Browsing by Subject "Insertional Mutagenesis"

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    Computational Techniques for Analyzing Tumor DNA Data
    (2016-06) Landman, Sean
    Cancer has often been described as a disease of the genome, and understanding the underlying genetics of this complex disease opens the door to developing improved treatments and more accurate diagnoses. The abundant availability of next-generation DNA sequencing data in recent years has provided a tremendous opportunity to enhance our understanding of cancer genetics. Despite having this wealth of data available, analyzing tumor DNA data is complicated by issues such as genetic heterogeneity often found in tumor tissue samples, and the diverse and complex genetic landscape that is characteristic of tumors. Advanced computational analysis techniques are required in order to address these challenges and to deal with the enormous size and inherent complexity of tumor DNA data. The focus of this thesis is to develop novel computational techniques to analyze tumor DNA data and address several ongoing challenges in the area of cancer genomics research. These techniques are organized into three main aims or focuses. The first focus is on developing algorithms to detect patterns of co-occurring mutations associated with tumor formation in insertional mutagenesis data. Such patterns can be used to enhance our understanding of cancer genetics, as well as to identify potential targets for therapy. The second focus is on assembling personal genomic sequences from tumor DNA. Personal genomic sequences can enhance the efficacy of downstream analyses that measure gene expression or regulation, especially for tumor cells. The final focus is on estimating variant frequencies from heterogeneous tumor tissue samples. Accounting for heterogeneous variants is essential when analyzing tumor samples, as they are often the cause of therapy resistance and tumor recurrence in cancer.
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    Identification and analysis of candidate MLL-AF9 cooperating genes in acute myeloid leukemia.
    (2010-07) Bergerson, Rachel Joy
    Human patients with a t(9;11) translocation (MLL-AF9) develop acute myeloid leukemia (AML) but evidence suggests that the product of the translocation requires additional cooperating genetic events for full-blown disease to develop. A retroviral insertional mutagenesis screen was performed in mice transgenic for the Mll-AF9 fusion oncogene, which also developed myeloid leukemia with a reduced latency compared to controls. We identified 88 candidate cancer genes near common sites of proviral insertion, including Fosb and Mn1. We found elevated expression of some candidate genes in leukemic tissues that were also upregulated in human AML harboring MLL gene translocations. A functional validation of several candidate genes was performed using RNAi lentiviral vectors in vitro and BMTT assays in vivo. We found the Open Biosystems libraries were not optimized for a hematopoietic system and shRNAs were not effective in all cells lines of causing gene knockdown or phenotype change. However, we still observed a requirement of FOSB for the maintenance the human U937 myeloid leukemia cell line. We also showed MN1 cooperated with Mll-AF9 in leukemogenesis in an in vivo bone marrow viral transduction and transplantation assay. To further investigate these leukemias, we transplanted bone marrow from the infected Mll-AF9 leukemic mice into recipient animals, which also succumb to myeloid leukemia. We established four AML cell lines from the recipient bone marrow with different signaling profiles and used them to test inhibitors against molecules in the receptor tyrosine kinase (RTK) and related pathways. The inhibitors were mostly ineffective in low doses but when cells were treated with combinations of drugs, dramatic changes in cell cycle and strong inhibitory effects on intracellular signaling were observed with variability for each cell line. The best combinations in all cell lines affected more biochemical targets and caused a prolonged apoptotic induction and inhibition of cell proliferation after three days of treatment. Our model of Mll-AF9 myeloid leukemia, induced with cooperating mutations provided by MuLV, helps define the genetic alterations in genes and pathway that are important in progression of leukemia with an MLL fusion. Furthermore, cell lines created from these leukemias are a valuable preclinical tool for assessment of cellular and biological response to inhibitors and therapeutic agents in AML cells with the Mll-AF9 translocation.