Dinotefuran exposure alters biochemical, metabolomic, gut microbiome, and growth responses in decapoda pacific white shrimp Penaeus vannamei.

Dinotefuran, a neonicotinoid insecticide, may impact nontarget organisms such as Decapoda P. vannamei shrimp with nervous systems similar to insects. Exposing shrimp to low dinotefuran concentrations (6, 60, and 600 µg/L) for 21 days affected growth, hepatosomatic index, and survival. Biomarkers erythromycin-N-demethylase, alanine aminotransferase, and catalase increased in all exposed groups, while glutathione S-transferase is the opposite; aminopyrin-N-demethylase, malondialdehyde, and aspartate aminotransferase increased at 60 and 600 µg/L. Concentration-dependent effects on gut microbiota altered the abundance of bacterial groups, increased potentially pathogenic and oxidative stress-resistant phenotypes, and decreased biofilm formation. Gram-positive/negative microbiota changed significantly. Metabolite differences between the exposed and control groups were identified using mass spectrometry and KEGG pathway enrichment. N-acetylcystathionine showed potential as a reliable dinotefuran metabolic marker. Weighted correlation network analysis (WGCNA) results indicated high connectivity of cruecdysone in the metabolite network and significant enrichment at 600 µg/L dinotefuran. The WGCNA results revealed a highly significant negative correlation between two key metabolites, caldine and indican, and the gut microbiota within co-expression modules. Overall, the risk of dinotefuran exposure to non-target organisms in aquatic environments still requires further attention.

A Secreted BBE-Like Enzyme Acting as a Drug-Binding Efflux Carrier Confers Microbial Self-Resistance to Mitomycin C.

The berberine bridge enzyme (BBE)-like flavoproteins have attracted continuous attention for their capability to catalyze various oxidative reactions. Here we demonstrate that MitR, a secreted BBE-like enzyme, functions as a special drug-binding efflux protein evolved from quinone reductase. Moreover, this protein provides self-resistance to its hosts toward the DNA-alkylating agent mitomycin C with a distinctive strategy, featured by independently performing drug binding and efflux.

Natural Products Inhibition of Cytochrome P450 2B6 Activity and Methadone Metabolism.

Methadone is cleared predominately by hepatic cytochrome P450 (CYP) 2B6-catalyzed metabolism to inactive metabolites. CYP2B6 also catalyzes the metabolism of several other drugs. Methadone and CYP2B6 are susceptible to pharmacokinetic drug-drug interactions. Use of natural products such as herbals and other botanicals is substantial and growing, and concomitant use of prescription medicines and non-prescription herbals is common and may result in interactions, often precipitated by CYP inhibition. Little is known about herbal product effects on CYP2B6 activity, and CYP2B6-catalyzed methadone metabolism. We screened a family of natural product compounds used in traditional medicines, herbal teas, and synthetic analogs of compounds found in plants, including kavalactones, flavokavains, chalcones and gambogic acid, for inhibition of expressed CYP2B6 activity and specifically inhibition of CYP2B6-mediated methadone metabolism. An initial screen evaluated inhibition of CYP2B6-catalyzed 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation. Hits were further evaluated for inhibition of racemic methadone metabolism, including mechanism of inhibition and kinetic constants. In order of decreasing potency, the most effective inhibitors of methadone metabolism were dihydromethysticin (competitive, K i 0.074 µM), gambogic acid (noncompetitive, K i 6 µM), and 2,2'-dihydroxychalcone (noncompetitive, K i 16 µM). Molecular modeling of CYP2B6-methadone and inhibitor binding showed substrate and inhibitor binding position and orientation and their interactions with CYP2B6 residues. These results show that CYP2B6 and CYP2B6-catalyzed methadone metabolism are inhibited by certain natural products, at concentrations which may be clinically relevant. SIGNIFICANCE STATEMENT: This investigation identified several natural product constituents which inhibit in vitro human recombinant CYP2B6 and CYP2B6-catalyzed N-demethylation of the opioid methadone. The most potent inhibitors (K i) were dihydromethysticin (0.074 µM), gambogic acid (6 µM) and 2,2'-dihydroxychalcone (16 µM). Molecular modeling of ligand interactions with CYP2B6 found that dihydromethysticin and 2,2'-dihydroxychalcone bound at the active site, while gambogic acid interacted with an allosteric site on the CYP2B6 surface. Natural product constituents may inhibit CYP2B6 and methadone metabolism at clinically relevant concentrations.

