INHIBITION OF PROLIFERATION AND SURVIVAL IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (T-ALL) CELLS REQUIRES BLOCKADE OF ALL THE CLASS I PI3Ks ISOFORMS

Introduction. The heterodimeric class I phosphatidylinositol 3-kinases (PI3Ks) regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3). PI3Ks are widely implicated in human cancers and in particular are upregulated in T-ALL, mainly due to the impairment of the lipid phosphatase PTEN function. At present different compounds which target single or multiple PI3K catalytic subunits have entered clinical trials. Here we have explored the role of the different PI3K isoforms in T-ALL in order to identify the most effective targeted therapy strategy. Methods. Both PTEN wt (ALLSIL and DND-41) and PTEN deleted (Jurkat and Loucy) T-ALL cell lines were treated with class I pan-PI3K inhibitors (BKM120 and ZSTK454) or p110\u3b1 (A-66), p110b (TGX-221), p110g (AS-605240), p110\u3b4 (CAL- 101) and p110g/\u3b4 (IPI-145) selective inhibitors (Selleck Chemicals, Houston TX, USA), and their effects were evaluated. Results. In all the tested cell lines, flow cytometric analysis evidenced a reduction in PIP3 levels after all treatments, documenting PI3K inhibition. However, the extent of PIP3 reduction varied in the different cell lines and did not correlate to drug cytotoxicity. Indeed, only pan-PI3K inhibition significantly affected cell viability and proliferation in a dose- and time-dependent manner, with IC50 values ranging between 1 and 3.4 \u3bcM, for both BKM120 and ZSTK454, whereas for all the other drugs IC50 values were not attained. These findings suggested that antiproliferative effects are not exclusively dependent on PIP3 reduction. Moreover, only Loucy cell line resulted sensitive to the p110g and g/\u3b4 inhibitors, suggesting that a more complex cellular background might influence cytotoxicity. Consistent with these results, Annexin V/PI staining analysis showed an increase of apoptosis after 48h treatment in both PTEN wt and deleted cell lines only after treatment with pan-PI3K inhibitors, whereas cell cycle progression was not affected. PI3K signaling pathway analysis revealed the complete inactivation of the downstream Akt pathway almost exclusively after treatment with pan-PI3K inhibitors, as evidenced by decrease of p-Akt, both at Thr308 and Ser473, p- P70S6K and p-S6RP. Of note, p110g and p110\u3b4 inhibition slightly affected these PI3K targets, but only in some cell lines. Furthermore, PI3K inhibition did not affect activity of PDK1 and PKC proteins. Overall, no differences emerged related to PTEN status. Conclusions. Here we demonstrated that, irrespective of PTEN status, only pan-class I PI3K inhibition is cytotoxic in T-ALL cells, implying that any isoform could sustain leukemic cell survival, and suggesting a redundant role played by each isoform. Therefore, our findings strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in TALL treatment