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WO2001034201A9 - Methodes de detection d'agents utiles pour inhiber la neurofibromatose de type 1 (nf1) - Google Patents

Methodes de detection d'agents utiles pour inhiber la neurofibromatose de type 1 (nf1)

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Publication number
WO2001034201A9
WO2001034201A9 PCT/US2000/030924 US0030924W WO0134201A9 WO 2001034201 A9 WO2001034201 A9 WO 2001034201A9 US 0030924 W US0030924 W US 0030924W WO 0134201 A9 WO0134201 A9 WO 0134201A9
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Prior art keywords
nfl
cells
kinase
ras
scf
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WO2001034201A3 (fr
WO2001034201A2 (fr
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Wade D Clapp
David A Ingram
Mark Steven Marshall
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Indiana University Research and Technology Corp
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Indiana University Research and Technology Corp
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Priority to EP00975626A priority Critical patent/EP1227846A2/fr
Priority to JP2001536198A priority patent/JP2003513939A/ja
Priority to AU13647/01A priority patent/AU1364701A/en
Priority to CA002388343A priority patent/CA2388343A1/fr
Publication of WO2001034201A2 publication Critical patent/WO2001034201A2/fr
Publication of WO2001034201A3 publication Critical patent/WO2001034201A3/fr
Anticipated expiration legal-status Critical
Publication of WO2001034201A9 publication Critical patent/WO2001034201A9/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • Neurofibromatosis type 1 is one of the most common genetic disorders in man and is caused by a mutation in the gene NFl.
  • NFl is a panethnic, autosomal dominant disorder with an incidence of 1 in 3000.
  • Patients with this disorder have multiple clinical sequelae including the development of benign and malignant tumors, particularly in the skin and brain, e.g., tumors in the skin called neurofibromas which are infiltrated with large numbers of mast cells, hype ⁇ igmentated areas of skin, bone disorders, e.g., curvature of the spine (scoliosis), learning disabilities, myeloid malignancies, high blood pressure, and numerous other complications in several organ systems.
  • Nonmalignant complications of NFl are very common, frequently debilitating, and a cause of decreased productivity and well-being to the affected patients.
  • there are no specific treatments for the learning disorders associated with NFl although some patients have been treated with anticonvulsants and others have been treated with medications that modulate hyperreactivity.
  • mast cell stabilizing agents are the only current therapy for the treatment of the growth of neurofibromas, it is controversial whether this treatment offers any efficacy at all.
  • Tumors in NFl patients e.g., tumors that impinge on other tissues such as the plexiform fibromas, have only been treated surgically. This therapy has sequelae because of difficulty in removing the tumor, and the surgery is oftentimes associated with morbidity.
  • Neurofibromin the protein encoded by NFl, is a tumor suppressor and functions within the cell (at least in part) through its influence of Ras proteins. Ras proteins cause cell growth in many different cell types.
  • Neurofibromin converts Ras from its active to its inactive form, thereby limiting cell growth, i.e., it negatively regulates Ras activity in mammalian cells.
  • GAP GTPase activating protein
  • Cutaneous neurofibromas are slow-growing tumors that are pathognomonic for ⁇ F1, and though mast cells have been implicated in the pathogenesis of neuro fibroma formation as well as other cutaneous tumors (Hirota et al., 1993; Ryan et al., 1994; Coussens et al., 1999), the cellular and biochemical consequences of alterations of p21 ras activity secondary to partial loss of neurofibromin is unknown.
  • the invention provides a method to inhibit, prevent or treat the clinical manifestations of NFl, e.g., in cells that are heterozygous or homozygous for a mutant NFl allele, by administering an agent that restores normal intracellular signaling as well as growth control.
  • Preferred agents inhibit the activity at least one protein selected from the group consisting of Raf, MEK, extracellular signal regulated kinase 1 (ERKl), extracellular signal regulated kinase 2 (ERK2), phosphatidylinositol triphosphate kinase (PI3K), p21 activated kinase 2 (PAK2), p21 activated kinase 3 (PAK3), Rac, CDC42, p21 activated kinase 1 (PAK1), protein kinase B (PKB), Akt, and c-jun N-terminal kinase (JNK).
  • Raf Raf
  • MEK extracellular signal regulated kinase 1
  • ERK2 extracellular signal regulated kinase 2
  • PI3K phosphatidylinositol triphosphate kinase
  • PAK2 p21 activated kinase 2
  • PAK3 p21 activated
  • the agents are thus useful to inhibit, prevent or treat clinical manifestations of NFl including but not limited to neurofibromas, cutaneous melanocyte hype ⁇ lasia, lytic bone lesions or other skeletal abnormalities, pruritis, plexiform fibromas, brain tumors, myeloid malignancies, e.g., juvenile myelomonocytic leukemia (JMML), and learning disorders or other disorders associated with alterations in small GTPases.
  • JMML juvenile myelomonocytic leukemia
  • Nfl +/- mice have increased numbers of peritoneal mast cells compared to wildtype littermates.
  • Bone marrow derived mast cells (BMMCs) cultured from Nfl +/- mice demonstrate increased proliferation and survival relative to wildtype cells in response to stem cell factor (SCF), a known mitogenic and survival factor for mast cells (Ingram et al., 2000). While these studies suggest that alterations in p21 ras activity can alter mast cell fates in vitro and in vivo, little is known about the specific role of p21 ras in controlling distinct mast cell functions.
  • SCF stem cell factor
  • results described herein offer the first physiological evidence that alterations in both signaling through the p21 ras -PI-3 kinase pathway and crosstalk between PI-3 kinase and the p2 l ras -Raf-Mek-ERK pathway can be linked to a cellular phenotype implicated in the pathogenesis of a human genetic disease.
  • the invention provides a therapeutic method for treating the clinical manifestations of NFl in a mammal.
  • the method comprises administering to a mammal in need to such treatment an effective amount of an agent that directly or indirectly inhibits the activity at least one protein selected from the group consisting of Raf, MEK, extracellular signal regulated kinase 1 (ERKl), extracellular signal regulated kinase 2 (ERK2), phosphatidylinositol triphosphate kinase (PI3K), p21 activated kinase 2 (PAK2), p21 activated kinase 3 (PAK3), Rac, CDC42, p21 activated kinase 1 (PAKl), protein kinase B (PKB), Akt, and c-jun N-terminal kinase (JNK), e.g., so as to inhibit abnormal cellular growth or pruritus due to NF-1.
  • an agent that directly or indirectly inhibits the activity at least
  • Abnormal cell growth includes but is not limited to abnormal growth characterized by neurofibromas, lytic bone lesions, plexiform fibromas, brain tumor, myeloid malignancy, optic glioma or pheochromocytoma.
