E2a-Pbx1 Direct Targets and Secondary Mutations in Leukemogenesis.

E2a-Pbx1 is expressed as a result of the t(1;19) chromosomal translocation in nearly 5% of cases of acute lymphoblastic leukemia. The E2a-Pbx1 chimeric transcription factor contains the N-terminal transactivation domain of E2A fused to the C-terminal DNAbinding homeodomain of Pbx1. Previews studies indicate that additional genetic events may be required for E2A-Pbx1-leukemic transformation, based on long incubation times and monoclonal nature of tumors observed in mouse models. While there is no doubt of its oncogenic potential, the mechanisms of E2a-Pbx1-mediated pre-B cell transformation and the nature of direct E2a-Pbx1 target genes and additional events that complement the fusion oncogene to create full-blown leukemia are still unclear. Herein we used chromatin immunoprecipitation (ChIP-chip) assays to identify direct targets of E2a-Pbx1, and we used gene and miRNA expression arrays of siRNA E2a-Pbx1-silenced cells to evaluate changes in expression induced by the fusion protein. To identify complementary genetic rearrangements, analyses of primary E2a-Pbx1 leukemias were performed to copy number changes and loss of heterozygosity which might identify mutations that synergize with the direct/functional E2a-Pbx1 targets to produce the leukemic phenotype. These arrays were analyzed in comparison to high-density gene promoter methylation arrays. Our data identified members of the WNT pathway as direct targets of E2a-Pbx1. Expression data from E2a-Pbx1 silenced cells support this finding as they demonstrate a functional regulation of this pathway. We further show a differential impact of E2a-Pbx1 silencing on the miRNA profile and identify E2a-Pbx1 dependent miRNAs. Using CGH arrays on primary E2a-Pbx1 samples we were able to pinpoint candidate secondary mutations as well as broad genetic categories: cases with 1q+, 1q+ combined with 9p-, and, separately, cases with +8. In summary we present direct and functional E2a-Pbx1 targets as well as candidate secondary mutations. We propose a model were direct and functional E2a- Pbx1 driven pathways that might include both genes and miRNAs might collaborate with identified auxiliary events to produce the E2a-Pbx1 leukemia.