3,4-Diarylmaleimides—a novel class of kinase inhibitors—effectively induce apoptosis in FLT3-ITD-dependent cells
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- Heidel, F.H., Mack, T.S., Razumovskaya, E. et al. Ann Hematol (2012) 91: 331. doi:10.1007/s00277-011-1311-3
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FLT3 kinase has become an attractive drug target in AML with up to 30% of cases harboring internal-tandem-duplication (ITD) mutations. For these, conferring a worse prognosis and decreased overall survival, several FLT3 tyrosine kinase inhibitors (TKIs) are currently being tested in clinical trials. However, when using these drugs as monotherapy, the problem of short duration of remissions and high incidence of TKI resistance has emerged. Here, we investigated two members of a novel class of tyrosine kinase inhibitors, 3,4-diarylmaleimides, for their efficacy on mutated FLT3 kinase. These compounds inhibit FLT3 kinase in an ATP-competitive manner and effectively inhibit phosphorylation of downstream targets. 3,4-Diarylmaleimides (DHF125 and 150) induce apoptosis in FLT3-ITD-dependent cells lines and patient blasts at low micromolar concentrations. They are retained in the cytoplasm of exposed cells for more than 24 h and synergize with chemotherapy and midostaurin. Both 3,4-diarylmaleimides show inhbition of FLT3-ITD-related kinase autophosphorylation at distinct tyrosine residues when compared to midostaurin. In conclusion, this novel group of compounds shows differential inhibition patterns with regard to FLT3 kinase and displays a promising profile for further clinical development. Currently, experiments evaluating toxicity in murine models and unraveling the exact binding mechanism are under way to facilitate a potential clinical application.
KeywordsAMLFLT3Tyrosine kinase inhibitorTyrosine phosphorylation
Acute myeloid leukemia (AML) is an aggressive malignant disease characterized by abnormal proliferation of immature hematopoietic cells. Despite advances in chemotherapeutic treatment within the last decade, only 30–45% of patients below the age of 60 can be cured. The majority of patients, above the age of 60, have a dismal prognosis with only 5–15% long-term survival [1–5]. Several prognostic factors have been established within the last decade. While cytogenetic changes are established prognostic markers survival and response to chemotherapy, several molecular markers have been defined and analyzed for their prognostic influence . Besides overexpression of the brain and acute leukemia gene , the v-ets erythroblastosis virus E26 oncogene [8, 9] and high meningioma 1 gene , mutation of nucleophosmin-1 (NPM1) , CAATT/enhancer binding protein , mixed-lineage-leukemia gene, Wilms’ tumor 1 gene  and FMS-related tyrosine kinase (FMS-like tyrosine kinase 3–internal tandem duplications (FLT3-ITD)) [14, 15] have shown prognostic influence.
The FLT3 gene is encoding a receptor tyrosine kinase that is part of the receptor tyrosine kinase III family (together with KIT, CSF-1R and PDGFRalpha and beta). FLT3 is expressed on the blasts of over 90% of AML cases. Mutations of FLT3 divide into length mutations (ITD) and point mutations. FLT3-ITD is a heterogenic group of genetic alterations found in up to 30% of AML cases [16–18]. These mutations confer a dismal prognosis with impaired progression free and overall survival [14, 15, 19, 20]. FLT3-ITDs lead to autophosphorylation and constitutive activation of the FLT3 receptor with consecutive phosphorylation of downstream signaling nodes such as STAT5, AKT, and ERK, presumably by disruption of the autoinhibitory region . Transfection of murine hematopoietic cells with FLT3-ITD leads to growth factor independent proliferation and development of a myeloproliferative phenotype in a murine bone marrow transplant model .
Small molecule tyrosine kinase inhibitors are a newly established class of therapeutic drugs. In AML, several tyrosine kinase inhibitors are currently tested in advanced clinical trials. Using these drugs as monotherapy has revealed remarkable efficacy. However, at the same time the problem of short duration of remissions has emerged indicating rapid development of secondary resistance [23, 24]. In addition, up to 30% of patients may show primary resistance to currently available FLT3-TKIs. This imposes a strong need to further develop treatment strategies: besides immunotherapeutic approaches , development of novel “second-generation” inhibitors using different mechanisms of action to achieve increased efficacy are clearly warranted.
