KIT Growth Factor Receptor Research Articles

Exposure of neuroblastoma cell lines to imatinib results in the upregulation of the CDK inhibitor p27KIP1 as a consequence of c-Abl inhibition

Imatinib mesylate is a tyrosine kinase inhibitor with selectivity for abelson tyrosine-protein kinase 1 (c-Abl), breakpoint cluster region (Bcr)-Abl fusion protein (Bcr-Abl), mast/stem cell growth factor receptor Kit (c-Kit), and platelet-derived growth factor receptor (PDGFR). Previous studies demonstrated that imatinib in the low micromolar range exerted antiproliferative effects on neuroblastoma cell lines. However, although neuroblastoma cells express c-Kit and PDGFR, the imatinib concentrations required to achieve significant growth inhibitory effects (≥10μM) are substantially higher than those required for inhibition of ligand-induced phosphorylation of wild type c-Kit and PDGFR (≤1μM), suggesting that additional mechanisms are responsible for the antitumor activity of imatinib on these cells. In this study, we show that treatment of neuroblastoma cell lines with 1–15μM imatinib resulted in a dose dependent inhibition of 5-bromo-2′-deoxyuridine (BrdU) incorporation into newly synthesized DNA. The antiproliferative effect of imatinib was dependent on the upregulation of the cyclin-dependent kinase (CDK) inhibitor p27KIP1 in the nuclear compartment as a result of increased p27KIP1 protein stability. We demonstrate that the mechanism of p27KIP1 stabilization relied on inhibition of p27KIP1 phosphorylation on tyrosine residues by c-Abl. We provide evidence that in neuroblastoma cell lines a significant fraction of cellular c-Abl is phosphorylated on Tyr-245, consistent with an open and active conformation. Notably, exposure to imatinib did not affect Tyr-245 phosphorylation. Given the low affinity of active c-Abl for imatinib, these data provide a molecular explanation for the relatively high imatinib concentrations required to inhibit neuroblastoma cell proliferation.

Kinase Genotype Analysis of Gastric Gastrointestinal Stromal Tumor Cytology Samples Using Targeted Next-Generation Sequencing

Gastric gastrointestinal stromal tumors (GISTs) usually contain the mast/stem cell growth factor receptor Kit gene (KIT) or platelet-derived growth factor receptor A (PDGFRA) mutations that can be targeted by, or mediate resistance to, imatinib. Diagnostic material often is obtained by endoscopic ultrasound-guided fine-needle aspiration, which often is unsuitable for molecular analysis. We investigated whether targeted next-generation sequencing (NGS) can be used in multiplex genotype analysis of cytology samples collected by endoscopic ultrasound-guided fine-needle aspiration. We used the Ion AmpliSeq V2 Cancer Hotspot NGS Panel (Life Technologies, Carlsbad, CA) to identify mutations in more than 2800 exons from 50 cancer-associated genes in GIST samples from 20 patients. We identified KIT mutations in 58% of samples (91% in exon 11 and 9% in exon 17) and PDGFRA mutations in 26% (60% in exon 18 and 40% in exon 12); 16% of samples had no mutations in KIT or PDGFRA. No pathogenic alterations were found in PIK3CA, BRAF, KRAS, NRAS, or FGFR3. We predicted that 32% of patients would have primary resistance to imatinib, based on mutations in exon 17 of KIT, exon 18 of PDGFRA (D842V), or no mutation in either gene. Targeted NGS of cytology samples from GISTs is feasible and provides clinically relevant data about kinase genotypes that can help guide individualized therapy.

Increased Risk of Malignant Melanoma in Patients with Systemic Mastocytosis?

Mastocytosis, a group of rare disorders that occur in both children and adults, is characterised by abnormal growth and pathological accumulation of mast cells in one or more organs, most commonly the skin (1). Urticaria pigmentosa (UP) is the most common cutaneous variant. In cases of extracutaneous involvement, systemic mastocytosis (SM) can be diagnosed on the basis of the criteria formulated by the WHO. The course of SM in most patients (90%) is indolent, with more aggressive presentation in only a few.The incidence of cutaneous melanoma is increasing and although this malignancy and mastocytosis originate from 2 different types of cells (melanocytes from the neural crest and mast cells from haematopoetic stem cells, respectively) they share certain similarities, including expression of the transcription factors MITF and STAT3, and dependence of the growth factor receptor KIT and its ligand stem cell factor for their growth and development (2, 3). We have found 5 published case reports that suggest a relationship between these 2 pathologies. In the first, published in 1979, a patient with nodular mastocytosis de -veloped both melanocytoma and mastocytoma (4). In the second, UP and SM preceded a metastatic melanoma (5) and the third involved combined mastocytoma-junctional naevus (6). In the fourth case, malignant melanoma was diagnosed prior to SM (7). And finally, a patient with telangiectasia macularis eruptive perstans (TEMP), a rare form of cutaneous mastocytosis, was found to have a malignant melanoma (8).Here, we describe our 4 additional cases and discuss possible associations between these 2 diseases.MATERIAL AND METHODS

