技术资料

The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) can modulate transcript abundance. Previous studies have identified several examples of such a regulation in developing neurons. However,the systems-level effects of AS-NMD in this context are poorly understood. We developed an R package,factR2,which offers a comprehensive suite of AS-NMD analysis functions. Using this tool,we conducted a longitudinal analysis of gene expression in pluripotent stem cells undergoing induced neuronal differentiation. Our analysis uncovers hundreds of AS-NMD events with significant potential to regulate gene expression. Notably,this regulation is significantly overrepresented in specific functional groups of developmentally downregulated genes. Particularly strong association with gene downregulation is detected for alternative cassette exons stimulating NMD upon their inclusion into mature mRNA. By combining bioinformatic analyses with CRISPR/Cas9 genome editing and other experimental approaches we show that NMD-stimulating cassette exons regulated by the RNA-binding protein PTBP1 dampen the expression of their genes in developing neurons. We also provided evidence that the inclusion of NMD-stimulating cassette exons into mature mRNAs is temporally coordinated with NMD-independent gene repression mechanisms. Our study provides an accessible workflow for the discovery and prioritization of AS-NMD targets. It further argues that the AS-NMD pathway plays a widespread role in developing neurons by facilitating the downregulation of functionally related non-neuronal genes. The online version contains supplementary material available at 10.1186/s13059-024-03305-8. View Publication

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor; GBM’s inevitable recurrence suggests that glioblastoma stem cells (GSC) allow these tumors to persist. Our previous work showed that FOSL1,transactivated by the STAT3 gene,functions as a tumorigenic gene in glioma pathogenesis and acts as a diagnostic marker and potential drug target in glioma patients. Accumulating evidence shows that STAT3 and NF-κB cooperate to promote the development and progression of various cancers. The link between STAT3 and NF-κB suggests that NF-κB can also transcriptionally regulate FOSL1 and contribute to gliomagenesis. To investigate downstream molecules of FOSL1,we analyzed the transcriptome after overexpressing FOSL1 in a PDX-L14 line characterized by deficient FOSL1 expression. We then conducted immunohistochemical staining for FOSL1 and NF-κB p65 using rabbit polyclonal anti-FOSL1 and NF-κB p65 in glioma tissue microarrays (TMA) derived from 141 glioma patients and 15 healthy individuals. Next,mutants of the human FOSL1 promoter,featuring mutations in essential binding sites for NF-κB were generated using a Q5 site-directed mutagenesis kit. Subsequently,we examined luciferase activity in glioma cells and compared it to the wild-type FOSL1 promoter. Then,we explored the mutual regulation between NF-κB signaling and FOSL1 by modulating the expression of NF-κB or FOSL1. Subsequently,we assessed the activity of FOSL1 and NF-κB. To understand the role of FOSL1 in cell growth and stemness,we conducted a CCK-8 assay and cell cycle analysis,assessing apoptosis and GSC markers,ALDH1,and CD133 under varying FOSL1 expression conditions. Transcriptome analyses of downstream molecules of FOSL1 show that NF-κB signaling pathway is regulated by FOSL1. NF-κB p65 protein expression correlates to the expression of FOSL1 in glioma patients,and both are associated with glioma grades. NF-κB is a crucial transcription factor activating the FOSL1 promoter in glioma cells. Mutual regulation between NF-κB and FOSL1 contributes to glioma tumorigenesis and stemness through promoting G1/S transition and inhibiting apoptosis. Therefore,the FOSL1 molecular pathway is functionally connected to NF-κB activation,enhances stemness,and is indicative that FOSL1 may potentially be a novel GBM drug target. The online version contains supplementary material available at 10.1007/s00018-024-05293-1. View Publication

Precise gene editing uncovers mis-splicing of BUBR1 and CDC27 in human SF3B1-mutant HSPCs,leading to activation of mitotic checkpoint and rendering the cells sensitive to CHK1 inhibitor prexasertib. View Publication

