Acknowledgments
This work was supported by National Institutes of Health grants R00HL103792 and R01NS094533 , University of Pennsylvania Neuro-oncology Innovation Award, and McCabe Award (to Y. Fan).
Glioma is the most common malignant primary tumor in the central nervous system, accounting for about 80% of total malignant brain tumors. The World Health Organization (WHO) classification divides glioma into 4 grades according to the degree of malignancy: anaplastic astrocytoma (WHO grade III) and well-differentiated astrocytoma (WHO grade I/II) have various median survivals from 2 to 7 years ; glioblastoma (GBM; WHO grade IV), which constitutes 54.9% of all gliomas, is the most serious and malignant form of glioma, with a median overall survival of 12 to 15 months.
Despite the aggressive standard-of-care treatment, which includes surgical resection, fractionated radiation, and temozolomide-based chemotherapy, the relapse of high-grade glioma is essentially universal, and the 5-year survival rate of patients with GBM is less than 10%. Multiple mechanisms contribute to treatment inefficacy: complete surgical removal is nearly impossible because of its location and infiltrative nature; the use of fractionated radiation is restricted because of the potential damage to normal brain tissue; the blood-brain barrier (BBB) blocks most chemotherapy drugs; and glioma cells develop primary and acquired resistance to chemotherapy. Therefore, the development of new therapies is urgently needed.
Angiogenesis, the formation of new blood vessels, plays a critical role in the growth and spread of cancer. Antiangiogenic therapy that primarily targets vascular endothelial growth factor (VEGF) has been an efficient therapeutic strategy in treating non–small cell lung, colorectal, renal, and ovarian cancers. Glioma is among the most vascularized tumors in humans. Recent studies show that bevacizumab, a monoclonal antibody against VEGF, increased progression-free survival (PFS) but not overall survival (OS) in newly diagnosed GBM, which may indicate some initial therapeutic efficacy that did not translate to long-term outcomes. In contrast, targeted molecular therapy has recently achieved remarkable success in various cancer types, including non–small cell lung cancer, breast cancer, and leukemia. Recent advances in identifying oncogenic signal pathways and deciphering metabolic, genomic, and epigenetic regulation in glioma cells have provided deep insights into the molecular pathogenesis of the malignancy, and more importantly, have shed light on the development of new targeted therapies in patients with glioma. This chapter discusses the potential targets of glioma therapy and their clinical efficacy, the potential therapeutic barriers, and the new direction and promise, with a focus on antiangiogenic and targeted molecular therapies.
Antiangiogenic therapy
Therapeutic Targets and Treatment Efficacy
Angiogenesis proceeds by endothelial cell (EC) sprouting and outgrowth from existing vessels. This process is subjected to spatiotemporal regulation: triggered by binding of angiogenic factors to their receptors and executed by sequent activation of downstream signal pathways. These signaling events eventually induce Rho GTPase–mediated and phosphatidylinositol 3-kinase (PI3K)–mediated cell migration and invasion, and lead to genetic and metabolic reprogramming to promote cell growth and proliferation. These ligands, receptors, kinases, and transcriptional factors in the regulatory network can serve as potential targets for antiangiogenic therapy ( Fig. 5.1 ).
Angiogenic factors
The most widely preferred approach for antiangiogenesis currently is the blockade of the pathways of angiogenic factors, including VEGF, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor/scatter factor (HGF/SF), and angiopoietins. Several of these factors are described here.
Vascular endothelial growth factor
VEGF and its receptor VEGFR2 have served as the primary therapeutic targets for antiangiogenic therapy in last 3 decades. Bevacizumab, the most widely used humanized VEGF antibody, has been approved for treating metastatic colorectal carcinoma, non–small cell lung cancer, metastatic renal cell carcinoma, breast cancer (in the European Union [EU]), ovarian cancer (in the EU), and recurrent GBM. Ranibizumab is another neutralizing antibody against VEGF, with a similar binding affinity. Bevacizumab, used as monotherapy or in combination with chemotherapy, slightly improved PFS but not OS in most clinical trials including patients with GBM. Ziv-aflibercept is a recombinant fusion protein consisting of VEGF-binding portions from the extracellular domains of VEGF1/2, therefore binding to circulating VEGF like a trap. Ziv-aflibercept has approximately 100-fold higher affinity than either bevacizumab or ranibizumab, showing markedly more potent blockade of VEGFR-1 or VEGFR-2 activation. Clinical trials showed that Ziv-aflibercept plus FOLFIRI (folinic acid, fluorouracil and irinotecan) improved PFS and OS in metastatic colorectal carcinoma with a median OS of 13.5 months, compared with a median OS of 12.06 months with FOLFIRI alone. The efficacy of Ziv-aflibercept needs further evaluation in patients with glioma.
