A multistep in silico approach identifies potential

glioblastoma drug candidates via inclusive

molecular targeting of glioblastoma stem cells
Nilambra Dogra (  nilambra@gmail.com )
Panjab University https://orcid.org/0000-0002-1682-638X
Parminder Singh
Panjab University
Ashok Kumar
Panjab University

Research Article

Keywords: GBM, GSC, DEGs, drug repurposing, molecular targets, cancer therapy

Posted Date: September 29th, 2023

DOI: https://doi.org/10.21203/rs.3.rs-3358531/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
Aims Glioblastoma (GBM) is the highest-grade of glioma for which no effective therapy is currently
available. Despite extensive research in diagnosis and therapy there has been no significant improvement
in GBM outcomes, with a median overall survival continuing at a dismal 15–18 months. In recent times,
glioblastoma stem cells (GSCs) have been identified as crucial drivers of treatment resistance and tumor
recurrence; and GBM therapies targeting GSCs are expected to improve patient outcomes. We used a
multistep screening strategy to identify repurposed candidate drugs against therapeutic molecular targets
in GBM with potential to concomitantly target GSCs. Main methods Common differentially expressed
genes (DEGs) were identified through analysis of multiple GBM and GSC datasets from the Gene
Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). For identification of target genes, we
further selected the genes with most significant effect on overall patient survival. The relative mRNA and
protein expression of the selected genes in TCGA control versus GBM samples was also validated and
their cancer dependency scores were assessed. Drugs targeting these genes and their corresponding
proteins were identified from LINCS database using Connectivity Map (CMap) portal, and by in silico
molecular docking against each individual target using FDA approved drugs library from DrugBank
database, respectively. The molecules thus obtained were further evaluated for their ability to cross blood
brain barrier (BBB) and their likelihood of resulting in drug resistance by acting as p-glycoprotein (p-Gp)
substrates. The effect of these final shortlisted compounds on a panel of GBM cell lines was examined
and compared with temozolomide through the drug sensitivity EC50 values and AUC from the PRISM
Repurposing Secondary Screen, and IC50 values obtained from GDSC portal. Key findings We identified
RPA3, PSMA2, PSMC2, BLVRA and HUS1 as molecular targets in GBM including GSCs with significant
impact on patient survival. Our results show GSK-2126458, linifanib, drospirenone, eltrombopag, nilotinib
and PD198306 as candidate drugs which can be further evaluated for their anti-tumor potential against
GBM. Significance Through this work we identified repurposed candidate therapeutics against GBM
utilizing a GSC inclusive targeting approach, which demonstrated high in vitro efficacy. These drugs have
the potential to be developed as individual or combination therapy to improve GBM outcomes.

Introduction
Gliomas are the most frequent and deadly primary brain tumors in adults. Among these, glioblastoma,
also known as glioblastoma multiforme (GBM), is one of the most biologically aggressive subtypes with
extremely poor prognosis.1,2 The gold standard treatment for GBM patients is surgical resection
combined with radiotherapy and adjuvant chemotherapy with the alkylating agent temozolomide
(TMZ)3,4. Despite extensive research in the area of diagnosis and therapy in the past few decades, the
maximal life expectancy for GBM continues to remain a meagre 12–18 months after standard
treatments. This dismal prognosis is associated with a high rate of relapse and resistance to currently
available therapies5,6. The primary cause of bleak outcomes in GBM is the high rate of tumor recurrence,
which is mainly attributed to its resistance to standard therapies. Thereby, the treatment of GBM remains

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a challenge despite the advances in therapeutic methods, and there is a need to identify relevant
therapeutic targets leading to the development of newer more effective therapy.

