Peter Besmer - US grants

The objectives of our Research program are: 1) to determine the basis of the differing biology of Abelson MuLV and the HZ-abl-FeSV; 2) to identify and characterize viral oncogenes in new FeSV strains. The HZ-3590-FeSV has been recently isolated in our laboratories from a multicentric fibrosarcoma. The characterization of the oncogene of this virus revealed homology with the oncogene abl of Abelson-MuLV. Like the A-MuLV it specifies a gag-abl protein with protein kinase activity. In order to understand the differing biology we will investigate the in vivo and in vitro target specificities of this virus and evaluate the contribution of the v-abl insert and of the LTR to the biological properties of this virus. We will clone the integrated HZ-abl-FeSV genome, and we will characterize the v-abl insert. We will determine the sequence relationship between the cat-v-abl and the mouse c-abl and v-abl boundaries. We will sequence the v-abl boundaries and the LTR of this virus. We will construct LTR-gage-abl chemeras in order to investigate whether LTR sequences determine the target specificities of the virus. We will investigate the targets for transformation and consequences of transformation by the abl-FeSV and the in vitro made recombinant viruses: by inoculation into neonate mice and kittens, by in vitro transformation assays, by analyzing the phenotype of the transformants. Three feline sarcoma viruses have been characterized extensively. Two of them contain the oncogene fes, which is related to the avian oncogene fps. A third contains the oncogene fms. We have recently characterized two new FeSV's, the PI-FeSV and the HZ-3590-FeSV. In the PI-FeSV sequences related to the Simian Sarcoma Virus (SSV) oncogene sis were identified. In the HZ-3590-FeSV sequences related to the oncogene abl of Abelson MuLV. More FeSV's are being isolated in W. D. Hardy's Laboratory. We therefore will continue our effort to characterize new FeSV's

The objectives of our proposal are twofold: a) to investigate the mechanism of c-kit receptor activation/function and b) to determine the molecular basis of the pleiotropic functions of c-kit, i.e. to elucidate the mechanism of the control of expression of the c-kit gene. The proto-oncogene c-kit is the normal cellular homolog of the oncogene v- kit of the HZ4 - feline sarcoma virus (Besmer et al., 1986) and encodes a transmembrane tyrosine protein kinase receptor. c-kit has been determined to be allelic with the dominant white spotting locus (W) of the mouse. Mutations at the W locus affect various aspects of hematopoiesis and the proliferation and/or migration of primordial germ cells and melanoblasts during development. The recent analysis of several dominant E mutations revealed that they are dominant negative mutations providing clues about the mechanism of kit mediated signal transduction. We will use an in vitro mutagenesis approach to further define the mechanism of activation of the c-kit tyrosine kinase receptor and of the P80gag-kit transforming protein. A most interesting aspect of the c-kit/W receptor system is its pleiotropy. c-kit function is the result of a combination of factors, namely, availability of ligand, the expression of the c-kit receptor and a functioning downstream signaling pathway. An important concern of our proposal is to further our understanding of the mechanism of the control of c-kit expression at the level of transcription and post transcriptional processing. Tissue specific regulatory sequences in c-kit will be identified by investigating transcription start sites, determining DNaseI hypersensitive sites, determining the transcription rates, by deletion mapping of the 5' flanking region of the c-kit gene with receptor gene constructs and by performing binding experiments to identify cell specific nuclear transacting factors. We will also attempt to create a mutation in a tissue specific promoter element of c-kit in the mouse germ line by use of homologous recombination technology and embryonic stem cells.

The genes at the W and S1 loci encode a tyrosine kinase receptor and its ligand, respectively. W and S1 mutations cause defects in development of primordial germ cells and later germ cells, as well as of hematopoietic cells and melanocytes. Dr. Bachvarova has shown that c-kit (encoded at the W locus) is expressed on the surface of oocytes from the stage of stored primordial oocytes through oocyte growth, meiotic maturation, and in development to the 2-cell stage. The principal aim here is to elucidate the role of c-kit in oocyte growth, survival, development and/or adhesion. Expression of ligand in the ovary will be examined by in situ hybridization, immunocytochemistry and functional assays. These results may elucidate what function is involved. She will determine whether functional c-kit is present on oocytes using an immunoprecipitation and autophosphorylation assay. Using in vitro culture systems, she will test whether addition of the ligand promotes aspects of oocyte development. The effect of adding KL to oocytes cultured under suboptimal conditions will be tested, as well as the effect of culture on cell lines expressing transfected or genetically mutated forms of KL. Effects on oocytes growth, survival, and time to undergo meiotic maturation will be examined. She will also determine whether added KL can induce the initiation of oocyte growth in vitro. Finally, she will determine whether blocking antibodies directed against either ligand or receptor can inhibit these processes.

