Adhesion GPCRs in Glioblastoma

Members of the adhesion family of G protein-coupled receptors (GPCRs) have received attention for their roles in health and disease, including cancer. Over the past decade, several members of the family have been implicated in the pathogenesis of glioblastoma. Here, we discuss the basic biology of adhesion GPCRs and review in detail specific members of the receptor family with known functions in glioblastoma. Finally, we discuss the potential use of adhesion GPCRs as novel treatment targets in neuro-oncology. DNA demethylating agent, restores BAI1 expression; (2) chromatin immunoprecipitation shows an enrichment of MBD2 at the BAI1 promoter region; and (3) shRNA-mediated knockdown of MDB2 leads to the re-activation of BAI1 expression in glioma cells. BAI1 can be cleaved at its GPS, autoproteolytically, resulting in a 120 kDa NTF (Vasculostatin-120), or at its MMP-14 site, resulting in a 40 kDa NTF (Vasculostatin-40). Both cleavage products have been shown to contribute to physiological processes within the brain. Vasculostatin-120 decreases intracranial glioma growth in vivo , while both Vasculostatin-120 and Vasculostatin-40 were suggested to increase anti-angiogenic and anti-tumorigenic effects in normal brain and GBM 68-70 . In orthotopic xenografts implanted in rats, Vasculostatin-120 reduces intracranial growth of malignant gliomas and tumor vascular density, even upon a pro-angiogenic stimulus 70 . In endothelial cells, the anti-angiogenic effect was suggested to be dependent on the surface molecule CD36 70 . Likewise, the antiangiogenic and anti-tumorigenic effect of full length BAI1 were shown in xenograft models in vivo 71 , independent of P53 expression within the tumor 65,72 . Taken together, these findings suggest a tumor suppressive role for BAI1 in GBM. The identification of agents that restore expression of BAI1 could potentially serve as a therapeutic tool for the treatment of GBM. implicated in the regulation of (AML) stem cells 88 . expression of the in a of including GBM 89 demonstrated direct involvement in a series of oncogenic processes, including cellular migration and invasion (CD97), stem cell self-renewal (GPR133), ECM remodeling (GPR124, GPR56, CD97), and vascularization (GPR124, BAI1, ELTD1). Given their expression profile, presence on the plasma membrane of tumor cells, potential “druggability” and essential roles in tumorigenesis, we propose that aGPCRs represent putative novel targets in GBM. With this therapeutic potential in mind, we review exisiting data on small molecules and biologics that modulate aGPCR function and suggest opportunities for therapy development.

A c c e p t e d M a n u s c r i p t ADGRA, ADGRB, ADGRC, ADGRD, ADGRE, ADGRF, ADGRG, ADGRL, and ADGRV, although new taxonomies have recently emerged 21 . In this review, we will primarly refer to the aGPCRs by their original names.
Like all GPCRs, members of the aGPCR family are structurally defined by seven conserved α-helical transmembrane loops , an intracellular C-terminus and an extracellular N-terminus. What distinguishes aGPCRs from other GPCRs, however, is their long N-terminus, which varies in length and functional subdomain composition based on the receptor subtype (Fig. 2). These functional domains have been shown to convey cell-cell or extracellular matrix (ECM) interactions, suggesting that these receptors have a dual role as cell adhesion and signaling proteins 20 . All aGPCRs, with the exception of GPR123, possess a conserved GPCR autoproteolysis-inducing (GAIN) domain in the N-terminus that catalyzes cleavage at a GPCR proteolysis site (GPS) to generate an N-terminal and a C-terminal fragment (NTF and CTF, respectively) 22 . The processes following proteolysis have not been fully elucidated, but there is evidence that the NTF and CTF may remain non-covalently bound to each other in the secretory pathway and dissociate after being trafficked to the plasma membrane. Immediately distal to the GPS lies an endogenous agonist sequence, named the Stachel sequence, which is responsible for activating canonical signaling. Soluble peptides derived from this tethered agonist sequence have been used to experimentally modulate aGPCR function 23-31 .
