Genetics of CNS Tumors

#### **Chapter 9**

## Annexin A1-Binding Carbohydrate Mimetic Peptide Targets Drugs to Brain Tumors

*Michiko N. Fukuda, Misa Suzuki-Anekoji and Motohiro Nonaka*

#### **Abstract**

Annexin A1 (Anxa1) is expressed specifically on the surface of the tumor vasculature. Previously, we demonstrated that a carbohydrate-mimetic peptide, designated IF7, bound to the Anxa1 N-terminal domain. Moreover, intravenously injected IF7 targeted the tumor vasculature in mouse and crossed tumor endothelia cells to stroma via transcytosis. Thus, we hypothesized that IF7 could overcome the blood–brain barrier to reach brain tumors. Our studies in brain tumor model mice showed that IF7 conjugated with the anti-cancer drug SN38 suppressed brain tumor growth with high efficiency. Furthermore IF7-SN38-treated mice mounted an immune response to brain tumors established by injected tumor cells and shrank those tumors in part by recruiting cytotoxic T-cells to the injection site. These results suggest that Anxa1-binding peptide IF7 represents a drug delivery vehicle useful to treat malignant brain tumors. This chapter describes the unique development of IF7-SN38 as a potential breakthrough cancer chemotherapeutic.

**Keywords:** carbohydrate mimetic peptides, phage display, annexin 1(Anxa1), blood–brain barrier (BBB), glioblastoma, chemotherapy, SN-38, CPT-11, geldanamycin (GA), cytotoxic T cell, CD8

#### **1. Introduction**

Brain malignancies are difficult to treat due to the blood–brain barrier (BBB), a layer of endothelial cells that separates the circulation from the brain and protects the central nervous system from pathogens and toxic materials [1, 2]. Although brain tumor cells cultured *in vitro* respond to several anti-cancer drugs, brain tumors *in vivo* do not due to the BBB. Chemotherapeutic drugs capable of passing the BBB are small lipopholic molecules of less than 500 Da [3], as exemplified by temozolomide [4–6]. Numerous investigators have attempted to overcome this hurdle using brain vasculature surface receptor-mediated proteins [7, 8], tumorpenetrating peptides [4, 9, 10], or nanoparticles [11, 12]. However, these attempts have not yet achieved clinically satisfactory results. On the other hand, tumor vasculature surrounding a brain tumor is chaotic, allowing large molecules and nanoparticles to overcome the BBB by passing through gaps between endothelial cells [13]. However, as this approach relies on passive diffusion, drugs must be administered at the maximum tolerable dose, causing adverse side effects.

Thus, efficient treatment of brain tumors requires both tumor vasculature targeting and penetration by a therapeutic to overcome the BBB. In this chapter, we describe the carbohydrate mimetic 7-mer peptide IF7, which serves as a highly specific tumor vasculature-targeting vehicle. When conjugated to the anti-cancer drug SN-38 and injected into mouse brain tumor models, IF7-SN38 has a potent anti-tumor effect. IF7-SN38 represents a potential breakthrough chemotherapeutic in brain malignancies.

#### **2. IF7 peptide: how was it identified?**

IF7 is a linear 7-mer peptide with the sequence IFLLWQR [14]. This peptide is considered a carbohydrate mimetic, as it was identified in studies of cancer cell surface carbohydrates [15, 16]. Epithelial cancer cells express complex carbohydrate antigens, and some serve as ligands for carbohydrate binding proteins known as selectins. We hypothesized that interaction between selectin and selectin ligand functions in carbohydrate-dependent tumor cells colonization to the lung [17], in a manner similar to that seen in selectin-dependent hematogenous liver metastasis [18, 19].

Our goal, however, was challenging, as chemical synthesis of oligosaccharides as elaborate as the selectin ligand involved tedious, time-consuming and therefore expensive steps. To overcome this problem, we used phage display technology to identify carbohydrate mimetic peptides that might function as an E-selectin ligand. However, initially when we used E-selectin as the target, we did not obtain a phage clone. We then took a different approach and screened peptides for ability to bind

#### **Figure 1.**

*E-selectin binding of linear 7-mer peptide sequences. Phage clones were selected by mouse monoclonal anti-Lewis A antibody (clone 7LE). Each cloned phage was added to microtiter plate wells coated with E-selectin-IgG chimeric protein. Phage binding to E-selectin was tested in the presence or absence of 1 mM CaCl2. The best binder, IELLQAR, was designated I-peptide.*

*Annexin A1-Binding Carbohydrate Mimetic Peptide Targets Drugs to Brain Tumors DOI: http://dx.doi.org/10.5772/intechopen.96517*

anti-carbohydrate antibodies that recognize E-selectin ligands or related carbohydrates. Using this approach, we succeeded in identifying a linear 7-mer peptide from a phage display library. Since carbohydrate antigen specificity is determined by 3–4 carbohydrate residues of 600–800 Da, we assumed that a 7-mer peptide of 770 Da would mimic a carbohydrate antigen. The phage library screening yielded a series of peptides with the consensus sequence IXLLXXR [15] (**Figure 1**).

Among those peptides, the strongest binder to E-selectin was IELLQAR, which we designated I-peptide. Chemically synthesized I-peptide inhibited hematogenous colonization of the tumor cells to the lung in mouse [15]. However, in E/P-selectin doubly-deficient mice, tumor cells expressing selectin ligand carbohydrate colonized the lung, and that colonization was inhibited by I-peptide [20]. These results indicated that I-peptide receptor is not an E- or P-selectin and raised the question of what receptor I-peptide bound to in lung vasculature?

To identify the I-peptide receptor, we injected mice intravenously with a biotinylation reagent plus I-peptide-displaying phage or controls. We then isolated lung tissue and immunoprecipitated lysates with rabbit anti-phage antibody or control IgG. When we resolved immunoprecipitates on SDS-PAGE, we detected two *in vivo* biotinylated proteins as 40 kDa and 20 kDa bands on a peroxidase avidin blot (**Figure 2**). We isolated respective candidate receptor proteins from lung membrane fractions using I-peptide affinity chromatography and proteomic analysis and

**Figure 2.**

*Detection of I-peptide receptor(s) on the surface of lung endothelial cells by* in vivo *biotinylation. Mice were injected intravenously with either PBS (lane 1) or a biotinylation reagent (lanes 2–6), followed by intravenous injection of I-peptide-displaying (lanes 3 and 4) or control (lanes 5 and 6) phage. After perfusion of mice with PBS, lungs were isolated and phage was immunoprecipitated with rabbit anti-phage antibody (lanes 4 and 6) or rabbit IgG (lane 3 and 5). Immunoprecipitates were resolved on SDS-PAGE and biotinylated proteins detected using a peroxidase avidin blot.*

#### *Central Nervous System Tumors*

found them to be the pre-mRNA splicing factor (Sfrs) and annexin A1 (Anxa1) [21]. Recombinant Sfrs proteins showed binding to I-peptide and a series of carbohydrates, whose structures overlapped with selectin ligand [21].

Full-length Anxa1 is 37 kDa; therefore, we considered the 20 kDa band seen in **Figure 2** to be a fragment of the full-length protein. By the time we identified Anxa1 fragments, Oh *et al.* had undertaken rigorous subtractive proteomics analysis and identified Anxa1 as a specific vasculature surface marker of malignant tumors [22]. Thus, we hypothesized that I-peptide or related peptides could serve as tumor vasculature-specific drug delivery vehicles via binding to Anxa1. We then rescreened a series of phage clones for tumor-targeting activity and identified an Anxa1-binding, but not Sfrs-binding, phage clone displaying IFLLWQR peptide (**Figure 3**). Moreover, tumor targeting was inhibited in the presence of an anti-Anxa1 antibody specific to the N-terminal region (**Figure 4**) [21].

#### **Figure 3.**

In vivo *phage targeting specificity to tumor and normal lung. Subcutaneous B16 tumor-bearing mice were intravenously injected with each phage clone, and phage number in the tumor and lung was determined by a colony-forming assay. Note that IFLLWQR- or IF7 peptide-displaying phage exclusively targeted the tumor but not lung tissue.*

#### **Figure 4.**

In vivo *tumor and organ targeting by IF7-peptide-displaying phage. IF7-displaying phage were injected intravenously into subcutaneous B16 tumor-bearing mice. Note that IF7-displaying phage targeted the tumor but not normal organs. Tumor targeting by IF7 phage was inhibited by pre-injection of mice with rabbit polyclonal anti-Anxa1 (N-19) antibody directed to the Anxa1 N-terminal domain but not by injection with control rabbit IgG.*

### **3. Targeting the tumor vasculature by IF7 peptide**

Next, to visualize tumor targeting by chemically synthesized IF7 peptide, we used intravital microscopy, in which tumor was implanted in a dorsal skinfold chamber [23] visualized tumor vasculature targeting of IF7 under fluorescence microscopy. Green fluorescent Alexa 488-labeled IF7 was injected intravenously and green fluorescent signals were recorded over time by video [14]. Fluorescence appeared in the tumor within 30 sec of injection and increased over time (**Figure 5**). Analysis of tumor tissue sections taken 15 minutes after A488-IF7 injection indicated fluorescent signals as a punctate staining pattern over endothelial cells (**Figure 6**, upper). By 40 min, green fluorescence had moved to the stroma (**Figure 6**, lower), suggesting that IF7 passed through endothelial cells and penetrated the tumor stroma where cancer cells reside. The punctate appearance of Alexa 488 staining suggests that IF7-bound to Anxa1 is internalized by endothelial cells, possibly in vesicles. Anxa1 on the tumor vasculature surface reportedly localizes in caveolae and, when bound by anti-Anxa1 antibody, the complex is internalized into vesicles transported to the basal surface via transcytosis [24]. Accordingly, we concluded that IF7 bound to Anxa1 on the tumor vasculature was transported from the luminal surface to the basal membrane via transcytosis through endothelial cells and likely released to the tumor stroma. Therefore, we asked whether IF7-conjugated chemotherapeutics could cross the BBB to deliver a cytotoxic drug to brain stroma.

