Aberrant activation of the PI3K/mTOR pathway promotes resistance to sorafenib in AML.

Therapy directed against oncogenic FLT3 has been shown to induce response in patients with acute myeloid leukemia (AML), but these responses are almost always transient. To address the mechanism of FLT3 inhibitor resistance, we generated two resistant AML cell lines by sustained treatment with the FLT3 inhibitor sorafenib. Parental cell lines carry the FLT3-ITD (tandem duplication) mutation and are highly responsive to FLT3 inhibitors, whereas resistant cell lines display resistance to multiple FLT3 inhibitors. Sanger sequencing and protein mass-spectrometry did not identify any acquired mutations in FLT3 in the resistant cells. Moreover, sorafenib treatment effectively blocked FLT3 activation in resistant cells, whereas it was unable to block colony formation or cell survival, suggesting that the resistant cells are no longer FLT3 dependent. Gene expression analysis of sensitive and resistant cell lines, as well as of blasts from patients with sorafenib-resistant AML, suggested an enrichment of the PI3K/mTOR pathway in the resistant phenotype, which was further supported by next-generation sequencing and phospho-specific-antibody array analysis. Furthermore, a selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation, and induced apoptosis in resistant cell lines. Gedatolisib significantly extended survival of mice in a sorafenib-resistant AML patient-derived xenograft model. Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway in FLT3-ITD-dependent AML results in resistance to drugs targeting FLT3.

The activation loop tyrosine 823 is essential for the transforming capacity of the c-Kit oncogenic mutant D816V.

Oncogenic c-Kit mutations have been shown to display ligand-independent receptor activation and cell proliferation. A substitution of aspartate to valine at amino acid 816 (D816V) is one of the most commonly found oncogenic c-Kit mutations and is found in >90% of cases of mastocytosis and less commonly in germ-cell tumors, core-binding factor acute myeloid leukemia and mucosal melanomas. The mechanisms by which this mutation leads to constitutive activation and transformation are not fully understood. Previous studies have shown that the D816V mutation causes a structural change in the activation loop (A-loop), resulting in weaker binding of the A-loop to the juxtamembrane domain. In this paper, we have investigated the role of Y823, the only tyrosine residue in the A-loop, and its role in oncogenic transformation by c-Kit/D816V by introducing the Y823F mutation. Although dispensable for the kinase activity of c-Kit/D816V, the presence of Y823 was crucial for cell proliferation and survival. Furthermore, mutation of Y823 selectively downregulates the Ras/Erk and Akt pathways as well as the phosphorylation of STAT5 and reduces the transforming capacity of the D816V/c-Kit in vitro. We further show that mice injected with cells expressing c-Kit/D816V/Y823F display significantly reduced tumor size as well as tumor weight compared with controls. Finally, microarray analysis, comparing Y823F/D816V cells with cells expressing c-Kit/D816V, demonstrate that mutation of Y823 causes upregulation of proapoptotic genes, whereas genes of survival pathways are downregulated. Thus, phosphorylation of Y823 is not necessary for kinase activation, but essential for the transforming ability of the c-Kit/D816V mutant.

The PI3-kinase isoform p110δ is essential for cell transformation induced by the D816V mutant of c-Kit in a lipid-kinase-independent manner.

PI3-kinase has a crucial role in transformation mediated by the oncogenic c-Kit mutant D816V. In this study, we demonstrate that the c-Kit/D816V-mediated cell survival is dependent on an intact direct binding of PI3-kinase to c-Kit. However, mutation of this binding site had little effect on the PI3-kinase activity in the cells, suggesting that c-Kit/D816V-mediated cell survival is dependent on PI3-kinase but not its kinase activity. Furthermore, inhibition of the lipid kinase activity of PI3-kinase led only to a slight inhibition of cell survival. Knockdown of the predominant PI3-kinase isoform p110δ in c-Kit/D816V-expressing Ba/F3 cells led to reduced cell transformation both in vitro and in vivo without affecting the overall PI3-kinase activity. This suggests that p110δ has a lipid-kinase-independent role in c-Kit/D816V-mediated cell transformation. We furthermore demonstrate that p110δ is phosphorylated at residues Y524 and S1039 and that phosphorylation requires an intact binding site for PI3-kinase in c-Kit/D816V. Overexpression of p110δ carrying the Y523F and S1038A mutations significantly reduced c-Kit/D816V-mediated cell survival and proliferation. Taken together, our results demonstrate an important lipid-kinase-independent role of p110δ in c-Kit/D816V-mediated cell transformation. This furthermore suggests that p110δ could be a potential diagnostic factor and selective therapeutic target for c-Kit/D816V-expressing malignancies.