**2. Concept 1: Glioblastoma subtypes**

There is an old adage that cancer is a hundred diseases masquerading in one. While this adage is based on clinical and pathologic observations, systemic genomic characterization of a large number of glioblastoma specimens (TCGA) confirms the notion that subtypes with distinct pathologic molecular events and therapeutic response.

The Cancer Genome Atlas (TCGA) is a major NIH initiative involving institutions spanning the continental U.S. with the goal of tumor specimen collection and molecular characterization 10. Glioblastoma was one of the first tumor types characterized in this effort. This vast wealth of data is unprecedented, and despite the enormous challenge to process and analyze this incoming information, correlations of such emerging 'genetic and expression profiles' or 'tumor landscapes' with tumor biology and clinico-pathological features of the patients including therapeutic responses are beginning to impact oncology.

This profiling approach 11 has led to the understanding that glioblastoma is but an umbrella term that encapsulates subtypes characterized by distinct molecular properties. Based on global transcript profiling, glioblastoma can be divided into three to four distinct subtypes 11, 12. Interestingly, each subtype harbor distinct genetic aberrations 12 and proteomic profiles 13. The recognition that glioblastoma consists of subtypes varying in molecular circuitry and biologic behavior suggests that no therapy can be universally efficacious. The major importance of this concept of heterogeneity is that meaningful therapeutic gain can only be attained by customizing the therapy to the underlying molecular circuit.

One subtype (termed classical by the TCGA and proliferative by Philips et al) is characterized by frequent amplification or mutations in the Epidermal Growth Factor Receptor (EGFR) gene 10, 11. In contrast, in another subtype, termed proneural by both groups, harbored frequent mutations in p53, Platelet Derived Growth Factor Receptor A (PDGFRA), and Isocitrate Dehydrogenase 1 (IDH1) 12. A third type, termed mesenchymal by both groups, is characterized by frequent mutations in the Neurofibromatosis type 1 gene (NF-1). Of note, these subtypes differ in their clinical responses to therapy. Patients afflicted with the classical (proliferative) or mesenchymal subtypes benefit from radiation and temozolomide treatment 12. Such benefit was not observed in patients afflicted with the proneural subtype.

Despite some progress in the clinical management of glioblastoma, prognosis of patients suffering from this deadly tumor remains dismal, and design of new and more effective therapies for glioblastoma is highly desirable. Arguably the most promising route to discoveries of innovative treatment strategies is to obtain better mechanistic insights into glioblastoma pathogenesis and biology. Indeed, recent research in this area of experimental and clinical oncology has identified the key signaling pathways, critical regulatory nodes, genes and their protein products, as well as their mutual cross-talks, thereby providing a solid molecular basis for selection of candidate therapeutic targets and drug discovery programs. These lines of investigation complement the recent efforts to sequence entire genomes of a growing number of human tumors including glioblastoma, formulation of new concepts and principles in tumor cell biology, and potential exploitation of these major advances for personalized disease management in oncology. Collectively, such efforts have begun to provide exciting leads to conceptual framework that afford innovative therapeutic strategies. This review will aim to review these critical concepts and their relevance for

There is an old adage that cancer is a hundred diseases masquerading in one. While this adage is based on clinical and pathologic observations, systemic genomic characterization of a large number of glioblastoma specimens (TCGA) confirms the notion that subtypes with

The Cancer Genome Atlas (TCGA) is a major NIH initiative involving institutions spanning the continental U.S. with the goal of tumor specimen collection and molecular characterization 10. Glioblastoma was one of the first tumor types characterized in this effort. This vast wealth of data is unprecedented, and despite the enormous challenge to process and analyze this incoming information, correlations of such emerging 'genetic and expression profiles' or 'tumor landscapes' with tumor biology and clinico-pathological features of the patients including therapeutic responses are beginning to impact oncology. This profiling approach 11 has led to the understanding that glioblastoma is but an umbrella term that encapsulates subtypes characterized by distinct molecular properties. Based on global transcript profiling, glioblastoma can be divided into three to four distinct subtypes 11, 12. Interestingly, each subtype harbor distinct genetic aberrations 12 and proteomic profiles 13. The recognition that glioblastoma consists of subtypes varying in molecular circuitry and biologic behavior suggests that no therapy can be universally efficacious. The major importance of this concept of heterogeneity is that meaningful therapeutic gain can only be

