Title: Investigation of RRM2 as a potential therapeutic target against glioblastoma Supervisors: Dr. Sven Nelander and Mr. Sathishkumar Baskaran. Department: Department of Immunology, genetics and pathology (IGP), Uppsala University   Glioblastoma (GBM) is the most malignant form of glioma and associated with high proliferation rate, necrosis and highly invasive nature. Current treatment includes tumor resection followed by combination of radiotherapy and chemotherapy with temozolomide (TMZ). Despite of combination therapy, GBM exhibits dismal prognosis and mean survival rate of patients is only 3.3 % at 2 years and 1.2 % at 3 years. Therefore, there is an increasing demand of identifying new therapeutic targets against GBM. In this project, we studied the function of RRM2 gene as a potential therapeutic target in two patients derived GBM cell lines (GC). By knocking down RRM2 using short interfering RNAs, the viability of cells and proliferation was significantly reduced in both the GC. The cause of cell death was due to induction of apoptosis by the treatment in GC. Treatment of COH29, an inhibitor of RNR, induced cell death at therapeutically relevant dose in GC. Our results indicate that RRM2 has a significant role in GBM cell growth/proliferation. More evaluation must be performed in both in-vitro and in-vivo to pursue RRM2 as a molecular therapeutic target against GBM.

Glioblastoma is the most common and aggressive type of adult brain tumour. It is currently incurable and requires more effective treatments. Tissue transglutaminase 2 (TGM2) has previously been suggested to have a role in glioblastoma. Previous studies focused on TGM2 expression and inhibition in glioblastoma cells. Here we were interested in TGM2 expression in glioblastoma-associated microglia/macrophages and in identifying the role it plays in the tumor microenvironment. Based on data from bioinformatics, cell culture experiments, immunohistochemistry and immunofluorescence on mouse samples and human samples, we have shown that glioblastoma-associated microglia/macrophages are the major source of TGM2 in the tumor microenvironment. We also identified a novel role for TGM2 in efferocytosis in glioblastoma; this suggests a role for TGM2 in the maintenance of an immunosuppressive environment in this cancer. With this, we hope that further studies will be designed to evaluate the use of TGM2 antagonists as therapeutic agents for glioblastoma.

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EGFR inhibitors, such as gefitinib, are examples of targeted anticancer drugs whose drug sensitivity is related to gene mutations that adds a pharmacogenetic [PG] dimension to any pharmacokinetic [PK] and pharmacodynamic [PD] analysis. The goal of this project was to characterize the PK/PD properties of gefitinib in tumors and then apply these results to design rational drug design regimens, and provide a foundation for future studies with EGFR inhibitors. Progressions of in vitro and in vivo studies were completed to understand the PK and PD behavior of gefitinib. In vitro cytotoxicity assays were first conducted to confirm the gefitinib sensitivity differences in a pair of human glioblastoma cell lines, LN229-wild-type EGFR and LN229-EGFRvIII mutant, an EGFR inhibitor-sensitizing mutation. Subsequent in vitro PD studies identified phosphorylated-ERK1/2 (pERK) as a common PD marker for both cell lines. To describe the most salient features of drug disposition and dynamics in the tumor, groups of mice bearing either subcutaneous LN229-wild-type EGFR or LN229-EGFRvIII mutant tumors were administered gefitinib at doses of 10 mg/kg intravenously (IV), 50 mg/kg intraarterially (IA) and 150 mg/kg orally (PO). In each group, gefitinib plasma and tumor concentrations were quantitated, as were tumoral pERK. Hybrid physiologically-based PK/PD models were developed for each tumor type, which consisted of a forcing function describing the plasma drug concentration-profile, a tumor compartment depicting drug disposition in the tumor, and a mechanistic target-response PD model characterizing pERK in the tumor. Gefitinib showed analogous PK properties in each tumor type, yet different PD characteristics consistent with the EGFR status of the tumors. Using the PK/PD model for each tumor type, simulations were done to define multiple-dose regimens for gefitinib that yielded equivalent PD profiles of pERK in each tumor type. Based on the designed PK/PD equivalent dosing regimens for each tumor type, gefitinib 150 mg/kg PO qd × 15 days and 65 mg/kg PO qd × 15 days multiple-dose studies were conducted in wild-type EGFR and EGFRvIII mutant tumor groups, respectively. In each tumor group, gefitinib plasma and tumor concentrations were measured on both day 1 and day 15, as were tumoral amounts of pERK. Different from single-dose model simulations, gefitinib showed nonlinear PK property in the wild-type tumor due to the down-regulation of membrane transporter ABCG2. Moreover, acquired resistance of tumoral pERK inhibition was observed in both tumor types. Nevertheless, gefitinib had an analogous growth suppression action in both tumor groups, supporting the equivalent PD dosing strategy. Overall, single-dose gefitinib PK/PD investigations in a pair of genetically distinct glioblastomas facilitated the development of hybrid physiologically-based PK/PD models for each tumor type, and further introduced a novel concept of PK/PD equivalent dosing regimens which could be applied in novel drug development paradigms. Preliminary multiple-dose gefitinib studies revealed more complex PK/PD characteristics that needed to be further explored. / Pharmaceutics