Highly efficient export of a disulfide-bonded protein to the periplasm and medium by the Tat pathway using CyDisCo in Escherichia coli.

High-value heterologous proteins produced in Escherichia coli that contain disulfide bonds are almost invariably targeted to the periplasm via the Sec pathway as it, among other advantages, enables disulfide bond formation and simplifies downstream processing. However, the Sec system cannot transport complex or rapidly folding proteins, as it only transports proteins in an unfolded state. The Tat system also transports proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Most of the studies related to Tat secretion have used the well-studied TorA signal peptide that is Tat-specific, but this signal peptide also tends to induce degradation of the protein of interest, resulting in lower yields. This makes it difficult to use Tat in the industry. In this study, we show that a model disulfide bond-containing protein, YebF, can be exported to the periplasm and media at a very high level by the Tat pathway in a manner almost completely dependent on cytoplasmic disulfide formation, by other two putative Tat SPs: those of MdoD and AmiC. In contrast, the TorA SP exports YebF at a low level.

Multidisciplinary Insights into the Structure-Function Relationship of the CYP2B6 Active Site.

Cytochrome P450 2B6 (CYP2B6) is a highly polymorphic human enzyme involved in the metabolism of many clinically relevant drugs, environmental toxins, and endogenous molecules with disparate structures. Over the last 20-plus years, in silico and in vitro studies of CYP2B6 using various ligands have provided foundational information regarding the substrate specificity and structure-function relationship of this enzyme. Approaches such as homology modeling, X-ray crystallography, molecular docking, and kinetic activity assays coupled with CYP2B6 mutagenesis have done much to characterize this originally neglected monooxygenase. However, a complete understanding of the structural details that make new chemical entities substrates of CYP2B6 is still lacking. Surprisingly little in vitro data has been obtained about the structure-function relationship of amino acids identified to be in the CYP2B6 active site. Since much attention has already been devoted to elucidating the function of CYP2B6 allelic variants, here we review the salient findings of in silico and in vitro studies of the CYP2B6 structure-function relationship with a deliberate focus on the active site. In addition to summarizing these complementary approaches to studying structure-function relationships, we note gaps/challenges in existing data such as the need for more CYP2B6 crystal structures, molecular docking results with various ligands, and data coupling CYP2B6 active site mutagenesis with kinetic parameter measurement under standard expression conditions. Harnessing in silico and in vitro techniques in tandem to understand the CYP2B6 structure-function relationship will likely offer further insights into CYP2B6-mediated metabolism. SIGNIFICANCE STATEMENT: The apparent importance of cytochrome P450 2B6 (CYP2B6) in the metabolism of various xenobiotics and endogenous molecules has grown since its discovery with many in silico and in vitro studies offering a partial description of its structure-function relationship. Determining the structure-function relationship of CYP2B6 is difficult but may be aided by thorough biochemical investigations of the CYP2B6 active site that provide a more complete pharmacological understanding of this important enzyme.

JMJD1C Regulates Megakaryopoiesis in In Vitro Models through the Actin Network.

