A Unique N-Phenylbenzamide Inhibitor Targeting Non-Receptor Tyrosine Kinases Potently Suppresses AML Cell Growth In Vitro and In Vivo

Abstract

Acute myelogenous leukemia (AML) is a hematologic cancer correlated with advancing age and poor prognosis. While diverse genetic changes are associated with AML, activating mutations in the Flt3 receptor tyrosine kinase are associated with nearly one-third of cases and include internal tandem duplication (ITD) of the regulatory juxtamembrane region. Additionally, recent studies have implicated several non-receptor tyrosine kinases in AML oncogenic signaling, including Fes, Syk and the myeloid Src-family members Fgr, Hck and Lyn. While selective Flt3 kinase inhibitors have been developed for clinical use, they have not produced durable responses, highlighting the need for new therapeutic approaches. In this study, we investigated the therapeutic potential of a multi-targeted tyrosine kinase inhibitor, TL02-59, which is based on an N-phenylbenzamide scaffold. Kinome-wide target specificity profiling (KINOMEscan) demonstrated a relatively narrow overall specificity profile for TL02-59, with only 23 potential targets out of 456 kinases when tested at a concentration of 1 µM. To confirm the results of the kinome-wide profiling against AML-associated kinases, we tested TL02-59 using in vitro kinase assays with recombinant Fes, Syk, Fgr, Hck, and Lyn as well as wild-type and ITD forms of the Flt3 kinase domain. TL02-59 displayed remarkable potency against the myeloid Src-family kinases Fgr and Lyn, with IC50values in the 100 pM range. TL02-59 also inhibited Fes, Syk, Hck and both forms of Flt3 in vitro, albeit with IC50 values in the 150-400 nM range. We then tested TL02-59 for growth-suppressive activity against Flt3-ITD+ AML cell lines, MV4-11 and MOLM-14, and the Flt3 wild-type cell line THP-1. Remarkably, TL02-59 inhibited the proliferation of MV4-11 cells with an IC50 value of 780 pM, with slightly lower potency against the MOLM-14 cells (IC50 = 3.5 nM). Quantitative real-time RT-PCR (qPCR) and immunoblot analysis confirmed that the AML-associated TL02-59 target kinases identified in vitro, including Fgr and Lyn, are expressed and active in MV4-11 and MOLM-14 cells. In contrast, TL02-59 had no effect on THP-1 cell growth up to 1 µM, consistent with transformation of these cells by kinase-independent mechanisms. We next examined the growth suppressive activity of TL02-59 in 26 bone marrow samples from AML patients, half of which expressed Flt3-ITD and half of which expressed wild-type Flt3. Twenty of the 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 all possible TL02-59 target kinases from the KINOMEscan analysis in these primary AML samples by qPCR. We discovered a striking correlation between TL02-59 sensitivity and expression levels of Fgr, Hck, and Lyn, supporting the idea that these Src-family members are the primary targets for TL02-59 in AML. In contrast, no correlation was observed with Flt3 expression, consistent with the observation that the four most sensitive patient samples were wild-type for Flt3. To test the efficacy of TL02-59 in vivo , we used immunocompromised (NSG) mice engrafted with MV4-11 AML cells as a model system. Two weeks after MV4-11 cell engraftment, mice were treated once daily with TL02-59 at 1 or 10 mg/kg by oral gavage for a total of 21 days. The mice were then sacrificed and evaluated for MV4-11 cells in their peripheral blood, spleen, and bone marrow by flow cytometry and IHC. At 1 mg/kg, TL02-59 reduced MV4-11 engraftment in the bone marrow by 20% and spleen engraftment by half, while suppressing the number of leukemia cells in the peripheral blood by 70% compared to vehicle control animals. At 10 mg/kg, TL02-59 reduced bone marrow AML cells by 70% and completely eliminated leukemic cells from the spleen and peripheral blood. Overall, these results demonstrate that TL02-59 is an effective inhibitor of AML cell growth in vitro and in vivo with a mechanism of action independent of Flt3 mutational status. The primary kinase targets for this inhibitor appear to be myeloid Src-family kinases, including Fgr, Lyn and Hck. DisclosuresNo relevant conflicts of interest to declare.