Small Molecule-Inducible and Photoactivatable Cellular RNA N1-Methyladenosine Editing.

N1-methyladenosine (m1A) modification is one of the most prevalent epigenetic modifications on RNA. Given the vital role of m1A modification in RNA processing such as splicing, stability and translation, developing a precise and controllable m1A editing tool is pivotal for in-depth investigating the biological functions of m1A. In this study, we developed an abscisic acid (ABA)-inducible and reversible m1A demethylation tool (termed AI-dm1A), which targets specific transcripts by combining the chemical proximity-induction techniques with the CRISPR/dCas13b system and ALKBH3. We successfully employed AI-dm1A to selectively demethylate the m1A modifications at A8422 of MALAT1 RNA, and this demethylation process could be reversed by removing ABA. Furthermore, we validated its demethylation function on various types of cellular RNAs including mRNA, rRNA and lncRNA. Additionally, we used AI-dm1A to specifically demethylate m1A on ATP5D mRNA, which promoted ATP5D expression and enhanced the glycolysis activity of tumor cells. Conversely, by replacing the demethylase ALKBH3 with methyltransferase TRMT61A, we also developed a controllable m1A methylation tool, namely AI-m1A. Finally, we caged ABA by 4,5-dimethoxy-2-nitrobenzyl (DMNB) to achieve light-inducible m1A methylation or demethylation on specific transcripts. Collectively, our m1A editing tool enables us to flexibly study how m1A modifications on specific transcript influence biological functions and phenotypes.

Oxygen Self-Supplied Perfluorocarbon-Modified Micelles for Enhanced Cancer Photodynamic Therapy and Ferroptosis.

Photodynamic therapy (PDT) and ferroptosis show significant potential in tumor treatment. However, their therapeutic efficacy is often hindered by the oxygen-deficient tumor microenvironment and the challenges associated with efficient intracellular drug delivery into tumor cells. Toward this end, this work synthesized perfluorocarbon (PFC)-modified Pluronic F127 (PFC-F127), and then exploits it as a carrier for codelivery of photosensitizer Chlorin e6 (Ce6) and the ferroptosis promoter sorafenib (Sor), yielding an oxygen self-supplying nanoplatform denoted as Ce6-Sor@PFC-F127. The PFCs on the surface of the micelle play a crucial role in efficiently solubilizing and delivering oxygen as well as increasing the hydrophobicity of the micelle surface, giving rise to enhanced endocytosis by cancer cells. The incorporation of an oxygen-carrying moiety into the micelles enhances the therapeutic impact of PDT and ferroptosis, leading to amplified endocytosis and cytotoxicity of tumor cells. Hypotonic saline technology was developed to enhance the cargo encapsulation efficiency. Notably, in a murine tumor model, Ce6-Sor@PFC-F127 effectively inhibited tumor growth through the combined use of oxygen-enhanced PDT and ferroptosis. Taken together, this work underscores the promising potential of Ce6-Sor@PFC-F127 as a multifunctional therapeutic nanoplatform for the codelivery of multiple cargos such as oxygen, photosensitizers, and ferroptosis inducers.

N6-methyladenosine facilitates mitochondrial fusion of colorectal cancer cells via induction of GSH synthesis and stabilization of OPA1 mRNA.

Mitochondria undergo fission and fusion that are critical for cell survival and cancer development, while the regulatory factors for mitochondrial dynamics remain elusive. Herein we found that RNA m6A accelerated mitochondria fusion of colorectal cancer (CRC) cells. Metabolomics analysis and function studies indicated that m6A triggered the generation of glutathione (GSH) via the upregulation of RRM2B-a p53-inducible ribonucleotide reductase subunit with anti-reactive oxygen species potential. This in turn resulted in the mitochondria fusion of CRC cells. Mechanistically, m6A methylation of A1240 at 3'UTR of RRM2B increased its mRNA stability via binding with IGF2BP2. Similarly, m6A methylation of A2212 at the coding sequence (CDS) of OPA1-an essential GTPase protein for mitochondrial inner membrane fusion-also increased mRNA stability and triggered mitochondria fusion. Targeting m6A through the methyltransferase inhibitor STM2457 or the dm6ACRISPR system significantly suppressed mitochondria fusion. In vivo and clinical data confirmed the positive roles of the m6A/mitochondrial dynamics in tumor growth and CRC progression. Collectively, m6A promoted mitochondria fusion via induction of GSH synthesis and OPA1 expression, which facilitated cancer cell growth and CRC development.

