INVOLVEMENT AND ROLE OF THE PI3K/AKT NETWORK IN DIFFERENT TYPES OF LEUKEMIA CELLS BEFORE AND AFTER IN VITRO TREATMENT WITH CONVENTIONAL OR NEWLY DEVELOPED DRUGS

Alterations in the PI3K/Akt network play important roles in the etiology, maintenance and progression of acute leukemia. The analysis of the intracellular signaling profile of leukemia patients could serve to reveal novel molecular targets for treatment of this disease and to identify critical biomarkers for accurate and clinically relevant diagnosis and prognosis. This research is aimed by investigating the signaling PI3K/Akt pathway in acute leukemia cells. In particular, this work aims at investigating enzymatic and genetic alterations of the PI3K/Akt network in AML and ALL. It will be useful for diagnosis, underlying mechanisms of alterations occurring in leukemia development and progression. The Akt expression levels were very high in acute leukemia patients and cell lines, with very high activation levels, detected by the phosphorylation on the serine 473 residue, in ALL cells. Powerful activation of Akt and some main downstream targets can be observed in ALL cell lines. Moreover, it is known that the bad prognosis of some patients depends on the activation of some signaling pathways, and a bad prognosis is associated to patients that have higher activation of Akt (Kornblau et al 2006). The administration of drugs directly acting on PI3K and Akt (Perifosine, ErPC and PI3K/Erk inhibitor) was able to induce a strong reduction of proliferation and induce apoptosis. These drugs occurred via PI3K/Akt, as demonstrated by the weakened phosphorylation levels of the Akt downstream targets GSK3, mTOR and p21, involved in critical cell processes; treatment with Doxorubicin does not inhibits phosphorylation of downstream target and activity of Akt. Sequencing analysis on the AML and ALL cell lines and patients, could contribute to identify the molecular alterations underlying the leukemogenesis. We found one recurrent intronic transition, 2 cell lines out of 6 and all 4 patients, the IVS4a+30g. This mutation may be involved in mechanisms of splicing of Akt. Data on Akt alterations in acute leukemias are scarce in literature, thus, more detailed analyses in a wider group of patients are necessary to confirm the frequency of the identified change and its potential functional meaning. In this study we also addressed the work on the role of Akt during the initial steps of the human erythroid differentiation. We have shown that EPO treatment activates Akt in K562 erythroleukemia cells and induces the translocation of Akt active form in the nuclear compartment in a rapid and transient manner, suggesting that Akt plays a crucial role in EPO mediated erythroid differentiation. In vivo Akt phosphorylation and in vitro Akt kinase activity were up-regulated by EPO administration to K562 cells. Both phosphorylation and activity of Akt were down-regulated by the pharmacological PI3K inhibitors, Ly294002 or Wortmannin, thus showing that Akt activation is in the PI3K signal transduction axis. Immunoprecipitable nuclear Akt activity was detectable after 10 minutes and showed an increase that reached a maximum after 15-20 minutes. This enhancement was similar but delayed of 5 to 10 minutes when compared to Akt activation in whole cells and may be explained by the time required by Akt for translocating into this cell fraction, where almost all Akt results phosphorylated in the sites of activation. Although it is now clear that Akt migrates to the nucleus as the result of treatment with growth factors, thus, Akt nuclear translocation may be attributable to an activation of PI3K. It is worthy to note that, when nuclear Akt kinase activity is inhibited erythroid maturation is not taking place. This work provided evidences for the up-regulation of the PI3K/Akt axis in acute leukemias, and in particular in ALL type. Important step in this field will be the identification of the molecular events produced by Akt activation during erythroleukemia cells differentiation. These findings may help identifying novel target into the nucleus essential for differentiation and thus useful for anticancer therapy of hematological malignancies.