One subtype (termed classical by the TCGA and proliferative by Philips et al) is characterized by frequent amplification or mutations in the Epidermal Growth Factor Receptor (EGFR) gene 10, 11. In contrast, in another subtype, termed proneural by both groups, harbored frequent mutations in p53, Platelet Derived Growth Factor Receptor A (PDGFRA), and Isocitrate Dehydrogenase 1 (IDH1) 12. A third type, termed mesenchymal by both groups, is characterized by frequent mutations in the Neurofibromatosis type 1 gene (NF-1). Of note, these subtypes differ in their clinical responses to therapy. Patients afflicted with the classical (proliferative) or mesenchymal subtypes benefit from radiation and temozolomide treatment 12. Such benefit was not observed in patients afflicted with the

glioblastoma therapeutic development.

proneural subtype.

**2. Concept 1: Glioblastoma subtypes** 

distinct pathologic molecular events and therapeutic response.

attained by customizing the therapy to the underlying molecular circuit.

### **3. Concept 2: Oncogene addiction**

The term "oncogene addiction" was initially coined by Dr. Bernard Weinstein to describe the phenomenon that some tumors exhibit exquisite dependence on a single oncogenic protein (or pathway) for sustaining growth and proliferation 14. Such dependence has been convincingly demonstrated in both tissue culture and transgenic mice systems for oncogenic versions of MYC 15-17 and RAS 18. Application of this concept to the clinical setting has achieved variable success in various cancer types, including chronic myelogeneous leukemia (CML) harboring the BCR-ABL translocation, Erb2 over-expression breast cancer, and Non-Small Cell Lung Cancer harboring selected EGFR mutations 19, 20. A simplistic application of this concept in glioblastoma would involve identification of the critical "addicted" oncogene followed by the inhibition of such oncogene(s). Unfortunately, the actual biology of glioblastoma is far more complex.

To understanding this complexity, a careful analysis of the fundamental notion of oncogenic addiction is needed. In some ways, the observation that tumors exhibit dependence on a particular oncogenic pathway at some point in its history is not surprising. However, taken in the context of the plethora of dynamic genetic changes that accumulated during cancer progression 21, it is somewhat anti-intuitive to suspect that any particular pathway would play a prominent role in maintaining cell viability. Moreover, inactivation of the normal counterpart of the addicted oncogenic protein is often tolerated in normal tissue. These observations suggest that the genetic circuitry of the cancer cell have been extensively reprogrammed to result in this "addicted" state 14.

The molecular nature of this re-programming remains poorly understood. Several hypotheses have been put forward. One hypothesis involves the notion of "genetic streamlining", where genetic instability in cancer cells is thought to mutationally or epigenetically inactivate certain signaling pathways that are operational in a normal cell but not required for growth in the cancer cell. In this "streamlined" state, the tumor cell becomes hyper-dependent on the oncogene driven processes 22. A more generalized form of this explanation involved the notion of synthetic lethality. Two genes are considered synthetically lethal if cells remain viable with inactivation of either gene. Simultaneous inactivation of both genes, on the other hand, results in cell death 23. It is thought that the cancer cells have accumulated mutations that are synthetically lethal with the absence of critical oncogenes. The main difference between this hypothesis and the "streamline" hypothesis is that the mutation in the former can result in a gain or loss of function, whereas the later specifically proposes a loss of function. A third hypothesis suggests that oncogenes reprogrammed the tumor cell by both pro-survival and pro-apoptotic signaling 22. With acute inactivation, the pro-survival signaling decayed faster than the pro-apoptotic signaling, resulting in tumor death.

