Systematic identification of anticancer drug targets reveals a nucleus-to-mitochondria ROS-sensing pathway.

Multiple anticancer drugs have been proposed to cause cell death, in part, by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs, exactly how the resultant ROS function and are sensed is poorly understood. It remains unclear which proteins the ROS modify and their roles in drug sensitivity/resistance. To answer these questions, we examined 11 anticancer drugs with an integrated proteogenomic approach identifying not only many unique targets but also shared ones-including ribosomal components, suggesting common mechanisms by which drugs regulate translation. We focus on CHK1 that we find is a nuclear H2O2 sensor that launches a cellular program to dampen ROS. CHK1 phosphorylates the mitochondrial DNA-binding protein SSBP1 to prevent its mitochondrial localization, which in turn decreases nuclear H2O2. Our results reveal a druggable nucleus-to-mitochondria ROS-sensing pathway-required to resolve nuclear H2O2 accumulation and mediate resistance to platinum-based agents in ovarian cancers.

Identification of chemotherapy targets reveals a nucleus-to-mitochondria ROS sensing pathway.

Multiple chemotherapies are proposed to cause cell death in part by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs exactly how the resultant ROS function and are sensed is poorly understood. In particular, it's unclear which proteins the ROS modify and their roles in chemotherapy sensitivity/resistance. To answer these questions, we examined 11 chemotherapies with an integrated proteogenomic approach identifying many unique targets for these drugs but also shared ones including ribosomal components, suggesting one mechanism by which chemotherapies regulate translation. We focus on CHK1 which we find is a nuclear H 2 O 2 sensor that promotes an anti-ROS cellular program. CHK1 acts by phosphorylating the mitochondrial-DNA binding protein SSBP1, preventing its mitochondrial localization, which in turn decreases nuclear H 2 O 2 . Our results reveal a druggable nucleus-to-mitochondria ROS sensing pathway required to resolve nuclear H 2 O 2 accumulation, which mediates resistance to platinum-based chemotherapies in ovarian cancers.

SULT1A1-dependent sulfonation of alkylators is a lineage-dependent vulnerability of liver cancers.

Adult liver malignancies, including intrahepatic cholangiocarcinoma and hepatocellular carcinoma, are the second leading cause of cancer-related deaths worldwide. Most individuals are treated with either combination chemotherapy or immunotherapy, respectively, without specific biomarkers for selection. Here using high-throughput screens, proteomics and in vitro resistance models, we identify the small molecule YC-1 as selectively active against a defined subset of cell lines derived from both liver cancer types. We demonstrate that selectivity is determined by expression of the liver-resident cytosolic sulfotransferase enzyme SULT1A1, which sulfonates YC-1. Sulfonation stimulates covalent binding of YC-1 to lysine residues in protein targets, enriching for RNA-binding factors. Computational analysis defined a wider group of structurally related SULT1A1-activated small molecules with distinct target profiles, which together constitute an untapped small-molecule class. These studies provide a foundation for preclinical development of these agents and point to the broader potential of exploiting SULT1A1 activity for selective targeting strategies.

NRF2 activation induces NADH-reductive stress, providing a metabolic vulnerability in lung cancer.

Multiple cancers regulate oxidative stress by activating the transcription factor NRF2 through mutation of its negative regulator, KEAP1. NRF2 has been studied extensively in KEAP1-mutant cancers; however, the role of this pathway in cancers with wild-type KEAP1 remains poorly understood. To answer this question, we induced NRF2 via pharmacological inactivation of KEAP1 in a panel of 50+ non-small cell lung cancer cell lines. Unexpectedly, marked decreases in viability were observed in >13% of the cell lines-an effect that was rescued by NRF2 ablation. Genome-wide and targeted CRISPR screens revealed that NRF2 induces NADH-reductive stress, through the upregulation of the NAD+-consuming enzyme ALDH3A1. Leveraging these findings, we show that cells treated with KEAP1 inhibitors or those with endogenous KEAP1 mutations are selectively vulnerable to Complex I inhibition, which impairs NADH oxidation capacity and potentiates reductive stress. Thus, we identify reductive stress as a metabolic vulnerability in NRF2-activated lung cancers.