To test this possibility we injected A488-IF7 through the tail vein into brain tumor-bearing mice and then prepared sections of mouse brain tissue 20 minutes later. Fluorescence microscopy analysis revealed bright fluorescence in tumor tissue [25]. At higher magnification A488-IF7 fluorescent signals were evident in cytoplasm and/or nuclei of cancer cells. Micrographs of representative organs from the same mouse showed no significant fluorescent signals in normal organs. Brain tumors and representative organs from an animal injected with A488-C(RR) peptide control showed background fluorescence. These results strongly suggest that intravenously-injected IF7 crosses the BBB to target and penetrate brain tumor vasculature and reach cancer cells [25].

#### **Figure 5.**

*Intravital microscopy of Alexa 488-conjugated IF7 peptide. Mouse lung carcinoma LL/2 tumors were inoculated in the skin of nude mice of a dorsal skin-fold chamber [23]. A488-IF7 was injected intravenously and green fluorescent signals were video-recorded and detected at the indicated times.*

#### **Figure 6.**

*Fluorescence micrograms of subcutaneous B16 tumor sections from mice intravenously-injected with A488-IF7. Tissue sections taken at 15 (upper) and 40 (lower) min after A488-IF7 injection were stained by anti-CD34 antibody plus red fluorescence-conjugated secondary antibody to mark endothelial cells. Note that at 15 min, green IF7 signals are seen as punctate signals over endothelial cells, whereas at 40 min IF7 signals are in the stroma and seen as diffuse green fluorescence.*

#### **4. The ANXA1 N-terminal domain is present on the tumor vasculature surface**

Several lines of evidence suggest that IF7 binds to human and mouse Anxa1 at the N-terminal domain. First, an IF7 peptide-displaying phage clone failed to bind full-length recombinant ANXA protein when the N-terminus was blocked by a His6-tag, whereas IF7 bound to full-length ANXA1 that was C-terminally tagged with His6 [14]. Second, IF7 bound to full-length ANXA1 but not to an N-terminal deletion delta 27 mutant [14]. Third, when IF7 peptide-displaying phage was injected intravenously into a tumor-bearing mouse, phage targeted the tumor

#### *Annexin A1-Binding Carbohydrate Mimetic Peptide Targets Drugs to Brain Tumors DOI: http://dx.doi.org/10.5772/intechopen.96517*

vasculature, but that binding was blocked in comparable mice injected with an antibody raised against the ANXA1 N-terminal domain (**Figure 4**) [14]. Finally, *in vitro* binding assays showed that synthetic IF7 bound to a synthetic peptide representing the ANXA1 N-terminal domain (designated MC16), which includes 15 amino acids from Met1 to Glu15, plus a C-terminal Cys [25]. IF7 bound to both mouse and human MC16 peptides.

The molecular weight of full-length Anxa1 is 37 kDa, but Western blotting of endothelial plasma membranes and caveolae isolated from tumors detected a 34 kDa band [24]. Proteomics analysis of this 34 kDa protein suggested that it may lack the N-terminal domain. To determine whether the Anxa1 N-terminal domain is on the tumor vasculature surface, we generated a mouse monoclonal antibody specific to the human ANXA1 N-terminal domain (or MC16 peptide). Immunohistochemical analysis of various clinical specimens with anti-MC16 antibody revealed positive signals located at endothelial cells lining malignant tumor tissues in specimens from prostate, breast, lung, liver, ovarian and brain cancers [25], indicating that the ANXA1 N-terminus is present on endothelial cells in many human malignancies. Immunostaining alone did not reveal whether the antigen was on the cell surface or in the cytoplasm.

We confirmed that the MC16 domain was present on the luminal side of the plasma membrane by *in vivo* biotinylation of mouse brain tumors followed by immunoprecipitation by anti-MC16 antibody and proteomics analysis [25]. Plate binding assays of precipitates indicated high levels of biotinylated materials bound by the anti-MC16 antibody in tumor lysates, whereas biotinylated materials in tumor lysates treated with control antibody or those from normal liver tissue lysates showed significantly lower levels of biotinylated material. However, when immunoprecipitates from these tumors were analyzed on a protein gel followed by an avidin blot, we did not detect biotinylated proteins, whereas proteomics analysis had revealed predominantly Anxa1 peptide fragments [25]. We conclude that the Anxa1 N-terminal domain is cleaved from the rest of the protein and displayed on the tumor vasculature surface as a peptide fragment too small to be detected on a Western blot.

#### **5. Therapeutic activity of IF7 conjugated to the anti-cancer drug SN-38 against brain tumors**

Both we and others have reported that IF7-conjugated drugs show efficient anti-tumor activity in mouse models of tumors other than brain tumors. Examples include IF7-conjugated geldanamycin (GA) against prostate, lung, and breast cancers as well as melanoma [21], IF7-SN38 against colon cancer [21], IF7-taxol against breast cancer [26] and IF7-conjugated10B with boron neutron capture therapy against bladder carcinoma [27]. Below we focus on our studies of the effect of IF7-SN38 on mouse brain tumor models.

To target brain tumors, we chose to conjugate IF7 to SN-38, the active component of irinotecan (CPT-11), which is used clinically to treat brain cancer [28, 29]. To compare IF7-SN38 dosages we employed a dual-tumor model, in which a single mouse receives luciferase gene-transfected cancer cells in brain and under the skin (**Figure 7**). Growth of tumors in both regions was quantitatively monitored using an IVIS imager to detect photon numbers produced by luciferase. Once tumors were formed in the brain and under the skin, the dual tumor model mice were injected intravenously with IF7-SN38 and tumor growth in both locations was assessed *in vivo* by photon number. IF7-SN38 treatment significantly suppressed tumor growth relative to buffer controls in both brain and under the skin at a dosage of

#### **Figure 7.**

*Analysis of whether IF7-SN38 overcomes the blood–brain barrier using a dual tumor mouse model. (upper) schematic showing that single mice established to harbor both brain and subcutaneous tumors are injected with IF7-conjugated drug through the tail vein. If the drug cannot overcome the BBB, only subcutaneous tumors are eradicated; if drug overcomes the BBB, growth of both is suppressed. (lower) graphs show that daily injection of IF7-SN38 (7.0 μmoles/kg) suppressed both brain (left) and subcutaneous (right) tumor growth. Moreover, CPT-11, the SN-38 pro-drug, suppressed growth of subcutaneous tumors at high dosage (50 μmoles/kg) [25].*

3.15 μmoles/kg. Moreover we found using either C6-Luc cells in SCID mice or B16- Luc cells in C57BL/6 mice, intravenously-injected IF7-SN38 significantly antagonized growth of brain and subcutaneous tumors relative to controls at a dosage of 7.0 μmoles/kg. When we performed similar experiments using the SN38 prodrug irinotecan alone at doses as high as 50 μmoles/kg, irinotecan suppressed subcutaneous tumor growth but only minimally suppressed brain tumor growth. Overall, these results indicate that in the mouse dual tumor models tested here, IF7-SN38 suppresses brain tumor growth as effectively as subcutaneous tumor growth.

For the dual tumor model experiments (**Figure 7**), we had dissolved IF7-SN38 in Cremophore EL, a non-ionic detergent used clinically to administer taxol, prior to injection. However, there are concerns about potential inflammatory effects of this detergent [30, 31]. Thus, we conducted experiments in which we dissolved

#### *Annexin A1-Binding Carbohydrate Mimetic Peptide Targets Drugs to Brain Tumors DOI: http://dx.doi.org/10.5772/intechopen.96517*

IF7-SN38 in 10% Solutol HS15, a non-ionic surfactant with low toxicity. The therapeutic effect of IF7-SN38 in 10% Solutol HS15 improved significantly relative to administration with Cremophore EL (**Figure 8A**): B16 brain tumors began shrinking during the first week of daily injections at dosages as low as 2.5 μmoles/ kg, continued shrinking during the second week without drug injection, and then completely disappeared. Mice survived for more than 3 months after cessation of drug treatment without showing signs of B16-Luc cell growth in brain or other parts of their body, suggesting complete remission and potential involvement of host immune systems.

