Changes in alternative splicing and associated neo-antigens due to therapy
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ABSTRACT:
PROVIDER: EGAS00001004524 | EGA |
REPOSITORIES: EGA
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Project description:Alternative splicing is a rich source of tumor-specific neoantigen targets for immunotherapy. This holds promise for glioblastomas (GBMs), the most common primary tumors of the adult brain, which are resistant to standard-of-care therapy. Although most clinical trials enroll patients at recurrence, most preclinical studies have been done with specimens from primary disease. There are limited expression data from GBMs at recurrence and surprisingly little is known about the evolution of splicing patterns under therapy. We profiled 29 primary-recurrent paired human GBM specimens via RNA sequencing. We describe the landscape of alternative splicing in GBM at recurrence and contrast that to primary and non-malignant brain-tissue specimens. By screening single-cell atlases, we identify cell-type specific splicing patterns and novel splicing events in cell-surface proteins that are suitable targets for engineered T-cell therapies. We identify recurrent-specific isoforms of mitogen-activated kinase pathway genes that enhance invasiveness and are preferentially expressed by stem-like cells. These studies shed light on gene expression in recurrent GBM and identify novel targets for therapeutic development.
2020-12-10 | GSE155434 | GEO
Project description:Treating recurrent GBM is a clinical challenge due to its highly resistant and aggressive nature. In order to develop new therapeutic targets for recurrent GBM a better understanding of its molecular landscape is necessary. Here we used a cellular model, developed in our lab which generates paired primary and recurrent samples from GBM cell lines and primary patient samples hence allowing us to compare the molecular differences between the two populations. Total RNA seq analysis of parent and recurrent population of two cell lines and one patient sample revealed a significant upregulation of Extracellular matrix interaction in recurrent population. Since matrix stiffness plays a pivotal role in cell-ECM interaction and downstream signaling, we developed a system that mimicked the brain like substrate stiffness by using collagen coated polyacrylamide-based substrate whose stiffness can be modified from normal brain (0.5kPa) to tumorigenic (10kPa). Using these substrates, we were able to capture the morphological and physiological differences between parent and recurrent GBM which were not evident on plastic surfaces (~1 GPa). On 0.5kPa, unlike circular parent cells, recurrent GBM cells showed two morphologies (circular and elongated). The recurrent cells growing on 0.5kPa also showed higher proliferation, invasion, migration and in-vivo tumorigenicity in orthotropic GBM mouse model, compared to parent cells. Furthermore, recurrent cells exhibited elevated velocity irrespective of substrate stiffness, which indicated that recurrent cells may possess inherent differential mechanosignalling ability which was reflected by higher expression of ECM proteins like Collagen IVA, MMP2 and MMP9. Moreover, mice brain injected with recurrent cells grown on 0.5kPa substrate showed higher Young’s modulus values suggesting that recurrent cells conditioned on 0.5kPa make the surrounding ECM stiffer. Importantly, inhibition of EGFR signaling, that is amplified with tissue stiffening in GBM resulted in decreased invasion, migration and proliferation in 0.5kPa recurrent cells, but interestingly survival remained unaffected, highlighting the importance of mimicking the physiological stiffness of the brain mimicking clinical scenario. Total RNA seq analysis of parent and recurrent cells grown on plastic and 0.5kPa substrate identified PLEKHA7 as significantly upregulated gene specifically in 0.5kPa recurrent sample. Higher protein expression of PLEKHA7 in recurrent GBM as compared to primary GBM was validated in patient biopsies. Accordingly, PLEKHA7 knockdown reduced invasion and survival of recurrent GBM cells. Together, these data provides a model system that captures the differential mechanosensing signals of primary and recurrent GBM cells and identifies a novel potential target specific for recurrent GBM.
2023-06-29 | E-MTAB-11293 | biostudies-arrayexpress
Project description:Glioblastoma (GBM) is characterized by an exceptionally high intratumoral heterogeneity. However, the molecular mechanisms underlying the origin of different GBM cell populations remain unclear. Here we found that the composition of ribosomes of GBM cells in the tumor core and edge differ due to alternative RNA splicing. The acidic pH in the core switches pre-mRNA splicing of the ribosomal gene RPL22L1 toward the RPL22L1b isoform. To elucidate the functions of RPL22L1 isoforms we identified proteins that interact with RPL22L1a and RPL22L1b. First, we generated GBM cells with stable expression of Fc-tagged RPL22L1 isoforms. Fc-RPL22L1a and Fc-RPL22L1b together with their binding partners were isolated from neurospheres using magnetic beads and analyzed by LC-MS/MS.
