Development of a combination strategy based on ER and oxidative stress in Acute Myeloid Leukemia

AML is a heterogeneous disease caused by different genetic aberrations that often result in the expression of oncogenic fusion or mutant proteins. At present, the only effective molecular targeted therapy for AML is based on retinoic acid (RA) and/or arsenic trioxide (ATO) in acute promyelocytic leukemia (APL), expressing the promyelocytic leukemia (PML)–retinoic acid receptor α (RARα) fusion protein. We previously showed that in the presence of RA APL cell lines and primary blasts are sensitive to amounts of ER stress not detrimental for the same cells in its absence. Furthermore, the same cells resulted highly sensitive to a combination of ER stress inducers with ATO that generates oxidative stress. Interestingly, we observed that ER stress caused increased amounts of disulphide-bound high molecular weight aggregates of PML-RARα, exacerbating the alteration of cellular proteostasis. Thus, also in other types of AML the presence of oncogenic proteins, that are easily prone to mis-folding because of their mutant structure, could render the cells particularly sensitive to low levels of ER and oxidative stress. We found that AML cell lines, bearing the MLL-AF6 fusion protein, or the fusion protein MLL-AF4 and FLT-3-ITD, are highly sensitive to the combination of drugs inducing ER and oxidative stress in the presence of RA, at doses showing no or low toxicity when used alone. In the cells undergoing ER and oxidative stress in combination, we found a strong activation of the unfolded protein response (UPR) and of the antioxidant response, as indicated by the expression of CHOP, BiP, sXBP1 and HMOX. Accordingly, the chemical chaperone 4-PBA and the antioxidant agent N-acetyl-cystenine strongly reduce the toxicity of the treatments. Furthermore, the use of inhibitors of the UPR PERK pathway suggests it could be an interesting molecular target. Importantly, the combination of ER and oxidative stress significantly reduces the colony forming capacity of primary leukemic blasts isolated from the bone marrow of FLT3-ITD positive patients, while not affecting normal hematopoietic progenitor cells. Altogether our data suggest that the combination of RA and low levels of ER and oxidative stress specifically leads to AML cell death.