Revisiting CDK Inhibitors for Treatment of Glioblastoma Multiforme

Despite extensive efforts and continual progress in research and medicine, outcomes for patients with high-grade glioma remain exceptionally poor. Over the past decade, research has revealed a great deal about the complex biology behind glioma development, and has brought to light some of the major barriers preventing successful treatment. Glioblastoma multiforme (GBM) (stage 4 astrocytoma) is a highly dynamic tumour and one of the most extreme examples of intratumoural heterogeneity, making targeting with specific therapeutics an inefficient and highly unpredictable goal. The cancer stem cell hypothesis offers a new view on the possible mechanisms dictating the heterogeneous nature of this disease and contributes to our understanding of glioma resistance and recurrence. Revealing cell division characteristics of initiating cell populations within GBM may represent novel treatment targets and/or the effective repurposing of existing therapies. In this review, we discuss the potential role of targeting the cyclin-dependent kinases (CDKs) driving this specific population. We also describe developments using multi-omic approaches that may aid in stratifying patient populations for CDK inhibitor therapy

The Atypical Cell Cycle Regulator Spy1 Suppresses Differentiation of the Neuroblastoma Stem Cell Population

Neuroblastoma is an aggressive pediatric cancer originating embryonically from the neural crest. The heterogeneity of the disease, as most solid tumors, complicates diagnosis and treatment. In neuroblastoma this heterogeneity is well represented in both primary tumours and derived cell lines and has been shown to be driven by a population of stem-like tumour initiating cells. Resolving the molecular mediators driving the division of this population of cells may indicate effective therapeutic options for neuroblastoma patients. This study has determined that the atypical cyclin-like protein Spy1, recently indicated in driving symmetric division of glioma stem cells, is a critical factor in the stem-like properties of neuroblastoma tumor initiating cell populations. Spy1 activates Cyclin Dependent Kinases (CDK) in a manner that is unique from classical cyclins. Hence this discovery may represent an important opportunity to design CDK inhibitor drugs to uniquely target subpopulations of cells within these aggressive neural tumours

Novel Approach in Resolving the Mechanism Behind Brain Tumour Heterogeneity and Therapy Response

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumour with 5-year survival rates of less than 10%. Data supports that select cell populations within the tumour mass, referred to as Brain Tumour Initiating Cells (BTICs), are the drivers of GBM growth. While the true origin of these cells is debatable, physiologically these cells possess immature properties of normal neural stem cells. They are highly resistant to drug treatment, radiation and form tumours at a high rate when transplanted into mice. Adding to GBM complexity is the fact that not all the tumours are the same; most patients can be grouped into at least 3 different ‘subtypes’ of GBM using modern genetic tools. This project builds on exciting data demonstrating that a unique cell cycle regulator, Spy1 (or RINGO by other groups) is found in normal neural stem cells during brain development, yet, it controls expansion of BTICs. Understanding which specific BTIC populations within the GBM heterogeneous mass are driven by Spy1 and whether this is subtype dependent, may represent novel and effective treatment strategies. Utilizing brain tumour patient-derived cells of genetically determined GBM subtype and based on expression of well defined BTIC markers, I established a BTIC bank through application of different cell sorting techniques. This approach allows me to further dissect the role of Spy1 in those dangerous cell populations, both in vitro and in vivo, to understand how specific BTICs grow, divide, and what role they play in each of the GBM subtypes, which is the aim of this project. Primary results revealed a strong association of specific combinations of markers within distinct GBM subtypes and significant correlation of Spy1 levels with specific BTIC populations, which sets a potential direction for assessment of new therapy targets and effective treatment strategies against GBM

Novel Regulation of the Cell Cycle During Cell Fate Decisions in the Central Nervous System

