Fresh Insights Into SLC25A26: Potential New Therapeutic Target for Cancers: A Review.

SLC25A26 is the only known human mitochondrial S-adenosylmethionine carrier encoding gene. Recent studies have shown that SLC25A26 is abnormally expressed in some cancers, such as cervical cancer, low-grade glioma, non-small cell lung cancer, and liver cancer, which suggests SLC25A26 can affect the occurrence and development of some cancers. This article in brief briefly reviewed mitochondrial S-adenosylmethionine carrier in different species and its encoding gene, focused on the association of SLC25A26 aberrant expression and some cancers as well as potential mechanisms, summarized its potential for cancer prognosis, and characteristics of mitochondrial diseases caused by SLC25A26 mutation. Finally, we provide a brief expectation that needs to be further investigated. We speculate that SLC25A26 will be a potential new therapeutic target for some cancers.

Circulating Tumor DNA Profiling Approach Based on In Silico Background Elimination Guides Chemotherapy in Nasopharyngeal Carcinoma.

Circulating tumor DNA (ctDNA) analysis increasingly provides a promising minimally invasive alternative to tissue biopsies in precision oncology. However, there are no ctDNA analysis approaches available in nasopharyngeal carcinoma (NPC) and current methods of ctDNA mutation profiling have limited resolution because of the high background noise and false-positive rate caused by benign variants in plasma cell-free DNA (cfDNA), majorly generated during clonal hematopoiesis. Although personalized parallel white blood cell genome sequencing suppresses the noise of clonal hematopoiesis variances, the system cost and complexity restrict its extensive application in clinical settings. We developed Matched WBC Genome sequencing Independent CtDNA profiling (MaGIC) approaches, which synergically integrated a ctDNA capturing panel for a hybrid capture cfDNA deep sequencing, in silico background elimination, and a reliable readout measurement. We profiled the ctDNAs of 80 plasma samples from 40 patients with NPC before and during chemotherapy by MaGICs. In addition, the public cfDNA sequencing data and The Cancer Genome Atlas project data were analyzed by MaGICs to evaluate their application in other scenarios of patient classification. The MaGIC version-2 has the ability to predict the chemosensitivity of patients with NPC with high accuracy by utilizing a single sample of liquid biopsy from each patient prior to a standardized treatment regimen. Moreover, both versions of MaGICs are of ideal performance in the diagnosis of patients with prostate cancer by liquid biopsy and prognosis prediction of multiple cancers by tissue biopsy. This study has the potential to enhance the sensitivity and expand the application scope of ctDNA detection, independently of other paired genome sequencing methods. As a result, it might further increase the clinical utilization of liquid biopsy based on ctDNA.

RNF8 depletion attenuates hepatocellular carcinoma progression by inhibiting epithelial-mesenchymal transition and enhancing drug sensitivity.

Despite substantial advances that have been made in understanding the etiology of hepatocellular carcinoma (HCC), the early-stage diagnosis and treatment of advanced-stage HCC remain a major challenge. RNF8, an E3 ligase important for the DNA damage response, has been proven to facilitate the progression of breast and lung cancer, but its role in HCC remains unclear. In this study, we find that the expression of RNF8 is up-regulated in HCC tissues and positively correlated with poor prognosis of HCC. Furthermore, silencing RNF8 by siRNAs attenuates the migration of HCC cells and inhibits epithelial-mesenchymal transition (EMT) by regulating the expressions of proteins including N-cadherin, ß-catenin, snail, and ZO-1. Moreover, Kaplan‒Meier survival analysis shows that high RNF8 expression predicts poor survival benefits from sorafenib. Finally, cell viability assay demonstrates that RNF8 depletion enhances the sensitivity of HCC cells to sorafenib and lenvatinib treatment. We hypothesize that the inhibitory role of RNF8 in EMT and its enhancing effects on anti-cancer drugs orchestrate the protective effects of RNF8 deficiency in HCC, which indicates its potential in clinical application.

Zbtb46 Controls Dendritic Cell Activation by Reprogramming Epigenetic Regulation of cd80/86 and cd40 Costimulatory Signals in a Zebrafish Model.

