Bulk RNA-Seq analysis of adoptively co-transferred PKM2 wildtype and PKM2 knockout CD8+ T cells into tumor-bearing mice subsequently treated with and without PD-1 checkpoint blockade
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ABSTRACT: In this study we investigated how manipulation of the glycolytic enzyme pyruvate kinase muscle 2 (PKM2) alters CD8+ T cell gene expression and responsiveness to PD-1 checkpoint blockade in orthotopic mouse lung adenocarcinoma. In this dataset, we performed adoptive co-transfers of antigen-specific OT-I+ Thy1.1+ CD8+ T cells genetically modified by CRISPR/Cas9 ribonucleoproteins to become PKM2 knockout (in Thy1.1+/- cells) or PKM2 wildtype (in Thy1.1+/+ cells). T cells were transferred into orthotopic mouse lung adenocarcinoma HKP1-ova-GFP tumor-bearing syngeneic C57BL/6 mice, and subsequently mice were treated with either IgG or anti-PD-1. Adoptively-transferred T cells were sorted based on Thy1.1 zygosity from tumors at two different time points and bulk RNA sequenced.
Project description:Alternative splicing of the Pkm gene product generates the PKM1 and PKM2 isoforms of pyruvate kinase, and PKM2 expression is closely linked to embryogenesis, tissue regeneration, and cancer. To interrogate the functional requirement for PKM2 during development and tissue homeostasis, we generated germline PKM2 null mice (Pkm2-/-). Unexpectedly, despite being the primary isoform expressed in most wild-type adult tissues, we found that Pkm2-/- mice are viable and fertile. Thus, PKM2 is not required for embryonic or postnatal development. Loss of PKM2 leads to compensatory expression of PKM1 in the tissues that normally express PKM2. Strikingly, PKM2 loss leads to spontaneous development of hepatocellular carcinoma (HCC) with high penetrance that is accompanied by progressive changes in systemic metabolism characterized by altered systemic glucose homeostasis, inflammation, and hepatic steatosis. Therefore, in addition to its role in cancer metabolism, PKM2 plays a role in controlling systemic metabolic homeostasis and inflammation, thereby preventing HCC by a non-cell-autonomous mechanism. RNA was isolated from flash frozen ground whole liver tissue of 35 week old PKM2 KO and WT mice. Three independent mice from each condition were used as biological replicates.
Project description:Oxidative stress is a major pathogenic mechanism in Parkinson’s disease (PD). As an important cellular antioxidant, glutathione (GSH) balances the production and incorporation of free radicals to protect neurons from oxidative damage. Unfortunately, the GSH level is greatly decreased in the brains of PD patients. Hence, clarifying the molecular mechanism of GSH deficiency may help deepen our knowledge of PD pathogenesis. Here we report that the astrocytic dopamine D2 receptor (DRD2) regulates GSH synthesis via PKM2-mediated Nrf2 transactivation. Besides, we find that pyridoxine can dimerize PKM2 to promote GSH biosynthesis. Further experiments show that pyridoxine supplementation increases the resistance of nigral dopaminergic neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in wild-type mice as well as in astrocytic Drd2 conditional knockout mice. Given that dopamine agonist treatment in PD is limited by their intolerable side effects and diminished effectiveness over time, we anticipate dimerizing PKM2 to be a new strategy for PD treatment.
Project description:Bulk RNA-Seq analysis of adoptively co-transferred PKM2 wildtype and PKM2 knockout CD8+ T cells into tumor-bearing mice subsequently treated with and without PD-1 checkpoint blockade
Project description:Purpose: In this study, we compared transcriptome profiling (RNA-seq) between normal mouse embryonic stem cell (E14) and Hexokinase2 (Hk2)/ Pyruvate Kinase M2 (Pkm2) overexpressed E14 cell. Result: Using an optimized data analysis workflow, we mapped over 4 billion sequence reads per sample to the mouse genome (build mm9) and identified 28698 transcripts in 5 samples. Conclusion: Our study represents the first detailed analysis of Hk2/ Pkm2 overexpressed E14 cell transcriptomes, generated by RNA-seq technology We compared transcriptome profiling (RNA-Seq) between normal mouse embryonic stem cell (E14) and E14 cells over-expressing Hexokinase2 (Hk2)/Pyruvate Kinase M2 (Pkm2)
Project description:We profiled global gene expression for two separate lines of mouse embryonic fibroblasts and find that deletion of PKM2 and expression of PKM1 does not alter global gene expression profiles. We used microarrays to futher characterize the effects of PKM1 expression compared to PKM2 expression on global gene expression in mouse embryonic fibroblasts. Primary mouse embryonic fibroblast cells were used for RNA extraction and hybridization on Affymetrix microarrays. Intensity files were RMA normalized using Affymetrix expression console.