Relevant to potential immunogenicity, when we injected cells of either one of two isogenic lines, B16-Luc or LL/2-Luc, subcutaneously into naïve C57BL/6 mice, both lines produced tumors at injected site. By contrast, when either of these lines were injected into mice that had recovered from brain B16-Luc tumors, LL/2-Luc tumors grew but B16-Luc tumors did not (**Figure 8B**). The presence of tumorinfiltrating lymphocytes, especially CD8<sup>+</sup> cytotoxic T cells, is correlates with better

#### **Figure 8.**

*IF7-SN38 treatment promotes complete remission of B16 brain tumors and a host immune response against tumor cells. (A) Effect of IF7-SN38 on B16-Luc brain tumors in isogeneic C57BL/6 mice. Drug dosage was 2.5 μmoles/kg each for IF7-SN38 and control C(RR)-SN38 diluted with 10% Solutol HS15 in water and administered daily for 7 days. Note that brain tumors continued shrinking after cessation of IF7-SN38 administration in C56BL/6 mice. (B) Growth of two syngeneic cancer lines in naïve and brain-tumorrecovered mice 4 days after subcutaneous injection of LL/2-Luc and B16-Luc cells. (C) Immunohistochemistry with anti-CD8 antibody of B16-Luc cells at subcutaneous injection sites, 20 hours after B16-Luc cell injection.*

prognosis of various cancers [32, 33]. Immunohistochemistry of subcutaneous B16-Luc injection site using an anti-CD8 antibody 20 hours after injection of naïve C57BL/6 mice with B16-Luc cells revealed a minimal number of CD8<sup>+</sup> T cells at challenged sites. By contrast, we observed significant CD8<sup>+</sup> cell infiltration at injection sites of B16-Luc cells in C57BL/6 mice that had recovered from B16-Luc brain tumors following IF7-SN38 treatment (**Figure 8C**). Thus it is likely that IF7-SN38 therapy leads to complete remission in part by promoting immunological rejection of tumor cells by the host, preventing tumor recurrence elsewhere in the body.

#### **6. Clinical application of IF7-SN38**

As described, in mouse the Anxa1 N-terminal domain is present on the surface of tumor vasculature as peptide fragments. Nonetheless, such fragments should serve an IF7 receptor, as either the first 15 amino acid residues of ANXA1 or synthetic MC16 peptide is sufficient for IF7 binding [25]. IF7 binds both human and mouse MC16 peptides equally, suggesting that our results with IF7 in mouse tumor models are relevant to humans.

Although IF7-conjugated drugs are effective in various cancer types [14, 26, 27], their effectiveness against brain malignancies may be particularly high as gene expression data indicates *ANXA1* overexpression in brain tumors [34, 35], a finding supported by immunohistochemistry with anti-MC16 antibody [25]. Moreover, as IF7 targets tumor vasculature and overcomes the BBB, IF7-conjugated drug would accumulate in brain tumor cells, a critical advantage over low molecular weight drugs like temozolomide, which does not target brain tumors and must be administered at high doses (**Figure 9**).

We found the effective dosage of IF7-SN38 in the mouse brain tumor models to be 2.5 μmoles (5.35 mg)/kg (**Figure 8**), which translates into a human equivalent [36] of 0.43 mg/kg or SN-38 0.079 mg. This dosage is considerably lower than that currently recommended for CPT-11 (the SN-38 pro-drug) administered to cancer patients, namely, 120 ~ 200 mg/m2 or 2.91 ~ 4.85 mg/kg [37, 38]. Anticipated doses of IF7-SN38 in humans are also unlikely to be toxic at pharmacologically active dosage.

#### **Figure 9.**

*Mode of action of chemotherapeutics directed against brain tumors. General chemotherapeutics do not penetrate brain tumors due to the BBB and thus are administered to patients at high dosage. Some low molecular weight chemotherapeutics such as temozolomide penetrates the brain but requires high dosage, because temozolomide does not target brain tumors. IF7-SN38 targets brain tumors and overcomes the BBB. At low dosage, IF7-SN38 becomes concentrated in tumors, including brain tumors, and exhibits therapeutic activity.*

### **7. Conclusions and future perspectives**

Historically, reagents like IF7 emerged with the advent of carbohydrate mimetic peptides [15, 16]. The surprising finding that Anxa1 is an I-peptide receptor led us to identify IF7 [14, 21]. When IF7 was injected intravenously into brain tumorbearing mice, it targeted tumor vasculature by binding the Anxa1 N-terminal domain and then crossed the vasculature via transcytosis, to overcome the BBB. Due to its highly specific tumor vasculature targeting activity, IF7-SN38 eradicated brain tumors at low dosage, initiating an immune reaction against cancer cells, followed by complete remission of brain tumors [25]. A similar host immune reaction was also found in IF7-conjugated boron neutron capture therapy in a mouse bladder carcinoma model [27].

IF7-SN38 is, however, susceptible to esterases and proteases. To circumvent stability issues, we have developed an ANXA1-binding D-peptide, designated dTIT7 [39]. We have found that GA-dTIT7, in which geldanamycin is conjugated to dTIT7 through an esterase-resistant linker, is orally administrable and suppresses brain tumor growth in the mouse.

Cancer treatments are increasingly expensive due to development of sophisticated diagnostics and therapies. IF7-SN38 can be chemically synthesized cost-effectively and is stable as a dry powder. Furthermore, orally-administrable peptide-conjugated drugs would be advantageous in societies that lack infrastructure required for costly treatment. Further development of peptide-conjugated drugs could reveal additional candidates with clinical applications against intractable cancers.

#### **Acknowledgements**

We thank Dr. Elise Lamar for her editing of the manuscript.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Michiko N. Fukuda\*, Misa Suzuki-Anekoji and Motohiro Nonaka Sanford-Burnham-Prebys Medical Discovery Institute, California, USA

\*Address all correspondence to: michiko@sbpdiscovery.org

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 10**

## DNA Damage Repair Genes and Noncoding RNA in High-Grade Gliomas and Its Clinical Relevance

*Tanvi R. Parashar, Febina Ravindran and Bibha Choudhary*

#### **Abstract**

Gliomas are the most common malignant tumors originating from the glial cells in the central nervous system. Grades III and IV, considered high-grade gliomas occur at a lower incidence (1.5%) but have higher mortality. Several genomic alterations like IDH mutation, MGMT mutation, 1p19q Codeletion, and p53 mutations have been attributed to its pathogenicity. Recently, several noncoding RNAs have also been identified to alter the expression of crucial genes. Current chemotherapeutic drugs include temozolomide targeting hypermethylated MGMT, a DNA repair protein; or bevacizumab, which targets VEGF. This book chapter delves deeper into the DNA damage repair pathway including its correlation with survival and the regulation of these genes by noncoding RNAs. Novel therapeutic drugs being developed are also highlighted.

**Keywords:** DDR in glioblastoma, noncoding RNA in gliomas, targeted therapy

#### **1. Introduction and epidemiology**

Gliomas are the brain's solid tumors that arise from the glial cells, which are the non-neuronal cells of the central nervous system (CNS). Neurons function in synaptic interactions, whereas glial cells provide protective and structural support to the neurons. According to the 2020 GLOBOCAN, cancer of the brain and central nervous system rank at 19th and 12th, respectively [1]. The age-standardized incidence of these tumors is 3.9 per 100,000 in males and 3.0 in females globally. In comparison, the mortality is 3.5 per 100,000 in males vs. 2.8 in females worldwide. These cancers are prevalent in countries with a high human development index [1]. In 2020 alone, 308102 worldwide brain and central nervous system cases were reported. More than half were reported from Asia (54.2%) [1]. The number of deaths reported in the same year was 251329 worldwide, pushing the mortality rate to 81.57% [1]. The survival rate of gliomas vary based on their grade; the median survival time for high-grade glioma is 14 to 16 months. It ranges from 3–15 years for low-grade gliomas [2].

One of the only risk factors identified for the development of high-grade gliomas is exposure to high-dose of ionizing radiation [3]. However, environmental factors, toxins, infections, cell phone usage, or head trauma have not been correlated to the development of gliomas. Only 5% of cases of brain tumors have been linked to

hereditary genetic syndromes [4]. Some of which are Li-Fraumeni cancer syndrome (associated with a germline mutation in the TP53 gene), neurofibromatosis, Turcot syndrome, and Lynch syndrome (constitutional mismatch repair deficiency), tuberous sclerosis, melanoma-neural system tumor syndrome, Ollier disease and Rubinstein-Taybi syndrome [4–7].