The establishment of an appropriate costimulatory phenotype is crucial for dendritic cells (DCs) to maintain a homeostatic state with optimal immune surveillance and immunogenic activities. The upregulation of CD80/86 and CD40 is a hallmark costimulatory phenotypic switch of DCs from a steady state to an activated one for T cell activation. However, knowledge of the regulatory mechanisms underlying this process remains limited. In this study, we identified a Zbtb46 homolog from a zebrafish model. Zbtb46 deficiency resulted in upregulated cd80/86 and cd40 expression in kidney marrow-derived DCs (KMDCs) of zebrafish, which was accompanied with a remarkable expansion of CD4+/CD8+ T cells and accumulation of KMDCs in spleen of naive fish. Zbtb46 -/- splenic KMDCs exhibited strong stimulatory activity for CD4+ T cell activation. Chromatin immunoprecipitation-quantitative PCR and mass spectrometry assays showed that Zbtb46 was associated with promoters of cd80/86 and cd40 genes by binding to a 5'-TGACGT-3' motif in resting KMDCs, wherein it helped establish a repressive histone epigenetic modification pattern (H3K4me0/H3K9me3/H3K27me3) by organizing Mdb3/organizing nucleosome remodeling and deacetylase and Hdac3/nuclear receptor corepressor 1 corepressor complexes through the recruitment of Hdac1/2 and Hdac3. On stimulation with infection signs, Zbtb46 disassociated from the promoters via E3 ubiquitin ligase Cullin1/Fbxw11-mediated degradation, and this reaction can be triggered by the TLR9 signaling pathway. Thereafter, cd80/86 and cd40 promoters underwent epigenetic reprogramming from the repressed histone modification pattern to an activated pattern (H3K4me3/H3K9ac/H3K27ac), leading to cd80/86 and cd40 expression and DC activation. These findings revealed the essential role of Zbtb46 in maintaining DC homeostasis by suppressing cd80/86 and cd40 expression through epigenetic mechanisms.

Application of Data Science in Circulating Tumor DNA Detection: A Promising Avenue Towards Liquid Biopsy.

The circulating tumor DNA (ctDNA), as a promising biomarker of liquid biopsy, has potential clinical relevance on the molecular diagnosis and monitoring of cancer. However, the trace concentration level of ctDNA in the peripheral blood restricts its extensive clinical application. Recently, high-throughput-based methodologies have been leveraged to improve the sensitivity and specificity of ctDNA detection, showing a promising avenue towards liquid biopsy. This review briefly summarizes the high-throughput data features concerned by current ctDNA detection strategies and the technical obstacles, potential solutions, and clinical relevance of current ctDNA profiling technologies. We also highlight future directions improving the limit of detection of ctDNA for better clinical application. This review may serve as a reference for the crosslinks between data science and ctDNA-based liquid biopsy, benefiting clinical translation in advanced cancer diagnosis.

Attenuation of Antiviral Immune Response Caused by Perturbation of TRIM25-Mediated RIG-I Activation under Simulated Microgravity.

Microgravity is a major environmental factor of space flight that triggers dysregulation of the immune system and increases clinical risks for deep-space-exploration crews. However, systematic studies and molecular mechanisms of the adverse effects of microgravity on the immune system in animal models are limited. Here, we establish a ground-based zebrafish disease model of microgravity for the research of space immunology. RNA sequencing analysis demonstrates that the retinoic-acid-inducible gene (RIG)-I-like receptor (RLR) and the Toll-like receptor (TLR) signaling pathways are significantly compromised by simulated microgravity (Sµg). TRIM25, an essential E3 for RLR signaling, is inhibited under Sµg, hampering the K63-linked ubiquitination of RIG-I and the following function-induction positive feedback loop of antiviral immune response. These mechanisms provide insights into better understanding of the effects and principles of microgravity on host antiviral immunity and present broad potential implications for developing strategies that can prevent and control viral diseases during space flight.

CRISPR-GEMM Pooled Mutagenic Screening Identifies KMT2D as a Major Modulator of Immune Checkpoint Blockade.

Immune checkpoint blockade (ICB) has shown remarkable clinical efficacy in several cancer types. However, only a fraction of patients will respond to ICB. Here, we performed pooled mutagenic screening with CRISPR-mediated genetically engineered mouse models (CRISPR-GEMM) in ICB settings, and identified KMT2D as a major modulator of ICB response across multiple cancer types. KMT2D encodes a histone H3K4 methyltransferase and is among the most frequently mutated genes in patients with cancer. Kmt2d loss led to increased DNA damage and mutation burden, chromatin remodeling, intron retention, and activation of transposable elements. In addition, Kmt2d-mutant cells exhibited increased protein turnover and IFNγ-stimulated antigen presentation. In turn, Kmt2d-mutant tumors in both mouse and human were characterized by increased immune infiltration. These data demonstrate that Kmt2d deficiency sensitizes tumors to ICB by augmenting tumor immunogenicity, and also highlight the power of CRISPR-GEMMs for interrogating complex molecular landscapes in immunotherapeutic contexts that preserve the native tumor microenvironment. SIGNIFICANCE: ICB is ineffective in the majority of patients. Through direct in vivo CRISPR mutagenesis screening in GEMMs of cancer, we find Kmt2d deficiency sensitizes tumors to ICB. Considering the prevalence of KMT2D mutations, this finding potentially has broad implications for patient stratification and clinical decision-making.This article is highlighted in the In This Issue feature, p. 1775.

Multiplexed activation of endogenous genes by CRISPRa elicits potent antitumor immunity.

Immunotherapy has transformed cancer treatment. However, current immunotherapy modalities face various limitations. In the present study, we developed multiplexed activation of endogenous genes as an immunotherapy (MAEGI), a new form of immunotherapy that elicits antitumor immunity through multiplexed activation of endogenous genes in tumors. We leveraged CRISPR activation (CRISPRa) to directly augment the in situ expression of endogenous genes, and thereby the presentation of tumor antigens, leading to dramatic antitumor immune responses. Deploying this as a cell-based vaccination strategy showed efficacy in both prophylactic and therapeutic settings. Intratumoral adeno-associated virus delivery of CRISPRa libraries elicited strong antitumor immunity across multiple cancer types. Precision targeting of mutated gene sets eradicated a large fraction of established tumors at both local and distant sites. This treatment modality led to alterations in the tumor microenvironment, marked by enhanced T cell infiltration and antitumor immune signatures. Multiplexed endogenous gene activation is a versatile and highly scalable strategy to elicit potent immune responses against cancer, distinct from all existing cancer therapies.

Systematic Immunotherapy Target Discovery Using Genome-Scale In Vivo CRISPR Screens in CD8 T Cells.

CD8 T cells play essential roles in anti-tumor immune responses. Here, we performed genome-scale CRISPR screens in CD8 T cells directly under cancer immunotherapy settings and identified regulators of tumor infiltration and degranulation. The in vivo screen robustly re-identified canonical immunotherapy targets such as PD-1 and Tim-3, along with genes that have not been characterized in T cells. The infiltration and degranulation screens converged on an RNA helicase Dhx37. Dhx37 knockout enhanced the efficacy of antigen-specific CD8 T cells against triple-negative breast cancer in vivo. Immunological characterization in mouse and human CD8 T cells revealed that DHX37 suppresses effector functions, cytokine production, and T cell activation. Transcriptomic profiling and biochemical interrogation revealed a role for DHX37 in modulating NF-κB. These data demonstrate high-throughput in vivo genetic screens for immunotherapy target discovery and establishes DHX37 as a functional regulator of CD8 T cells.

Avenues Toward microRNA Detection In Vitro: A Review of Technical Advances and Challenges.