Here, we investigated two novel TKIs (3,4-diarylmaleimides, DHF125 and DHF150) previously described as angiogenesis inhibitors [26–28], to determine their mechanism of action and efficacy in FLT3-ITD-positive cell lines as well as in primary ITD-positive AML blasts.
Materials and methods
DNA constructs and generation of transfected cells
A human FLT3-ITD construct (amino acid position, N598; amino acid sequence, NEYFYVDFREYE), subcloned into the pAL expression vector under control of the 5′ long terminal repeat of the Moloney murine sarcoma virus and the plasmid pMAM/BSD were used as previously described . The pAL-vector construct was stably transfected into the murine IL3-dependent myeloid cell line 32D by electroporation.
Cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS), 20 mM Hepes (pH 7.3), 50-mM β-mercaptoethanol, and 2 mM l-glutamine.
Protein extract preparation, immunoprecipitation, and Western blotting
Cells at 2 × 106 were incubated in the presence of different inhibitor concentrations and in combination of different inhibitors for 1 h at 37°C. Preparation of cellular lysates was performed as described previously . Protein lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on nitrocellulose membrane (Amersham, Freiburg, Germany). The following antibodies were used: anti-phospho-FLT3 (pTyr591), anti-phospho-STAT5 (pTyr694/699), anti-phospho AKT (pSer473), anti-phospho ERK (pThr202/pTyr204) (all Cell Signaling Technology™, Frankfurt, Germany), antiphosphotyrosine (Biomol, Hamburg, Germany; and Santa Cruz, Heidelberg, Germany), anti-FLT3, anti-STAT5 (Santa Cruz, Heidelberg, Germany), anti-AKT (Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin. Densitometric analysis was performed using the program Gel-Pro Analyzer®. For immunoprecipitation, 1 × 107 inhibitor/DMSO-treated MV4;11 cells were used for protein extraction; 100 μL of protein lysates were incubated with 20 μL of FLT3 antibody (sc-479, Santa Cruz, Heidelberg, Germany) for 24 h at 4°C. AG beads were added for 1 h at 4°C, followed by multiple washing steps. Finally, AG beads were covered with 30-μL β-mercaptoethanol-containing loading dye, heated for 5 min at 95°C followed by a quick spin-down. The supernatant was subjected to SDS-PAGE gel electrophoresis and Western blotting. Phospho-specific antibodies were produced and applied for Western blotting experiments as previously described .
For determination of in vitro FLT3 kinase activity, we used HTScan™ FLT3 Kinase Assay Kit (Cell Signaling Technology, Inc., Danvers, MA, USA). Analysis was performed according to the manufacturer’s protocol. For ATP competition experiments 10-fold of ATP concentration (200 μM instead of 20 μM) was used. Absorbance was measured using a standard ELISA reader at 450 nm.
A second kinase assay was performed on a commercial basis by Millipore UK Ltd. (Gemini Crescent; Dundee Technology Park, Dundee DD2 1SW, UK). Details of assay protocols can be found on Millipore’s website at www.millipore.com/drugdiscovery/dd3/assayprotocols.
Transfected murine 32D cells (1 × 105 cells/well) were incubated in 2 ml RPMI 1640 medium with different inhibitor concentrations for 48 h at 37°C. Primary human cells (2 × 105 cells/well) were incubated 72 h at 37°C. After incubation, cells were washed with ice-cold PBS, pelleted, and mixed with 300 μl of propidium-iodide-buffer (containing 50 μg/ml PI in 0.1% sodium citrate plus 0.1% Triton × 100, Sigma) for 30 min at 4°C. Cell cycle analysis was performed as described previously  using FACSCanto™ flow cytometers (BD Biosciences, Heidelberg, Germany). For AnnexinV/SytoxRed staining, treated 32D cells were washed with ice-cold PBS, resuspended in AnnexinV buffer and stained with AnnexinV-FITC and SytoxRed (Invitrogen) for 30 min on ice.