During the last decade, glioblastoma stem cells (GSCs) have been shown to play an indispensable role in
the formation, maintenance and recurrence of GBM tumors, revealing them as crucial therapeutic targets
for potentially improved outcomes. GSCs are capable of initiating tumor growth in vivo and have the
potential to differentiate into astrocytes, oligodendrocytes, and neurons. GSCs are characterized by their
ability of self-renewal, pluripotency and neurosphere formation in addition to proliferation, angiogenesis,
invasion, modulation of immune response and high motility. 7

After DNA damage, normal stem cells become quiescent and stop proliferating. However, GSCs respond
to cancer treatment procedures by overexpressing various cell survival promoting genes. These include
major drug resistance proteins like MGMT, ABCG2 and MDR1, and various antiapoptotic factors, such as
BCL-2, BCL-XL, FLIP and cIAP1.7 GSCs display resistance to chemo and radiotherapy through their
assumed quiescence in response to DNA damage, ability of extensive DNA repair, relatively higher
mitochondrial reserves and their location in hypoxic niches 8–10.

The concept that GSCs could be the key originators of GBM resistance to chemotherapeutic
drugs/radiation has led to a rethinking in drug development with newer therapeutic agents being directed
at eliminating these cells or modulating their stemness. Currently, research strategies involve the
development of drugs that abrogate GSC stemness by targeting multiple molecules either alone or in
combination.7,11 In the present study, we analyzed five GBM datasets from the Gene Expression Omnibus
(GEO) and GBM data from the Cancer Genome Atlas (TCGA) to screen out differentially expressed genes
(DEGs). Three GSC datasets obtained from GEO were also analyzed for DEGs. A protein-protein
interaction (PPI) network was then created using common DEGs from all datasets. Subsequent
enrichment analysis identified biological functions or pathways of the over-expressed genes. Network
analysis showed a strong association of the DEGs with pathways involved in cell cycle process, cellular
response to DNA damage and protein folding. Survival analysis of the identified common genes was
used to select genes with significant association between their expression levels and survival. Over
expression of the protein products of shortlisted genes was confirmed in TCGA data. Upregulated genes
with the most significant difference in overall patient survival were selected as potential therapeutic
targets. Finally, drugs targeting the selected genes were obtained from the Library of Integrated Network-
based Cellular Signatures (LINCS) database through the Connectivity Map (CMap) portal. Virtual
screening using molecular docking against their encoded proteins was also performed using the FDA
approved drugs from DrugBank database. These protein targeting drugs were ranked on the basis of
binding energies and compounds targeting all five proteins were screened out. The top gene
downregulating and protein binding compounds were further screened on the basis of their ability to
cross blood brain barrier (BBB) as well as their possibility of causing drug resistance on the basis of p-Gp
binding. The screened compounds were finally assessed for their efficacy on a panel of GBM cell lines
from the Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) Repurposing Secondary Screen
and Genomics of Drug Sensitivity in Cancer (GDSC) portals. (Fig.1) Our study includes GSC population in
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identifying repositioned drugs against GBM, resulting in selected compounds effectively inhibiting
multiple GBM cell lines at significantly lower IC50 values than that of TMZ. These candidate drugs have
the potential to be developed as effective therapeutics for GBM.

Methods
GBM datasets

Five GBM expression profiling datasets - GSE4290 [Affymetrix Human Genome U133 Plus 2.0 Array (HG-
U133_Plus_2)], GSE14805 [Affymetrix HT Human Genome U133A Array (HT_HG-U133A)], GSE35493 (HG-
U133_Plus_2), GSE50161 (HG-U133_Plus_2), and GSE116520 (Illumina HumanHT-12 V4.0 expression
beadchip) were obtained from the Gene Expression Omnibus (GEO) for analysis. Within these datasets a
total of 190 GBM samples and 57 control samples were analyzed. In addition, 166 GBM and 5 control
samples from TCGA were also included in the study. (Table 1) Three glioblastoma stem cell (GSC)
datasets GSE179882 (Illumina HiSeq 2500), GSE154958 (Illumina HiSeq 4000) and GSE119834 (Illumina
HiSeq 2500) with a total of 73 GSC and 30 control samples were also analyzed. (Table 2)

