**4. Targets for anti-angiogenics**

#### **4.1. VEGF receptor blockers**

#### *4.1.1. Bevacizumab*

Bevacizumab (Avastin) is a recombinant humanized monoclonal antibody that targets VEGF. It was the first anti-angiogenesis agent to be approved by the United States Food and Drug Administration (FDA) in 2004. Bevacizumab was initially approved for use in metastatic colorectal cancer, but its clinical use has been extended to other cancer types (Van Meter and Kim 2010). Bevacizumab has six VEGF binding residues that neutralize the ability of VEGF to bind to its target receptors on endothelial cells. This neutralization has been shown to have efficacy not only in *in vitro* studies, but also in *in vivo* ones.

Recently, two phase III clinical trials investigating Bevacizumab as a first-line treatment for newly diagnosed GBM tumors were completed. Unfortunately, both trials were consistent in showing no statistically-significant prolongation of overall survival time (OS) but there was a slight improvement in progression-free survival time (PFS). The two trials had a similar design, namely double-blinded prospective trials where newly diagnosed GBM patients were randomized to either standard of care with Bevacizumab or with placebo; the standard of care consisted of radiation therapy with adjuvant and concomitant Temozolomide. A total of 637 and 921 adult participants were randomized in the Radiation Therapy Oncology Group (RTOG) and AVAglio trials, respectively. The median OS was 16.1 vs. 15.7 months, in the RTOG trial (*p* = 0.11). The median PFS was longer in patients who received Bevacizumab, 7.3 vs. 10.7 months (*p* = 0.004) and 6.2 vs. 10.6 months (p<0.0001) in the RTOG and Avaglio trials, respectively. In addition, the results also showed a higher incidence of adverse reactions in the Bevacizumab arm, including neutropenia, hypertension, and deep vein thromboembolism and pulmonary emboli (Gilbert, Dignam et al. 2013). The AVAglio trial noted delayed time to definitive deterioration in terms of health-related quality of life (p<0.0001) and Karnofsky Performance Scale, and increased time to corticosteroid initiation (HR 0.71, 95% CI 0.57-0.88; median 12.3 vs. 3.7 months) (Henriksson, Bottomley et al. 2013). These results are discouraging and do not justify the use of Bevacizumab in a GBM patient who has had a reasonable surgical resection.

The data support the idea that Bevacizumab may be reserved until the time of recurrence as several prospective phase II clinical trials have shown prolongation of the 6-month PFS rates ranging from 25 to 42.6 percent and median OS times from 6.5 to 9.2 months. However, a significant limitation of these trials is that the comparison was made to historical controls (Friedman, Prados et al. 2009; Kreisl, Kim et al. 2009; Raizer, Grimm et al. 2010).

#### *4.1.2. VEGF-trap*

et al. 1994; Risau 1997; Kurz, Korn et al. 2001; Plate, Scholz et al. 2012). In this model, a vascular sprout is led by tip cells toward an angiogenic stimulus that is produced by tumor cells. This sprout then elongates via dividing stalk cells. The newly formed vessel undergoes remodeling to create a vascular lumen that allows blood flow (Plate, Scholz et al. 2012). There is evidence to support that both tip and stalk cell phenotypes co-exist in the glioblastoma vasculature (Plate, Breier et al. 1994; Broholm and Laursen 2004;

**2.** *Vascular co-option*. This is the process by which tumor cells infiltrate into normal tissue and adopt pre-existing vasculature (Holash, Wiegand et al. 1999; Plate, Scholz et al. 2012). This pathway seems to be enhanced through activity of pro-angiogenic molecules, like VEGF

**3.** *Myeloid cell-driven angiogenesis*. Tumor-associated macrophages contribute to angiogene‐ sis by secreting pro-angiogenic factors such as fibroblast growth factor 2 (FGF2), VEGF, and matrix metalloproteinases (MMPs) (Plate, Scholz et al. 2012). Tumor-associated macrophages may also assist two vascular sprouts to form a direct connection through a

