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.

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Macromolecular interactions play very important roles in regulation of all levels of biological processes. Aberrant macromolecular interactions often result in diseases. By applying a combination of spectroscopy, calorimetry, computation and other techniques, the protein-protein interactions in the system of the Shaw2 Kv channel and the protein-RNA interactions in West Nile virus RNA-cellular protein TIAR complex were explored. In the former system, the results shed light on the local structures of the key channel components and their potential interaction mediated by butanol, a general anesthetic. In the later studies, the binding modes of TIAR RRM2 to oligoU RNAs and West Nile virus RNAs were investigated. These findings provided insights into the basis of the specific cellular protein–viral RNA interaction and preliminary data for the development of strategies on how to interfere with virus replication

Acquired tamoxifen resistance develops in the majority of hormone responsive breast cancers and frequently involves overexpression of the PI3K/AKT axis. Here, breast cancer cells, with elevated endogenous AKT or overexpression of activated AKT exhibited tamoxifen-stimulated cell proliferation and enhanced cell motility. To gain mechanistic insight on AKT-induced endocrine resistance, gene expression profiling was performed to determine the transcripts that are differentially expressed post-tamoxifen therapy under conditions of AKT overexpression. Consistent with the biological outcome, many of these transcripts function in cell proliferation and cell motility networks and were quantitatively validated in a larger panel of breast cancer cells. Moreover, ribonucleotide reductase M2 (RRM2) was revealed as a key contributor to AKT-induced tamoxifen resistance. Inhibition of RRM2 by RNAi-mediated approaches significantly reversed the tamoxifen-resistant cell growth, inhibited cell motility, and activated pro-apoptotic pathways. In addition, treatment of tamoxifen-resistant breast cancer cells with the small molecule RRM2 inhibitor Didox significantly reduced cell growth in vitro and in vivo. To further establish a functional association between RRM2 expression and tamoxifen resistance in breast cancer cells, gain of function studies were performed by overexpressing RRM2 in MCF-7 cells. Overexpression of RRM2 profoundly reduced tamoxifen sensitivity and down-regulated ER-&agr; in otherwise tamoxifen sensitive breast cancer cells. Furthermore, breast cancer cells with high RRM2 had elevated Her-2 and EGFR expression, modulated ER-&agr; signaling and NFκB expression. These findings also indicate that it may be possible to use RRM2 as a prognostic factor in breast cancer patients under tamoxifen therapy, and can be considered a potential therapeutic target in tumors that have acquired resistance to tamoxifen. Finally, inhibition of RRM2 by drug Didox effectively eradicates the tamoxifen resistant population, revealing a potential beneficial effect of combination therapy that includes RRM2 inhibition to delay or abrogate tamoxifen resistance. In conclusion, the findings of this work delineate the important role of RRM2 in Akt induced and acquired tamoxifen resistance in breast cancer. It also provides a preclinical rationale for evaluating tamoxifen in combination with Didox for breast cancer treatment.