METTL3 promotes cellular senescence of colorectal cancer via modulation of CDKN2B transcription and mRNA stability.

Cellular senescence plays a critical role in cancer development, but the underlying mechanisms remain poorly understood. Our recent study uncovered that replicative senescent colorectal cancer (CRC) cells exhibit increased levels of mRNA N6-methyladenosine (m6A) and methyltransferase METTL3. Knockdown of METTL3 can restore the senescence-associated secretory phenotype (SASP) of CRC cells. Our findings, which were confirmed by m6A-sequencing and functional studies, demonstrate that the cyclin-dependent kinase inhibitor 2B (CDKN2B, encoding p15INK4B) is a mediator of METTL3-regulated CRC senescence. Specifically, m6A modification at position A413 in the coding sequence (CDS) of CDKN2B positively regulates its mRNA stability by recruiting IGF2BP3 and preventing binding with the CCR4-NOT complex. Moreover, increased METTL3 methylates and stabilizes the mRNA of E2F1, which binds to the -208 to -198 regions of the CDKN2B promoter to facilitate transcription. Inhibition of METTL3 or specifically targeting CDKN2B methylation can suppress CRC senescence. Finally, the METTL3/CDKN2B axis-induced senescence can facilitate M2 macrophage polarization and is correlated with aging and CRC progression. The involvement of METTL3/CDKN2B in cell senescence provides a new potential therapeutic target for CRC treatment and expands our understanding of mRNA methylation's role in cellular senescence.

An Activatable Dual Polymer Nanosystem for Photoimmunotherapy and Metabolic Modulation of Deep-Seated Tumors.

Nanomedicine in combination with immunotherapy has shown great potential in the cancer treatment, but phototherapeutic nanomaterials that specifically activate the immunopharmacological effects in deep tumors have rarely been developed due to limited laser penetration depth and tumor immune microenvironment. Herein, this work reports a newly synthesized semiconducting polymer (SP) grafted with imiquimod R837 and indoxmid encapsulated micelle (SPRIN-micelle) with strong absorption in the second near infrared window (NIR-II) that can relieve tumor immunosuppression and enhance the photothermal immunotherapy and catabolic modulation on tumors. Immune agonists (Imiquimod R837) and immunometabolic modulators (indoxmid) are covalently attached to NIR-II SP sensors via a glutathione (GSH) responsive self-immolation linker and then loaded into Pluronic F127 (F127) micelles by a temperature-sensitive critical micelle concentration (CMC)-switching method. Using this method, photothermal effect of SPRIN-micelles in deep-seated tumors can be activated, leading to effective tumor ablation and immunogenic cell death (ICD). Meanwhile, imiquimod and indoxmid are tracelessly released in response to the tumor microenvironment, resulting in dendritic cell (DC) maturation by imiquimod R837 and inhibition of both indoleamine 2,3-dioxygenase (IDO) activity and Treg cell expression by indoxmid. Ultimately, cytotoxic T-lymphocyte infiltration and tumor metastasis inhibition in deep solid tumors (9 mm) are achieved. In summary, this work demonstrates a new strategy for the combination of photothermal immunotherapy and metabolic modulation by developing a dual functional polymer system including activable SP and temperature-sensitive F127 for the treatment of deep solid tumors.

Isothermal Amplification-Based Detection of Single-Base RNA N6-Methyladenosine.

N6-methyladenosine (m6A) has recently gained much attention due to its diverse biological functions. Currently, the commonly used detection methods for locus-specific m6A marks are complicated to operate, it is difficult to quantify the methylation level, and they have high false-positive levels. Here, we report a new method for locus-specific m6A detection based on the methylate-sensitive endonuclease activity of MazF and the simultaneous amplification and testing (SAT) method, termed "m6A-MazF-SAT". Mechanically, MazF fails to cleave the A (m6A) CA motif; therefore, the undigested template can be SAT-amplified using specific probes targeting the upstream and downstream of sites of interest. Fluorescent signals of SAT amplification can be detected by real-time PCR, and therefore, they achieve the detection of m6A existence. After the condition optimization, m6A-MazF-SAT can significantly, accurately, and rapidly detect the m6A-modified sites in mRNA, rRNA, and lncRNA at the fmol level, as well as 10% m6A at the fmol level. In addition, m6A-MazF-SAT can quantify the abundance of target m6A in biological samples and can be used for the inhibitor selection of m6A-related enzymes. Together, we offer a new approach to detect locus-specific m6A both qualitatively and quantitatively; it is easy to operate, results can be obtained rapidly, and it has low false-positive levels and high repeatability.

