An in vivo model of leukemogenesis: Analysis of signaling pathways contributing to malignant transformation

To analyze individual factors that may contribute to leukemic transformation in vivo, we have developed a murine model of leukemogenesis based on the murine early hematopoietic precursor cell FL5.12. FL5.12 cells are IL-3 dependent for growth, proliferation, and survival. Relative resistance to cell death following IL-3 withdrawal can be conferred by either overexpression of the Bcl-x_L apoptotic inhibitor, or constitutive activation of the serine/threonine kinase Akt. The ability of Bcl-x_L or a constitutively-active myristylated Akt to promote leukemic transformation of FL5.12 cells was compared in athymic nu+/nu + mice. Bcl-x_L alone could not promote leukemic transformation, but mice injected with FL5.12 cells overexpressing Bcl-x_L and a dominant negative p53 construct developed leukocytosis and blastic infiltration of lymph nodes, spleen, and liver with features of a high-grade lymphoid malignancy. In contrast to the cells injected into these animals, cell lines derived from the mice were able to proliferate in the absence of IL-3. A constitutively active Akt construct introduced into FL5.12 cells promoted similar clonal expansion in vivo, with emergence of clonal IL-3 independent proliferation. Bcl-x_L and Akt appeared to function cooperatively in this model, enhancing rapid clonal outgrowth in vivo relative to Akt alone. All four FL5.Bcl-x_L.DD clones, isolated from independent mice, were found to have constitutively-activated Akt. In two lines, this constitutive activation was associated with alterations of PTEN. Some of the leukemic clones also demonstrated concurrent constitutive upregulation of Erk activity. The upregulation of Erk in these clones appears to be MEK-dependent and not due to mutations in HRAS1, NRAS, KRAS2, SHP-2 or NF1. Activation of the P13K/Akt/mTOR and Ras/MEK/Erk pathways were shown to contribute to the IL-3-independent proliferation of all four subclones. Exposure to rapamycin resulted in a reduced proliferation in the absence of IL-3, suggesting that mTOR activation by Akt is required for maximal IL-3 independent proliferation of these lines. UO126 also inhibited proliferation in a dose-dependent manner in all clones, suggesting that MEK activation is important for maximal IL-3 independent proliferation. These results implicate activated Akt and growth factor independence in leukemogenic transformation, and demonstrate the potential for in vivo analysis of genetic determinants of leukemogenesis.*; *This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: Adobe Acrobat.