Although platinum-based chemotherapy is the frontline regimen for colorectal cancer (CRC),drug resistance remains a major challenge affecting its therapeutic efficiency. However,there is limited research on the correlation between chemotherapy resistance and lipid metabolism,including PIK3CA mutant tumors. In this present study,we found that PIK3CA-E545K mutation attenuated cell apoptosis and increased the cell viability of CRC with L-OHP treatment in vitro and in vivo . Mechanistically,PIK3CA-E545K mutation promoted the nuclear accumulation of SREBP1,which promoted the transcription of Apolipoprotein A5 (APOA5). APOA5 activated the PPARγ signaling pathway to alleviate reactive oxygen species (ROS) production following L-OHP treatment,which contributed to cell survival of CRC cells. Moreover,APOA5 overexpression enhanced the stemness-related traits of CRC cells. Increased APOA5 expression was associated with PIK3CA mutation in tumor specimens and poor response to first-line chemotherapy,which was an independent detrimental factor for chemotherapy sensitivity in CRC patients. Taken together,this study indicated that PIK3CA-E545K mutation promoted L-OHP resistance by upregulating APOA5 transcription in CRC,which could be a potent target for improving L-OHP chemotherapeutic efficiency. Our study shed light to improve chemotherapy sensitivity through nutrient management in CRC. View Publication

Chimeric antigen receptor (CAR) T cell therapy has emerged as a powerful therapeutic approach against a range of hematologic malignancies. While the incorporation of CD28 or 4-1BB costimulatory signaling domains into CARs revolutionized immune responses,there is an exciting prospect of further enhancing CAR functionality. Here,we investigated the design of CD19 CARs enriched with distinct Toll-like receptor 4 (TLR4),myeloid differentiation primary response 88 (MyD88),or Toll/IL-1 domain-containing adaptor-inducing interferon (IFN)-β (TRIF) costimulatory domains. Screening of various designs identified several candidates with no tonic activity but with increased CD19 target cell-dependent interleukin (IL)-2 production. Human T cells transduced with the selected CAR construct exhibited augmented hIL-2 and hIFN-γ induction and cytotoxicity when cocultured with CD19-positive lymphoma and solid-tumor cell lines. RNA sequencing (RNA-seq) analysis demonstrated the upregulation of some genes involved in the innate immune response and T cell activation and proliferation. In experiments on a xenogeneic solid-tumor mice model,MyD88 and TLR4 CAR T cells exhibited prolonged remission. This study demonstrates that the integration of a truncated TLR4 signaling costimulatory domain could provide immunotherapeutic potential against both hematologic malignancies and solid tumors. View Publication

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer’s Disease (AD),but the mechanism underlying this link is poorly understood. In particular,the relevance of direct interactions between apoE and amyloid-β (Aβ) remains controversial. Here,single-molecule imaging shows that all isoforms of apoE associate with Aβ in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aβ co-aggregates account for ~50% of the mass of diffusible Aβ aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aβ tune disease-related functions of Aβ aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how,in an isoform- and lipidation-specific way,apoE modulates the aggregation,clearance and toxicity of Aβ. Selectively removing non-lipidated apoE4-Aβ co-aggregates enhances clearance of toxic Aβ by glial cells,and reduces secretion of inflammatory markers and membrane damage,demonstrating a clear path to AD therapeutics. Subject terms: Protein aggregation,Nanoscale biophysics View Publication

Adult T-cell leukemia/lymphoma (ATL) occurs after human T-cell leukemia virus type-1 (HTLV-1) infection with a long latency period exceeding several decades. This implies the presence of immune evasion mechanisms for HTLV-1-infected T cells. Although ATL cells have a CD4 + CD25 + phenotype similar to that of regulatory T cells (Tregs),they do not always possess the immunosuppressive functions of Tregs. Factors that impart effective immunosuppressive functions to HTLV-1-infected cells may exist. A previous study identified a new CD13 + Treg subpopulation with enhanced immunosuppressive activity. We,herein,describe the paired CD13 − (designated as MT-50.1) and CD13 + (MT-50.4) HTLV-1-infected T-cell lines with Treg-like phenotype,derived from the peripheral blood of a single patient with lymphoma-type ATL. The cell lines were found to be derived from HTLV-1-infected non-leukemic cells. MT-50.4 cells secreted higher levels of immunosuppressive cytokines,IL-10 and TGF-β,expressed higher levels of Foxp3,and showed stronger suppression of CD4 + CD25 − T cell proliferation than MT-50.1 cells. Furthermore,the CD13 inhibitor bestatin significantly attenuated MT-50.4 cell growth,while it did not for MT-50.1 cells. These findings suggest that CD13 expression may be involved in the increased Treg-like activity of MT-50.4 cells. Hence,MT-50.4 cells will be useful for in-depth studies of CD13 + Foxp3 + HTLV-1-infected cells. Subject terms: Cancer,Microbiology,Oncology View Publication

