MODULATING TUMOR MICROENVIRONMENT USING COMBINATORIAL THERAPY

Abstract

Glioblastoma multiforme (GBM) is an aggressive brain cancer with a poor prognosis. Currently, standard radiotherapy and chemotherapy is the only treatment option with adverse outcomes and low survival rate. Thus, advancements in the treatment of GBM are of utmost importance, which can be achieved in recent decades. However, despite having advancements in therapeutic strategies recurrence is inevitable, and the overall survival rate of patients is impossible to achieve. Currently, researchers across the globe target signaling events along with tumor microenvironment (TME) through different drug molecules to inhibit the progression of GBM, but clinically they failed to demonstrate much success. Additionally, the main therapeutic difficulties in treating hypoxia induced-(GBM) are toxicity of current treatments and resistance brought on by microenvironment. More effective therapeutic alternatives are urgently needed to reduce tumor lethality. Hence, we screened plant-based natural product panels intending to identify novel drugs without elevating drug resistance. We explored GEO for hypoxia GBM model and compared hypoxic genes to non-neoplastic brain cells. A total of 2429 differentially expressed genes expressed exclusively in hypoxia were identified. The functional enrichment analysis demonstrated genes associated with GBM, further PPI network was constructed, and biological pathways associated with them were explored. Seven webtools, including GEPIA2.0, TIMER2.0, TCGA-GBM, and GlioVis, were used to validate 32 hub genes discovered using Cytoscape tool in GBM patient samples. Four GBM-specific hypoxic hub genes-LYN, MMP9, PSMB9, and TIMP1-were connected to the TME using TIMER analysis. 11 promising hits demonstrated positive drug-likeness with non toxic characteristics and successfully crossed blood-brain barrier and ADMET analysis. Top ranking hits have stable intermolecular interactions with MMP9 protein, according to molecular docking, MD simulation, MM-PBSA, PCA, and DCCM analysis. Herein, we have viii | P a g e reported flavonoids: 7, 4'-dihydroxyflavan, (3R)-3-(4-Hydroxybenzyl)-6-hydroxy-8-methoxy 3,4-dihydro-2H-1-benzopyran, and 4'-hydroxy-7-methoxyflavan to inhibit MMP9, a novel hypoxia gene signature that could serve as promising predictors in various clinical applications, including GBM diagnosis, prognosis, and targeted therapy. Moreover, we highlighted the importance of BMP1, CTSB, LOX, LOXL1, PLOD1, MMP9, SERPINE1, and SERPING1 in GBM etiology. Further, we demonstrated the positive relationship between the E2 conjugating enzymes (Ube2E1, Ube2H, Ube2J2, Ube2C, Ube2J2, and Ube2S), E3 ligases (VHL and GNB2L1) and substrate (HIF1A). Additionally, we reported the novel HAT1-induced acetylation sites of Ube2S (K211) and Ube2H (K8, K52). Structural and functional characterization of Ube2S and Ube2H have identified their association with protein kinases. Lastly, our results found a putative therapeutic axis HAT1-Ube2S(K211)-GNB2L1-HIF1A and potential predictive biomarkers (CTSB, HAT1, Ube2H, VHL, and GNB2L1) that play a critical role in GBM pathogenesis. We also investigated the GEO dataset to compare the genes in the Peritumoral Brain Zone (PT) and tumor core (TC) with non-neoplastic brain cells to find significantly differentially expressed genes that are only involved in the growth of GBM tumor. Concurrently, protein targets of FDA-approved atypical antipsychotic drugs were examined. Through computational analysis and bioinformatics tools, we have found potential drug combinations for top-ranked atypical antipsychotic drugs and their associated significant cell cycle and calcium pathways. We quetiapine and clozapine as promising combination therapy. Molecular signatures connected to these pathways were CDK2, CCNA2, DRD4, GABRA5, CHRM1, ADRA1B, and HTR2A can act as biomarkers and therapeutic targets and have a significant impact on lowering the tumor burden and reducing pathogenesis of GBM.