  • Preferred agents for use in the method of the invention include an agent that inhibits Raf, PI-3 kinase, e.g., LY294002, PAK3, PAK2, PAKl, ERK, MEK, e.g., PD98059, or an agent that inhibits crosstalk between the PI3-K and the Ras- Raf-MEK-ERK pathway.
  • the method comprises measuring, detecting or determining the ability of the agent to block or inhibit stem cell factor (SCF)-induced hype ⁇ roliferation and/or survival and/or degranulation of Nfl +/- mammalian mast cells.
  • SCF stem cell factor
  • the cells are derived from skin, fetal liver or from bone marrow of a mammal.
  • the agent is contacted with a mouse comprising Nfl +/- mast cells.
  • the method comprises measuring, detecting or determining the ability of the agent to reduce ERK2 activity in Nfl +/- mammalian cells following stimulation with SCF. For example, the ERKl phosphorylation of an ELK- 1 fusion protein is measured.
  • the cellular growth which is treated is mast cell growth.
  • an agent that alters the Ras-Raf-ERK-MEK pathway in a mammal for the preparation of a medicament for the treatment or prevention of at least one clinical manifestation of NFl, e.g., to prevent or treat cutaneous neurofibromas or learning disorders.
  • an agent that directly or indirectly suppresses Ras signalling cascade hyperactivation for the preparation of a medicament for the treatment or prevention of indications associated with abnormal intracellular signalling and mast cell growth in a mammal.
  • FIG. 1 Dose response curves of progenitor colony growth in response to cytokines. Growth of Scal + lin-/dim cells from wildtype (Nfl +/+), heterozygous (Nfl +/-) and homozygous (Nfl -/-) mice is shown at the indicated concentrations of mGM-CSF (a), mIL-3 (b), mGM-CSF and 10 ng/ml SCF (c), mIL-3 and 10 ng/ml mSCF (d) or mSCF (e). The data are expressed as percent of maximal numbers of CFU-GM colonies (Zhang et al., 1998).
  • FIG. 3 ERK kinase activity in Nfl -I- and Nfl +/+ primary c-kit + cells.
  • C-kit + bone marrow cells from mice previously reconstituted with Nfl -I- or Nfl +/+ fetal liver cells were isolated, cultured with either MEK inhibitor or vehicle for 1 hour prior to stimulation with a maximum stimulating capacity of SCF and IL3. Cells were lysed 5 minutes after stimulation with cytokines. The autoradiographs and activities of 1 of 3 representative experiments is shown. Cytokine and MEK inhibitor treatment of cells is indicated by + or -.
  • the bar on the far right panel represents 30 microns.
  • C Representative ear biopsies stained for cutaneous mast cells from individual mice of the four Nfl and PF genotypes. Specimens were stained with hematoxylin-eosin to assess routine histology and Giemsa to identify mast cells. Ear biopsies were stained with Fontana-Masson to differentiate melanin- containing cells from mast cells. Cutaneous mast cells (Giemsa-positive, Fontana-Masson-negative) were quantitated in a blinded fashion by counting the distal 5 mm of ears. Dark arrows indicate Giemsa-positive mast cells and open arrows indicate Fontanna-Masson melanin containing cells. The bar on the far right panel represents 35 microns.
  • FIG. 5 Effect of haploinsufficiency of Nfl and Won the survival and proliferation of bone marrow derived mast cells (BMMCs) in response to stem cell factor (SCF).
  • A Proliferation of BMMCs from mice of the four Nfl and W genotypes in response to recombinant murine SCF. Following deprivation of growth factors for 24 hours, 2 x 10 5 cells/ml were plated in triplicate in 24 well dishes in RPMI containing 1% glutamine, 10% fetal bovine serum and 100 ng/ml of SCF in a total volume of 1 ml. After 1 and 3 days, viable cells were counted using a hemocytometer, and expressed as the percent of input cells.
  • Asterisk indicates P ⁇ 0.05 for comparison of Nfl +/-; ⁇ ⁇ 1 to +/+; r ⁇ /W 41 and Nfl +/-; +1+ to +/+; +/+ cells by Student's paired t-test.
  • B Percent survival of bone marrow derived mast cells (BMMCs) of the four Nfl and W genotypes. Following deprivation of growth factors for 24 hours, 3 x 10 5 cells of each genotype were plated in RPMI containing 1% bovine serum albumin and 100 ng/ml of recombinant murine SCF. The number of surviving cells was determined by trypan blue exclusion and expressed as a percentage of input cells.
  • Asterisk indicates P ⁇ 0.05 for comparison of Nfl +/-; W* 1 / ⁇ 1 to +/+; r ⁇ 1 / ⁇ 1 and Nfl +/-; +/+ to +/+; +/+ cells by Student's paired t-test.
  • FIG. 6 Analysis of ERK kinase activity from bone marrow derived mast cells stimulated with SCF in the four Nfl and W genotypes. Activation of ERK kinase was determined by depriving cells of growth factors for 24 hours followed by stimulation with SCF for 5 and 15 minutes. Autoradiography and quantitative densitometry of the phosphorylation of Elk 1 fusion protein by ERK kinase from lysates obtained from SCF stimulated BMMCs are shown. Data represents 1 of 3 independent experiments.
  • Figure 7. Analysis of Akt kinase activity from bone marrow derived mast cells stimulated with SCF in Nfl +/- and Nfl +/+ genotypes.
  • Akt kinase Activation of ERK kinase was determined by depriving cells of growth factors for 24 hours followed by stimulation with SCF for 5 and 15 minutes. Autoradiography and quantitative densitometry of the phosphorylation of histone 2b fusion protein by Akt kinase from lysates obtained from SCF stimulated BMMCs are shown. One of 3 independent experiments with similar results are shown.
  • Figure 8 Schematic representation of c-kit mediated Ras-Raf-ERK and PI-3-kinase signaling pathways and potential cross talk between them.
  • Figure 9 Effect of LY294002, a specific PI-3 kinase inhibitor, on DNA synthesis of Nfl +/- and Nfl +/+ BMMCs. Data represents the mean of triplicate cultures in one representative experiment. Six other experiments were conducted with similar results. Asterisk indicates significant reduction in DNA synthesis after addition of LY294002 to the cultures compared to cultures incubated with vehicle alone.
