Simple SummaryTumor cell survival depends on the inter-connected activation of multiple pathways. Here, we show that acute myeloid leukemia cell lines benefit from an important inter-play between mTORC1-ERK and AKT, explaining why mTOR inhibition is rather ineffective in killing those cells. In fact, ERK1/2 activation, caused by the inhibition of the mTORC1-P70S6K dependent inhibitory loop, did not allow an efficient killing of AML cell lines. However, even ERK1/2 inhibition, together with that of mTORC1, caused a partial dephosphorylation of 4EBP1, a mTORC1 target, depending on AKT activation. Notably, AKT inhibition resulted in ERK1/2 activation. Thus, the concomitant inhibition of ERK1/2 and AKT caused a stronger 4EBP1 de-phosphorylation and was required to induce a more efficient cytotoxicity.AbstractmTOR is constitutively activated in acute myeloid leukemia (AML) cells, as indicated by the phosphorylation of its substrates, 4EBP1 and P70S6K. Here, we found that quercetin (Q) and rapamycin (Rap) inhibited P70S6K phosphorylation, partially dephosphorylated 4EBP1, and activated ERK1/2 in U937 and THP1, two leukemia cell lines. ERK1/2 inhibition by U0126 induced a stronger dephosphorylation of mTORC1 substrates and activated AKT. The concomitant inhibition of ERK1/2 and AKT further dephosphorylated 4EBP1 and further increased Q- or Rap-mediated cytotoxicity, compared to the single ERK1/2 or AKT inhibition in cells undergoing Q- or Rap-treatments. Moreover, quercetin or rapamycin reduced autophagy, particularly when used in combination with the ERK1/2 inhibitor, U0126. This effect was not dependent on TFEB localization in nuclei or cytoplasm or on the transcription of different autophagy genes, but did correlate with the reduction in protein translation due to a strong eIF2α-Ser51 phosphorylation. Thus, ERK1/2, by limiting 4EBP1 de-phosphorylation and eIF2α phosphorylation, behaves as a paladin of protein synthesis. Based on these findings, the combined inhibition of mTORC1, ERK1/2, and AKT should be considered in treatment of AML.