Kunhua Hu

Pancreatic ductal adenocarcinoma (PDAC) radiotherapy (RT) resistance is frequently mediated by an immunosuppressive tumor microenvironment (TIME). Utilizing an in vivo CRISPR-Cas9 metabolic enzyme screen, we identified fucosyltransferase 2 (FUT2) as a potent non-catalytic enhancer of RT response. Mechanistically, FUT2 scaffolds the E3 ubiquitin ligase FBXO2, facilitating K362 site-specific ubiquitination and proteasomal degradation of the transcription factor NR2F2. This degradation suppresses expression of the immunosuppressive factor Lipocalin-2 (LCN2), which drives CD8⁺ T cell exhaustion and impedes NK cell infiltration, fostering a radioresistant TIME. Interestingly, we observed that RT could reduce FUT2 transcript levels via an METTL14-mediated m⁶A RNA methylation, while NR2F2 was identified to transcriptionally upregulate METTL14, establishing a feedforward inhibitory loop that sustains FUT2 suppression. Clinically, FUT2 expression positively correlates with CD8⁺ T cell infiltration and prolonged survival in RT-treated PDAC patients. Preclinically, combining RT with LCN2-neutralizing antibodies elicited synergistic anti-tumor immunity. These results unveil FUT2 as a regulator of PDAC radiosensitivity via the FUT2-FBXO2-NR2F2-LCN2 axis, offering a promising therapeutic target to overcome RT resistance.

RNA splicing is pivotal in neural development, yet the role of isoform diversity across cell types remains unclear. Here, we combined metabolic RNA labeling and single-cell full-length transcriptome sequencing to capture transcriptional dynamics in developing mouse cortices. We observed predetermined cell states supported by nascent RNAs and characterized the driving isoforms of transcription factors that regulated the development of deep- and upper-layer neurons. Additionally, we investigated isoform regulation associated with autism spectrum disorder (ASD) during the embryonic development of BTBR T + Itpr3tf mice. Our findings indicated premature emergence of callosal projection neurons (CPNs) with an immature identity in ASD-affected cortices. These CPNs exhibited abnormal transcript usage, and the related RNA binding proteins included nearly 60% that have been reported to be ASD risk genes. We identified isoform switching events modulating neurogenesis and ASD development. Finally, we observed reduced isoform diversity in ASD potentially linked to dysregulated H3K27ac levels. Collectively, our study represents a significant advancement in understanding the molecular basis of cortical development and functions.

Transcriptome profiling of RNA modifications is essential for uncovering and characterizing novel post-transcriptional regulatory mechanisms. However, most of the known RNA modifications remain poorly explored due to technological limitations. Nanopore direct RNA sequencing (DRS) provides an advantageous solution for transcriptome-wide RNA modification profiling, enabling simultaneous identification of any modification type in native RNA, with full-length coverage and single-molecule resolution. We developed RMPore (https://rmpore.renlab.cn/), a comprehensive database of single-molecule RNA modifications detected from 958 DRS samples across 34 species. We constructed a practical analytical pipeline integrating 20 detection tools and categorized all detected sites into three confidence levels (high, medium, and low) based on the prediction thresholds and reproducibility of tools, datasets, and other technologies, identifying a total of 65 025 784 modification sites spanning 25 modification types. To further investigate the characteristics of these modification sites and elucidate the regulatory relationships among different modification types, we performed single-molecule advanced analyses of correlated sites and haplotype-biased sites. Meanwhile, we also incorporated extensive molecular event annotations of modification sites in RMPore, including splicing events, RNA-binding protein interactions, RNA–RNA interactions, and circular RNAs. We expect that RMPore will advance single-molecule epitranscriptomics research, bridging critical gaps in the field of RNA modification research.

Rapid development of resistance to sorafenib and subsequent hyperprogression in patients with advanced hepatocellular carcinoma (HCC) pose significant challenges, with the underlying mechanisms still largely unknown. Herein, sorafenib‐induced TRIB3 is identified as a liver‐specific determinant driving secondary resistance to sorafenib by facilitating the accumulation of protumorigenic neutrophils within tumors. Mechanistically, TRIB3, triggered by the sorafenib‐elicited ROS‐ER stress axis, operates in an NF‐κB‐dependent manner to upregulate CXCR1/2 ligands, subsequently promoting neutrophil recruitment into tumors. These enriched neutrophils enhance epithelial‐mesenchymal transition processes in malignant cells through the oncostatin M‐STAT3 pathway, thereby repurposing the therapeutic efficacy of sorafenib away from anti‐angiogenesis and toward lung metastasis. Clinically, elevated TRIB3 expression indicates inferior survival and unfavorable clinical efficacy of sorafenib in HCC patients. Correspondingly, strategies that either inhibiting TRIB3 upregulation or blocking its downstream signaling successfully augment the therapeutic efficacy of sorafenib and prevent sorafenib‐induced hyperprogression in vivo. The study thus identifies a pivotal mechanism of sorafenib resistance in HCC, centered on the TRIB3‐mediated recruitment of protumorigenic neutrophils and subsequent disease hyperprogression.