  • FIG. 11 Effect of PD98059, a specific inhibitor of ERK activation, on DNA synthesis of Nfl +1- and Nfl +1+ BMMCs. Asterisk indicates significant reduction in DNA synthesis after addition of PD98059 to the cultures compared to cultures incubated with vehicle alone. A 15% reduction in DNA synthesis was observed in Nfl +/+ cells cultured with PD98059, while a 2.5 fold greater reduction in DNA synthesis was noted in Nfl +/- mast cells cultured with PD98059.
  • FIG. 12 Analysis of p21 Activated Kinase (PAK) activity in N 7 +/- and Nfl +/+ BMMCs stimulated with SCF. Activation of PAK was deteraiined by serum starving cells for 18 hours followed by stimulation with 10 ng/ml SCF for 30, 60, and 120 seconds. Autoradiography and quantitative densitometry of the phosphorylation of myelin basic protein (MBP) by PAK from lysates obtained from SCF stimulated BMMCs are shown. Nfl +1- cells have a 2-3 fold higher PAK activity at all time points tested.
  • Figure 13 Analysis of p21 Activated Kinase (PAK) activity in N 7 +/- and Nfl +/+ BMMCs stimulated with SCF. Activation of PAK was deteraiined by serum starving cells for 18 hours followed by stimulation with 10 ng/ml SCF for 30, 60, and 120 seconds. Autoradiography and quantitative densitometry of the phosphoryl
  • Akt kinase activity in the presence or absence of 100 nM of wortmannin was determined from Akt immunoprecipitates and an in vitro kinase assay was performed using histone 2b as a substrate. Autoradiography and quantitative densitometry of the phosphorylation of histone 2b are shown. Western blots for total Akt are shown. Data are representative of 4 independent experiments for each assay. Similar inhibition of Akt kinase was observed in cells preincubated in 25 ⁇ M of LY294002, a second PI-3 kinase inhibitor (data not shown).
  • Figure 14 Effect of heterozygosity of Nfl on mast cell survival, D ⁇ A synthesis, and activation of downstream effectors of c-kit in response to SCF in the presence or absence of MEK or PI-3 kinase inhibitors.
  • A Survival of Nfl +/- and wildtype BMMCs. After deprivation of growth factors for 24 hours, 3 x 10 5 cells of each genotype were plated in RPMI containing 1% BSA and SCF in the presence of 0, 5, 10, or 25 ⁇ M of LY294002. The number of surviving cells after 24 hours of culture was determined by trypan blue exclusion and expressed as the percentage of input cells.
  • FIG. 15 Analysis of MEK and Raf-1 kinase activity and phosphorylation of MEK 1/2 serine residues 217/221 in Nfl +/- and wildtype BMMCs following stimulation with SCF in the presence or absence of a PI-3K inhibitor.
  • Raf-1 and MEK kinase activities were determined from immunoprecipitates from cell lysates and in vitro kinase assays using GST-MEK as the substrate for Raf-1 and GST-ERK p42 fusion protein as the substrate for MEK.
  • FIG. 16 Effect of heterozygosity of Nfl on PAK, Cdc42 and Rac activity and PAK phosphorylation sites on Raf-1 and MEK in BMMCs following stimulation with SCF.
  • A PAK activity in Nfl +/- and wildtype mast cells following stimulation with SCF for 2 minutes. PAK activity was determined in PAK immunoprecipitates from cell lysates by in vitro kinase assay using myelin basic protein as the substrate. Western blots for total PAK are shown. Autoradiography and quantitative densitometry o the phosphorylated substrate are shown.
  • Cdc42 and D Rac activity in Nfl +/- and wildtype BMMCs.
  • GTP-bound Cdc42 or Rac levels were determined by incubating cell lysates with PAK binding domain agarose beads, fractionation by SDS-P AGE and probing with either anti-Cdc42 or anti-Rac antibodies. Immunoblots and quantitative densitometry for the respective Cdc42-GTP and Rac-GTP levels, and Westerns of total Rac and Cdc42, are shown. Data are representative of 3 independent experiments for each assay.
  • FIG. 17 Effect of heterozygosity of Nfl on F-actin content, phosphorylation of cofilin and chemotaxis of BMMCs in response to SCF.
  • A F-actin content in BMMCs. Mast cells were stimulated with 10 ng/ml SCF and fixed, at the time points indicated, by addition of formaldehyde. Wildtype and Nfl +/- cells were examined in triplicate. Results are expressed as mean channel fluorescence (MCF) and represent the mean MCF of 4 independent experiments per genotype. *P ⁇ 0.001 for all comparisons between genotypes using a Student's unpaired t-test.
  • B Fluorescent photomicrograph of F-actin content in SCF stimulated BMMCs.
  • Affected individuals are predisposed to the development of a wide array of malignant and nonmalignant clinical manifestations.
  • Malignant manifestations occur in up to 3-4% of NFl patients and include optic gliomas, neurofibrosarcomas, solid tumors and a predisposition to develop childhood myeloid leukemias. Consistent with Knudson's "two hit" model of tumor suppressor genes (Knudson, 1985), the leukemia and malignant solid tumors are frequently acquired after a somatic mutation and loss of function of the normal NE allele.
  • Neurofibromas are composed of Schwann cells, endothelial cells, fibroblasts, and mast cells (reviewed, Riccardi, 1992). Though numerous investigators have examined different aspects of neurofibroma biology (Stark et al., 1995; Sawada et al., 1996; Colman et al., 1995; Badache et al., 1998; Hirota et al., 1993; Rosenbaum et al., 1995), a comprehensive understanding of the genetic, biochemical and cellular mechanisms leading to the formation of neurofibromas has not been clear.
  • Some neurofibromas have a loss of heterozygosity of the normal NFl allele (Colman et al., 1995) consistent with the "two hit" tumor suppressor model, however, the majority of these lesions retain the normal NE7 allele (Sawada et al, 1996; Colman et al., 1995; Stark et al.,
  • neurofibroma samples are comprised of mixed populations of cells where only a small fraction of cells have loss of heterozygosity (LOH).
  • LHO heterozygosity
  • Mast cells have been implicated in the pathogenesis of neurofibromas and other skin malignancies in both human and murine systems (Hirota et al., 1993; Galli et al., 1993; Ryan et al., 1994; Coussens et al., 1999; Galli 1993; Kerdel et al., 1987). Mast cells release mediators of inflammation including histamine, serotonin, proteoglycans, and leukotrienes subsequent to crosslinking of the high affinity IgE receptor (Fc ⁇ RI) and the c-kit receptor (Galli et al., 1993; Serve et al., 1995).