DEG identification

DEG analysis was performed using NetworkAnalyst which is a offers comprehensive gene expression
profiling, meta-analysis, biological network analysis and data visualization (12–14). The datasets were
uploaded in the ‘multiple gene expression data’ option and analyzed according to the sequential steps.
These included data processing for Official Gene Symbol/Entrez ID/Genbank ID, annotation check, PCA
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plot generation, confirmation of data normalization, individual DEG analysis and data summary. For
determination of DEGs, the cutoff p-value was adjusted to 0.01. Integrity of all datasets was ensured for
data merging by effect size (ES) combination, which could generate biologically relevant DEGs by
incorporation of both the magnitude and direction of expression fold change. Cochran’s Q test on the
integrated data suggested random-effects model (REM) since the Q values deviated significantly from a
chi-squared distribution. Based on this model, the GBM and GSC datasets were first processed separately
to identify the DEGs in each set. The common DEGs from these two datasets were then selected for
further analysis.

Construction of the protein–protein interaction (PPI) network and identification of significant module

Search Tool for the Retrieval of Interacting Genes (STRING) was used to construct the DEG PPI network
(15,16). A confidence score > 0.4 was set as the criterion of judgment. Next, the most significant modules
of the network map were identified using Molecular Complex Detection (MCODE) (version 1.5.1,
Cytoscape plug-in) using the default parameters.

Analysis and identification of hub genes

Hub genes were excavated with the cut-off value for degrees ≥10. Subsequently, Kyoto Encyclopedia of
Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were used to functionally annotate the
hub genes.

Enrichment analysis

Gene enrichment analysis of DEGs was done using Metascape 17 with all parameters set to default.
Metascape is a platform which simplifies the process of gene enrichment analysis by integrating multiple
data sources. It integrates various databases and resources, including GO, KEGG pathways, protein-
protein interaction networks and transcription factor-target interactions. For the given gene list, pathway
and process enrichment analysis were from KEGG Pathway, GO Biological Processes, Reactome Gene
Sets, Canonical Pathways, CORUM, and WikiPathways. All genes in the genome were used as the
enrichment background. Terms with a p-value < 0.01, a minimum count of 3, and an enrichment factor >
1.5 (the ratio between the observed counts and the counts expected by chance) were collected and
grouped into clusters based on their similarities. Specifically, the pathways with p-value less than 0.01
were considered, and minimal combined effect size (CES) of selected upregulated genes was kept at 1.

Weighted Gene Co-expression Network Analysis (WGCNA)

WGCNA of the DEGs in GBM as well as common DEGs in GBM and GSCs was performed using Integrated
Differential Expression and Pathway analysis (iDEP1.0) tool 18. After network construction, iDEP 1.0 was
used to identify modules or clusters of co-expressed genes. In order to explore the biological relevance of
the modules in GBM, functional enrichment analysis within each module was also performed and
visualized using this tool.

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DEG confirmation by Gene Expression Profiling Interactive Analysis (GEPIA)

The expression profiles of common genes showing differential expression between GBM and control
samples were further validated in TCGA data using the web server GEPIA. 19

Dependency scores

Dependency scores and essentiality of the chosen target genes were obtained from Project Score portal
of the Cancer Dependency Map. 20

Identification of gene expression inhibitors against the selected targets using Connectivity Map (CMap)

CMap database was used to query for inhibitors against the specific shortlisted target genes. For this, the
gene symbols were entered into the CMap search interface and small molecules that induce gene
expression changes that are opposite to the provided gene signature were obtained. These small
molecules were ranked by their connectivity scores. Higher negative connectivity scores indicate a higher
likelihood of the compound being a potential inhibitor or modulator of the genes of interest. LINCS Data
Portal and the Xena browser were also used for identifying target gene downregulating drugs.

In silico identification of target protein inhibitors using virtual screening based on molecular docking.

