Differential Gene and MicroRNA Expression In a HOXA9-MEIS1 Model of Leukemia

Abstract 64

Altered expression of the HOX-MEIS axis is associated with leukemia, particularly AML, where it plays a role in increased proliferation and impaired differentiation. Particular focus has been placed on the role of HOX-MEIS in leukemia with MLL gene rearrangements and MLL-fusion induced models that represent this poor prognostic subgroup. However more recently, we and others have identified altered expression of HOX-MEIS in AML with normal karyotype (AML-NK) that accounts for the vast majority of patients with this disease. The prognostic outcome of this large subgroup of patients is varied and may depend on other molecular aberrations such as NPM-1 mutation, FLT3-ITD or CEBPB status. A murine model of leukemia using co-overexpression of HOXA9-MEIS1 was developed to investigate their role in an AML-NK setting. Expression levels of the collaborating oncogenes was limited by combination of a bicistronic ires targeting vector, low titre retroviral infection and limiting dilution assay (LDA). HOXA9 and MEIS1 expression was measured using quantitiative PCR (qPCR) and levels obtained in the mouse were similar to those observed for AML-NK patients. The resultant leukemia was transplantable leading to premature death in non-irradiated recipient mice within 30 days (10⁴ cells transplanted). Observed pathology included circulating blasts in the periphery, splenomegaly, tissue infiltration of blast cells (kidney, lung, liver) and enlarged lymph nodes, indicative of leukemia. Blast cells were identified as Kit⁺, Gr-1⁺, Mac-1⁺, CD150^lo consistent with a myeloid progenitor phenotype. To evaluate downstream events associated with the leukaemic phenotype TaqMan-based Low Density Arrays (TLDAs) and Open Arrays (Biotrove) were used to obtain gene and microRNA (mIR) expression profiles. The majority of genes assayed 699/821 were either not expressed or showed no appreciable change in expression (ΔΔC_T < 2) between the leukaemic and normal bone marrow control samples. Of those genes that showed a measurable change in expression, 111/122 assessed in the pluripotent stem cell, transcription factor and immune arrays were downregulated in the HOXA9-MEIS1 induced leukemia samples compared to control mice (n=4 per group). Individual assays were designed for a subset of candidate genes and validated against the TLDA results. Several genes identified as consistently downregulated in the leukaemic cells, including Smad1 and E2f7, were associated with negative regulation of cell proliferation using DAVID Bioinformatics Resources 6.7. Consistent increased expression of HOXA9 and MEIS1 was confirmed by individual assays as was elevated Elf5, Cd34 and Cd28 levels. mIRs regulate the expression of protein-coding genes in part through cleavage of targeted transcripts by partial or complete base pairing. A growing number of mIRs are differentially regulated in normal and malignant hematopoiesis, where increased mIR levels are associated with downregulation of target gene subsets. mIR expression profiles were compared between leukemic and control mice to investigate their association with the global downregulation of gene expression observed in the leukaemic model. Approximately 16% (48/303) of the mIRs showed measurable changes in expression levels (ΔΔC_T >2) between the two models, with >70% (34/48) demonstrating increased expression, up to 360-fold, in the leukemia samples. Candidate mIRs were further examined using the mirDB and/or miRecords target prediction databases. Several of the leukaemia-associated upreglated mIRs putatively target members of the validated subset of downregulated genes e.g. mIR-466c and mIR-181c are predicted to target Smad1 and E2F7 respectively. This study suggests that HOXA9-MEIS1 leukemogenesis occurs in part due to increased expression of a subset of mIRs that are predicted to target genes associated with inhibition of proliferation in an AML-NK model.