C-kit Receptor Tyrosine Kinase Research Articles

Epigenetics and mutations in chronic myeloproliferative neoplasms

They share substantial phenotypic mimicry, can undergo phenotypic shifts (from PV to ET and vice versa) as well as evolution to myelofibrosis (post- PV/post-ET myelofibrosis), and all eventually progress to leukemia. The hypothesis that hypersensitivity of hematopoietic stem and progenitor cells to cytokines might largely account for the pathogenesis of myeloprolif - erative neoplasms has been corroborated by the discovery of mutations that affect cytoplasmic proteins involved in cytokine signaling, either resulting in a gain-of-function (JAK2 and MPL) or a loss-of-function (CBL and LNK). Dysregulation of tyrosine kinases is a recurrent theme in chronic myeloid neoplasms, as exemplified by the consti- tutive activation of ABL caused by oligomerization of the BCR-ABL fusion protein in chronic myelogenous leukemia, the gain-of-function mutation of the tyrosine kinase receptor c-KIT in mastocytosis, and the activation of platelet-derived growth factor receptor- α or - b and fibroblast growth factor receptor in hypereosinophilic dis - orders. However, high-throughput genomic analyses of myeloproliferative neoplasms have recently identified a second group of mutations that affect proteins involved in the epigenetic regulation of transcription, such as TET2, ASXL1 and EZH2. 2 These abnormalities can occur in asso - ciation and/or with mutations targeting tyrosine kinases. Figure 1. Known functions of EZH2, TET2, ASXL1 and JAK2 in epigenetic regulation of gene expression. The upper part of each box schemat - ically depicts the main known function of each protein, while the effects of abnormal proteins are shown in the lower part. TET2 normally converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosines (5hmC). EZH2 is the catalytic subunit of the PRC2 complex and trimethylates Lys-27 of histone H3 (H3K27) leading to transcriptional repression of target genes. ASXL1 exists in chromatin-associated multiprotein com - plexes, together with PcG and TrxG proteins, involved in modifications of chromatin configuration that result in repressed and enhanced tran - scription, respectively, in a cellular context-specific manner. Described loss-of-function mutations of EZH2, TET2 and ASXL1 presumably lead to suppression of catalytic activity of these enzymes. Mutant JAK2, but not the wild-type protein, phosphorylates protein arginine methyl- transferase 5 (PRMT5), causing inhibition of its arginine methyltransferase activity on H2A and H4 (H2AR3me and H4R3me). JAK2 also phos - phorylates Tyr 41 (Y41) on histone H3 leading to decreased HP1 α binding to chromatin; the displacement of HP1 α is magnified after enhanced H3Y41 phosphorylation due to JAK2V617F.

Structural Basis for c-KIT Inhibition by the Suppressor of Cytokine Signaling 6 (SOCS6) Ubiquitin Ligase

The c-KIT receptor tyrosine kinase mediates the cellular response to stem cell factor (SCF). Whereas c-KIT activity is important for the proliferation of hematopoietic cells, melanocytes and germ cells, uncontrolled c-KIT activity contributes to the growth of diverse human tumors. Suppressor of cytokine signaling 6 (SOCS6) is a member of the SOCS family of E3 ubiquitin ligases that can interact with c-KIT and suppress c-KIT-dependent pathways. Here, we analyzed the molecular mechanisms that determine SOCS6 substrate recognition. Our results show that the SH2 domain of SOCS6 is essential for its interaction with c-KIT pY568. The 1.45-Å crystal structure of SOCS6 SH2 domain bound to the c-KIT substrate peptide (c-KIT residues 564-574) revealed a highly complementary and specific interface giving rise to a high affinity interaction (K(d) = 0.3 μm). Interestingly, the SH2 binding pocket extends to substrate residue position pY+6 and envelopes the c-KIT phosphopeptide with a large BG loop insertion that contributes significantly to substrate interaction. We demonstrate that SOCS6 has ubiquitin ligase activity toward c-KIT and regulates c-KIT protein turnover in cells. Our data support a role of SOCS6 as a feedback inhibitor of SCF-dependent signaling and provides molecular data to account for target specificity within the SOCS family of ubiquitin ligases.