The role of the remaining four mechanisms in glioma angiogenesis is not yet fully understood. Briefly, *vessel intussusception* is the process by which a new vessel is formed by internal division of the pre-existing capillary plexus without sprouting through a series of steps that include vascular invagination, intra-luminar pillar formation and remodeling, and splitting (Djonov, Schmid et al. 2000; Plate, Scholz et al. 2012). *Vasculogenic mimicry* refers to the process by which cancer cells form *de novo* vasculature as a result of their high plasticity (Plate, Scholz et al. 2012; Seftor, Hess et al. 2012). *Bone marrow-derived vasculogenesis* refers to the process by which circulating endothelial precursor cells are recruited to the tumor and are incorporated into the vessel wall (Plate, Scholz et al. 2012; Huang, Peng et al. 2013). *Cancer stem-like derived vasculo‐ genesis* is the process by which tumor-derived cells trans-differentiate into endothelial cells (Ricci-Vitiani, Pallini et al. 2010; Plate, Scholz et al. 2012). It is not the goal of this chapter to study these mechanisms in detail, but instead to provide an overview of angiogenesis in glioma and discuss key molecules involved and possible therapeutic options that target them.

Bevacizumab (Avastin) is a recombinant humanized monoclonal antibody that targets VEGF. It was the first anti-angiogenesis agent to be approved by the United States Food and Drug Administration (FDA) in 2004. Bevacizumab was initially approved for use in metastatic colorectal cancer, but its clinical use has been extended to other cancer types (Van Meter and Kim 2010). Bevacizumab has six VEGF binding residues that neutralize the ability of VEGF to

Dieterich, Mellberg et al. 2012; Plate, Scholz et al. 2012)

180 Tumors of the Central Nervous System – Primary and Secondary

and Angiopoeitin-2 (Holash, Wiegand et al. 1999).

**4. Targets for anti-angiogenics**

**4.1. VEGF receptor blockers**

*4.1.1. Bevacizumab*

process referred to as anastomosis (Plate, Scholz et al. 2012).

VEGF-Trap (drug name Aflibercept) is a recombinant fusion protein that acts as a decoy receptor for VEGF, thereby blocking its interaction with its normal receptors and interrupting the VEGF signaling pathway (Holash, Davis et al. 2002). VEGF-Trap was developed by incorporating domains of both VEGF receptor 1 and VEGF receptor 2 fused to the constant region of human immunoglobulin G1. VEGF Trap has a high affinity for all isoforms of VEGF-A, as well as for PlGF, another pro-angiogenic agent that primarily acts on VEGF receptor 1 (Holash, Davis et al. 2002; Gomez-Manzano, Holash et al. 2008; de Groot, Lamborn et al. 2011). Preclinical studies demonstrated efficacy of VEGF-trap in glioma animal models (Haapa-Paananen, Chen et al. 2013). de Groot et al. conducted a Phase II study of Aflibercept in recurrent malignant glioma; unfortunately, their results revealed that Aflibercept had minimal activity as a single-agent against recurrent GBM (de Groot, Lamborn et al. 2011).

#### *4.1.3. Sunitinib*

Sunitinib is a small-molecule inhibitor of VEGF receptors 1 and 2, PDGFR alpha and beta, stemcell factor receptor (SCFR), fms-like tyrosine kinase 3 (FLT-3), colony-stimulating factor-1 receptor (CSF-1R), and the *RET* oncogene tyrosine kinase (*RET*) (Chow and Eckhardt 2007; Kreisl, Smith et al. 2013). It has FDA approval for use in metastatic renal-cell carcinoma, gastorintestical stromal tumors refractory to imatinib mesylate, and advanced pancreatic neuroendocrine neoplasms (Kreisl, Smith et al. 2013). Recently, a phase II clinical trial was completed investigating the role of continuous daily Sunitinib in recurrent GBM in both Bevacizumab exposed and Bevacizumab naïve patients (Kreisl, Smith et al. 2013). Unfortu‐ nately, the results did not demonstrate an improvement in PFS in either population. Recent evidence by Costa et al suggests that silencing of micro-RNA 21 (miR-21), a small, non-coding RNA that regulates gene expression, may enhance the anti-tumoral effect of Sunitinib (Costa, Cardoso et al. 2013).

proliferation, survival, and differentiation of cells that form the vasculature (Hynes, Bader et al. 1999; Tchaicha, Mobley et al. 2010). The αv integrin subfamily has five members- αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8- and the αvβ8 member, in particular, has been shown in mouse models to be a central regulator of angiogenesis in the developing brain (McCarty, Monahan-

