Dual protein kinase and nucleoside kinase modulators for rationally designed polypharmacology.

Masitinib, a highly selective protein kinase inhibitor, can sensitise gemcitabine-refractory cancer cell lines when used in combination with gemcitabine. Here we report a reverse proteomic approach that identifies the target responsible for this sensitisation: the deoxycytidine kinase (dCK). Masitinib, as well as other protein kinase inhibitors, such as imatinib, interact with dCK and provoke an unforeseen conformational-dependent activation of this nucleoside kinase, modulating phosphorylation of nucleoside analogue drugs. This phenomenon leads to an increase of prodrug phosphorylation of most of the chemotherapeutic drugs activated by this nucleoside kinase. The unforeseen dual activity of protein kinase inhibition/nucleoside kinase activation could be of great therapeutic benefit, through either reducing toxicity of therapeutic agents by maintaining effectiveness at lower doses or by counteracting drug resistance initiated via down modulation of dCK target.

Signal transduction by several KIT juxtamembrane domain mutations.

Mutations of KIT receptor tyrosine kinase are found in the majority of patients with mastocytosis and in most gastrointestinal stromal tumors. Oncogenic KIT mutations in GISTs are located in the KIT juxtamembrane domain (JMD), while codon 816 in the KIT kinase domain is mutated in systemic mastocytosis. We describe and characterize a mutation in the KIT-JMD named Kdelta27. We show that Kdelta27 mutant is constitutively dimerized and phosphorylated. Kdelta27 ectopic expression renders both the Ba/F3 cell line and primary cultures of bone marrow mast cells independent of cytokines for proliferation and cell survival. The classical signaling pathways activated by wild-type KIT upon ligand stimulation are constitutively activated by Kdelta27 and other JMD mutations. However, a side-to-side comparison revealed differences between the wild-type and JMD mutations. First, in vitro kinase assays reveal a change in peptide substrate specificity. Second, STAT proteins are preferentially phosphorylated by KIT mutants. Third, inhibitors of KIT kinase are more efficient on JMD mutations than on WT KIT. We conclude that Kdelta27 is a new oncogenic KIT mutation showing constitutive activation of downstream signaling pathways, and suggest that specific pathways are activated by oncogenic KIT.

Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit receptors found in mast cell neoplasms.

Systemic mastocytosis (SM) is a rare disease caused by an abnormal mast cell accumulation in various tissues. Two classes of constitutive activating c-kit mutations are found in SM. The most frequent class occurs in the catalytic pocket coding region with substitutions at codon 816 and the other in the intracellular juxtamembrane coding region. Therefore, kinase inhibitors that block mutated c-kit activity might be used as therapeutic agents in SM. Here, we show that STI571 inhibits both wild-type and juxtamembrane mutant c-kit kinase activity, but has no effect on the activity of the D816 V mutant. Accordingly, STI571 selectively decreases the survival of normal mast cell and of mast cell lines either with juxtamembrane c-kit mutations, but not that of tumoral mast cell from patient with SM or of mast cell lines with the D816 V mutation. Therefore, STI571 is not a good candidate to treat SM and specific kinase inhibitors should be designed to inhibit constitutive activating mutations at codon 816.

Masitinib combined with standard gemcitabine chemotherapy: in vitro and in vivo studies in human pancreatic tumour cell lines and ectopic mouse model.

BACKGROUND: Tyrosine kinases are attractive targets for pancreatic cancer therapy because several are over-expressed, including PDGFRalpha/beta, FAK, Src and Lyn. A critical role of mast cells in the development of pancreatic cancer has also been reported. Masitinib is a tyrosine kinase inhibitor that selectively targets c-Kit, PDGFRalpha/beta, Lyn, and to a lesser extent the FAK pathway, without inhibiting kinases of known toxicities. Masitinib is particularly efficient in controlling the proliferation, differentiation and degranulation of mast cells. This study evaluates the therapeutic potential of masitinib in pancreatic cancer, as a single agent and in combination with gemcitabine. METHODOLOGY/FINDINGS: Proof-of-concept studies were performed in vitro on human pancreatic tumour cell lines and then in vivo using a mouse model of human pancreatic cancer. Molecular mechanisms were investigated via gene expression profiling. Masitinib as a single agent had no significant antiproliferative activity while the masitinib/gemcitabine combination showed synergy in vitro on proliferation of gemcitabine-refractory cell lines Mia Paca2 and Panc1, and to a lesser extent in vivo on Mia Paca2 cell tumour growth. Specifically, masitinib at 10 microM strongly sensitised Mia Paca2 cells to gemcitabine (>400-fold reduction in IC(50)); and moderately sensitised Panc1 cells (10-fold reduction). Transcriptional analysis identified the Wnt/beta-catenin signalling pathway as down-regulated in the cell lines resensitised by the masitinib/gemcitabine combination. CONCLUSIONS: These data establish proof-of-concept that masitinib can sensitise gemcitabine-refractory pancreatic cancer cell lines and warrant further in vivo investigation. Indeed, such an effect has been recently observed in a phase 2 clinical study of patients with pancreatic cancer who received a masitinib/gemcitabine combination.

Masitinib (AB1010), a potent and selective tyrosine kinase inhibitor targeting KIT.

BACKGROUND: The 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 (IC(50)) of 200+/-40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC(50) 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. CONCLUSIONS: Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity.