Insulin-stimulated glucose uptake is central to global carbohydrate metabolism, yet metabolites that enhance glucose uptake independently of insulin remain undefined. Here, we identify L-lactate as an insulin-independent...Insulin-stimulated glucose uptake is central to global carbohydrate metabolism, yet metabolites that enhance glucose uptake independently of insulin remain undefined. Here, we identify L-lactate as an insulin-independent regulator of glucose uptake that mitigates hyperglycemia. Loss of LDHA in muscle reduces lactate production, impairing glucose homeostasis in mice. By contrast, lactate administration or genetic upregulation of lactate production improves glucose control. Knockout of the lactate receptor GPR81 in skeletal muscle worsens glucose tolerance, whereas its ectopic expression or pharmacological activation enhances carbohydrate metabolism. Mechanistically, GPR81 recruits FARP1 to activate RAC1, promoting GLUT4 translocation independently of insulin signaling. Notably, the expression of LDHA, GPR81, and FARP1 is upregulated after exercise, and GPR81 variants are highly correlated with fasting insulin levels in humans, underscoring the synergy of the GPR81-FARP1-GLUT4 axis with insulin in glucose regulation. Our findings suggest that targeting GPR81 represents a potential insulin-independent strategy for the treatment of hyperglycemia.
R-loops are pervasive genomic structures that link epigenetic modification and transcriptional regulation. However, the functional roles and regulatory mechanisms of R-loops during preimplantation development in mammals...R-loops are pervasive genomic structures that link epigenetic modification and transcriptional regulation. However, the functional roles and regulatory mechanisms of R-loops during preimplantation development in mammals remain unexplored. Here, we reveal that the reprogramming of R-loops across developmental stages depends on CG density, with CG-poor R-loops more stage specific and strongly associated with early embryonic development. Loss of CG-poor R-loops causes severe defects in the maternal-to-zygotic transition (MZT) and preimplantation embryo development. This abnormal maintenance of CG-poor R-loops promotes premature activation of major zygotic genome activation (ZGA) genes. CG-poor R-loops inhibit DDX21 helicase activity on the 7SK/HEXIM1 snRNP complex, restricting CDK9 release and subsequent phosphorylation of Ser2 at the C-terminal domain of RNA polymerase II (RNAPII S2p) - the biochemical hallmark of pause release - thus enforcing RNAPII accumulation at major ZGA gene promoters to ensure productive transcription. These findings establish R-loops as direct modulators of RNAPII pause release, promoting the temporal fidelity of gene expression during the MZT.
Wang Q, Zeng X, Yang H
… +20 more, Lu H, Jiang L, Xu L, Li J, Li J, Han Y, Wu X, Zhou Y, Chen X, Zhao Y, Shi J, Luo Y, Ni F, Sun J, Zhao Q, Yang F, Xia P, Jiang H, Huang H, Qian P
Achieving long-term ex vivo expansion of functional hematopoietic stem cells (HSCs) is essential for advancing HSC-based clinical therapies. Although mechanosensitive ion channels are known to play key roles in the hemat...Achieving long-term ex vivo expansion of functional hematopoietic stem cells (HSCs) is essential for advancing HSC-based clinical therapies. Although mechanosensitive ion channels are known to play key roles in the hematopoietic system, their involvement in HSC expansion remains unclear. Here, we show that Piezo1 is highly expressed in HSCs. Both genetic deletion and prolonged chemical activation of Piezo1 impair cultured HSC function, indicating that transient mechanical activation of Piezo1 is required for maintenance of HSCs in culture. To achieve this, we screened various microspheres and found that PS500 (500-nm polystyrene microspheres) significantly enhanced ex vivo expansion of mouse bone marrow HSCs with long-term repopulating capacity. PS500 also expanded human umbilical cord blood HSCs capable of engraftment in immunodeficient mice. Mechanistically, PS500 activates Piezo1, triggering Ca-dependent expression of proliferative cytokines and subsequent STAT3 activation, which support HSC self-renewal and proliferation. Together, these findings show that PS500 enables transient Piezo1 activation and efficient, non-toxic expansion of functional HSCs, offering a promising approach for the generation of transplantable HSCs for clinical use.
Jiang B, Xing W, Xu X
… +22 more, Chen S, Feng H, Shao R, Sun J, Zhang Y, Xie Z, Wang W, Yin X, Wang Y, Wang M, Li L, Zhang Z, Gao B, Suo J, Hu X, Wang L, Sun J, Zhou B, Zhou BO, Greenblatt MB, Le R, Zou W
Periosteum contains abundant Ctsk-lineage skeletal stem cells (P-SSCs) that are key drivers of intramembranous ossification during bone development and maintenance. However, P-SSCs regenerate fractured bones by mediating...Periosteum contains abundant Ctsk-lineage skeletal stem cells (P-SSCs) that are key drivers of intramembranous ossification during bone development and maintenance. However, P-SSCs regenerate fractured bones by mediating endochondral ossification, raising the question of whether distinct P-SSCs subsets separately mediate steady-state bone formation and fracture repair. Here we uncover the heterogeneity of P-SSCs, identifying an Angptl7-expressing quiescent P-SSCs subset, which is restricted to the fibrous-layer of periosteum and barely contributes to postnatal bone development. After bone fracture, these cells largely contribute to bone healing by dedicating to endochondral ossification, regenerating the entire bone architecture. Dysfunction of Angptl7-lineage P-SSCs strongly impairs the bone healing process but does not affect steady-state bone formation. Multimodal analysis reveals that these cells can be immediately activated under the regulation of TNF-α/NF-κB signaling, subsequently acquiring osteogenic capacity. Together, our findings unravel an injury-specified P-SSCs subpopulation, providing a model that there are tissue-resident stem cells specialized for injury repair, while parallel stem cells maintain homeostasis.
Chen GY, Zhu WJ, Li Z
… +19 more, Hu YW, Luo XS, Mai ZQ, Pan Y, Shi YX, Li ZY, Huang J, Yuan PD, Xiao ZQ, Chen Q, Xie YY, Huang HX, Chen YX, Lu Y, Wang MZ, Xia YW, Chen XQ, Kuang DM, Liang D
Robust mitochondrial ROS production induces extensive double-strand breaks (DSBs) in telomeric DNA of effector T cells, where the DNA repair machinery is rapidly hyper-evoked to sense and ligate DSBs during the respirato...Robust mitochondrial ROS production induces extensive double-strand breaks (DSBs) in telomeric DNA of effector T cells, where the DNA repair machinery is rapidly hyper-evoked to sense and ligate DSBs during the respiratory burst. However, whether effector T cells can exploit the DNA repair system to simultaneously potentiate their functional activation remains largely unknown, especially in the context of autoimmunity. Here, we demonstrate that non-homologous end joining (NHEJ), a predominant mechanism of DNA repair, is highly activated in pathogenic T helper 17 (pTh17) cells and exerts a previously unrecognized effect on shaping the pathogenic nature of pTh17s to trigger autoimmunity. Mechanistically, the perception of DSBs by KU proteins facilitates auto-phosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which stabilizes RORγt to bind to the promoters of effector-gene loci, thus initiating the pTh17 effector program to induce autoimmunity. Using mass spectrometry and transcriptome analyses, we identified IER2 as a novel NHEJ factor that potentiates DNA-PKcs kinase activity in response to IL-23R stimulation, which is necessary for shaping Th17 pathogenicity. Therefore, targeting the immuno-pattern of the NHEJ system shows potential for the treatment of autoimmune diseases.
Mitophagy, an evolutionarily conserved quality-control process, selectively removes damaged mitochondria to maintain cellular homeostasis. Recent advances in our understanding of the molecular machinery underlying mitoph...Mitophagy, an evolutionarily conserved quality-control process, selectively removes damaged mitochondria to maintain cellular homeostasis. Recent advances in our understanding of the molecular machinery underlying mitophagy - from receptors and stress-responsive triggers to lysosomal degradation - illustrate its key role in maintaining mitochondrial integrity and adapting mitochondrial function to ever-changing physiological demands. In this review, we outline the fundamental mechanisms of mitophagy and discuss how dysregulation of this pathway disrupts mitochondrial function and metabolic balance, driving a wide range of disorders, including neurodegenerative, cardiovascular, metabolic, and immune-related diseases, as well as cancer. We explore the dual role of mitophagy as both a disease driver and a therapeutic target, highlighting the efforts and challenges of translating mechanistic insights into precision therapies. Targeting mitophagy to restore mitochondrial homeostasis may be at the center of a large range of translational opportunities for improving human health.
Ma WH, Li WY, Chen T
… +19 more, Jing L, Chen YH, Li K, Xu ZL, Shen RF, He Y, Mou T, Luo TY, Sun X, Wu ZK, Wang LJ, Liu HJ, Qiu X, Gao Y, Bai X, Wang W, Wu D, Li G, Zhou WJ
Colorectal cancer (CRC) remains largely refractory to immune-checkpoint blockade, with adenomatous polyposis coli (APC) mutations present in 80%-90% of cases. Loss of APC was previously thought to promote tumor progressi...Colorectal cancer (CRC) remains largely refractory to immune-checkpoint blockade, with adenomatous polyposis coli (APC) mutations present in 80%-90% of cases. Loss of APC was previously thought to promote tumor progression mainly through deregulated Wnt/β-catenin signaling. Here, we report that APC loss leads to inhibition of CD8 T cell infiltration and CRC immune evasion through the dephosphorylation of signal transducers and activators of transcription 1 (STAT1) by protein tyrosine phosphatase non-receptor type 13 (PTPN13), independently of β-catenin. Peptides containing the last 11 C-terminal amino acid (aa) residues of APC (APC11) bind directly to PTPN13 to block PTPN13-STAT1 interactions and facilitate STAT1 phosphorylation, interferon regulatory factor-1 (IRF1) expression, major histocompatibility complex (MHC) class I antigen presentation, and T cell intratumoral infiltration, all of which eventually inhibit tumor progression and enhance the effects of programmed cell death 1 (PD1) blockade. Thus, we have identified a previously unknown APC/PTPN13/STAT1-dependent tumor immune-suppressive mechanism. The potent tumor-suppressing effect of combining anti-PD1 antibodies with APC11 peptides provides a compelling target and rationale for future development of anti-tumor drugs for patients with CRC.
Resistance to anti-PD-1/PD-L1 immune checkpoint blockade continues to be a critical challenge undermining its therapeutic efficacy in clinical applications. Most of the resistance mechanisms characterized to date have pr...Resistance to anti-PD-1/PD-L1 immune checkpoint blockade continues to be a critical challenge undermining its therapeutic efficacy in clinical applications. Most of the resistance mechanisms characterized to date have predominantly involved external factors beyond PD-L1. Here, we unexpectedly discovered that PD-L1 itself possesses E3 ubiquitin ligase activity to induce β2m ubiquitylation and subsequent degradation, which notably reduces MHC-I levels on the surface of tumor cells and antigen-presenting cells, thereby contributing to tumor cell evasion of recognition by CD8 T cells and ultimately resulting in resistance to anti-PD-1/PD-L1 immunotherapy, particularly in tumors with low basal β2m expression. Disrupting the E3 ubiquitin ligase activity of PD-L1 or interfering with the PD-L1-β2m interaction dramatically enhanced the sensitivity of tumor cells to PD-L1 blockade therapy. Our study reveals a previously unknown function of PD-L1 in the immune evasion of tumor cells, expanding our understanding of intrinsic resistance mechanisms to immune checkpoint blockade therapy.