MoS2-Based Memristor: Robust Resistive Switching Behavior and Reliable Biological Synapse Emulation.

Memristors are recognized as crucial devices for future nonvolatile memory and artificial intelligence. Due to their typical neuron-synapse-like metal-insulator-metal(MIM) sandwich structure, they are widely used to simulate biological synapses and have great potential in advancing biological synapse simulation. However, the high switch voltage and inferior stability of the memristor restrict the broader application to the emulation of the biological synapse. In this study, we report a vertically structured memristor based on few-layer MoS2. The device shows a lower switching voltage below 0.6 V, with a high ON/OFF current ratio of 104, good stability of more than 180 cycles, and a long retention time exceeding 3 × 103 s. In addition, the device has successfully simulated various biological synaptic functions, including potential/depression propagation, paired-pulse facilitation (PPF), and long-term potentiation/long-term depression (LTP/LTD) modulation. These results have significant implications for the design of a two-dimensional transition-metal dichalcogenides composite material memristor that aim to mimic biological synapses, representing promising avenues for the development of advanced neuromorphic computing systems.

Orally-Delivered, Cytokine-Engineered Extracellular Vesicles for Targeted Treatment of Inflammatory Bowel Disease.

The use of orally-administered therapeutic proteins for treatment of inflammatory bowel disease (IBD) has been limited due to the harsh gastrointestinal environment and low bioavailability that affects delivery to diseased sites. Here, a nested delivery system, termed Gal-IL10-EVs (C/A) that protects interleukin 10 (IL-10) from degradation in the stomach and enables targeted delivery of IL-10 to inflammatory macrophages infiltrating the colonic lamina propria, is reported. Extracellular vesicles (EVs) carrying IL-10 are designed to be secreted from genetically engineered mammalian cells by a plasmid system, and EVs are subsequently modified with galactose, endowing the targeted IL-10 delivery to inflammatory macrophages. Chitosan/alginate (C/A) hydrogel coating on Gal-IL10-EVs enables protection from harsh conditions in the gastrointestinal tract and favorable delivery to the colonic lumen, where the C/A hydrogel coating is removed at the diseased sites. Gal-IL10-EVs control the production of reactive oxygen species (ROS) and inhibit the expression of proinflammatory cytokines. In a murine model of colitis, Gal-IL10-EVs (C/A) alleviate IBD symptoms including inflammatory responses and disrupt colonic barriers. Taken together, Gal-IL10-EVs (C/A) features biocompatibility, pH-responsive drug release, and macrophage-targeting as a therapeutic platform for oral delivery of bioactive proteins for treating intestinal diseases.

Anti-Tumor Immunity Induced by a Ternary Membrane System Derived From Cancer Cells, Dendritic Cells, and Bacteria.

Cancer vaccines generally are limited by insufficient tumor-specific cellular immunogenicity. Herein, a potent "ABC" ternary membrane-derived vaccine system blended from antigen-presenting mature dendritic cell membranes ("A"), bacterial E. coli cytoplasmic membranes ("B"), and cancer cell membranes ("C") is developed using a block-copolymer micelle-enabled approach. The respective ABC membrane components provide for a source of cellular immune communication/activation and enhanced accumulation in lymph nodes (A), immunological adjuvant (B), and tumor antigens (C). The introduction of dendritic cell (DC) membranes enables multiple cell-to-cell communication and powerful immune activation. ABC activates dendritic cells and promotes T-cell activation and proliferation in vitro. In vivo, ABC is 14- and 304-fold more immunogenic than binary (BC) and single (C) membrane vaccines, and immunization with ABC enhances the frequency of tumor-specific cytotoxic T lymphocytes, leading to an 80% cure rate in tumor-bearing mice. In a surgical resection and recurrence model, ABC prevents recurrence with vaccination from autologous cancer membranes, and therapeutic effects are observed in a lung metastasis model even with heterologous cancer cell membranes. ABCs formed from human cancer patient-derived tumor cells activate human monocyte-derived dendritic cells (moDC). Taken together, the ternary ABC membrane system provides the needed functional components for personalized cancer immunotherapy.

