Molecular Genetic Analysis of Cooperative Gene Regulation in Leukemia Transformation

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biomedical Genetics, 2011.Malignant transformation is a complex process, often involving the activity of many genes within multiple pathways. Elucidating the mechanisms central to transformation remains challenging. Studies have identified hundreds of mutations associated with leukemia; however, determining whether these mutations are fundamental to leukemia transformation or simply a consequence of the process remains largely unknown. To investigate the genetic events important in leukemia transformation we employed an animal model leukemia induced by co-expression of BCR-ABL and NUP98-HOXA9 in primitive hematopoietic cells. In doing so, we identified a subset of cooperatively regulated genes (CRGs) in primary acute leukemia. These leukemia CRGs (LCRGs) were identified using genome wide transcriptional profiling and their relevance to leukemia was determined by a targeted RNAi approach. Using a custom lentiviral RNAi library screen, we showed that knockdown of expression in over 60% of the up-regulated LCRGs reduced in vivo growth of primitive leukemia. Further, the RNAi screen results were confirmed by independent validation of several genes identified for importance in leukemia. Given that the LCRG signature represented genes with many distinct cellular functions, we investigated the outcome of targeting multiple LCRGs simultaneously on leukemia survival. Using the Connectivity Map (CMAP), we identified several compounds predicted to inhibit the expression of genes in the LCRG signature. Intriguingly, treatment of both murine and human primary specimens with these compounds led to selective toxicity of leukemia cells. These data suggest the importance of the LCRGs in leukemia growth and survival both at the single gene level as well as in combination. Next, given that leukemia is generally accepted to be a cancer stem cell disorder, we investigated the role of the LCRGs in leukemia stem cell transformation with a particular focus on SerpinB2. Using a knockout mouse model of SerpinB2, we demonstrated that both LSC transformation and frequency was reduced in the absence of SerpinB2 expression, suggesting expression of SerpinB2 contributes to LSC transformation. Taken together, these results demonstrate the utility of using oncogene cooperativity as a means of identifying genes central to leukemogenesis and thereby providing a novel platform for future research toward more effective therapeutic strategies to treat leukemia