Gliomas are diagnosed when the patients become symptomatic, exhibiting recurrent headaches, the onset of seizures, personality changes, weakness in limbs, or language disturbances [8]. Elevated intracranial pressure is also a common feature in gliomas [9]. Infantile spasms and seizures have also been noted in infants [9]. Gliomas are generally diagnosed by computed tomography (CT), and Magnetic Resonance Imaging (MRI) scans [10]. The current treatment regimen is based on the tumor grade and includes either or combinations of surgical resection, radiation, and chemotherapy [11]. The chemotherapeutic drugs used for glioma treatment fall under the category of alkylating agents that induce double-stranded breaks in the DNA, thereby inhibiting tumor proliferation [12]. The standard chemotherapeutic drug used for high-grade glioma is temozolomide (TMZ), and for low-grade gliomas are carmustine, procarbazine, and lomustine [13]. Metastasis of malignant gliomas is rare, primarily due to the low survival of the patients and also due to the blood–brain barriers [14]. However, in certain rare cases of highgrade gliomas, metastasis to the lung, pleura, lymph nodes, bone, and liver have been reported [15]. Recurrence post-treatment is reported in most gliomas and can be attributed mainly to surgical brain injury (SBI) and TMZ chemoresistance [16].

The following sections describe the glioma subtypes, their molecular characterization, and their deregulated signaling pathways. This chapter's primary focus is on the DNA damage response (DDR) pathway, and noncoding RNAs in high-grade glioma called glioblastoma multiforme (GBM). The role of noncoding RNAs affecting chemosensitivity and other novel therapeutic drugs being developed for gliomas are also highlighted.

#### **2. Glioma classification**

The Glial cells are classified as astrocytes, oligodendrocytes, and ependymal cells [17]. The astrocytes function in providing mechanical support to the neurons; oligodendrocytes are involved in myelin production, a component of the myelin sheath and ependymal cells play essential roles in the transport of CSF and brain homeostasis [18]. Based on the cellular origins, gliomas are classified as astrocytoma (derived from astrocytes), oligodendrogliomas (derived from oligodendrocytes), and ependymoma [2].

Until 2016, the World Health Organization (WHO) had categorized gliomas entirely based on histological features and graded them according to their malignancy profile [19]. **Table 1** represents this WHO grading of gliomas where grades I and II are considered low-grade gliomas (LGGs) that are slow-growing with a better prognosis. The Grade I tumors are mainly diagnosed in children and curable with just surgical resection. On the contrary, the most aggressive tumors are referred to as high-grade gliomas (grade III and IV). Grade III tumors are termed 'anaplastic' as they have lost their characteristic cellular features to become malignant. The grade IV in this category, which accounts for 90% of gliomas, is GBM, the most aggressive and deadly tumor of all gliomas, with an abysmal survival rate. About 90% of GBM cases are de novo and develop in older patients [20]. On the contrary, secondary GBM, which arises from LGG, manifests mostly in younger patients and has a better prognosis [20].

*DNA Damage Repair Genes and Noncoding RNA in High-Grade Gliomas and Its Clinical… DOI: http://dx.doi.org/10.5772/intechopen.97074*


**Table 1.**

*Glioma classification based on histology and malignancy scale.*

#### **2.1 Molecular classification of gliomas**

A more recent WHO classification in 2016 includes genetic screening to histopathological analysis, which integrates the tumor's morphological and genetic considerations [21]. The status of the following molecular alterations has been incorporated in this classification and are critical to diagnosis and further treatment.

IDH mutation: The most prevalent genetic mutation is the Isocitrate dehydrogenase (IDH) mutation accounting for a single point mutation in around 80% of glioma cases [22]. It is identified to be one of the earliest mutations for gliomagenesis and has been implemented primarily to classify gliomas as either IDH mutant or IDH wildtype. IDH mutation is considered to be a favorable prognostic marker with increased survival [23]. It is a metabolic enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and produces NADPH from NADP without the Kreb cycle's involvement. This mutated IDH produces high levels of 2-hydroxyglutarate (2-HG) instead of the α-KG which is implicated in glioma invasion as well in epigenetic alterations leading to a glioma CpG island methylator (G-CIMP) phenotype (G-CIMP) [24].

Codeletion 1p19q: Post IDH mutation status, the gliomas are further classified based on this chromosomal co-deletion of 1p19q where the short arm chromosome 1 (1p) and the long arm of chromosome 19 (19q) are lost. It is observed in more than 70% oligodendrogliomas and 50% mixed oligoastrocytomas [25]. Clinically, IDH mutants with co-deletion 1p19q are linked to better prognosis and chemotherapy response [26].

TERT promoter mutations: Telomerase reverse transcriptase (TERT) promoter mutations are reported in several cancers leading to enhanced activity of TERT resulting in tumor cell survival and its progression [27]. It is present in 55% GBM and its prevalence is inversely correlated with IDH mutation [27, 28]. This TERT mutation serves as a prognostic biomarker and is associated with poor survival [29].

MGMT promoter methylation: MGMT (O[6]-methylguanine-DNA methyltransferase) is a DNA damage repair protein that removes alkyl groups added to nucleotides preventing mutation. Chemotherapeutic drugs like TMZ blocks cell growth by alkylating DNA. Hypermethylation of MGMT promoter regions renders this enzyme inactive and is reported in 40% GBM cases [30]. IDH mutant-MGMT promoter methylation cases are associated with increased PFS (Progression-free

survival) whereas MGMT promoter methylation with TP53 mutation has favorable outcome irrespective of IDH status [31].

ATRX mutation: The alpha thalassemia/mental retardation syndrome X-linked (ATRX) is a chromatin remodeling enzyme involved in incorporating histone H3.3 at telomeres and pericentromeric heterochromatin. Loss of function mutations of ATRX is reported in gliomas which correspond to alternative lengthening of telomeres (ALT) phenotype [32]. ATRX and TERT mutations occur in 90% diffuse IDH mutant gliomas with both being mutually exclusive which confer better progression-free and overall survival [33].

H3K27M mutations: H3K27M (methionine substitution of lysine at residue 27 of histone H3) are mutations that occur in Histone 3 of H3F3A or HIST1H3B/C gene. These mutations are predominantly present in pediatric cases with IDH-wildtype and lack 1p/19q co-deletion and are associated with poor prognosis [34]. The H3K27M mutant protein has a dominant-negative effect on EZH2 protein, a histone methyltransferase impacting the epigenetic landscape of tumor genes [35].

Besides the above, other somatic and germline mutations are also reported in gliomas. More than 25 gene loci are linked to an increased risk of development of gliomas. Somatic mutations of cyclin-dependent kinase inhibitor 2A and B (CDKN2A, CDKN2B), epidermal growth factor receptor (EGFR), pleckstrin homology-like domain family B member 1 (PHLDB1), and regulator of telomere elongation helicase 1 (RTEL1) are reported in gliomas [36]. In case of GBM, the frequent genetic alterations in the decreasing order are LOH 10q (69%), EGFR amplification (34%), TP53 mutations (31%), p16INK4a deletions (31%) and PTEN mutations (24%) [37].

#### **3. Deregulated pathways in glioblastomas**

GBMs are the most fatal of all glial cancers. Secondary GBMs arising from LGG constitute 10% whereas the remaining 90% GBMs arise de novo. The genomic alterations of oncogenes and tumor suppressors are the fundamental cause of cancer development. These alterations further lead to deregulation of several signaling pathways aiding in tumor progression manifesting in metastasis and chemoresistant cancers. GBMs were one of the first tumors to be studied by the TCGA [38] and some of the key signaling pathways reported to be deregulated are as follows:

RTK/RAS/PI3K pathway: This pathway is majorly involved in growth and proliferation and is dysregulated in 88% of GBM cases. This dysregulation occurs by amplification and mutational activation of receptor tyrosine kinase (RTK) genes – EGFR, ERBB2, PDGFRA, MET. A variant of the protein – EGFRvIII that occurs due to intragenic deletions is also a common feature. Activation of the phosphatidylinositol 3-kinase (PI3K) pathway are achieved by PTEN deletion, activating mutations in PIK3CA or PIK3R, AKT3 amplification, NF1 mutation, RAS mutation, FOXO mutation.

p53 pathway: Inactivation of the p53 pathway occurs in about 87% of the GBM cases. TP53, termed as "the guardian of the genome", is a tumor suppressor gene and is frequently mutated or deleted in most cancers [39, 40]. The pathway is involved in several processes like cell cycle arrest, DNA repair, apoptosis, autophagy, differentiation, senescence, and self-renewal [41]. Mutations in the TP53 gene lead to nonfunctional proteins. Several missense mutations, particularly in IDH-wildtype GBM (primary GBM), have been reported, resulting in accumulating the protein in the nucleus [42]. Additionally, deletions in ARF (ADP-ribosylation factor) at 55%, amplification of MDM2 (Mouse double minute 2 homolog)at 11%, and amplification of MDM4 (Double minute 4 protein) at 4% contribute to the inactivation of the P53 pathway [38]. TP53 is the most frequent and the earliest detectable alteration in the transition from low grade to high-grade [43].

*DNA Damage Repair Genes and Noncoding RNA in High-Grade Gliomas and Its Clinical… DOI: http://dx.doi.org/10.5772/intechopen.97074*

Rb pathway: This retinoblastoma (Rb) pathway is dysregulated in 78% of GBM cases and is a vital regulator of the cell cycle and controls progression through the G1 to S phase of the cell cycle at the G1 checkpoint [44]. The Rb gene promoter is methylated frequently in secondary than primary GBMs and is associated with its low gene expression. There are two significant genetic alterations seen in the pathway– deletion of the CDKN2A/CDKN2B locus on chromosome 9p21 and the amplification of the CDK4 locus [38]. Such a loss of CDKN2A, RB or CDK4 amplification disrupts the p16INK4A-CDK4-RB tumor suppressor pathway. It has been shown to correlate with decreased expression and survival.