Basic fibroblast growth factor
bFGF/fibroblast growth factor receptor (FGFR) induces angiogenesis by promoting extracellular matrix degradation, altering intercellular adhesion, enhancing cell motility, and stimulating cell growth in ECs. Pazopanib is a second-generation tyrosine kinase inhibitor that targets FGFR, VEGFR, platelet-derived growth factor receptor (PDGFR), and c-Kit. Pazopanib has been approved by the US Food and Drug Administration (FDA) for treating soft tissue sarcoma. Pazopanib did not prolong PFS but showed in situ biological activities indicated by radiographic responses in a phase II trial for patients with recurrent GBM. The combination of pazopanib and lapatinib (epidermal growth factor receptor [EGFR] inhibitor) was evaluated in a phase I/II trial, but was terminated early because of the poor 6-month PFS rate. Other clinical trials are ongoing to evaluate the combination of pazopanib with temozolomide in newly diagnosed GBM ( NCT02331498 ) and the combination of pazopanib with topotecan in recurrent GBM ( NCT01931098 ).
Platelet-derived growth factor
Aberrant activation of PDGF/PDGFR signaling is one of the hallmarks of glioma biology. Overexpression of PDGF/PDGFR has been found in glioma cells and surrounding ECs; coexpression of ligand receptor in these cells allows both autocrine and paracrine forms of activation, resulting in vessel formation and glioma cell migration, survival, and invasion. Both sorafenib and sunitinib are multitargeted angiogenesis inhibitors that target PDGFR, VEGFR, mast/stem cell growth factor (c-Kit) and FMS-like tyrosine kinase 3 (FLT-3). They have been approved for the treatment of multiple cancer types, including renal cell carcinoma, hepatocellular carcinoma, pancreatic neuroendocrine tumor, and gastrointestinal stromal tumors. A phase II trial of sunitinib alone in recurrent anaplastic astrocytoma and recurrent GBM failed to show significant antitumor activity; no partial responses (PRs) or complete responses (CRs) were observed in the cohort. A phase I trial of the combination of sorafenib with temozolomide and radiation therapy showed that sorafenib was well tolerated. Combination of sorafenib and temsirolimus (mammalian target of rapamycin [mTOR] inhibitor) showed poor efficacy with a 6-month PFS rate of 0%. Further clinical trials of different combinations of sorafenib with other agents are now under study ( NCT01434602 , NCT01817751 ).
Hepatocyte growth factor
HGF is often highly expressed in GBM, which may lead to increased glioma cell invasion. HGF/c-Met also stimulates EC proliferation and migration and induces angiogenesis. Cabozantinib is an orally bioavailable inhibitor that targets HGF, c-Met, rearranged during transfection (RET), and VEGFR2. Cabozantinib has been approved for treating medullary thyroid cancer. A phase I trial of cabozantinib with temozolomide and radiotherapy in newly diagnosed patients with GBM showed that cabozantinib was well tolerated at a dose of 40 mg daily. Several phase II trials of cabozantinib in patients with malignant gliomas were completed ( NCT01068782 , NCT00704288 ), but the results have not been published yet.
Angiopoietins
Angiopoietins and their receptor Tie2 are important for angiogenesis induction. Regorafenib is an oral multikinase inhibitor that targets several protein kinases, including those involved in the regulation of tumor angiogenesis (VEGFR, Tie2, PDGFR, and FGFR) and oncogenesis (c-Kit, RET, rapidly activated fibrosarcoma 1 (RAF1), BRAF, and BRAF V600E ). Regorafenib has been approved for treating metastatic colorectal cancer and gastrointestinal stromal tumor. Large randomized clinical trials revealed that regorafenib provides a significantly improved PFS in metastatic gastrointestinal stromal tumors. Although the antitumor efficacy of regorafenib in malignant glioma cells was shown in a preclinical study, the clinical effects of regorafenib have not been validated in patients with glioma.
Antivascular agents
Thalidomide and its derivatives, lenalidomide and pomalidomide, are synthetic derivatives of glutamic acid with multiple properties, including immunomodulatory, antiinflammatory, and antiangiogenesis effects. These agents can inhibit EC proliferation, block biological functions induced by proangiogenic factors such as VEGF and bFGF, and induce antitumor activity. They have been approved for treating multiple myeloma. Clinical trials revealed that thalidomide had a limited efficacy in patients with recurrent or newly diagnosed GBM. Combinations of thalidomide with irinotecan or carmustine or conventional therapy have also failed to achieve sufficient efficacy in several phase II trials. Furthermore, phase I trials showed that lenalidomide was tolerable in both pediatric and adult patients with glioma. A phase II trial is ongoing to evaluate the antitumor effects of lenalidomide in patients with glioma ( NCT01553149 ). Pomalidomide shows effective anticancer activities in hematologic malignancies such as multiple myeloma and acute myeloid leukemia, but the efficacy of pomalidomide in malignant glioma has not been fully investigated. A phase I trial of pomalidomide in recurrent gliomas is ongoing ( NCT02415153 ). The antiangiogenic effects of these agents in gliomas needs further evaluation in clinical trials.
Potential Therapeutic Barriers
Antiangiogenic therapy, albeit initially groundbreaking, has encountered difficulties and failures in most malignant cancers. Current angiogenic therapies that mainly target VEGF pathways fail to produce a permanent response in most patients, usually showing a transient response initially with impressive radiographic responses followed by tumor regrowth and disease progression. Moreover, certain patients show no response after the antiangiogenic treatment in multiple cancer types, including GBM, suggesting both primary (intrinsic) and acquired (treatment-induced) mechanisms existing for the treatment resistance.
