Tyrosine Kinase Inhibitors Only Partially Inhibit AKT-Dependent Signaling in Cells Expressing Oncogenic KIT, FLT3 or ABL Kinases. A Potential Model for Leukemia Cell Resistance to TKIs.

Activating mutations of the KIT, FLT3 and ABL tyrosine kinases (TKs) are found in association with hematologic malignancies including subsets of acute leukemias, systemic mastocytosis, and chronic myelogenous leukemia. TK inhibitors (TKIs) have proven clinical activity for treatment of these diseases, but responses are largely partial or incomplete. We and others have previously demonstrated that the small molecule TKIs imatinib (KIT, BCR/ABL), dasatinib (KIT, BCR/ABL), sunitinib (KIT, FLT3), and tandutinib (KIT, FLT3) are potent inhibitors of oncogenic kinases relevant to hematologic malignancies. We now report that inhibition of TK phosphorylation (a surrogate for kinase activation), potently silences STAT and MAPK1/2 signaling - but incompletely inhibits activation of the AKT pathway: Using mastocytosis, leukemia and hematopoietic cell lines with endogenous or exogenous mutant KIT (V560G, D816V), FLT3 (ITD, D835V) or BCR/ABL isoforms, oncogenic TK-dependent downstream pathways were evaluated by immunoblotting. In addition, we determined the effect of treatment with specific TKIs +/– rapamycin on cellular proliferation and the induction of apoptosis. (Auto)phosphorylation of KIT, FLT3 or BCR/ABL results in activation of downstream pathways important for cell viability and cell survival. This includes the MAP Kinase (MAPK), STAT and PI3K/AKT pathways. All tested TKIs were able to potently decrease phosphorylation of members of the MAPK1/2, STAT3 and AKT pathways in cells harboring a corresponding TKI-sensitive oncogenic kinase. Notably, TKI treatment abolished phosphorylation of AKT at the Ser-473 site, whereas the Thr-308 phospho-site remained constitutively phosphorylated. This partial inhibition of AKT activity was insufficient to completely inhibit downstream kinases, such as p70S6Kinase, a known substrate of mTOR (Target Of Rapamycin). The addition of low nanomolar doses of rapamycin completely abrogated p70S6K phosphorylation alone or in combination with TK-inhibition. To determine the biological effect of more completely inhibiting AKT-dependent signaling, we tested TKI in physiological concentrations in combination with low dosage rapamycin (5–20nM). While rapamycin had only weak anti-proliferative effects when administered as monotherapy, combining rapamycin with each TKI demonstrated a strong additive to synergistic inhibitory potency with an up to 4 fold increase of antiproliferative as well as enhanced pro-apoptotic effects when compared to TKI monotherapy. We conclude that TKI inhibition of mutant KIT, FLT3 or ABL signaling is insufficient to globally silence AKT-driven pathways that may contribute to therapy failure. The addition of inhibitors targeting the AKT pathway, such as rapamycin, may improve clinical efficacy of TKI.