**2.1. Genetic alterations of RTK in primary GBM**

**1. Introduction**

32 Glioma - Contemporary Diagnostic and Therapeutic Approaches

GBM with IDH-wild-type.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Recently, based on mutations in the gene encoding isocitrate dehydrogenase enzyme 1/2(IDH1/2), GBMs were separated into three main groups (2016 WHO classification of CNS tumors): (1) IDH-wild-type GBMs (about 90% of cases); (2) IDH-mutant GBMs (about 10% of cases); and (3) not otherwise specified (NOS) GBMs. Among these, IDH-mutant phenotype is strongly associated with secondary GBM, younger age, and better outcome, while IDHwild-type with primary GBM. Typical molecular alterations in primary GBM include mutations in genes regulating receptor tyrosine kinase (RTK)/rat sarcoma (RAS)/phosphoinositide

There have been identified approximately 58 mammalian RTKs, which contain an intracellular catalytic protein tyrosine kinase domain and regulatory sequences, transmembrane domain, and an extracellular ligand-binding domain [1]. In response to environmental cues, RTKs are crucial regulators of the growth factor signaling that controls cellular processes including proliferation, metabolism, survival, etc. RTK activation triggers complex signaling network through Ras/Raf/ MEK/ERK, PI3K/Akt and other intracellular pathways in both physiological and pathological conditions; RTK dysregulation through mutation and amplification often occurs in a wide range of cancers including GBMs. RTKs such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), c-Met, Tie, Axl, discoidin domain receptor 1 (DDR1), erythropoietin-producing human hepatocellular carcinoma (Eph) and others play a major role in human GBM pathobiology [2]. Therefore, RTK-targeted agents including tyrosine kinase inhibitors and antibod-

The tumor microenvironment (TME) in malignant glioma is a dynamic entity that consists, besides glioma cells [including glioma stem cells (GSCs)], of an intricate network that encompasses various cell types (e.g., endothelial cells, astrocytes, microglia, and pericytes), stromal components, soluble factors, as well as the extracellular matrix (ECM) [3, 4]. Together, these TME elements play an important role in facilitating the integration of tumor cells with their surrounding environment maintaining features of tumor malignancy [3]. Initially, tumor cells actively exploit their stromal environment through the recruitment of nonmalignant cells and elements that may provide physiological resources to facilitate rapid tumor growth. In time, these recruited cells become a major source of secreted factors to mobilize further inflammatory cells into the microenvironment until the entity becomes steady and strong to progression [5]. In the meantime, rapid proliferation of the malignant cells per se has a metabolic effect on the TME, which is rapidly deprived of glucose and oxygen, becoming acidic and hypoxic [6]. Overall, both tumor cells and the TME are adaptive and undergo evolution from time to time during tumor progression. Human brain tumor bears unique TME in that the tumor rarely metastasizes to other parts of the body [7]. Currently, almost in each type of cancer, TME has drawn much

attention regarding the mechanisms of cancer biology and novel therapeutic strategies.

In this chapter, we consider the interaction and role of RTKs and TME during GBM progression; especially their close interactions in GBM biology and targeted therapies. We then discuss recent improvements on approaches of targeting RTKs and TME mainly in primary

ies are currently used in preclinical and clinical settings in cancers including GBM.

3-kinase (PI3K), p53, and retinoblastoma protein (RB) signaling.

Aberrant RTK activation frequently occurs during glioma initiation and progression and that the associated activation cascades may cooperate through multiple signaling cross-talks in the malignant transformation of cells, tumor growth and progression, treatment resistance, and disease relapse. In 2008, the Cancer Genome Atlas project (TCGA) reported significant alterations in three core signaling pathways, including RTK/RAS/PI3K (88%), p53 (87%), and retinoblastoma protein (78%), in the collected samples from patients with primary GBM, which may represent the majority of human GBM [8]. 60% of the primary GBM harbors RTK amplifications and/or mutations, among them, EGFR amplifications and/or mutations were observed more than 50% of the disease. About half of GBM with EGFR amplification had an in-frame deletion of exons 2–7 from the extracellular ligand-binding domain of EGFR resulting in a mutant protein with ligandindependent receptor activity (designated delta-EGFR or EGFRvIII) [9]. Therefore, EGFRvIII is commonly expressed in a subset of EGFR-amplified cells. Only a small portion (7%) of the tumor showed EGFR genetic alterations in combination with other RTK lesions. Amplification of platelet-derived growth factor receptor alpha polypeptide (PDGFRA) occurs in 13% of GBMs; ErbB2 (HER2/Neu) belongs to the EGFR receptor family that includes the other three members: EGFR, ErbB3, and ErbB4. Activation of ErbB2 depends on the patterns of dimerization within other family members [10]. ErbB2 mutation was observed in 8% GBM tested in a TCGA study [11, 12]. MET amplifications and fibroblast growth factor receptor (FGFR) mutations, including fusion genes, occur in about 2% of the GBMs [1]. Additionally, overexpression of ligand and/or receptor and co-expression of both (autocrine loop formation) are frequent events in cancers, including GBM, and many have been associated with increased malignancy and worse patient outcome.

### **2.2. Cooperation of RTKs and their downstream signaling pathways**

RTK alterations usually coexist with mutations that activate other core regulatory pathways, including intracellular Ras/MAPK and PI3K/Akt pathways, as well as tumor suppressor pathways in certain types of GBM. Furthermore, the frequent co-occurrence of mutations in *PI3K* and deletion of *PTEN*, in addition to the co-occurrence of mutations and/or deletion of cyclin-dependent kinase inhibitor 2A (CDKN2A; encoding both INK4A and ARF) were observed within all of the detectable RTK alterations in primary GBM. This is consistent with the required cooperation of multiple core pathways for tumor formation in genetically engineered mouse models of GBM. Besides, phosphorylated tyrosine kinases of RTK provided PLC-γ1 docking sites for PLCG1 SH2 domains, leading to phosphorylation of tyrosine kinases on PLC-γ1 and signaling activation pathways [13, 14]. JAK/STAT3 signaling was reported associated with EGFR and EGFRvIII signaling [15].

Given that individual tumor cells express multiple RTKs, it is reasonable to speculate that these RTKs are actively interacting with each other. For example, the phosphorylation of c-Met receptor is strongly correlated with functional levels of EGFRvIII, suggesting the presence of cross-talk between these two RTK signaling, although the intermediary molecules were not elucidated [16]. The Axl RTK follows a similar phosphorylation response as a function of EGFRvIII levels [16]. EGFRvIII expressed in glioma cells stimulates upregulation of TGFα and HB-EGF, which stimulate in turn wild-type EGFR forming an autocrine loop [17]. It was previously reported that EGFR and EphA2 are both expressed in GBM cells and co-localize to the cell surface. EphA2 phosphorylation is dependent on EGFR activity, and EphA2 downregulation inhibits EGFR phosphorylation, downstream signaling, and EGF-induced cell viability [17]. HGF indirectly activates alternative RTKs such as EGFR by upregulating expression of EGFR ligands such as TGF-α and HB-EGF [2]. Previous studies report that EphA4, whose expression is correlated with increasing glioma grade, forms a heteroreceptor complex with fibroblast growth factor receptor 1(FGFR1) in glioma cells and that the EphA4-FGFR1 complex potentiated FGFR-mediated downstream signaling such as Akt/MAPK, Rac1, and Cdc42 pathways, resulting in the promotion of invasion [18]. A few other reports suggest that Tie2 activation regulates angiogenesis in a highly context and tissue-dependent manner and closely collaborates with VEGF and other angiogenesis regulators [19, 20].