Primary resistance
Angiogenic pathway redundancy
There is a plethora of proangiogenic factors expressed in solid tumors inducing persistent, simultaneous activation of multiple RTK-mediated signal pathways. This abundance may explain why current therapies that target VEGF or several other angiogenic factors, such as PDGF and bFGF, individually have limited efficacy. This limited efficacy may be overcome by a combination of multiple angiogenic inhibitors. Molecular diagnosis that examines the activation panel of signal pathways specific to vascular ECs of tumor biopsy samples may further ensure the success of the combined therapies in individual patients.
Microenvironment-dependent protection
It has become increasingly recognized that the stromal cells including circulation-derived progenitor cells and myeloid cells in the tumor microenvironment contribute to the treatment resistance in ECs. CD11b + Gr1 + myeloid cells that express proangiogenic factors and other cytokines can infiltrate the tumor, which is critical for the anti-VEGF resistance in ECs. Studies show that targeting these cells by either antibody-based deletion or PI3K inhibition significantly reverses the treatment resistance in immunocompetent preclinical models of RT2 primitive neuroectodermal tumor and other murine cancers. Recent studies showed that macrophages are a critical determinant for glioma progression. These results suggest that targeting tumor-associated macrophage may serve as a promising strategy to sensitize antiangiogenic treatment in glioma.
Vascular transformation
Our recent work reveals that GBM-associated ECs undergo mesenchymal transformation to acquire fibroblast phenotypes, including enhanced cell migration and proliferation, leading to aberrant angiogenesis. This new concept is different from the previously proposed endothelial-mesenchymal transition in a breast cancer model: instead of EC generation of tumor-associated fibroblasts de novo, transformed ECs still retain vascular functions, including vessel formation and absorption of acylated low-density lipoprotein (acLDL). More importantly, transformation-induced downregulation of VEGFR2, and possibly its downstream signaling, renders the EC resistant to anti-VEGF treatment. Therefore, this newly identified mechanism may provide an explanation for primary resistance to anti-VEGF therapy and serve as an alternative target for antiangiogenic therapy in gliomas.
Acquired resistance
Compensatory activation of angiogenic pathways
Among other cancer types, GBM relapse after antiangiogenic therapy with the VEGFR inhibitor cediranib was associated with the reactivation of tumor angiogenesis, suggesting the tumor development of resistance mechanisms to evade the VEGF/VEGFR2 blockade. Compensatory upregulation of the other angiogenic factor pathways may contribute to this resistance, as indicated by increased bFGF/FGFR system in experimental tumor models and in patients with cancer. Therefore, an efficient antiangiogenic therapy may require the temporally precise targeting of multiple angiogenic pathways.
Pericyte-mediated vessel protection
Pericytes play a critical role in supporting EC function under physiologic conditions and inducing vascular abnormalities in cancer settings. Previous studies show that VEGF inhibition reduces vascularity and selectively eliminates the ECs that have no pericyte coverage, suggesting a protective role for pericytes. A further study reveals that tumor-associated pericytes secrete angiogenic factors, including VEGF, to support EC survival after antiangiogenic treatment. As such, targeting pericytes seems to enhance the efficacy of antiangiogenic therapy in a mouse model of pancreatic islet cancer, which needs to be clinically evaluated in patients with glioma.
Hypoxia-induced treatment resistance
Theoretically, antiangiogenic therapy designed to destroy the tumor vasculature can induce vascular shutdown, leading to hypoxia caused by lack of sufficient oxygen. Hypoxia stimulates tumor progression through a hypoxia-inducible factor (HIF) 1α/2α–dependent mechanism, by which HIFs transcribe multiple growth factors to enhance angiogenesis and tumor cell survival as well as metabolic enzymes to allow tumor cells to adapt to the poorly oxygenated environment. Furthermore, hypoxia promotes the stemness of cancer stem cells, which is critical for their self-renewal and survival, and contributes to cancer recurrence and tumor resistance to cytotoxicity treatment. In addition, HIF-1α is also activated in GBM-associated ECs, likely contributing to the acquired resistance in tumor ECs to antiangiogenic therapy. Thus, combination with HIF-targeted therapy may offer new opportunities for overcoming the resistance of ECs to antiangiogenic therapy and tumor cells to chemotherapy. However, the concept of antiangiogenesis-induced tumor hypoxia is still controversial. In contrast, antiangiogenesis therapy targeting VEGF and VEGFR2 alleviates hypoxia, because of its vessel normalization effects. Clinical data in patients with glioma show that anti-VEGF treatment induces transiently enhanced tumor blood perfusion and oxygenation. Note that patients with recurrent and newly diagnosed GBM who show increased tumor oxygenation response to anti-VEGF therapy survive 6 to 9 months longer than those showing no responses, suggesting that vessel normalization may induce survival benefits. This could be, at least partially, explained by the role of VEGF in vascular abnormalities. Further examination of the vessel normalization function of antiangiogenesis by using thalidomide, or its derivatives, and the inhibitors targeting other angiogenic factors in preclinical models and patients with cancer is needed.