  • IgE receptor Fc ⁇ RI
  • c-kit receptor the high affinity IgE receptor
  • mast cells release inflammatory mediators secondary to integrin attachment to the extracellular matrix (Serve et al., 1995). Since Fc ⁇ RI, integrins and c-kit signal through Ras proteins, aberrations in the release of mast cell molecules could exist where Ras in hyperactivated. Thus, NFl mast cells could release abnormal levels of mediators that act locally upon Schwann cells, endothelial cells and fibroblasts to influence their biologic functions (Skuse et al., 1989). Interestingly, in at least two independent clinical series, a significant number of neurofibromas in NFl patients were associated with pruritus at the site of development (Huson et al., 1994).
  • mast cell stabilizers are associated with a reduction in pruritus in some patients with NFl, and represents the only known medical treatment that alters the growth of neurofibromas (Huson et al., 1994).
  • Children with NFl are at markedly increased risk of developing malignant myeloid disorders, particularly JMML, and comprise as many as 10% of de novo cases of preleukemia in the pediatric age group (Johnson et al., 1993; Shannon et al., 1992; Neubauer et al., 1991; Miles et al., 1995; Brön 1994; Shannon et al., 1994).
  • myeloid progenitors colony-forming unit-granulocyte macrophage, CFU-GM
  • CFU-GM colony-forming unit-granulocyte macrophage
  • Neurofibromin the protein encoded by Nfl negatively regulates Ras activity in NFl -I- murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting locus, W).
  • SCF and its receptor c-kit, are components of a signaling pathway that is essential for murine hematopoiesis, melanogenesis and gametogenesis.
  • These proteins are encoded by the Dominant White Spotting (W) and Steel (SI) loci, respectively, and ligand binding to c-kit activates Ras in myeloid lineage cells.
  • the c-kit/SCF signaling pathway influences the development of cell lineages such as mast cells and melanocytes, which are implicated in the common, nonmalignant sequelae of NFl.
  • Ras proteins The biochemical output of Ras proteins is tightly regulated by their ability to cycle between a guanosine triphosphate bound state (Ras-GTP), and an inactive guanosine diphosphate bound state (Ras-GDP) (Hall 1992; Bourne et al., 1990; Bourne et al., 1991).
  • Ras activation is an essential component of proliferative responses induced after receptor binding by a variety of growth factors including interleukin-3 (IL-3), granulocyte macrophage colony stimulating factor (GM-CSF), and SCF.
  • IL-3 interleukin-3
  • GM-CSF granulocyte macrophage colony stimulating factor
  • Ras-GTP recruits the serine-threonine kinase Raf-1 to the plasma membrane and Raf-1, in turn, activates a series of downstream effectors such as ERKs (p42/p44 MAPkinase) (Sokoe et al., 1994; Leevers et al., 1994) that are important in the mitogenic response to extracellular stimuli.
  • ERKs p42/p44 MAPkinase
  • Ras-GTP has recently been shown to activate other downstream effectors, including phosphatidylinositol triphosphate kinase (PI3K) which influences multiple cell functions including survival.
  • GTPase activating proteins GAPs regulate Ras output by stimulating the slow intrinsic Ras GTPase (Satoh et al., 1992; Rodenhuis 1992; Miyauchi et al., 1994). Because Ras-GTP transduces signals to multiple downstream effectors, GAPs act (at least in part) as important negative regulators of Ras function.
  • Two GAPs, pi 20 GAP and neurofibromin (the protein encoded by the NFl gene) regulate Ras output in mammalian cells by promoting the conversion of Ras-GTP to Ras GDP (Satoh et al., 1991).
  • Agents useful in the practice of the invention include agents that inhibit, reduce or prevent one or more of the clinical manifestations of NFl, e.g., neurofibromas, cutaneous melanocyte hype ⁇ lasia, lytic bone lesions or other skeletal abnormalities, pruritis, plexiform fibromas, brain tumors, myeloid malignancies, e.g., juvenile myelomonocytic leukemia (JMML), and learning disorders or other disorders associated with alterations in small GTPases.
  • NFl e.g., neurofibromas, cutaneous melanocyte hype ⁇ lasia, lytic bone lesions or other skeletal abnormalities, pruritis, plexiform fibromas, brain tumors, myeloid malignancies, e.g., juvenile myelomonocytic leukemia (JMML), and learning disorders or other disorders associated with alterations in small GTPases.
  • JMML juvenile myelomonocytic leukemia
  • BMMCs can be cultured as described by Serve et al., 1995. Homogeneity of BMMCs can be determined by Giemsa staining. Cells can also be stained with alcian blue and safranin to confirm they are mast cells. 2. In vitro mast cell survival assay
  • a survival assay may be conducted in which, before the addition of a growth factor varying amounts of an agent are mixed in the presence of mast cells previously deprived of growth factors.
  • a range of known concentrations of an agent e.g., a kinase inhibitor
  • a defined number e.g. 10 4 - 10 6
  • murine BMMCs are contacted with a defined number (e.g., 10 4 - 10 6 ) of murine BMMCs and plated in individual wells of a tissue culture dish in serum-free RPMI containing 1% glutamine and 100 ng/ml of recombinant murine SCF in a total volume of 1 ml.
  • Cells are then incubated for 48, 72, and 96 hours in a 37°C, 5% CO 2 , humidified incubator.
  • Any method suitable for counting cells can be used, for example, counting with a hemocytometer or FACS analysis.
  • the number of surviving cells are determined by trypan blue exclusion.
  • the number of apoptotic cells are calculated using a TUNEL assay as described in other studies (Haneline et al., 1998).
  • mast cell proliferation assay Agents may also be screened in a proliferation assay in which mast cells previously deprived of growth factors are mixed with or without growth factors. Proliferation of mast cells is determined adding tritiated thymidine to the culture and measuring ⁇ emission. For example, a defined number of cells (e.g., 10 4 - 10 6 ) are plated in 96 well dishes in 200 ml of RPMI containing 1% glutamine, 10% fetal bovine serum (Hyclone Laboratories) and 100 ng/ml of rmSCF or no growth factors and incubated in a 37°C, 5% CO 2 , humidified incubator.
  • agents may be screened in a chemotaxis assay, in which varying amounts of a growth factor are mixed with cells in the presence of a known chemoattractant.
  • murine BMMCs are serum starved for a period of time, e.g., 16-18 hours.
  • a defined number e.g., 10 4 - 10 6
  • BMMCs are resuspended in a volume of serum free RPMI containing 1% glutamine and 0.5% BSA (Sigma).
  • BMMCs are then loaded onto each transwell filter (8- ⁇ m pore filter Transwell, 24-well cluster; Costar, Boston, MA).
  • Mast cell motility assays in which cellular motility is observed by time lapse imaging of cells exposed to a gradient of growth factor, may also be employed to screen agents.
