BackgroundThe stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT.Methodology/Principal Findings In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC50) of 200±40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC50 of 150±80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant.ConclusionsMasitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity.

In the current study, we examined the types and frequency of KIT mutations in mast cell tumors from 191 dogs. Sequencing of reverse transcription-PCR products revealed alterations in 50 (26.2%) of the dogs. Most mutations were in exon 11 (n = 32), and of these, most were internal tandem duplications (n = 25) between residues 571 and 590. Within exon 11, there were two hotspots for mutations at codons 555-559 and 571-590. In addition, nine dogs had mutations in exon 8 and eight had mutations in exon 9. We selected the two most common mutants and two representative exon 11 mutants for further analysis. When expressed in Ba/F3 cells, they were constitutively tyrosine phosphorylated and induced growth factor -independent cell proliferation. AG1296, a tyrosine kinase inhibitor, dose dependently inhibited both the tyrosine phosphorylation of these mutants and their induction of growth factor -independent proliferation. This study shows that activating mutations in not only exon 11 but also exons 8 and 9 are common in canine mast cell tumors. These results also show that Ba/F3 cells can be used for the direct characterization of canine KIT mutants, eliminating the need to make equivalent mutations in the mouse or human genes.

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Two new anil molecules exhibiting photochromism in the crystalline state, N-(4-hydroxy)-salicylideneamino-4-(methylbenzoate) (2) and N-(3,5-di-tert-butylsalicylidene)-4-aminopyridine (3), are obtained. Upon irradiation in the UV, the yellow crystals change color to red, owing to enol-keto intramolecular tautomerism. The red color disappears, when crystals are left in the dark or irradiated with visible light. 3 has the most stable keto form among all anil-type photochromic compounds (τ ) 460 days at room temperature). Both exhibit nonlinear optical (NLO) properties and show powder second harmonic generation (SHG) of respectively 10 and 3 times vs urea. X-ray diffraction shows acentric structures where molecules line up "head-to-tail" through hydrogen bonds for 2 (space group Pc), or form a chiral helix 3 (space group P3 2 ). Evidence of reversible structural change is given for 3, and we demonstrate the functionality of this crystal as an NLO switching material, as SHG can be photomodulated by about 30%.

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Suppressor of cytokine signaling (SOCS) proteins are a family of Src homology 2-containing adaptor proteins. Cytokine-inducible Src homology domain 2-containing protein, SOCS1, SOCS2, and SOCS3 have been implicated in the down-regulation of cytokine signaling. The function of SOCS4, 5, 6, and 7 are not known. KIT receptor signaling is regulated by protein tyrosine phosphatases and adaptor proteins. We previously reported that SOCS1 inhibited cell proliferation in response to stem cell factor (SCF Cytokines and growth factors regulate the survival, proliferation, differentiation, and migration of hematopoietic cells. Binding of these factors to transmembrane receptors induces receptor activation, which in turn results in the recruitment of signaling complexes in the vicinity of the plasma membrane. The kinetics and magnitude of signal transduction are tightly regulated by multiple mechanisms. Among proteins that modulate signaling, members of the suppressor of cytokine signaling (SOCS) 1 family have been shown to down-regulate the function of cytokines or growth factors (1-3).The eight members of the SOCS family, SOCS1-7 and CIS (cytokine-inducible Src homology domain (SH2)-containing protein), are structurally characterized by a SH2 domain followed by a conserved C-terminal motif, the SOCS box (4). The N-terminal region of SOCS proteins is variable both in length and in the primary amino acid sequence. Although many reports including knock-out studies shed light on the function of CIS (5, 6), SOCS1 (7, 8), SOCS2 (9, 10), and SOCS3 (11-13), very little is known regarding the function of SOCS4, SOCS5, SOCS6, and SOCS7.The mechanisms whereby CIS, SOCS1, and SOCS3 inhibit signaling by classical cytokine receptor (i.e. receptors without catalytic activity that associate with JAK tyrosine kinases) are the best characterized. All three are involved in the downregulation of the JAK/STAT pathway. SOCS1 has a dual function as a direct potent JAK kinase inhibitor (14 -17) and as a component of an E3 ubiquitin-ligase complex recruiting substrates to the protein degradation machinery (18 -20). SOCS3 also inhibits JAK activity but indirectly through recruitment to the cytokine receptors (1, 21). More recently, SOCS3 has been suggested to compete with SHP2 for the same binding sites on glycoprotein 130 (22, 23), erythropoietin receptor (21), and leptin receptor (24). CIS binds to cytokine receptors at STAT5-docking sites, which impairs recruitment of STAT5 to the receptor signaling complex and results in the down-regulation of STAT5 activation (6,25).Mice lacking SOCS6 have been generated, and they developed normally with the exception of a 10% reduction in weight compared with wild-type littermates (26). SOCS6 mRNA was induced by erythropoietin in cell lines (27) and was ubiquitously expressed in murine tissues (26). SOCS6 does not interact with JAKs, but the interaction with elongins B and C suggests that, as all SOCS proteins, it might be part of an E3 ubiquitin-ligase complex (28). Yet, there is no evidence so far suggesti...

Mastocytosis is a rare neoplastic disease characterized by a pathologic accumulation of tissue mast cells (MCs). Mastocytosis is often associated with a somatic point mutation in the Kit protooncogene leading to an Asp/Val substitution at position 816 in the kinase domain of this receptor. The contribution of this mutation to mastocytosis development remains unclear. In addition, the clinical heterogeneity presented by mastocytosis patients carrying the same mutation is unexplained. We report that a disease with striking similarities to human mastocytosis develops spontaneously in transgenic mice expressing the human Asp816Val mutant Kit protooncogene specifically in MCs. This disease is characterized by clinical signs ranging from a localized and indolent MC hyperplasia to an invasive MC tumor. In addition, bone marrow–derived MCs from transgenic animals can be maintained in culture for >24 mo and acquire growth factor independency for proliferation. These results demonstrate a causal link in vivo between the Asp816Val Kit mutation and MC neoplasia and suggest a basis for the clinical heterogeneity of human mastocytosis.