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.
University of Minnesota Ph.D. dissertation. July 2010. Major: Microbiology, Immunology and Cancer Biology. Advisor: David Largaespada. 1 computer file (PDF); xiii, 198 pages.
Bergerson, Rachel Joy.
Identification and analysis of candidate MLL-AF9 cooperating genes in acute myeloid leukemia..
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