Treatment Options in Recurrent GBM Research

Treatment Options in Re circulating(prenominal) GBM Re look toStrategies for clinical applicationsThe multi-omics entropy may also reveal primal leads for therapeutic applications. A rattling recent review on GBM, reported outcomes of clinical trials investigating current preaching options in recurrent GBM, including anti-angiogenic, prognosticateing bridle-path blockade and immun contrastingapy ground memory accesses (1). However the factortic and cellular heterobrokerity reflects in the modest results obtained so far. This necessitates identification and validation of better therapeutic targets and active strategies to combat GBM. roughly sweet strategies argon showing promise in Phase II trials and prior in mannikination is becoming operational, such as, EGFRvIII peptide vaccine, Rindopepimut CD95 targeted monoclonal antibody, APG100 and multi-targeted tyrosine kinase inhibitor cabozantinib (1). A multi-pronged ascend targeting a panel of proteins may thus hold t he key to eliciting a synergistic response and prove more beneficial than current sermon modalities targeting individual markers. When it comes to circulatory or plasm-establish biomarkers, in view of the technical limitations encountered in deep and direct plasm psychoanalysis as discussed earlier, alternate methods which would throw overboard prediction of neoplasm link up molecules and their targeted exploration would be highly expendful. one(a) of the outcomes of the get a line was the identification of pieceive strategies for selective instruction analysis and integration, facilitated by the bioinformatics tools available today. It shows experimental identification of proteins passed with the screen to ensure analytical awkwardness and functional relevance as above (Stage 1). Biologically important and authorisation tumour specific proteins identified in verbal expression studies be because assessed for their secretory authorization based on com arrogateatio nal prediction algorithms for signal peptide and transmembrane domain containing proteins, such as, SignalP and TMHMM, observeively and via non-classical secretory mechanisms apply SecretomeP. These proteins atomic number 18 hike up prioritized based on their detectability and occurrence in proteomic data for secretome, CSF and plasma analysis (normal or patient) (Stage 2). The potential secretory basisdidates are then explored in plasma in a targeted manner (Stage 3). Interestingly, some of these proteins were identified in analysis of plasma or CSF from GBM patients (2, 3). Once bioinformatically scrutinized as above and compiled, the scene biomarker panels, can be subjected to validation and experimentation in cohorts of tissue sections, blood plasma/serum specimens from patients (Stage 4). We believe construction of such high confidence protein panels would be a valuable paradigm for studies in larger cohorts in clinical experimental designs.High confident lead candidates for experimental applicationGBM Secreted proteinsSecreted proteins squander an integral eccentric in GBM tumourigenesis through cell offset, migration, invasion, and angiogenesis besides world important in normal physiological processes and thus instrumental to the baring of cancer biomarkers. Besides being useful as markers for typing the tumor, their front end in easily accessible body fluids makes them useful for monitoring the malady progression or treatment response and recurrence. A thorough study of all available literature was done to key the several candidate biomarkers experience been reported in serum or plasma of GBM patients and these are shown in Table 1 in Chapter 1. However, such potential and lustrous new biomarkers are yet to be rigorously evaluated for application against this unmet need. Non-invasive methods based on circulatory biomarkers would be useful for monitoring not only when GBM patients but also for lower grades Grade II and III tumors th at usher longer survival periods.Further, some new reports on circulating tumor DNA (ctDNA) that commit identified in the plasma of GBM patients such as mutated IDH1 DNA (4), methylated MGMT DNA (5) and EGFRvIII mutant DNA (6). The highly fine sequencing based methods for detection of circulatory tumor DNA (ctDNA) in patients plasma are under progress (7). These ctDNA markers shed by dead tumor cells may surface in future to be reasonable indicators for tumor diagnostics.Kinases in GBM Identification of GnRH signaling pathway using an alternate approachAs mentioned in Chapter 2, I used alternate approaches to enhance pathway views by targeting specific protein families, i.e. kinases. Protein kinases (PKs) are well known therapeutic targets in contrasting cancers and a family of proteins that are major components of signal transduction pathways acting as membrane sense organs (RTKs) or as intracellular signaling mediators (non-receptor PKs) and several protein kinases give birt h been regard in gliomagenesis (8, 9). Several studies gravel also shown altered expression of protein kinases in GBM and targeted therapies directed towards RTKs using kinase inhibitors are in clinical trials (10, 11). in that respect is renewed optimism in the use of kinase inhibitors to treat GBM (12). New therapeutic strategies have emerged that use multi-targeted kinase inhibitors to simultaneously disrupt multiple kinases (13). The GBM data was found to be enriched with several kinases. A total of 102 kinases were present tense in GBM datasets 77 different kinases in transcriptomics data and 30 kinases in proteomics data with 26 in putting surface between them. Pathway analysis using these kinases revealed GnRH signaling as the top pathway