Differential Contributions of c-Kit Activating Mutations to Promotion of AML1-ETO Associated Neoplasia

AbstractAbstract 4195The t(8;21) AML1-ETO translocation is one of the most frequent cytogenetic abnormalities observed in acute myeloid leukemia (AML), occurring in 10–12% of cases. The t(8;21) fuses the DNA binding domain of AML1 with the transcriptional repressor gene ETO. AML1-ETO dominantly blocks wild-type AML1 function, preventing activation of AML1 target genes and impairing myeloid differentiation. However, expression of AML1-ETO in mice does not promote AML in the absence of additional mutations. Another commonly mutated gene detected in AML1-ETO+ patient samples is the receptor tyrosine kinase c-KIT. Two classes of activating c-KIT mutations have been reported; one where mutations like D814V occur in the tyrosine kinase domain and the other involving a deletion within Exon 8 (E8D419) in a region that mediates receptor dimerization. These mutations are not randomly distributed with t(8;21) or with other chromosomal translocations found in AML. For instance, c-KITD814V is observed in 11–44% of t(8;21) patients, whereas c-KITE8Æ419 with t(8;21) is reported in 2–13% of cases. These observations suggest a degree of specificity with which activating c-KIT mutations can cooperate with AML1-ETO to promote AML. To test this hypothesis, we used a retroviral transduction/transplantation model to co-express AML1-ETO with c-KitD814V or c-KitE8Æ419 in murine bone marrow (BM) progenitor cells used to reconstitute lethally irradiated mice. Analysis of reconstituted mice has shown that simultaneous expression of AML1-ETO with c-KitD814V results in three non-overlapping phenotypes. The most common phenotype, occurring in 50% of animals, is a transplantable AML characterized by a relatively short disease latency that may or may not present with an associated granulocytic sarcoma. Second, a rapidly fatal non-transplantable myeloproliferative phenotype was observed in 25% of animals. Lastly, several mice (25%) developed a lethal, short latency B-cell leukemia that was transplantable to secondary recipient animals. The heterogeneity of disease phenotypes that develop in AML1-ETO;c-KITD814V mice may reflect the specific hematopoietic progenitor cell type that was initially transduced and capable of reconstituting the recipient mouse or differences in AML1-ETO and/or c-KitD814V expression levels. In contrast, co-expression of AML1-ETO with c-KitE8Æ419 promoted a transplantable AML phenotype characterized by a relatively long disease latency in a fraction (43.7%) of reconstituted mice. AML1-ETO; c-KitE8Æ419–expressing cells could persist for months without showing signs of selective expansion, which suggests that additional genetic changes are necessary for overt transformation to AML in this context. These observations indicate that when overlaid on the AML1-ETO genetic lesion, c-KitD814V promotes a more aggressive disease phenotype than c-KitE8Æ419, which displays a longer latency and reduced penetrance. These differences may account for the observed disparity in the frequencies of each c-KIT mutation concurrent with AML1-ETO in human AML cases. Disclosures:No relevant conflicts of interest to declare.

Tumor-intrinsic and -extrinsic roles of c-Kit: mast cells as the primary off-target of tyrosine kinase inhibitors

c-Kit tyrosine kinase receptor and its ligand stem cell factor have multiple functions during development, whereas in adulthood they are mostly needed for stem cell (SC) maintenance and mast cell (MC) biology. c-Kit plays an essential tumor-cell-intrinsic role in many types of cancer, either providing the tumorigenic force when aberrantly activated or conferring stem-like features characterizing the most aggressive variants. A tumor-cell-extrinsic role occurs through c-Kit-dependent accessory cells (such as MCs) that infiltrate tumors and deeply influence their progression. c-Kit-targeted therapy with tyrosine kinase inhibitors (TKIs) may ideally work against both tumor and stromal cells. Here, we summarize the tumor-intrinsic and -extrinsic roles of c-Kit in cancer and discuss TKIs with their on- and off-targets, with a special emphasis on MCs as paradigmatic c-Kit-dependent accomplices for tumor progression.

