Acute myelogenous leukemia (AML) is a hematologic cancer associated with advancing age and poor prognosis. While diverse genetic changes are associated with AML, activating mutations in the Flt3 receptor tyrosine kinase are fairly common and include internal tandem duplication (ITD) of the regulatory juxtamembrane region. Selective Flt3 kinase inhibitors developed for clinical use have not produced durable responses, highlighting the need for new therapeutic approaches. Recent studies have implicated non-receptor tyrosine kinases downstream of Flt3-ITD signaling in AML cells, including c-Fes and the myeloid Src-family members Hck and Lyn. Development of a multi-targeted inhibitor with activity against Flt3 and these downstream kinases may enhance anti-AML potency while reducing the risk of acquired resistance. To test this idea, we screened a small collection of phenoxyquinazoline tyrosine kinase inhibitors and identified a compound with triple-digit nM activity against Flt3-ITD, Fes, Hck and Lyn in vitro. This multi-targeted tyrosine kinase inhibitor, TL02-59, was then tested for growth-suppressive activity against AML cells lines expressing either an active Flt3-ITD mutant (MV4-11 cells) or wild-type Flt3 (THP-1). Remarkably, TL02-59 inhibited the proliferation of Flt3-ITD+ MV4-11 cells with an IC50 value in the 100 pM range, suggesting that its multi-targeted activity against Flt3-ITD and these associated kinases may be responsible. Quantitative real-time RT-PCR (qPCR) and immunoblot analysis confirmed that the four TL02-59 target kinases identified in vitro are expressed and active in MV4-11 cells. In contrast, proliferation of THP-1 cells was unaffected by TL02-59 even at a concentration as high as 3 μM, consistent with the absence of the Flt3-ITD driver mutation in this cell line. We next examined the growth suppressive activity of TL02-59 in primary AML bone marrow samples. Fourteen patient samples were identified with either wild-type or mutant (ITD; D835Y) Flt3 alleles and otherwise normal cytogenetics and tested for their sensitivity to TL02-59. Nine of the 14 primary AML samples responded to TL02-59 treatment with growth inhibitory IC50 values in the nM range. We then determined the relative expression levels of possible TL02-59 target kinases in these primary AML samples by qPCR, and discovered a striking correlation between TL02-59 sensitivity and expression level of the Src-family kinases Hck, Lyn and Fgr. This observation suggested that members of the Src kinase family may represent key targets for TL02-59 in AML. To test this idea further, we compared the sensitivity of MV4-11 cells to the pan-SFK inhibitor, A-419259, which has previously been shown to block AML cell growth in vivo. MV4-11 cells were about 250-fold less sensitive to A-419259 than TL02-59, suggesting that additional kinase targets are inhibited by TL02-59 in MV4-11 cells. To identify these additional kinases, we performed kinome-wide KINOMEscan profiling for both inhibitors, and identified several kinases that are potential targets for TL02-59 but not A-419259. These include kinases previously linked to Flt3-ITD oncogenic signaling, such as Syk and Fes, as well as several Ser/Thr kinases not previously investigated in the context of Flt3-ITD+ AML. Ongoing studies are exploring the expression of these kinases in AML and their contribution to the anti-AML activity of TL02-59.
Citation Format: Mark C. Weir, Sabine Hellwig, Ravi Patel, Nathanael S. Gray, Thomas E. Smithgall. Multi-targeted tyrosine kinase inhibitors potently suppress FLT3-ITD+ AML cell growth. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B180.