General control nonderepressible 2 (GCN2), a primary sensor of amino acid insuffciency and activator of the integrated stress response (ISR), plays an integral role in liver metabolism. The objective of this work was to examine the impact of feeding a low protein quality (LPQ) diet on diurnal rhythms in liver metabolism. We hypothesized that activation of the ISR by GCN2 maintains metabolic rhythms within the liver during LPQ feeding. To address our objective, wild type (WT) and whole body Gcn2 knockout (GCN2KO) mice were housed in total darkness and provided free access to either a Control or a LPQ diet (devoid of leucine) for 8-days and then killed at 2 circadian time (CT) points 12-hours apart (CT3, CT15). WT mice fed a Control diet showed oscillations in hepatic ISR activation that were lost in the livers of GCN2KO mice. Diurnal patterns in the liver transcriptome including genes regulating triglyceride metabolism were also dampened in GCN2KO mice. WT mice fed LPQ showed increased hepatic ISR activation and greater abundance of ISR target genes relative to Control only at CT15 (p&lt;0.05 by two factor ANOVA). GCN2KO mice fed LPQ failed to show oscillations in ISR activation and instead displayed reductions in liver intracellular amino acids alongside significant upregulation of genes encoding gluconeogenesis and amino acid catabolism at CT15 (q&lt;0.05, Log2FC&gt;1). In conclusion, GCN2 is required for diurnal ISR activation and execution in the liver. LPQ feeding increases hepatic ISR activation in a time dependent manner. Sensing of LPQ by GCN2 is necessary for the maintenance of rhythms in liver macronutrient metabolism. NIH DK109714, Pilot award from the NJ IFNH Center for Human Nutrition, Exercise, and Metabolism (NExT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Molecular Toxicity of Popular Toothpaste Formulations on Post-Juveniles of Clarias gariepinus

The widespread use of toothpaste containing various chemical formulations has raised concern regarding their potential impact on aquatic ecosystems. This research examines the molecular toxicity of popular toothpaste brands on post-juveniles of Clarias gariepinus from a commercial fish farm in Akure, Ondo State, Nigeria. Specifically assessing the impact of these toothpaste brands on the mRNA expression levels of the heat shock protein (HSP70), interleukin (IL-1β), melatonin receptors (MEL1C), and growth hormone in comparison to a control group. The results revealed a significant upregulation of HSP70, IL-1β, and MEL1C genes in the exposed group, indicating a potential stress response and immune system activation. Intriguingly, the growth hormone mRNA expression remained unaffected in the treated group compared to the control. These findings underscore the need for further exploration into the potential molecular consequences of common toothpaste ingredients on aquatic organisms, raising important questions about environmental safety and consumer product development.

Altered serine metabolism promotes drug tolerance in Mycobacterium abscessus via a WhiB7-mediated adaptive stress response.

Mycobacterium abscessus is an emerging opportunistic pathogen responsible for chronic lung diseases, especially in patients with cystic fibrosis. Treatment failure of M. abscessus infections is primarily associated with intrinsic or acquired antibiotic resistance. However, there is growing evidence that antibiotic tolerance, i.e., the ability of bacteria to transiently survive exposure to bactericidal antibiotics through physiological adaptations, contributes to the relapse of chronic infections and the emergence of acquired drug resistance. Yet, our understanding of the molecular mechanisms that underlie antibiotic tolerance in M. abscessus remains limited. In the present work, a mutant with increased cross-tolerance to the first- and second-line antibiotics cefoxitin and moxifloxacin, respectively, has been isolated by experimental evolution. This mutant harbors a mutation in serB2, a gene involved in L-serine biosynthesis. Metabolic changes caused by this mutation alter the intracellular redox balance to a more reduced state that induces overexpression of the transcriptional regulator WhiB7 during the stationary phase, promoting tolerance through activation of a WhiB7-dependant adaptive stress response. These findings suggest that alteration of amino acid metabolism and, more generally, conditions that trigger whiB7 overexpression, makes M. abscessus more tolerant to antibiotic treatment.