N6-Methyladenosine Promotes Translation of VEGFA to Accelerate Angiogenesis in Lung Cancer.

Angiogenesis is hijacked by cancer to support tumor growth. RNA modifications such as N6-methyladenosine (m6A) can regulate several aspects of cancer, including angiogenesis. Here, we find that m6A triggers angiogenesis in lung cancer by upregulating VEGFA, a central regulator of neovasculature and blood vessel growth. m6A-sequencing and functional studies confirmed that m6A modification of the 5'UTR (untranslated region) of VEGFA positively regulates its translation. Specifically, methylation of a 5'UTR internal ribosome entry site (IRES) recruited the YTHDC2/eIF4GI complex to trigger cap-independent translation initiation. Intriguingly, the m6A methylation site A856 of the 5'UTR was located within the conserved upstream open reading frame (uORF) of VEGFA IRES-A, which overcomes uORF-mediated translation suppression while facilitating G-quadruplex-induced translation of VEGFA. Targeted specific demethylation of VEGFA m6A significantly decreased expression of VEGFA and reduced lung cancer cell-driven angiogenesis. In vivo and clinical data confirmed the positive effects of m6A modification of VEGFA on angiogenesis and tumor growth of lung cancer. This study not only reveals that the m6A/VEGFA axis is a potential target for lung cancer therapy but also expands our understanding of the impact of m6A modification of IRES in the 5'UTR of mRNA on translation regulation. SIGNIFICANCE: Methylation of the 5'UTR IRES of VEGFA mRNA increases cap-independent translation via recruitment of the YTHDC2/eIF4GI complex, which stimulates angiogenesis to promote lung tumor growth.

Rolling circle extension-assisted loop-mediated isothermal amplification (Rol-LAMP) method for locus-specific and visible detection of RNA N6-methyladenosine.

N6-methyladenosine (m6A) is the most prevalent RNA modification in eukaryotic mRNAs. Currently available detection methods for locus-specific m6A marks rely on RT-qPCR, radioactive methods, or high-throughput sequencing. Here, we develop a non-qPCR, ultrasensitive, isothermal, and naked-eye visible method for m6A detection based on rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP), named m6A-Rol-LAMP, to verify putative m6A sites in transcripts obtained from the high-throughput data. When padlock probes hybridize to the potential m6A sites on targets, they are converted to circular form by DNA ligase in the absence of m6A modification, while m6A modification hinders the sealing of padlock probes. Subsequently, Bst DNA polymerase-mediated RCA and LAMP allow the amplification of the circular padlock probe to achieve the locus-specific detection of m6A. Following optimization and validation, m6A-Rol-LAMP can ultra-sensitively and quantitatively determine the existence of m6A modification on a specific target site as low as 100 amol under isothermal conditions. Detections of m6A can be performed on rRNA, mRNA, lincRNA, lncRNA and pre-miRNA from biological samples with naked-eye observations after dye incubation. Together, we provide a powerful tool for locus-specific detection of m6A, which can simply, quickly, sensitively, specifically, and visually determine putative m6A modification on RNA.

Metal coordination micelles for anti-cancer treatment by gene-editing and phototherapy.

