LS104, a Novel Kinase Inhibitor, Induces Apoptosis, Synergizes with Cytostatic Drugs and Is Targeting the Receptor Tyrosine Kinase FLT3.
Authors: Kasper Stefan , Kindler Thomas , Heidel Florian , Breitenbuecher Frank , Hoehn Yvette , Lipka Daniel , Thomas Helmut , Huber Christoph , Fischer Thomas 
Published: April 2020

Abstract
Fms-like tyrosine kinase 3 (FLT3), a member of the class III tyrosine kinase receptor family, is expressed in up to 90% of acute myeloid leukemia (AML). Activating mutations like internal tandem duplication (ITD) of the juxtamembrane domain and kinase domain point mutations are found in approximately 35% of AML-cases and are considered to represent an attractive therapeutic target. In this study, we report that the novel hydroxystyryl-acrylonitrile compound LS104 induces potent cytotoxic effects in FLT3 ITD-positive leukemic cells. As a cellular model to investigate FLT3-ITD specific effects we used 32D myeloid cells stably transfected with FLT3-ITD and wt-FLT3, respectively. In MTS assays, pronounced inhibition of cell growth was seen at nanomolar concentration (IC50=50nM) which could be partially rescued by addition of IL3. LS104 at a concentration ranging from 3–10μM readily induced apoptosis as evaluated by cell cycle analysis, annexin-V assays and PARP cleavage. Throughout the experiments, FLT3-ITD expressing cells showed significantly higher sensitivity towards LS104 than cells expressing wt-FLT3. Similar results were observed evaluating the cytotoxic effects of LS104 in the human myeloid-leukemia derived FLT3-ITD harbouring MV4;11 and the wt-FLT3 expressing lymphoid-leukemia derived RS4;11 cell line. Immunoblot analysis demonstrated that LS104 inhibits tyrosine phosphorylation of FLT3-ITD and of its downstream target STAT5. This points out that FLT3-ITD is a molecular target of LS104. Interestingly, efficacy of LS104 to induce apoptosis was significantly reduced in 32D cells transfected with a FLT3-ITD isoform (N676K) previously reported to be associated with clinical resistance of FLT3-ITD to the kinase inhibitor PKC412 (Heidel et al., ASH 2004). This result strongly suggests that the mechanism of action of LS104 is indeed inhibition of FLT3 kinase activity rather than inhibition of other kinases targeted by LS104. As clinical development of FLT3 kinase inhibitors in AML likely will be in combination with chemotherapy, we evaluated the in vitro effects of combining LS104 with the cytostatic drugs cytosine arabinosid (Ara-C) and daunorubicin (DNR). In 32D FLT3-ITD cells, cell cycle analysis showed strong synergy in induction of apoptosis upon incubation with LS104 (1–3μM) plus Ara-C (1μM) and DNR (10nM), respectively. Using primary blasts from AML patients, we further evaluated cytotoxicity of LS104 at various concentrations and in combination with Ara-C and DNR, respectively. LS104 as a single agent was shown to induce apoptosis in primary AML blasts and combination of LS104 with cytostatic drugs resulted in additive and synergistic effects in leukemic blasts from 4 out of 6 patients investigated. Western-blotting of primary AML blasts revealed inhibition of FLT3-ITD and STAT5 tyrosine phosphorylation by LS104 in a dose-dependent way (3–50μM). Interestingly, LS104 showed intrinsic fluorescence activity. Using FACS analysis and fluorescence microscopy we could demonstrate a rapid and dose-dependent cellular uptake of LS104 in leukemic cell lines and in primary blasts. After removal of LS104 from medium, enhanced fluorescence corresponding to intracellular LS104 was detectable up to 24 hours. This property could be used to monitor drug uptake of primary AML blasts in vivo. In conclusion, our data provide a preclinical framework for clinical trials of LS104 in FLT3-ITD positive leukemia.