that has still not investigated in the context of GBM. We refer an overall enrichment of about 129 entities from omics datasets of which 26 kinases and 57 non-kinase members are coming from the consonant (n=711) transcriptome and prot eome dataset. The 26 concordant kinases along with their fold changes are shown in the finger 48 below. A large proportion of GnRH pathway entities include kinases (MAPKs, CAMKs, and RTKs) that enabled its identification as a top pathway using this approach. A targeted search of other non-kinase members of the pathway resulted in additional members of the pathway in omics datasets that foster increased its significance value.In GBM, it has been shown that human GnRH receptors are expressed in tumor cells and receptor activation affects apoptosis, adhesion and angiogenesis to promote tumorigenesis. GnRH signalling as a possible therapeutic target in cancer has already been suggested and put together with my observations it strongly concomitants this possibility in the context of GBM. The expanded hand-curated routine of GnRH signaling is a valuable resource for the scientific community.Expression of GnRH and GnRH receptor has been reported in GBM cell lines and tissue samples at both informational RNA and protein levels concordant with clinical data obtained using GBM tumor tissues and treatment with GnRH agonists resulted in anti-proliferative activity (14-16).There is also evidence that the analogues can cross the blood- wizardry barrier, indicating suitability for treatment of malignant glioblastomas (17). Given the significance of this pathway in cancers and GBM, further savvy the molecular interplay involving GnRH signalling pathway in light of my findings leave behind reveal is use as a potential molecular and therapeutic target. Glioma Amplicon and Risk RegionsThe protein cryptanalytics genes implicated in Glioma and other cancers were clustered based on their chromosomal locations using Gene Set Enrichment Analysis tools to compute overlaps with positional gene sets from Molecular Signatures database and further clustered based on proximity to other known oncogenes from Atlas of Genetics and Cytogenetics in Oncology and Haematology data resour ce, to identify colocalized gene clusters on Chr. 12 and other chromosomes as shown in Chapter 3. An important finding was that larger number of overexpressed differential regulated genes in glioma datasets mapped to 2 significant regions the glioma amplicon (n=37) in 12q13-15 region and the glioma susceptibility (n=16) in the 12p13 region implicated as a major happen region in patients with a family history of gliomas. The discovery of these two clusters of overexpressed genes provides a biological validation of mass-spectrometry derived data. unconnected from these two essential regions, several genes from the glioma dataset were found to cluster around amplicons on other chromosomes and other known cancer associated genes that were not identified in GBM datasets but present in close proximity to them. These can be investigated in a more targeted manner in glioma. Many studies have been done to understand the biological significance of these amplicon regions in gliomas that in dicate that these amplifications are more frequent in gliomas than previously thought and have different distribution patterns in low grade versus high grade tumors (18, 19). Overall, a relative high degree of amplifications and deletions are seen in GBM that have implications on the expression of the genes involved and contribute to relevant pathogenic genes (20).Novel genes and isoforms preference splicing increases the repertoire of protein functionality and heterogeneity and aberrant splicing events have been a great deal seen in several cancers, including GBM and increasing evidence now points to their important role in tumor initiation and progression. The concept of proteogenomics has emerged rapidly as a valuable approach to integrate mass spectrometry (MS)-derived proteomic data with transcriptomic data to identify fresh draw variants. However, the role of alternative splicing in GBM is still nascent and needs to be explored as potential biomarkers or molecular targets.A s detailed in Chapter 4, the identification of a novel variant of NCAM1, using a proteogenomics approach with 5 peptide evidences from MS data spanning a novel coding DNAic region, is very significant finding in GBM. NCAMs are well characterized glycoproteins that mediate cell-cell or cell-matrix adhesion among neurons and between neurons and muscle. Several hook up with variants of NCAM1 have been identified (21, 22) and alterations in these have been found in serum and tissues of brain tumors (23, 24). NCAM1 has 5 known isoforms and also exhibits glycoforms as it can be post-translationally modified by the addition of polysialic acid (PSA), which is thought to abrogate its homophilic stick properties and affect the adhesive properties of NCAM (25). Further, PSA conjugated NCAM, was shown to potentiate migration via FGFR signaling apparent from its adhesion capability (26). The following observations may be noted with respect to this novel variantThe observation is supported in transcriptomics data in 18 out of 25 RNAseq samples.Multiple gene modelling software program such as Augustus, GenScan, AceView and Ensemble support the presence of this novel exon in their gene models and a high degree of conservation was seen as expected for an exonic region.This variant was also separately identified in MS-derived kind Proteome and IvyGAP RNAseq datasetsNCAM1 is upregulated in several cancers however, in GBM both transcript and protein data support its down regulation. We discovered two known