  • BMMC motility is directly observed by time lapse imaging of cells exposed to a gradient of 10 ng/ml rmSCF in a Dunn chemotaxis chamber (Dunn and Zicha, Dunn & Jones, 1997; Weber Scientific Ltd., Surrey, England).
  • a defined number (e.g., 10 - 10 ) of BMMCs are resuspended in a volume of medium, e.g., lO ⁇ l RPMI, and are applied to fibronectin fragment H-296 covered glass coverslips.
  • the coverslips are mounted on the Dunn chamber, the inner well of which is filled with RPMI and the outer well is filled with RPMI/rmSCF.
  • the chamber is sealed and mounted on the stage of a Nikon Diaphot 300 inverted microscope equipped with DIC optics. Chamber temperature is maintained at 37°C using a stage heater (Instec Instruments Inc., Boulder, CO).
  • a stable gradient to form, and images are recorded digitally at 15-second intervals using a 40x LWD objective and a Spot RT cooled CCD camera and analyzed using Metamo ⁇ h software (Universal Imaging, Brandywine, PA).
  • the centroid coordinates of cells at each timepoint is determined on calibrated images and is used to calculate the speed and direction of migration.
  • F-actin quantitation may also be performed to screen agents of the invention.
  • flow cytometry is used to quantitate the amount of filamentous actin per mast cell.
  • a defined number of mast cells e.g., 10 6
  • growth factor e.g., SCF, or carrier.
  • cells are fixed by the addition of 10 volumes of PBS containing 4.6% formaldehyde and 0.1% BSA.
  • mast cell kinase assays may also be utilized to screen agents of the invention.
  • Mast cells deprived of serum and growth factors are stimulation with a growth factor for varying lengths of time.
  • murine BMMCs are deprived of serum and growth factors for 24 hours.
  • BMMCs are then stimulated by contacting then with an amount of growth factor, e.g., 10 ng/ml rmSCF, for various amounts of time.
  • Cells are washed with PBS containing 1 mM sodium orthovanadate.
  • Cells are then lysed in nonionic lysis buffer (20 mM Tris/HCl, 137 mM NaCI, 1 mM EGTA, 1% Triton X-100, 10% Glycerol, 1.5 mM MgCl 2 , and COMPLETE protease inhibitors (Amersham) as previously described (Bollag et al., 1996a). Lysate protein concentration is quantitated, e.g., using the BCA assay (Pierce Chemical Co.). A volume of each lysate is subjected to SDS-P AGE, and equal loading of kinases in these assays is confirmed by Western blot. Gels are transferred to PVDF filters. Gels and PNDF filters are dried and subjected to autoradiography.
  • ERK and PAK kinase immunoprecipitations can be carried out with an anti- ERK-2 (C-14) antibody and an anti-PAK(C-19) antibody (Santa Cruz), respectively.
  • Raf-1 and Mek kinase immunoprecipitations can be carried out with an anti-Raf-1 (C-12) antibody (Santa-Cruz) and an anti-Mek-1 (C-18) antibody (Santa Cruz), respectively.
  • Akt kinase immunoprecipitations can be carried out with an anti-Akt antibody (Cell Signaling Technology).
  • the kinase immune complex assay can also be performed as previously described (Bollag et al., 1996a).
  • Elk-1 fusion protein New
  • Agents of the invention may also be screened by Ras, Cdc42 and Rac activation assays, in which mast cells are stimulated with a growth factor and activation is determined via a commercially available assay kit.
  • BMMCs are deprived of serum and growth factors for 24 hours and stimulated at various amounts of time with a known concentration of growth factor, e.g., 10 ng/ml rmSCF.
  • Ras, Cdc42 and Rac activation is determined using Ras, Cdc42, and Rac activation assay kits (Upstate Biotechnology) according to the manufacturer's protocol and as previously described in Gille and Downward (1999). 9.
  • Ras, Cdc42 and Rac activation assay kits Upstate Biotechnology
  • PI3K, PAKl, PAK3, Rac, CDC42, PKB, Akt or JNK is to inject a selected growth factor into the skin of an animal in the presence of absence of an agent of the invention.
  • animals are sacrificed and the number of mast cells in animals exposed to the agent is compared to the number of mast cells in animals exposed to growth factor alone, e.g., by quantitative immunofluorescence, relative to control animals.
  • a continuous infusion of various doses of a growth factor e.g., rmSCF, or vehicle (PBS) may be delivered from micro-osmotic pumps (Alzet) placed under the dorsal back skin of adult Nfl +/- or wildtype mice.
  • Osmotic pumps may be surgically placed under light avertin anesthesia. Growth factor or vehicle is released over a period of time at a set rate, e.g., 7 days at 0.5 ⁇ l/hr. Osmotic pumps are then surgically removed following sacrifice. To accurately identify cutaneous sections for quantitating changes in mast cell numbers in response to growth factor, the dorsal skin of the animal is stained with a drop of India ink at the point of exit of growth factor from the osmotic pump prior to removal of the pump. Sections of skin marked with India ink are removed, fixed in buffered formalin and processed in paraffin-embedded sections.
  • Specimens are stained with hematoxylin-eosin to assess routine histology and with Giemsa stain to identify mast cells. Cutaneous mast cells can be quantitated in a blinded fashion by counting 2 mm 2 sections in proximity to the India ink stain. II. Dosages. Formulations and Routes of Administration of the Agents of the Invention
  • the therapeutic agents of the invention are preferably administered so as to inhibit, treat or prevent at least one of the clinical manifestations of NFl.
  • the amount administered will vary depending on various factors including, but not limited to, the agent chosen, whether prevention or treatment is to be achieved, and if the agent is modified for bioavailability and in vivo stability.
  • Administration of the therapeutic agents in accordance with the present invention may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the pu ⁇ ose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of the agents of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.
  • One or more suitable unit dosage forms comprising the therapeutic agents of the invention can be administered by a variety of routes including oral, or parenteral, including by rectal, buccal, vaginal and sublingual, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathoracic, intrapulmonary and intranasal routes.
  • the formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to pharmacy. Such methods may include the step of bringing into association the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.
  • the therapeutic agents of the invention are prepared for oral administration, they are preferably combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form.
  • a pharmaceutically acceptable carrier diluent or excipient to form a pharmaceutical formulation, or unit dosage form.
  • the total active ingredients in such formulations comprise from 0.1 to 99.9% by weight of the formulation.
  • pharmaceutically acceptable it is meant the carrier, diluent, excipient, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
  • the active ingredient for oral administration may be present as a powder or as granules; as a solution, a suspension or an emulsion; or in achievable base such as a synthetic resin for ingestion of the active ingredients from a chewing gum.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, douches, lubricants, foams or sprays containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
  • Formulations suitable for rectal administration may be presented as suppositories.