#### **4. Significance of DDR pathway in glioblastoma**

Recent studies have implicated the DNA damage response (DDR) pathway in modulating GBM chemoresistance. GBMs being the most aggressive gliomas with the least survival rate with treatment options being only radiation and chemotherapy using TMZ. These tumors ultimately gain resistance, leading to cancer relapse. This chemoresistant phenotype is attributed to enhanced DDR with alterations in DNA-repair and cell-cycle genes [12]. DNA repair mechanisms have evolved to counteract this damage based on the type of damage the DNA experiences (**Figure 1**). Some of the commonly observed damage and repair mechanisms are:


#### **Figure 1.**

*Genes involved in the various types of DDR.*

#### **4.1 Frequently mutated genes of DDR pathway in glioblastoma**

Besides mutations in IDH, TP53, and TERT promoter in GBMs, the mutation in genes that function in various DDR pathways have been reported:

MGMT-mediated DNA repair: As previously explained, MGMT is a DNA repair enzyme involved in DNA damage repair induced by alkylating drugs like TMZ. It is involved in the repair of DNA lesions. MGMT enzyme reverses O-alkylated DNA lesions of the alkylated bases [45]. MGMT is mostly hypermethylated in GBM; ~1.6% of the patient's mutation is observed (The results are in whole or part based upon data generated by the TCGA Research Network: https://www.cancer. gov/tcga).

Base excision repair: BER corrects base damage that does not cause significant distortions to the DNA helix. The enzymes involved in repair are DNA glycosylase, AP endonuclease, POL β, DNA ligase 1, or a complex of DNA ligase 3 and XRCC1 [46]. Unlike direct repair by MGMT, there are very few BER machinery components that showed a mutation in GBM.

Nucleotide excision repair: NER is the pathway chosen to remove bulky lesions. The damage is sensed by XPC complexed with RAD23B and CETN2. The other pathway proteins are the UV–DDB complex consisting of DDB1, DDB2, and TFIIH complex. Endonuclease XPF–ERCC1 and XPG, the replicative proteins PCNA, RFC, POL δ, POL ε or POL κ, and LIG1, XRCC1–LIG3 [47]. Of these genes, 5.6% of the cases had a mutation in POLE [48].

Mismatch repair (MMR): The mismatches incorporated during replication are recognized by MutSα heterodimer (MSH2/MSH6) or MutSβ heterodimer (MSH2/MSH3). The other proteins involved are POL δ, RFC, HMGB1, and LIG1 [49]. Of these, 3.8% of patients had a mutation in MSH6 and 1.6% in the MSH2 gene [48].

Double-strand breaks repair: The Double-Stranded Breaks (DSBs) are majorly repaired by nonhomologous end-joining (NHEJ) [50] and homologous recombination (HR) [51]. The alternate less-characterized pathway is microhomologymediated end joining (MMEJ) or alternative end-joining (AEJ) [52]. While HR is restricted to the cell-cycle S and G2 phases, NHEJ and MMEJ are free to get employed in any cell cycle phase [53]. In response to DSBs, three proteins of the phosphoinositide 3-kinase-related kinase (PIKK) family are activated – ATM, ATR, and DNA-PK, downstream they phosphorylate other substrates, activating them [12]. The additional factors that are subsequently recruited include XRCC4, XLF, DNA ligase IV (LIG4), ARTEMIS, and PAXX which plays a key role in stabilizing the complex chromatin [54]. Other proteins that facilitate the pathway are DNA polymerases like POLM and POLL. Multiple proteins in this pathway are mutated in GBM. The ATR gene is mutated in 4.5% patients followed by 2.9% in PRKDC (DNA-PK), 2.5% in ATM, 1.9% ARTEMIS, 1.94% in XRCC5 (Ku80) and POLL [48].

The HR preferentially repairs the DSBs, which occur at the replication fork [55]. The pre-requisite for the homologous recombination repair pathway is the end-processing of DSBs by helicases and nucleases to produce single-stranded DNA. ATM, CtIP, MRN complex(MRE11-RAD50-NBS1) is involved in generating ssDNA [56]. This ssDNA binds with the RecA/RAD51 complex, stimulated by RPA, promotes DNA pairing and strand exchange in an ATP-dependent fashion [57]. Additionally, the tumor suppressor proteins – BRCA1, BRCA2, and PALB2 are involved in HR [58]. In GBM patients, 3.55% BRCA1, 1.86% MRE11A and RAD50, 1.4% NBN, and ~ 1% RPA1 mutations have been reported [48].

The MMEJ pathway is promoted by PARP-1, Ligase III, CtIP, and Mre11. It uses the same machinery as the HR pathway to form a 3′ single-stranded overhang at the *DNA Damage Repair Genes and Noncoding RNA in High-Grade Gliomas and Its Clinical… DOI: http://dx.doi.org/10.5772/intechopen.97074*

region of DSB [52, 59]. Mutations in Ligase III (3.49%) PARP1 (3.33%) and CtIP (2.5%) have been reported in GBM patients [48].

Single strand annealing (SSBR): The single-strand breaks are detected by PARP1, followed by end-processing by PE1, PNKP, and APTX. FEN1 acts as an endonuclease to create a gap. POL β, in combination with POL δ/ε, fills the gap and is ligated by LIG1 [60]. Mutations, although at a much lower frequency, have been reported in all the components of SSBR, APTX (1.17%), FEN1 and PNKP (0.78%), and POLB (0.39%) [48].

Inter-strand crosslink repair (ICL): ICLs are resolved by complex FANCM and FAAP24. MFH stimulates the remodeling of the replication fork. The RPA protein binds to ssDNA and activates ATR, CHK1, FANCE, FANCD2, FANCI, and MRN consecutively. Further, excision is carried out by PF-ERCC1, MUS8-EME1, SLX4- SLX1, FAN1, SNM1A/SNM1B. The polymerase which acts to repair includes POL ι, POL κ, POL ν, and REV1 [61]. 4.42% mutations in FANCD2, 2.26% in FANCI, 1.61% in FANCE, 2.7% and 1.91% in SNM1A and SNM1B, respectively have been reported in GBM patients [48].

Depending on the type of damage a cell encounters, any of these pathways can be activated to restore the damage sites. One of the most deleterious repairs found in cancer cells is MMEJ which results in large deletions and translocations, destabilizing the genome. In GBM, HR and c-NHEJ have higher mutation rates than in MMEJ, making MMEJ the preferred pathway for DNA repair. **Figure 2** represents the frequently mutated genes of the various DDR pathways along with their impact

on overall survival obtained from NCI - GDC Database [62]. As can be observed, the mutations in these genes reduce patients' survival in GBM (14–16 months).

#### **4.2 Altered gene expressions of DDR pathway genes in glioblastoma**

The various genomic mutations like the overexpression of oncogenes and under expression of tumor suppressor genes lead to altered genomic and epigenomic changes favoring cancer growth. In GBM several genes that encode proteins in the DNA repair pathway have altered expression. **Figure 3** represents some of the altered gene expressions in the different DDR pathways in GBMs. This data is obtained from GEPIA database which compares normal patient samples with GBM tumor samples [63].

**Figure 3.** *Altered gene expressions in the various DDR pathway in glioblastoma.*

*DNA Damage Repair Genes and Noncoding RNA in High-Grade Gliomas and Its Clinical… DOI: http://dx.doi.org/10.5772/intechopen.97074*

The DDR genes are significantly upregulated and include HR factors - RAD51 recombinase, the chromatin remodelers RAD54B and RAD54L, enzymes in the HOLLIDAY JUNCTION resolution (EME1/MUS81 complex), NER (ERCC3 (XPB), ERCC4 (XPF). Also, expression of genes encoding DNA glycosylase NEIL3, Fanconi Anemia factors (FANCD2, UBE2T), the ubiquitin-protein ligase UBE3B, and two specialized DNA polymerases POLM and POLQ in the NHEJ pathway are increased significantly [64]. Coincident with the least mutation, MMEJ transcripts show relatively higher expression than other pathways. Closer observation shows elevated MMR transcripts, but a higher mutation rate has been observed of some of the genes like MSH2 and MSH6 in GBM. Among HR gene expression, PDS5B is highly expressed, which is required for proper segregation.

Additionally, these signatures also suggest the sensitivity of the tumor to therapeutic drugs. Upregulation of the TOP2A gene, which encodes topoisomerase II, might be more sensitive to topoisomerase II inhibitors like etoposide. Similarly, the decreased expression of NER genes like ERCC3/XPB and ERCC4/XPF can be more sensitive to cisplatin. Cisplatin acts by causing inter-strand crosslinking, and its repair requires NER [64]. Targeting RAD51 is also a potential therapeutic option that can either target the HR pathway or sensitize the cancer cells to irradiation and chemotherapeutic agents that cause DSBs [65].

