Comparative Effects of PP242 and Rapamycin on mTOR Signalling and NOTCH Signalling in Leukemia Cells

Aim: PP242 is a compound which inhibits both mammalian target of rapamycin complex-1 (mTORC1) and mTORC2. We examined the effects of PP242 and rapamycin on mTOR signalling and evaluated potential crosstalk with the NOTCH signalling in eight leukemia cell lines. Materials and Methods: We examined the effects of treatment with these inhibitors on cell growth and protein expression. Results: PP242 suppressed growth more potently than did rapamycin. In two cell lines poorly sensitive to PP242, PP242 failed to inhibit v-akt murine thymoma viral oncogene homolog (AKT) phosphorylation. Suppression of mTOR phosphorylation was weaker in myeloid cell lines. Rapamycin induced eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) hyper- phosphorylation in three cell lines. Phosphorylation of both isoforms (p70 and p85) of S6 kinase (S6K) was suppressed in three cell lines; only p70 was suppressed in the others. NOTCH1 expression and activation were up-regulated by PP242 in one cell line but down-regulated in another. Conclusion: PP242 is a candidate for molecular-targeted leukemia therapy, although its effects must be evaluated on a case-by-case basis. Crosstalk was found between the mTOR and NOTCH signalling pathways. Abnormal activation of the mammalian target of rapamycin (mTOR) pathway is involved in the growth of leukemia cells (1). mTOR resides in two multiprotein complexes, mTORC1 and mTORC2. The former contains a regulatory-associated protein of mTOR (RAPTOR), and its activation leads to the phosphorylation of S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The latter contains a rapamycin-insensitive companion of mTOR (RICTOR), and its activation leads to the phosphorylation of v-akt murine thymoma viral oncogene homolog (AKT) protein. In both cases, mTOR activation engenders protein synthesis and cell growth (Figure 1). Therefore, mTOR inhibitors have potential utility as drugs for molecular-targeted therapy against various malignancies, including leukemia. An allosteric inhibitor, rapamycin, generally inhibits the activity of mTORC1 by binding to the FK506-binding protein (FKBP)-rapamycin-binding domain of mTOR protein, resulting in the dissociation of mTOR from RAPTOR. A small-molecule compound, PP242, is a second- generation mTOR inhibitor which inhibits mTOR catalytic activity in the context of both mTORC1 and mTORC2 (2). In this study, we examined the effects of these inhibitors on cell growth and mTOR signalling proteins in various leukemia cell lines. NOTCH signalling is also involved in the growth of