The main reason for revisiting the framework of oncogene addiction is that mechanism by which the cells can evolve to avoid such addiction. For instance, in the context of synthetic lethality, EGFR inhibition may be cytotoxic to glioblastoma cells only in the appropriate genetic context. Indeed, therapeutic effects of EGFR inhibition were observed only in patients with tumors harboring an oncogenic form of EGFR and an intact PTEN tumor suppressor gene 24. To complicate the matter, recent studies demonstrate that glioblastomas harbor activation of multiple oncogenic Receptor Tyrosine Kinases (RTKs), such that inactivation of any single oncogene merely diverts signaling through other active oncogenes 25. In these contexts, it is evident that meaningful therapy will require simultaneous inhibition of multiple oncogenes or identification of the fitting genetic context.

Key Principles in Glioblastoma Therapy 57

that within a total population of glioblastoma cells, there appears to be a small subpopulation of cells that are highly tumorigenic (hence the term "tumor initiating cells" or "TICs") with tremendous capacity for self-renewal 32, 33. To the extent that glioblastoma tumor initiating cells share many common properties when compared to neural stem cells, it is proposed that the TICs originated from stem cells. While there are some data supporting

Protein markers to prospectively identify and isolate these putative TICs such as the transmembrane glycoprotein CD133 (prominin-1) in glioblastomas have been identified 5. However, the value of CD133 as a single marker of glioblastoma TICs remains controversial, partly because also CD133-negative glioblastoma cells could give rise to tumors in an intracranial mouse xenograft model 34-36. These uncertainties motivate an ongoing search for additional candidate TIC markers. Candidate cell surface molecules suggested in this context include the adhesion glycoprotein L1CAM 37, surface carbohydrate antigen CD15 (SSEA-1) 38, surface marker A2B5 39, and integrin 6 40. Currently, there are no generally accepted cell surface markers for defining TIC. The definition of TICs remains a functional one as defined by the ability of a tumor cell to sustain self-renewal and initiate glioblastoma

Arguably, the most important aspect of the concept of TICs is that this population appeared particularly resistant to conventional radiation and chemotherapy 32. In this context, TICs may be responsible for glioblastoma recurrence after conventional therapy. Given such properties, it is understandable that glioblastoma research has recently focused on identification and development of potential anti-TIC therapies. Two of these strategies, namely targeting the TICs as part of a vascular niche, and attempts to overcome their therapeutic resistance, will be discussed in the following sections on glioblastoma angiogenesis and the role of DNA damage response pathways, respectively. Here, we briefly consider strategies that are emerging as potentially fruitful approaches to treat

The first strategy reflects the efforts to identify suitable cell surface markers to reliably identify glioblastoma TICs – with the hope of conjugating the corresponding antibody to cytotoxic compounds as therapeutic agents. The second strategy is based on observations that some TICs, like neural stem cells, can be induced into a differentiated state whereby the self-renewal properties are lost. Among the suggested agents to induce such TIC differentiation, the bone morphogenetic proteins (BMPs) appear promising 41. The third strategy involves modulating specific signaling pathways required for maintaining the TIC state. Pathways targeted include those mediated by EGFR, Wnt-beta catenin, STAT3, Sonic Hedgehog-Gli, and Notch pathways 42. To the extent that these pathways are also regulated by miRNAs such as miR-21 43, such miRNA constitute therapeutic targets in this strategy. Finally, normal neural stem cells have been shown to migrate toward and track TICs. Based on this principle, neural stem cells have been as delivery vehicles to increase local

In this chapter, we have discussed key principles underlying current development of glioblastoma therapeutics. Emphasis was placed on conceptual framework rather than specific drugs or targets. These frameworks should serve as the basis for translating

this hypothesis 5, the universality of this hypothesis remain controversial.

formation in immuno-compromised xenograft models.

concentration of therapeutic agents in the vicinity of TICs 44.

fundamental biologic tenets into clinically useful therapeutic strategies.

glioblastoma through targeting TICs.

**6. Summary** 