Virtual screening is a computational technique used in drug discovery to search libraries of small
molecules in order to identify those structures which are most likely to bind to a drug target, typically a
protein receptor or enzyme. It is an important tool in the discovery of novel drugs, representing a cost-
effective and efficient method to screen lakhs of compounds for target specific binding. For virtual
screening based identification of inhibitors, compound library in sdf format was downloaded from the
Drugbank database 21,22. Virtual screening was done by AutoDock Vina version 4 using the PyRx
interface (9). Crystal structures of the target proteins were downloaded from PDB in .pdb format. Each
structure was uploaded in PyRx as macromolecule and converted to pdbqt format. Then library of FDA
approved drugs was downloaded from the DrugBank database as in .sdf format. This file was also
converted to .pdbqt format and uploaded as ligands in PyRx. A grid was designed in PyRx as “Vina
Search Space” around active site of the protein which was identified through PDBsum as the active
amino acid residues involved in protein-protein/ protein-ligand interactions. The exhaustiveness in search
parameters in Vina wizard was set to 8, so that each compound could be modeled into the target site with
different conformations. Virtual screening was then executed between the protein and the compounds
library (both in pdbqt formats) in the defined search space. Scores in the form of binding energies
between the compound and the active site of the protein were obtained. The lower the binding energy, the
higher the affinity of the compound for the target site of the protein. The protein-ligand complexes were
visualized using Chimera 23.

Evaluation of BBB permeability and p-Gp binding of drugs

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BBB permeability of the shortlisted compounds was evaluated using three different tools, namely –
SwissADME 24, LightBBB 25 and Cbligand BBB predictor 26. Predicted p-Gp substrate status was obtained
from SwissADME 27.

Effect of selected drugs on a panel of GBM cell lines

AUC and EC50 values of GSK-2126458, Linifanib, Drospirenone, Eltrombopag, Nilotinib, PD198306 and
Temozolomide against a panel of 30 GBM cell lines were obtained from the Profiling Relative Inhibition
Simultaneously in Mixtures (PRISM) database 28. IC50 values of GSK-2126458, linifanib, nilotinib and
temozolomide were obtained from the Genomics of Drug Sensitivity in Cancer (GDSC) portal. 29

Results
Identification of common differentially expressed genes (DEGs) from GBM and GSC datasets

Gene expression datasets from GEO database were used to analyze DEGs in GBM and GSC versus
normal samples. A total of 356 GBM and 73 GSC samples were included for analysis. DEGs between
control and GBM, and control and GSC populations were identified using the NetworkAnalyst server using
random-effects model (REM) 12,13. REM takes into account sampling error and differences in study
characteristics, such as the experimental design, sample size, or study population which are considered
as “random effects”, and which contribute to the overall variability in effect sizes across studies. It
considers both within-study and between-study variability when estimating the overall effect size (ES).
REM assumes that the ESs of individual studies are not identical but follow a distribution, and assigns
weights to each study based on the sample size and estimated variances, which allows for a more robust
and consistent conclusion regarding gene expression patterns across multiple studies. The results
revealed a total of 5733 genes to be significantly differentially expressed in GBM, including 3450 up-
regulated and 2283 down-regulated genes. 351 genes were found to be upregulated in GSC datasets
while 413 genes were found to be downregulated. (Fig S1, S2) Overall, 117 genes were found upregulated
and 76 genes were found to be downregulated in common in all GBM and GSC datasets which were
analyzed. (Fig.2A & B) Upregulated genes with > 1 combined effect size were selected for further
analyses.

Common genes network and signaling pathways

GBM and GSC common genes were analyzed by creating genes network using NetworkAnalyst (Fig.3A).
Top hub genes with high degree of connectivity were obtained (Fig.S3). Gene enrichment analysis of
common upregulated genes was performed using Metascape. Metascape is a platform for RNA seq data
analyses and can identify enriched biological pathways and functional modules within the provided gene
lists. Enriched signaling pathways regulated by common upregulated genes included cell cycle
checkpoints, mitosis, Rap1 signaling, mRNA splicing, DNA damage response and protein folding. (Fig.3B,

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C and Table S1) Top enriched terms for MCODE networks were mRNA splicing and neddylation (Fig.3D,
E).