#### **4.3 Drugs targeting DDR kinases**

In tumors treated with DNA damaging agents, efficient DNA repair systems become the primary cause for treatment failure. GBM's ability to resist DNA insults is directly attributable to its upregulation of DNA repair pathways. Hence, along with the standard care regimen, DDR kinase inhibitors are being investigated to overcome chemo- and radio-resistance. **Table 2** represents inhibitors that are being developed to target kinases in the DNA damage response pathway.


**Table 2.** *List of drugs developed targeting DDR kinases in gliomas.*

#### **4.4 miRNAs involved in DDR**

MicroRNAs are a group of noncoding RNAs ~18–22 nucleotides in length. miRNA regulates gene expression at both transcriptional and post-transcriptional levels. It modulates transcription by binding to the 5' UTR of the gene. The binding of miRNA at 3' UTR regions (untranslated regions) reduces mRNA stability or inhibits translation [72, 73]. Dysregulated miRNA expression is one of the hallmarks of cancer. They have been shown to affect several crucial processes like proliferation, invasion, and metastasis [74]. Hence, they are potential biomarkers and targets for therapeutic intervention. The aberrant expression of miRNAs in GBM is well documented. 256 upregulated miRNAs and 95 downregulated miRNAs are reported in GBM compared to normal brain tissue [72]. Here, we focus on the deregulated miRNAs involved in DDR pathways leading to chemoresistant or chemosensitive phenotype (**Table 3**).


*DNA Damage Repair Genes and Noncoding RNA in High-Grade Gliomas and Its Clinical… DOI: http://dx.doi.org/10.5772/intechopen.97074*


#### **Table 3.**

*Deregulated miRNAs involved in DNA damage response in GBM.*

#### **4.5 lncRNAs in gliomas**

The noncoding RNAs are a diverse group of transcribed RNAs, with long-non coding RNA or lncRNA being the largest sub-type in this category [106]. Long noncoding RNA can regulate gene expression by binding to the gene's promoter and recruiting activators or repressors, or chromatin modifiers and activating or repressing transcription, respectively [106, 107]. Alternatively, they can work as antisense and bind to the transcripts, thereby inhibiting translation or destabilizing the transcript. They can also act as miRNA sponges, altering gene expression posttranscriptionally [108]. LncRNA deregulation is involved in cancer development, progression, and metastasis. It is a potential target for therapeutic interventions. Their expression pattern in response to chemotherapeutic treatment has prognostic value and serves as predictive biomarkers [106, 107].

lncRNAs are abundantly expressed in the brain as compared to other parts of the body [109]. Glioma subclassification has also been done based on the lncRNA profile into three groups: (i) astrocytic tumor with high EGFR amplification (ii) neuronal-type tumor (iii) oligodendrocytic tumor enriched with an IDH-1 mutation and 1p19q co-deletion. Such a classification has been shown to correspond to patient survival where lncRNAs like PART1, MGC21881, MIAT, GAS5, and PAR5 were correlated with prolonged survival. At the same time, KIAA0495 was associated with poor survival [109]. **Table 4** represents the lncRNAs studied in gliomas that are involved in chemoresistance or chemosensitivity.

#### **4.6 Circular RNAs in gliomas**

Circular RNA is yet another group of noncoding RNA produced from pre-mRNA back-splicing [137]. They inhibit miRNA and upregulate the expression of genes at the transcriptional and post-transcriptional levels [138, 139]. CircRNAs have also been shown to bind to different proteins to form circRNA-protein complexes (circRNPs) that regulate the action of associated proteins, the subcellular localization of proteins, and the transcription of parental or related genes [140]. circRNAs play significant roles in tumor growth, metastasis, EMT transformation, and therapy resistance [141]. circRNAs are the most abundant in the brain and play a crucial role in the brain's functioning [142]. In glioma, they are expressed aberrantly and play a key role in tumor initiation and progression [143]. In GBM, several studies have identified the upregulated and the down-regulated circRNAs. Identifying these circRNAs is valuable for further understanding the molecular mechanism of glioma and developing novel targeted treatments [144]. **Table 5** represents the circRNAs studied in gliomas with their targets.


#### **Table 4.**

*lncRNAs in glioma involved in chemoresistance or chemosensitivity.*


#### **Table 5.**

*circRNAs involved in chemoresistance/chemosensitivity in gliomas.*

*DNA Damage Repair Genes and Noncoding RNA in High-Grade Gliomas and Its Clinical… DOI: http://dx.doi.org/10.5772/intechopen.97074*

#### **5. Novel therapeutic drugs being developed for gliomas**

The standard chemotherapeutic drugs used for gliomas are alkylating agents (TMZ, procarbazine, vincristine, carmustine). More recently, GLIADEL wafer containing carmustine is approved for GBM as an adjunct to surgery and radiation [152]. Humanized monoclonal IgG1 antibody Bevacizumab targeting VEGF is used for recurrent GBM [153]. Surpassing the blood–brain barrier makes treating gliomas difficult [154]. Several inhibitors targeting enzymes like topoisomerase II, [155], immunotherapeutic agents like α-type-1 dendritic cell vaccine [156], autologous cytokine-induced killer cell immunotherapy [157], autologous dendritic cell vaccine [158], and immunomodulatory drugs [159] are in clinical trials phases I and II. Additionally, many of these drugs in combination with the standard chemotherapeutic drug are also in trials, including Giladel wafers with dendritic cell vaccine [160], Lomustine-temozolomide [160, 161], Bevacizumab + radiation therapy + temozolomide [162], Irinotecan + bevacizumab + temozolomide [163]. The **Table 6** lists some of the drugs which are in phase 3 trial for glioma treatment.


#### **Table 6.**

*Novel drugs in clinical trials for glioma treatment.*

#### **6. Conclusion**

Gliomas are the most common malignant brain cancers constituting 80% of all brain & central nervous system cancers. Even though gliomas represent a small percentage of all cancers, they account for disproportionally high morbidity and mortality. Despite the emphasis on new therapeutic interventions, the standard care regimen has not changed drastically. However, there has been more emphasis on understanding molecular pathogenesis and its clinical relevance. Emerging preclinical and clinical data points to a shift towards more personalized therapies, and targeting the DDR pathway and its related noncoding genes is on the horizon. **Figure 4** summarizes the interplay of noncoding in DDR and drug resistance in gliomas.

**Figure 4.** *Representative genes and non-coding RNAs in glioblastomas.*

### **Acknowledgements**

We acknowledge the support from the Department of Science and Technology Fund for Improvement of S&T Infrastructure in Higher Educational Institutions (Grant no. SR/FST/LSI-5361/2012), the Department of Biotechnology, India, Glue grant (BTIPR23078/MED/29/1253/2017), and the Departments Information Technology, Biotechnology and Science and Technology, Government of Karnataka, India.

### **Author details**

Tanvi R. Parashar, Febina Ravindran and Bibha Choudhary\* Institute of Bioinformatics and Applied Biotechnology, Bangalore, India

\*Address all correspondence to: vibha@ibab.ac.in

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*DNA Damage Repair Genes and Noncoding RNA in High-Grade Gliomas and Its Clinical… DOI: http://dx.doi.org/10.5772/intechopen.97074*

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#### **Chapter 11**

## The Dynamic m6 A Epitranscriptome in Glioma Stem Cell Plasticity and Function

*David Karambizi and Nikos Tapinos*

#### **Abstract**

Glioblastoma multiforme is one of the most aggressive tumors of the central nervous system. The current standard-of-care includes maximal resection followed by chemotherapy, radiation and more recently, tumor treating fields (TTFs). Despite this multimodal approach, glioblastoma remains refractory to therapy. Glioblastoma resistance, recurrence and malignancy are believed to be driven by a subpopulation of glioma stem cells (GSCs) within the tumor bulk which are characterized by the retention of self-renewal potential as well as the capacity to recapitulate tumor heterogeneity. Within the dynamic intratumoral niche, GSCs demonstrate a high degree of cellular plasticity, reversibly interconverting between stem-like states and more differentiated states as a result of environmental cues/signaling fluctuations. Such plastic adaptive properties are mostly driven by multiple dynamic, reversible epigenetic modifications. We posit that reversible post-transcriptional methylation of RNA transcripts at the m6 A position may be one such regulatory mechanism employed by GSCs to efficiently maintain plasticity and adaptive phenotypic transitions. In this section, we discuss the concept of cellular plasticity, introduce dynamic m6 a epitranscriptomic mechanisms as potential key regulators of GSC plasticity and finally propose epigenetic based therapeutics as a mean of attenuating glioblastoma plasticity to improve patient outcome.

**Keywords:** glioma stem cell, plasticity, epigenetic landscape, epitranscriptome, cellular states, glioblastoma

#### **1. Introduction**

Glioblastoma is one of the most lethal malignant tumors of the central nervous system. Its treatment involves maximal resection followed by chemotherapy, radiation and tumor treating fields [1]. Despite this multimodal approach, GBM remains uniformly lethal, with a median survival of 15 to 16 months [1]. Histologically, GBM presents as a heterogenous mass with multifocal necrosis, hypervascularization, hemorrhage, pleiomorphic cells with notable mitotic activity and pseudopalisading nuclei [2, 3]. Recent advances in whole genome sequencing allowed for better GBM characterization to compliment current medical knowledge.

