Targeting Tyrosine Kinase Drug Resistance Mechanisms and Metastatic Pathways in Brain Tumors

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Background: The goal of this dissertation is to improve our understanding of two important processes in carcinogenesis: First, the resistance mechanisms to tyrosine kinase inhibitors in glioblastoma (GBM). Second, the mechanisms involved in the metastatic spread of non-small cell lung cancer (NSCLC) to the brain. Malignant gliomas have a poor prognosis, as recurrence remains nearly inevitable despite aggressive treatment. Gliomas are histologically graded as I–IV, and grade IV GBM is the most common and lethal malignant glioma, with a median survival time of just over one year. Primary GBM usually displays amplification or mutation of at least one receptor tyrosine kinase, most often the epidermal growth factor receptor (EGFR). Targeting EGFR for inhibition often provides an initial tumor response in patients, but recurrence is a main limitation of these therapies because secondary mutations emerge that lead to drug resistance. In this work, we identified a receptor tyrosine kinase, proto-oncogene ROS1 fusion, that drives the resistance to therapy and leads to tumorigenesis in GBM. In parallel, the aggressive metastatic phenotype is one of the hallmarks of recurrent tumors, and its estimated that 90% of all cancer deaths arise from the metastatic spread of primary tumors. Of all the processes involved in carcinogenesis, local invasion and the formation of metastases are clinically the most relevant, but the least well understood at the molecular level. Therefore, a detailed biochemical, molecular, and functional analyses of anoikis-resistant cells may provide insight into the biology of cancer metastasis and help identify novel targets for preventing cancer dissemination. Objective: The objective of this dissertation is to improve our understanding tumorigenesis and highlight novel oncogenic pathways that drive resistance to therapy, which leads to the progression and dissemination of cancer, specifically in GBM and NSCLC. In addition, this dissertation highlights the process of the dissemination of cancer cells from the primary tumor to form metastasis and identifies therapeutically targetable mechanisms to prevent the metastatic process of cancer. This work also provides evidence for the use of circulating tumor cells as a biomarker for the metastatic spread of cancer, specifically in lung cancer. Methods and Results: To evaluate GBM-specific mechanisms of resistance to the EGFR tyrosine kinase inhibitor, EGFR-overexpressing U87 cells were treated with increasing doses of gefitinib. RNA sequencing of gefitinib-resistant cells revealed overexpression of the receptor tyrosine kinase ROS1, which was confirmed in multiple resistant clones by western blot. A cell viability assay showed gefitinib-resistant cells to be sensitive to treatment with a combination of gefitinib and a small molecule inhibitor of ROS1, which caused cell cycle arrest in S phase, followed by apoptosis. To verify that our findings were not isolated to the U87-EGFR cells, we generated gefitinibresistant 1048 cells, which were early-passage primary GBM cells with EGFR amplification and confirmed ROS1 overexpression and sensitivity to an ROS1 inhibitor. To uncover the molecular changes that govern the transition from a primary lung tumor to a secondary metastasis and specifically the mechanisms by which circulating tumor cells (CTCs) survive in circulation, we carried out whole genome sequencing of normal lung tissue, primary tumors, and the corresponding brain metastases from five patients with progressive metastatic NSCLC. We also isolated CTCs from patients with metastatic cancer and subjected them to whole genome amplification and Sanger sequencing of genes of interest. Whereas the primary tumors showed mutations in genes associated with cell adhesion and motility, brain metastases acquired mutations in adaptive, cyto