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Cilengitide, a selective inhibitor of αvβ3 and αvβ5 integrins, demonstrated preclinical activity against angiogenesis in GBMs and it is also being investigated clinically (MacDonald, Taga et al. 2001; Onishi, Kurozumi et al. 2013). A phase II study of Cilengitide conducted by Reardon et al. was associated with a median survival of 9.9 months and a PFS rate of 15% in recurrent glioma patients. Unfortunately, the CENTRIC phase III trial revealed that Cilengitide failed to prolong PFS or OS in patients with newly diagnosed GBM and a methylated MGMT promoter

Notch signaling in host endothelial cells is important for angiogenesis. Recent evidence has shown that delta-like ligand 4 (DLL4), a member of the Notch ligand family, is expressed in tumor cells and can activate Notch signaling in host endothelial cells and can therefore affect the vascular function of tumors. In fact, DLL4 expression appears to be regulated by VEGF and the tumor's hypoxic microenvironment (Patel, Li et al. 2005; Li, Gong et al. 2012). Increased levels of VEGF lead to an up-regulation of DLL4 expression which results in endothelial cells expressing Notch receptors to down-regulate VEGF-induced vessel sprouting and branching and ultimately resulting in productive and efficient angiogenesis (Li, Gong et al. 2012). Furthermore, it has also been demonstrated that blockade of DLL4 can result in non-productive angiogenesis by causing tumor growth inhibition and a decrease in tissue perfusion (Scehnet, Jiang et al. 2007; Li, Gong et al. 2012). Li et al. recently conducted a study to investigate the role of DLL4 in malignant gliomas, specifically in terms of vascular quantity and quality and showed that DLL4 expression was significantly up-regulated in malignant human gliomas as compared to normal brain tissue. Additionally, they also demonstrated that DLL4-positive malignant glioma tissues have increased proliferation of vascular endothelial cells and pericyte recruitment, as compared to DLL4-negative malignant glioma tissue, and that DLL4-positive tissues had a higher vessel maturation index (VMI). These results provide evidence that DLL4 inhibition may alter glioma vessel maturity and, in turn, may improve the effects of anti-

Gamma secretase is a pre-senilin dependent protease that acts as a regulator of angiogenesis through a series of complex steps that are beyond the scope of this chapter. However, part of its role in angiogenesis is related to Notch signaling (Jain, di Tomaso et al. 2007; Boulton, Cai et al. 2008). RO4929097 is a potent and selective gamma secretase inhibitor of Notch signaling that is being investigated as an anti-tumor agent. Phase I studies have demonstrated safety

Earley et al. 2002; Zhu, Motejlek et al. 2002; Tchaicha, Reyes et al. 2011).

(Onishi, Kurozumi et al. 2013).

angiogenic agents (Li, Gong et al. 2012).

*4.3.2. Gamma secretase*

**4.3. Notch signaling**

*4.3.1. Ligands*

#### *4.1.4. Nintedanib*

Nintedanib (BIBF 1120) is a small, orally available triple angio-kinase inhibitor that targets VEGF receptors 1-3, FGFRs 1-3, and PDGFR alpha and beta. It is still in phase III development, but preclinical models demonstrated effective growth inhibition of both endothelial and perivascular cells when the above listed pathways were simultaneously interrupted (Hilberg, Roth et al. 2008; Muhic, Poulsen et al. 2013). Phase I/II clinical trial results have demonstrated tumor stabilization rates of 46-76%, when tested in various tumor types (Mross, Stefanic et al. 2010; Okamoto, Kaneda et al. 2010; Richeldi, Costabel et al. 2011; Muhic, Poulsen et al. 2013) Muhic et al. conducted an uncontrolled phase II trial assessing the efficacy of single-agent Nintedanib in patients with recurrent GBM who had previously failed 1-2 lines of therapy; unfortunately, this study was stopped prematurely secondary to futility (Muhic, Poulsen et al. 2013).

#### *4.1.5. Vandetanib*

Vandetanib is a multi-targeted tyrosine kinase inhibitor of VEGF receptor 2, epidermal growth factor receptor (EGFR) 2, and the rearranged-during-transfection oncogene that results in the simultaneous blocking of several pathways, including angiogenesis (Kreisl, McNeill et al. 2012). Preclinical rat and mice glioma xenografts have shown anti-tumor effects of Vandetanib (Sandstrom, Johansson et al. 2004; Rich, Sathornsumetee et al. 2005; Kreisl, McNeill et al. 2012). Kreisl et al. conducted a phase I/II trial of Vandetanib in patients with recurrent malignant glioma and found that it did not have activity as a single agent in this population (Kreisl, McNeill et al. 2012).