To date, there are numerous publications that provide data on aGPCR canonical signaling via G proteins. Coupling to G αs , G αi , G α12/13 , or G αq proteins has been shown for many of the receptors 20,26, 29,32,33 . G protein-independent non-canonical signaling has also been reported for aGPCRs. The most prominent examples are the BAI family of aGPCRs and GPR124 (ADGRA2), which are involved in Rac-1-mediated signaling [34][35][36] and Wnt pathways 37,38 , respectively.
aGPCRs play pivotal roles in physiological cellular processes, such as establishing cell shape and polarity, mediating cell adhesion and migration, and transmitting mechanical stimuli [39][40][41][42][43][44] . At the organismal level, aGPCRs have been implicated in the immune response, endocrine and nervous system function, as well as tumorigenesis 20,21, 45,46 . Moreover, aGPCRs are involved in brain development, establishment of the blood-brain barrier (BBB) and regulation of brain angiogenesis, and may contribute to the stemness of GBM stem cells A c c e p t e d M a n u s c r i p t migration, brain invasion, cellular proliferation, stem cell self-renewal, and angiogenesis ( Fig.   3).

Specific Adhesion GPCRs in Glioblastoma
Several aGPCRs have been implicated in GBM tumorigenesis. Here, we will focus on some of the specific aGPCRs that our analysis indicates are upregulated in our patient-derived GBM cultures (Fig. 1A) and that are most prominent in GBM research. These include GPR124, BAI1, GPR133, CD97, EMR2, GPR56, and ELTD1. Some aGPCRs, which demonstrate similar expression patterns, for example members of the family of cadherin EGF LAG seven-pass G-type receptors (CELSR), are purposely left out of the review due to a lack of relevant literature implicating them in GBM. For the aGPCRs reviewed here, we will discuss the structural and functional properties of each receptor and provide up-to-date information regarding their implication in the oncogenic process, with a particular focus on GBM. Since several of these aGPCRs are expressed in both tumor and endothelial cells, we will review their function in both cellular contexts where appropriate.
According to recent taxonomy arrangements, it belongs to subfamily III of aGPCRs 20 .
GPR124's serine/threonine-rich N-terminus is characterized by leucine-rich repeats (LRR), a leucine-rich repeat C-terminal domain (LRRCT), an immunoglobulin (Ig) domain and a hormone binding domain (HBD) (Fig. 2). Thrombin-induced shedding cleaves the receptor at the HBD into an NTF and CTF 47 . The association/dissociation of NTF and CTF is protein disulfide isomerase (PDI)-dependent 47 . Thrombin-induced cleavage exposes an RGD motif which mediates cell adhesion by binding integrins 47,48 . A more recent study also suggests that GPR124 is involved in cell adhesion via the interaction with the Rhoguanine exchange factors (GEFs) Elmo/Dock and intersectin (ITSN) through its C-terminus 49 .
Numerous publications highlighted GPR124's involvement in Wnt signaling in the brain endothelium and the receptor's key role in angiogenesis and development of the brain vasculature 37, 38,50,51 . Both in vitro and in vivo studies suggested that GPR124 serves as a coactivator for canonical Wnt signaling via Frizzled and Lrp receptors and Wnt7a/7b 37,38 .
The impact of GPR124 on adult forebrain angiogenesis and the establishment of the BBB were further investigated by producing an inducible conditional knockout in mouse endothelial cells. Endothelial GPR124 deficiency led to BBB disruption, increased tumor A c c e p t e d M a n u s c r i p t hemorrhage and decreased survival in a GBM mouse model 59 . Interestingly, the proliferation capacity of cultured GBM cells was significantly reduced by both tumor-cell specific overexpression and knockdown of the receptor 60 . Transcript levels of GPR124 are not detected in RNA-Seq datasets from the Allen Brain Atlas in normal brain cells (Fig. 1B), while it is moderately expressed in patient-derived GBM cell lines (Fig. 1A).
Collectively, most data suggest that GPR124 is mainly expressed in tumor vasculature, is upregulated in GBM, and plays a major role in Wnt signaling, an important pathway for brain angiogenesis and BBB formation. GPR124 may merit further investigation as a target in GBM, since suppression of its pro-angiogenic function in predicted to inhibit tumor growth and progression.