Mitotically active cells at the sites of neurogenesis and primitive cells of the sympathetic nervous system are speculated to act as a source of stem-like tumor initiating cells (TICs) in neural cancers like glioma and neuroblastoma. Understanding how normal neural stem and progenitor cells regulate their growth and differentiation decisions throughout life is of high priority. Spy1 (Speedy, Spdya, RINGO) is a unique cyclin-like protein that enhances proliferation by activating the cyclin dependent kinase 2 (CDK2) and promoting the degradation of the CDK inhibitor p27 Kip1 . Spy1 levels are tightly regulated during developmental processes and Spy1 upregulation has been reported in several types of cancer including human glioma. Spy1 effectors, CDK2 and p27 Kip1 , play a regulatory role in many developmental events including neurogenesis and these effectors are aberrantly regulated in several aggressive forms of cancer. My study sought to investigate the role of Spy1 in regulating proliferation, self-renewal and differentiation processes of neural progenitors and to determine the implications of this protein in neural cancers. My work demonstrates that Spy1 is an important driver of the CD133+ brain tumour initiating cell (BTIC) population. Spy1 controls the ability of BTICs to symmetrically divide and the amplification of the SPDYA gene loci correlates with poor patient prognosis. We show that Spy1 is expressed at high levels in neurogenic regions of the adult brain and the overexpression of Spy1 significantly increases cell proliferation parameters including the number and longevity of neurospheres. These data demonstrate for the first time that the Spy1/RINGO family plays an important role in neural fate decisions and that overexpression of Spy1 is a driving factor in specific forms of neural cancer

The Role of the Microenvironmental Landscape in Brain Cancer Progression and Therapy Resistance

Glioblastoma multiforme (GBM) is a type of brain tumour that is categorized as having the highest degree of aggressiveness, invasiveness, and metastatic potential. GBM accounts for 60% of brain tumours in adults, and patient prognosis is very poor - ranging from only 14 to 15 months following diagnosis. Despite extensive chemo- and radio-therapy treatments, patients relapse. Thus, better understanding of GBM biology is crucial to the advent of novel and effective therapeutic interventions. The tumour niche, also known as the cancer stroma, is composed of the extracellular matrix and several types of recruited cells including astrocytes, fibroblasts and immune cells. Astrocytes, as well as fibroblasts, secrete diverse molecules of structural nature which were found, in other types of cancer, to contribute to the maintenance of malignant characteristics of the tumour mass. Therefore, we hypothesize that the brain tumour niche, as well as the activation of its fibroblasts and astrocytic components plays a crucial role in the aggressiveness of GBM. My project is the first to study the role and activation of normal fibroblasts and astrocytes in the progression of GBM. We will first study the content and characteristics of the fibroblast populations in sections obtained from GL261 glioma cell line-derived brain tumours, in comparison to normal brain tissue, using immunohistochemistry. We will further establish mouse normal primary astrocyte cell lines and employ commercially available mouse embryonic fibroblasts to establish co-cultures with GL261 cells in-vitro. Both monolayer and 3D culture models will be utilized to study the activation and the role of the astrocyte/fibroblast component in the control of GBM progression and therapy resistance. In summary, my project will not only contribute to a better understanding of the mechanisms regulating GBM microenvironment, but it will also identify potential novel treatment approaches

Role of Mechanical and structural properties of GBM microenvironment in tumour aggressiveness and therapy resistance

Glioblastoma Multiforme (GBM) is the most common and most aggressive type of brain cancer, accounting for 12 to 15% of all intracranial tumours. With a median survival of three to six months for patients with recurrent GBM, there is an urgent need for advancements in the study, diagnosis, and treatment of GBM. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is a method of non-invasive tumour analysis that can create a description of mechanical and fluid stress properties of a tumour. DCE-MRI has the potential to couple static mechanical descriptions of analyzed tumours to the flow and fluxes of the fluids in the tumour, thus generating a complete picture of the local stresses, pressure, and flows in and around an embedded tumour. By studying the expression of Vinculin and Hyaluronic Acid (HA), notable stress response markers, in the brain tumour sections; it is the aim of this project to establish protein signatures that correlate with DCE-MRI readings to provide a tool for more effective and detailed diagnosis of GBM. Furthermore, in order to study the tumour with its stress responses to changes in extracellular matrix pressures in a dynamic, three-dimensional setting, we employed brain tumour organoid (BTO) cultures. BTOs provide an in-vitro modelling method that more accurately represents in vivo tissue organization. Through control of microenvironmental factors which an organoid is suspended in, solid stresses can be manipulated and responses studied. As a result, the organoid model can reinforce characterization of cellular responses found when studying tumours. Preliminary results suggest that varying extracellular matrix stiffness results in quantifiable and correlated changes in invasion, aggressiveness and marker protein levels. Defying the local stress factors and their effects on tumour proliferation, aggressiveness marker expression and drug treatment response can help in designing better diagnostic tools and more effective therapeutic strategies