CRISPR-Cas9 is a central focus of the emerging field of gene editing and photodynamic therapy (PDT) is a clinical-stage ablation modality combining photosensitizers with light irradiation. But metal coordination biomaterials for the applications of both have rarely been investigated. Herein, Chlorin-e6 (Ce6) Manganese (Mn) coordination micelles loaded with Cas9, termed Ce6-Mn-Cas9, were developed for augmented combination anti-cancer treatment. Manganese played multiple roles to facilitate Cas9 and single guide RNA (sgRNA) ribonucleoprotein (RNP) delivery, Fenton-like effect, and enhanced endonuclease activity of RNP. Histidine (His)-tagged RNP could be coordinated to Ce6 encapsulated in Pluronic F127 (F127) micelles by simple admixture. Triggered by ATP and endolysosomal acidic pH, Ce6-Mn-Cas9 released Cas9 without altering protein structure or function. Dual guide RNAs were designed to target the antioxidant regulator MTH1 and the DNA repair protein APE1, resulting in increased oxygen and enhanced PDT effect. In a murine tumor model, Ce6-Mn-Cas9 inhibited tumor growth with the combination therapy of PDT and gene editing. Taken together, Ce6-Mn-Cas9 represents a new biomaterial with a high degree of versatility to enable photo- and gene-therapy approaches.

RNA m6 A methylation in cancer.

N6 -methyladenosine (m6 A) is one of the most abundant internal modifications in eukaryotic messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). It is a reversible and dynamic RNA modification that has been observed in both internal coding segments and untranslated regions. Studies indicate that m6 A modifications play important roles in translation, RNA splicing, export, degradation and ncRNA processing control. In this review, we focus on the profiles and biological functions of RNA m6 A methylation on both mRNAs and ncRNAs. The dynamic modification of m6 A and its potential roles in cancer development are discussed. Moreover, we discuss the possibility of m6 A modifications serving as potential biomarkers for cancer diagnosis and targets for therapy.

N1-methyladenosine modification in cancer biology: Current status and future perspectives.

Post-transcriptional modifications in RNAs regulate their biological behaviors and functions. N1-methyladenosine (m1A), which is dynamically regulated by writers, erasers and readers, has been found as a reversible modification in tRNA, mRNA, rRNA and long non-coding RNA (lncRNA). m1A modification has impacts on the RNA processing, structure and functions of targets. Increasing studies reveal the critical roles of m1A modification and its regulators in tumorigenesis. Due to the positive relevance between m1A and cancer development, targeting m1A modification and m1A-related regulators has been of attention. In this review, we summarized the current understanding of m1A in RNAs, covering the modulation of m1A modification in cancer biology, as well as the possibility of targeting m1A modification as a potential target for cancer diagnosis and therapy.

Manganese Coordination Micelles That Activate Stimulator of Interferon Genes and Capture In Situ Tumor Antigens for Cancer Metalloimmunotherapy.

Cancer immunotherapy holds great promise but is generally limited by insufficient induction of anticancer immune responses. Here, a metal micellar nanovaccine is developed by the self-assembly of manganese (Mn), a stimulator of interferon genes (STING) agonist (ABZI) and naphthalocyanine (ONc) coordinated nanoparticles (ONc-Mn-A) in maleimide-modified Pluronic F127 (malF127) micelles. Owing to synergy between Mn and ABZI, the nanovaccine, termed ONc-Mn-A-malF127, elevates levels of interferon-ß (IFNß) by 324- and 8-fold in vivo, compared to use of Mn or ABZI alone. As such, the activation of the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-STING pathway induces sufficient dendritic cell (DC) maturation, eventually resulting in the death of CD8+ T cell-sensitive tumors and CD8+ T cell-resistant tumors by simultaneously promoting cytotoxic CD8+ T cells and NK cells, respectively. Furthermore, with ONc used as a Mn chelator and an efficient photosensitizer, photoinduced immunogenic cell death (ICD) of tumor cells releases damage-associated molecular patterns (DAMPs) and neoantigens from dying primary tumor cells upon laser irradiation, which are captured in situ by malF127 in tumor cells and then transported to DCs. After laser treatment, in addition to the photothermal therapy, immune responses characterized by the level of IFNß are further elevated by another 4-fold. In murine cancer models, ICD-based metalloimmunotherapy using the ONc-Mn-A-malF127 nanovaccine in a single dose by intravenous injection achieved eradication of primary and distant tumors. Taken together, ONc-Mn-A-malF127 offers a nanoplatform to enhance anticancer efficacy by metalloimmunotherapy and photoinduced ICD based immunotherapy with strong abscopal effect.

Colistin Crosslinked Gemcitabine Micelles to Eliminate Tumor Drug Resistance Caused by Intratumoral Microorganisms.