Isolation of primary AML blasts and cell culture
Bone marrow and peripheral blood samples with heparin as anticoagulant were obtained from AML patients (patient characteristics are indicated below) or donors with no evidence of malignant bone marrow infiltration after informed consent in a study approved by the local ethics committee. Mononuclear cells (MNC) were isolated immediately by means of Ficoll-Hypaque (Seromed, Berlin, Germany) density gradient centrifugation. For immunoblotting freshly isolated MNCs were either lysed directly or after incubation in RPMI 1640 medium supplemented with 20 mM Hepes (pH 7.3), 50-mM β-mercaptoethanol and 2 mM l-glutamine containing varying amounts of different tyrosine kinase inhibitors (midostaurin (protein kinase C (PKC)412), DHF125, and DHF150). For cell cycle analysis, MNCs were maintained in RPMI 1640 medium supplemented as above plus 10% FCS.
% of Blasts
FLT3 mutation status was confirmed using standard diagnostic primer as follows: FLT3-ITD-fw 5-GCAATTTAGGTATGAAAGCCAGC-3 and FLT3-ITD-rev 5-CTTTCAGCATTTTGACGGCAACC-3.
% of Blasts
Bone marrow MNCs were isolated as indicated above. Bone marrow samples were obtained from patients in complete remission after leukemia or lymphoma treatment and without evidence of any malignant bone marrow infiltration; 1 × 106 cells were plated in 1.1 ml Methocult™ (GF H4534) “complete” methylcellulose medium with recombinant cytokines (StemCell Technologies, Vancouver, Canada) and incubated for 10 days at 37°C. Colony formation was counted on day 10.
3,4-Diarylmaleimides (academically developed at the Department of Pharmacy, Johannes-Gutenberg-University Mainz [26–28] and PKC412A (kindly provided by J. Roesel, Novartis Inc.) were dissolved and diluted in DMSO (Sigma, Munich, Germany). The chemotherapeutic drugs cytarabine and daunorubicin have been diluted in water. Equivalent doses of DMSO were added when used for combination treatment.
Detection of autofluorescence and cellular uptake
To determine uptake and persistence of 3,4-diarylmaleimides in AML blasts, primary cells were incubated at 37°C in a 5% CO2-humified incubator with either compound in RPMI 1640 medium supplemented with 10% FCS. Uptake was determined by FACS analysis (FACSCanto, BD Biosciences, Heidelberg, Germany) by mean fluorescence intensity (DHF125, PE-Cy5-A mean; DHF150, PE-A mean). For determination of persistence of 3,4-diarylmaleimides in primary blasts, cells were incubated for 10 min with either compound, washed twice with PBS, and incubated at 37°C in RPMI 1640 medium supplemented with 10% FCS for 24 h. Autofluorescence was detected as indicated above.
Fluorescence of 3,4-diarylmaleimides was detected by confocal laser microscopy using a Zeiss Axiovert 100 M microscope attached to an LSM confocal unit. 3,4-Diarylmaleimides were added to murine 32D cells or human AML-blasts 5 min prior to microscope imaging. Images were edited using Zeiss LSM Image Examiner (Version 22.214.171.124).
Quantification of synergism and antagonism in drug combinations
For definition of synergy and/or antagonism in drug combinations we used the CompuSyn™ software (Chou, TC; ComboSyn, Inc. Paramus, NJ, USA) as previously described .
Statistical analysis was performed using GraphPad PrismTM v4.00. All experiments were performed in triplicates unless otherwise stated. For statistical comparison of colony formation upon diarylmaleimide treatment, a paired Student’s t test was applied. Statistical significance was assumed for p < 0.05.