#### **4.2. Integrins**

Integrins are cell surface receptors that play key roles in mediating the migration of endothelial cells. They are receptors for many different extracellular matrix (ECM) ligands and they play an important role in angiogenesis via the processes of integrin-mediated adhesion, migration, proliferation, survival, and differentiation of cells that form the vasculature (Hynes, Bader et al. 1999; Tchaicha, Mobley et al. 2010). The αv integrin subfamily has five members- αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8- and the αvβ8 member, in particular, has been shown in mouse models to be a central regulator of angiogenesis in the developing brain (McCarty, Monahan-Earley et al. 2002; Zhu, Motejlek et al. 2002; Tchaicha, Reyes et al. 2011).

Cilengitide, a selective inhibitor of αvβ3 and αvβ5 integrins, demonstrated preclinical activity against angiogenesis in GBMs and it is also being investigated clinically (MacDonald, Taga et al. 2001; Onishi, Kurozumi et al. 2013). A phase II study of Cilengitide conducted by Reardon et al. was associated with a median survival of 9.9 months and a PFS rate of 15% in recurrent glioma patients. Unfortunately, the CENTRIC phase III trial revealed that Cilengitide failed to prolong PFS or OS in patients with newly diagnosed GBM and a methylated MGMT promoter (Onishi, Kurozumi et al. 2013).

#### **4.3. Notch signaling**

#### *4.3.1. Ligands*

*4.1.3. Sunitinib*

182 Tumors of the Central Nervous System – Primary and Secondary

Cardoso et al. 2013).

*4.1.4. Nintedanib*

al. 2013).

*4.1.5. Vandetanib*

**4.2. Integrins**

(Kreisl, McNeill et al. 2012).

Sunitinib is a small-molecule inhibitor of VEGF receptors 1 and 2, PDGFR alpha and beta, stemcell factor receptor (SCFR), fms-like tyrosine kinase 3 (FLT-3), colony-stimulating factor-1 receptor (CSF-1R), and the *RET* oncogene tyrosine kinase (*RET*) (Chow and Eckhardt 2007; Kreisl, Smith et al. 2013). It has FDA approval for use in metastatic renal-cell carcinoma, gastorintestical stromal tumors refractory to imatinib mesylate, and advanced pancreatic neuroendocrine neoplasms (Kreisl, Smith et al. 2013). Recently, a phase II clinical trial was completed investigating the role of continuous daily Sunitinib in recurrent GBM in both Bevacizumab exposed and Bevacizumab naïve patients (Kreisl, Smith et al. 2013). Unfortu‐ nately, the results did not demonstrate an improvement in PFS in either population. Recent evidence by Costa et al suggests that silencing of micro-RNA 21 (miR-21), a small, non-coding RNA that regulates gene expression, may enhance the anti-tumoral effect of Sunitinib (Costa,

Nintedanib (BIBF 1120) is a small, orally available triple angio-kinase inhibitor that targets VEGF receptors 1-3, FGFRs 1-3, and PDGFR alpha and beta. It is still in phase III development, but preclinical models demonstrated effective growth inhibition of both endothelial and perivascular cells when the above listed pathways were simultaneously interrupted (Hilberg, Roth et al. 2008; Muhic, Poulsen et al. 2013). Phase I/II clinical trial results have demonstrated tumor stabilization rates of 46-76%, when tested in various tumor types (Mross, Stefanic et al. 2010; Okamoto, Kaneda et al. 2010; Richeldi, Costabel et al. 2011; Muhic, Poulsen et al. 2013) Muhic et al. conducted an uncontrolled phase II trial assessing the efficacy of single-agent Nintedanib in patients with recurrent GBM who had previously failed 1-2 lines of therapy; unfortunately, this study was stopped prematurely secondary to futility (Muhic, Poulsen et

Vandetanib is a multi-targeted tyrosine kinase inhibitor of VEGF receptor 2, epidermal growth factor receptor (EGFR) 2, and the rearranged-during-transfection oncogene that results in the simultaneous blocking of several pathways, including angiogenesis (Kreisl, McNeill et al. 2012). Preclinical rat and mice glioma xenografts have shown anti-tumor effects of Vandetanib (Sandstrom, Johansson et al. 2004; Rich, Sathornsumetee et al. 2005; Kreisl, McNeill et al. 2012). Kreisl et al. conducted a phase I/II trial of Vandetanib in patients with recurrent malignant glioma and found that it did not have activity as a single agent in this population

