Introduction of Constitutivelly Active Akt in Primary CLL B-Cells Results in Upregulation of Mcl-1, Bcl-XL and Cyclin D3 and Promotes Leukemic Cell Survival.

The PI3K/Akt and Raf/MEK/ERK pathways are key regulators of various cellular responses, including proliferation, survival, differentiation, migration and malignant transformation. These pathways are activated in chronic lymphocytic leukemia B-cells by a number of survival or growth stimulatory signals, such as immobilized anti-IgM antibodies, interleukin-4, phorbol-ester, CXCL12, or stimulatory CpG oligonucleotides. Moreover, enhanced activation of Akt has been implicated in the pathogenesis of the CLL-like disorder that develops in mice transgenic for the TCL1 oncogene. To further delineate the relative contribution of the PI3K/Akt and Raf/MEK/ERK pathways in regulating leukemic cell growth and survival, we introduced constitutively active Akt or constitutively active MEK2 in primary CLL B-cells by nucleofection. Expression of constitutively active Akt consistently promoted survival, as evidenced by a higher percentage of Annexin V/PI negative cells after 48 hours in culture (median 52%) compared to samples transfected with control vector (median 31%). Immunoblot analysis of several important antiapoptotic proteins revealed that enforced activation of Akt upregulates Mcl-1 and Bcl-xL, whereas no changes were observed in the levels of Bcl-2. Expression of constitutively active Akt also induced an increase in size and granularity of the leukemic cells, indicating increased metabolic activity. These changes were associated with significant induction of cyclin D3, indicating that activation of Akt is required for both leukemic cell survival and cell cycle progression. In contrast, introduction of constitutively active MEK2 induced sustained activation of ERK, but showed only a modest increase in the percentage of viable CLL B-cells (median 36%) and no significant changes in the levels of any of the investigated antiapoptotic proteins. These experiments provide direct evidence that sustained activation of Akt promotes leukemic cell survival and upregulates Mcl-1, an antiapoptotic protein that has been associated with resistance to chemotherapy in patients with CLL.