Gastrointestinal stromal tumours (GIST)

Background-aim Gastrointestinal stromal tumours (GIST) are the most common mesenchymal neoplasms of the gastrointestinal tract, yet extremely rare since they account for less than 1% of all GI tumours, which may arise virtually in any part of the gastrointestinal tract. GISTs are typically defined as a group of heterogeneous gastrointestinal mesenchymal neoplasms that are characterized by the expression of c-KIT receptor tyrosine kinase (called CD117 antigen) and often also the CD34 antigen. There is an equal gender distribution, with a peak incidence between the fifth and sixth decade of life. Twenty percent of GISTs are asymptomatic. Clinical symptoms are non-specific including abdominal pain, ileus and weight loss, while the most common symptom at presentation is GI bleeding. Due to the non-specific presenting symptoms, diagnosis of GIST is often delayed. Useful diagnostic tools include imaging, endoscopy and histological examinations. GISTs are prone to metastasize. Prognostic factors include tumour size, mitotic activity and tumour location. The optimal treatment is radical surgery, while treatment with tyrosine kinase inhibitor imatinib has significantly improved the outcome of the disease. In the current study, we present five cases of GISTs treated in our department during the period 2007–2009, followed by a brief review of the literature.

Mutations in the c-Kit Gene Disrupt Mitogen-Activated Protein Kinase Signaling during Tumor Development in Adenoid Cystic Carcinoma of the Salivary Glands

The Ras/mitogen-activated protein kinase (MAPK) pathway is considered to be a positive regulator of tumor initiation, progression, and maintenance. This study reports an opposite finding: we have found strong evidence that the MAPK pathway is inhibited in a subset of adenoid cystic carcinomas (ACCs) of the salivary glands. ACC tumors consistently overexpress the receptor tyrosine kinase (RTK) c-Kit, which has been considered a therapeutic target. We performed mutational analysis of the c-Kit gene (KIT in 17 cases of ACC and found that 2 cases of ACC had distinct missense mutations in KIT at both the genomic DNA and messenger RNA levels. These mutations caused G664R and R796G amino acid substitutions in the kinase domains. Surprisingly, the mutations were functionally inactive in cultured cells. We observed a significant reduction of MAPK (ERK1/2) activity in tumor cells, as assessed by immunohistochemistry. We performed further mutational analysis of the downstream effectors in the c-Kit pathway in the genes HRAS, KRAS, NRAS, BRAF, PIK3CA, and PTEN. This analysis revealed that two ACC tumors without KIT mutations had missense mutations in either KRAS or BRAF, causing S17N K-Ras and V590I B-Raf mutants, respectively. Our functional analysis showed that proteins with these mutations were also inactive in cultured cells. This is the first time that MAPK activity from the RTK signaling has been shown to be inhibited by gene mutations during tumor development. Because ACC seems to proliferate despite inactivation of the c-Kit signaling pathway, we suggest that selective inhibition of c-Kit is probably not a suitable treatment strategy for ACC.

The role of c‐kit‐positive interstitial cells in mediating phasic contractions of bladder strips from streptozotocin‐induced diabetic rats