BAI1 (ADGRB1)
Subfamily VII of the aGPCR family comprises the three brain-specific angiogenesis inhibitor (BAI) genes: BAI1, BAI2, and BAI3 21,61 . Like all BAIs, BAI1 harbors thrombospondin type 1 repeat (TSR) domains and a hormone binding domain (HBD) within its N-terminus, as well as a C-terminal PDZ domain 61 . Of all three BAIs, only BAI1 contains an N-terminal RDG motif, an MMP-14 site, and a C-terminal proline-rich region (PRR) (Fig. 2). BAI1 is involved in both canonical G protein signaling via G αq and G α12/13 26 and non-canonical signaling leading to Rho pathway activation, phosphorylation of ERK and β-arrestin binding 62 .
Recently, peptides derived from BAI1's endogenous Stachel sequence were designed and used to activate the receptor in neurons, where it binds Neuroligin-1, a cell-adhesion molecule found at synapses 63 . The Stachel peptide-induced activation resulted in Rac-1 activation and synapse development, highlighting the role of BAI1 in synaptogenesis 63 .
The first evidence for BAI1's involvement in GBM was given in 1997, when Nishimori and colleagues found that the receptor is expressed in normal brain cells, but its transcript is significantly decreased in established GBM cell lines 64 . Several other studies agreed with those findings, observing repeated detection of BAI1 in normal glial cells at both the transcript and protein level, while failing to detect its presence in GBM cells 65 . Consistent with these findings is the observation that BAI1 expression decreases with rising malignancy grades in glioma tumors 66 . RNA-seq data from our lab shows only moderate BAI1 expression in patient-derived GBM cells in vitro (Fig. 1A), while it is one of the top 5 detected transcripts in normal brain cells from the Allen Brain Atlas (Fig. 1B). A recent study suggests that BAI1 is epigenetically downregulated in GBM by hypermethylation of its promoter region 67 . In this study, the following evidence suggested that MBD2 (methyl-CpG- Both cleavage products have been shown to contribute to physiological processes within the brain. Vasculostatin-120 decreases intracranial glioma growth in vivo, while both Vasculostatin-120 and Vasculostatin-40 were suggested to increase anti-angiogenic and anti-tumorigenic effects in normal brain and GBM [68][69][70] . In orthotopic xenografts implanted in rats, Vasculostatin-120 reduces intracranial growth of malignant gliomas and tumor vascular density, even upon a pro-angiogenic stimulus 70 . In endothelial cells, the anti-angiogenic effect was suggested to be dependent on the surface molecule CD36 70 . Likewise, the antiangiogenic and anti-tumorigenic effect of full length BAI1 were shown in xenograft models in vivo 71 , independent of P53 expression within the tumor 65,72 . Taken together, these findings suggest a tumor suppressive role for BAI1 in GBM. The identification of agents that restore expression of BAI1 could potentially serve as a therapeutic tool for the treatment of GBM.
In addition to the GAIN domain and the GPS, GPR133's N-terminal ectodomain contains a laminin G/pentraxin (LMN/PTX) domain (Fig. 2). As shown in other aGPCRs, the C-terminal sequence immediately following the cleavage site within the GPS represents the endogenous tethered Stachel agonist, which is responsible for activating GPR133 as confirmed by mutational studies 23 . Deleting the NTF of GPR133 leads to increased receptor activity 23 . Initial insights into GPR133 canonical signaling and G protein binding were produced by a few recent studies. Upon GPR133 heterologous expression in Cos-7 and HEK293T cells, cAMP levels increase significantly, an effect that is eliminated with G αs subunit knockdown [73][74][75] . This indicates that the GPR133 receptor couples with the G αs subunit upon activation. GPR133 signaling is increased by administering soluble peptides derived from the endogenous Stachel sequence to Cos-7 cells expressing the receptor 23 .