Integrins are cell surface receptors that play key roles in mediating the migration of endothelial cells. They are receptors for many different extracellular matrix (ECM) ligands and they play an important role in angiogenesis via the processes of integrin-mediated adhesion, migration, Notch signaling in host endothelial cells is important for angiogenesis. Recent evidence has shown that delta-like ligand 4 (DLL4), a member of the Notch ligand family, is expressed in tumor cells and can activate Notch signaling in host endothelial cells and can therefore affect the vascular function of tumors. In fact, DLL4 expression appears to be regulated by VEGF and the tumor's hypoxic microenvironment (Patel, Li et al. 2005; Li, Gong et al. 2012). Increased levels of VEGF lead to an up-regulation of DLL4 expression which results in endothelial cells expressing Notch receptors to down-regulate VEGF-induced vessel sprouting and branching and ultimately resulting in productive and efficient angiogenesis (Li, Gong et al. 2012). Furthermore, it has also been demonstrated that blockade of DLL4 can result in non-productive angiogenesis by causing tumor growth inhibition and a decrease in tissue perfusion (Scehnet, Jiang et al. 2007; Li, Gong et al. 2012). Li et al. recently conducted a study to investigate the role of DLL4 in malignant gliomas, specifically in terms of vascular quantity and quality and showed that DLL4 expression was significantly up-regulated in malignant human gliomas as compared to normal brain tissue. Additionally, they also demonstrated that DLL4-positive malignant glioma tissues have increased proliferation of vascular endothelial cells and pericyte recruitment, as compared to DLL4-negative malignant glioma tissue, and that DLL4-positive tissues had a higher vessel maturation index (VMI). These results provide evidence that DLL4 inhibition may alter glioma vessel maturity and, in turn, may improve the effects of antiangiogenic agents (Li, Gong et al. 2012).

#### *4.3.2. Gamma secretase*

Gamma secretase is a pre-senilin dependent protease that acts as a regulator of angiogenesis through a series of complex steps that are beyond the scope of this chapter. However, part of its role in angiogenesis is related to Notch signaling (Jain, di Tomaso et al. 2007; Boulton, Cai et al. 2008). RO4929097 is a potent and selective gamma secretase inhibitor of Notch signaling that is being investigated as an anti-tumor agent. Phase I studies have demonstrated safety and phase II studies are underway to assess its role in recurrent GBM when given alone and in combination with Bevacizumab (Tolcher, Messersmith et al. 2012).

invasive, as well as anti-angiogenic properties in several human cancer cell lines including glioblastoma (Ge, Rempel et al. 2000; Fiorio Pla, Grange et al. 2008; Karmali, Maxuitenko et al. 2011). In initial clinical development, CAI was shown to have poor bioavailability, limited efficacy, and high toxicity. CTO, however, appears to have much better bioavailability and less

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TRC105 is a novel, first-in-class antibody against endoglin (CD 105), an endothelial cell receptor that is essential to angiogenesis and acts primarily through its effects on TGF-β and BMP-9 signaling. A phase I trial conducted by Rosen et al. demonstrated that this drug is welltolerated at clinically relevant doses and multiple phase II trials are ongoing to evaluate its potential role in other malignancies, including malignant glioma (Rosen, Hurwitz et al. 2012).

Thalidomide and its analogue Lenalidomide have both been shown to have anti-angiogenic and anti-tumor effects in preclinical models (D'Amato, Loughnan et al. 1994; Short, Traish et al. 2001). The anti-angiogenic effects are thought to be related to a hepatic metabolite that inhibits endothelial growth, although the exact mechanism is unclear (Short, Traish et al. 2001). Additionally, an early clinical trial conducted by Baumann et al. showed that the combination of Thalidomide with Temozolomide appeared to be more effective than Thali‐ domide alone in the treatment of GBM (Baumann, Bjeljac et al. 2004). Additional studies are underway to examine the role of Thalidomide in combination with other anti-glioma agents.

