CD28-CBCA driven PKM splicing licenses CD8 T cell glycolysis and effector function
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA714096 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13953633_1.fastq.gz | Fastqsanger.gz | |||
| SRR13953633_2.fastq.gz | Fastqsanger.gz | |||
| SRR13953634_1.fastq.gz | Fastqsanger.gz | |||
| SRR13953634_2.fastq.gz | Fastqsanger.gz | |||
| SRR13953635_1.fastq.gz | Fastqsanger.gz |
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Project description:T cells stimulated in the absence of CD28 costimulation are functionally anergic, characterized by reduced glucose metabolism and ability to produce effector cytokines. Surprisingly, previous studies have shown relatively few CD28-specific changes in gene transcription upon costimulation. Using mice expressing mutations in the CD28 cytoplasmic tail, we show a major effect of CD28 costimulation is alternative splicing of numerous genes including the glycolytic enzyme pyruvate kinase (Pkm), which is preferentially spliced from Pkm1 to Pkm2 upon stimulation. PKM2 is dispensable for T cell activation, but necessary for CD8+ T cells to utilize aerobic glycolysis, produce effector cytokines, and provide anti-tumor immunity. Mechanistically, CD28 regulates expression of the Cap-Binding Complex adaptor protein ARS2, which binds Pkm splicing factors and facilitates their interaction with the elongating transcript. Data suggest a major function of CD28 costimulation is control of RNA biogenesis in support of T cell transcriptome remodeling and acquisition of effector function.
2023-09-01 | GSE168824 | GEO
Project description:Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM isoform, effectively constraining lower glycolysis. Here, we report the discovery of novel PKM activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the non-essential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress. A549 cancer cells were treated with compound-16 for up to 24 hours in the presence and absence of serine in the media.
2012-12-20 | E-GEOD-40191 | biostudies-arrayexpress
Project description:Metabolic reprogramming dictates the fate and function of stimulated T cells, yet these pathways can be suppressed in T cells subjected to unique microenvironments, such as in a tumor. We previously showed that glycolytic and mitochondrial adaptations directly contribute to reduced effector functions of Renal Cell Carcinoma (RCC) CD8 tumor infiltrating lymphocytes (TIL). Here we define the role of these metabolic pathways in the activation and effector functions of RCC CD8 TIL. CD28 co-stimulation plays a key role to augment T cell activation and metabolism and is antagonized by inhibitory and checkpoint immunotherapy receptors CTLA4 and PD-1. While RCC CD8 TIL activated poorly when stimulated through the T cell receptor alone, addition of CD28 co-stimulation greatly enhanced activation, function, and proliferation. CD28 co-stimulation reprogrammed RCC CD8 TIL metabolism with increased glycolysis and mitochondrial oxidative metabolism, in part through upregulation of GLUT3. Mitochondria also became more fused, with higher membrane potential and overall mass. These phenotypes were dependent on glucose metabolism, as the glycolytic inhibitor 2-deoxyglucose both prevented changes to mitochondria and suppressed RCC CD8 TIL activation and function. These data show that CD28 co-stimulation can restore RCC CD8 TIL metabolism and function through rescue of T cell glycolysis that supports mitochondrial mass and activity.
2020-08-10 | GSE151669 | GEO
Project description:Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM isoform, effectively constraining lower glycolysis. Here, we report the discovery of novel PKM activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the non-essential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress.
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Project description:Pyruvate kinase M isoform 2 (PKM2) is preferentially expressed in nearly all cancers. It primarily functions as the last enzyme in glycolysis, but has other reported non-canonical functions, including recruiting transcription factors to oncogenes, and phosphorylating proteins. We previously described antisense oligonucleotides that disrupt alternative splicing of PKM pre-mRNA (PKM-ASOs), resulting in PKM2-to-PKM1 isoform switching in hepatocellular carcinoma (HCC), which reduces HCC growth. PKM1 has higher enzymatic activity than PKM2, which potentially drives metabolism away from macromolecule synthesis, and may explain decreased HCC growth upon PKM-ASO treatment. As PKM-ASOs also reduce PKM2 levels, how PKM splice-switching inhibits HCC cell proliferation was unclear. Here, we characterized the individual consequences of altering PKM1 or PKM2 protein levels in HCC, and observed that reducing PKM2 alone was sufficient to decrease HCC cell proliferation, whereas overexpressing PKM1 had no effect. Moreover, increasing PK activity via a small-molecule PKM2 activator had no effect on HCC cell proliferation, suggesting that PKM-ASOs affect PKM2’s non-metabolic functions. Transcriptomic and RT-qPCR analyses of HCC cells treated with PKM-ASO or PKM2-siRNA revealed upregulation of dual-specificity phosphatase 2 (DUSP2) and other related DUSPs, which act directly on ERK1/2 in the MAPK signaling pathway. Luciferase reporter assays demonstrated that PKM-ASO treatment activated the DUSP2 promoter, which correlated with decreased ERK1/2 phosphorylation. Lenvatinib is a second-line HCC therapy that indirectly reduces ERK1/2 phosphorylation, and combined treatment with PKM-ASOs inhibited proliferation of HCC cells more than either treatment alone. In summary, our results reveal a mechanism by which PKM-ASOs affect PKM2 dependency in HCC.