The histone demethylase JMJD1C is associated with human platelet counts. The JMJD1C knockout in zebrafish and mice leads to the ablation of megakaryocyte-erythroid lineage anemia. However, the specific expression, function, and mechanism of JMJD1C in megakaryopoiesis remain unknown. Here, we used cell line models, cord blood cells, and thrombocytopenia samples, to detect the JMJD1C expression. ShRNA of JMJD1C and a specific peptide agonist of JMJD1C, SAH-JZ3, were used to explore the JMJD1C function in the cell line models. The actin ratio in megakaryopoiesis for the JMJDC modulation was also measured. Mass spectrometry was used to identify the JMJD1C-interacting proteins. We first show the JMJD1C expression difference in the PMA-induced cell line models, the thrombopoietin (TPO)-induced megakaryocyte differentiation of the cord blood cells, and also the thrombocytopenia patients, compared to the normal controls. The ShRNA of JMJD1C and SAH-JZ3 showed different effects, which were consistent with the expression of JMJD1C in the cell line models. The effort to find the underlying mechanism of JMJD1C in megakaryopoiesis, led to the discovery that SAH-JZ3 decreases F-actin in K562 cells and increases F-actin in MEG-01 cells. We further performed mass spectrometry to identify the potential JMJD1C-interacting proteins and found that the important Ran GTPase interacts with JMJD1C. To sum up, JMJD1C probably regulates megakaryopoiesis by influencing the actin network.

Bioinformatic Analyses of Broad H3K79me2 Domains in Different Leukemia Cell Line Data Sets.

A subset of expressed genes is associated with a broad H3K4me3 (histone H3 trimethylated at lysine 4) domain that extends throughout the gene body. Genes marked in this way in normal cells are involved in cell-identity and tumor-suppressor activities, whereas in cancer cells, genes driving the cancer phenotype (oncogenes) have this feature. Other histone modifications associated with expressed genes that display a broad domain have been less studied. Here, we identified genes with the broadest H3K79me2 (histone H3 dimethylated at lysine 79) domain in human leukemic cell lines representing different forms of leukemia. Taking a bioinformatic approach, we provide evidence that genes with the broadest H3K79me2 domain have known roles in leukemia (e.g., JMJD1C). In the mixed-lineage leukemia cell line MOLM-13, the HOXA9 gene is in a 100 kb broad H3K79me2 domain with other HOXA protein-coding and oncogenic long non-coding RNA genes. The genes in this domain contribute to leukemia. This broad H3K79me2 domain has an unstable chromatin structure, as was evident by enhanced chromatin accessibility throughout. Together, we provide evidence that identification of genes with the broadest H3K79me2 domain will aid in generating a panel of genes in the diagnosis and therapeutic treatment of leukemia in the future.

Lysine demethylase 1B (Kdm1b) enhances somatic reprogramming through inducing pluripotent gene expression and promoting cell proliferation.

Lysine demethylase 1B (Kdm1b) is known as an epigenetic modifier with demethylase activity against H3K4 and H3K9 histones and plays an important role in tumor progression and tumor stem cell enrichment. In this study, we attempted to elucidate the role of Kdm1b in somatic cell reprogramming. We found that exogenous expression of Kdm1b in human dermal fibroblasts (HDFs) can influence the epigenetic modifications of histones. Subsequent analysis further suggests that the overexpression of Kdm1b can promote cell proliferation, reprogram metabolism and inhibit cell apoptosis. In addition, a series of multipotent factors including Sox2 and Nanog, and several epigenetic factors that may reduce epigenetic barriers were upregulated to varying degrees. More importantly, HDFs transfected with the combination of Oct4 (POU5F1), Sox2, Klf4 and c-Myc and Kdm1b (OSKMK) achieved higher reprogramming efficiency. Therefore, we suggest that Kdm1b is an important epigenetic factor associated with pluripotency.

Jmjd1c demethylates STAT3 to restrain plasma cell differentiation and rheumatoid arthritis.

