ABSTRACT
T cell exhaustion limits anti-tumor immunity and responses to immunotherapy. Here, we explored the microenvironmental signals regulating T cell exhaustion using a model of chronic lymphocytic leukemia (CLL). Single-cell analyses identified a subset of PD-1hi, functionally impaired CD8+ T cells that accumulated in secondary lymphoid organs during disease progression and a functionally competent PD-1int subset. Frequencies of PD-1int TCF-1+ CD8+ T cells decreased upon Il10rb or Stat3 deletion, leading to accumulation of PD-1hi cells and accelerated tumor progression. Mechanistically, inhibition of IL-10R signaling altered chromatin accessibility and disrupted cooperativity between the transcription factors NFAT and AP-1, promoting a distinct NFAT-associated program. Low IL10 expression or loss of IL-10R-STAT3 signaling correlated with increased frequencies of exhausted CD8+ T cells and poor survival in CLL and in breast cancer patients. Thus, balance between PD-1hi, exhausted CD8+ T cells and functional PD-1int TCF-1+ CD8+ T cells is regulated by cell-intrinsic IL-10R signaling, with implications for immunotherapy.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunotherapy/methods , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Receptors, Interleukin-10/metabolism , T-Lymphocyte Subsets/immunology , Animals , Cell Line, Tumor , Cells, Cultured , Cellular Microenvironment , Hepatocyte Nuclear Factor 1-alpha/metabolism , Humans , Immunity , Mice , Mice, Inbred C57BL , NFATC Transcription Factors/metabolism , Programmed Cell Death 1 Receptor/metabolism , Receptors, Interleukin-10/genetics , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Signal Transduction , Transcription Factor AP-1/metabolismABSTRACT
BACKGROUND: Glioblastomas (GBM) continue to remain one of the most dreaded tumours that are highly infiltrative in nature and easily preclude comprehensive surgical resection. GBMs pose an intricate etiology as they are being associated with a plethora of genetic and epigenetic lesions. Misregulation of the PI3 kinase pathway is one of the most familiar events in GBM. While the PI3 kinase signalling regulated pathways and genes have been comprehensively studied, its impact on the miRNome is yet to be explored. The objective of this study was to elucidate the PI3 kinase pathway regulated miRNAs in GBM. METHODS: miRNA expression profiling was conducted to monitor the differentially regulated miRNAs upon PI3 kinase pathway abrogation. qRT-PCR was used to measure the abundance of miR-326 and its host gene encoded transcript. Proliferation assay, colony suppression assay and wound healing assay were carried out in pre-miR transfected cells to investigate its role in malignant transformation. Potential targets of miR-326 were identified by transcriptome analysis of miR-326 overexpressing cells by whole RNA sequencing and selected targets were validated. Several publically available data sets were used for various investigations described above. RESULTS: We identified several miRNA that were regulated by PI3 kinase pathway. miR-326, a GBM downregulated miRNA, was validated as one of the miRNAs whose expression was alleviated upon abrogation of the PI3 kinase pathway. Overexpression of miR-326 resulted in reduced proliferation, colony suppression and hindered the migration capacity of glioma cells. Arrestin, Beta 1 (ARRB1), the host gene of miR-326, was also downregulated in GBM and interestingly, the expression of ARRB1 was also alleviated upon inhibition of the PI3 kinase pathway, indicating similar regulation pattern. More importantly, miR-326 exhibited a significant positive correlation with ARRB1 in terms of its expression. Transcriptome analysis upon miR-326 overexpression coupled with integrative bioinformatics approach identified several putative targets of miR-326. Selected targets were validated and interestingly found to be upregulated in GBM. CONCLUSIONS: Taken together, our study uncovered the PI3 kinase regulated miRNome in GBM. miR-326, a PI3 kinase pathway inhibited miRNA, was demonstrated as a tumour suppressor miRNA in GBM.
Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Glioblastoma/genetics , Glioblastoma/metabolism , MicroRNAs/genetics , Phosphatidylinositol 3-Kinases/genetics , Signal Transduction , Transcriptome , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Cluster Analysis , Computational Biology/methods , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Ontology , Genes, Tumor Suppressor , Humans , Phosphatidylinositol 3-Kinases/metabolism , RNA Interference , beta-Arrestin 1/geneticsABSTRACT
Chronic lymphocytic leukemia (CLL) is a B-cell malignancy mainly occurring at an advanced age with no single major genetic driver. Transgenic expression of TCL1 in B cells leads after a long latency to a CLL-like disease in aged Eµ-TCL1 mice suggesting that TCL1 overexpression is not sufficient for full leukemic transformation. In search for secondary genetic events and to elucidate the clonal evolution of CLL, we performed whole exome and B-cell receptor sequencing of longitudinal leukemia samples of Eµ-TCL1 mice. We observed a B-cell receptor stereotypy, as described in patients, confirming that CLL is an antigen-driven disease. Deep sequencing showed that leukemia in Eµ-TCL1 mice is mostly monoclonal. Rare oligoclonality was associated with inability of tumors to develop disease upon adoptive transfer in mice. In addition, we identified clonal changes and a sequential acquisition of mutations with known relevance in CLL, which highlights the genetic similarities and therefore, suitability of the Eµ-TCL1 mouse model for progressive CLL. Among them, a recurrent gain of chromosome 15, where Myc is located, was identified in almost all tumors in Eµ-TCL1 mice. Interestingly, amplification of 8q24, the chromosomal region containing MYC in humans, was associated with worse outcome of patients with CLL.
Subject(s)
Clonal Evolution , Gain of Function Mutation , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins/metabolism , Animals , Chromosomes , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Proto-Oncogene Proteins/geneticsABSTRACT
The major part of the genome that was previously called junk DNA has been shown to be dynamically transcribed to produce non-coding RNAs. Among them, the long non-coding RNAs (lncRNA) play diverse roles in the cellular context and are therefore involved in various diseases like cancer. LncRNA transcript profiling of glioblastoma (n = 19) and control brain samples (n = 9) identified 2,774 and 5,016 lncRNAs to be upregulated and downregulated in GBMs respectively. Correlation analysis of differentially regulated lncRNAs with mRNA and lncRNA identified several lncRNAs that may potentially regulate many tumor relevant mRNAs and lncRNAs both at nearby locations (cis) and far locations (trans). Integration of our data set with TCGA GBM RNA-Seq data (n = 172) revealed many lncRNAs as a host as well as decoy for many tumor regulated miRNAs. The expression pattern of seven lncRNAs- HOXD-AS2, RP4-792G4.2, CRNDE, ANRIL, RP11-389G6.3, RP11-325122.2 and AC123886.2 was validated by TCGA RNA-Seq data and RT-qPCR. Silencing ANRIL, a GBM upregulated lncRNA, inhibited glioma cell proliferation and colony growth. Cox regression analysis identified several prognostic lncRNAs. An lncRNA risk score derived from five lnRNAs-RP6-99M1.2, SOX21-AS1, CTD-2127H9.1, RP11-375B1.3 and RP3-449M8.9 predicted survival independent of all other markers. Multivariate cox regression analysis involving G-CIMP, IDH1 mutation, MGMT promoter methylation identified lncRNA risk score to be an independent poor predictor of GBM survival. The lncRNA risk score also stratified GBM patients into low and high risk with significant survival difference. Thus our study demonstrates the importance of lncRNA in GBM pathology and underscores the potential possibility of targeting lncRNA for GBM therapy.
ABSTRACT
Chromothripsis and chromoanasynthesis are catastrophic events leading to clustered genomic rearrangements. Whole-genome sequencing revealed frequent complex genomic rearrangements (n = 16/26) in brain tumors developing in mice deficient for factors involved in homologous-recombination-repair or non-homologous-end-joining. Catastrophic events were tightly linked to Myc/Mycn amplification, with increased DNA damage and inefficient apoptotic response already observable at early postnatal stages. Inhibition of repair processes and comparison of the mouse tumors with human medulloblastomas (n = 68) and glioblastomas (n = 32) identified chromothripsis as associated with MYC/MYCN gains and with DNA repair deficiencies, pointing towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements.
Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/pathology , DNA Damage/genetics , DNA Repair/genetics , Genome , Animals , Apoptosis/genetics , Cell Line, Tumor , DNA End-Joining Repair/genetics , DNA-Binding Proteins/metabolism , Gene Amplification , Gene Rearrangement/genetics , Homologous Recombination/genetics , Humans , Karyotyping , Mice , N-Myc Proto-Oncogene Protein/genetics , Neural Stem Cells/metabolism , Neural Stem Cells/pathology , Proto-Oncogene Proteins c-myc/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
BACKGROUND: Glioma is the most common of all primary brain tumors with poor prognosis and high mortality. The 2016 World Health Organization classification of the tumors of central nervous system uses molecular parameters in addition to histology to redefine many tumor entities. The new classification scheme divides diffuse gliomas into low-grade glioma (LGG) and glioblastoma (GBM) as per histology. LGGs are further divided into isocitrate dehydrogenase (IDH) wild type or mutant, which is further classified into either oligodendroglioma that harbors 1p/19q codeletion or diffuse astrocytoma that has an intact 1p/19q loci but enriched for ATRX loss and TP53 mutation. GBMs are divided into IDH wild type that corresponds to primary or de novo GBMs and IDH mutant that corresponds to secondary or progressive GBMs. To make the 2016 WHO subtypes of diffuse gliomas more robust, we carried out Prediction Analysis of Microarrays (PAM) to develop DNA methylation signatures for these subtypes. RESULTS: In this study, we applied PAM on a training set of diffuse gliomas derived from The Cancer Genome Atlas (TCGA) and identified DNA methylation signatures to classify LGG IDH wild type from LGG IDH mutant, LGG IDH mutant with 1p/19q codeletion from LGG IDH mutant with intact 1p/19q loci and GBM IDH wild type from GBM IDH mutant with an accuracy of 99-100%. The signatures were validated using the test set of diffuse glioma samples derived from TCGA with an accuracy of 96 to 99%. In addition, we also carried out additional validation of all three signatures using independent LGG and GBM cohorts. Further, the methylation signatures identified a fraction of samples as discordant, which were found to have molecular and clinical features typical of the subtype as identified by methylation signatures. CONCLUSIONS: Thus, we identified methylation signatures that classified different subtypes of diffuse glioma accurately and propose that these signatures could complement 2016 WHO classification scheme of diffuse glioma.
Subject(s)
Brain Neoplasms/classification , DNA Methylation , Glioma/classification , Brain Neoplasms/genetics , CpG Islands , Databases, Genetic , Epigenesis, Genetic , Glioma/genetics , Humans , Isocitrate Dehydrogenase/genetics , Oligonucleotide Array Sequence Analysis , Tumor Suppressor Protein p53/genetics , World Health Organization , X-linked Nuclear Protein/geneticsABSTRACT
Neurological disorders are known to show similar phenotypic manifestations like anxiety, depression, and cognitive impairment. There is a need to identify shared genetic markers and molecular pathways in these diseases, which lead to such comorbid conditions. Our study aims to prioritize novel genetic markers that might increase the susceptibility of patients affected with one neurological disorder to other diseases with similar manifestations. Identification of pathways involving common candidate markers will help in the development of improved diagnosis and treatments strategies for patients affected with neurological disorders. This systems biology study for the first time integratively uses 3D-structural protein interface descriptors and network topological properties that characterize proteins in a neurological protein interaction network, to aid the identification of genes that are previously not known to be shared between these diseases. Results of protein prioritization by machine learning have identified known as well as new genetic markers which might have direct or indirect involvement in several neurological disorders. Important gene hubs have also been identified that provide an evidence for shared molecular pathways in the neurological disease network.