Lysosomes are essential for cellular homeostasis, serving as degradative organelles that recycle nutrients. Whether and how lysosomes maintain membrane integrity under energy stress is poorly understood. Here, we found t...Lysosomes are essential for cellular homeostasis, serving as degradative organelles that recycle nutrients. Whether and how lysosomes maintain membrane integrity under energy stress is poorly understood. Here, we found that the uptake of lipid droplets by lysosomes during glucose starvation provokes disruption of lysosomal membranes. We identified tectonin beta-propeller repeat-containing protein 1 (TECPR1) as a critical mediator of lysosomal repair during glucose starvation or LLOMe-induced lysosomal membrane permeabilization. TECPR1 is recruited to damaged lysosomes via interaction with PI4P on damaged lysosomal membranes. It interacts with KIF1A to facilitate tubule formation from damaged lysosomes, enabling the removal of damaged membrane components and promoting lysosomal repair. Our in vitro reconstituted tubulation process provided further evidence that TECPR1 coordinates with KIF1A to drive tubulation from PI4P-enriched giant unilamellar vesicles. TECPR1-mediated lysosomal repair is essential for maintaining lipid metabolism and cellular survival during an energy crisis, as TECPR1 deficiency exacerbates starvation-induced liver damage in a high-fat diet-induced MAFLD mouse model. Our findings demonstrate a previously unrecognized role of TECPR1 in lysosomal repair, revealing its critical contributions to energy stress adaptation and liver protection. This work provides new insight into mechanisms of lysosomal repair and their implications for metabolic and lysosome-related disorders.
Fu F, Shang J, Yan Y
… +21 more, Jiang H, Han H, Yuan H, Gao Z, Yang J, Gao J, Wang J, Pan Y, Lin Y, Ye T, Zhang Y, Zhang Y, Xiang J, Hu H, Cao Z, Zheng Y, Li Y, Zhang Y, Jin L, Shi L, Chen H
Lung adenocarcinoma (LUAD) progresses from pre-invasive to invasive stages, as well as from ground-glass opacities (GGOs) to solid nodules. However, the dynamic genomic and transcriptomic changes underlying LUAD progress...Lung adenocarcinoma (LUAD) progresses from pre-invasive to invasive stages, as well as from ground-glass opacities (GGOs) to solid nodules. However, the dynamic genomic and transcriptomic changes underlying LUAD progression are incompletely understood. Here, we performed whole-genome and transcriptome sequencing on 1008 LUAD samples from 954 patients who underwent surgery at Fudan University Shanghai Cancer Center, with comprehensive follow-up data. There was one atypical adenomatous hyperplasia, 42 adenocarcinomas in situ, 116 minimally invasive adenocarcinomas, and 849 invasive adenocarcinomas spanning all pathological stages. EGFR was the most frequently mutated gene in the study cohort, followed by TP53, RBM10, KRAS, and KMT2D. Mutation frequencies of tumor suppressor genes, such as TP53, RB1, MGA, KEAP1, and STK11, increased as the disease progressed to higher stages. A higher level of genomic instability was seen in LUAD compared with AAH/AIS/MIA samples, characterized by a higher tumor mutation burden, increased somatic copy number alteration burden, and increased structural variation burden. Notably, MAP2K1 E102-I103 deletion was frequently observed in pre-invasive samples, which endowed alveolar type II cells with increased growth potential and initiated tumor formation, suggesting that it is a potential driver mutation of LUAD. In summary, our study highlights key molecular changes during the stepwise progression of LUAD, provides insights into the identification of novel therapeutic targets, and helps to define the curative time window for this disease.
Hepatocellular carcinoma (HCC) remains a major therapeutic challenge. Although targeting tumor-specific antigens represents a cornerstone of cancer immunotherapy, current approaches focus predominantly on mutation-derive...Hepatocellular carcinoma (HCC) remains a major therapeutic challenge. Although targeting tumor-specific antigens represents a cornerstone of cancer immunotherapy, current approaches focus predominantly on mutation-derived neoantigens, which offer limited population coverage. Through an integrative analysis of multi-omics data from 279 HCC patients, we demonstrate that aberrant splicing (AS) events occur at a > 59-fold higher frequency than somatic mutations and generate substantially more immunogenic peptides with broader patient applicability (50.94% vs 4.40% population coverage). Focusing on AS transcripts, our stringent selection pipeline identified 34 neoantigens, prioritizing strong immunogenicity for effective vaccine development. Proof-of-concept in vivo experiments demonstrated the efficacy of mRNA vaccines encoding these neoantigens, resulting in significant tumor regression and enhanced intra-tumor infiltration of neoantigen-reactive T cells. We also address the challenge of transporter-associated antigen processing (TAP) deficiency in HCC by proposing the use of TAP-independent AS-derived neoantigens to circumvent immune evasion. Our findings establish AS as a promising source of neoantigens for off-the-shelf mRNA vaccines in HCC and underscore the need to overcome antigen-presentation barriers for effective immunotherapy.