Comparative transcriptome analysis reveals the impact of the daily rhythm on the hemolymph of the Chinese mitten crab (Eriocheir sinensis).

The daily rhythm affects a series of physiological functions in crustaceans. To study its effect on the physiological function in Eriocheir sinensis, a crustacean species of high economic value, we analyzed the hemolymph transcriptome during the daily rhythm by high-throughput sequencing. We sampled the hemolymph from crabs at four time points in a single day (06:00, 12:00, 18:00, and 24:00 h) and identified 3,01,661 and 1,03,998 transcripts and unigenes, respectively; some of the unigenes were annotated as core clock genes. Moreover, 15,564 differentially expressed genes (DEGs) were divided into nine different clusters. Functional enrichment analysis of DEGs indicated that the molting, metabolism, and immunity processes in E. sinensis were impacted by its daily rhythm. In addition, we mapped the DEGs involved in the daily entrainment pathway. To the best of our knowledge, this is the first comparative transcriptome analysis of crustacean hemolymph during the day-night cycle, and provides multi-level information for unraveling the finer regulatory effects of the daily cycle in crustaceans.

Mutant IDH Inhibits IFNγ-TET2 Signaling to Promote Immunoevasion and Tumor Maintenance in Cholangiocarcinoma.

Isocitrate dehydrogenase 1 mutations (mIDH1) are common in cholangiocarcinoma. (R)-2-hydroxyglutarate generated by the mIDH1 enzyme inhibits multiple α-ketoglutarate-dependent enzymes, altering epigenetics and metabolism. Here, by developing mIDH1-driven genetically engineered mouse models, we show that mIDH1 supports cholangiocarcinoma tumor maintenance through an immunoevasion program centered on dual (R)-2-hydroxyglutarate-mediated mechanisms: suppression of CD8+ T-cell activity and tumor cell-autonomous inactivation of TET2 DNA demethylase. Pharmacologic mIDH1 inhibition stimulates CD8+ T-cell recruitment and interferon γ (IFNγ) expression and promotes TET2-dependent induction of IFNγ response genes in tumor cells. CD8+ T-cell depletion or tumor cell-specific ablation of TET2 or IFNγ receptor 1 causes treatment resistance. Whereas immune-checkpoint activation limits mIDH1 inhibitor efficacy, CTLA4 blockade overcomes immunosuppression, providing therapeutic synergy. The findings in this mouse model of cholangiocarcinoma demonstrate that immune function and the IFNγ-TET2 axis are essential for response to mIDH1 inhibition and suggest a novel strategy for potentiating efficacy. SIGNIFICANCE: Mutant IDH1 inhibition stimulates cytotoxic T-cell function and derepression of the DNA demethylating enzyme TET2, which is required for tumor cells to respond to IFNγ. The discovery of mechanisms of treatment efficacy and the identification of synergy by combined CTLA4 blockade provide the foundation for new therapeutic strategies. See related commentary by Zhu and Kwong, p. 604. This article is highlighted in the In This Issue feature, p. 587.

Effects of Corrosion on Compressive Arch Action and Catenary Action of RC Frames to Resist Progressive Collapse Based on Numerical Analysis.

Many negative factors can influence the progressive collapse resistance of reinforced concrete (RC) frame structures. One of the most important factors is the corrosion of rebar within the structure. With increasing severity of corrosion, the duration, robustness, and mechanical performance can be greatly impaired. One specific side effect of rebar corrosion is the significant loss of protection against progressive collapse. In order to quantify the effects of rebar corrosion on load-resisting mechanisms (compressive arch action (CAA) and tensile catenary action (TCA)) of RC frames, a series of numerical investigations were carried out in this paper. The previous experimental results reported in the literature provide a benchmark for progressive collapse behavior as a sound condition and validate the proposed numerical model. Furthermore, based on the verified numerical model, the CAA and TCA with increasing corrosion and an elapsed time from 0 to 70 years are investigated. Comparing with the conventional empirical model, the proposed numerical model has shown the ability and feasibility in predicting the collapse resistance capacity in structures with corroded rebar. Therefore, this numerical modeling strategy provides comprehensive insights into the change of load-resisting mechanisms in these structures, which can be beneficial for optimizing the design.