New Direction and Promise
Endothelial metabolism
Emerging evidence indicates endothelial metabolism as a new and promising target for antiangiogenic therapy. ECs are quiescent for years but sprout to generate new vasculature (ie, angiogenesis) after receiving angiogenic stimulation; a metabolic switch seems requisite for this angiogenic activation. A recent study identified 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) as a major glycolytic enzyme in ECs, which regulates lamellipodia formation and cell migration, and is therefore critical for sprouting angiogenesis. Consistently, PFKFB3 inhibition by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one efficiently blocks inflammation-induced angiogenesis. Furthermore, peroxisome proliferator–activated receptor gamma coactivator-1 alpha (PGC-1α) inhibits notch activity in ECs, maintaining EC quiescence and inhibiting sprouting angiogenesis in diabetes. A recent study revealed that carnitine palmitoyltransferase 1 (CPT1A), a rate-limiting enzyme of fatty acid oxidation, is required for EC proliferation and vascular sprouting. These works have identified several promising metabolic targets, including PFKFB3, PGC-1α, and CPT1A, for the inhibition of sprouting angiogenesis, which may skew the vasculature toward a homeostatic, quiescent state. However, the roles of these metabolic targets in tumor angiogenesis remain largely unclear.
Vascular detransformation
Our recent work reveals endothelial-mesenchymal transformation as a driving force for vascular abnormalities and aberrant vascularization in gliomas. As proof of principle, inhibition of the mesenchymal transformation by Met deletion in ECs reduces vascularity, normalizes vessels, and sensitizes tumors to temozolomide chemotherapy in a genetically engineered murine GBM model. These results suggest that vascular detransformation may serve as a new strategy for antiangiogenesis and vessel normalization therapy. The authors expect that detransformation may not only structurally normalize tumor-associated vessels but may also recondition the abnormal tumor microenvironment, considering the increasingly recognized importance of ECs as a major source of growth factors and cytokines in the microenvironment. Therefore, vascular detransformation therapy may break the tumor resistance barrier and allow the reactivation of the host tumor immunity. Identification of the critical regulatory mechanisms is necessary for further evaluation of its therapeutic potential in preclinical models and patients with glioma.
Targeted molecular therapy
Therapeutic Targets and Treatment Efficacy
Multiple pathways have served as potential therapeutic targets for treating glioma, including EGF, PDGF, and transforming growth factor-beta (TGF-β) pathways ( Fig. 5.2 ) and developmental signal pathways ( Fig. 5.3 ).
Receptor tyrosine kinases
Receptor tyrosine kinases (RTKs) control fundamental cellular events, including cell survival, proliferation, migration, metabolism, differentiation, and apoptosis. RTKs are frequently mutated in gliomas, and the constitutively active mutations drive activation of oncogenic pathways leading to uncontrolled cell growth and tumorigenesis. Recent studies have revealed a critical role of RTKs, including EGFR, EGFR variant III (EGFR vIII), PDGFR, c-Met, and erythropoietin-producing human hepatocellular carcinoma (Eph) in glioma cell proliferation and invasion. Among these RTKs, EGFR, EGFR vIII, and platelet-derived growth factor receptor (PDGFR)-A are frequently activated in patients with glioma: EGFR gene amplification occurs in about 40% of patients with GBM and the EGFR vIII mutant is found in approximately 30% to 50% of these EGFR-amplified gliomas ; PDGFRA gene amplification is observed in about 16% patients with GBM. These mutation-mediated activations of RTKs induce the recruitment of PI3K and rat sarcoma (RAS) to the cell membrane, triggering downstream pro-oncogenic signal transduction cascades, eventually leading to cell malignancy. The Cancer Genome Atlas (TCGA) data reveal that up to 88% of patients with GBM harbor genetic mutations of the RTK/RAS/PI3K pathway. These RTKs therefore represent promising targets for antineoplastic treatment in glioma (see Fig. 5.2 ).
Epidermal growth factor receptor
Extensive studies have shown that activated EGFR and mutant EGFR vIII promote pro-oncogenic signal transduction and induce tumor progression. EGFR-targeted therapies have proved effective in other cancers, including lung cancer. The effectiveness of the anti-EGFR agents has recently been evaluated in gliomas, both preclinically and clinically. (1) Gefitinib is the first EGFR inhibitor that was tested in recurrent GBM. In a phase II trial, 55 patients with GBM were recruited for treatment with gefitinib. The results show a median PFS of only 8.1 weeks and a median OS of 39 weeks. There was no significant improvement in the clinical outcomes compared with the historical control data. (2) Erlotinib is another orally available EGFR inhibitor that shows good permeability across the BBB. In several phase II trials, patients with recurrent GBM received erlotinib treatment. These patients had a PFS of 6 months (3%–20%) and an OS of 6 to 8.6 months. No significant survival benefits were observed. Although the clinical efficacy of these EGFR inhibitors was limited, other pharmacologic inhibitors, including lapatinib, are under evaluation in patients with GBM in ongoing clinical trials ( NCT00727506 ; NCT00977431 ). (3) Cetuximab is a chimeric monoclonal antibody against EGFR. An open-label phase II trial showed that, in 55 patients with recurrent GBM treated with cetuximab, those patients with EGFR gene amplification tended to have a longer OS, but the difference was insignificant. (4) Nimotuzumab is an irreversible EGFR antibody. A randomized controlled study revealed that patients with malignant glioma who received nimotuzumab in addition to radiation and chemotherapy had a significantly longer OS of 16.5 months compared with 10.5 months in the control group. These data suggest for the possibility of antibody neutralization treatment of EGFR in gliomas.