Over the decades, the biological role of microRNAs (miRNAs) in the post-transcriptional regulation of gene expression has been discovered in many cancer types, thus initiating the tremendous expectation of their application as biomarkers in the diagnosis, prognosis, and treatment of cancer. Hence, the development of efficient miRNA detection methods in vitro is in high demand. Extensive efforts have been made based on the intrinsic properties of miRNAs, such as low expression levels, high sequence homology, and short length, to develop novel in vitro miRNA detection methods with high accuracy, low cost, practicality, and multiplexity at point-of-care settings. In this review, we mainly summarized the newly developed in vitro miRNA detection methods classified by three key elements, including biological recognition elements, additional micro-/nano-materials and signal transduction/readout elements, their current challenges and further applications are also discussed.

Highly Effective and Low-Cost MicroRNA Detection with CRISPR-Cas9.

MicroRNAs have been reported as related to multiple diseases and have potential applications in diagnosis and therapeutics. However, detection of miRNAs remains improvable, given their complexity, high cost, and low sensitivity as of currently. In this study, we attempt to build a novel platform that detects miRNAs at low cost and high efficacy. This detection system contains isothermal amplification, detecting and reporting process based on rolling circle amplification, CRISPR-Cas9, and split-horseradish peroxidase techniques. It is able to detect trace amount of miRNAs from samples with mere single-base specificity. Moreover, we demonstrated that such scheme can effectively detect target miRNAs in clinical serum samples and significantly distinguish patients of non-small cell lung cancer from healthy volunteers by detecting the previously reported biomarker: circulating let-7a. As the first to use CRISPR-Cas9 in miRNA detection, this method is a promising approach capable of being applied in screening, diagnosing, and prognosticating of multiple diseases.

Evolutionary implication of B-1 lineage cells from innate to adaptive immunity.

The paradigm that B cells mainly play a central role in adaptive immunity may have to be reevaluated because B-1 lineage cells have been found to exhibit innate-like functions, such as phagocytic and bactericidal activities. Therefore, the evolutionary connection of B-1 lineage cells between innate and adaptive immunities have received much attention. In this review, we summarized various innate-like characteristics of B-1 lineage cells, such as natural antibody production, antigen-presenting function in primary adaptive immunity, and T cell-independent immune responses. These characteristics seem highly conserved between fish B cells and mammalian B-1 cells during vertebrate evolution. We proposed an evolutionary outline of B cells by comparing biological features, including morphology, phenotype, ontogeny, and functional activity between B-1 lineage cells and macrophages or B-2 cells. The B-1 lineage may be a transitional cell type between phagocytic cells (e.g., macrophages) and B-2 cells that functionally connects innate and adaptive immunities. Our discussion would contribute to the understanding on the origination of B cells specialized in adaptive immunity from innate immunity. The results might provide further insight into the evolution of the immune system as a whole.

Characterization of surface phenotypic molecules of teleost dendritic cells.

Dendritic cells (DCs) are among the most important professional antigen-presenting cells (APCs) that participate in various biological activities in mammals. However, evidence of the existence of DCs in teleost fish and other lower vertebrates remains limited. In this study, phenotypic and functional characteristics of teleost DCs were described in a zebrafish model. An improved method to efficiently enrich DCs was established. Immunofluorescence staining revealed that the surface phenotypic hallmarks of mammalian DCs, including MHC-II, CD80/86, CD83, and CD209, were distributed on the surfaces of zebrafish DCs (DrDCs). Functional analysis results showed that DrDCs could initiate antigen-specific CD4(+) T cell activation, in which MHC-II, CD80/86, CD83, and CD209 are implicated. Hence, teleost DCs exhibit conserved immunophenotypes and functions similar to those of their mammalian counterparts. Our findings contributed to the current understanding of the evolutionary history of DCs and the DC-regulatory mechanisms of adaptive immunity.

B cells in teleost fish act as pivotal initiating APCs in priming adaptive immunity: an evolutionary perspective on the origin of the B-1 cell subset and B7 molecules.