2023-10-24 | PXD035767 | Pride
Project description:Glioblastoma multiforme (GBM) is the most lethal brain malignant neoplasm, associated with poor prognosis and high recurrence. The mechanisms involved in GBM development and recurrent remains largely unknown. Hence, it’s considerably imperative to explore the potential targets and unravel the mechanism implicated in GBM tumorigenesis and recurrence. In our study, mass spectrometry-based label-free quantitative proteomics was employed to explore the proteomic profiling in non-paired samples of primary and recurrent GBM. Bioinformatic analysis of differentially expression proteins (DEPs) were applied to screen the hub genes. Subsequently, hub genes expression was verified by western blot and immunohistochemistry (IHC) in GBM tissues, followed by its biological effect on GBM at a cellular level determined. We contoured the divergent landscape of proteome between primary and recurrent GBM, with only ten overlapped DEPs included. The several hub genes screened (e.g., CKAP4 and CANX) and enriched pathways correlated with primary GBM such as post-translation, metabolism, energy pathways and cell growth were profiled. Furthermore, CKAP4 was implicated in proliferation, migration and invasion of A172 and T98G cell lines. However, UQCRC1, together with relative enriched pathways such as oxidative phosphorylation, were screened in recurrent GBM. Establishment of the proteomics and several candidate proteins identified could pose considerable access to understand complex biochemical processes and obtain a better picture of GBM biology.
2021-06-06 | PXD026503 |
Project description:Glioblastoma (GBM) is characterized by an exceptionally high intratumoral heterogeneity. However, the molecular mechanisms underlying the origin of different GBM cell populations remain unclear. Here we found that the composition of ribosomes of GBM cells in the tumor core and edge differ due to alternative RNA splicing. The acidic pH in the core switches pre-mRNA splicing of the ribosomal gene RPL22L1 toward the RPL22L1b isoform. To elucidate the functions of RPL22L1 isoforms we identified proteins that interact with RPL22L1a and RPL22L1b. First, each isoform was expressed in E. coli and immobilized on magnetic beads. The beads were incubated with GBM cell lysates and proteins bound to the beads were identified using LC-MS/MS.
2023-10-24 | PXD035849 | Pride
Project description:The aim of this study was to obtain a landscape of alternative splicing (AS) in human hepatocellular carcinoma (HCC). We applied hybrid sequencing by integrating Pacbio long-read and Illumina short-read RNA sequencing data from 8 patient-derived HCC cell lines, and used an immortalized hepatocyte cell line as reference. This study revealed features of annotated and novel splice variants in HCC cells, including transcript length, AS type and protein changes. We also identified novel isoforms and tumor specific isoforms, which may represent potential biomarkers and targets for therapeutic development.
| EGAS00001002697 | EGA
Project description:Changes in alternative splicing are associated with several pathological conditions, including cancer. Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array platform was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events. The array consists of exon probes and thermodynamically balanced junction probes. Suboptimal probes are tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was first validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms for 8000 genes in lung cancer samples compared to matched normal lung tissue. The expression of lung cancer-associated splice isoforms was validated by RT-PCR. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer. In conclusion, this highly accurate methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies. 20 normal/tumor paired specimens. Tumor samples are from non-small cell lung cancer (NSCLC) whereas normals are from adjacent normal lung tissue.
2010-06-21 | E-GEOD-18346 | biostudies-arrayexpress
Project description:This study sought to determine whether an endometrial gland specific transcriptome and splicing profile are altered in the window of implantation (days 21-23 of the menstrual cycle) in women with recurrent pregnancy loss. Our data provide a comprehensive catalogue of gene isoforms and relative exon usage in endometrial glands. The endometrial gland targets identified in this study could be used to identify a perturbed endometrium, isolate causes of recurrent pregnancy loss and develop targeted therapies in personalised medicine.
2021-09-23 | GSE183555 | GEO
Project description:Glioblastoma (GBM) is the most common and devastating malignant brain tumor in adults. The mortality rate is very high despite different treatments. New therapeutic targets are therefore highly needed to improve patient care. Cell-surface proteins represent attractive targets due to their accessibility, their involvement in essential signaling pathways, and their dysregulated expression in cancer. Moreover, they are potential targets for CA-based immunotherapy or mRNA vaccine strategies. However, cell-surface proteins are often underrepresented in standard proteomic data sets, due to their poor solubility and lower expression levels compared to intracellular proteins. In this context, we investigated GBM-associated surfaceome by comparison to healthy astrocytes surfaceome to identify new specific targets to GBM. For this purpose, biotinylation of cell surface proteins has been carried out in GBM and healthy astrocytes cell lines. Biotinylated proteins were purified on streptavidin beads and analyzed by shotgun proteomics. After filtering our data with Cell Surface Proteins Atlas (CSPA) and Gene Ontology, 78 overexpressed or exclusive in GBM have been identified. Validation has been performed using Human Protein Atlas. In this context, we identified 21 specific potential targets for GBM including 5 mutated proteins (RELL1, CYBA, EGFR, and MHC I proteins). Taken together, we identified potential targets for immune therapy strategies in GBM.
2021-11-03 | PXD027110 | Pride
Project description:Nonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge. To identify and analyze endogenous targets of NMD, we applied cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identified, most derive from alternative exon usage. The isoform-aware analysis revealed many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3΄UTR and for mRNAs with a termination codon in the last exon. The length of the 3΄UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, but a main function is most likely to rid the transcriptome of isoforms resulting from spurious splicing events. Long-read sequencing enabled the identification of many novel NMD-sensitive mRNAs and revealed both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications.
2021-07-20 | E-MTAB-10452 | biostudies-arrayexpress