Molecular genetic aberrations in MDS/CMML and their progression to acute myeloid leukemia

The molecular pathogenesis is the development and progression of myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) to secondary acute myeloid leukemia (sAML) are not well defined. We have been extensively examining the known pathogenic gene mutations in patients with MDS/CMML. Systematic analyses of the genetic and epigenetic abnormalities on matched paired samples at both MDS/CMML and sAML phases will provide better understanding of the transforming events that convert MDS/CMML to sAML. New powerful technologies becoming available, can help us to fully document the genetic and epigenetic alterations in MDS/CMML. In this renewal project, our specific aims are (1) to analyze the mutation status of newly identified mutated genes including ASXL1, C-CBL and TET2 in patients with MDS and CMML, (2) to explore the oncogenic events that transform MDS/CMML to sAML, (3) to analyze comprehensively the genetic aberrations on sAML samples using whole genome methodology with single nucleotide polymorphism (SNP) array analysis and exon resequencing to search for both known and unknown pathogenic mutations at AML transformation, (4) to study genome-wide identification of hypermethylation pattern in matched paired MDS/CMML and sAML samples for individual patients in order to characterize the epigenetic changes associated with AML progression, and (5) to study the biological effect of selected target genes in leukemogenesis. Mutational analysis of ASXL1, C-CBL and TET2 genes will be performed by DNA/cDNA polymerase chain reaction (PCR) followed by direct sequencing or screened by denaturing high-performance liquid chromatography with sequencing confirmation. We will use SNP-array technology to detect the genomic alterations including microdeletion, duplication or uniparental disomy (UPD) on sAML samples. Screening for candidate genes located in the minimal affected UPD regions may identify novel mutations. We are going to do comprehensively mutational analysis using high throughout resequencing technology to selectively resequence exons. Our collaborator Dr. Ogawa’s team will prepare custum SureSelect beads to collect genomic regions of interests, e.g. common deletions or amplifications. We will use the methylated-CpG island recovery technology (MIRA) in combination with genome-wide CpG island array to identify epigenetic molecular markers. We will use bisulfite-pyrosequencing to measure the methylation levels of candidate genes selected by MIRA array. We will examine the potential of cooperating mutations of RUNX1 and N-RAS, RUNX1 and C-CBL, or C-CBL and JAK2V617F by retroviral transduction/transplantation mouse model. In this renewal project using new powerful technologies can help us to comprehensive search for genetic and epigenetic alterations in MDS/CMML and sAML. Understanding the total complement of genomic abnormalities that underlies the two phases of disease will help better characterize the transforming events. This will open new opportunities to develop new targeted treatment in the future for this dismal myeloid neoplasms.

Project ID:PG10201-0115
External Project ID:NHRI-EX102-10003NI