The objectives of our proposal are twofold: a) to investigate the mechanism of c-kit receptor activation/function and b) to determine the molecular basis of the pleiotropic functions of c-kit, i.e. to elucidate the mechanism of the control of expression of the c-kit gene. The proto-oncogene c-kit is the normal cellular homolog of the oncogene v- kit of the HZ4 - feline sarcoma virus (Besmer et al., 1986) and encodes a transmembrane tyrosine protein kinase receptor. c-kit has been determined to be allelic with the dominant white spotting locus (W) of the mouse. Mutations at the W locus affect various aspects of hematopoiesis and the proliferation and/or migration of primordial germ cells and melanoblasts during development. The recent analysis of several dominant E mutations revealed that they are dominant negative mutations providing clues about the mechanism of kit mediated signal transduction. We will use an in vitro mutagenesis approach to further define the mechanism of activation of the c-kit tyrosine kinase receptor and of the P80gag-kit transforming protein. A most interesting aspect of the c-kit/W receptor system is its pleiotropy. c-kit function is the result of a combination of factors, namely, availability of ligand, the expression of the c-kit receptor and a functioning downstream signaling pathway. An important concern of our proposal is to further our understanding of the mechanism of the control of c-kit expression at the level of transcription and post transcriptional processing. Tissue specific regulatory sequences in c-kit will be identified by investigating transcription start sites, determining DNaseI hypersensitive sites, determining the transcription rates, by deletion mapping of the 5' flanking region of the c-kit gene with receptor gene constructs and by performing binding experiments to identify cell specific nuclear transacting factors. We will also attempt to create a mutation in a tissue specific promoter element of c-kit in the mouse germ line by use of homologous recombination technology and embryonic stem cells.

The objectives of our proposal are twofold: a) to investigate the mechanism of c-kit receptor activation/function and b) to determine the molecular basis of the pleiotropic functions of c-kit, i.e. to elucidate the mechanism of the control of expression of the c-kit gene. The proto-oncogene c-kit is the normal cellular homolog of the oncogene v- kit of the HZ4 - feline sarcoma virus (Besmer et al., 1986) and encodes a transmembrane tyrosine protein kinase receptor. c-kit has been determined to be allelic with the dominant white spotting locus (W) of the mouse. Mutations at the W locus affect various aspects of hematopoiesis and the proliferation and/or migration of primordial germ cells and melanoblasts during development. The recent analysis of several dominant E mutations revealed that they are dominant negative mutations providing clues about the mechanism of kit mediated signal transduction. We will use an in vitro mutagenesis approach to further define the mechanism of activation of the c-kit tyrosine kinase receptor and of the P80gag-kit transforming protein. A most interesting aspect of the c-kit/W receptor system is its pleiotropy. c-kit function is the result of a combination of factors, namely, availability of ligand, the expression of the c-kit receptor and a functioning downstream signaling pathway. An important concern of our proposal is to further our understanding of the mechanism of the control of c-kit expression at the level of transcription and post transcriptional processing. Tissue specific regulatory sequences in c-kit will be identified by investigating transcription start sites, determining DNaseI hypersensitive sites, determining the transcription rates, by deletion mapping of the 5' flanking region of the c-kit gene with receptor gene constructs and by performing binding experiments to identify cell specific nuclear transacting factors. We will also attempt to create a mutation in a tissue specific promoter element of c-kit in the mouse germ line by use of homologous recombination technology and embryonic stem cells.

The objectives of this proposal are threefold: a) to investigate-aspects of c-kit mediated signaling in hematopoietic stem cell populations, b) to investigate the respective role of the soluble and the cell membrane forms of the ligand KL of the c-kit receptor and c) to determine the significance of co-expression of the c-kit receptor and kit-ligand, KL i.e. autocrine mechanisms. C-kit is the cellular homolog of the oncogene v-kit and encodes a receptor tyrosine kinase which belongs to the platelet-derived growth factor (PDGF) receptor subfamily (Besmer et al., 1986). We and others have identified and characterized the ligand of the c-kit receptor we designated KL. The KL gene encodes a cell surface molecule which may be proteolytically processed to produce a soluble product similar or identical to the one we had purified. While the c-kit gene is allelic with the murine white spotting locus (W), the KL gene was shown to be allelic with the murine steel locus. Mutations at both the W and the Sl loci affect various aspects of hematopoiesis, including the stem cell hierarchy, and the proliferation and/or migration of primordial germ cells and melanoblasts during development. Based on the analysis of mutant phenotypes the cellular responses the c-kit receptor appears to mediate are quite diverse and include: cell proliferation, cell survival, cell migration, cell differentiation and other post mitotic functions. The elucidation of the mechanisms of these different responses is of fundamental importance. In part, the current proposal is designed to address issues concerning the mechanism of c*it mediated survival and proliferation (self renewal) in hematopoietic stem cell populations. The cell-membrane bound nature of KL poses important questions about its function and mode of action. The current proposal is designed to address some of these issues. The goals toward this end include: l). An analysis of KL processing and elucidation of the function of soluble and membrane bound KL. 2) The in vivo analysis of mutant versions of KL in transgenic and chimeric mice. 3) The construction of mice expressing KL-2 but not KL- 1 by germline modification. 4) The characterization of the hematopoietic stem cell compartment in Sl mutant mice. 5) The derivation of transgenic mice expressing human versions of KL to facilitate engraftment and development of human hematopoietic cells in mouse models. 6) The evaluation of autocrine mechanisms involving cell membrane associate and soluble forms of KL in in vitro cell culture systems and in in vivo models.