Appropriate regulation of B cell differentiation into plasma cells is essential for humoral immunity while preventing antibody-mediated autoimmunity; however, the underlying mechanisms, especially those with pathological consequences, remain unclear. Here, we found that the expression of Jmjd1c, a member of JmjC domain histone demethylase, in B cells but not in other immune cells, protected mice from rheumatoid arthritis (RA). In humans with RA, JMJD1C expression levels in B cells were negatively associated with plasma cell frequency and disease severity. Mechanistically, Jmjd1c demethylated STAT3, rather than histone substrate, to restrain plasma cell differentiation. STAT3 Lys140 hypermethylation caused by Jmjd1c deletion inhibited the interaction with phosphatase Ptpn6 and resulted in abnormally sustained STAT3 phosphorylation and activity, which in turn promoted plasma cell generation. Germinal center B cells devoid of Jmjd1c also acquired strikingly increased propensity to differentiate into plasma cells. STAT3 Lys140Arg point mutation completely abrogated the effect caused by Jmjd1c loss. Mice with Jmjd1c overexpression in B cells exhibited opposite phenotypes to Jmjd1c-deficient mice. Overall, our study revealed Jmjd1c as a critical regulator of plasma cell differentiation and RA and also highlighted the importance of demethylation modification for STAT3 in B cells.

A Novel Necroptosis-Related lncRNA Signature for Predicting Prognosis and Immune Response of Glioma.

Glioma is one of the most common intracranial malignancies that plagues people around the world. Despite current improvements in treatment, the prognosis of glioma is often unsatisfactory. Necroptosis is a form of programmed cell death. As research progresses, the role of necroptosis in tumors has gradually attracted the attention of researchers. And lncRNA is regarded as a critical role in the development of cancer. Therefore, this study is aimed at establishing a prognostic model based on necroptosis-associated lncRNAs to accurately assess the prognosis and immune response of patients with glioma. The RNA sequences of glioma patients and normal brain samples were downloaded from The Cancer Genome Atlas (TCGA) and GTEx databases, respectively. The coexpression analysis was performed to identify the necroptosis-related lncRNAs. Then, we utilized LASSO analysis following univariate Cox analysis to construct a prognostic model. Subsequently, we applied the Kaplan-Meier curve, time-dependent receiver operating characteristics (ROC), and univariate and multivariate Cox regression analyses to assess the effectiveness of this model. And the functional enrichment analyses and immune-related analyses were employed to investigate the potential biological functions. A validation set was obtained from the Chinese Glioma Genome Atlas (CGGA) database. And qRT-PCR was employed to further validate the expression levels of selected necroptosis-associated lncRNAs. Seven necroptosis-related lncRNAs (FAM13A-AS1, JMJD1C-AS1, LBX2-AS1, ZBTB20-AS4, HAR1A, SNHG14, and LINC00900) were determined to construct a prognostic model. The area under the ROC curve (AUC) was 0.871, 0.901, and 0.911 at 1, 2, and 3 years, respectively. The risk score was shown to be an important independent predictor in both univariate and multivariate Cox regression analyses. Through functional enrichment analyses, we found that the differentially expressed genes (DEGs) were mainly enriched in protein binding and signaling-related biological functions and immune-associated pathways. In conclusion, we established and validated a novel necroptosis-related lncRNA signature, which could accurately predict the overall survival of glioma patients and serve as potential therapeutic targets.

JMJD1C-regulated lipid synthesis contributes to the maintenance of MLL-rearranged acute myeloid leukemia.

Mixed Lineage Leukemia rearranged acute myeloid leukemia (MLLr AML) predicts a poor prognosis. Histone demethylase JMJD1C is a potential druggable target of MLLr AML. However, little is known about how JMJD1C contributes to MLLr AML. Here we found that JMJD1C regulates lipid synthesis-associated genes including FADS2, SCD in MLLr AML cells. Lipid synthesis-associated protein FABP5 was identified as a specific interacting protein of JMJD1C and binds to the jumonji domain of JMJD1C. FABP5 also regulates JMJD1C mRNA and protein expression. JDI-10, a small molecular inhibitor of JMJD1C identified by us, represses MLLr AML cells, induces apoptosis, and decreases JMJD1C-regulated lipid synthesis genes. Moreover, JDI-10 mediated suppression of MLLr AML cells can be rescued by palmitic acid, oleic acid, or recombinant FABP5. In summary, we identified that JMJD1C-regulated lipid synthesis contributes to the maintenance of MLLr AML. Lipid synthesis repression may represent a new direction for the treatment of MLLr AML.