• To investigate the role of c-kit-positive interstitial cells (ICCs) in mediating muscarinic receptor-induced phasic contractions of isolated bladder strips from streptozotocin(STZ)-induced diabetic rats and to confirm the expression and location of ICCs in the rat bladder. • Bladders were removed from STZ-induced diabetic rats at 1, 4 and 12 weeks after induction of diabetes and from age-matched controls. • To investigate the functional role of ICCs in mediating phasic contractions, bladder strips were isolated from control and diabetic rats and mounted in tissue baths. • Strips were stimulated with low concentrations of the muscarinic receptor agonist carbachol (CCH; 0.1 µm) to induce phasic contractions and the effect of increasing concentrations (1-50 µm) of imatinib (Glivec® or Gleevec®, formerly STI571), a c-kit tyrosine kinase inhibitor, was then investigated. • For molecular studies, to detect expression of the c-kit tyrosine kinase receptor (c-kit), total cellular RNA was extracted from rat bladders and reverse-transcribed to obtain complementary DNA (cDNA). • Reverse transcription-polymerase chain reaction (RT-PCR) was then performed using primers specific to the c-kit sequence and amplified products separated by agarose gel electrophoresis. • Amplified PCR products were excised from the gel, sequenced and compared with the known c-kit sequence to confirm their identity. • For immunohistochemical detection, whole mount preparations of control rat bladders were fixed in acetone and labelled using antibodies directed to the ICC marker c-kit. • In functional studies, CCH induced phasic contractions in bladder strips from control and diabetic rats. Bladder strips from 1-week diabetic rats showed CCH-induced phasic contractions, which were greater in amplitude, but had lower frequency, than the controls, whilst no such differences were apparent at later time points of diabetes. • Imatinib decreased the amplitude and the frequency of the CCH-induced phasic contractions in both control and diabetic tissues in a concentration-dependent manner, although in diabetic tissues this effect was only seen at the higher concentrations of imatinib. RT-PCR of bladder cDNA yielded a single amplicon of 480 bp. • The sequence of this amplicon showed a 98% homology with the published c-kit sequence, thus confirming c-kit mRNA expression in both control and 1-week diabetic rat bladder. • Expression of c-kit protein was also detected in a network of cells on the edge of and between smooth muscle bundles of control rat bladders by positive immunoreactivity to c-kit specific antibodies. • These data show the presence of c-kit-positive ICCs in rat urinary bladder and their importance in mediating muscarinic receptor-induced phasic contractions of bladder strips from control and diabetic rats. The role of these ICCs does not seem to be significantly altered by the diabetic state.

A novel pathway of haematopoiesis revealed after experimental malaria infection

A novel pathway of haematopoiesis revealed after experimental malaria infection

Oncogenic c-kit transcript is a target for binase

Mutational activation of c-Kit receptor tyrosine kinase is common in acute myelogenous leukemia (AML). One such activating point mutation is the N822K replacement in the c-Kit protein. Here we investigate the selective cytotoxic effect of binase - RNase from Bacillus intermedius - on FDC-P1-N822K cells. These cells were derived from myeloid progenitor FDC-P1 cells, in which ectopic expression of N822K c-kit gene induces interleukin-3 independent growth. In order to determine whether the sensitivity of these cells to binase is caused by the expression of c-kit oncogene, the cytotoxicity of the RNase was studied in the presence of selective inhibitor of mutated c-Kit imatinib (Gleevec). Inhibition of mutated c-Kit protein leads to the loss of cell sensitivity to the apoptotic effect of binase, while the latter still decreases the amount of cellular RNA. Using green fluorescent protein as an expression marker for the c-Kit oncoprotein, we demonstrate that the elimination of c-Kit is the key factor in selective cytotoxicity of binase. Quantitative RT-PCR with RNA samples isolated from the binase-treated FDC-P1-N822K cells shows that binase treatment results in 41% reduction in the amount of с-kit mRNA. This indicates that the transcript of the activated mutant c-kit is the target for toxic action of binase. Thus, the combination of inhibition of oncogenic protein with the destruction of its mRNA is a promising approach to eliminating malignant cells.

Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT+ Cancers

Oncogenic mutations of the receptor tyrosine kinase c-KIT play an important role in the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis, and some acute myeloid leukemias (AML). Although juxtamembrane mutations commonly detected in gastrointestinal stromal tumor are sensitive to tyrosine kinase inhibitors, the kinase domain mutations frequently encountered in systemic mastocytosis and AML confer resistance and are largely unresponsive to targeted inhibition by the existing agent imatinib. In this study, we show that myeloid cells expressing activated c-KIT mutants that are imatinib sensitive (V560G) or imatinib resistant (D816V) can inhibit the tumor suppressor activity of protein phosphatase 2A (PP2A). This effect was associated with the reduced expression of PP2A structural (A) and regulatory subunits (B55alpha, B56alpha, B56gamma, and B56delta). Overexpression of PP2A-Aalpha in D816V c-KIT cells induced apoptosis and inhibited proliferation. In addition, pharmacologic activation of PP2A by FTY720 reduced proliferation, inhibited clonogenic potential, and induced apoptosis of mutant c-KIT(+) cells, while having no effect on wild-type c-KIT cells or empty vector controls. FTY720 treatment caused the dephosphorylation of the D816V c-KIT receptor and its downstream signaling targets pAkt, pSTAT5, and pERK1/2. Additionally, in vivo administration of FTY720 delayed the growth of V560G and D816V c-KIT tumors, inhibited splenic and bone marrow infiltration, and prolonged survival. Our findings show that PP2A inhibition is essential for c-KIT-mediated tumorigenesis, and that reactivating PP2A may offer an attractive strategy to treat drug-resistant c-KIT(+) cancers.

Can a familial gastrointestinal tumour syndrome be allelic with Waardenburg syndrome?

Familial gastrointestinal stromal tumours (GISTs) are rare but otherwise well-characterized tumour syndromes, most commonly occurring on a background of germline-activating mutations in the tyrosine kinase receptor c-KIT. The associated clinical spectrum reflects the constitutive activation of this gene product across a number of cell lines, generating gain-of-function phenotypes in interstitial cells of Cajal (GIST and dysphagia), mast cells (mastocytosis) and melanocytes (hyperpigmentation). We report a three-generation kindred harbouring a c-KIT germline-activating mutation resulting in multifocal GISTs, dysphagia and a complex melanocyte hyperpigmentation and hypopigmentation disorder, the latter with features typical of those observed in Waardenburg type 2 syndrome (WS2F). Sequencing of genes known to be causative for WS [microphthalmia transcription factor (MITF), Pax3, Sox10, SNAI2 ] failed to show any candidate mutations to explain this complex cutaneous depigmentation phenotype. Our case report conclusively expands the clinical spectrum of familial GISTs and shows a hitherto unrecognized link to WS. Possible mechanisms responsible for this novel cause of WS2F will be discussed.

The receptor tyrosine kinase c-Kit controls IL-33 receptor signaling in mast cells

AbstractMembers of the Toll/interleukin-1 receptor (TIR) family are of importance for host defense and inflammation. Here we report that the TIR-family member interleukin-33R (IL-33R) cross-activates the receptor tyrosine kinase c-Kit in human and murine mast cells. The IL-33R–induced activation of signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase B (PKB), and Jun NH2-terminal kinase 1 (JNK1) depends on c-Kit and is required to elicit optimal effector functions. Costimulation with the c-Kit ligand stem cell factor (SCF) is necessary for IL-33–induced cytokine production in primary mast cells. The structural basis for this cross-activation is the complex formation between c-Kit, IL-33R, and IL-1R accessory protein (IL-1RAcP). We found that c-Kit and IL-1RAcP interact constitutively and that IL-33R joins this complex upon ligand binding. Our findings support a model in which signals from seemingly disparate receptors are integrated for full cellular responses.