Construction of co-expression networks and identification of modules using Weighted Gene Co-
expression Network Analysis (WGCNA)

WGCNA is a widely used systems biology tool for identifying groups or modules of genes that are co-
expressed across multiple samples in high-throughput gene expression data. This method assumes that
genes that are co-expressed in a similar fashion across different samples are functionally related and are
likely to be involved in the same biological processes. WGCNA uses a correlation-based network
approach to identify co-expressed gene modules. The resulting modules can then be further analyzed to
identify their biological significance and potential roles in various cellular processes and diseases.

iDep1.0 server was used to perform WGCNA analysis. We constructed two co-expression networks – one
each of GBM data, and combined GBM and GSC data. Hierarchical clustering analysis conducted using
WGCNA provided clustering results segmented to obtain different gene modules. (Fig.4A & B) Notably, the
combined GBM and GSC data showed modules additionally enriched in MAP kinase signaling pathway,
cell adhesion molecules (CAMs) and ECM receptor interaction besides commonly enriched pathways like
cell cycle, neuroactive ligand-receptor interaction and calcium signaling pathway. (Fig.4C & D) MAPK
signaling pathway is known to play a key role in GBM through hyperactivation and enhancement of
tumorigenic processes like migration, proliferation and survival.30 CAMs have been reported to be
deregulated in GBM infiltration and their targeting could inhibit proliferation, migration and invasion in
GBM cell lines as well as neurosphere formation from GSCs.31 The unique ECM of GBM provides a
distinct therapeutic target for which antibodies/ligands, RNA interference and drugs are currently being
developed.32 Overall, this data indicates the advantage of developing GSC inclusive GBM therapeutic
strategies.

Target genes selection for GBM

In order to identify the putative target genes out of the identified common genes, we determined their
effect on patient survival (Fig.5A). The survival plots and hazards ratio of the genes were obtained from
GEPIA server (Fig.5B). Nine upregulated genes were found to have significant effect on patient survival
with p value < 0.05 and hazards ratio > 1 (Fig.5C). A hazard ratio greater than 1 indicates an increased
risk in the high expression group.

Out of these, seven genes were found to be significantly upregulated in TCGA samples at mRNA levels
and five at the protein level as seen in data obtained from the UALCAN server 33,34 (Fig.6A & B). These
included RPA3, PSMA2, PSMC2, BLVRA and HUS1. These five genes were, therefore, selected for further
studies. Expression levels of these genes in all datasets analyzed in the study are shown in Fig. S4. TCGA
data analysis also indicated mutual co-occurrence of these genes (Table S2). Apart from survival
analysis, previous in vitro studies also revealed the involvement of these genes in proliferation, invasion

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and chemoresistance of GSCs and their potential to be used as markers for poor prognosis of GBM 35–37
Gene interaction networks of the selected genes were constructed and visualized using GeneMania
(Fig.6C).38 Individual gene interaction maps of all five selected genes were also created using GeneMania
(Fig. S5 A, B, C, D & E).

Dependency scores for relevance of the selected target genes

The dependence of cancer cells on specific genes or pathways for their survival and growth can be
measured by their cancer dependency scores. Cancer dependency scores are generated by analyzing the
screening data obtained following knock down of individual genes and assigning a score to each gene
based on its impact on cancer cell fitness. Genes that, when targeted, significantly impair cancer cell
survival or growth, are assigned high dependency scores, indicating that the cancer cells are highly reliant
on those genes. It is a useful tool to assess the vulnerability of cancer cells to the inhibition or disruption
of particular molecular targets for the development of targeted cancer therapies. We obtained the cancer
dependency scores of our shortlisted target genes from the Project Score portal of the Cancer
Dependency Map at Sanger 20. Our acquired data showed RPA3, PSMA2, and PSMC2 to be essential
genes across a panel of GBM cell lines in terms of dependency scores. HUS1 was also found to have
significant scores for multiple GBM cell lines. (Table 3)

Table 3. Dependency scores and essentiality of selected target genes in a panel of GBM cell lines.

