Identification and analysis of candidate MLL-AF9 cooperating genes in acute myeloid leukemia.

Abstract

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.