3,4-Diarylmaleimides-a novel class of kinase inhibitors-effectively induce apoptosis in FLT3-ITD-dependent cells.

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.

qPCR in gastrointestinal stromal tumors: Evaluation of reference genes and expression analysis of KIT and the alternative receptor tyrosine kinases FLT3, CSF1-R, PDGFRB, MET and AXL.

BACKGROUND: Gastrointestinal stromal tumors (GIST) represent the most common mesenchymal tumors of the gastrointestinal tract. About 85% carry an activating mutation in the KIT or PDGFRA gene. Approximately 10% of GIST are so-called wild type GIST (wt-GIST) without mutations in the hot spots. In the present study we evaluated appropriate reference genes for the expression analysis of formalin-fixed, paraffin-embedded and fresh frozen samples from gastrointestinal stromal tumors. We evaluated the gene expression of KIT as well as of the alternative receptor tyrosine kinase genes FLT3, CSF1-R, PDGFRB, AXL and MET by qPCR. wt-GIST were compared to samples with mutations in KIT exon 9 and 11 and PDGFRA exon 18 in order to evaluate whether overexpression of these alternative RTK might contribute to the pathogenesis of wt-GIST. RESULTS: Gene expression variability of the pooled cDNA samples is much lower than the single reverse transcription cDNA synthesis. By combining the lowest variability values of fixed and fresh tissue, the genes POLR2A, PPIA, RPLPO and TFRC were chosen for further analysis of the GIST samples. Overexpression of KIT compared to the corresponding normal tissue was detected in each GIST subgroup except in GIST with PDGFRA exon 18 mutation. Comparing our sample groups, no significant differences in the gene expression levels of FLT3, CSF1R and AXL were determined. An exception was the sample group with KIT exon 9 mutation. A significantly reduced expression of CSF1R, FLT3 and PDGFRB compared to the normal tissue was detected. GIST with mutations in KIT exon 9 and 11 and in PDGFRA exon 18 showed a significant PDGFRB downregulation. CONCLUSIONS: As the variability of expression levels for the reference genes is very high comparing fresh frozen and formalin-fixed tissue there is a strong need for validation in each tissue type. None of the alternative receptor tyrosine kinases analyzed is associated with the pathogenesis of wild-type or mutated GIST. It remains to be clarified whether an autocrine or paracrine mechanism by overexpression of receptor tyrosine kinase ligands is responsible for the tumorigenesis of wt-GIST.

Coverage-dependent self-assembly of rubrene molecules on noble metal surfaces observed by scanning tunneling microscopy.

Coverage-dependent self-assembly of rubrene molecules on different noble metal surfaces, Au(111) and Au(100), Ag(111) and Ag(100), is presented. On Au(111), the homochiral supramolecular assemblies evolve with increasing rubrene coverage from very small structures composed of a few molecules, to honeycomb islets, and to one-dimensional chains of supramolecular pentamers. At higher coverage, the racemic mixture of molecules forms close-packed islands. On Au(100), chains of pentamers and two different types of densely packed islands are formed. On the Ag surfaces, exclusively close-packed islands are created, independently of the rubrene coverage. Moreover, the role of the chiral nature of the molecules in the self-assembly process is discussed, as well as the existence of different molecular conformers depending on the supramolecular assembled phase. The observed differences and similarities reflect the influence of the electronic properties and the geometric structure of the various substrates on molecular self-assembly.

Three-dimensional chirality transfer in rubrene multilayer islands on Au(111).

The growth of rubrene (C(42)H(28), 5,6,11,12-tetraphenylnaphthacene) multilayer islands up to a thickness of six layers on a Au(111) surface has been investigated by scanning tunneling microscopy. The molecules self-organize in parallel twin rows, forming mirror domains of defined local structural chirality. Each layer is composed of twin-row domains of the same structural handedness rotated by 120 degrees with respect to each other. Moreover, this structural chirality is transferred to all successive layers in the island, resulting in the formation of three-dimensional objects having a defined structural chirality. The centered rectangular surface unit cell differs from the one characteristic for the single-crystal orthorhombic phase.

LS104, a non-ATP-competitive small-molecule inhibitor of JAK2, is potently inducing apoptosis in JAK2V617F-positive cells.

The activating JAK2V617F mutation has been described in the majority of patients with BCR-ABL-negative myeloproliferative disorders (MPD). In this report, we characterize the small-molecule LS104 as a novel non-ATP-competitive JAK2 inhibitor: Treatment of JAK2V617F-positive cells with LS104 resulted in dose-dependent induction of apoptosis and inhibition of JAK2 autophosphorylation and of downstream targets. Activation of these targets by JAK2 was confirmed in experiments using small interfering RNA. LS104 inhibited JAK2 kinase activity in vitro. This effect was not reversible using elevated ATP concentrations, whereas variation of the kinase substrate peptide led to modulation of the IC50 value for LS104. In line with these data, combination treatment using LS104 plus an ATP-competitive JAK2 inhibitor (JAK inhibitor I) led to synergistically increased apoptosis in JAK2V617F-positive cells. Furthermore, LS104 strongly inhibited cytokine-independent growth of endogenous erythroid colonies isolated from patients with JAK2V617F-positive MPD in vitro, whereas there was no significant effect on growth of myeloid colonies obtained from normal controls. Based on these data, we have recently started a phase I clinical trial of LS104 for patients with JAK2V617F-positive MPDs. To the best of our knowledge, this is the first report on a non-ATP-competitive kinase inhibitor being tested in a clinical trial.

Fluorescence and phosphorescence from individual molecules excited by local electron tunneling.

Using the highly localized current of electrons tunneling through a double barrier scanning tunneling microscope junction, we excite luminescence from a selected C60 molecule in the surface layer of fullerene nanocrystals grown on an ultrathin NaCl film on Au(111). In the observed fluorescence and phosphorescence spectra, pure electronic as well as vibronically induced transitions of an individual C60 molecule are identified, leading to unambiguous chemical recognition on the single-molecular scale.