The long-held paradigm that B cells cannot uptake nonspecific particulate Ags for the initiation of primary adaptive immunity has been challenged by the recent discovery that teleost B cells have potent phagocytic and microbicidal abilities. This discovery provides preliminary clues that primitive B cells might act as initiating APCs in priming adaptive immunity. In this study, zebrafish B cells clearly showed a potent Ag-presenting ability to both soluble Ags and bacterial particles to prime naive CD4(+) T cell activation. This finding demonstrates the innate-like nature of teleost B cells in the interface of innate and adaptive immunity, indicating that they might consist of a major population of initiating APCs whose performance is similar to that of dendritic cells. Given the functional similarities between teleost B cells and the mammalian B-1 subset, we hypothesize that B-1 lineage and teleost B cells might originate from a common ancestor with potent phagocytic and initiating APC capacities. In addition, CD80/86 and CD83 costimulatory signals were identified as being essential for B cell-initiated adaptive immunity. This result suggests that the costimulatory mechanism originated as early as the origin of adaptive immunity and is conserved throughout vertebrate evolution. In fish, only a single CD80/86 copy exists, which is similar to mammalian CD86 rather than to CD80. Thus, CD86 might be a more primordial B7 family member that originated from fish. This study provides valuable insights into the evolutionary history of professional APCs, B cell lineages, and the costimulatory mechanism underlying adaptive immunity as a whole.

Conserved inhibitory role of teleost SOCS-1s in IFN signaling pathways.

The suppressor of cytokine signaling 1 (SOCS-1) protein is a critical regulator in the immune systems of humans and mammals, which functions classically as an inhibitor of the IFN signaling pathways. However, data on functional characterisation of SOCS-1 in ancient vertebrates are limited. In this study, we report the function of teleost SOCS-1s in IFN signaling in fish models (zebrafish and Tetraodon) and human cells. Structurally, teleost SOCS-1s share conserved functional domains with their mammalian counterparts. Functionally, teleost SOCS-1s could be significantly induced upon stimulation with IFN stimulants and zebrafish IFNφ1. Overexpression of teleost SOCS-1s could dramatically suppress IFNφ1-induced Mx, Viperin and PKZ activation in zebrafish, and IFN-induced ISG15 activation in HeLa cells. Furthermore, a SOCS-1 variant that lacks the KIR domain was also characterised. This study demonstrates the conserved negative regulatory role of teleost SOCS-1s in IFN signaling pathways, providing perspective into the functional conservation of SOCS-1 proteins during evolution.

Essential role of IL-4 and IL-4Rα interaction in adaptive immunity of zebrafish: insight into the origin of Th2-like regulatory mechanism in ancient vertebrates.

The roles of IL-4 and IL-4Rα in Th2-mediated immunity have been well characterized in humans and other mammals. In contrast, few reports have been documented in ancient vertebrates. Several putative IL-4- and IL-4Rα-like molecules were identified recently from a few fish species, providing preliminary insight into the occurrence of Th2-type immunity in teleosts. However, functional determination still is required to address this hypothesis. To this end, these two molecules were characterized functionally in zebrafish (Danio rerio). Besides the identification of a full-length IL-4Rα molecule and an isoform lacking most of the cytoplasmic region as predicted previously, two novel alternatively spliced soluble variants with the extracellular domain only also were identified. Zebrafish IL-4Rα (DrIL-4Rα) shared overall conserved structural features of the IL-4Rα family. Immunofluorescence staining showed that DrIL-4Rα distributed on B cells. In vitro binding assays demonstrated that zebrafish IL-4 (DrIL-4) can bind specifically to DrIL-4Rα. In vivo administration of DrIL-4 significantly upregulated B cell proliferation and Ab production. These DrIL-4-elicited immune responses were downregulated by the administration of zebrafish soluble IL-4Rα or by DrIL-4Rα blockade using anti-DrIL-4Rα Abs. In addition, Th2-related cytokines or transcription factors were upregulated by DrIL-4. The DrIL-4-DrIL-4Rα interaction promoted CD40 expression on B cells and enhanced the CD154-CD40 costimulatory response, both of which are crucial for the initiation of Th2-type immunity. To our knowledge, this is the first report showing that a possible Th2-mediated regulatory mechanism may have appeared before the divergence of teleosts and mammals. These results add greater insight into the evolutionary history of adaptive immunity.