Project description:Alternative splicing of the Pkm gene product generates the PKM1 and PKM2 isoforms of pyruvate kinase, and PKM2 expression is closely linked to embryogenesis, tissue regeneration, and cancer. To interrogate the functional requirement for PKM2 during development and tissue homeostasis, we generated germline PKM2 null mice (Pkm2-/-). Unexpectedly, despite being the primary isoform expressed in most wild-type adult tissues, we found that Pkm2-/- mice are viable and fertile. Thus, PKM2 is not required for embryonic or postnatal development. Loss of PKM2 leads to compensatory expression of PKM1 in the tissues that normally express PKM2. Strikingly, PKM2 loss leads to spontaneous development of hepatocellular carcinoma (HCC) with high penetrance that is accompanied by progressive changes in systemic metabolism characterized by altered systemic glucose homeostasis, inflammation, and hepatic steatosis. Therefore, in addition to its role in cancer metabolism, PKM2 plays a role in controlling systemic metabolic homeostasis and inflammation, thereby preventing HCC by a non-cell-autonomous mechanism.
Project description:The study identified Pkm2 to be highly expressed in human embryonic stem cells and hepatocellular carcinoma cells, and targeted by a microRNA that is highly expressed in human primary hepatocytes. Gene expression patterns were compared from total RNA obtained from human embryonic stem cells, human primary hepatocytes, and hepG2 and hep3B.
Project description:During myocardial infarction (MI), energy production decreases as hypoxia inhibits oxidative metabolism. Our lab has identified an ischemia-regulated alternative splicing event of the glycolytic enzyme pyruvate kinase (muscle, PKM), reducing PKM1 expression and increasing PKM2 expression. We hypothesized the upregulation of PKM2 aids in energy production to maintain heart function after MI. We utilized high-throughput sequencing to evaluate altered gene expression and identify pathways that may be regulated by PKM2.
Project description:Pyruvate kinase M2 (PKM2), the rate-limiting enzyme of glycolysis, plays a critical role in macrophage activation and a broad spectrum of chronic liver diseases. However, whether PKM2 contributes to the pathogenesis of acute liver injury (ALI) remains largely unexplored. By bioinformatic screening and analysis of ALI liver, we found that PKM2 was significantly upregulated in the liver tissues of ALI patients and mice. Immunofluorescence staining further demonstrated that PKM2 was markedly upregulated in macrophages during ALI progression. Notably, macrophage PKM2 depletion effectively alleviated acetaminophen (APAP)- and lipopolysaccharide/D-galactosamine (LPS/D-GalN)-induced ALI, as demonstrated by ameliorated immune cells infiltration, pro-inflammatory mediators, and hepatocellular cell death. PKM2-deficient macrophages showed M2 anti-inflammatory polarization in vivo and in vitro. Furthermore, PKM2 deletion limited HIF-1α signaling and aerobic glycolysis of macrophages, which thereby attenuated macrophage pro-inflammatory activation and hepatocyte injury. Pharmacological PKM2 antagonist efficiently ameliorated liver injury and prolonged the survival of mice in APAP-induced ALI model. Our study highlights the pivotal role of macrophage PKM2 in advancing ALI, and therapeutic targeting of PKM2 may serve as a novel strategy to combat ALI.
Project description:We performed 10x single cell RNA-seq on sorted populations: T cell (7AAD- Calcein blue+ CD45+ THY1.1- TCRb+), fibroblasts (7AAD- Calcein Blue+ CD45- THY1.1- CD31- PDPN+), myeloid cells (7AAD- Calcein Blue+ CD45+ THY1.1- CD11b+) and tumor cells (7AAD- Calcein Blue+ CD45- THY1.1+) from cells in an orthotopic EMT6 tumor model 7 days after treatment initiation in four experimental groups: 1) Control 2) aPD-L1 3) aTGFb 4) aPD-L1 and aTGFb.