Roles of Raf/MEK/ERK and PI3K/Akt/mTOR Signaling and p53 Pathways on Apoptosis, Drug Resistance and Therapeutic Sensitivity of Early Hematopoietic Precursor Cells

The interleukin-3 (IL-3) dependent FL5.12 hematopoietic precursor cell line was isolated from murine fetal liver, an important site of early hematopoiesis, by panning with an anti-AA4 antibody and culturing the cells in medium containing IL-3. The AA4 cell antigen (human analogue, Clq Receptor) is a key cell surface marker expressed on early hematopoietic stem cells along with the IL-3R (CD123) and others. Upon Hoechst-33342 staining and flow cytometric analysis, FL5.12 cells normally have approximately 0.4% of the cells present in side population (SP) which are proposed to possess “stem cell like characteristics”. Some leukemic stem cells often display drug resistance properties. FL5.12 cells are normally sensitive to chemotherapeutic drugs such as doxorubicin, however, drug resistant cells (FL/Doxo) were isolated by subcloning the cells in doxorubicin. These drug resistant cells contain approximately 2-fold more SP cells than drug sensitive FL5.12 cells. Drug resistance was associated with 6-fold increased ERK activation and 11-fold decreased p53 induction after doxorubicin treatment. Furthermore the drug resistant cells displayed decreased apoptosis (1.8 fold) and caspase 3, 8 and 10 activation (4.2, 2.3, 2 fold respectively) upon culture with doxorubicin than the parental cells. A 3-fold higher level of proteasomal degradation of p53 was observed in the drug resistant cells which was due to 8-fold higher levels of MDM2. Drug resistant FL/Doxo cells were more sensitive to proteosome inhibitors (IC₅₀ of 75 nM compared to 125 nM for parental cells). Treatment with proteosome inhibitors resulted in 12-fold and 2-fold more p53 detection in FL/Doxo and FL5.12 cells respectively. Synergistic responses to doxorubicin and proteosome inhibitor treatment were observed as the IC₅₀ for doxorubicin was reduced 16- and 5-fold respectively with FL/Doxo and FL5.12 respectively. FL/Doxo cells were also highly sensitive to mTOR inhibitors and synergistic effects were observed upon combining doxorubicin and rapamycin as the IC₅₀s decreased 40- and 6-fold for FL/Doxo and FL5.12 cells respectively, documenting the importance of the mTOR pathway in their drug resistance. Introduction of dominant negative (DN) p53 or activated MEK1 (MEK1 CA) further increased the resistance of the FL/Doxo cells to doxorubicin approximately 18- and 34-fold respectively. The DN p53 or MEK1 CA transduced cells contained 3.3- and 5.4-fold more SP positive cells than parental FL5.12 cells respectively. While both DN p53 and MEK1 CA increased drug resistance and the frequency of SP cells, this occurred by different mechanisms. DN p53 suppressed Bax induction after doxorubicin treatment and decreased the sensitivity of the cells to proteosome (1.6-fold), MEK (1.2-fold), MDM- 2 (5-fold) and mTOR (3-fold) inhibitors compared to the parental FL5.12 cells. MEK1 CA also decreased the sensitivity to proteosome inhibitors 3-fold, yet the cells remained sensitive to MEK, mTOR and MDM2 inhibitors. Thus while DN p53 or MEK1 CA both increased the frequency of SP hematopoietic “leukemia stem” like cells, they do this by different mechanisms which also alters the sensitivities of small molecule inhibitors. Mutation of p53 or activation of the Raf/MEK/ERK pathway in leukemia stem cells may render them resistant to chemotherapy as well as certain targeted therapeutic approaches.