Two novel lead-based coordination polymers for luminescence sensing of anions, cations and small organic molecules.

Two novel lead-based coordination polymers, namely [Pb(cbdcp)]·0.5H2O·0.5CH3OH (1) and [Pb(cbdcp)] (2), have been solvothermally constructed by using a zwitterionic ligand 4-carboxy-1-(3,4-dicarboxy-benzyl)-pyridinium chloride (abbreviated as H3cbdcpCl). Compound 1 has a three-dimensional framework displaying a valence-bonded SrAl2 topology with the 42·63·8 symbol, while compound 2 has a two-dimensional sheet structure that can be simplified into a three-dimensional ππ interaction-connected topology with the {44·62}2{48·615·85} symbol. Notably, compound 1 proved to be a promising potential luminescent sensor capable of selectively detecting anions, cations and small organic molecules, especially Cr2O72-, CrO42-, Fe3+ and nitrobenzene.

Metabolomics Investigation of Voriconazole-Induced Hepatotoxicity in Mice.

Voriconazole (VCZ) is a widely used triazole drug for the treatment of serious incidence of invasive fungal infections (IFIs), and its most commonly reported clinical side effect is hepatotoxicity. The mechanism of VCZ-induced hepatotoxicity is unclear, and no specific marker can be used for prediction and diagnosis. This study aims to apply the targeted metabolomics approach to identify specific VCZ-induced metabolites related to hepatotoxicity via liquid chromatography-triple quadrupole mass spectrometry (LC-QqQ-MS) in a C57BL/6 mouse model. Mice treated with three repeated doses of 40 mg/kg VCZ by tail vein injection to induce hepatotoxicity (VCZ-induced hepatotoxicity group, n = 8) were compared with mice without treatment (control group, n = 10). Both liver tissue and plasma were collected and analyzed to propose underlying mechanisms associated with VCZ-induced hepatotoxicity. The results indicated that the metabolites associated with oxidative stress were altered, and alterations in the metabolites involved in glutathione biosynthesis were noticed. The ratio of glutamine to glutamate showed a significant reduction in the VCZ-induced hepatotoxicity group compared to the control group, suggesting that glutamine might be transformed into glutamate for glutathione biosynthesis. Accordingly, we proposed that VCZ-induced hepatotoxicity is associated with oxidative stress to cause cell dysfunction, leading to alterations in energy metabolism, the urea cycle, and nucleoside metabolism. To the best of our knowledge, this is the first study to apply metabolomics for investigating the mechanism of VCZ-induced hepatotoxicity.

The MAP Kinase CfPMK1 Is a Key Regulator of Pathogenesis, Development, and Stress Tolerance of Colletotrichum fructicola.

The Ascomycetes fungus Colletotrichum fructicola causes severe diseases on a wide range of crops, fruits, and vegetables. Its pathogenic mechanisms, however, remain poorly understood. Mitogen-activated protein kinases (MAPKs) are conserved regulators of fungal development and pathogenesis. In this study, a Fus3/Kss1-related MAPK from C. fructicola was functionally characterized via gene deletion. On potato dextrose agar (PDA) and oatmeal agar media, the CfPMK1 gene deletion mutants (ΔCfPMK1) were slightly reduced in radial growth rate, severely limited in aerial hyphal differentiation and hyphal melanization, and formed deformed perithecia that were smaller in size and more compactly organized relative to wild type. When artificially inoculated on plants, conidia of these mutants failed to differentiate appressoria or penetrate cuticle, and their pathogenicity defect could not be rescued by wounding plant tissue prior to inoculation. On PDA, ΔCfPMK1 mutants were hypersensitive to osmotic stresses, but were more tolerant to membrane and cell wall stresses. Genetic complementation rescued all phenotypic changes associated with CfPMK1 gene deletion. Based on GFP fusion expression, CfPMK1 protein accumulation was detected at all life stages, and the accumulation level was higher in nascent appressoria relative to conidia. Overall, this study identified CfPMK1 as a key regulator of appressorium and sexual development, pathogenesis, and stress tolerance in C. fructicola.