3,4-Diarylmaleimides inhibit FLT3 kinase in an ATP-dependent manner and reduce phosphorylation of its downstream signaling molecules
To directly address inhibition of FLT3 kinase activity, we performed FLT3 kinase assays. DHF150 and DHF125 effectively inhibited FLT3 kinase with an IC50 of ~350 and ~450 nM, respectively. As suggested by the initial kinase screen, we aimed to confirm the ATP-competitive mechanism of 3,4-diarylmaleimides. As inhibitors binding in the ATP-pocket can be outcompeted by escalation of ATP , we applied a tenfold ATP concentration (Fig. 1b, left panel). These experiments revealed that both DM compounds could be antagonized by ATP escalation, indeed indicating an ATP-competitive mechanism of action. Midostaurin used as a positive control, also revealed ATP-dependent inhibition of FLT3 kinase (data not shown). This FLT3 kinase-specific mechanism of inhibition was confirmed using a commercially available FLT3 kinase assay performed by Millipore. In this assay, DHF125 showed inhibition of FLT3 kinase with an IC50 of 166 nM (Fig. 1b, right panel). The differences between the two kinase assays applied may be best explained by the different methodology: antibody-based array technology versus detection of kinase activity by radioactive labeled ATP, respectively.
Thus, we were able to provide first evidence that both 3,4-diarylmaleimides inhibit FLT3 kinase and its downstream targets in an ATP-competitive manner.
3,4-Diarylmaleimides (DHF compounds) effectively induce apoptosis in FLT3-ITD-dependent cell lines
To determine the effects of 3,4-diarylmaleimides on mutated FLT3 tyrosine kinase in a cellular context, we transfected the murine IL3-dependent hematopoietic 32D cells with an FLT3 length mutation (FLT3-ITD)- or FLT3-wildtype (FLT3-WT)-cDNA construct. While FLT3-ITD-positive cells gain growth factor-independent proliferation, FLT3-WT cells generally remain IL3 dependent. 32D-FLT3-ITD-positive cells are known to undergo apoptosis upon incubation with FLT3 inhibitors like PKC412 in the absence of IL-3 . However, when IL-3 is added, induction of apoptosis can be prevented bypassing FLT3-ITD dependency via growth factor receptor. Therefore, we incubated these cells with either DHF compound in the presence or absence of IL-3.
Induction of apoptosis was detectable at low micromolar concentrations (Fig. 2a), as detected by flow cytometry analysis using AnnexinV (as an early apoptosis marker) and SytoxRed (as a dead cell stain). Analyzing the subG1 fraction in cell cycle analysis upon incubation with DHF125 and DHF150, we were able to define an IC50 of 375 and 850 nM, respectively (Fig. 2b, c). To confirm the FLT3 specificity of these effects, FLT3-ITD-positive cells were also cultured in the presence of IL-3 allowing the cells to bypass FLT3-ITD dependency by activation of alternative signaling pathways. As depicted in Fig. 2b, induction of apoptosis was reversible in both cases, confirming FLT3-ITD specificity of the effects observed. Induction of apoptosis was further confirmed by Western blotting experiments as indicated by caspase-3 cleavage (Fig. 2d).
As 32D cells transfected with an FLT3-WT construct are cultured in growth factor containing medium, any induction of cell death would potentially display toxic off-target effects. To elucidate the “maximal tolerable” or toxic dose for 32D transfectants, we incubated 32D-FLT3-WT cells with increasing concentrations of either compound until cell death occurred. 32D-FLT3-WT cells showed no significant increase in apoptosis up to 10 μM of either DHF compound (Fig. 2e; displayed is data for DHF125). However, higher doses of up to 50 μM revealed increasing rates of cell death in terms of cellular toxicity. This provides first evidence for a possible therapeutic range of this novel class of inhibitors.
To evaluate a potential broader spectrum of kinase inhibition, we analyzed the effects of DHF compounds on JAK2-kinase that had not been included in previous screens. Ba/F3 cells harboring the V617F mutation leading to constitutive activation of JAK2-kinase were incubated with increasing concentrations of either inhibitor. No significant induction of apoptosis could be demonstrated using doses up to 5 μM while apoptosis could be induced using JAK inhibitor (data not shown).