Glioblastoma Multiforme (GBM) is the most common and malignant form of adult brain cancer, with 95% of patients succumbing to the disease within 5 years of diagnosis. An important contributing factor to this poor prognosis is upregulation of the transmembrane protein system xc- (SXC) found on GBM cells. Approximately 50% of GBM patients have tumors with upregulated levels of SXC, and these patients experience faster disease progression than patients with tumors expressing moderate levels of SXC. SXC is a sodium-independent antiporter and is comprised of a light chain catalytic subunit (xCT) bound to a heavy chain regulatory subunit (4f2hc/CD98) via a disulfide bond. The xCT subunit is responsible for the equimolar exchange of extracellular cystine for intracellular glutamate. Clinical studies have shown areas immediately surrounding the tumor, known as the peritumoral region, reach glutamate concentrations over 100 times that of the normal brain, creating an excitotoxic environment in which neurons cannot survive. In addition to neuronal excitotoxicity, excess glutamate release has also been shown to promote GBM cell invasion, as well as contributing to the clinical presentation of seizures in patients. Moreover, cystine is a component of the antioxidant glutathione, which confers protection to the cells from alkylating therapeutics such as temozolomide (TMZ). In an effort to identify novel targets that regulate SXC function, I investigated the relationship between SXC and two signaling molecules known to promote GBM progression: CD44 and the epidermal growth factor receptor (EGFR). I experimentally manipulated the CD44-hyaluronic acid (HA) interaction and EGFR to determine if these two signaling molecules were involved in regulating SXC expression and function in two patient-derived GBM cell lines. Experimental data led me to conclude that the tumorigenic potential conferred to GBM cells by CD44 is not related to an interaction with SXC. However, I found that knocking down EGFR led to a significant reduction in SXC expression. These findings are important to the field, as combinatorial therapies become more actively pursued in clinical trials. Inhibition of EGFR may provide quality of life benefits to patients who suffer from tumor-associated epilepsy through downregulating xCT-mediated glutamate release. / Doctor of Philosophy / Glioblastoma multiforme (GBM) is an advanced and aggressive form of brain cancer. Incidence of this disease in the United States of America is approximately 3.19 per 100,000 individuals, which translates to more than 13,000 expected annual diagnoses. These tumors arise from genetic mutations that instruct cells to replicate and migrate abnormally. Despite an aggressive medical armamentarium that includes maximal surgical resection, chemotherapy, and radiation, GBM patients have an expected survival period of 12-15 months after diagnosis. Previous studies have shown that approximately 50% of GBM patients have unusually high expression levels of the System xc- (SXC) protein. SXC is a protein transporter located at the membrane of GBM cells, and facilitates the exchange of the excitatory neurotransmitter glutamate for the amino acid dimer cystine. SXC exports glutamate out of the tumor cell, where it can then bind to glutamate receptors on surrounding neurons. In the brain, the concentration of extracellular glutamate must be tightly regulated to prevent hyperexcitability of neurons, which may lead to cell death and the induction of seizures. In patients whose tumors highly express SXC, studies have shown that glutamate levels can rise to concentrations over 100 times greater than the levels seen in normal brain tissue. Additionally, glutamate has been shown to stimulate GBM cells to migrate within the brain and establish secondary tumor sites. The medical and scientific community is justifiably interested in discovering novel methods for regulating or inhibiting SXC-mediated glutamate release. While SXC inhibitors have been identified, clinical studies have determined they are not appropriate for the clinical treatment of GBM. Thus the focus of this project was to identify novel molecular regulators of SXC. To that end, I explored two signaling molecules that are known to promote GBM pathogenesis: CD44 and the epidermal growth factor receptor (EGFR). I found no evidence to support a role for CD44 in regulating SXC in GBM. However, I was able to determine, through genetic and pharmacologic manipulation of patient-derived GBM cells, that EGFR regulates SXC expression and function. The results of these experiments confirmed EGFR as a key signaling protein involved in orchestrating SXC-mediated glutamate release, and may inform future clinical studies investigating combinatorial therapies for GBM patients.

Glioblastoma is a deadly brain cancer with discouraging standard of care. New methods like convection enhanced delivery and chimeric antigen receptor T cells (CAR-T) are promising treatments that can be translated to glioblastoma. In this study, CAR-T cell flow through a hydrogel was explored in the context of in-vitro convection enhanced delivery. A culture method to create large spheroids mimicking tumors from preexisting glioblastoma stem cell lines was fabricated, a convection enhanced delivery system for in-vitro testing was designed, and characterization of the CAR-T cells using the in-vitro system took place. The spheroid culture method was successfully optimized to produce spheroids large enough to act as a sufficient tumor in little time, the in-vitro set-up successfully administered treatment, and CAR-T cells were found to increase their velocities through a medium as their injection velocity increased. It was discovered that the density of the spheroid plays a crucial role in treatment delivery, often times driving how treatment will move through the spheroid. This system can be used in the future studies to test the killing potential of CAR-T cells to a tumor in-vitro. / Master of Science / Glioblastoma is a deadly brain cancer with current treatments that are discouraging at best. New methods must be utilized to aid in patient recovery. Chimeric antigen receptor T-Cells (CAR-T) are a promising treatment that can be used in glioblastoma. In this study, CAR-T cell behavior is defined in the context of in-vitro convection enhanced delivery. A large spheroid, or sphere of cells, mimicking a tumor was created, a convection enhanced delivery system set-up for in-vitro testing was designed, and characterization of CAR-T cell behavior using the in-vitro system took place. The spheroids were successfully cultured to act as a sufficient tumor, the in-vitro set-up successfully administered treatment, and CAR-T cells were found to increase their velocities in a gel as their injection velocity increases. It was discovered that the density of the spheroid plays a crucial role in treatment delivery, often times driving how treatment will move through the spheroid. This system can be used in the future studies to test the killing potential of CAR-T cells to a tumor in-vitro.