Effects of codon optimization, N-terminal truncation and gene dose on the heterologous expression of berberine bridge enzyme.

Morphine, sanguinarine and chelerythrine are benzylisoquinoline alkaloids (BIAs), and these compounds possess strong biological activities. (S)-scoulerine is a commonly shared precursor of these compounds, and berberine bridge enzyme (BBE) is a key rate-limiting enzyme in the synthesis of (S)-scoulerine. We isolated the BBE gene from Macleaya cordata (McBBE) and used CEN.PK2-1C as a chassis strain. We compared the catalytic efficiency of five codon-optimized McBBE genes in Saccharomyces cerevisiae and finally obtained a yeast strain (YH03) that exhibited a 58-fold increase in yield (1.12 mg/L). Then, we truncated the N-terminus of McBBE by 8 and 22 amino acids and found that with the increase in the number of N-terminal truncated amino acids, the production of (S)-scoulerine gradually decreased. Additionally, we used CRISPR-Cas9 to integrate the McBBE gene at the delta site of the S. cerevisiae genome to achieve stable genetic inheritance and found that the yield of (S)-scoulerine was not significantly increased in the integrated strain. In conclusion, our work achieved high-efficiency expression of McBBE in S. cerevisiae, explored the influence of N-terminal truncation on the yield of (S)-scoulerine, and obtained a genetically stable S. cerevisiae strain with high McBBE expression. This study provides a reference for further complex metabolic engineering optimization and lays a foundation for the efficient biosynthesis of BIAs.

Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase.

N-demethylases have been reported to remove the methyl groups on primary or secondary amines, which could further affect the properties and functions of biomacromolecules or chemical compounds; however, the substrate scope and the robustness of N-demethylases have not been systematically investigated. Here we report the recreation of natural evolution in key microdomains of the Thermomicrobium roseum sarcosine oxidase (TrSOX), an N-demethylase with marked stability (melting temperature over 100 °C) and enantioselectivity, for enhanced substrate scope and catalytic efficiency on -C-N- bonds. We obtained the structure of TrSOX by crystallization and X-ray diffraction (XRD) for the initial framework. The natural evolution in the nonconserved residues of key microdomains-including the catalytic loop, coenzyme pocket, substrate pocket, and entrance site-was then identified using ancestral sequence reconstruction (ASR), and the substitutions that accrued during natural evolution were recreated by site-directed mutagenesis. The single and double substitution variants catalyzed the N-demethylation of N-methyl-L-amino acids up to 1800- and 6000-fold faster than the wild type, respectively. Additionally, these single substitution variants catalyzed the terminal N-demethylation of non-amino-acid compounds and the oxidation of the main chain -C-N- bond to a -C=N- bond in the nitrogen-containing heterocycle. Notably, these variants retained the enantioselectivity and stability of the initial framework. We conclude that the variants of TrSOX are of great potential use in N-methyl enantiomer resolution, main-chain Schiff base synthesis, and alkaloid modification or degradation.

Molecular insights into the unusually promiscuous and catalytically versatile Fe(II)/α-ketoglutarate-dependent oxygenase SptF.

Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis revealed that SptF catalyzes several continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced unique cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF revealed the molecular basis of the enzyme reactions, and suggested that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions.

Berberine bridge enzyme-like oxidase-catalysed double bond isomerization acts as the pathway switch in cytochalasin synthesis.

