FLT3-ITD is the most frequent tyrosine kinase mutation in acute myeloid leukemia 
(AML) associated with poor prognosis. We previously reported that activation of 
STAT5 confers resistance to PI3K/Akt inhibitors on the FLT3-ITD-positive AML 
cell line MV4-11 and 32D cells driven by FLT3-ITD (32D/ITD) but not by FLT3 
mutated in the tyrosine kinase domain (32D/TKD). Here, we report the involvement 
of Pim kinases expressed through STAT5 activation in acquisition of this 
resistance. The specific pan-Pim kinase inhibitor AZD1208 as well as PIM447 in 
combination with the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 
cooperatively downregulated the mTORC1/4EBP1 pathway, formation of the 
eIF4E/eIF4G complex, and Mcl-1 expression leading to activation of Bak and Bax 
to induce caspase-dependent apoptosis synergistically in these cells. These 
cooperative effects were enhanced or inhibited by knock down of mTOR or 
expression of its activated mutant, respectively. Overexpression of Mcl-1 
conferred the resistance on 32D/ITD cells to combined inhibition of the PI3K/Akt 
pathway and Pim kinases, while the Mcl-1-specific BH3 mimetic A-1210477 
conquered the resistance of MV4-11 cells to GDC-0941. Furthermore, 
overexpression of Pim-1 in 32D/TKD enhanced the mTORC1/Mcl-1 pathway and 
partially protected it from the PI3K/Akt inhibitors or the FLT3 inhibitor 
gilteritinib to confer the resistance to PI3K/Akt inhibitors. Finally, AZD1208 
and GDC-0941 cooperatively inhibited the mTORC1/Mcl-1 pathway and reduced viable 
cell numbers of primary AML cells from some FLT3-ITD positive cases. Thus, Pim 
kinases may protect the mTORC1/4EBP1/Mcl-1 pathway to confer the resistance to 
the PI3K/Akt inhibitors on FLT3-ITD cells and represent promising therapeutic 
targets.