PI3 kinase is indispensable for oncogenic transformation by the V560D mutant of c-Kit in a kinase-independent manner

Abstract

Oncogenic mutants of c-Kit are often found in mastocytosis, gastrointestinal stromal tumors (GISTs) and acute myeloid leukemia (AML). The activation mechanism of the most commonly occurring mutation, D816V in exon 17 of c-Kit, has been well-studied while other mutations remain fairly uncharacterized in this respect. In this study, we show that the constitutive activity of the exon 11 mutant V560D is weaker than the D816V mutant. Phosphorylation of downstream signaling proteins induced by the ligand for c-Kit, stem cell factor (SCF), was stronger in cKit/V560D expressing cells than in cells expressing c-kit/D816V. Although cells expressing cKit/V560D showed increased ligand-independent proliferation and survival compared to wildtype c-Kit expressing cells, these biological effects were weaker than in c-Kit/D816V expressing cells. In contrast to cells expressing wild-type c-Kit, cells expressing c-Kit/V560D were independent of Src family kinases for downstream signaling. However, the independence of Src family kinases was not due to a Src-like kinase activity that c-Kit/D816V displayed. Point mutations that selectively block the association of PI3 kinase with c-Kit/V560D inhibited ligandindependent activation of the receptor, while inhibition of the kinase activity of PI3 kinase with pharmacological inhibitors didn't affect the kinase activity of the receptor. This suggests a lipidkinase independent key role of PI3 kinase in c-Kit/V560D-mediated oncogenic signal transduction. Thus, PI3 kinase is an attractive therapeutic target in malignancies induced by c-Kit mutations independent of its lipid kinase activity.

Germline mutations of KIT in gastrointestinal stromal tumor (GIST) and mastocytosis

“…Ligand-independent constitutive activation is considered as a cause of cell transformation induced by KIT mutants. However, recent studies suggest that KIT mutants gain extra activity in addition to the constitutive activation [ 30 ], and the activation mechanism, as well as downstream signaling pathways, are different compared to that of wild-type KIT [ 31 – 34 ].…”

Loss of PI3 kinase association improves the sensitivity of secondary mutation of KIT to Imatinib

“…It has been reported that PI3 kinase is crucial for KIT/D816V induced cell transformation [46]. Unlike that the important role of PI3 kinase relies on its lipid kinase activity in cancers, we previously found that the key role of PI3 kinase in KIT mutation mediated cell transformation is independent on the lipid kinase activity of PI3 kinase [27,28]. In this study, we further showed that the ligand-independent activation of secondary mutation of KIT relies on its association with PI3 kinase as well, and the oncogenic role of PI3 kinase in the secondary mutation of KIT mediated tumorgenesis is not dependent on the lipid kinase activity of PI3 kinase, loss of PI3 kinase increases the sensitivity of secondary mutation of KIT to Imatinib.…”

“…In contrary to that, PI3 kinase inhibitor did not affect KIT activation ( Fig. 2a, b), suggesting that the inhibition of the ligandindependent activation of secondary mutations of KIT by loss of PI3 kinase association is independent of the lipid kinase activity of PI3 kinase, which is similar to the primary mutates of KIT [27,28].…”

“…PI3 kinase association is important for the ligand-independent activation of secondary mutation of KIT independent of the lipid kinase activity of PI3 kinase PI3 kinases are a group of lipid kinases that play important roles in KIT mediated cell survival and proliferation [34][35][36]. In our previous studies, we found that loss of PI3 kinase association, but not inhibition of the lipid kinase activity of PI3 kinase, inhibits the ligand-independent activation of the often occurred KIT mutation V560D and D816V, and loss of PI3 kinase association inhibits both KIT/V560D and KIT/D816V mediated cell transformation [27,28]. In addition, it has been reported that loss of PI3 kinase association blocks GISTs tumor development in knockin mice carrying germline KIT mutant [37].…”

“…Among the downstream signaling pathways, activation of Src family kinases and PI3 kinase is crucial for KIT signaling and KIT mediated biological response such as cell survival and proliferation [26]. Oncogenic mutations of KIT identified in malignancies such as mastocytosis and GISTs can be autoactivated without ligand stimulation, and the ligand-independent activation of KIT mutations is dependent on the PI3 kinase association with KIT, loss of PI3 kinase association dramatically inhibits the ligand-independent activation of KIT mutations [27,28]. In addition to the ligand-independent activation, KIT mutations can activate different downstream signaling pathways compared with wild-type KIT.…”