The Novel Role of GABAB and CXCR4 in Medulloblastoma

Medulloblastoma (MB) is the most common malignant pediatric brain tumor and it occurs in 16-25% of diagnosed cases, with a higher incidence in children aged 1 to 9 years old. The current standard of care consists of multiple stages of therapy including surgery, irradiation, and chemotherapy. However, a subset of tumors with a still devastating prognosis remains. Metabotropic receptors are G-protein coupled receptors (GPCRs) that act as second messengers. Γ-aminobutyric acid B receptors (GABAB) and C-X-C chemokine receptor type 4 (CXCR4) are metabotropic receptors that belong to the C-family of GPCRs and are activated by the neurotransmitters, γ-aminobutyrate (GABA) and stromal-cell derived factor; SDF-1 (CXCL12), respectively. GABAB receptors are heterodimers where GABA binds to a B1 subunit, and the B2 subunit is coupled to G-proteins regulating activities of the Ca2+ channels, K+ channels, and adenylyl cyclase (AC). Previous studies showed that CXCR4 is highly expressed by glial and neuronal cells in the central nervous system (CNS) and GABAergic neurons. Evidence shows that CXCR4 is overexpressed in MB and upon administration of a CXCR4 antagonist significantly decreased the cell proliferation rate in Type II MB. Evidence also proved that CXCR4 and GABAB­ can crosstalk and GABAB was found to be highly expressed in Type II – MB showing increased Ca2+ levels and protein receptor localization. Current results show that upon administration of the GABAB agonist; baclofen increased cell proliferation in Type II MB cells. Moreover, immunofluorescence showed increased levels of GABAB during mitotic division. In conclusion, by administering the antagonist; phaclofen would enhance the efficacy of chemotherapeutic treatments on MB patients by decreasing the proliferation rate of the aggressive tumors

The Role of Spy1 in Glioblastoma Multiforme Initiation and Progression

Glioblastoma Multiforme (GBM) represents the most common and aggressive form of brain tumour. The success of therapeutic approach has been hindered by the extreme heterogeneity observed not only between individual patients but also within a single tumour. It has been shown recently that certain cell populations within the tumour possess stem cell properties contributing to the heterogeneity, aggressive character and therapy resistance of GBM, a phenomenon known as cancer stem cell hypothesis. Deregulation of the cell cycle control network plays a critical role in maintaining proliferation and stem like characteristics of cancer cells in GBM. In addition, mutations and/or deletions of tumour suppressors p53 and pTEN are some of the most common features of stem like cell population of brain tumours. Speedy (Spy1) is a cyclin-like protein that has been shown to enhance cellular proliferation and stem cell self-renewal in several systems, including brain. Moreover, Spy1 has been shown to be up-regulated in GBM and elevated levels of Spy1 are indicative of poor prognosis of patient outcome in GBM. A mouse model, termed NTA-Spy1, was used in order to over-express Spy1 in the specific stem cell population in the brain. To date, NTA-Spy1 mice have shown no spontaneous tumour formation. When these cells or their controls are combined with knockdown of either p53 (shp53) or drug inhibition of pTEN (pTENi) alone, or in combination, NTA-Spy1 cells increase the rate of tumoursphere formation in soft agar cell cultures. It also has been shown that NTA-Spy1 cells in combination with shp53 or pTEN inhibitor increases gene expression of GBM cancer stem cell markers. This work will help explain the role of Spy1 in susceptibility to brain tumour initiation and progression and the importance of its over-expression in face of other aberrant. This research may provide insight into novel targeted therapies that could be designed against GBM