The Cancer Genome Atlas (TCGA) initiative generated DNA, RNA and methylation sequencing data on multiple GBMs and lower grade gliomas [4], shedding light onto GBM specific structural, mutational and methylation alterations. It was shown that NF1, IDH, PDGFRA and PARK2 were mutated and that AKT3 and EGFR were amplified in GBMs [5, 6]. Additionally, the vast majority of GBMs were shown to activate the RB, p53 and RTK/RAS/PI3K pathways [5]. Using tumor gene expression signatures, patients could be categorized into discrete subtypes, namely mesenchymal, proneural and classical [6]. However, subtyping did not directly relate to long term survival [7]. Tendencies towards survival were only observed when the data was restricted to patients with lowest simplicity score [7].

The TCGA derived data supplied useful information, but it simultaneously raised new questions. First, it was noted that 8% of the samples did not discretely fit within defined TCGA subtypes, but instead scored for multiple subtypes [6–8]. Second, tumors were shown to undergo subtype switching following recurrence [9]. Third, even with low mutational burden, GBM exhibited significant intra and inter tumoral heterogeneity. GBM's aggressiveness and recurrence is believed to be driven by a small subpopulation of stem like cells within the tumor niche [10–13]. These cells, generally referred to as glioma stem cells (GSCs), possess the ability to self-renew and can fully recapitulate the tumor bulk with fidelity to parental tissue properties following xenotransplantation [14]. Recent developments have helped to catapult GSCs at the nexus of GBM tumorigenesis. It has been shown that the adult human brain is not an entirely post-mitotic tissue and to possess specific regions with an enrichment for cells with stem like properties or neural stem cells (NSCs) [14, 15]. Interestingly, NSC markers such as CD133 and Nestin are frequently expressed in GSCs [16]. Such homology raised questions on GSCs relation to NSCs. Thus, "the cell of origin" theory emerged. The theory posited that GSCs, which are mutated NSCs are the cells of origin of GBM. Spatial studies demonstrated that GBMs exhibited a growth bias for the subventricular zone (SVZ), a region known to be enriched with NSCs [14]. Furthermore, multiple studies showed that *de novo* GBM tumorigenesis could be achieved by inducing tumor initiating mutations within the SVZ [8]. Together, these findings cemented GSCs as initiators and drivers of GBM, hence placing them center stage as key targets in GBM therapy. However, most therapies targeted at GSC continue to fail, likely due to GSCs' high adaptability potential and tendency to continuously fuel tumor niche dynamic heterogeneity by undergoing reversible multilineage differentiation.

The aforementioned complex cell dynamics likely rely on coordinated genetic and epigenetic processes. Here, we focus on epigenetic processes, more specifically post transcriptional chemical decorum on mRNA adenosine or mRNA m6A. This chemical modification has widely been explored in dynamic processes ranging from neurogenesis, memory formation to various pathophysiological processes including cancers [17–19]. We discuss what is known at the m6A/plasticity interface in GBM and finally postulate/propose ways in which epitranscritpomics can function as a predictive or therapeutic tool to affect clinical outcome.

#### **2. Cellular plasticity in glioblastoma**

GBM exhibits a high degree of intertumoral and intratumoral heterogeneity. Such heterogeneity is sustained by constant, dynamic interconversion between cellular states. Differentiated glioma stem cells (DGCs) undergo spontaneous de-differentiation to primordial states or back to GSCs and vice versa in response to fluctuating microenvironmental cues [20–23]. It is likely that this tumor hijacks highly conserved genetic and epigenetic programming generally associated with stemness multipotency and early embryonic development in order to rapidly adapt to and evade various therapeutic strategies. Therefore, glioma cancer cells leverage such plasticity to maintain an adaptive, shifting cell state population equilibrium

#### *The Dynamic m6 A Epitranscriptome in Glioma Stem Cell Plasticity and Function DOI: http://dx.doi.org/10.5772/intechopen.96792*

that is not amenable to therapy. For example, radiotherapy and temozolamide induce adaptive, spontaneous de-differentiation of DGCs to GSCs, thereby increasing and replenishing the cancer stem pool [24]. Such a shift in cell population distribution towards increased stemness forcibly translates to a more refractory tumor organ.

Recent work shows that GSCs clones are able to readily undergo reversal phenotypic transition between clonal populations [21]. The authors also demonstrated the reversible nature of the cellular equilibria assumed by GSCs in the face of hypoxia as the cells return to a naïve, pre-hypoxia exposure following normoxia [21]. In a fashion reminiscent of the Waddington landscape, GSCs inherently possess a high cellular plasticity potential, thus exist in thermodynamically poise cellular states, and can adaptively differentiate to assume multiple population equilibria in response to external perturbation [21]. These rapid processes entail myriad cellular epigenetic regulatory mechanisms, one of which is the dynamic regulation of m6A.

#### **3. The epitranscriptome in glioblastoma**

Currently, there are over 170 possible chemical modifications on RNA species [25]. The majority occurs on highly abundant non-coding RNA species such as rRNAs, tRNAs and snRNAs and consequently influence RNA stability and RNA secondary/tertiary structure [26]. Most of these modifications are challenging to study in mRNA due to their sparsity and relatively higher abundance in rRNA and tRNA, hence imposing a detection problem in coding RNA [25, 27]. Conversely, N6-methyladenosine (m6A) is highly enriched in mRNA, but sparse in rRNA and absent in tRNAs. The occurrence of m6A on mRNA and its effector role on mRNA stability were established in the 1970s [28, 29]. Since, a set of complexes responsible for 1) placement of m6A on transcripts (m6A methyltransferases or "writers") 2) removal of m6A (m6A demethylases or "erasers" 3) "interpretation" or effector function of m6A marks (readers) have been identified. Readers include the YTH domain containing YTHDF1-F3 and YTHDC1-C2. YTHDC1 and YTHDC2 bind methylated nuclear transcript, while YTHDF1, YTHDF2 and YTHDF3 bind methylated cytoplasmic transcript [30–35]. Methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14) and Wilm's-tumor-1-associated protein (WTAP) form a multimeric methyltransferase complex responsible for m6A mark transcript placement [36–38]. The removal of m6A is mediated by the fat massand-obesity-associated protein (FTO) and alkylation repair homolog protein 5 (ALKBH5) [39, 40]. The discovery of these m6A RNA demethylases implied possible reversibility or dynamism inherent to the epitranscriptome. Once placed on transcripts, m6A has been shown to modulate mRNA stability, splicing and translation and thus ultimately influence gene expression kinetics and outcome [32–34]. Following the refinement of m6A detection techniques, m6A has been widely studied in physiologic processes, such as early development, and in pathophysiologic processes, ranging from psychiatric disorders to cancers. Here, we focus our attention to the glioblastoma/m6A interface as it pertains to plasticity.

#### **3.1 m6A writers in glioblastoma**

The most common way the m6A code has been probed in GBM is via enzymatic inhibition or transcript level perturbation of the m6A machinery. The majority of works on the role of writers in GBM suggest an oncogenic role for METTL3/METTL14. The methyltransferase METTL3 has been shown to be essential for sustenance of GSCs, radioresistance and GBM oncogenic signaling [41–44]. Yet, METTL3 and

METTL14 have been shown in an overexpression-based study to reduce GSCs tumorigenicity and stemness potential, suggesting a potential tumor suppressive function [45]. The reasons for these discrepancies pertaining to m6A writers are unclear and necessitate additional clarifying studies. Ultimately, these results could possibly reflect GBM heterogeneity/complexity and hence dissuade against generalizations on m6A in GBM.

#### **3.2 m6A erasers in glioblastoma**

So far, the known m6A erasers exhibit oncogenic tendencies in GBM. Inhibition of FTO demethylase activity has been shown by two independent groups to inhibit stemness propensity in GSCs [45, 46]. In another study, ALKBH5 was shown to be highly expressed in GSCs and functioned to promote tumorigenicity via FOXM1 transcript stabilization [47].

#### **3.3 m6A readers in glioblastoma**

The functional role of the YTH readers in GBM had been unknown until very recently. Two recent studies show that YTHFD2 promotes GBM aggressiveness, albeit through different proposed mechanisms [48, 49]. One study finds that YTHDF2, though previously shown to destabilize transcripts, does however stabilize *MYC* and *VEGFA* transcripts in GSCs in an m6A-dependent manner [48]. The other study shows that the EGFR/SRC/ERK pathway functions to stabilize YTHDF2 via protein phosphorylation and YTHDF2 consequently destabilizes transcripts implicated in cholesterol dysregulation and invasive GBM growth [49]. Again, these differences may suggest context dependence given GBM's high levels of heterogeneity and plasticity.