This suggests that 3,4-diarylmaleimides effectively induce apoptosis in FLT3-ITD-dependent murine cell lines at low micromolar concentrations. These effects are specific for mutated FLT3 kinase and unspecific effects do not occur until doses above 50-fold the IC50.
3,4-Diarylmaleimides show synergy with chemotherapy and PKC412
Applied as monotherapy, various tyrosine kinase inhibitors have shown promising responses. However, in most cases, these responses were short lived and insufficient for disease control. Currently, advanced clinical trials evaluate tyrosine kinase inhibitors in combination with chemotherapy. As anthracyclines and cytarabine are standard drugs for the treatment of AML, we investigated the efficacy of a combination therapy with DHF inhibitors. Synergy was calculated using Chou–Talalay plots (CI Index).
Recent reports suggest that FLT3-TKI can display distinct mechanisms of action [33, 34]. This offers a potential for combination therapy. Thus, we combined both DHF compounds with midostaurin to investigate potential synergy. Interestingly, co-incubation of PKC412 with DHF125 or DHF150, using a constant ratio design, revealed synergy (CI index, <1) at all concentrations applied (Fig. 3b). PKC412 doses used in this assay varied from 0.5 to 7 nM. In clinical phase 2 trials, PKC412 trough levels of up to 40 nM have been shown to be reached and have shown limited clinical efficacy with tolerable toxicity [24, 35, 36]. Thus, our results offer a rationale to clinically investigate TKI combination treatment in order to reach increased efficacy. Moreover, this suggests a distinct mechanism of action for DHF compounds, as identical binding would rather lead to competition of both compounds and potentially to antagonistic effects.
3,4-Diarylmaleimides induce apoptosis in primary AML blasts harboring FLT3-ITD mutations in vitro while leaving colony formation of normal stem and progenitor cells unaffected
In contrast to cell culture models, primary blasts may display a variety of genetic and epigenetic modifications, leading to increased resistance to apoptosis or proliferation.
To approach a possible therapeutic dose range, we investigated bone marrow cells of healthy donors or patients in stable remission after chemotherapy treatment. Toxicity was evaluated as impairment of colony formation. Usually, stem and progenitor cells in healthy human bone marrow have the ability to form colonies when plated in methylcellulose while further differentiated cells lack that ability. Therefore, choosing a colony formation assay seemed to be a relevant approach to investigate potential hematopoietic toxicity that would arise from impaired stem or progenitor compartments.
Addition of diarylmaleimide compounds showed no significant reduction in colony formation up to doses of 5 μM (p = 0.39 for DHF125—5 μM compared to DMSO control; p = 0.33 for DHF150—5 μM compared to DMSO control) (Fig. 4b). However, dose escalation of up to 10 μM revealed markedly impaired colony formation as readout for hematopoietic toxicity. This indicates that normal human stem and progenitor cells may not be affected by exposure to 3,4-diarylmaleimides concentrations of up to 5 μM of either compound (Fig. 4b). As we had investigated efficacy of a combination therapy using DM compounds with midostaurin (Fig. 3), we aimed to assess the impact of a combination therapy on colony formation of normal bone marrow. All doses investigated did not reveal any significant reduction in colony formation when compared to DMSO-treated controls (Fig. 4c)
These data are consistent with our results observed in murine cell lines (as described above). As FLT3-ITD-specific induction of apoptosis in murine cell lines and primary patient blasts occur at low micromolar concentration, these results can be interpreted as a first indicator for a potential therapeutic window.
3,4-Diarylmaleimides show rapid uptake and prolonged intracellular persistence in transfected cell lines and primary AML blasts
Exposure of malignant cells to tyrosine kinase inhibitors is one of the crucial issues in TKI treatment. Recent publications provide first evidence that prolonged exposure of kinases to the corresponding inhibitors is a crucial prerequisite for induction of cell death. Therefore, we aimed to investigate the retention of 3,4-diarylmaleimides in both, murine 32D cells and primary patient blasts. As 3,4-diarylmaleimides show autofluorescence that can be used as a correlate for inhibitor binding, we performed confocal fluorescence microscopy and flow cytometry analysis.
