Genetic and Genomic Abnormalities in Acute Myeloid Leukemia with MLL Rearrangements and Cooperating Mutations in Leukemogenesis

Background: We have recently characterized the fusion transcripts of MLL rearrangement (MLL-R) in a large cohort of de novo acute myeloid leukemia (AML) patients. Two-hit model (collaboration of class I and class II mutations) of leukemogenesis has been proposed in AML. MLL-R belongs to class II. In the ongoing study of cooperating mutations in MLL-R, we found that class I mutations occurred in 65% of AML with MLL-PTD and in 58% of MLL/t11q23. Search for other genetic aberrations in the remaining 35-40% of MLL-R AML is warranted.We also observed that 25% of MLL-AF10 were associated with K-Ras mutations. We identified a PTPN11 mutation in one patient with MLL-AF10 and a novel c-FMS mutation in another MLL-AF10 patient. The leukemogenic potential of cooperating mutations in MLL-AF10 will be investigated. Recently, overexpressions of EVI1, BAALC, and ERG genes have been described to be associated with adverse outcome in adult AML with normal karyotypes. Other investigators, in a small series, found 8 of 14 patients with MLL/t11q23 had overexpression of EVI1. However, no data of EVI1 expression on patients with MLL-PTD have been reported. The implications of BAALC and ERG expressions in patients with MLL-R remain to be defined. Hypermethylation of FHIT gene, a putative tumor suppression gene, has recently been described in adult AML. However, the methylation status and expression of FHIT in adult AML patients with MLL-R are unclear. These prompt us to conduct this study to compare the expression levels of EV11, BAALC, ERG and FHIT genes between patients with MLL-PTD and those with MLL/t11q23, as well as the methylation status of FHIT gene in these two subgroups. ManyAML patients harbor submicroscopic genetic abnormalities which can not be detected by cytogenetic analysis or FISH, and only discernible by molecular genetic techniques. Recently, a robust technology, a genome-wide analysis with single nucleotide polymorphism (SNP) chip using high resolution array based technology in concert with bioinformatics allows identification of submicroscopic deletion or duplication, loss of heterozygosity, and uniparental disomy (UPD) which can not be detected by standard methods. In this study, we will perform SNP array analysis in patients with MLL-PTD which are more hemogeneous as compared with those with MLL/t11q23 that are very heterogeneous with multiple partner genes. Purpose: The specific aims of this study are (1) to measure the expression of EVI1, BAALC, FHIT, and ERG genes in AML patients with MLL-R and to compare the expression levels between MLL-PTD and MLL/t11q23 subgroups; (2) to explore the patterns of additional genomic abnormalities in de novo AML patients with MLL-PTD; (3) to investigate the leukemogenesis of cooperating mutations in patients with MLL-AF10. 2 Materials and methods: The mononuclear cells of bone marrow samples from AML patients with MLL-R (N=140) are collected and freshly frozen at -70C or in liquid nitrogen. For aim #1: RNAs will be extracted and reverse transcribed to cDNA. The expression levels of EVI1, BAALC, FHIT and ERG will be measured by using real-time quantitative RT-PCR assay with TaqMan probe. Methylation-specific PCR of bisulfite-treated DNA will be used for FHIT methylation status determination. For aim #2: Molecular allelokaryotyping of leukemic cells carrying MLL-PTD will be performed by using Affymetrix Genome-wide Human SNP Nsp/StyAssay Kit 5.0 with a SNP-chip platform. Data analysis of deletion, duplication and UPD will be accomplished using copy-number analysis for Affymetrix GeneChips (CNAG) and allele-specific copy-number analysis using anonymous reference (AsCNAR) programs. Size, position and location of genes will be identified with UCSC Genome Browser (http://genome.ucsc.edu/). For aim #3: Leukemogenesis of cooperation of MLL-AF10 and class I mutations will be investigated in a retroviral transduction/transplantation system. Wide-type and mutants of K-Ras (Gly13His), c-FMS (Asn572Thr), and PTPN11 (Gly503Ala) will be subcloned into the retroviral vectors with hygromycin resistance. 5-FU enriched murine hematopoietic progenitor cells will be transduced by retroviruses with single mutant or in combination with MLL-AF10 and then cultured. The immortalized transduced hematopoietic cells will be injected into sublethally irradiated syngeneic mice to evaluate the leukemogenesis. Significance. The results of this study will provide information on the expression levels of EVI1, BAALC, FHIT and ERG genes in AML with MLL-R as well as the difference in the expression between AML with MLL-PTD and MLL/t11q23. The results will be incorporated into our previous mutation data to get more insight into the role of EVI1, BAALC, FHIT and ERG genes in the leukemogenesis of MLL-PTD and MLL/t11q23 AML. By molecular allelokaryotyping using SNP array analysis, we expect to explore the patterns of genomic abnormalities in patients with MLL-PTD and find candidate genes that warrant in depth investigation in the future. After accomplishment of study on leukemogenesis of cooperating mutations in a murine model, we will be able to define the latency and phenotypes in leukemia development, in the presence of single or cooperation of two mutation classes in MLL-AF10 AML. It will be a breakthrough in the understanding of leukemogenesis of cooperation of MLL-AF10 and c-FMS (Asn572Thr).

Project ID:PC9709-0459
External Project ID:NSC97-2314-B182-011-MY3