Platelet-derived growth factor receptor
The amplified or mutated PDGFR induces activation of the PI3K/mTOR and RAS/MAPK pathways, therefore serving as an important therapeutic target in glioma. Imatinib is a well-known small-molecule inhibitor of PDGFR, c-KIT, and breakpoint cluster region fused with Abl1 (BCR-ABL). Imatinib has been successfully used to treat various cell types, including chronic myeloid lymphoma. However, the results of trials for GBM have not been promising. A multicenter, randomized, phase III clinical trial showed that combined treatment with imatinib and hydroxyurea in patients with recurrent GBM only achieved a limited 6-month PFS rate (5%) and median OS (5.3 months), and had no survival benefit compared with those treated with hydroxyurea alone. Another PDGFR-targeted agent named dasatinib, which also inhibits c-KIT, ABL, and Src family kinase, showed no significant effect in monotherapy or combined with lomustine. The combinations of dasatinib with standard chemoradiotherapy or bevacizumab are being evaluated in ongoing clinical trials ( NCT00869401 and NCT00892177 ).
Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway
The PI3K/Akt/mTOR signal pathway is critical for cell survival, growth, migration, and metabolism, which is aberrantly activated in most patients with GBM, because of the activation of upstream RTKs and/or a loss-of-function mutation of phosphatase and tensin homolog (PTEN) and phosphatase. Activated PI3K pathway is associated with increased proliferation and survival in glioma cells.
Phosphatidylinositol 3-kinase
Buparlisib is a pan-PI3K inhibitor. Monotherapy with buparlisib showed no significant benefit in a phase II clinical trial in patients with recurrent GBM. However, the efficacy of the combinations of buparlisib with bevacizumab or carboplatin plus lomustine are currently under investigation in clinical trials ( NCT01349660 , NCT01934361 ). PX-866 is a semisynthetic derivative of wortmannin, which can irreversibly inhibit PI3K. In a phase II trial with PX-866, 33 patients with recurrent GBM showed that PX-866 was well tolerated, and the 6-month PFS rate is 17%. Although 28% of patients showed stable disease (SD), the outcome was modest.
Mammalian target of rapamycin
mTOR is a major downstream target of the PI3K pathway. Multiple mTOR inhibitors, including temsirolimus, sirolimus, everolimus, and ridaforolimus, have been investigated in several trials. Although these agents were well tolerated by patients, monotherapies failed to show any clinical benefit in patients with malignant glioma. There are several novel inhibitors, including INK128, CC115, and CC223, that can inhibit both mammalian target of rapamycin complex 1 (mTORC1) and mTORC2, showing great potential in preclinical studies. Clinical trials with these agents are currently in progress ( NCT02133183 , NCT01353625 , and NCT01177397 ). Furthermore, recent studies indicated that novel inhibitors, including NVP-BEZ235 and XL765, which dually target PI3K and mTOR, displayed robust antitumor activities in glioma cells, glioma stem cells, and animal models, but further clinical investigation is needed of their efficacy in patients with glioma.
Rat sarcoma/rapidly activated fibrosarcoma/mitogen-activated erk kinase/extracellular signal-regulated kinase pathway
Rat sarcoma (RAS) belongs to a class of small GTPases. RAS is usually activated by RTKs and/or by the inactivation of neurofibromatosis 1 (NF1), followed by the downstream signal transduction of rapidly activated fibrosarcoma (RAF), mitogen-activated erk kinase (MEK), and extracellular signal-regulated kinase (ERK). Farnesyl transferase (FT) posttranslationally modifies RAS proteins to activate their downstream signal transduction, serving as an important therapeutic target. Tipifarnib is an FT inhibitor. In a phase II trial with 67 patients with recurrent GBM treated with tipifarnib, only 7% of patients had a radiographic response and the 6-month PFS was 9%. Similar results were observed with another FT inhibitor, lonafarnib. A phase Ib trial with 34 patients with recurrent GBM treated with lonafarnib combined with temozolomide showed that the CR and PR rate was 24% and the 6-month PFS was 38%.
Transforming growth factor-beta pathway
The TGF-β pathway is essential for glioma cell proliferation, invasion, differentiation, and survival. Trabedersen (AP12009) is a synthetic antisense phosphorothioate oligodeoxynucleotide complementary to the messenger RNA of the human TGF-β2 gene. In a phase IIb trial, 145 patients with recurrent/refractory GBM or anaplastic glioma were treated with trabedersen or standard chemotherapy. Patients treated with 2.48mg/cycle trabedersen had significantly better 14-month survival rates compared with other groups. Galunisertib (LY2157299) is a novel kinase inhibitor of TGF-β1. A first-in-human dose study showed that galunisertib was well tolerated and no adverse cardiac events were observed. Seven of 56 patients with malignant glioma achieved CR or PR and 5 patients had stable disease (SD) greater than or equal to 6 cycles of treatment. Further clinical trials with galunisertib are ongoing ( NCT02304419 , NCT02304419 ).