Besides analysis of plasma levels in drug development, pharmacokinetics on a cellular level moves also into focus. As cells can retain inhibitors or even accumulate them in the cytoplasm, drug exposure can be prolonged and more effective. Therefore we aimed to address that specific question by incubating primary patient blasts for 20 min with 1 μM of either inhibitor, followed by washout and analysis of fluorescence levels compared to unstained controls.
For all patients investigated, a rapid uptake within 5–10 min with increase of mean fluorescence intensity of up to 2,600% of baseline values could be demonstrated. Of note, there was a large variability in uptake and accumulation of inhibitors as detected by increase of fluorescence between the different patient samples investigated. After washout, fluorescence levels decreased, but remained elevated up to 200–500% of baseline control within 24 h (Fig. 5c). This was detectable for both compounds with a comparable patient variability. However, none of the samples investigated showed a drop below 100% of baseline values.
In summary, these data show prolonged availability of both diarylmaleimides in the cytoplasm of leukemic cells for a sustained period of up to 24 h. This could be a first indicator for a promising clinical profile, as 3,4-diarylmaleimide may be retained or even accumulated in malignant blasts and thereby ensure a prolonged exposure of mutated FLT3 kinase.
3,4-Diarylmaleimides show a distinct profile for inhibition of FLT3-ITD-induced autophosphorylation
Activity of FLT3 tyrosine kinase inhibitors has been frequently measured using reduction of ITD-induced autophosphorylation as readout in Western blotting experiments. However, up to date few data exist on detailed analysis of the phosphorylation status of individual tyrosine residues upon inhibitor treatment. For example, it is not known, whether specific inhibition of autophosphorylation at distinct residues is required for inhibition of downstream targets and induction of apoptosis. The majority of publications investigating established FLT3 kinase inhibitors used total tyrosine phosphorylation (using the 4G10 antibody) as a correlate for kinase inhibition [32, 37]. In addition, analysis of Y591 phosphorylation has also been used. Recently, phosphorylation of tyrosine residues Y589, Y591, Y599, Y726, Y768, and Y955 has been shown to be involved in constitutive activation of FLT3 kinase induced by presence of ITD length mutations .
In order to investigate ITD-relevant tyrosine residues, we performed additional FLT3-ITD immunoprecipitation experiments in 32D-FLT3-ITD cells. Interestingly, investigating seven additional tyrosine residues, we found residues affected (1) by midostaurin treatment but not by 3,4-diarylmaleimides, (2) affected by neither group of compounds or (3) affected by both, midostaurin and 3,4-diarylmaleimides (Fig. 6b). None of the tyrosine residues was affected by 3,4-diarylmaleimides but not by midostaurin treatment.
In detail, tyrosine residue Y589 showed reduced phosphorylation upon midostaurin but was not affected by 3,4-diarylmaleimide treatment. Apparently, residues Y842, Y726, Y768, Y599, and Y955 were not significantly affected by any tyrosine kinase inhibitor treatment applied potentially indicating non-informative tyrosine residues in terms of TKI treatment. Residue Y793 showed reduction of autophosphorylation upon incubation with both 3,4-diarylmaleimides and PKC412. Total phosphorylation of FLT3, as determined using the 4G10 phospho-tyrosine antibody revealed a clear inhibitory effect of midostaurin as well as a minor reduction using DHF inhibitors (Fig. 6b). This effect could be seen best in the Y793 panel as no residual activity would be detected after stripping off pY793 and re-probing with 4G10.