Comparative analysis of the mitochondrial genomes of Colletotrichum gloeosporioides sensu lato: insights into the evolution of a fungal species complex interacting with diverse plants.

BACKGROUND: The fungal species complex Colletotrichum gloeosporioides sensu lato contains over 20 plant-interacting species. These species exhibit different life styles (e.g., endophytes, foliar and fruit pathogens) and show considerable variation in host and tissue adaptation strategies. Accurate species delimitation in C. gloeosporioides s.l. is very challenging due to nascent lineage boundaries and phenotypic plasticity, which strongly impedes studies of the complex's host-interaction biology. In this study, we first sequenced and compared nine mitogenomes belonging to four C. gloeosporioides s.l. species lineages (C. gloeosporioides, C. fructicola, C. aenigma, and C. siamense s.l.), and evaluated the usefulness of mitogenome sequence in complementing prevailing nuclear markers for species delimitation. RESULTS: The C. gloeosporioides s.l. mitogenomes ranged between 52,671 and 58,666 bp in size, and each contained an identical set of genes transcribed in the same direction. Compared with previously reported Colletotrichum mitogenomes, these mitogenomes were uniquely featured by: (1) significantly larger genome size due to richer intron content and longer intergenic region; (2) striking GC content elevation at the intergenic region; and (3) considerable intron content variation among different species lineages. Compared with nuclear DNA markers commonly used in phylogeny, the mitogenome nucleotide diversity was extremely low, yet the mitogenome alignment contained the highest number of parsimony informative sites, which allowed the generation of a high-resolution phylogeny recognizing all taxonomic lineages, including ones belonging to the very nascent C. siamense s.l. complex. The tree topology was highly congruent with the phylogeny based on nuclear marker concatenation except for lineages within C. siamense s.l. Further comparative phylogenetic analysis indicated that lineage-specific rapid divergence of GS and SOD2 markers confounded concatenation-based species relationship inference. CONCLUSIONS: This study sheds light on the evolution of C. gloeosporioides s.l. mitogenomes and demonstrates that mitogenome sequence can complement prevailing nuclear markers in improving species delimitation accuracy. The mitogenome sequences reported will be valuable resources for further genetic studies with C. gloeosporioides s.l. and other Colletotrichum species.

Evaluation of disulfide scrambling during the enzymatic digestion of bevacizumab at various pH values using mass spectrometry.

Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure the quality and function of protein pharmaceuticals. There are, however, problems associated with maintaining disulfide linkages in the conventional procedures that are used to digest a protein. In order to preserve enzyme activity during the digestion of a protein, it is commonly carried out at neutral to basic environment which increases the possibilities of disulfide bond scrambling. However, it is not easy to differentiate whether the scrambled disulfide linkages are initiated by the sample itself or whether they are induced during the protease digestion process. In this study, the optimum pH for minimizing disulfide bond rearrangements during the digestion process was determined. Three sets of proteases, trypsin plus Glu-C, Lys-C and thermolysin were used, followed by dimethyl labeling and mass spectrometry for a bevacizumab (Avastin) disulfide linkage analysis. No disulfide linkage scrambling was detected at pH6 when Lys-C or trypsin plus Glu-C were used as enzymes. When thermolysin was applied, some scrambled disulfide bonds were identified at pH5, 6 and 7. Nevertheless, there was less disulfide bond scrambling at a lower pH. All correct disulfide bonds on bevacizumab could be identified using this approach. The results demonstrated that by choosing the proper enzymes, using a lower pH environment for the digestion could reduce the degree of artifact disulfide scrambling.