CHCHD10S59L/+ mouse model: Behavioral and neuropathological features of frontotemporal dementia

CHCHD10-related disease causes a spectrum of clinical presentations including mitochondrial myopathy, cardiomyopathy, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We generated a knock-in mouse model bearing the p.Ser59Leu (S59L) CHCHD10 variant. Chchd10S59L/+ mice have been shown to phenotypically replicate the disorders observed in patients: myopathy with mtDNA instability, cardiomyopathy and typical ALS features (protein aggregation, neuromuscular junction degeneration and spinal motor neuron loss). Here, we conducted a comprehensive behavioral, electrophysiological and neuropathological assessment of Chchd10S59L/+ mice. These animals show impaired learning and memory capacities with reduced long-term potentiation (LTP) measured at the Perforant Pathway-Dentate Gyrus (PP-DG) synapses. In the hippocampus of Chchd10S59L/+ mice, neuropathological studies show the involvement of protein aggregates, activation of the integrated stress response (ISR) and neuroinflammation in the degenerative process. These findings contribute to decipher mechanisms associated with CHCHD10 variants linking mitochondrial dysfunction and neuronal death. They also validate the Chchd10S59L/+ mice as a relevant model for FTD, which can be used for preclinical studies to test new therapeutic strategies for this devastating disease.

Membrane Lipids Augment Cell Envelope Stress Signaling via the MadRS System to Defend Against Antimicrobial Peptides and Antibiotics in Enterococcus faecalis.

Enterococci have evolved resistance mechanisms to protect their cell envelopes against bacteriocins and host cationic antimicrobial peptides (CAMPs) produced in the gastrointestinal environment. Activation of the membrane stress response has also been tied to resistance to the lipopeptide antibiotic daptomycin. However, the actual effectors mediating resistance have not been elucidated. Here, we show that the MadRS (formerly YxdJK) membrane antimicrobial peptide defense system controls a network of genes, including a previously uncharacterized three gene operon (madEFG) that protects the E. faecalis cell envelope from antimicrobial peptides. Constitutive activation of the system confers protection against CAMPs and daptomycin in the absence of a functional LiaFSR system and leads to persistence of cardiac microlesions in vivo. Moreover, changes in the lipid cell membrane environment alter CAMP susceptibility and expression of the MadRS system. Thus, we provide a framework supporting a multilayered envelope defense mechanism for resistance and survival coupled to virulence.

Abstract LB411: Investigating the dependency of androgen receptor-positive prostate cancer on class I myosins

Abstract Prostate adenocarcinoma is characterized by high expression and activity of the androgen receptor (AR). AR is a nuclear hormone receptor that stimulates anabolic cell metabolism. We hypothesized that the high activity of AR in prostate cancer creates unique dependencies on cellular machinery to support this high rate of anabolism. We observed that androgen treatment of patients with metastatic castration-resistant prostate cancer resulted in increased abundance of MYO1E, a class I myosin that facilitates intracellular vesicular trafficking, in tumor biopsies. Similarly, MYO1E protein increased in AR+ prostate cancer cell lines upon treatment with androgens. Notably, a specific small molecule inhibitor of MYO1E, pentachloropseudilin (PClP), induced cell death of AR-positive prostate cancer cell lines with an IC50 of 2-4uM, but was non-toxic to AR-negative prostate cancer cell lines. Expression of AR in these AR-negative lines conferred sensitivity to PClP, while deletion of AR in an AR-positive line conferred resistance to PClP, indicating that AR expression was required and sufficient for sensitivity of prostate cancer cell lines to PClP. Previous studies have suggested that AR globally increases protein synthesis and proteotoxic stress, and we asked whether this may create a dependency on MYO1E for secretion of protein-containing vesicles to manage proteotoxic stress. Initial experiments suggest that AR increases global rates of protein synthesis, as assessed by puromycin-incorporation assays, and inhibition of MYO1E increases phosphorylation of EIF2A, suggestive of activation of the integrated stress response. Further experiments will determine the essential function of MYO1E in AR-positive prostate cancer. Altogether our studies suggest that the class I myosin MYO1E is an essential protein in AR-positive cell lines and may represent a novel drug target for prostate adenocarcinoma. Citation Format: Anoushka Kadam, Shivani Kumar, Varsha Vakkala, Rajendra Kumar, Angelo M. De Marzo, Edward J. Pearce, Laura A. Sena. Investigating the dependency of androgen receptor-positive prostate cancer on class I myosins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB411.

