Suppression of ITPKB degradation by Trim25 confers TMZ resistance in glioblastoma through ROS homeostasis

Temozolomide(TMZ)represents a standard-of-care chemotherapeutic agent in glioblastoma(GBM).However,the development of drug resistance constitutes a significant hurdle in the treatment of malignant glioma.Although specific innovative approaches,such as immunotherapy,have shown favorable clinical outcomes,the inherent invasiveness of most gliomas continues to make them challenging to treat.Consequently,there is an urgent need to identify effective therapeutic targets for gliomas to overcome chemoresistance and facilitate drug development.This investigation used mass spectrometry to examine the proteomic profiles of six pairs of GBM patients who underwent standard-of-care treatment and surgery for both primary and recurrent tumors.A total of 648 proteins exhibiting significant differential expression were identified.Gene Set Enrichment Analysis(GSEA)unveiled notable alterations in pathways related to METABOLISM_OF_LIPIDS and BIOLOGICAL_OXIDATIONS between the primary and recurrent groups.Validation through glioma tissue arrays and the Xiangya cohort confirmed substantial upregulation of inositol 1,4,5-triphosphate(IP3)kinase B(ITPKB)in the recurrence group,correlating with poor survival in glioma patients.In TMZ-resistant cells,the depletion of ITPKB led to an increase in reactive oxygen species(ROS)related to NADPH oxidase(NOX)activity and restored cell sensitivity to TMz.Mechanistically,the decreased phosphorylation of the E3 ligase Trim25 at the S100 position in recurrent GBM samples accounted for the weakened ITPKB ubiquitination.This,in turn,elevated ITPKB stability and impaired ROS production.Furthermore,ITPKB depletion or the ITPKB inhibitor GNF362 effectively overcome TMZ chemoresistance in a glioma xenograft mouse model.These findings reveal a novel mechanism underlying TMZ resistance and propose ITPKB as a promising therapeutic target forTMZ-resistant GBM.

CDK11 negatively regulates Wnt/β-catenin signaling in the endosomal compartment by affecting microtubule stability

Objectives:Improper activation of Wnt/β-catenin signaling has been implicated in human diseases.Beyond the well-studied glycogen synthase kinase 3p(GSK3p)and casein kinase 1(CK1),other kinases affecting Wnt/β-catenin signaling remain to be defined.Methods:To identify the kinases that modulate Wnt/β-catenin signaling,we applied a kinase small interfering RNA(siRNA)library screen approach.Luciferase assays,immunoblotting,and real-time polymerase chain reaction(PCR)were performed to confirm the regulation o f the Wnt/β-catenin signaling pathway by cyclin-dependent kinase 11(CDK11)and to investigate the underlying mechanism.Confocal immunofluorescence,coimmunoprecipitation(co-IP),and scratch wound assays were used to demonstrate colocalization,detect protein interactions,and explore the function of CDK11.Results:CDK11 was found to be a significant candidate kinase participating in the negative control of Wnt/P-catenin signaling.Down-regulation of CDK11 led to the accumulation of Wnt/β-catenin signaling receptor complexes,in a manner dependent on intact adenomatosis polyposis coli(APC)protein.Further analysis showed that CDK11 modulation of Wnt/P-catenin signaling engaged the endolysosomal machinery,and CDK11 knockdown enhanced the colocalization of Wnt/β-catenin signaling receptor complexes with early endosomes and decreased colocalization with lysosomes.Mechanistically,CDK11 was found to function in Wnt/β-catenin signaling by regulating microtubule stability.Depletion of CDK11 down-regulated acetyl-a-tubulin.Moreover,co-IP assays demonstrated that CDK11 interacts with the a-tubulin deacetylase SIRT2,whereas SIRT2 down-regulation in CDK11-depleted cells reversed the accumulation of Wnt/(3-catenin signaling receptor complexes.CDK11 was found to suppress cell migration through altered W nt/β-catenin signaling.Conclusions:CDK11 is a negative modulator of Wnt/β-catenin signaling that stabilizes microtubules,thus resulting in the dysregulation of receptor complex trafficking from early endosomes to lysosomes.