Cytochalasans (CYTs), as well as their polycyclic (pcCYTs) and polymerized (meCYTs) derivatives, constitute one of the largest families of fungal polyketide-nonribosomal peptide (PK-NRP) hybrid natural products. However, the mechanism of chemical conversion from mono-CYTs (moCYTs) to both pcCYTs and meCYTs remains unknown. Here, we show the first successful example of the reconstitution of the CYT core backbone as well as the whole pathway in a heterologous host. Importantly, we also describe the berberine bridge enzyme (BBE)-like oxidase AspoA, which uses Glu538 as a general acid biocatalyst to catalyse an unusual protonation-driven double bond isomerization reaction and acts as a switch to alter the native (for moCYTs) and nonenzymatic (for pcCYTs and meCYTs) pathways to synthesize aspochalasin family compounds. Our results present an unprecedented function of BBE-like enzymes and highly suggest that the isolated pcCYTs and meCYTs are most likely artificially derived products.

High-Throughput-Methyl-Reading (HTMR) assay: a solution based on nucleotide methyl-binding proteins enables large-scale screening for DNA/RNA methyltransferases and demethylases.

Epigenetic therapy has significant potential for cancer treatment. However, few small potent molecules have been identified against DNA or RNA modification regulatory proteins. Current approaches for activity detection of DNA/RNA methyltransferases and demethylases are time-consuming and labor-intensive, making it difficult to subject them to high-throughput screening. Here, we developed a fluorescence polarization-based 'High-Throughput Methyl Reading' (HTMR) assay to implement large-scale compound screening for DNA/RNA methyltransferases and demethylases-DNMTs, TETs, ALKBH5 and METTL3/METTL14. This assay is simple to perform in a mix-and-read manner by adding the methyl-binding proteins MBD1 or YTHDF1. The proteins can be used to distinguish FAM-labelled substrates or product oligonucleotides with different methylation statuses catalyzed by enzymes. Therefore, the extent of the enzymatic reactions can be coupled with the variation of FP binding signals. Furthermore, this assay can be effectively used to conduct a cofactor competition study. Based on the assay, we identified two natural products as candidate compounds for DNMT1 and ALKBH5. In summary, this study outlines a powerful homogeneous approach for high-throughput screening and evaluating enzymatic activity for DNA/RNA methyltransferases and demethylases that is cheap, easy, quick, and highly sensitive.

Physio-pathological effects of m6A modification and its potential contribution to melanoma

Methylation of N6-adenosine (m6A) is the most prevalent internal RNA modification and is especially common among the messenger RNAs. These m6A modifications regulate splicing, translocation, stability and translation of RNA through dynamic and reversible interactions with m6A-binding proteins, namely the writers, erasers and readers. RNA methyltransferases catalyze the m6A modifications, while demethylases reverse this methylation. Deregulation of the m6A modification process has been implicated in human carcinogenesis, including melanoma—which carries one of the highest mutant rates. In this review, we provide an up-to-date summary of m6A regulation and its biological impacts on normal and cancer cells, with emphasis on the deregulation of m6A modification and m6A regulators in melanoma. In addition, we highlight the prospective potential of exploiting m6A modification in the treatment of melanoma and non-cancer diseases. (AU)

The comprehensive interactomes of human adenosine RNA methyltransferases and demethylases reveal distinct functional and regulatory features.

N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two abundant modifications found in mRNAs and ncRNAs that can regulate multiple aspects of RNA biology. They function mainly by regulating interactions with specific RNA-binding proteins. Both modifications are linked to development, disease and stress response. To date, three methyltransferases and two demethylases have been identified that modify adenosines in mammalian mRNAs. Here, we present a comprehensive analysis of the interactomes of these enzymes. PCIF1 protein network comprises mostly factors involved in nascent RNA synthesis by RNA polymerase II, whereas ALKBH5 is closely linked with most aspects of pre-mRNA processing and mRNA export to the cytoplasm. METTL16 resides in subcellular compartments co-inhabited by several other RNA modifiers and processing factors. FTO interactome positions this demethylase at a crossroad between RNA transcription, RNA processing and DNA replication and repair. Altogether, these enzymes share limited spatial interactomes, pointing to specific molecular mechanisms of their regulation.