GPR133, whose ligands remain unknown, was recently shown to be necessary for tumor growth in GBM 76,77 . Knockdown of GPR133 by shRNA results in reduced cell proliferation and tumorsphere formation in vitro. Furthermore, GPR133 knockdown impairs orthotopic tumor xenograft initiation in vivo 76 . RNA-Seq data from GBM cells show GPR133 transcript expression (Fig. 1A), while it is not detected in neurons, astrocytes and oligodendrocyte progenitor cells (OPCs) (Fig. 1B). Frenster et al. used immunohistochemistry to show that A c c e p t e d M a n u s c r i p t GPR133 is essentially de novo expressed in GBM, since it is absent from normal brain tissue (Fig. 4) 78 . Importantly, GPR133 expression was detected in both IDH wild type and IDH mutant tumors 78 . Furthermore, the same study suggested a positive correlation between GPR133 expression and the WHO grade of gliomas, raising the possibility that GPR133 is a marker of anaplasia in the glioma family.
GPR133 is enriched in the most hypoxic regions of GBM, also known as areas of pseudopalisading necrosis. This phenomenon is mediated by transcriptional upregulation of GPR133 in hypoxia via direct binding of hypoxia-inducible factor 1α (HIF1α) to its promoter 76 . The finding suggests that GPR133 is not only a necessary component of GBM growth, but it may also mediate the tumor's cellular response to hypoxia. Collectively, these data suggest that GPR133 merits further consideration as a potential target in GBM. It is therefore necessary to identify inhibitory ligands and small molecule inhibitors of GPR133 or to engineer antibody-drug conjugates as therapeutics for GBM treatment.

CD97 (ADGRE5) & EMR2 (ADGRE2)
CD97 is an aGPCR from subfamily II, consisting of five total EGF-TM7 receptors, aptly named for a series of epidermal growth factor (EGF) repeats found in the N-terminal ectodomain 79 (Fig. 2) Overall, CD97 and EMR2 may represent exciting drug targets due to their high expression levels in multiple solid tumors, including GBM. Existing evidence suggests that the two receptors promote cellular migration and invasion, a phenotype of GBM cells linked to their aggressive behavior and poor patient prognosis. Nevertheless, growing evidence suggests that CD97 also regulates other processes, such as maintenance of the stem cell hierarchy and facilitation of cellular adhesion. Further research is needed to elucidate the function of CD97 and the impact that targeting the receptor may have on cancer progression, including in GBM.

GPR56 (ADGRG1)
GPR56 belongs to the aGPCR subfamily VIII 20 and is arguably the most broadly studied aGPCR within an oncological context. The receptor contains a Pentraxin/Laminin/neurexin/sex-hormone-binding-globulin-like (PLL) domain within its Nterminus (Fig. 2), shown to be essential for ligand binding 95 . Alternative splicing of GPR56 generates multiple isoforms, one of which is termed splice variant 4 (S4) and completely A c c e p t e d M a n u s c r i p t lacks the PLL domain 95 . The receptor also contains a series of N-and O-linked glycosylation sites along its extracellular domain 96 .
Ligands of GPR56 include the ECM components collagen-III and transglutaminase-2 (TG2) 97 . Both of these proteins have been found to facilitate NTF dissociation after receptor cleavage. The binding of TG2 to the NTF of GPR56 causes the receptor-ECM complex to be internalized and degraded by the cell 98 . GPR56, therefore, may play a role in ECM remodeling (reviewed in 99 ), which is an essential aspect of GBM cell migration and invasion.
The receptor also binds heparin, a glycosaminoglycan that interacts with other ECM components 100 . GPR56 activation has been observed in both a Stachel-dependent and Stachel-independent manner 101 . The receptor is known to couple with the G α12/13 subunit to activate the Rho signaling pathway 102 . Non-canonical signaling by GPR56 includes modulation of the PI3K/AKT 103 and β-catenin 96 pathways. Though GPR56 has mainly been implicated in oncogenic processes such as cellular adhesion, migration, and ECM remodeling, the receptor also seems to promote an anti-angiogenic response by reducing VEGF secretion 104 .
RNA-seq data from our lab show that GPR56 is the most abundantly expressed aGPCR in patient-derived GBM cells (Fig. 1A), whereas single cell SMART-seq data from the Allen Brain Atlas suggest that GPR56 expression is low in neurons and moderate in astrocytes and OPCs from normal brain tissue (Fig. 1B). From the developmental point of view, GPR56 plays a crucial role in brain development, neural progenitor migration and differentiation in the oligodendrocyte lineage, and has been linked with polymicrogyria 40,105-109 .