Tandutinib (MLN0518) is an active inhibitor of type III receptor kinases with activity against PDGF receptors alpha and beta, FLT3, and c-KIT. Its anti-angiogenic effects appear to be mediated by interruption of PDGF/PDGFR. It is currently being investigated in combination

Our immune system can be viewed as an intricate balance of opposing functions that lead to either immunity or tolerance. Perturbations that disrupt this stable equilibrium could lead to autoimmune disease or tolerance to malignant cells. In general, the immune system has the ability to recognize and to react to foreign antigens, which leads to their removal as well as to the destruction of cells that express them. Before attempting immunotherapy for cancer, one needs to understand the crucial balancing acts of the immune response that eventually lead to a desired outcome; in addition, the central nervous system has unique features that require

therapy with other agents against malignant glioma (Boult, Terkelsen et al. 2013).

**5. Immunotherapy for malignant gliomas**

toxicity (Grover, Kelly et al. 2007; Karmali, Maxuitenko et al. 2011).

*4.7.2. TRC105*

*4.7.4. Tandutinib*

special considerations.

*4.7.3. Thalidomide and lenalidomide*

#### **4.4. Transforming growth factor beta (TGF-β)**

TGF-β is a multifunctional protein that is involved in the regulation of proliferation, differen‐ tiation, and survival of many cells, including glioma cells and endothelial cells (Bertolino, Deckers et al. 2005). TGF-β1 and TGF-β2, members of the TGF-β family, stimulate expression of VEGF, the plasminogen activator inhibitor, and some metalloproteinases that are involved in vascular remodeling, angiogenesis, and degradation of the extracellular matrix. Animal models demonstrate that inhibitors of TGF-β signaling reduce viability and invasion of gliomas (Kaminska, Kocyk et al. 2013). Fresolimumab, a human monoclonal antibody that inactivates all forms of TGF-β, is being investigated as a potential therapeutic for glioma (Trachtman, Fervenza et al. 2011).

#### **4.5. Topoisomerase I inhibitors**

Topoisomerase I is critical for efficient DNA replication and cell division. Topoisomerase I activity is increased in malignant gliomas and inhibitors of topoisomerase I activity, such as Camptothecin, Irinotecan, and the indolocarbazoles, have been tested as potential glioma therapies (Pommier 2006; Feun and Savaraj 2008; Vredenburgh, Desjardins et al. 2009; Lampropoulou, Manioudaki et al. 2011). Recently, Lampropoulou et al. have shown that inhibition of topoisomerase I activity by the pyrrolo[2,3-α]carbazole derivatives may be linked to a decrease in the number of viable glioma and endothelial cells *in vitro* and may also be related to inhibition of angiogenesis *in vivo* (Lampropoulou, Manioudaki et al. 2011)*.*

#### **4.6. Oncoproteins**

B-cell specific Moloney murine leukemia virus integration site 1 (Bmi-1) is an oncoprotein that plays a role in the development and progression of cancers including breast, lung, prostate, and interestingly, brain (Jagani, Wiederschain et al. 2010). Bmi-1 is a member of the Polycomb gene family of proteins that function as epigenetic silencers of genes that control self-renewal, differentiation, and proliferation; dysregulation of Bmi-1 has been associated with cancer cell proliferation, invasion, and repression of apoptosis or senescence (Jiang, Song et al. 2013). In particular, Bmi-1 promotes growth and survival of glioma tumor cells (Li, Gong et al. 2010; Jiang, Song et al. 2013); furthermore, Bmi-1 promotes angiogenesis of gliomas by activating the NF-κB signaling pathway *in vitro* as well as *in vivo* (Jiang, Song et al. 2013). Thus, targeting Bmi-1 is a promising aim in gliomas.

#### **4.7. Other potential therapeutics in development**

#### *4.7.1. Carboxyamidotriazole orotate (CTO)*

CTO is a triazole orotate formulation of carboxyamidotriazole (CAI), which is an inhibitor of receptor-operated calcium channel-mediated calcium influx. CTO has anti-proliferative, antiinvasive, as well as anti-angiogenic properties in several human cancer cell lines including glioblastoma (Ge, Rempel et al. 2000; Fiorio Pla, Grange et al. 2008; Karmali, Maxuitenko et al. 2011). In initial clinical development, CAI was shown to have poor bioavailability, limited efficacy, and high toxicity. CTO, however, appears to have much better bioavailability and less toxicity (Grover, Kelly et al. 2007; Karmali, Maxuitenko et al. 2011).