In the tumor microenvironment, there exist microorganisms that metabolize anticancer drugs, leading to chemotherapy failure. To solve this problem, herein, we develop antibiotic and anticancer drug co-delivery micelles, termed colistin crosslinked gemcitabine micelle (CCGM). A self-immolative linker enables colistin and gemcitabine to be released on demand without affecting their antibacterial and anticancer effects. Once CCGM is delivered to the tumor microenvironment, intracellular glutathione triggers the release of colistin and gemcitabine, inhibiting the growth of microbes in the tumor, thus eliminating the microbe-induced drug resistance of tumor.

RNA m1A methylation regulates glycolysis of cancer cells through modulating ATP5D.

Studies on biological functions of RNA modifications such as N6-methyladenosine (m6A) in mRNA have sprung up in recent years, while the roles of N1-methyladenosine (m1A) in cancer progression remain largely unknown. We find m1A demethylase ALKBH3 can regulate the glycolysis of cancer cells via a demethylation activity dependent manner. Specifically, sequencing and functional studies confirm that ATP5D, one of the most important subunit of adenosine 5'-triphosphate synthase, is involved in m1A demethylase ALKBH3-regulated glycolysis of cancer cells. The m1A modified A71 at the exon 1 of ATP5D negatively regulates its translation elongation via increasing the binding with YTHDF1/eRF1 complex, which facilitates the release of message RNA (mRNA) from ribosome complex. m1A also regulates mRNA stability of E2F1, which directly binds with ATP5D promoter to initiate its transcription. Targeted specific demethylation of ATP5D m1A by dm1ACRISPR system can significantly increase the expression of ATP5D and glycolysis of cancer cells. In vivo data confirm the roles of m1A/ATP5D in tumor growth and cancer progression. Our study reveals a crosstalk of mRNA m1A modification and cell metabolism, which expands the understanding of such interplays that are essential for cancer therapeutic application.

A novel inhibitor of N 6-methyladenosine demethylase FTO induces mRNA methylation and shows anti-cancer activities.

N 6-methyladenosine (m6A) modification is critical for mRNA splicing, nuclear export, stability and translation. Fat mass and obesity-associated protein (FTO), the first identified m6A demethylase, is critical for cancer progression. Herein, we developed small-molecule inhibitors of FTO by virtual screening, structural optimization, and bioassay. As a result, two FTO inhibitors namely 18077 and 18097 were identified, which can selectively inhibit demethylase activity of FTO. Specifically, 18097 bound to the active site of FTO and then inhibited cell cycle process and migration of cancer cells. In addition, 18097 reprogrammed the epi-transcriptome of breast cancer cells, particularly for genes related to P53 pathway. 18097 increased the abundance of m6A modification of suppressor of cytokine signaling 1 (SOCS1) mRNA, which recruited IGF2BP1 to increase mRNA stability of SOCS1 and subsequently activated the P53 signaling pathway. Further, 18097 suppressed cellular lipogenesis via downregulation of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and C/EBPß. Animal studies confirmed that 18097 can significantly suppress in vivo growth and lung colonization of breast cancer cells. Collectively, we identified that FTO can work as a potential drug target and the small-molecule inhibitor 18097 can serve as a potential agent against breast cancer.

Effective Genome Editing Using CRISPR-Cas9 Nanoflowers.

CRISPR-Cas9 as a powerful gene-editing tool has tremendous potential for the treatment of genetic diseases. Herein, a new mesoporous nanoflower (NF)-like delivery nanoplatform termed Cas9-NF is reported by crosslinking Cas9 and polymeric micelles that enables efficient intracellular delivery and controlled release of Cas9 in response to reductive microenvironment in tumor cells. The flower morphology is flexibly tunable by the protein concentration and different types of crosslinkers. Cas9 protein, embedded between polymeric micelles and protected by Cas9-NF, remains stable even under extreme pH conditions. Responsive cleavage of crosslinkers in tumor cells, leads to the traceless release of Cas9 for efficient gene knockout in nucleus. This crosslinked nanoparticle exhibits excellent capability of downregulating oncogene expression and inhibiting tumor growth in a murine tumor model. Taken together, these findings pave a new pathway toward the application of the protein-micelle crosslinked nanoflower for protein delivery, which warrants further investigations for gene regulation and cancer treatment.