Role for the Proapoptotic Factor BIM in Mediating Imatinib-induced Apoptosis in a c-KIT-dependent Gastrointestinal Stromal Tumor Cell Line

The c-KIT receptor tyrosine kinase is constitutively activated and oncogenic in the majority of gastrointestinal stromal tumors. The identification of selective inhibitors of c-KIT, such as imatinib, has provided a novel therapeutic approach in the treatment of this chemotherapy refractory tumor. However, despite the clinical importance of these findings and the potential it provides as a model system for understanding targeted therapy, this approach has not yielded curative outcomes in most patients, and the biochemical pathways connecting c-KIT inhibition to cell death are not completely understood. Here, we show that inhibition of c-KIT with imatinib in gastrointestinal stromal tumors (GISTs) triggered the up-regulation of the proapoptotic protein BIM via both transcriptional and post-translational mechanisms. The inhibition of c-KIT by imatinib increased levels of the dephosphorylated and deubiquitinated form of BIM as well as triggered the accumulation of the transcription factor FOXO3a on the BIM promoter to activate transcription of BIM mRNA. Furthermore, using RNA interference directed against BIM, we demonstrated that BIM knockdown attenuated the effects of imatinib, suggesting that BIM functionally contributes to imatinib-induced apoptosis in GIST. The identification and characterization of the pathways that mediate imatinib-induced cell death in GIST provide for a better understanding of targeted therapy and may facilitate the development of new therapeutic approaches to further exploit these pathways.

Abstract 3953: C-Kit and Mitf signaling: Novel involvement of the PI3K/Akt and p38 pathways

Abstract The development of melanocyte from melanoblast is regulated by the tyrosine kinase receptor c-Kit and the basic-helix-loop-heliz-leucine zipper transcription factor Mitf. It is established that Mitf activity is controlled by c-Kit activation and subsequent phosphorylation of the Erk/Mapk pathway. Serine phosphorylation (activation) of Mitf not only leads to upregulation of Mitf target gene expression but also ubiquitin dependent proteosomal degradation of Mitf. There is little knowledge surrounding the dysregulation between c-Kit and Mitf signaling that is often found in melanocyte progression to melanoma. The aim of our study is, therefore, to identify the signal transduction pathways involved in c-Kit mediated regulation of Mitf. In our study, we found c-Kit tyrosine phosphorylation sites and novel signaling pathways involved in the regulation of Mitf. Phosphorylation of c-Kit at Tyr721 has been shown to activate the PI3K pathway and Tyr568 and Tyr568/570 are responsible for Src kinase mediated activation of Erk/Mapk pathway. We demonstrate that Y721F, Y568F and Y568/570F mutation of c-Kit prevented Mitf mobility shift, indicating that these tyrosine phosphorylation sites are involved in Mitf regulation. Further, we observe that treatment of c-Kit and Mitf transfected HEK293T cells with LY294002 (PI3K inhib.), Akt IV (Akt inhib.), SU6656 (Src kinase inhib.), SB203580 (p38 inhib.) or U0126 (Mek inhib.) prevented c-Kit mediated Mitf mobility shift. Similar results were also observed in the murine melanocyte, Melan- A cell line where Mitf and c-Kit are endogenously expressed. In order to further verify c-Kit's role as Mitf regulator, we examined tyrosinase (downstream target of Mitf) promoter activity using a luciferase reporter construct. Ligand stimulation of c-Kit increased the Mitf target promoter activity. In conclusion, for the first time, we demonstrate that the signal transduction between c-Kit and Mitf is dependent on, 1) c-Kit Tyr721/PI3K/Akt pathway, 2) p38 pathway and 3) c-Kit Tyr568 or Tyr568/570 / Src kinase mediated Erk pathway. C-Kit activation leading to downstream were also shown to promote the transcriptional activity of Mitf. Our results reveal novel mechanisms by which c-Kit signaling regulates Mitf, with implication for understanding of both melanocyte development and melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3953.