Clinical studies have suggested that tirzepatide may also possess hepatoprotective effects; however, the molecular mechanisms underlying this association remain unclear. In our study, we performed biochemical analyses of serum and histopathological examinations of liver tissue in mice. To preliminarily explore the molecular mechanisms of tirzepatide on metabolic dysfunction-associated fatty liver disease (MAFLD), liquid chromatography-mass spectrometry (LC-MS) was employed for comprehensive metabolomic, lipidomic, and proteomic analyses in MAFLD mice fed a high-fat diet (HFD). The results demonstrated that tirzepatide significantly reduced serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), as well as hepatic triglycerides (TG) and total cholesterol (TC), indicating its efficacy in treating MAFLD. Further findings revealed that tirzepatide reduced fatty acid uptake by downregulating Cd36 and Fabp2/4, as well as enhance the mitochondrial-lysosomal function by upregulating Lamp1/2. In addition, tirzepatide promoted cholesterol efflux and reduced cholesterol reabsorption by upregulating the expression of Hnf4a, Abcg5, and Abcg8. These results suggest that tirzepatide exerts its therapeutic effects on MAFLD by reducing fatty acid uptake, promoting cholesterol excretion, and enhancing mitochondrial-lysosomal function, providing a theoretical basis for a comprehensive understanding of tirzepatide.

The vast neuronal diversity in the human neocortex is vital for high-order brain functions, necessitating elucidation of the regulatory mechanisms underlying such unparalleled diversity. However, recent studies have yet to comprehensively reveal the diversity of neurons and the molecular logic of neocortical origin in humans at single-cell resolution through profiling transcriptomic or epigenomic landscapes, owing to the application of unimodal data alone to depict exceedingly heterogeneous populations of neurons. In this study, we generated a comprehensive compendium of the developing human neocortex by simultaneously profiling gene expression and open chromatin from the same cell. We computationally reconstructed the differentiation trajectories of excitatory projection neurons of cortical origin and inferred the regulatory logic governing lineage bifurcation decisions for neuronal diversification. We demonstrated that neuronal diversity arises from progenitor cell lineage specificity and postmitotic differentiation at distinct stages. Our data paves the way for understanding the primarily coordinated regulatory logic for neuronal diversification in the neocortex.

Background: Uncovering functional genetic variants from an allele-specific perspective is of paramount importance in advancing our understanding of gene regulation and genetic diseases. Recently, various allele-specific events, such as allele-specific gene expression, allele-specific methylation, and allele-specific binding, have been explored on a genome-wide scale due to the development of high-throughput sequencing methods. RNA secondary structure, which plays a crucial role in multiple RNA-associated processes like RNA modification, translation and splicing, has emerged as an essential focus of relevant research. However, tools to identify genetic variants associated with allele-specific RNA secondary structures are still lacking. Results: Here, we develop a computational tool called ‘AStruct’ that enables us to detect allele-specific RNA secondary structure (ASRS) from RT-stop based structuromic probing data. AStruct shows robust performance in both simulated datasets and public icSHAPE datasets. We reveal that single nucleotide polymorphisms (SNPs) with higher AStruct scores are enriched in coding regions and tend to be functional. These SNPs are highly conservative, have the potential to disrupt sites involved in m6A modification or protein binding, and are frequently associated with disease. Conclusions: AStruct is a tool dedicated to invoke allele-specific RNA secondary structure events at heterozygous SNPs in RT-stop based structuromic probing data. It utilizes allelic variants, base pairing and RT-stop information under different cell conditions to detect dynamic and functional ASRS. Compared to sequence-based tools, AStruct considers dynamic cell conditions and outperforms in detecting functional variants. AStruct is implemented in JAVA and is freely accessible at: https://github.com/canceromics/AStruct.

Wei Qin, Lin Yang, Xiaotong Chen, Shanyu Ye, Aijun Liu, Dongfeng Chen, Kunhua Hu. IJSC -0001;0:. https://doi.org/10.15283/ijsc22046

Tumor cells surviving hypoxic stress acquire the ability to drive cancer progression. To explore the contribution of dehydrogenases to the low oxygen concentration response, we used siRNAs targeting 163 dehydrogenase-coding genes and discovered that glutamate dehydrogenase 1 (GDH1) plays a critical role in regulating colorectal cancer (CRC) cell survival under hypoxia. We observed that GDH1 deficiency had an inhibitory effect on CRC occurrence and impaired hypoxia-inducible factor 1-alpha (HIF-1α) stability even under hypoxia. Mechanistically, hypoxia triggered p300 recruitment to GDH1, promoting its acetylation at K503 and K527. GDH1 acetylation at K527 induced the formation of a GDH1 complex with EGLN1/HIF-1α; in contrast, GDH1 acetylation at K503 reinforced its affinity for α-ketoglutarate (αKG), and glutamate production. In line with this view, αKG is a product of GDH1 under normoxia, but hypoxia stimulation reversed GDH1 enzyme activity and αKG consumption by the EGLN1/HIF-1α complex, increasing HIF-1α stability and promoting CRC progression. Clinically, hypoxia-modulated GDH1 AcK503/527 can be used as a biomarker of CRC progression and is a potential target for CRC treatment.