Regulation of CD8^(+)T memory and exhaustion by the mTOR signals

CD8^(+)T cells are the key executioners of the adaptive immune arm,which mediates antitumor and antiviral immunity.Naïve CD8^(+)T cells develop in the thymus and are quickly activated in the periphery after encountering a cognate antigen,which induces these cells to proliferate and differentiate into effector cells that fight the initial infection.Simultaneously,a fraction of these cells become long-lived memory CD8^(+)T cells that combat future infections.Notably,the generation and maintenance of memory cells is profoundly affected by various in vivo conditions,such as the mode of primary activation(e.g.,acute vs.chronic immunization)or fluctuations in host metabolic,inflammatory,or aging factors.Therefore,many T cells may be lost or become exhausted and no longer functional.Complicated intracellular signaling pathways,transcription factors,epigenetic modifications,and metabolic processes are involved in this process.Therefore,understanding the cellular and molecular basis for the generation and fate of memory and exhausted CD8^(+)cells is central for harnessing cellular immunity.In this review,we focus on mammalian target of rapamycin(mTOR),particularly signaling mediated by mTOR complex(mTORC)2 in memory and exhausted CD8^(+)T cells at the molecular level.

scRNA-seq profiling of neonatal and adult thymus-derived CD4+ T cells by a T cell origin-time tracing model

It is well documented that the neonatal thymus-derived (neonatal-TD) regulatory T cells (Treg) are essential to prevent lethal autoimmune diseases and allergies, and neonatal and adult thymus possesses distinct output potentials for naïve T cells, including Treg. However, the molecular features and detailed functional differences between neonatal-TD and adult thymus-derived (adult-TD) T cells in terms of their ability to maintain immune homeostasis during long-term environmental influences are still largely unknown, partially due to the lack of appropriate animal models to precisely trace these cells at specific time points. In this study, neonatal-TD and adult-TD CD4+ T cells from the spleen and Peyer's patches were traced for 9 weeks by a T cell origin-time tracing mouse model and analysed by single-cell RNA sequencing. More Treg but fewer naïve T cells were found in neonatal-TD CD4+ T cells from both tissues than those from adult-TD counterparts. Interestingly, the neonatal-TD Treg in both the spleen and Peyer's patches exhibited augmented expression of Foxp3, Gata3, Ctla4, Icos, Il2ra, Tgfb1, and Nrp1, as well as enriched Gene Ontology terms like T cell activation and tolerance induction, indicating an enhanced immunosuppressive function. These results were further confirmed by flow cytometry analysis and in vitro immune suppression assays. Flow cytometry also revealed a significantly higher proportion of neonatal-TD Treg in total Treg than that of adult-TD counterparts, suggesting the longer lifespan of neonatal-TD Treg. To investigate the intrinsic features of neonatal-TD and adult-TD CD4+ T cells, a shortened tracing time was performed. Surprisingly, the neonatal-TD and adult-TD CD4+ T cells had similar proportions of Treg and did not exhibit significant differences in Foxp3, Gata3, Ctla4, Icos, Il2ra, and Tgfb1 expression levels after tracing for 12 days. On the other hand, neonatal-TD Treg present an increased Nrp1 expression level compared with adult-TD counterparts, indicating the enhanced stability. Together, our work reveals that the neonatal-TD Treg are more immunosuppressive, which is likely shaped primarily by environmental factors.