Abstract 1439: De novo expression of c-kit in prostate cancer cells interacting with the bone microenvironment

Abstract Specific cellular and molecular interactions between prostate cancer (PC) cells and the bone microenvironment lead to expansion of the metastatic deposit, disease progression, and ultimately significant morbidity and mortality. We recently reported an association between expression of the tyrosine kinase receptor c-kit and clinical PC progression, with the highest expression seen in clinical metastatic bone specimens. Moreover, we found that intraosseous tumors formed by otherwise c-kit negative PC3 cells injected into human bone xenografts in SCID mice (SCID-hu model) expressed c-kit. To determine the relevance of these findings, we examined additional human PC cell lines, namely LNCaP, C42b, and DU145, for the expression of c-kit in vitro and in vivo. While none of the cultured PC cells expressed c-kit at the message or protein level, immunostaining for c-kit in intraosseous tumors produced by these cells in the SCID-hu model revealed a distinctive and specific reactivity in the cancer cell compartment. The induction of c-kit expression in PC cells growing within the bone microenvironment was emulated by co-culture of PC3 cells and bone-derived cells embedded in Matrigel, as demonstrated by RT-PCR analysis. In an attempt to mimic the de novo c-kit expression observed in the SCID-hu model and to identify potential signaling pathways triggered by c-kit activation by its ligand stem cell factor (SCF), normally expressed by the bone microenvironment, we transiently transfected PC3 cells with wild-type c-kit. Activation of c-kit was confirmed by immunoblotting for phosphorylated c-kit. Using the whole human genome Agilent microarray, we found that 220 genes were differentially expressed when SCF-treated and non-treated c-kit-transfected PC3 cells were compared, resulting in discrete functional sub-networks that were coordinately up or down-regulated. Most notably, pathway and genetic network analysis revealed that c-kit activation resulted in an up-regulated fibroblast growth factor-2-mediated sub-network. Taken together, these results suggest that c-kit induction in PC cells in the bone microenvironment modulates cell-cell interaction pathways and growth factor signaling systems that ultimately lead to expansion of bone metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1439.

Abstract 351: Mutations of genes in the c-Kit receptor tyrosine kinase signaling pathway are inactive in adenoid cystic carcinoma of the salivary glands: Implications for c-Kit targeted therapy

Abstract Adenoid cystic carcinoma (ACC) is a malignant salivary gland tumor well-known for spreading in wide local area and invasion of vital structures of the head and neck. Unfortunately, conventional therapies have not improved longer-term survival, resulting in significant morbidity and a very low overall survival rate. Improved systemic therapies are clearly needed for this population. ACC tumors overexpress the receptor tyrosine kinase c-Kit, which is thought to be a therapeutic target. However, the role of c-Kit in the pathogenesis of ACC is not currently understood. We addressed this question through a functional genomic study with 17 sporadic ACC tumor specimens. We found heterogeneous missense mutations in three genes in the c-Kit signaling pathway, although mutations were identified at a low frequency. Two ACC tumors had distinct missense mutations in the gene KIT, resulting in G664R and R796G amino acid substitutions in the c-Kit kinase domain. Furthermore, two ACC tumors without KIT mutations had missense mutations in either KRAS or BRAF, causing S17N K-Ras and V590I B-Raf mutants. KRAS and BRAF are downstream effectors of c-Kit. We explored the functional consequences of these amino acid substitutions and found that all of c-Kit, K-Ras and B-Raf mutations in our ACC samples were inactive. These observations suggest that the c-Kit signaling pathway must be dispensable for maintaining established ACC. As a result, we believe that selective c-Kit inhibition is not a suitable treatment for the patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 351.

Le carcinome nasopharyngé en Afrique du Nord et dans le Sud-Est asiatique : entre similitude et différence