Jurgelaitis E, Zagorskaitė E, Kopūstas A
… +10 more, Asmontas S, Manakova E, Dalgėdienė I, Tylenytė U, Silanskas A, Toliusis P, Grybauskas A, Tutkus M, Venclovas Č, Zaremba M
Present in all three domains of life, Argonaute proteins use short oligonucleotides as guides to recognize complementary nucleic acid targets. In eukaryotes, Argonautes are involved in RNA silencing, whereas in prokaryot...Present in all three domains of life, Argonaute proteins use short oligonucleotides as guides to recognize complementary nucleic acid targets. In eukaryotes, Argonautes are involved in RNA silencing, whereas in prokaryotes, they function in host defense against invading DNA. Here, we show that SPARDA (short prokaryotic Argonaute, DNase associated) systems from Xanthobacter autotrophicus (Xau) and Enhydrobacter aerosaccus (Eae) function in anti-plasmid defense. Upon activation, SPARDA nonspecifically degrades both invader and genomic DNA, causing host death, thereby preventing further spread of the invader in the population. X-ray structures of the apo Xau and EaeSPARDA complexes show that they are dimers, unlike other apo short pAgo systems, which are monomers. We show that dimerization in the apo state is essential for inhibition of XauSPARDA activity. We demonstrate by cryo-EM that activated XauSPARDA forms a filament. Upon activation, the recognition signal of the bound guide/target duplex is relayed to other functional XauSPARDA sites through a structural region that we termed the "beta-relay". Owing to dramatic conformational changes associated with guide/target binding, XauSPARDA undergoes a "dimer-monomer-filament" transition as the apo dimer dissociates into the guide/target-loaded monomers that subsequently assemble into the filament. Within the activated filament, the DREN nuclease domains form tetramers that are poised to cleave dsDNA. We show that other SPARDAs also form filaments during activation. Furthermore, we identify the presence of the beta-relay in pAgo from all clades, providing new insights into the structural mechanisms of pAgo proteins. Taken together, these findings reveal the detailed structural mechanism of SPARDA and highlight the importance of the beta-relay mechanism in signal transduction in Argonautes.
Hu HH, Wang X, Lan B
… +21 more, Cheng H, Wen H, Chen F, Wu J, Li M, Chen J, Zhang J, Chen D, Lin S, Lin J, Yang M, Wu Z, Zheng ZZ, Chen F, Zhou J, Chen G, Chen Y, Deng X, Zhang CS, Liu J, Lin SC
Dysregulated metabolism in tumor tissues and para-tumor tissues alike can lead to immunosuppression, which may underlie cancer development. However, metabolic intervention as a therapeutic strategy has been of no avail....Dysregulated metabolism in tumor tissues and para-tumor tissues alike can lead to immunosuppression, which may underlie cancer development. However, metabolic intervention as a therapeutic strategy has been of no avail. In this study, we explored the anti-cancer therapeutic effect of aldometanib, which specifically targets lysosome-associated aldolase to mimic glucose starvation and thereby activates lysosomal AMP-activated protein kinase (AMPK), a master regulator of metabolic homeostasis. We show that aldometanib inhibits the growth of hepatocellular carcinoma (HCC) in an AMPK-dependent manner, allowing hepatoma-bearing mice to survive to mature ages, although aldometanib does not possess cytotoxicity toward HCC or normal cells. Intriguingly, aldometanib exerts anti-cancer effects only in immune-competent host mice, but not in immune-defective mice. We also found that HCC tissues in aldometanib-treated mice were massively infiltrated with CD8 T cells, which was not seen in mice with liver-specific knockout of AMPKα. Our findings thus suggest that the metabolic regulator AMPK rebalances the tumor microenvironment to allow cytotoxic immune cells inside the body to eliminate cancer cells and effectively contain the tumor tissues. The finding that metabolic intervention can make cancer a lifelong manageable disease may usher in a new era of cancer therapy.