The development of regorafenib and its current and potential future role in cancer therapy

Regorafenib is a novel multikinase inhibitor that has demonstrated broad antitumor activity across various solid tumor types, in preclinical and clinical studies. Preclinical data show inhibitory activity of angiogenic, stromal and oncogenic tyrosine kinases through the targeting of vascular endothelial growth factor receptors 1, 2 and 3, tyrosine-protein kinase receptor TIE-2, platelet-derived growth factor receptor β, fibroblast growth factor receptor 1, proto-oncogene tyrosine-protein kinase receptor Ret, mast/stem cell growth factor receptor Kit, RAF proto-oncogene serine/threonine-protein kinase and wild-type and V600E mutant serine/threonine-protein kinase B-raf. Phase I trials have shown that the drug is relatively well tolerated at doses of 160 mg daily on a 3-weeks-on/1-week-off schedule, or 100 mg daily on a continuous schedule, with adverse effects typical of other multikinase inhibitors. Phase II studies demonstrated clinical benefit in a variety of tumor types, mostly associated with prolonged stable disease. Phase III studies include the CORRECT trial, which ultimately led to FDA approval of the drug in the setting of metastatic colorectal cancer previously treated with standard therapies. There is still much work to be done to determine the role of regorafenib in the future of cancer therapy. This review will focus on the development of regorafenib, from early preclinical work through phase I, II and III trials, as well as highlighting the current role and potential future directions of this novel agent.

Lantrunculin B-induced filamentous actin disassembly enables human mast cell degranulation by SCF alone (177.7)

Abstract The release of inflammatory mediators from mast cells following antigen/IgE-mediated FcϵRI aggregation induces allergic reactions including anaphylaxis. Stem cell factor (SCF), the ligand for the mast cell growth factor receptor KIT, potentiates such release. However, despite inducing similar early signaling events as antigen, SCF alone induces minimal, if any, degranulation in the absence of other stimuli. We therefore investigated whether the process underlying these differences may lie in receptor-induced distal events involving the reorganization of the actin cytoskeleton. To explore this issue, we utilized latrunculin B, an inhibitor of actin polymerization which is known to potentiate FcϵRI-mediated mast cell degranulation. As expected, latrunculin B moderately potentiated IgE/antigen-induced human mast cell degranulation, This response was, however, markedly enhanced in the presence of SCF (100 ng/ml), especially at lower antigen concentrations, suggesting that the SCF dependent component was more sensitive to latrunculin B than the antigen component. Furthermore, although SCF alone failed to induce degranulation, in the presence of latrunculin B, it induced degranulation in a concentration-dependent manner to a similar extent as that produced by IgE/antigen. These data thus suggest that SCF provides inadequate signals for the necessary remodeling of the actin filament network for degranulation which is rectified by latrunculin B.

Hematopoietic stem cell transplantation without irradiation

Hematopoietic stem cell (HSC) transplantation is limited by histocompatibility barriers and by lack of space in bone marrow niches. These obstacles prevent in vivo analysis of histoincompatible mutant stem cells and of HSC functions in non-irradiated mice. By genetically combining immunodeficiency with impaired function of the growth factor receptor Kit in mice, we generated a 'universal' HSC recipient that efficiently accepts long-term histocompatible and histoincompatible HSCs without prior irradiation.

The tyrosine kinase network regulating mast cell activation

Mast cell mediator release represents a pivotal event in the initiation of inflammatory reactions associated with allergic disorders. These responses follow antigen-mediated aggregation of immunoglobulin E (IgE)-occupied high-affinity receptors for IgE (Fc epsilon RI) on the mast cell surface, a response which can be further enhanced following stem cell factor-induced ligation of the mast cell growth factor receptor KIT (CD117). Activation of tyrosine kinases is central to the ability of both Fc epsilon RI and KIT to transmit downstream signaling events required for the regulation of mast cell activation. Whereas KIT possesses inherent tyrosine kinase activity, Fc epsilon RI requires the recruitment of Src family tyrosine kinases and Syk to control the early receptor-proximal signaling events. The signaling pathways propagated by these tyrosine kinases can be further upregulated by the Tec kinase Bruton's tyrosine kinase and downregulated by the actions of the tyrosine Src homology 2 domain-containing phosphatase 1 (SHP-1) and SHP-2. In this review, we discuss the regulation and role of specific members of this tyrosine kinase network in KIT and Fc epsilon RI-mediated mast cell activation.

Imatinib as a Treatment Option for Systemic Non-Langerhans Cell Histiocytoses

Systemic non-Langerhans cell histiocytoses are disorders characterized by the accumulation of histiocytes that do not meet the criteria for Langerhans cells in various organs. So far, no causative treatment is known. Herein, we report the case of a 41-year-old man with Rosai-Dorfman disease, a form of systemic non-Langerhans cell histiocytoses, with histiocytic infiltrations in the skin, bone marrow, liver, and spleen. Histiocytes were positive for the imatinib target proteins platelet-derived growth factor receptor beta and KIT. The disease completely responded to treatment with 400 to 600 mg daily of imatinib for more than 7 months. This case shows that imatinib is a powerful treatment option for patients with non-Langerhans cell histiocytoses.