  • compositions containing the therapeutic agents of the invention can be prepared by procedures known in the art using well known and readily available ingredients.
  • the agent can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like.
  • excipients, diluents, and carriers that are suitable for such formulations include the following fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose, HPMC and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone; moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for retarding dissolution such as paraffin; reso ⁇ tion accelerators such as quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adso ⁇ tive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.
  • fillers and extenders such as starch, sugars, mannitol, and silicic derivatives
  • binding agents such as carboxymethyl cellulose, HPMC and other
  • tablets or caplets containing the agents of the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate.
  • Caplets and tablets can also include inactive ingredients such as cellulose, pregelatinized starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, and zinc stearate, and the like.
  • Hard or soft gelatin capsules containing an agent of the invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil.
  • enteric coated caplets or tablets of an agent of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum.
  • the therapeutic agents of the invention can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes.
  • the pharmaceutical formulations of the therapeutic agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.
  • the therapeutic agent may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative.
  • the active ingredients may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water
  • These formulations can contain pharmaceutically acceptable vehicles and adjuvants which are well known in the prior art.
  • organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol”, polyglycols and polyethylene glycols, C ⁇ -C 4 alkyl esters of short-chain acids, preferably ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name "Miglyol”, isopropyl myristate, animal, mineral and vegetable oils and polysiloxanes.
  • solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol”, polyglycols and polyethylene glycols, C ⁇ -C 4 alkyl esters of short-chain acids, preferably ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the
  • compositions according to the invention can also contain thickening agents such as cellulose and/or cellulose derivatives. They can also contain gums such as xanthan, guar or carbo gum or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites, and the like. It is possible to add, if necessary, an adjuvant chosen from antioxidants, surfactants, other preservatives, film-forming, keratolytic or comedolytic agents, perfumes and colorings. Also, other active ingredients may be added, whether for the conditions described or some other condition.
  • thickening agents such as cellulose and/or cellulose derivatives. They can also contain gums such as xanthan, guar or carbo gum or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites, and the like. It is possible to add, if necessary, an adjuvant chosen from antioxidants, surfactants, other preservatives, film-forming, keratolytic or comedolytic agents, perfumes
  • the galenical forms chiefly conditioned for topical application take the form of creams, milks, gels, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, or alternatively the form of aerosol formulations in spray or foam form or alternatively in the form of a cake of soap.
  • the agents are well suited to formulation as sustained release dosage forms and the like.
  • the formulations can be so constituted that they release the active ingredient only or preferably in a particular part of the intestinal or respiratory tract, possibly over a period of time.
  • the coatings, envelopes, and protective matrices may be made, for example, from polymeric substances, such as polylactide-glycolates, liposomes, microemulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes, and protective matrices are useful to coat indwelling devices, e.g., stents, catheters, peritoneal dialysis tubing, and the like.
  • the therapeutic agents of the invention can be delivered via patches for transdermal administration. See U.S. Patent No. 5,560,922 for examples of patches suitable for transdermal delivery of a therapeutic agent.
  • Patches for transdermal delivery can comprise a backing layer and a polymer matrix which has dispersed or dissolved therein a therapeutic agent, along with one or more skin permeation enhancers.
  • the backing layer can be made of any suitable material which is impermeable to the therapeutic agent.
  • the backing layer serves as a protective cover for the matrix layer and provides also a support function.
  • the backing can be formed so that it is essentially the same size layer as the polymer matrix or it can be of larger dimension so that it can extend beyond the side of the polymer matrix or overlay the side or sides of the polymer matrix and then can extend outwardly in a manner that the surface of the extension of the backing layer can be the base for an adhesive means.
  • the polymer matrix can contain, or be formulated of, an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer.
  • an adhesive polymer such as polyacrylate or acrylate/vinyl acetate copolymer.
  • Examples of materials suitable for making the backing layer are films of high and low density polyethylene, polypropylene, polyurethane, polyvinylchloride, polyesters such as poly(ethylene phthalate), metal foils, metal foil laminates of such suitable polymer films, and the like.
  • the materials used for the backing layer are laminates of such polymer films with a metal foil such as aluminum foil. In such laminates, a polymer film of the laminate will usually be in contact with the adhesive polymer matrix.
  • the backing layer can be any appropriate thickness which will provide the desired protective and support functions.
  • a suitable thickness will be from about 10 to about 200 microns.
  • those polymers used to form the biologically acceptable adhesive polymer layer are those capable of forming shaped bodies, thin walls or coatings through which therapeutic agents can pass at a controlled rate.
  • Suitable polymers are biologically and pharmaceutically compatible, nonallergenic and insoluble in and compatible with body fluids or tissues with which the device is contacted. The use of soluble polymers is to be avoided since dissolution or erosion of the matrix by skin moisture would affect the release rate of the therapeutic agents as well as the capability of the dosage unit to remain in place for convenience of removal.
  • Exemplary materials for fabricating the adhesive polymer layer include polyethylene, polypropylene, polyurethane, ethylene/propylene copolymers, ethylene/ethylacrylate copolymers, ethylene/vinyl acetate copolymers, silicone elastomers, especially the medical-grade polydimethylsiloxanes, neoprene rubber, polyisobutylene, polyacrylates, chlorinated polyethylene, polyvinyl chloride, vinyl chloride-vinyl acetate copolymer, crosslinked polymethacrylate polymers (hydrogel), polyvinylidene chloride, poly(ethylene terephthalate), butyl rubber, epichlorohydrin rubbers, ethylenvinyl alcohol copolymers, ethylene- vinyloxyethanol copolymers; silicone copolymers, for example, polysiloxane- polycarbonate copolymers, polysiloxane-polyethylene oxide copolymers, polysiloxane-pol
  • a biologically acceptable adhesive polymer matrix should be selected from polymers with glass transition temperatures below room temperature.
  • the polymer may, but need not necessarily, have a degree of crystallinity at room temperature.
  • Cross-linking monomenc units or sites can be inco ⁇ orated into such polymers.
  • cross-linking monomers can be inco ⁇ orated into polyacrylate polymers, which provide sites for cross-linking the matrix after dispersing the therapeutic agent into the polymer.
  • Known cross- linking monomers for polyacrylate polymers include polymethacrylic esters of polyols such as butylene diacrylate and dimethacrylate, trimethylol propane trimethacrylate and the like.