Chen L, Bu X, Sun Y
… +23 more, Huang D, Chen Y, Hou T, Hu X, Wang J, Yan P, Qi Y, Jiang W, Xiong Y, Liu J, Gao Y, Huan M, Wang B, Liu Q, Dai X, Dang F, Asara JM, Fujimoto M, Inuzuka H, Jin J, Zhang J, Freeman GJ, Wei W
Immune checkpoints serve as regulatory pathways that are essential for regulating immune response and homeostasis. As such, many components along the pathway have emerged as pivotal targets in cancer therapy. To overcome...Immune checkpoints serve as regulatory pathways that are essential for regulating immune response and homeostasis. As such, many components along the pathway have emerged as pivotal targets in cancer therapy. To overcome the treatment resistance and limited efficacy encountered by current immune checkpoint therapies, there is an urgent need for new immunotherapeutic targets and strategies. V-domain Ig suppressor of T cell activation (VISTA) is an immune checkpoint protein with a unique expression pattern and has emerged as a novel therapeutic target in anti-tumor immunotherapy; however, the precise role of VISTA and its regulatory mechanisms in tumor cells remain incompletely understood. Here, we identify a novel strategy targeting VISTA for cancer immunotherapy, enhancing therapeutic outcomes. Mechanistically, we show that VISTA undergoes anaphase-promoting complex/cyclosome (APC/C)/CDH1-mediated ubiquitination and subsequent proteasomal degradation, a process that can be reversed by the deubiquitinase USP2. Therapeutically, the USP2 inhibitor MS102 significantly reduces VISTA protein abundance in vitro and in vivo, enhances T cell responses, and synergizes with anti-PD-1 immunotherapy to improve survival in syngeneic mouse tumor models. Our findings reveal a regulatory network for VISTA stability control and support the combination of USP2 inhibitors with anti-PD-1 immunotherapy to enhance anti-tumor immune responses.
Sun Y, Zhang Z, Li H
… +25 more, Bu X, Chen L, Wang X, Fan L, Chen B, Kong L, Dai P, Song W, Xiao X, Shi J, Xiang B, He C, Yao Y, Xiong W, Yu H, Jiang C, Qian Q, Liu H, Tian S, Qing G, Yang Z, Wei W, Freeman GJ, Zhu H, Zhang J
The limited success of current immunotherapies emphasizes the need for new targets and combination treatments. V-domain Ig suppressor of T cell activation (VISTA) is a promising immune checkpoint target in cancer immunot...The limited success of current immunotherapies emphasizes the need for new targets and combination treatments. V-domain Ig suppressor of T cell activation (VISTA) is a promising immune checkpoint target in cancer immunotherapy, but its regulatory mechanism is poorly understood. Through CRISPR knockout screening and proteomic analysis, we identify tripartite motif containing 25 (TRIM25) as a positive regulator for VISTA largely through antagonizing its degradation signaling. Moreover, ERK-mediated phosphorylation of VISTA at Thr284 enhances its interaction with TRIM25, leading to VISTA stabilization. A VISTA-derived phospho-peptide competitively disrupts TRIM25-VISTA interaction, thereby reducing VISTA expression and potentiating the anti-tumor efficacy of PD-1/PD-L1 blockade. Moreover, single-cell RNA sequencing analysis shows that tumor-infiltrating cytotoxic CD8 T cells are increased in mice with T cell-specific knockout of Trim25. Of note, genetic ablation of Trim25 in T cells not only improves anti-PD-L1 immunotherapy, but also significantly ameliorates CAR T anti-tumor activity in various mouse tumor models. Collectively, this study unveils a mechanism for VISTA regulation in T cells and highlights targeting TRIM25-VISTA as a potential strategy to enhance tumor immunotherapy.