Nasopharyngeal carcinoma (NPC) is an unusual head and neck cancer because of its unequal geographical distribution and its consistent association with the Epstein-Barr virus (EBV). This malignant tumor poses a serious public health problem in many countries, especially in Southeast Asia and North Africa where the recorded incidence are highest. During the past decade, a growing number of studies were undertaken to define the molecular basis of NPC. However, the analysis of several clinical and biological parameters of North African and Southeast Asian NPCs has shown notable differences, suggesting that they could result from a distinct combination of etiological factors. One intriguing characteristic of North African NPC, concerns its bimodal age distribution with a secondary peak of incidence in the range of 15-25 years, not observed in Asian NPC. In this juvenile form of NPC, immuno-histochemistry assay has shown that the two key proteins controlling the apoptotic-survival balance p53 and Bcl-2 are less frequently expressed whereas the transmembrane tyrosine-kinase receptor c-kit and the main EBV oncoprotein LMP1 were more abundant. In addition, the EBV serological alterations are less informative for the diagnosis of the juvenile compared to the adult form. In addition, most North African NPCs contain EBV strains with genetic polymorphisms distinct from those described in the Southeast Asia series (predominance of F, D, H1-H2, XhoI+ and f, C, H, XhoI– respectively). In contrast, studies relating on tumor chromosomal alterations or aberrant promoter methylation result in data very similar to those obtained from the Southeast Asia series, supporting the concept of a common molecular basis for all NPC regardless of patient geographic origin.

Dasatinib Inhibits the Growth of Molecularly Heterogeneous Myeloid Leukemias

Dasatinib is a dual Src/Abl inhibitor recently approved for Bcr-Abl+ leukemias with resistance or intolerance to prior therapy. Because Src kinases contribute to multiple blood cell functions by triggering a variety of signaling pathways, we hypothesized that their molecular targeting might lead to growth inhibition in acute myeloid leukemia (AML). We studied growth factor-dependent and growth factor-independent leukemic cell lines, including three cell lines expressing mutants of receptor tyrosine kinases (Flt3 or c-Kit) as well as primary AML blasts for responsiveness to dasatinib. Dasatinib resulted in the inhibition of Src family kinases in all cell lines and blast cells at approximately 1 x 10(-9) mol/L. It also inhibited mutant Flt3 or Kit tyrosine phosphorylation at approximately 1 x 10(-6) mol/L. Mo7e cells expressing the activating mutation (codon 816) of c-Kit were most sensitive to growth inhibition with a GI(50) of 5 x 10(-9) mol/L. Primary AML blast cells exhibited a growth inhibition of <1 x 10(-6) mol/L. Cell lines that showed growth inhibition at approximately 1 x 10(-6) mol/L showed a G(1) cell cycle arrest and correlated with accumulation of p21 and p27 protein. The addition of rapamycin or cytotoxic agents enhanced growth inhibition. Dasatinib also caused the apoptosis of Mo7e cells expressing oncogenic Kit. Although all of the precise targets for dasatinib are not known, this multikinase inhibitor causes either growth arrest or apoptosis in molecularly heterogeneous AML. The addition of cytotoxic or targeted agents can enhance its effects.

Colony stimulating factor-1 receptor as a target for small molecule inhibitors

Imatinib, dasatinib, sunitinib, CEP-701, and PKC-412, ATP-competitive small molecule inhibitors of type III receptor tyrosine kinases c-KIT and/or FLT3, were evaluated for binding to the closely related receptor, FMS, by docking into models of inactive and active conformations of the FMS kinase domain. To confirm the docking predictions, the drugs were tested for their activity and selectivity in inhibiting cell proliferation and FMS phosphorylation upon stimulation by the FMS ligand, CSF-1. All five drugs inhibited FMS activity. Imatinib, dasatinib and CEP-701 represent three different types of interactions determining drug potency and selectivity.

The complexity of the complicity of mast cells in cancer

Mast cells are evolutionarly ancient cells of the immune cells which can secrete a variety of effector molecules. Animal and pathologic studies suggest that mast cells may promote tumor growth in some cancer types but may act in an opposite manner in others. In several mouse models a critical role of mast cells for tumor promotion was demonstrated. In humans mast cells are dependent upon the tyrosine kinase receptor c-Kit. This receptor is inhibited by many of the new anti-cancer tyrosine kinase inhibitors including Pazopanib, Imatinib and Masitinib. These drugs probably ablate some tumor mast cells, in addition to their other known antitumor effects. Understanding the complex roles of mast cells in cancer should aid in understanding mechanisms of current tyrosine kinase inhibitors, and the development of innovative anti-cancer therapies.