Janus kinase/signal transducer and activator of transcription pathway
The Janus kinase (JAK) signal transducer and activator of transcription (STAT) signal pathway play an important role in preventing apoptosis and promoting the proliferation and the invasion of tumor cells. WP1066 is an orally bioavailable inhibitor of JAK2 with potential antineoplastic activity. A preclinical study revealed that systemic intraperitoneal administration of WP1066 inhibited the growth of subcutaneous malignant glioma xenografts in mice. A clinical trial of WP1066 is ongoing ( NCT01904123 ). LLL3 and LLL12 are STAT3 inhibitors that directly inhibit the phosphorylation of STAT3 and expression of downstream STAT3-target genes. Both agents inhibited growth of glioma cells and GBM xenografts, which needs further clinical investigation for their efficacy in patients with glioma.
Notch pathway
Notch signaling is activated via transmembrane ligands and receptors such as Delta-like ligand 1 (DLL1), DLL3, and DLL4, followed by the release of the intracellular domains into the nucleus and transcription activation in a gamma-secretase–dependent manner. The Notch pathway is critical for maintenance of stemness properties in cancer stem cells and tumor angiogenesis. RO4929097 is a gamma-secretase inhibitor. Preclinical data showed that RO4929097 had limited effects as a single agent, but enhanced efficacy was observed when combined with DNA-interfering agents in GBM xenografts. Clinical trials designed to investigate the clinical efficacy of RO4929097 as monotherapy or combined therapy with chemoradiation in patients with glioma are underway ( NCT01269411 , NCT01122901 , and NCT01119599 ).
Hedgehog pathway
The hedgehog (HH) ligands bind to the patched (PTCH) transmembrane receptors to release Smoothened (SMO), leading to the activation of transcriptional factors, including glioma-associated oncogene homologue (GLI) 1 and GLI2. Most of the target genes, such as GLI1, GLI2, and PTCH1, are essential for self-renewal and the survival of cancer stem cells. Vismodegib is a Hedgehog inhibitor that blocks the activities of the Hedgehog-ligand cell surface receptors PTCH and/or SMO. Vismodegib was proved to be clinically effective in patients with Recurrent Sonic Hedgehog–subgroup medulloblastoma. The clinical trial with vismodegib in treating patients with recurrent GBM is ongoing ( NCT00980343 ).
WNT/beta-catenin pathway
WNT/beta-catenin is the canonical WNT pathway. Translocation of beta-catenin induces activation of beta-catenin–T cell–specific transcription factor (TCF)/lymphoid enhancer–binding factor (LEF) complex, which is vital for tumor development, progression, and invasion. WNT/beta-catenin is important for stemness functions in glioma cancer stem cells. Several small-molecule inhibitors of this pathway, including PNU 7465431 and 2,4-diamino-quinazoline, have recently been identified by high-throughput screening, but their antitumor activities in gliomas remain unclear.
Isocitrate dehydrogenase 1/2
Tumor sequencing studies have identified high frequencies of isocitrate dehydrogenase 1 (IDH1)/IDH2 mutation in most secondary GBMs and approximately 60% to 70% of grade II and grade III gliomas. Wild-type (IDH mutant negative) promotes oncogenic activity. Mutated IDH1 (mIDH1) was identified in 46% of the patients and was significantly correlated to a good survival in both univariate (HR 0.24, 95% CI 0.11 – 0.53) and in multivariate analysis (HR 0.40, 95% CI 0.17 – 0.91). AGI-5198 is a novel inhibitor of the IDH1 mutant, which selectively inhibits IDH-1 R132H and depletes 2-HG production. A recent study showed that AGI-5198 reduced glioma cell growth in vitro. A similar effect on glioma cells was induced by AG 120, another IDH inhibitor. The clinical trial with AG 120 in IDH1-mutated multiple solid tumors, including glioma, is ongoing ( NCT02073994 ).
Proteasome
The ubiquitin proteasome system is an essential metabolic constituent that controls intracellular protein concentrations and tightly regulates multiple cellular functions, including cell growth, survival, and metabolism and the cell cycle. Proteasome inhibition has proved effective in myeloma. Bortezomib is a proteasome inhibitor that has been evaluated in malignant gliomas. A phase I trial of bortezomib showed limited benefit in survival time (6 months median OS) in patients with recurrent high-grade gliomas. Another phase I trial of bortezomib combined with temozolomide and radiotherapy showed that patients with newly diagnosed GBM treated with bortezomib had a median OS of 16.9 months, slightly longer than the historical control (14.4 months). More phase II studies are needed to evaluate the therapeutic effect of proteasome inhibitors.
Histone deacetylase
Aberrant epigenetic function leads to altered gene expression and malignant cellular transformation, contributing to cancer development and progression. Central to epigenetic regulation is the histone acetylation mainly controlled by histone deacetylases (HDACs) and acetyltransferases. Inhibition of HDAC seems to reduce cell division and promote cancer cell apoptosis. The FDA has approved 2 HDAC inhibitors, vorinostat and romidepsin, as anticancer agents. Phase I trials of vorinostat combined with temozolomide or chemoradiation showed that vorinostat was well tolerated. A phase II trial evaluated the monotherapy of vorinostat in patients with recurrent GBM and showed modest activity with a median OS of 5.7 months. A phase II trial of combined therapy in newly diagnosed GBM is ongoing ( NCT00731731 ).