These results implicate several novel findings: when investigating distinct tyrosine residues for their involvement in inhibitory activity of FLT3 kinase inhibitors, several residues involved in autophosphorylation caused by the presence of ITD mutations were not affected by midostaurin. Thus it is tempting to speculate that these residues (namely Y842, Y726, Y768, Y599, and Y955) do not play a crucial role in midostaurin-mediated reduction of kinase activity. Moreover, 3,4-diarylmaleimides seem to carry out their inhibitory activity without affecting tyrosine residues Y589 and Y591, which are believed to play a pivotal role in FLT3-ITD-mediated transformation. Thus, in future studies, the phosphorylation status of these residues will be investigated using a broad spectrum of kinase inhibitors to gain a deeper insight into TKI-induced regulation of ITD-mediated autophosphorylation.
AML is a malignant disease with high biological and genetic heterogeneity and survival rates of 30–40% in patients <60 years upon myelosuppressive chemotherapy [2–4]. However, the majority of patients affected by AML are above the age of 60, with a long-term disease-free survival of only 5–15% after myelosuppressive chemotherapy treatment. Besides cytogenetic risk-stratification, molecular markers—such as NPM1 or FLT3 mutations—have shown prognostic impact [14, 15, 19]. Therefore, inhibition of mutated FLT3 tyrosine kinase may improve response rates to chemotherapy and overall survival. Currently, advanced clinical studies are testing this hypothesis, using FLT3 tyrosine kinase inhibitors such as PKC412 or lestaurtinib (CEP-701) in combination with myelosuppressive chemotherapy. The majority of tyrosine kinase inhibitors currently investigated in clinical phase 2–3 trials for FLT3-ITD-positive AML originally have not been designed as “FLT3-inhibitors.” Examples are PKC412 being developed as an inhibitor of PKC, BAY 43-9006 (Sorafenib) as an inhibitor of Ras/Raf or CEP-701 (Lestaurtinib) as an inhibitor of Trkl. As a ‘beneficial side effect’, these compounds have shown to inhibit FLT3 kinase mostly at low micromolar or nanomolar concentrations. Currently, second-generation inhibitors, developed and designed for inhibition of FLT3 kinase are being tested in clinical and pre-clinical settings.  . Here, we describe a novel class of inhibitors that was initially detected in a screening approach for angiogenesis inhibitors. These 3,4-diarylmaleimides are obviously potent inhibitors of mutant FLT3 kinase and display several characteristics that make them a promising group for further clinical development: (1) 3,4-diarylmaleimides are retained in the cytoplasm of exposed cells and suggest a prolonged intracellular exposure of mutant FLT3 kinase, (2) they synergize with chemotherapeutic drugs and notably with midostaurin which may be explained (3) by a distinct inhibition pattern with regard to the phosphorylation status of specific FLT3 tyrosine residues.
Exposure of malignant cells to tyrosine kinase inhibitors is one of the crucial issues in TKI treatment. Several inhibitors did not reach advanced clinical studies due to pharmacokinetic/-dynamic problems and inadequate exposure of target cells for TKI. In addition, uptake into malignant cells displays an important issue in the clinical use of tyrosine kinase inhibitors. Recently, irreversible commitment to apoptosis upon short-term high-dose exposure to TKIs has been suggested as a general mechanism .
Using a combination therapy of DM compounds with midostaurin, we could achieve synergy and decent activity against 32D-FLT3-ITD cells. The concentrations applied for PKC412 are achievable, even as trough levels (12.4–157 nM corrected for 99% protein binding) during long-term treatment . Although no studies have been conducted using 3,4-diarylmaleimides in vivo so far, other FLT3 kinase inhibitors (such as Sorafenib) have been shown to reach peak plasma levels up to 10 μM (when given twice daily at 400 mg) , suggesting a potential therapeutic window.
More importantly, our experiments provide first evidence for prolonged retention of both diarylmaleimide inhibitors investigated in leukemic cells for a period of up to 24 h, resulting in apoptotic cell death. Thus, both compounds investigated seem to meet the requirements for sufficient intracellular target inhibition.
