Roles of Raf/MEK/ERK and PI3K/Akt/mTOR Pathways in Prevention of Apoptosis and Induction of Drug Resistance in Myeloid Hematopoietic Cells.

The roles of signaling and apoptotic pathways in apoptosis and drug resistance of p53 wild-type (WT) hematopoietic cells were investigated in cytokine-dependent early myeloid (FL5.12) and Flt-3 mutant AML (MOLM-13) cells. Drug resistant lines were isolated by limiting dilution in doxorubicin, a drug frequently used in AML therapy. Drug resistant FL/Doxo cells had higher levels of activated Raf/MEK/ERK and PI3K/Akt signaling than parental FL5.12 cells as 5.4-, 6.8-, and 3-fold more active MEK, ERK and Akt respectively were detected upon doxorubicin treatment whereas similar levels were detected in the absence of doxorubicin. The increased levels of activated MEK, ERK and Akt were essential for their drug resistance as MEK and mTOR inhibitors eliminated drug resistance. Introduction of dominant negative (DN) MEK1 increased drug sensitivity of FL5.12 and FL/Doxo cells 6.8-, and 5.3-fold respectively, while introduction of constitutively-active (CA) MEK1 increased resistance 2.5-, and 5.3-fold. MEK and mTOR inhibitors also synergized with doxorubicin and paclitaxel to reduce the IC₅₀ for these drugs as determined by combination indexes. Drug resistance was associated with altered induction of anti- and pro-apoptotic molecules as 5.8-fold higher levels of anti-apoptotic Mcl-1 and 14.3-, and 7.7-fold lower levels of pro-apoptotic Puma and Bok respectively were detected in resistant cells upon doxorubicin treatment. Doxorubicin-induced apoptosis was altered in resistant cells as 4-fold less caspase 3 activation and 8-fold less PARP cleavage were observed than in sensitive cells. Approximately 2.3-, and 2-fold lower levels of caspase 8 and 10 cleavage were detected in resistant than in sensitive cells indicating that there was decreased activation of the extrinsic apoptotic pathway in the resistant cells following drug treatment. Drug sensitive cells displayed 25-fold more induction of p53 when they were cultured with doxorubicin than resistant cells. DN p53 increased drug resistance of the FL5.12 and FL/Doxo cells 2.1-, and 2.3-fold. However, both cell types were sensitive to the MDM-2 antagonist Nutlin 3A. Drug resistant MOLM-13 cells displayed 5.3-fold elevated ERK signaling which was associated with resistance as treatment with Raf and MEK inhibitors eliminated their resistance. Introduction of DN-p53 into MOLM-13 cells increased their doxorubicin resistance and sensitivity to Raf but not MEK inhibitors suggesting that the endogenous mutant Flt-3 protein could also signal through other kinases including PI3K/Akt to induce resistance in presence of DN p53. Indeed, mTOR inhibitors synergized with doxorubicin to induce death. These results provide roles for Raf/MEK/ERK and PI3K/Akt pathways in drug resistance and suggest that targeting these pathways may be effective in leukemia therapy.