Potential Therapeutic Barriers
Despite recent breakthroughs in glioma biology, most of the current targeted molecular therapies for malignant gliomas show only poor to modest therapeutic effects in clinical trials. There are several therapeutic barriers that limit their clinical efficacy.
Treatment-resistant glioma cancer stem cells
Cancer stem cells (CSCs), also known as tumor-initiating cells or tumor-propagating cells, are highly tumorigenic and able to differentiate asymmetrically to orchestrate a heterogeneous tumor mass. Importantly, CSCs are refractory to radiation and chemotherapy, and therefore contribute significantly to therapy resistance and tumor relapse. Recent studies have identified a prominent population of CSCs in brain tumors, including GBM, which are pluripotent and radioresistant and have the ability to repopulate tumors. Radiation induces robust enrichment of the CD133 + CSC population in human GBM and mouse xenograft tumors. Multiple mechanisms generally contribute to the treatment resistance in CSCs, including cell dormancy, increased drug efflux and detoxification, activation of antiapoptotic signal pathways, and enhanced activities for DNA repair.
Intratumoral heterogeneity
Gliomas, including GBM, are well characterized by a high degree of intratumoral heterogeneity, which contributes to the tumor resistance to molecular targeted therapies. Malignant gliomas consist of cells with different phenotypes, genotypes, and epigenetic states. According to the TCGA gene expression data, GBM has been classified into 4 molecular subtypes: proneural, neural, classic, and mesenchymal. Although each subtype shows a different mutated gene expression pattern, single-cell RNA sequencing revealed that the established GBM subtype classifiers are variably expressed across individual cells within a single tumor. Coamplification of 3 RTKs (EGFR, MET, PDGFRA) was observed in different cells of 34 GBM samples (7.3% TCGA GBM). This may contribute significantly to the resistance to monotargeted therapy because other RTKs could compensate by maintaining the pathway activation when only 1 RTK is inhibited. The complex intratumoral heterogeneity may be the major reason for the failure of most targeted therapies in malignant gliomas.
Signaling pathway redundancy
Redundancy of multiple signaling pathways may be another possible explanation for the limited efficacy of the targeted agents. Major pathways identified by TCGA data, including RTK/RAS/PI3K, p53, and Rb, have complicated interplay networks. Crosstalk and feedback loops were also found in the aforementioned pathways. A single inhibitor may not be able to suppress the signal transduction of the pathway because of the interactive network.
Blood-brain barrier
The BBB is an anatomic and biochemical barrier that protects the brain from potentially harmful substances. The BBB ECs are characterized by the absence of fenestrations, more extensive tight junctions, and sparse pinocytic vesicular transport. EC tight junctions limit the paracellular flux of hydrophilic molecules across the BBB. Although disruption of the BBB can be found in most patients with GBM, there are still regions of the tumor with an intact BBB. Most lipophilic small-molecule inhibitors are thought to be able to penetrate the BBB by passive diffusion, but tight junctions hold the ECs together on the BBB and limit the delivery of targeted agents into the tumor.
New Direction and Promise
New molecular targets
Discovery of new therapeutic targets will be crucial for next-generation therapies in gliomas. For example, the FGFR-ATCC fusion gene, expressed in about 3% of GBM, promotes tumor progression. An FGFR inhibitor significantly prolonged the survival of mice harboring intracranial GBM xenografts. BGJ398, a pan FGFR inhibitor, is under evaluation in a phase II clinical trial for patients with FGFR-ATCC + recurrent GBM ( NCT01975701 ). Importantly, the development of new therapies that are effective at eradicating glioma CSCs is urgently needed. The combination of traditional cytotoxic chemotherapy and inhibition of the aforementioned developmental signal pathways (see Fig. 5.3 ) may offer opportunities to eliminate both CSCs and glioma cells. Recent studies have revealed several potential therapeutic targets for CSC-focused treatment, including BMP/Gremlin1, Ephrins, iNOS, iron transporter, MELK, TGF-β, transcription factors Ascl1 and Myc, and the epigenetic modifier MLL. Therapeutic strategies that target these molecules have great promise to overcome CSC-mediated treatment resistance to radiation and chemotherapy and to prevent glioma relapse.
Combined targeted molecular therapies
The development of combination antiretroviral therapy (cART) to combat acquired immune deficiency syndrome (AIDS) has been one of the most impressive achievements in medical sciences. The combination regimen of different inhibitors of nucleoside analogue reverse transcriptase successfully reduces human immunodeficiency virus infection to a manageable chronic disease. As indicated by the cART in AIDS treatment, the combination of targeted molecular therapies with other novel therapeutic modalities may represent a promising way to significantly prolong the survival of patients with glioma and reduce gliomas to chronic diseases.