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Effect of RPA3 and BLVRA silencing in NPCs:

We could obtain the gene signatures of neural progenitor cells (NPCs) after RPA3 and BLVRA

silencing from the integrative LINCS (iLINCS) database. The combined differentially regulated

genes from these signatures were subjected to pathway analysis on Metascape. 39

Results of gene set enrichment analysis showed that the genes downregulated in these signatures were
involved in cell cycle, FoxM1 pathway, Hedgehog GLI and p53 regulation pathway. (Fig.7)

RPA3 is part of the three- subunit replication protein A (RPA) complex involved in homologous
recombination and double strand break repair, and has been implicated in glioblastoma survival
outcomes. 40 FoxM1 has been reported to promote stemness, radioresistance and mesenchymal
transition in GBM. Radioresistance in GBM is known to be conferred by glioblastoma stem-like cells and
is a key factor of GBM treatment resistance. Hence, inhibition of this pathway has emerged as a crucial

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strategy to increase the sensitivity of GBM to radiotherapy. 41–44 Similarly, inhibition of Hegdehog-GLI
pathway has also been shown to induce apoptosis in glioma stem cells and sensitize GBM to
chemotherapy. 45,46 Hence, the data suggests that inhibition of our selected genes is likely to improve
GBM outcomes by co-targeting GSC population.

Identification of candidate drugs against selected molecular targets and encoded proteins

Drug repurposing, also known as drug repositioning, has emerged as a potent approach in drug discovery
and development as it can save time and resources compared to developing new drugs from scratch. It
refers to the process of identifying new therapeutic uses for existing drugs that have already been
approved by regulatory authorities for the treatment of other diseases or conditions.

We used a two-pronged approach for identifying repurposed candidate drugs against our selected
molecular targets at the mRNA and protein level. For identification of target mRNA inhibitors, we used
LINCS and CMap databases. The CMap contains data on the transcriptional responses of human cells to
over 20,000 small molecules, including FDA-approved drugs, experimental drugs, and bioactive
compounds. It is a powerful tool for identifying potential drug targets and for repurposing existing drugs
for new indications. By comparing the gene expression profiles induced by different drugs, drugs that
have similar or opposite effects on gene expression can be identified from this resource. The CMap has
also been integrated into the LINCS portal. LINCS provides a comprehensive set of cellular responses to
various perturbations, such as drug treatments and genetic manipulations. LINCS generates large-scale
data sets that provide changes in gene expression, protein levels and other cellular features in response
to different perturbations. These data sets can be accessed through various online resources, such as the
LINCS Data Portal and the Xena browser. The drugs identified through CMap and LINCS portals were
further assessed for their BBB permeability and substrate specificity for p-Gp. (Table 4 A, B & C)

Further, for identifying inhibitors against proteins encoded by the selected target genes, FDA approved
library from DrugBank was screened against the protein structures of all five targets individually.
DrugBank is a publicly available database of over 14000 FDA approved and experimental drugs that can
be used for drug discovery and development. The structures of all the shortlisted targets were
downloaded in pdb format from the RCSB PDB database and the DrugBank compounds library was
screened against the target proteins individually using PyRx tool for virtual screening. The binding
energies for the drugs common for all five target proteins are listed in Table 5A.

Evaluation of BBB permeability and predicted p-Gp binding of the screened compounds

The drugs obtained from both the mRNA and protein targeting approaches were further subjected to
filtering based on the criteria of BBB permeability and their possibility of being p-Gp substrates. BBB
permeability is a measure of how easily a compound can cross the blood-brain barrier and access the
central nervous system - properties essential for a GBM drug candidate. p-Gp, on the other hand, is a drug
efflux transporter protein that is highly expressed in BBB cells as well as in GSCs and actively pumps out
a wide range of substances, including certain drugs, from the brain back into the bloodstream, which can
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contribute to drug resistance and hinder the efficacy of anticancer treatments. Both BBB permeability and
p-Gp substrate status are crucial considerations in oncology research and drug development. The final
shortlisted mRNA downregulating drugs were GSK-2126458 and linifanib from the LINCS database, PD-
198306 from CMap portal, and rofecoxib, chlorzoxazone, haloperidol and valproic acid from the Xena
browser. The protein targeting drugs from the DrugBank database which fulfilled all criteria were nilotinib,
eltrombopag, conivaptan and drospirenone (Table 5B and Fig.8). Effect of these drugs on the viability of
GBM cell lines was then evaluated.