### *4.7.2. TRC105*

and phase II studies are underway to assess its role in recurrent GBM when given alone and

TGF-β is a multifunctional protein that is involved in the regulation of proliferation, differen‐ tiation, and survival of many cells, including glioma cells and endothelial cells (Bertolino, Deckers et al. 2005). TGF-β1 and TGF-β2, members of the TGF-β family, stimulate expression of VEGF, the plasminogen activator inhibitor, and some metalloproteinases that are involved in vascular remodeling, angiogenesis, and degradation of the extracellular matrix. Animal models demonstrate that inhibitors of TGF-β signaling reduce viability and invasion of gliomas (Kaminska, Kocyk et al. 2013). Fresolimumab, a human monoclonal antibody that inactivates all forms of TGF-β, is being investigated as a potential therapeutic for glioma (Trachtman,

Topoisomerase I is critical for efficient DNA replication and cell division. Topoisomerase I activity is increased in malignant gliomas and inhibitors of topoisomerase I activity, such as Camptothecin, Irinotecan, and the indolocarbazoles, have been tested as potential glioma therapies (Pommier 2006; Feun and Savaraj 2008; Vredenburgh, Desjardins et al. 2009; Lampropoulou, Manioudaki et al. 2011). Recently, Lampropoulou et al. have shown that inhibition of topoisomerase I activity by the pyrrolo[2,3-α]carbazole derivatives may be linked to a decrease in the number of viable glioma and endothelial cells *in vitro* and may also be

related to inhibition of angiogenesis *in vivo* (Lampropoulou, Manioudaki et al. 2011)*.*

B-cell specific Moloney murine leukemia virus integration site 1 (Bmi-1) is an oncoprotein that plays a role in the development and progression of cancers including breast, lung, prostate, and interestingly, brain (Jagani, Wiederschain et al. 2010). Bmi-1 is a member of the Polycomb gene family of proteins that function as epigenetic silencers of genes that control self-renewal, differentiation, and proliferation; dysregulation of Bmi-1 has been associated with cancer cell proliferation, invasion, and repression of apoptosis or senescence (Jiang, Song et al. 2013). In particular, Bmi-1 promotes growth and survival of glioma tumor cells (Li, Gong et al. 2010; Jiang, Song et al. 2013); furthermore, Bmi-1 promotes angiogenesis of gliomas by activating the NF-κB signaling pathway *in vitro* as well as *in vivo* (Jiang, Song et al. 2013). Thus, targeting

CTO is a triazole orotate formulation of carboxyamidotriazole (CAI), which is an inhibitor of receptor-operated calcium channel-mediated calcium influx. CTO has anti-proliferative, anti-

in combination with Bevacizumab (Tolcher, Messersmith et al. 2012).

**4.4. Transforming growth factor beta (TGF-β)**

184 Tumors of the Central Nervous System – Primary and Secondary

Fervenza et al. 2011).

**4.6. Oncoproteins**

**4.5. Topoisomerase I inhibitors**

Bmi-1 is a promising aim in gliomas.

*4.7.1. Carboxyamidotriazole orotate (CTO)*

**4.7. Other potential therapeutics in development**

TRC105 is a novel, first-in-class antibody against endoglin (CD 105), an endothelial cell receptor that is essential to angiogenesis and acts primarily through its effects on TGF-β and BMP-9 signaling. A phase I trial conducted by Rosen et al. demonstrated that this drug is welltolerated at clinically relevant doses and multiple phase II trials are ongoing to evaluate its potential role in other malignancies, including malignant glioma (Rosen, Hurwitz et al. 2012).

#### *4.7.3. Thalidomide and lenalidomide*

Thalidomide and its analogue Lenalidomide have both been shown to have anti-angiogenic and anti-tumor effects in preclinical models (D'Amato, Loughnan et al. 1994; Short, Traish et al. 2001). The anti-angiogenic effects are thought to be related to a hepatic metabolite that inhibits endothelial growth, although the exact mechanism is unclear (Short, Traish et al. 2001). Additionally, an early clinical trial conducted by Baumann et al. showed that the combination of Thalidomide with Temozolomide appeared to be more effective than Thali‐ domide alone in the treatment of GBM (Baumann, Bjeljac et al. 2004). Additional studies are underway to examine the role of Thalidomide in combination with other anti-glioma agents.