Histone demethylase JMJD1C promotes the polarization of M1 macrophages to prevent glioma by upregulating miR-302a.

Glioma is regarded as an aggressive lethal primary brain tumor. Jumonji domain containing 1C (JMJD1C) is a H3K9 demethylase which participates in the progression of various tumors, but its specific function and underlying mechanism in glioma development remain undefined, which is the purpose of our work. We initially assessed JMJD1C expression in glioma tissues and cells using the assays of RT-qPCR and immunohistochemistry. Meanwhile, the H3K9 level at the microRNA (miR)-302a promoter region was measured by chromatin immunoprecipitation assay, while luciferase-based reporter assay was performed for validation of the binding affinity between miR-302a and methyltransferase-like 3 (METTL3). The effect of METTL3 on suppressor of cytokine signaling 2 (SOCS2) was subsequently analyzed by MeRIP-RT-qPCR. Finally, a xenograft tumor model was established in nude mice, followed by measurement of tumor-associated macrophages using flow cytometry. JMJD1C was poorly expressed in glioma tissues. Furthermore, JMJD1C increased miR-302a expression through promoting H3K9me1 demethylation at the miR-302a promoter region. miR-302a was identified to target METTL3, which could inhibit SOCS2 expression via m6A modification. JMJD1C promoted M1 macrophage polarization and suppressed the growth of glioma xenografts through the miR-302a/METTL3/SOCS2 axis both in vivo and in vitro. In conclusion, JMJD1C could enhance M1 macrophage polarization to inhibit the onset of glioma, bringing a new insight into the contribution of JMJD1C to the pathobiology of glioma, with possible implications for targeted therapeutic method.

AR-negative prostate cancer is vulnerable to loss of JMJD1C demethylase.

Prostate cancer is a leading cause of cancer-related mortality in men. The widespread use of androgen receptor (AR) inhibitors has generated an increased incidence of AR-negative prostate cancer, triggering the need for effective therapies for such patients. Here, analysis of public genome-wide CRISPR screens in human prostate cancer cell lines identified histone demethylase JMJD1C (KDM3C) as an AR-negative context-specific vulnerability. Secondary validation studies in multiple cell lines and organoids, including isogenic models, confirmed that small hairpin RNA (shRNA)-mediated depletion of JMJD1C potently inhibited growth specifically in AR-negative prostate cancer cells. To explore the cooperative interactions of AR and JMJD1C, we performed comparative transcriptomics of 1) isogenic AR-positive versus AR-negative prostate cancer cells, 2) AR-positive versus AR-negative prostate cancer tumors, and 3) isogenic JMJD1C-expressing versus JMJD1C-depleted AR-negative prostate cancer cells. Loss of AR or JMJD1C generates a modest tumor necrosis factor alpha (TNFα) signature, whereas combined loss of AR and JMJD1C strongly up-regulates the TNFα signature in human prostate cancer, suggesting TNFα signaling as a point of convergence for the combined actions of AR and JMJD1C. Correspondingly, AR-negative prostate cancer cells showed exquisite sensitivity to TNFα treatment and, conversely, TNFα pathway inhibition via inhibition of its downstream effector MAP4K4 partially reversed the growth defect of JMJD1C-depleted AR-negative prostate cancer cells. Given the deleterious systemic side effects of TNFα therapy in humans and the viability of JMJD1C-knockout mice, the identification of JMJD1C inhibition as a specific vulnerability in AR-negative prostate cancer may provide an alternative drug target for prostate cancer patients progressing on AR inhibitor therapy.