Immunohistochemistry against GPR56 reveals its increased abundance within GBM tissue compared to normal brain tissue 96 . The aGPCR seems to be particularly concentrated at membrane extensions (such as filopodia) and co-localizes with actin filaments at focal adhesion points within GBM cells in vitro 96 (Fig. 2). One variant of the receptor is truncated at the Cterminal end 111 . Currently, ELTD1 remains an orphan receptor and little is known about its post-translational processing and signaling. In our bulk RNA-seq data, we find only modest ELTD1 expression in patient-derived GBM cells, however, we have included it in this review due to extensive available literature implicating the receptor in GBM biology and associated angiogenesis (Fig. 1A).
ELTD1 has emerged as an angiogenic biomarker, co-regulated with other angiogenic factors, such as VEGF, NOTCH1, and DLL4 113,114 . ELTD1 is transcriptionally upregulated in blood vessels of high grade glioma tumors compared to vessels from low grade gliomas and from non-malignant control tissue. Immunohistochemical analysis confirmed expression of ELTD1 in vascular-associated cells 113   A c c e p t e d M a n u s c r i p t As an alternative to antibodies, monobodies are a novel biologic platform for targeting aGPCRs (Fig. 5). Monobodies are synthetic binding proteins based on a fibronectin type III domain with an immunoglobulin fold, but without any disulfide bonds 123  Targeting the ligands of aGPCRs may also be a viable approach (Fig. 5) 128,129 ). These examples help demonstrate the utility of targeting ligands toward modulating aGPCR function.
As discussed previously, peptides derived from the Stachel sequence have been successfully used as aGPCR agonists, modulating signaling and receptor function in vitro 21,23-31 (Fig. 5). In principle, such peptides could be mutated to inhibit aGPCR activation.
However, their hydrophobic character, low solubility and low potency currently limit possible clinical applications.
The conventional pharmacologic strategy to modulate aGPCR signaling in GBM involves small molecules, typically identified via high-throughput screening (Fig. 5). To date, GPR56 and GPR114 have been successfully inhibited by the small molecule antagonist dihydromunduletone in vitro 130 . A small molecule partial agonist of GPR56 was found to mediate G α13 activation 31 . Moreover, decylubiquinone, which modulates the ROS/P53/BAI1 signaling pathway and increases BAI1 expression, reduces breast cancer growth and metastasis in a mouse model. Together, these studies suggest that the use of small molecule drugs to modulate aGPCR signaling and function is a promising approach in the treatment of cancer 131 .
In conclusion, we review compelling evidence that several aGPCRs are de novo expressed in GBM and serve primarily pro-tumorigenic roles, with the exception of BAI1, whose functional profile suggests tumor suppressive properties. Specific aGPCRs have A c c e p t e d M a n u s c r i p t  Cortex Areas SMART-seq data. The ranking of aGPCRs is identical to that in Figure 1A.
Data represent averaged log 2 (CPM) values from layer 1-6 cortical astrocytes (n=966), layer 1-6 cortical OPCs (n=773) and excitatory and inhibitory neuronal clusters (n=7382). The gene expression heatmaps were generated with R.   and an IDH-wild-type GBM (B). The subependymal zone around the brain ventricular system contains progenitor cells that may represent the putative cell-of-origin for glioma. A c c e p t e d M a n u s c r i p t Figure 5. Approaches to modulating aGPCRs as targets toward novel therapeutics. A.
Antibodies that interfere with receptor-ligand interactions can modulate receptor function and signaling. Alternatively, antibodies that target the receptor and lead to internalization may be used to deliver cytotoxic cargo in ADC approaches. B. Similar to antibodies, monobodies can modulate receptor activity or deliver cytotoxic agents upon internalization, but with the added advantage of smaller size. C,D. Small peptide agonists and antagonists (C), derived from endogenous Stachel sequences, and small molecules (D) also represent viable approaches to modulating aGPCR signaling.