Single-agent treatment
There are several pharmacologic inhibitors that can target multiple kinases or signaling pathways. Vandetanib, the dual EGFR and VEGFR inhibitor, when combined with temozolomide, has been shown to shrink glioma xenografts. However, the phase I trial showed a marginal effect of vandetanib in patients with recurrent GBM with a median OS of 6.3 months after vandetanib treatment. Both sunitinib and sorafenib target VEGFR, PDGFR, c-KIT, and FLT-3. Sunitinib failed to show a significant outcome in a phase II clinical trial, but a phase I trial of sorafenib in primary or recurrent high-grade gliomas showed a significant effect with a median OS of 18 months. Further clinical evaluation of these agents with multiple targets is needed.
Multiple-agent treatment
Combinations of different targeted agents are expected to have synergistic effects. However, phase II clinical trials of 2 combinations, gefitinib plus everolimus and erlotinib plus sirolimus, had negligible efficacy in patients with recurrent GBM, with median OS of 5.8 months and 8.5 months, respectively. Other combinations, including dasatinib plus erlotinib, vorinostat plus bortezomib, and pazopanib plus lapatinib, also showed limited clinical efficacy in different trials. A new combination of perifosine (AKT inhibitor) and temsirolimus (mTOR inhibitor) is under investigation in a phase I/II clinical trial ( NCT01051557 ). These results suggest that an appropriate combination may be crucial for a favorable outcome, which should be based on the genetic and epigenetic diagnosis of individual tumors.
Combination of multiple therapeutic modalities
Antiangiogenesis plus targeted molecular therapy
Antiangiogenesis therapy that targets VEGF/VEGFR can normalize tumor-associated blood vessels within a certain therapeutic window, which may promote vessel-based drug delivery to the tumor and therefore enhance the outcome of targeted molecular therapy. Moreover, vessel normalization can reduce intratumoral hypoxia by temporally increasing perfusion, leading to less hypoxia-dependent treatment resistance to the targeted molecular therapy. Considering the important role of tumor ECs as a niche for CSCs and glioma cells, antiangiogenic therapy may recondition the tumor microenvironment to make it less malignancy permissive for glioma cells, enhancing the efficacy of the targeted molecular therapy. Cumulatively, a significantly better clinical outcome is expected for combined antiangiogenesis plus targeted molecular therapy in patients with glioma.
Immunotherapy plus targeted molecular therapy
Recent studies have highlighted the potential of immunotherapy for glioma treatment. Cancer immunotherapy includes passive immunotherapy such as administration of antibodies or activated immune cells, and active immunotherapy that attempts to stimulate the immune system by presenting antigens in a way that triggers an immune response; both show promising results for glioma therapy in preclinical models. Monoclonal antibodies against immune modulators such as ipilimumab (CTLA-4 inhibitor) and nivolumab (PD-1 inhibitor) are currently being evaluated in a phase II clinical trial for patients with recurrent GBM ( NCT02017717 ). However, most immunotherapy targets specific antigens or certain subgroups of tumor cells. Combined targeted molecular therapy can work as a complementary treatment, when designed to kill additional off-target cells based on the molecular signature of the tumor.
Proton radiation plus targeted molecular therapy
Proton therapy is one of the newer radiation treatment modalities and, compared with conventional x-ray photon radiation, proton beams can be applied to small, precise areas with minimal lateral scattering in tissue, ensuring that little to no radiation is delivered to healthy tissue surrounding the tumor. This property makes proton therapy an excellent option for treating gliomas in order to minimize neurocognitive deficits in normal brain tissue. Clinical trials investigating the therapeutic effect of proton therapy in different grades of glioma are ongoing ( NCT02671981 and NCT01358058 ). Note that recent studies by others and our group show that proton radiation causes significantly greater cytotoxic damage in the radiation-resistant, stem cell–like tumor cells in non–small cell lung cancer and GBM than conventional photon radiation. Therefore, proton therapy may further enhance the efficacy of targeted molecular therapy by eliminating CSCs that are refractory to conventional targeted treatments, improving the survival of patients with glioma.
Improvement of drug delivery through the blood-brain barrier
Drug penetration of the BBB is a major challenge for targeted molecular therapy in gliomas. There are several potential strategies that help these agents penetrate the BBB. Focus ultrasonography can temporally open the tight conjunctions between ECs by transcranial delivery of low-frequency ultrasound waves, thereby enhancing delivery of therapeutic agents into the brain. Certain compounds, such as histamine, leukotrienes, and bradykinin, can disrupt tight junctions in BBB ECs by transiently increasing cytosolic Ca 2+ levels and inducing cytoskeleton reorganization. Furthermore, elacridar, a dual inhibitor of drug efflux transporters P-gp and ATP-binding cassette sub-family G member 2 (ABCG2), when combined with other potential targeted agents, may improve drug delivery through the BBB ECs. Together, these BBB-targeting approaches in combination with targeted molecular therapy may improve drug delivery to tumors, leading to better clinical outcomes.
Related posts:
Principles and Tenets of Radiation Treatment in Glioblastoma
Radiographic Detection and Advanced Imaging of Glioblastoma
Awake Craniotomy for Glioblastoma
Brain Plasticity and Reorganization Before, During, and After Glioma Resection
Lessons Learned: Clinical Trials and Other Interventions for Glioblastoma
Recurrent Glioblastoma
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