FLT3 and KIT Isoforms Activate PI3K/AKT Signaling Which Is Potently Blocked by the Novel Dual PI3K/Mtor Inhibitors NVP-BEZ235 and NVP-BGT226.

Abstract 1740

Poster Board I-766

In ∼40% of de novo cases of acute myeloid leukemia (AML), autoactivating mutations in the class III receptor tyrosine kinases (RTK) KIT and FLT3 are observed and are closely linked to leukemogenesis. We and others previously demonstrated that leukemia-driving gain-of-function mutations of KIT and FLT3 consecutively activate AKT, MAPK1/2 (ERK1/2) and STAT signalling. We show that global AKT phosphorylation can be detected in AML patients. In contrast, RTK-inhibition only partially blocks PI3K/AKT/p70S6K signaling in our in vitro leukemia models, which suggests the existence of an escape mechanism that is not yet understood. Combination of tyrosine kinase inhibitors (TKI) with rapamycin, a specific MTOR inhibitor acting upstream of p70S6K, potentiates the antitumor activity of TKI. However, we show that upregulation of AKT-phophorylation – possibly by feedback loops – is observed in western blot analysis. In an attempt to globally block AKT signaling we tested the dual PI3K/MTOR inhibitors NVP-BEZ235 and NVP-BGT226 with regard to their antiproliferative and proapoptotic potential in a variety of mutant-KIT/FLT3 leukemia cell lines. Inhibition of PI3K and MTOR phosphorylation sites in the upper nanomolar range consequently lead to dephosphorylation of the threonine 308 as well as the serine 473 AKT phospho-sites - as well as the phospho p70S5kinase phospho-sites with both tested inhibitors. Moreover, global blockage of the PI3K/AKT/MTOR/p70S6K signal transduction pathway translated into a potent antitumor activity on the cellular level - with NVP-BGT226 being the more potent agent in antiproliferation and apoptosis assays with IC50s in the nanomolar range. Moreover, we also tested the blast crisis CML cell line K562 – which also revealed sensitivity towards PI3K/MTOR inhibition in the same dose range. To study the influence of different mutant isoforms of KIT/FLT3 on sensitivity to TKI, we created FLT3, KIT and BCR/ABL transfectants in a Ba/F3 cell line background. Both inhibitors displayed the most profound activity in cells transfected with an autoactivating FLT3 or KIT isoform with IC50s between 250-500 nanomolar. In contrast, the FLT3 wildtype isoform displayed decreased sensitivity towards both inhibitors. No sensitivity to NVP-BEZ235 and NVP-BGT226 was observed in BCR/ABL transfectants and the Ba/F3 parental cell line up to 5-10 micromolar. As expected, combination of the dual PI3K/MTOR inhibitors with TKI (Imatinib, Dasatinib, and Sunitinib) potentiated the antiproliferative and proapoptotic effects observed for the single agents.

We conclude that the PI3K/AKT pathway is activated by mutant FLT3 and KIT isoforms - but not by BCR/ABL. In acute leukemias, including CML blast crisis, the PI3K/AKT pathway is further activated due to other yet unknown mechanisms. Thus, the PI3K/AKT pathway may provide an escape mechanism from TKI therapy contributing to the moderate/shorter response rates in acute forms of leukemia (including CML blast crisis) compared to TKI therapy in CML. NVP-BEZ235 and NVP-BGT226 display potent antiproliferative as well as proapoptotic effects in leukemia in vitro models alone and in combination with TKI and warrant clinical evaluation.