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock,but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma,we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction,abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent,synergistic therapeutic potential agnostic of glioma subtype,leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy,opening avenues for combinatorial treatment in these lethal malignancies. Subject terms: CNS cancer,Paediatric cancer,Radiotherapy View Publication

Malaria parasite invasion to host erythrocytes is mediated by multiple interactions between merozoite ligands and erythrocyte receptors that contribute toward the development of disease pathology. Here,we report a novel antigen Plasmodium prohibitin “ Pf PHB2” and identify its cognate partner “Hsp70A1A” in host erythrocyte that plays a crucial role in mediating host-parasite interaction during merozoite invasion. Using small interfering RNA (siRNA)- and glucosamine-6-phosphate riboswitch (glmS) ribozyme-mediated approach,we show that loss of Hsp70A1A in red blood cells (RBCs) or Pf PHB2 in infected red blood cells (iRBCs),respectively,inhibit Pf PHB2-Hsp70A1A interaction leading to invasion inhibition. Antibodies targeting Pf PHB2 and monoclonal antibody therapeutics against Hsp70A1A efficiently block parasite invasion. Recombinant Pf PHB2 binds to RBCs which is inhibited by anti- Pf PHB2 antibody and monoclonal antibody against Hsp70A1A. The validation of Pf PHB2 to serve as antigen is further supported by detection of anti- Pf PHB2 antibody in patient sera. Overall,this study proposes Pf PHB2 as vaccine candidate and highlights the use of monoclonal antibody therapeutics for future malaria treatment. Subject areas: biochemistry,molecular biology,immunology View Publication

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor,CDC20,preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However,the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression,we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for,gastrointestinal atrophy is detrimental. Remarkably,deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome,while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect,identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast,only overexpression of anti-apoptotic BCL2,but none of the BH3-only protein deficiencies mentioned above,can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo. View Publication

As a critical mitotic regulator,Aurora kinase A (AURKA) is aberrantly activated in a wide range of cancers. Therapeutic targeting of AUKRA is a promising strategy for the treatment of solid tumors. In this study,we evaluated the preclinical characteristics of JAB-2485,a small-molecule inhibitor of AURKA currently in Phase I/IIa clinical trial in the US ( NCT05490472 ). Biochemical studies demonstrated that JAB-2485 is potent and highly selective on AURKA,with subnanomolar IC 50 and around 1500-fold selectivity over AURKB or AURKC. In addition,JAB-2485 exhibited favorable pharmacokinetic properties featured by low clearance and good bioavailability,strong dose–response relationship,as well as low risk for hematotoxicity and off-target liability. As a single agent,JAB-2485 effectively induced G2/M cell cycle arrest and apoptosis and inhibited the proliferation of small cell lung cancer,triple-negative breast cancer,and neuroblastoma cells. Furthermore,JAB-2485 exhibited robust in vivo antitumor activity both as monotherapy and in combination with chemotherapies or the bromodomain inhibitor JAB-8263 in xenograft models of various cancer types. Together,these encouraging preclinical data provide a strong basis for safety and efficacy evaluations of JAB-2485 in the clinical setting. View Publication

STAT3 is constitutively activated in many cancer types,including lung cancer,and can induce cancer cell proliferation and cancer stem cell (CSC) maintenance. STAT3 is activated by tyrosine kinases,such as JAK and SRC,but the mechanism by which STAT3 maintains its activated state in cancer cells remains unclear. Here,we show that PRMT5 directly methylates STAT3 and enhances its activated tyrosine phosphorylation in non-small cell lung cancer (NSCLC) cells. PRMT5 expression is also induced by STAT3,suggesting the presence of a positive feedback loop in cancer cells. Furthermore,methylation of STAT3 at arginine 609 by PRMT5 is important for its transcriptional activity and support of tumour growth and CSC maintenance. Indeed,NSCLC cells expressing the STAT3 mutant which R609 was replaced to alanine (R609K) show significantly impaired tumour growth in nude mice. Overall,our study reveals a mechanism by which STAT3 remains activated in NSCLC and provides a new target for cancer therapeutic approaches. Subject terms: Oncogenes,Non-small-cell lung cancer,Growth factor signalling View Publication