  • Water-soluble polyols are generally suitable for this pu ⁇ ose. Inco ⁇ oration of a humectant in the formulation allows the dosage unit to absorb moisture on the surface of skin which in turn helps to reduce skin irritation and to prevent the adhesive polymer layer of the delivery system from failing.
  • Therapeutic agents released from a transdermal delivery system must be capable of penetrating each layer of skin.
  • a transdermal drug delivery system In order to increase the rate of permeation of a therapeutic agent, a transdermal drug delivery system must be able in particular to increase the permeability of the outermost layer of skin, the stratum corneum, which provides the most resistance to the penetration of molecules.
  • the fabrication of patches for transdermal delivery of therapeutic agents is well known to the art.
  • the therapeutic agents of the invention are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the composition may take the form of a dry powder, for example, a powder mix of the therapeutic agent and a suitable powder base such as lactose or starch.
  • a powder mix of the therapeutic agent and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatine or blister packs from which the powder may be administered with the aid of an inhalator, insufflator or a metered-dose inhaler.
  • the therapeutic agent may be admimstered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered- dose inhaler.
  • a liquid spray such as via a plastic bottle atomizer or metered- dose inhaler.
  • Typical of atomizers are the Mistometer (Wintrop) and the Medihaler (Riker).
  • the local delivery of the therapeutic agents of the invention can also be by a variety of techniques which administer the agent at or near the site of disease. Examples of site-specific or targeted local delivery techniques are not intended to be limiting but to be illustrative of the techniques available. Examples include local delivery catheters, such as an infusion or indwelling catheter, e.g., a needle infusion catheter, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct applications.
  • the therapeutic agents may be formulated as is known in the art for direct application to a target area.
  • Conventional forms for this pu ⁇ ose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols, as well as in toothpaste and mouthwash, or by other suitable forms, e.g., via a coated condom.
  • Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
  • the active ingredients can also be delivered via iontophoresis, e.g., as disclosed in U.S. Patent Nos. 4,140,122; 4,383,529; or 4,051,842.
  • the percent by weight of a therapeutic agent of the invention present in a topical formulation will depend on various factors, but generally will be from 0.01% to 95% of the total weight of the formulation, and typically 0.1-25% by weight.
  • the above-described formulations can be adapted to give sustained release of the active ingredient employed, e.g., by combination with certain hydrophilic polymer matrices, e.g., comprising natural gels, synthetic polymer gels or mixtures thereof.
  • Drops such as eye drops or nose drops, may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents.
  • Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure.
  • the therapeutic agent may further be formulated for topical administration in the mouth or throat.
  • the active ingredients may be formulated as a lozenge further comprising a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; mouthwashes comprising the composition of the present invention in a suitable liquid carrier; and pastes and gels, e.g., toothpastes or gels, comprising the composition of the invention.
  • a flavored base usually sucrose and acacia or tragacanth
  • pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia
  • mouthwashes comprising the composition of the present invention in a suitable liquid carrier
  • pastes and gels e.g., toothpastes or gels, comprising the composition of the invention.
  • compositions and compositions described herein may also contain other ingredients such as antimicrobial agents, or preservatives.
  • active ingredients may also be used in combination with other therapeutic agents, for example, oral contraceptives, bronchodilators, anti-viral agents, steroids and the like.
  • oral contraceptives for example, oral contraceptives, bronchodilators, anti-viral agents, steroids and the like.
  • Nfl +/- mice were used to examine the effects of homozygous disruption of Nfl.
  • Nfl -I- embryos die in utero at dl2.5-13.5 p.c. from apparent hydrops fetalis secondary to complex developmental abnormalities of the cardiac system, including overgrowth of the ventricular outflow tracts (Jack et al., 1994).
  • Fetal liver hematopoietic cells were isolated from Nfl -I- embryos prior to their death at day 12.5-13.5 p.c.
  • FIG. 1 shows that cultures of Nfl -I- cells yielded more CFU-GM colonies at low GM-CSF concentrations than cells from wild-type (Nfl +/+) or heterozygous (Nfl +/-) littermates.
  • the dose-response relationship for myeloid colony formation was similar for Nfl +/+, Nfl +/-, and Nfl -I- cells in the presence of IL-3 ( Figure 1 A).
  • hematopoiesis is hierarchical and has multiple compartments, it is possible that neurofibromin is only required to regulate growth of specific subpopulations of cells in response to a restricted number of hematopoietic growth factors.
  • Previous studies in human and murine cells examined only the responsiveness of relatively differentiated lineage restricted progenitors from mixed populations of hematopoietic cells, and cultures were established using single growth factors.
  • Nfl -I- c-kit+ cells were isolated from the bone marrows of mice previously irradiated and reconstituted with fetal liver cells from dl3.5 gestation Nfl -I- and Nfl +1+ littermates. It was determined that the Nfl -I- c-kit+ cells isolated from transplant recipients had a similar hypenesponsiveness to GM-CSF, SCF, or SCF and IL-3 in clonogenic assays as Nfl -I- fetal liver cells (data not shown).
  • c-kit+ cells were tested for activation of ERKs following growth factor stimulation.
  • Cells were maintained in liquid cultures overnight without exogenous growth factors to induce quiescence.
  • aliquots of cells were incubated with either a MEK inhibitor, which inhibits ERK activation, or vehicle alone to confirm that ERK activation results from activation of the Ras-MEK-ERK pathway.
  • Cells were then stimulated with SCF and IL-3 for 5 or 60 minutes. Three independent experiments yielded similar results. Data from one of these is shown in Figure 3.
  • ERK activity was observed in unstimulated c-kit + cells isolated from the Nfl -I- recipients.
  • this kinase activity increased significantly in Nfl -I- cells after exposure to SCF + IL-3 and persisted above baseline at 5 minutes.
  • c-kit + cells isolated from Nfl +/+ recipients had lower baseline activity and a much less pronounced increase in ERK activity 5 minutes after stimulation compared to Nfl -I- cells.
  • a high concentration of a MEK inhibitor which in turn blocks activation of ERKs, inhibited ERK activity in both Nfl -I- and +/+ cells.
  • mice were generated with mutations at both W and Nfl to examine potential genetic and biochemical interactions in vivo and in vitro, and to understand the biological effect of heterozygous inactivation of Nfl in these specific cell lineages that are involved in the pathogenesis of NFl.
  • C-kit is known to be important in skin pigmentation and mast cell biology, and mast cells and skin melanocytes are implicated in the nonmalignant complications of NFl.
  • mice display varying degrees of sterility, mast cell hypoplasia, anemia, and coat color deficiency conelating with the residual kinase activity of the mutant receptor (Blouin et al., 1993; Nocka et al., 1990).
