Abstract A25: Dual inhibition of Flt3 and Fes tyrosine kinases potently blocks proliferation of AML cells expressing an active Flt3 mutant.
Authors: Weir Mark , Hellwig Sabine , Gray Nathanael S. , Smithgall Thomas E. 
Published: August 2015

Abstract
Acute myelogenous leukemia (AML) is a hematologic cancer associated with a relatively poor prognosis, especially in the elderly. Although the diverse genetic changes associated with AML make treatment a challenge, about one-third of cases involve activating mutations in the receptor tyrosine kinase, Flt3. While many Flt3 tyrosine kinase inhibitors have been tested in the clinic, most have performed poorly as single agents indicating that additional signaling pathways contribute to the transformed phenotype. Previous studies have linked the Fes protein-tyrosine kinase to Flt3 in AML, and RNAi-knockdown studies suggest that Fes may be required for Flt3 function. In the present study, we tested the phenoxyquinazoline Fes kinase inhibitor, TL02-59, for its growth-suppressive activity against MV4-11 myeloid leukemia cells. These cells have an activating mutation in Flt3 (internal tandem duplication or ITD) that causes constitutive Fes activation downstream. TL02-59 blocked MV4-11 cell proliferation with an IC50 value of 25 nM, rivaling the potency of the clinical Flt3 inhibitors lestaurtinib and tandutinib in this cell line. In contrast, TL02-59 had no effect on the growth of THP-1 cells, which are wild-type for Flt3. We next explored the inhibitory activity of TL02-59 against Fes using an in vitro kinase assay. Surprisingly, TL02-59 inhibited recombinant Fes with an IC50 value of only 1.5 µM, suggesting that it may have additional kinase targets in AML cells. In vitro kinase assays with recombinant Flt3 support this idea, where TL02-59 also blocked Flt3 kinase activity with an IC50 value of 0.6 µM. This observation suggests that the potency of TL02-59 in MV4-11 cells may be the result of dual inhibition of both Fes and Flt3-ITD kinase activities, a possibility confirmed by anti-phosphotyrosine immunoblotting of Fes and Flt3 immunoprecipitates from TL02-59-treated cells. We next investigated downstream targets linked uniquely to Fes by engineering mutants of Flt3-ITD that are resistant to TL02-59 based on clinical cases of acquired drug resistance to other Flt3 inhibitors. These mutants were used to transform human TF-1 myeloid cells to cytokine independence, resulting in constitutive activation of endogenous Fes and sensitization to growth suppression by TL02-59 (IC50 = 24 nM). Interestingly, TF-1 cells transformed with TL02-59-resistant mutants of Flt3-ITD were less sensitive to the growth-suppressive actions of the inhibitor, consistent with its dual specificity. Using these cell lines, we will be able to identify downstream phosphorylation targets unique to Fes, because TL02-59 treatment will only affect Fes and not Flt3-ITD directly. Preliminary experiments performed in TF-1 cells transformed with one TL02-59-resistant mutant of Flt3-ITD, show inhibitor treatment still results in inhibition of STAT5 phosphorylation. These results support a critical role for Fes in downstream signaling from Flt3-ITD+ AML, and suggest that dual inhibition of both Flt3-ITD and Fes may provide a therapeutic advantage for the treatment of this subtype of AML.
Citation Format: Mark Weir, Sabine Hellwig, Nathanael S. Gray, Thomas E. Smithgall. Dual inhibition of Flt3 and Fes tyrosine kinases potently blocks proliferation of AML cells expressing an active Flt3 mutant. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A25.