Abstract LB171: First-in-class inhibitors of the tRNA methyltransferase METTL1 for the treatment of cancer

Abstract METTL1 is an RNA methyltransferase that catalyzes N7-methylation on guanine at position 46 (m7G46) in a subset of transfer RNAs (tRNAs). These tRNAs are stabilized by m7G46, and this increases the translation of mRNAs containing the cognate codons. METTL1 is often over-expressed in cancer tissue, and its gene locus is frequently amplified in specific tumor types. Recent studies have shown that reducing METTL1 expression leads to tumor growth inhibition highlighting this enzyme as a potential novel target for anti-cancer drugs. We optimized small molecule inhibitors of METTL1 from a HTS hit using structure-guided medicinal chemistry. Representative compounds of two distinct chemical series exhibit potent in vitro METTL1 inhibition at low nanomolar concentrations while displaying exquisite selectivity over other RNA and protein methyltransferases. The effects of inhibiting METTL1 were measured by cellular mechanistic assays and modification-induced misincorporation tRNA sequencing (mim-tRNAseq). As expected, pharmacological inhibition led to decreased m7G46 methylation, lower levels of a subgroup of tRNAs and reduced cancer cell proliferation.The efficacy of METTL1 inhibition in a panel of cancer cell lines representing a wide range of solid and hematologic tumor types was determined. Cell proliferation was impaired in a subset of cell lines originating from various cancer types, including lymphoma, sarcoma, esophageal squamous cell carcinoma, glioblastoma, colorectal and lung adenocarcinoma. Importantly, viability was not affected in non-transformed cell lines, indicating a highly selective mechanism of action. The phenotypic consequences of METTL1 inhibition in selected cell lines were further investigated by flow cytometry and western blotting. Cell lines sensitive to METTL1 inhibition displayed reduced cell cycle progression and lowered expression of cell cycle regulators. RNA sequencing and proteome analyses identified molecular changes at both transcript and protein level. METTL1 inhibition led to induction of ATF4 expression and activation of the integrated stress response, as well as global changes to the translation machinery. Studies on cell-line derived xenograft or syngeneic in vivo mouse models were conducted to assess tumor growth inhibition after oral or intraperitoneal administration of the potent METTL1 small molecule inhibitor STM9005. Inhibition of METTL1 by this compound reduced tumor growth in both immune-deficient and immune-competent mouse models, effects accompanied by dose-dependent changes in pharmacodynamic biomarkers. Here, we describe the discovery of first-in-class inhibitors of METTL1 tRNA methyltransferase. To our knowledge, our data provide the first evidence that pharmacological inhibition of a tRNA methyltransferase reduces tumor growth in vivo. Citation Format: Alexandra Sapetschnig, Beth Thomas, Eliza Yankova, Harry Fischl, Aleksandra Azevedo, Sarah Bucknell, Richard Fosbeary, Sapphire Sawyer, Sian Evans, Carmen Livi, Byron Andrews, Jack Rogan, Natalie Webster, Matthew Fyfe, Konstantinos Tzelepis, Oliver Rausch. First-in-class inhibitors of the tRNA methyltransferase METTL1 for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB171.

Transcriptome analysis revealed that Arabidopsis model plant invokes the activation of heat shock proteins and ER stress response against cesium stress

Transcriptome analysis revealed that Arabidopsis model plant invokes the activation of heat shock proteins and ER stress response against cesium stress

Tigilanol tiglate is an oncolytic small molecule that induces immunogenic cell death and enhances the response of both target and non-injected tumors to immune checkpoint blockade

BackgroundTigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that...