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Project description:CD28 Co-stimulation Drives Tumor-Infiltrating T Cell Glycolysis to Promote Inflammation
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Project description:During persistent antigen stimulation, such as in chronic infections and cancer, T cells differentiate into a hypofunctional exhausted state to survive. Exhausted PD-1+ CD8 T cell responses are sustained by TCF-1+ precursors (Tpex) that self-renew or differentiate into exhausted T cells (Tex) that retain some effector function. Tpex have high expression of the costimulatory molecule CD28 and are the cells that respond to PD-1 targeted immunotherapy. Here, we demonstrate that abrogation of CD28 signaling impairs maintenance of anti-viral T cell responses during chronic infection. Low-level CD28 signaling was required to preserve mitochondrial fitness and self-renewal of Tpex, whereas stronger CD28 signaling enhanced glycolysis and promoted Tpex differentiation. Our findings show that CD28 signaling is essential for long-term maintenance of antigen-specific T cells during chronic stimulation, and suggest that the activation status of antigen presenting cells determines Tpex differentiation into more functional effector-like Tex cells.
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Project description:In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we ask whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e., ER-excluded) polyribosome interactor and investigate how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels–providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode for lysine and glutamate enriched tracts. Additionally, PKM binding to ribosomes causes translational stalling near these lysine and glutamate encoding sequences. Lastly, we find that PKM recruitment to polysomes is dependent on poly-ADP ribosylation. Overall, our study uncovers a novel role for PKM in posttranscriptional gene regulation, linking cellular metabolism and mRNA translation, and provides the first reported evidence of nascent chains being co-translationally modified with poly-ADP ribose.
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Project description:The adoptive transfer of chimeric antigen receptor- (CAR) modified T cells is revolutionizing the treatment of B cell malignancies and has the potential to be applied to other diseases. CARs redirect T cell specificity by linking an antigen recognition domain to T cell signaling modules comprised of CD3z to provide signal 1, and CD28 or 4-1BB to provide costimulation. CD28/CD3z and 4-1BB/CD3z CARs confer differences in effector function and cell fate that affect clinical efficacy and toxicity. These differences may result from activation of divergent transcriptional programs. To gain this insight, we analyzed changes in gene expression in stimulated and resting CD28/CD3z or 4-1BB/CD3z CAR T cells. CD28/CD3z CAR stimulation initiated more marked early transcriptional changes with greater fold increases in the expression of effector molecules including GZMB, IFNG, IL2, TNF, and IL6. Direct comparison of CD28/CD3z and 4-1BB/CD3z samples stimulated for 6 hours identified 1,673 differentially expressed genes. Of these, the memory T cell-associated genes KLF2, IL7R, and FAM65B were expressed at lower levels in CD28/CD3z CAR T cells. KLF2 and IL7R are FOXO transcription factor family targets and we found that FOXO4 expression was similarly reduced in CD28/CD3z CAR T cells. CD28/CD3z CAR stimulation induces an effector T cell-like transcriptional profile that may underlie the decreased persistence and increased risks of toxicities observed with CD28/CD3z CAR T cells in early clinical trials.
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Project description:Bladder cancer (BCa) is one of the most common malignant tumors of urinary system and has high incidence rate and mortality but there is a lack of effective treatment. Therefore, an in-depth study of the molecular mechanisms involved in BCa is of great significance for improving the survival of patients with advanced BCa. We found that the expression of RBMX was significantly declined in BCa, especially in muscle-invasive BCa. BCa patients with low levels of RBMX had poor prognoses. By in vitro and in vivo experiments, RBMX was shown to inhibit BCa cells growth and metastasis. Co-IP coupled with MS and GO analysis identified hnRNP A1 as a RBMX-binding protein. Mechanistically, RBMX competitively bond to the RGG box of hnRNP A1 and antagonized the hnRNP A1-mediated regulation of PKM splicing by blocking the binding of the RGG motif of hnRNP A1 to the sequences flanking PKM exon 9, resulting in downregulation of PKM2 and upregulation of PKM1. By decreasing the PKM2/PKM1 ratio, RBMX suppressed BCa cells aerobic glycolysis, which further inhibited tumorigenicity and progression of BCa.
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