The mTOR inhibitor, RAD001, displays higher cytotoxicity in leukemias with hyperactivated PI3K/AKT/mTOR pathway

Acute myelogenous leukemia (AML) is a disease resulting from the clonal expansion and accumulation of hematopoietic stem cells arrested at various stages of development. B-cell Acute lymphoblastic leukemia (B-ALL) is a form of leukemia characterized by lymphoblast abnormal increase. B-ALL is the most common form of cancer in childhood with a peak of incidence at 2-5 years of age, and another peak in the elderly. The overall cure rate in children is about 80%, and about 38% to 60% of adults have long-term disease-free survival. Although the prognosis of AML and ALL has improved in the last decades, the outcome of relapsed and chemoresistant AML and ALL is still poor, especially in adults, with only a 35-40% survival at 5 years. Therefore, major efforts are being made to develop rationally targeted therapies against alterated signaling cascades that sustain AML and ALL cell proliferation, survival, and drug-resistance. A signaling pathway that is frequently upregulated in AML and ALL is the PI3K/Akt/mTOR network. To explore whether this pathway could represent a potential pharmacological target in AML and ALL, we evaluated the effects of the mTOR inhibitor, RAD001, on the HL60 AML cell line and on the SEM B-ALL cell line.RAD001decreased viability in AML and ALL cell lines, as demonstrated by MTT experiments. The values of IC50 at 24 and 48 hours in HL60 cell line was 8μM, while in SEM cell line, that displays an hyperactivation of the PI3K/Akt/mTOR pathway, the IC50 range was from 4,7 to 6,8 μM, thus showing an higher sensitivity of this cell line to this targeted therapy. As documented by Western Blotting, RAD001 showed a concentration-dependent induction of apoptosis with a relevant increment of PARP, Caspase 3, 8 and 9 cleavage in both cell lines. The ALL cell line SEM, with hyperactivated PI3K/Akt/mTOR pathway, displayed a much higher Caspase and PARP cleavage dependent apoptosis.PI3K/Akt/mTOR downstream targets were also partially dephosphorylated by RAD001, in particular GSK3β and 4E-BP1. RAD001 induced no significant changes in Akt dephosphorylation in HL60 and SEM cell lines, and this is in agreement with several reports showing no relevant changes in the phosphorylation of Akt. The cells always expressed unchanged total Akt. This indicates that PI3K/Akt/mTOR inhibition could be an attractive target to develop innovative therapeutic strategies directed towards AML and ALL leukemia cells