The overall objectives of this proposal is to investigate the role of PI 3- kinase in c-kit receptor mediated processes in vivo and in vitro. The proto-oncogene c-kit, encodes a receptor tyrosine kinase (RTK) which belongs to a family of receptors that includes the platelet- derived growth factor (PDGF). The ligand of the c-kit receptor, designated KL, encodes a cell surface molecule which may be processed to produce a soluble product. Whereas the c-kit gene is allelic with the white spotting locus (W) in the mouse, kit-ligand (KL) is allelic with the steel locus (Sl) in the mouse. W and Sl mutations affect various aspects of hematopoiesis, including the stem cell compartment, gametogenesis during embryonic development and in the adult animal, causing macrocytic anemia, mast cell deficiency anemia, mast cell deficiency, spotting, male and female sterility and megacolon. Based on mutant phenotypes the cellular responses mediated by the c- kit receptor are quite diverse and include: cell proliferation, cell survival, cell adhesion, cell migration, secretion of mediators, cell differentiation and other post mitotic functions. The elucidation of the mechanisms of these different responses is of fundamental importance. Similar to other RTK's Kit mediates its action through a variety of cytoplasmic signal transducers including PI 3-kinase in KIT mediated adhesion of BMMC to a fibronectin matrix and potentiation of IgE mediated secretion in BMMC. To study Kit/PI 3-kinase mediated processes in other cell systems we will modify the c-kit gene in the germ line to mutate the PI 3-kinase binding site in the Kit receptor by using a cre-lox based strategy and derive mice homozygous for this mutation. We then will study the effect of the mutation on various targets of W and SI mutations, the dynamics of hematopoietic cell populations and their behavior in situations that require cell adhesion and migration, i.e. homing to the marrow compartment and mobilization of stem cells into the periphery. We will also modify tyrosine residues in the juxtamembrane region of the Kit receptor in ES cells which affect KL mediated cell proliferation but not suppression of apoptosis and investigate the consequences of this mutation in vivo. Finally, we will investigate the mechanism of Kit mediated suppression of radiation and factor deprivation induced apoptosis in mast cells.

The overall objective of this proposal is to continue our investigations into the roles of soluble and membrane Kit-ligand (KL) in vivo and the mechanism of the formation of soluble KL in vivo and in vitro. Membrane growth factors which are processed to produce soluble ligands may function both as soluble factors and as membrane factors. Important questions concerning membrane growth factors include the role of soluble and membrane forms in vivo and the elucidation of mechanisms governing their expression. The membrane growth factor Kit-Ligand (KL), the ligand of the Kit receptor tyrosine kinase, is encoded at the Sl locus and mice carrying SIS mutations have defects in hematopoiesis, gametogenesis and melanogenesis. Two alternatively spliced KL transcripts encode two cell associated KL protein products, KL-1 and KL-2. The KL-2 protein lacks the major proteolytic cleavage site for the generation of soluble KL, thus representing a more stable cell-associated form of KL. In this application in Specific Aim 1 we propose to investigate the regulation of KL-1 and KL- 2 mRNA expression in vivo during embryogenesis and in the postnatal animal by tagging KL-1 and KL-2 mRNAs using a novel knock-in strategy. In Specific Aim 2 we will continue our investigation of the in vivo role of membrane and soluble KL using knock-in mouse models. A) For this purpose mice expressing a membrane obligatory KL-2 protein by knock-in technology will be made and phenotypes of heterozygous and homozygous mutant mice will be characterized with particular emphasis on the determination of the dynamics of hematopoietic cell populations in mutant mice. B) By using knock-in technology mice expressing KL-1 exclusively will be obtained and the phenotypes of hetero-and homozygous mutant mice will be characterized. Specific Aim 3 addresses the role of cytoplasmic domain sequences of KL in KL/Kit function. In these studies we will examine the consequences, in vivo, a) of replacement of cytoplasmic domain sequences with those of CSF- 1 and b) of deletion of cytoplasmic domain sequences of KL by using knock- in strategy. Furthermore we propose to use fetal liver derived stromal cell lines capable of supporting long-term hematopoiesis in vitro to investigate role of cytoplasmic domain using cell biological approaches. Specific Aim 4 focuses on the elucidation of the mechanism of proteolytic processing of KL-1 and KL-2 and Kit receptor products. In these studies we will evaluate in vitro KL-1, KL-2 and Kit peptides as target sequences for proteolytic cleavage by disintegrins TACE, MDC9 and MDC15. Furthermore, in order to evaluate the in vivo role of disintegrins in the production of soluble KL and Kit in serum knock-out mouse models will be used.

