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.

Activation of the μ-opioid receptor (μOR) alleviates pain but also elicits adverse effects through diverse G proteins and β-arrestins. The structural details of μOR complexes with G and β-arrestins have not been determined, impeding a comprehensive understanding of μOR signaling plasticity. Here, we present the cryo-EM structures of the μOR-G and μOR-βarr1 complexes, revealing selective conformational preferences of μOR when engaged with specific downstream signaling transducers. Integrated receptor pharmacology, including high-resolution structural analysis, cell signaling assays, and molecular dynamics simulations, demonstrated that transmembrane helix 1 (TM1) acts as an allosteric regulator of μOR signaling bias through differential stabilization of the G-, G-, and βarr1-bound states. Mechanistically, outward TM1 displacement confers structural flexibility that promotes G protein recruitment, whereas inward TM1 retraction facilitates βarr1 recruitment by stabilizing the intracellular binding pocket through coordinated interactions with TM2, TM7, and helix8. Structural comparisons between the G-, G-, and βarr1-bound complexes identified a TM1-fusion pocket with significant implications for downstream signaling regulation. Overall, we demonstrate that the conformational and thermodynamic heterogeneity of TM1 allosterically drives the downstream signaling specificity and plasticity of μOR, thereby expanding the understanding of μOR signal transduction mechanisms and providing new avenues for the rational design of analgesics.

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.

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.

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.

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.

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.

MDA5 is a RIG-I-like receptor (RLR) that recognizes viral double-stranded RNA (dsRNA) to initiate the innate immune response. Its activation requires filament formation along the dsRNA, which triggers the oligomerization of N-terminal caspase activation and recruitment domains. The ATPase activity of MDA5 is critical for immune homeostasis, likely by regulating filament assembly. However, the molecular basis underlying this process remains poorly understood. Here, we show that MDA5 operates as an ATP-hydrolysis-driven motor that translocates along dsRNA in a one-dimensional (1D) manner. Multiple MDA5 motors can cooperatively load onto a single dsRNA, but their movements rarely synchronize, inhibiting spontaneous filament formation and activation. LGP2, a key regulator of MDA5 signaling, recognizes MDA5 motors and blocks their movement, thereby promoting filament assembly through a translocation-directed mechanism. This unique assembly strategy underscores the role of 1D motion in higher-order protein oligomerization and reveals a novel mechanism for maintaining immune homeostasis.

Lineage plasticity, the ability of cells to transition to an alternative phenotype as a means for adaptation, is an increasingly recognized mechanism of tumor evolution and a driver of resistance to anticancer therapies. The most extensively described clinical settings impacted by such molecular phenomena include neuroendocrine transformation in androgen receptor-dependent prostate adenocarcinoma, and adenocarcinoma-to-neuroendocrine and adenocarcinoma-to-squamous transdifferentiation in epidermal growth factor receptor-driven lung adenocarcinoma, affecting 10%-20% of patients treated with targeted therapy. Recent analyses of human tumor samples and in vivo models of histological transformation have led to insights into the biology of lineage plasticity, including biomarkers predictive of high risk of transformation. However, no clinically available therapies aimed to prevent or revert plasticity are currently available. In the present review, we will provide a biological and therapeutic overview of the current understanding of common and divergent molecular drivers of neuroendocrine and squamous transdifferentiation in tumors from different origins, including descriptive analysis of previously known and recently described molecular events associated with histological transformation, and propose evidence-based alternative models of transdifferentiation. A clear definition of the commonalities and differences of transforming tumors in different organs and to different histological fates will be important to translate molecular findings to the clinical setting.

The KCNQ1 + KCNE1 potassium channel complex produces the slow delayed rectifier current (I) critical for cardiac repolarization. Loss-of-function mutations in KCNQ1 and KCNE1 cause long QT syndrome (LQTS) types 1 and 5 (LQT1/LQT5), accounting for over one-third of clinical LQTS cases. Despite prior structural work on KCNQ1 and KCNQ1 + KCNE3, the structural basis of KCNQ1 + KCNE1 remains unresolved. Using cryo-electron microscopy and electrophysiology, we determined high-resolution (2.5-3.4 Å) structures of human KCNQ1, and KCNQ1 + KCNE1 in both closed and open states. KCNE1 occupies a pivotal position at the interface of three KCNQ1 subunits, inducing six helix-to-loop transitions in KCNQ1 transmembrane segments. Three of them occur at both ends of the S4-S5 linker, maintaining a loop conformation during I gating, while the other three, in S6 and helix A, undergo dynamic helix-loop transitions during I gating. These structural rearrangements: (1) stabilize the closed pore and the conformation of the intermediate state voltage-sensing domain, thereby determining channel gating, ion permeation, and single-channel conductance; (2) enable a dual-PIP2 modulation mechanism, where one PIP2 occupies the canonical site, while the second PIP2 bridges the S4-S5 linker, KCNE1, and the adjacent S6', stabilizing channel opening; (3) create a fenestration capable of binding compounds specific for KCNQ1 + KCNE1 (e.g., AC-1). Together, these findings reveal a previously unrecognized large-scale secondary structural transition during ion channel gating that fine-tunes I function and provides a foundation for developing targeted LQTS therapy.

The innate immune system adapts its behavior based on previous insults, mounting an enhanced response upon re-exposure. Hematopoietic progenitors in the bone marrow and peripheral innate immune cells can undergo epigenetic and metabolic reprogramming, establishing an innate immune memory known as trained immunity. The concept of trained immunity recently gained relevance in our understanding of how innate immunity is regulated in various diseases. This review explores the role of trained immunity in infections, autoimmune disease, cardiovascular disease, cancer, and neurodegenerative disease. We discuss how trained immunity can provide heterologous protection against infections, as it has been induced for decades by the Bacillus Calmette Guérin vaccine, how it can help counteract immunosuppression, and how it can be inappropriately induced leading to chronic inflammation. By understanding how trained immunity is involved in processes leading to health and disease, novel therapeutic strategies can be developed.