3,4-Diarylmaleimides Effectively Inhibit Proliferation of FLT3-ITD-Positive Leukemic Cells, Induce Apoptosis and Show Additive Effects in Combination with Chemotherapeutic Drugs.
Authors: Heidel Florian H. , Lipka Daniel B. , Mirea Fian K. , Markova Boyka , Peifer Christian , Kramp Jan-Peter , Plutizki Stanislav , Huber Christoph , Dannhardt Gerd , Fischer Thomas 
Published: April 2020

Abstract
Internal tandem duplication (ITD) mutations of FLT3 are present in leukemic blasts of approximately 30% of AML patients. ITD-mutations of FLT3 confer a worse prognosis and decreased overall survival. Therefore, FLT3-tyrosine kinase is considered an attractive drug target in AML and several FLT3-tyrosine kinase inhibitors (TKIs) are currently being tested in clinical trials (CEP701, MLN518, Sorafenib, PKC412). However, using these drugs as monotherapy, against the setting of remarkable efficacy has emerged the problem of short duration of remission indicating rapid development of secondary resistance. In addition, up to 30% of patients may show primary resistance to currently available FLT3-TKIs. Therefore, to overcome these limitations further clinical development requires novel approaches (e.g. combination with chemotherapy) and development of more efficacious FLT3-TKI. Here, we investigated two novel TKIs (3,4-Diarylmaleimides, compounds 53 and 80) previously described as angiogenesis inhibitors (Peifer, J. Med. Chem. 2006), to determine their mechanism of action and efficacy in FLT3-ITD transfected 32D cells as well as in primary ITD-positive AML blasts. Western Blotting experiments of 32D-FLT3-ITD cells confirmed dose dependent inhibition/dephosphorylation of downstream targets as STAT5 (pTyr694/699), ERK (pThr202/pTyr204) and AKT (pSer473) upon incubation with either compound using nanomolar concentrations. Both compounds (cpd.) showed induction of apoptosis with an IC50 of 350nM (cpd. 53) and 800nM (cpd. 80), respectively, as detected by sub G1-fraction in cell cycle analysis. Using colony assays we defined a possible therapeutic window of these inhibitors: Bone-marrow cells of healthy donors were incubated with increasing concentrations of both inhibitors revealing no significant inhibition of colony formation up to a dose of 5 μM. As clinical studies in AML will evaluate FLT3 inhibitors in combination with chemotherapy, we tested both cpds. in vitro in combination with cytosine arabinosid (Ara-C) and daunorubicine (DNR). Within a range of 1–50nM DNR and 0.3–10μM Ara-C, both cpds. showed additive effects in combination with chemotherapy in 32D-FLT3-ITD cells as revealed by apoptosis assays. Primary AML blasts harboring the FLT3-ITD length mutation were also investigated to determine the efficacy in patient material. These experiments revealed apoptosis rates of 10–30% upon incubation with 3,4-Diarylmaleimides for 72 hours. Cellular up-take of cpd. 53 in primary AML blasts could be demonstrated within 5 minutes of incubation using FACS analysis. Increased fluorescence (200–400% of baseline values) could be detected for more than 24 hours suggesting stable intracellular concentration of the inhibitor in AML blasts within this time period. To evaluate signaling targets of 3,4-Diarylmaleimides in detail, we applied a kinomics peptide chip containing a set of 960 kinase substrates (human sequences only) in triplicate on a slide. Phosphorylation of peptide substrates was detected by autoradiography. Results of evaluation will be presented. In conclusion, 3,4-Diarylmaleimides show efficacy in FLT3-ITD-positive cells alone as well as in combination with chemotherapy. Currently, experiments evaluating toxicity in murine models are planned to explore a possible clinical application.