In general, monotherapy treatment of relapsed or refractory leukemia using tyrosine kinase inhibitors in early clinical trials showed short-lived responses. However, first results from trials using TKI in combination with chemotherapy revealed promising duration of remission rates and suggested further development in clinical phase 3 studies. These trials are currently under way and underline the importance of potential synergistic effects when applied simultaneously or sequentially. 3,4-Diarylmaleimides revealed synergistic effects with both, cytarabine and daunorubicin, which can be considered standard therapeutics for AML treatment. The concept of combining different kinase inhibitors could be a novel approach to overcome resistance in AML therapy.
Recently, several tyrosine residues of FLT3 have been investigated for their differential phosphorylation in FLT3-WT, FLT3-D835Y, and FLT3-ITD kinase . While tyrosine residues Y589, Y591, Y599, Y726, Y768, and Y955 are phosphorylated sites in FLT3-ITD as well as in FLT3-D835Y mutants and FLT3-WT cells upon stimulation with its physiological ligand (Flt3 ligand), Y793 and Y842 seem to be exclusively phosphorylated in FLT3-D835Y mutants . Moreover, functional analysis of tyrosine residues within an FLT3-ITD background has already been performed [41, 42]. Tyrosine to phenylalanine substitution of the FLT3 tyrosine residues Y589 and Y591 on an ITD background led to significant reduction of STAT5 phosphorylation while not affecting tyrosine kinase activity or whole FLT3 tyrosine phosphorylation. As tyrosines Y589 and Y591 were identified as two candidate STAT5 SH2 docking phosphorylation sites in the juxtamembrane (JM) domain, this finding raised the possibility that they might display occult STAT5 binding sites and that ITD mutations expose these sites by conformational disruption of the JM domain. Single amino acid exchanges from Y to F at known tyrosine phosphorylation sites (Y591F, Y726F, Y955F, and Y969F) and even some combined exchanges (such as Y726F/Y955F and Y955F/Y969F) did not lead to significant reduction of STAT5 phosphorylation . Consistent with this finding, single Y to F mutations of Y589, Y591 or Y597 in the ITD background did not reduce FLT3-transforming potential of ITD mutations . However, combined mutations of certain phosphorylation sites, showed an influence on phosphorylation of STAT5 or reduced transforming capacity of ITD mutation (Y589F/Y591F, Y589F/Y597F, Y589F/Y599F, Y591F/Y597F, and Y591F/Y599F).
In our hands, DM inhibitors clearly inhibited or even abrogated phosphorylation of FLT3 downstream targets (such as STAT5) and inhibited FLT3 kinase activity. However, inhibition of phosphorylation at residues affected by midostaurin (such as FLT3-Y591 and FLT3-Y598) was not evident in Western Blottting. This indicates that residual phosphorylation of single tyrosine residues may have no influence on transforming potential or phosphorylation status of important downstream targets such as STAT5. Our data suggest that even Y591 which is thought to be involved in STAT5 binding within an FLT3-ITD background and which has been shown to reduce phosphorylation of downstream targets when mutated to phenylalanine in combination with cooperating phosphorylation sites may not be crucial for kinase inhibitor function.
Further studies investigating efficacy in cells resistant to currently available FLT3-TKIs and evaluation of toxicity in mouse models are clearly warranted and currently under way. Additional studies investigating differential inhibition patterns of FLT3 autophosphorylation at distinct tyrosine residues and experiments focusing on the exact binding mechanism of 3,4-diarylmaleimides are future aims but were beyond the scope of this report.
We thank Fian Mirea and Ann-Kathrin Borrmann for technical assistance and Dr. D. Strand (Department of Gastroenterology, University Hospital Mainz) for support with confocal microscopy. This work was supported by grants from the Johannes-Gutenberg-University (MAIFOR, Mainz Research Funding no. 9728249, to F.H.H.) and the German Cancer Aid (DKH 108218 and DKH 108401 (TP6) to T.F.).
F. Heidel, J.-P. Kramb, S. Plutitzki, G. Dannhardt, and T. Fischer filed a patent on the use of 3,4-diarylmaleimides in leukemia.