Epidermal Growth Factor (EGF) Receptor Ligands in the Chicken Ovary: I. Evidence for Heparin-Binding EGF-Like Growth Factor (HB-EGF) as a Potential Oocyte-Derived Signal to Control Granulosa Cell Proliferation and HB-EGF and Kit Ligand Expression

There is increasing evidence that epidermal growth factor (EGF) receptor (EGFR) ligand and Kit ligand (KL) play critical roles in controlling follicular development in mammals. Because little is known about their expressions in the ovary of nonmammalian vertebrate, our study aimed to examine the expression, hormonal regulation, and interaction of HB-EGF and KL in the chicken ovary. Using semiquantitative RT-PCR, we demonstrated that ovarian HB-EGF expression increased dramatically with the posthatching ovarian growth. In line with this finding, HB-EGF was shown to be produced primarily by the growing oocytes and capable of stimulating the proliferation of granulosa cells in prehierarchal (3 mm) and preovulatory follicles (F5 and F1). Although HB-EGF expression is mainly restricted to the oocytes, its expression in cultured granulosa cells could be transiently yet strongly induced by HB-EGF and other EGFR ligands including EGF and TGF-alpha. And the inducing effect of HB-EGF was completely abolished by AG1478 (10 microM) or PD98059 (100 microM), indicating that the action of HB-EGF is mediated by EGFR and intracellular MAPK/ERK signaling pathway. Unlike mammals, only KL-1, not the other three isoforms identified (KL-2, -3, and -4), was detected to be predominantly expressed in the chicken ovary. Interestingly, KL expression in undifferentiated and differentiated granulosa cells could be transiently down-regulated by HB-EGF, implying an intrafollicular communication between growing oocyte and surrounding granulosa cells through the interplay of EGFR ligand and KL. Collectively, our data suggest that HB-EGF is likely a paracrine signal from the oocyte to regulate granulosa cell proliferation and HB-EGF and KL expression during ovarian follicular development.

Pleomorphic characteristics of a germ-line KIT mutation in a large kindred with gastrointestinal stromal tumors, hyperpigmentation, and dysphagia.

Somatic mutations that result in the activation of the growth factor receptor KIT are commonly found in gastrointestinal stromal tumors (GISTs). Six families have been reported in which a germ-line mutation in KIT is associated with an autosomal dominant predisposition to the development of GISTs. Hyperpigmentation, urticaria pigmentosa, and dysphagia have been described in some, but not all, families. Preliminary correlations between the site of mutation and the clinical phenotype have been proposed, but the strength of these associations is not defined. A large kindred with multiple GISTs, hyperpigmentation, and dysphagia was identified after the index case presented with multiple GISTs. A germ-line mutation in KIT (W557R) was identified in an affected cousin, after which a large family meeting was held and testing offered. Clinical data were obtained by interview and, whenever possible, medical record documentation. To date, 19 individuals have been tested, and the mutation has been shown to cosegregate with the syndrome. The phenotypic expression, however, is variable. GISTs, often presenting as upper gastrointestinal bleeding, and hyperpigmentation are common, but not diagnosed in all documented or obligate carriers. Dysphagia is a less prevalent complaint. The diagnosis of GISTs appears to be made at a younger age in more recent generations. Metastatic disease is uncommon. A germ-line mutation in KIT resulting in an amino acid substitution in the juxtamembrane region is associated with a syndrome of GIST, hyperpigmentation, and dysphagia, although the prominence of each component varies.

Two domains distantly related to protein-tyrosine kinases in the vesicular stomatitis virus polymerase

We have carried out an exhaustive search for amino acid sequence similarities between vesicular stomatitis virus (VSV) proteins and database entries. Unexpectedly, we found that the L polymerase protein contains two blocks of sequence (residues 725–1102 and 1291–1671) with distant but statistically significant similarity to the catalytic domain of tyrosine-specific protein kinases. The first kinase-like region is most similar to members of the AN subfamily, Fes and Fps (26.6% and 27.3% identity, respectively), whereas the second region is closest to members of the platelet-derived growth factor receptor (PDGFR) subfamily, PDGFR and Kit (30.4% and 25.9% identity, respectively). Multiple alignment of the catalytic domain of these kinases to all three rhabdovirus L protein sequences available (VSV Indiana, VSV New Jersey, and rabies) revealed that the polymerases contain many but not all residues well conserved in the protein kinase family. Similarity was highest for VSV Indiana and lowest for rabies. We conclude that the kinase-like regions in the rhabdoviral L proteins are probably very distantly related to the protein kinase family. The similarities could either reflect contemporary protein kinase activity or represent some other function(s) associated with these large multifunctional polymerase proteins. Our findings also shed new light on questions of the origins and evolution of RNA viruses.