Effect of selected drugs on viability of GBM cell lines

We finally determined the effect of the selected drugs on a panel of GBM cell lines from the Profiling
Relative Inhibition Simultaneously in Mixtures (PRISM) drug repurposing resource. PRISM is a database
that provides comprehensive pharmacological profiling of anticancer drugs in preclinical models.28 It
offers detailed drug response data for a wide range of anticancer compounds across a large panel of
cancer cell lines.

The AUC and EC50 values were used to compare the efficacy of drugs on multiple GBM cell lines. A
higher AUC value and lower EC50 value indicates a greater overall response of the cells to the drug, while
a lower AUC and higher EC50 value indicates a weaker response. All selected drugs showed inhibitory
activity against GBM cell lines with EC50 values below 5uM for most cases. (Table 6 and Table S5)

We could also obtain the IC50 values of GSK2126458, linifanib and nilotinib from the GDSC portal and
compared these values with that of temozolomide 29 (Table 7). Remarkably, all three drugs showed
significantly lower IC50 values in GBM cell lines as compared to temozolomide, indicating their potential
as GBM candidate drugs (Fig.9). GSK2126458/omipalisib was observed to have the lowest IC50 values
in all cell lines.

Discussion
Cancer stem cells can self-renew, initiate tumor growth and are also responsible for tumor cell
heterogeneity and induction of systemic immunosuppression. GSCs are well established as key elements
in GBM resistance and management and have now been identified as key therapeutic targets because of
their ability to drive resistance to pharmacology, radiation and surgery. Integral to their role as tumor
initiators and sustainers is the ability of GSCs to thrive in harsh, complex microenvironmental niches,
unrestrained by proliferation and survival constraints that impede their normal counterparts. Although
specific pathways that contribute to the resilience of GSCs have been described, effective therapies
remain elusive. Currently, there are limited drugs specifically developed and approved for targeting GSCs.
This limited repertoire of therapeutic options debilitates the ability to effectively treat this aggressive form
of brain cancer. In addition, some drugs developed against GSCs may have off-target effects or cause
toxicity to healthy brain tissue. Maintaining a balance between therapeutic efficacy and minimizing

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damage to normal cells is a significant challenge in cancer therapy. Drug repurposing is a potent strategy
to counter this issue.

Targeting common differentially expressed genes in cancer stem cells can be combined with other
treatment modalities, such as chemotherapy or radiation therapy, to enhance the overall efficacy of the
treatment. By targeting both the bulk tumor cells and the cancer stem cells, the treatment can potentially
achieve a more comprehensive and long-lasting response. Keeping this in mind, we utilized a target
prediction approach considering the common genes from GBM and GSCs that could be mediators of
GBM progression as evaluated by their expression in tumors and effect on patient survival. We obtained
the common upregulated genes in GBM and GSC samples and then examined their effect on overall
patient survival. The mRNA and protein level expression of these genes was also validated in TCGA GBM
samples. The genes RPA3, BLVRA, PSMA2, PSMC2 and HUS1 showed significant upregulation at both
mRNA and protein levels and their elevated expression was also found to be associated with significantly
reduced patient survival. These genes were therefore considered potential GBM targets. We screened for
mRNA downregulating and protein targeting drugs against the predicted targets. CMap, which is an
effective tool for drug discovery and repurposing by identifying compounds with the desired effect on
gene expression patterns, was used to identify target mRNA down-regulating drugs. Potential protein
targeting repositioned drugs were identified through molecular docking by virtual screening of the FDA
approved drugs from DrugBank database against the predicted targets. The drugs were subjected to
further filtering on the basis of their ability to cross blood-brain-barrier (BBB) and also their being putative
p-Gp substrates. Finally, the effect of the screened drugs was determined on a panel of GBM cell lines by
evaluating their AUC and EC50 values obtained from PRISM data using DepMap database. IC50 values
of GSK2126458, linifanib, nilotinib were also obtained from the GDSC portal and compared with that of
TMZ. All the screened drugs showed inhibitory activity against GBM cell lines at lower concentrations
than the standard GBM drug TMZ in most cases. GSK2126458/omipalisib, in particular, was seen to be a
potent inhibitor at very low doses. GSK2126458 is a known PI3K/mTOR inhibitor. PI3K/mTOR pathway
has been shown to specifically contribute to the increased chemo-resistance of GBM stem cell-like cells
specifically, while it contributes to the motility of the differentiated GBM cells. 47,48 This finding further
strengthens the case for evaluating GSK2126458 as effective GBM therapy individually or in combination
with other drugs. Overall, our work identifies potential GBM drug candidates which may prove to be
effective GBM therapeutics by simultaneously targeting GSCs.

In conclusion, the target genes identified in this work can be explored for their potential in diagnosis,
prognosis and targeted therapy through further molecular studies. Additional in vitro and in vivo
experimental studies can conceivably develop the identified candidate GBM drugs as effective therapy
either alone or in combination with existing drugs.

Declarations
Authors' contributions

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Nilambra Dogra: Conceptualization, Methodology, Investigation, Analysis, Visualization, Supervision,
Writing – original draft, review & editing. Parminder Singh: Data Curation, Investigation & Formal
Analysis. Ashok Kumar: Resources. All authors read and approved the final manuscript.

Ethics approval: NA

Consent to participate: NA

Consent for publication: NA

Availability of data and materials: NA

Competing interests: The authors have no relevant financial or non-financial interests to disclose.

Funding: This work was supported by funding from CSIR (to ND).

Acknowledgements: We thankfully acknowledge support by SERB (to PS & AK).

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Table 4 To 7
Table 4 To 7 are available in the Supplementary Files section.

Figures

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Figure 1

Workflow employed for the identification of GBM target genes and candidate drugs.

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Figure 2

A. Heatmap of DEGs from the GBM and GSC datasets; B. Venn diagrams showing the common
upregulated and downregulated genes

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Figure 3

3A. GBM and GSC common gene network; B. Enriched pathways in GBM and GSC common upregulated
genes; C. Clustered enriched pathways from B; D, MCODE analysis for closely connected proteins; E. GO
enriched terms for identified MCODE networks with top p-values.

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Figure 4

A. WGCNA analysis of DEGs from GBM datasets; B. WGCNA analysis of common DEGs from GBM and
GSC datasets; C. Enriched pathways in co-expressed modules from DEGs of GBM datasets; D. Enriched
pathways in co-expressed modules from common DEGs of GBM and GSC datasets.

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Figure 5

A. Survival map of common upregulated genes (GBM & GSC) in TCGA GBM samples; B. Survival plots of
genes showing significant difference in survival associated with overexpression in TCGA GBM patients;
C. Significant p-values and hazards ratio of common upregulated genes in GBM and GSC datasets.

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Figure 6

A. mRNA expression levels of selected genes in TCGA GBM samples (* significant difference); B. protein
levels of selected genes in TCGA GBM samples with p-values. C.Gene interactions of the selected target
genes.

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Figure 7

Gene set enrichment for downregulated genes from RPA3 & BLVRA knockdown signatures in NPCs
obtained from iLINCS. Gene lists colored by p-values.

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Figure 8

Protein-drug complexes obtained after docking of DrugBank compounds with target proteins.
[RPA3:2Z6K, BLVRA:2H63, HUS1:6J8Y(Chain B), PSMA2:5GJQ(ChainD), PSMC2:5GJQ(Chain N)]

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Figure 9

Graphical representation of cell lines with their corresponding IC50 values.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.

SupplementaryInformation.docx
Table4To7.docx

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