Paradoxical effects of DNA tumor virus oncogenes on epithelium-derived tumor cell fate during tumor progression and chemotherapy response

Epstein-Barr virus(EBV)and human papillomavirus(HPV)infection is the risk factors for nasopharyngeal carcinoma and cervical carcinoma,respectively.However,clinical analyses demonstrate that EBV or HPV is associated with improved response of patients,although underlying mechanism remains unclear.Here,we reported that the oncoproteins of DNA viruses,such as LMP1 of EBV and E7 of HPV,inhibit PERK activity in cancer cells via the interaction of the viral oncoproteins with PERK through a conserved motif.Inhibition of PERK led to increased level of reactive oxygen species(ROS)that promoted tumor and enhanced the efficacy of chemotherapy in vivo.Consistently,disruption of viral oncoprotein-PERK interactions attenuated tumor growth and chemotherapy in both cancer cells and tumor-bearing mouse models.Our findings uncovered a paradoxical effect of DNA tumor virus oncoproteins on tumors and highlighted that targeting PERK might be an attractive strategy for the treatment of NPC and cervical carcinoma.

A novel reporter mouse line for studying alveolar macrophages

Alveolar macrophages(AMs)are self-maintained immune cells that play vital roles in lung homeostasis and immunity.Although reporter mice and culture systems have been established for studying macrophages,an accurate and specific reporter line for alveolar macrophage study is still not available.Here we reported a novel Rspo1-tdTomato gene reporter mouse line that could specifically label mouse AMs in a cell-intrinsic manner.Using this reporter system,we visualized the dynamics of alveolar macrophages intravitally under steady state and characterized the alveolar macrophage differentiation under in vitro condition.By performing ATAC-seq,we found that insertion of the tdTomato cassette in the Rspo1 locus increased the accessibility of a PPARE motif within the Rspo1 locus and revealed a potential regulation by key transcription factor PPAR-γfor alveolar macrophage differentiation in vitro and in vivo.Consistently,perturbation of PPAR-γby its agonist rosiglitazone or inhibitor GW9662 resulted in corresponding alteration of tdTomato expression in alveolar macrophages together with the transcription of PPAR-γdownstream target genes.Furthermore,global transcriptomic analyses of AMs from the wild type mice and the Rspo1-tdTomato mice showed comparable gene expression profiles,especially those AM-specific genes,confirming that the insertion of the tdTomato cassette in the Rspo1 locus does not impact the cell identity and biological function of AMs under normal condition.Taken together,our study provides an alternative tool for in vivo and in vitro labeling of alveolar macrophages with high specificity which could also be utilized as an indicator of PPAR-γactivity for future development of PPAR-γspecific targeting drugs.

Comments on‘MAP3K2-regulated intestinal stromal cells define a distinct stem cell niche’

For a long period of time,intestinal mesenchymal stromal cells(IMSC)have been considered as a relatively simple and homogeneous group of cells.These cells could effectively regulate intestinal homeostasis and epithelium integrity via producing growth factors and cytokines(Powell et al.,2011).With the help of single-cell transcriptomics studies,it has now been clear that IMSC are quite complex and heterogeneous(Kinchen et al.,2018).

Applying artificial intelligence for cancer immunotherapy

Artificial intelligence(AI)is a general term that refers to the use of a machine to imitate intelligent behavior for performing complex tasks with minimal human intervention,such as machine learning;this technology is revolutionizing and reshaping medicine.AI has considerable potential to perfect health-care systems in areas such as diagnostics,risk analysis,health information administration,lifestyle supervision,and virtual health assistance.In terms of immunotherapy,AI has been applied to the prediction of immunotherapy responses based on immune signatures,medical imaging and histological analysis.These features could also be highly useful in the management of cancer immunotherapy given their ever-increasing performance in improving diagnostic accuracy,optimizing treatment planning,predicting outcomes of care and reducing human resource costs.In this review,we present the details of AI and the current progression and state of the art in employing AI for cancer immunotherapy.Furthermore,we discuss the challenges,opportunities and corresponding strategies in applying the technology for widespread clinical deployment.Finally,we summarize the impact of AI on cancer immunotherapy and provide our perspectives about underlying applications of AI in the future.