Summary of the role of various components of the m6A RNA methylation machinery in glioblastoma is presented in **Figure 1**. Though hinting at plasticity, most of these studies do not explicitly determine m6A dynamics in the context of GBM cell state transition.

#### **3.4 Role of m6A in cellular plasticity in glioblastoma**

Recent findings in neuroscience pertaining to neurogenesis and gliogenesis emphasize the centrality of m6A in dictating cell fate/state specification and plasticity events during early brain development. Stem cells of the nervous system, known as radial glia cells (RGCs) or neural progenitor cells, which are responsible for neurogenesis and gliogenesis, show m6A dependency [50]. As per one study, conditional KO of *Mettl3/Mettl14* in embryonic mouse brain resulted in premature activation of later stage differentiation specific transcripts that are normally kept low in RGCs [50]. Consequently, m6A depleted RGCs could not undergo appropriate multilineage differentiation and expectedly formed abnormal brain tissue [50]. Another study shows that the process of glial specification relies on m6A [51]. Depletion of Prrc2a, which is a gene coding for the Olig2 stabilizing m6A reader PRRC2A, results in hypomethylation and cognitive defect secondary to Olig2 transcription factor destabilization [51]. These studies demonstrate the key role of the m6A code in driving cell fate specification, differentiation and hence plasticity via transcriptional regulation during neurogenesis. From these data, a corollary can be drawn that GSCs, which are mutated NSCs, could exhibit significant m6A dependence during differentiation, de-differentiation, tumorigenesis and in response to external perturbation such as radiation and chemotherapy. However, this m6A/ plasticity axis in GSCs and GBM niche remains poorly understood. Recently, we

*The Dynamic m6 A Epitranscriptome in Glioma Stem Cell Plasticity and Function DOI: http://dx.doi.org/10.5772/intechopen.96792*

#### **Figure 1.**

*Summary of key findings for the role of the various components of the m6A machinery in glioblastoma.*

#### **Figure 2.**

*miRNAs direct FTO/AGO1/ILF3 complex on RRACH m6A motifs to induce RNA demethylation and increase in nascent translation during the transition of GSCs to differentiated progenies.*

performed an integrated whole genome meRIP-seq, RNA-seq and ribo-seq analysis in three patient-derived GSCs and differentiated progenies [52]. This allowed for the interrogation of transcriptional, epitranscriptional and translational kinetics during

cell state transition [52]. In the study, we deliberately avoid m6A machinery perturbation and simply attempt to unravel what happens in one of the most basic process of GSCs plasticity (differentiation) in the context of m6A dynamics. We discovered that a set of clinically relevant transcripts which experience the greatest increase in translation efficiency during differentiation also show significant loss in m6A peaks. This pattern occurred independently of glioblastoma subtypes. We found that these common, highly translated transcripts during GSC differentiation share a consensus m6A motif (the RRACH motif) that overlap a specific set of miRNA sequences. In addition, we discovered a corresponding striking increase in expression of some of these miRNAs with GSC differentiation. Subsequently, we asked whether these findings implicate miRNA at the m6A/translation interface during differentiation. Through a series of mechanistic studies, we propose a mechanism whereby miRNAs can facilitate the formation of a transcript stabilizing FTO-ILF3-AGO1 complex. This results in more efficient association with the ribosome, thus promoting an increase in nascent translation (**Figure 2**).

#### **4. The epitranscriptome as a therapeutic target in glioblastoma**

Though in its infancy, the field of epitranscriptomics holds significant promise for the development of novel epigenetic therapies against GBM. Currently, strategies for targeting the m6A machinery in glioblastoma are directed at the inhibition of enzymatic activity [45, 46]. Recently, targeting of the m6A erasers as well as YTHDF2 have shown some encouraging results for GBM treatment [48]. Specifically, it was shown that high levels of YTHDF2 correlate with increased sensitivity to Linsitinib, an inhibitor of the YTHDF2 downstream effector IGFBP3 [48].

Another emerging avenue is the fusion of m6A machinery components to RNA targeting CRISPR complexes [53, 54]. The deployment of the m6A machinery-CRISPR complex can allow for the specific activation or deactivation of specific transcripts via m6A manipulation [56, 57]. The safe and reversible target specific stabilization or destabilization of coding and/or non-coding RNA species represents an exciting frontier in the development of RNA based therapeutics.

However, these findings leave more avenues for inquiry. For example, what role does the epitrancriptome play in de-differentiation of GSCs, in therapeutic evasion and in microstate transitions of GSCs and differentiated progenies? Are there specific "m6A codes" associated with specific cellular microstates? And how do m6A processes work in synergy with other cellular machineries such as miRNAs, long non-coding RNAs, or well established GBM tumor promoting/suppressing signaling pathways to maintain plasticity? Are m6A dynamics driving or secondary events in GBM microstate transitions? Evidently, more work needs to be done to probe the m6A/plasticity interface in GSCs in order to aid in the discovery of novel epigenetic therapies targeting GSC plasticity.

#### **5. A systems approach towards an epigenetic landscape in glioblastoma**

Recent advancements in single cell RNA sequencing, which include integrated single cell multi-omics analysis, as well as the application of novel algorithms such as pseudotime and RNA velocity have allowed for better characterization of the dynamics within the heterogenous GBM tumor niche [21, 55, 56]. Initial single cell analysis demonstrated that GBM cancer cells exist in a cell state continuum

#### *The Dynamic m6 A Epitranscriptome in Glioma Stem Cell Plasticity and Function DOI: http://dx.doi.org/10.5772/intechopen.96792*

with polarization towards specific fates [55]. Additionally, projection of single cell transcriptomics onto a fetal neurodevelopmental roadmap identified previously unidentified glioma stem cell properties and established GSC at the apex of the glioblastoma tumor hierarchy [21]. These rapidly cycling apical progenitor cancer stem cells were found to have a transcriptional profile that overlapped glial progenitor cells [21]. Furthermore, RNA velocity analysis showed apical stem cell transcriptional adjacency and velocity vector flow towards the more differentiated tumor cell lineages [21]. Collectively, such findings hint at clear plasticity/fluidity within the tumor. The integration of epitranscritpomics with single cell multi-omics technology could help unveil the yet undiscovered mechanism of how dynamic m6A changes play a role in driving plasticity within the tumor niche.

It has been posited that a stem cell may exist at a high or even maximal cellular state of entropy and can readily shift states in the face of perturbation [57]. Our view of the cancer stem cell state in glioblastoma agrees with the theory put forward 20 years ago [58] suggesting that the glioma stem cell is a cellular state or function rather than an entity and this state of maximum cellular entropy is influenced by the constantly adaptable microenvironment of the tumor. In this context, distribution of species would represent heterogeneity, which single cell RNA sequencing adequately captures. Quantum states would equate probability distribution of discrete cell state occupancy bias. In other words, if we looked across a large set of samples and performed, for instance, m6A, ATAC-seq, RNA-seq integrated multiomics single cell analysis, it is possible to generate cell state probability occupancy distribution and ultimately identify discrete, preferred transcriptomic and epitranscriptomic cell state occupancies or quanta states. This will allow to construct an individualized transcriptomic/epitranscritomic landscape and to find patterns within the seemingly stochastic, chaotic environment that is the tumor.

Can we integrate multiple epigenetic "landscapes" with observed clinical outcomes and use this information on a training predictive model to identify discrete favorable and unfavorable cellular microstates? And ultimately can we target plasticity-based processes to convert the microstate cellular make up of a highly malignant tumor bulk into a less aggressive cellular composition? It is plausible that m6A regulatory processes may represent a key target in this endeavor.

#### **6. Conclusion**

In this chapter, we introduce GBM in the context of early genetic characterization and suggest that limitations in discrete classification hinted at an inherent cell state fluidity or plasticity. This plasticity may stand as a key function utilized by GSCs and differentiated cancer cells to rapidly and constantly respond to natural and non-natural/therapy induced microenvironmental fluctuations. Epitranscriptomic dynamic changes are explored as a new frontier in epigenetic based adaptation mechanisms. Additionally, single cell multi-omic technology and its yet to occur application to m6A can pave the way for improvement in GBM characterization and patient management. Lastly, we theorize that the integration of multi-omic cell technology and m6A using massive, high dimensional patient data can aid in the characterization of plasticity through the identification of GBM cell states distribution and quantum state occupancy bias. In the future, such works can be used to develop a Waddington like epigenetic landscape predicting favorable cell state distribution and thus help in the development of plasticity-based therapy to convert glioblastoma into a non-adaptable therapeutic target.

*Central Nervous System Tumors*

### **Conflict of interest**

"The authors declare no conflict of interest."

#### **Author details**

David Karambizi1 and Nikos Tapinos1,2\*

1 Laboratory of Cancer Epigenetics and Plasticity, Brown University, Rhode Island Hospital, Providence, RI, USA

2 Department of Neurosurgery, Brown University, Providence, RI, USA

\*Address all correspondence to: nikos\_tapinos@brown.edu

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Dynamic m6 A Epitranscriptome in Glioma Stem Cell Plasticity and Function DOI: http://dx.doi.org/10.5772/intechopen.96792*

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Section 6