forms of NCAM1 as well as the novel form to be down regulated.It is interesting to note that the miRNA (hsa-mir-30a-5p) that regulates NCAM1 is upregulated in GBM indicating the deregulation of a putative oncogenic cascade.In summary, our findings demonstrate the usefulness of compounding omics approaches to identify novel putative candidates in GBM. Although, it is not clear if the novel splice variant represents a major or minor form of NCAM1. At the transcript level, it seems to be a minor component however, preliminary assessment at the protein level is suggestive of it being a plethoric form. Regardless, it would be interesting to explore the biological significance of the novel splice variant of NCAM1 and examine its role in GBM tumorigenesis. Hence, in the light of this observation my identification of novel NCAM1 splice variant through proteogenomics analysis using GBM RNAseq data is a very important finding in GBM. The effect of this novel variant on cell-cell adhesion and migration in GBM needs to be further investigated in a targeted manner.Disease implications and targeted analysisStudies suggest that gliomas symbolise a rapidly progressing neurodegenerative disease caused by the malignant growth of glial cells that nourish neurons, resulting in a loss of brain function. Glutamate excitotoxicity is observed in several neurological diseases, which is also utilized by gliomas to gain growth advantage (27). My observations that neurologi cal conditions like Alzhiemers and Parkinsons disease share many common genes with gliomas possible indicate shared molecular mechanisms inducing neurodegeneration. Further, the chromosomal interpret of glioma differentials revealed two clusters one around 12p13 implicated as a glioma risk region and another around 12q13-15 region harboring a glioma amplicon with several overexpressed and amplified genes. Hence, extracting gene/disease associations and generation of a glioma-centric functional and diseasome network is important for correspondence GBM tumorigenesis. Further, this region was found to be enriched in several cancers including other brain neoplasms and neurological diseases that may share disease genes and processes with gliomas. Only 22 of the observed 108 disease genes in the diseasome network were identified in our proteomic analysis. The other 86 disease genes implicated in gliomas but not identified in our dataset can be investigated in a more targeted manner in g liomas, providing a global view of linkages between disease phenotypes. Additionally, the finding that chromosomal proximity of genes may have an impact on their functions can be used to explore the functions of missing proteins mapping within functional cassettes of related protein/genes. Such investigations offer newer paradigms that may be valuable to investigate and present clinically important targets.Future ScopeMetabolomic data integration and potentialCompared to the genome and proteome, metabolome represents the phenotypic changes more closely and has already been investigated for malignancies such as breast, ovarian, colon, bland and esophageal cancers. This line of investigation has been extended to gliomas albeit on a littler scale, revealing novel insights into the role of metabolites in GBM tumorigenesis (reviewed in ref. (28)). Previous studies have revealed how mutations can lead to generation of oncometabolites such as 2-hydroxyglutarate (2-HG) specifically in IDH1 mutated gliomas (29). The discovery by Otto Warburg that cancer cells prefer to metabolize glucose through a seemingly inefficient process of aerobic glycolysis led to the application of 18-FDG-PET imaginativeness to predict the histological grade of gliomas. Using this technique we could now bed low grade gliomas that have low specific uptake (SUV) set from grade III and IV that have higher SUVs. One study performed global metabolic profiling using mass-spectrometry coupled to liquid/ bodge chromatography on patient derived tumor samples and found increased levels of glutathione, tryptophan and metabolites associated with phentose orthophosphate and nucleotide synthesis and glycolytic intermediates such as phosphoenolpyruvate (PEP) and 3-phosphoglycerate (30). These studies have collectively provided a window of opportunity for further investigation and integrating these changes with the changes at proteomic, transcriptomic and genomic levels will be the next big step in to stu dy the underlying biology of these tumors.Improving pathway analysis with phosphoproteomics dataProtein phosphorylation plays a interchange role in transmitting the signal from outside the cell through a cascade effect into an intracellular signal to control the biochemical pathways in all living cells. This mechanism of activation or defusing can be orchestrated by protein kinases via phosphorylation and phosphatases via dephosphorylation. Modifications to these signaling networks via mutations or anomalous protein expression or post-translational modifications may underlie both development and progression of tumorigenesis.Glioma RepositoryIn order to facilitate annotation of key foothold and manage the allurement of high-throughput data coming from different omics technologies and platforms and make it easier to salt away and retrieve large amounts of information, I proposed to a synopsis for data annotation, collection and deposition. The data will be stored in the backen d, in separate tables in a relational database (RDBMS), to enable effortless retrieval of key information for particular candidates of interest and also allow for complex querying. The outline for the schema is given below.Figure 49 Schema for development of a glioma deposit