Synergistically Regulated Genes and Pathways in Leukemias Induced by Co-Expression of BCR-ABL and Nup98-HoxA9.

Abstract 3473

Poster Board III-410

Leukemia arises when normal hematopoiesis is altered causing an over-production of immature blood cells. In most cases, mutations appear to occur early in hematopoietic development affecting the more primitive stem/progenitor cell compartment, generating a leukemia stem cell (LSC). The BCR-ABL translocation is a common abnormality linked to chronic myeloid leukemia (CML), predisposing patients to the acute form of the disease, blast crisis (bcCML). The genetic interplay between BCR-ABL and the secondary mutations required for acute leukemia is poorly understood. Therefore, to better define the transformation process, we adapted a strategy previously described by McMurray et al in which genes synergistically regulated by two cooperating oncogenes are identified. This approach was shown to identify key targets of cancer cell survival and potential targets for therapeutic intervention. To implement this strategy for bcCML we employed a mouse model in which primitive (lineage depleted) mouse bone marrow cells are engineered through retroviral transduction to express BCR-ABL alone or in combination with another oncogene associated with human leukemia; Nup98-HoxA9. We generated leukemic mice and isolated bone marrow cells using fluorescence activated cell sorting (FACS). Each oncogene was tagged with a distinct fluorescent marker (green fluorescent protein for BCR-ABL and yellow fluorescent protein for Nup98-HoxA9) allowing for isolation of purified cell populations. From six independent cohorts, 4 distinct primitive (lineage negative) cell populations were purified: 1) BCR-ABL⁺, 2) Nup98-HoxA9⁺, BCR-ABL⁺/Nup98/HoxA9⁺, and BCR-ABL⁻/Nup98HoxA9⁻ (i.e endogenous normal cells). Probes from each cell population were analyzed using mouse 430 Plus microarray chips from Affymetrix. Genome wide microarray analysis was performed by first comparing gene expression between non-malignant (endogenous normal) and bcCML (both oncogenes) cells providing a list of genes differentially expressed in the disease state. This gene-set was further refined by comparison to cells expressing each oncogene independently, identifying 78 genes that are synergistically regulated by both BCR-ABL and Nup98-HoxA9 in primitive leukemic cells. We term this gene set, CRGs, for cooperation response genes, the nomenclature previously established by McMurray et al. The aberrant expression of 8 specific CRGs (serpinB2, AREG, EREG, pla2g7, csf2, selp, irf8, mycn) was verified using quantitative real-time PCR, and found to be in close agreement with the gene array data. Next, pathway analysis of the CRG gene set was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Pathways identified by this analysis include activation of JAK-STAT, Hedgehog, and Erb2 signaling, as well as increased xenobiotic metabolism. Interestingly, our data set suggests that activation of Erb2 signaling by AREG and EREG acts through STAT5, thereby influencing cell survival. In addition, drug metabolism through cytochrome P450 is observed to be up-regulated in primitive leukemia cells, indicating an enhanced ability to tolerate therapeutic agents. Taken together, the analyses performed thus far have identified gene targets implicated as central to the growth of primitive leukemia cells. Going forward, we will evaluate how the 78 synergistically-regulated genes are modulated in response to various forms of therapy, and determine whether perturbation of specific genes/pathways can selectively inhibit/eradicate LSC.