Rescue of Methionine Dependence by Cobalamin in a Human Colorectal Cancer Cell Line.

Methionine dependence is a characteristic of most cancer cells where they are unable to proliferate when the essential amino acid methionine is replaced with its precursor homocysteine in the growing media. Normal cells, on the other hand, thrive under these conditions and are referred to as methionine-independent. The reaction that adds a methyl group from 5-methyltetrahydrofolate to homocysteine to regenerate methionine is catalyzed by the enzyme methionine synthase with the cofactor cobalamin (vitamin B12). However, decades of research have shown that methionine dependence in cancer is not due to a defect in the activity of methionine synthase. Cobalamin metabolism has been tied to the dependent phenotype in rare cell lines. We have identified a human colorectal cancer cell line in which the cells regain the ability to proliferation in methionine-free, L-homocystine-supplemented media when cyanocobalamin is supplemented at a level of 1 µg/mL. In human SW48 cells, methionine replacement with L-homocystine does not induce any measurable increase in apoptosis or reactive oxygen species production in this cell line. Rather, proliferation is halted, then restored in the presence of cyanocobalamin. Our data show that supplementation with cyanocobalamin prevents the activation of the integrated stress response (ISR) in methionine-deprived media in this cell line. The ISR-associated cell cycle arrest, characteristic of methionine-dependence in cancer, is also prevented, leading to the continuation of proliferation in methionine-deprived SW48 cells with cobalamin. Our results highlight differences between cancer cell lines in the response to cobalamin supplementation in the context of methionine dependence.

Single Cell RNAseq Analysis of Cytokine-Treated Human Islets: Association of Cellular Stress with Impaired Cytokine Responsiveness.

Pancreatic β-cells are essential for survival, being the only cell type capable of insulin secretion. While they are believed to be vulnerable to damage by inflammatory cytokines such as interleukin-1 beta (IL-1β) and interferon-gamma, we have recently identified physiological roles for cytokine signaling in rodent β-cells that include the stimulation of antiviral and antimicrobial gene expression and the inhibition of viral replication. In this study, we examine cytokine-stimulated changes in gene expression in human islets using single-cell RNA sequencing. Surprisingly, the global responses of human islets to cytokine exposure were remarkably blunted compared to our previous observations in the mouse. The small population of human islet cells that were cytokine responsive exhibited increased expression of IL-1β-stimulated antiviral guanylate-binding proteins, just like in the mouse. Most human islet cells were not responsive to cytokines, and this lack of responsiveness was associated with high expression of genes encoding ribosomal proteins. We further correlated the expression levels of RPL5 with stress response genes, and when expressed at high levels, RPL5 is predictive of failure to respond to cytokines in all endocrine cells. We postulate that donor causes of death and isolation methodologies may contribute to stress of the islet preparation. Our findings indicate that activation of stress responses in human islets limits cytokine-stimulated gene expression, and we urge caution in the evaluation of studies that have examined cytokine-stimulated gene expression in human islets without evaluation of stress-related gene expression.

Do oral and gut microbiota communicate through redox pathways? A novel asset of the nitrate-nitrite-NO pathway.

Nitrate may act as a regulator of •NO bioavailability via sequential reduction along the nitrate-nitrite-NO pathway with widespread health benefits, including a eubiotic effect on the oral and gut microbiota. Here, we discuss the molecular mechanisms of microbiota-host communication through redox pathways, via the production of •NO and oxidants by the family of NADPH oxidases, namely hydrogen peroxide (via Duox2), superoxide radical (via Nox1 and Nox2) and peroxynitrite, which leads to downstream activation of stress responses (Nrf2 and NFkB pathways) in the host mucosa. The activation of Nox2 by microbial metabolites is also discussed. Finally, we propose a new perspective in which both oral and gut microbiota communicate through redox pathways, with nitrate as the pivot linking both ecosystems.