DESCRIPTION (provided by applicant): The overall objective of this proposal is to continue our investigations into the mechanisms of Kit receptor signaling in vitro and in vivo with emphasis on hematopoiesis. Furthermore, we will develop mouse models to investigate roles for Kit in oncogenesis. The Kit receptor tyrosine kinase encoded at the murine W locus functions in gametogenesis, hematopoiesis and melanogenesis Normal Kit receptor mediated functions include cell proliferation, cell survival, cell adhesion, cell migration, secretory response and differentiation. In human neoplasia oncogenic activation of Kit is thought to have roles in mastocytosis/mast cell leukemia, acute myelogenous leukemia, gastro intestinal stromal tumors (GlST) and germ cell tumors. Kit receptor functions are mediated by kinase activation, receptor autophosphorylation and association with various signaling molecules, We had investigated the role of PI 3-kinase and Src kinases in Kit mediated cell proliferation, suppression of apoptosis, cell adhesion and secretory responses. Analysis of Kit-/- BMMC expressing mutant Kit receptors indicated that both pathways contribute to the proliferative and the cell survival responses and that elimination of both pathways abolishes them. These studies also revealed that the P1 3-kinase and Src kinase signaling pathways converge to activate Raci and JNK. Moreover, recruitment and activation of Fl 3-kinase was shown to play a critical role in mediatingn cell adhesion and secretory responses. To investigate the consequences in vivo of blocking Kit mediated Fl 3-kinase activation we have mutated tyrosine 719 in the mouse c-kit gene (KitY719F), a known binding site for the p85 subunit of Fl 3-kinase. Analysis of homozygous mutant KitY719F/KitY719F mice indicated essential roles for Kit induced PIl 3-Kinase activity in mouse gametogenesis. In hematopoiesis phenotypes were minimal showing an effect on peritoneal mast cell numbers and no other phenotypes. These findings emphasize the importance of the cellular context for Kit receptor signaling in vivo. The purpose of this application is twofold: 1) to investigate more precisely the mechanism and the consequences of Kit mediated Fl 3-kinase and Kit mediated src signaling in vivo in hematopoiesis, and 2) to construct mouse models for investigating the role of Kit in oncogenesis (hematopoietic malignancies and gastrointestinal stromal tumors) and to elucidate the mechanisms of signaling by oncogenically activated Kit receptors.

DESCRIPTION (provided by applicant): The overall objective of this proposal is to investigate: 1) mechanisms leading to the formation of gastrointestinal stromal tumor (GIST) and 2) the consequences of therapeutic intervention, by using a mouse model we have recently developed for this disease. The Kit receptor tyrosine kinase encoded at the murine W locus. Kit loss of function mutations result in major deficiencies in several major cell systems during embryogenesis and in the postnatal animal including gametogenesis, hematopoiesis, melanogenesis, and interstitial cells of Cajal (ICC) in the gastrointestinal tract. Normal Kit receptor mediated functions include cell proliferation, cell survival, cell adhesion, cell migration, secretory responses, and differentiation. In addition, in human neoplasia oncogenic activation of Kit is thought to have roles in gastro intestinal stromal tumors (GIST), mastocytosis/mast cell leukemia, acute myelogenous leukemia, and germ cell tumors. Activating Kit mutations are found in all of these neoplasms. Based on the finding of familial cases of GIST syndrome we have developed a mouse model for familial GIST syndrome by targeted mutation of the Kit receptor tyrosine kinase gene using a knock-in strategy. We now propose to use this mouse model to investigate mechanism of the development of GIST, to study the consequences of therapeutic intervention in mice with GIST and to elucidate mechanisms of signaling by oncogenetically activated Kit receptors. We furthermore propose to investigate the effect of the KitV558del mutation on the development of ICC networks during embryonic development and on embryonic heart development.