  • Mice homozygous for a point mutation in the cytoplasmic domain of c-kit (W* ) have a partial inactivation of the kinase activity of the c-kit receptor resulting in an abnormal mottled, white coat color (Nocka et al., 1990).
  • W* x and Nfl mice both of which had been backcrossed onto a C57BL/6 background for greater than 10 generations, were crossed, a very su ⁇ rising result was observed.
  • mice homozygous for different mutant W alleles have reduced numbers of peritoneal and cutaneous mast cells conelating with the residual kinase activity of the mutant receptor (Blouin et al., 1993; Paulson et al., 1996).
  • numbers of peritoneal mast cells harvested from Nfl +/-; W ⁇ ,x IW animals were compared to those taken from singly mutant mice (+/+;W il /W il ).
  • Peritoneal mast cell numbers in Nfl +/-; +/+ and wildtype mice were also compared.
  • Giemsa-stained biopsies obtained from a second site revealed similar differences in cutaneous mast cell numbers between the genotypes (data not shown).
  • the increased numbers of peritoneal and cutaneous mast cells in Nfl +/- mice provides additional evidence that haploinsufficiency at Nfl augments signaling through the c-kit receptor in vivo.
  • Asterisk indicates P ⁇ 0.05 for comparison of Nfl +/+ and Nfl +/- in both ff ⁇ '/J 41 and wildtype genetic backgrounds by Student's paired t-test.
  • Nfl +/- cells of both W genotypes showed greater prohferative responses to SCF after 24 and 72 hours in culture than the conesponding Nfl +/+ populations ( Figure 5 A).
  • thymidine inco ⁇ oration assays gave similar results (data not shown).
  • a total of 9 independently derived heterozygous and wildtype lines were evaluated with similar results.
  • haploinsufficiency at Nfl is associated with an increased prohferative response of wildtype and W* X IW* BMMCs in liquid cultures containing 10% FCS and exogenous SCF.
  • Nfl +/-; +/+ BMMCs had a 2-fold greater increase in ERK activity from baseline in comparison to wildtype mast cells that was sustained at both tested time points (Figure 6).
  • NE7 +/- mast cells had increased proliferation and enhanced survival in response to SCF compared to wildtype mast cells in vitro.
  • Nfl heterozygous mice on a mutant IF background have an increased number of cutaneous mast cells, unstressed, singly mutant, Nfl +/- mice have only a modest increase in cutaneous mast cells, analogous to NFl patients.
  • investigators have shown that mast cells increase dramatically in neoplastic lesions that involve Schwann cells, such as neurofibromas and Schwannomas (Hirota et al., 1993; Ryan et al., 1994), and mast cells cluster around normal peripheral nerves, particularly during times of nerve injury and repair (Iemura et al., 1994).
  • Kitamura et al. have demonstrated that the amount of SCF mRNA was greater in neurofibroma tissue, suggesting that SCF and the c-kit receptor are associated with the increase in mast cells found in neurofibroma tissues (Hirota et al., 1993).
  • Schwann cells and fibroblasts two principal components of neurofibromas, secrete SCF in response to many different stimuli (Iemura et al., 1994).
  • mast cell stabilizing drugs have been preliminarily shown to decrease the size and pruritus associated with neurofibromas (Huson et al., 1994). This increase in the number of mast cells in vivo could be the result of increased proliferation, survival or a combination of both proliferation and survival in NFl patients.
  • Piebaldism is an autosomal dominant genetic disorder that occurs secondary to a mutation in the c-kit receptor and results in a decrease in c-kit receptor tyrosine kinase activity.
  • the most distinctive clinical features of patients with piebaldism is cutaneous hypopigmentation, though some patients also have learning disorders and hypoplastic anemia.
  • a striking feature of rare patients diagnosed with both piebaldism and neurofibromatosis type 1 was that none of the patients had neurofibromas.
  • apoptosis is a common mechanism for reducing the size of mast cell populations in vivo (Iemura et al., 1994).
  • Nfl +/- mast cells may also release abnormal amounts of inflammatory mediators influencing the local growth of fibroblasts, Schwann cells and endothelial cells in vivo.
  • PI-3 kinase is a major downstream effector of c-kit activation which has an important role in both proliferation and survival of wildtype mast cells.
  • Ras-GTP has been shown to activate PI3K by numerous investigators. Since in vitro studies demonstrated that Nfl +/- cells have both increased proliferation and survival compared to wildtype cells, Akt activity, a downstream effector of PI3K, was measured in Nfl +/- and wildtype mast cells, as an indication of PI3K activity.
  • the c-kit SCF signaling pathway is known to be essential to the regulation of hematopoiesis (including mast cell development), melanogenesis and gametogenesis.
  • W receptor deficiency
  • SI ligand deficiency
  • Nfl may modulate c-kit signaling and activated downstream effectors of c-kit in a gene dose dependent fashion.
  • Nfl +/- mast cells may mediate diverse alterations in the function of these cells including proliferation, survival, differentiation, degranulation, and cytoskeletal rearcangement.
  • the pu ⁇ ose of these studies is to evaluate the cellular and biochemical consequences of heterozygosity of Nfl in mast cells in vitro and in vivo. Evaluation of in vivo proliferation of cutaneous mast cells in Nfl +/- and +/+ mice. Mast cells have a complex developmental pattern of differentiation in that they are derived from progenitor cells found in the bone marrow (reviewed, Galli 1993).
  • Mast cell precursors migrate via the circulation to interstitial tissues, serosal cavities and epithelial cells, and then complete their program of maturation, and differentiation within these tissues.
  • Preliminary data indicate that heterozygosity of Nfl modulates the proliferation of BMMCs in vitro in response to stem cell factor.
  • a murine model was employed that was originally developed by Galli who demonstrated that local subcutaneous injection of SCF increases the proliferation (Tsai et al., 1991) and survival (Iemura et al., 1994) of mast cell precursors located within the skin.
  • the cutaneous injection site is excised, fixed in Carnoy's fixative, and embedded in paraffin.
  • Four micron sections are cut, placed on polylysine-coated slides and stained for counting of both total numbers of mast cells and the percentage of total cells that are actively proliferating.
  • Two types of dermal mast cells are readily identified by conventional staining methods. Mast cells that retain a capacity for local tissue proliferation are identified by alcian blue staining, while terminally differentiated mast cells are identified by staining with the heparin-binding fluorescent dye berberine sulfate (alcian-blue negative). Both types of mast cells are quantitated to compare total numbers of mast cells at the local injection site between the four genotypes.
  • Nfl +/- cells will have a higher percentage of BrdU positive mast cells than Nfl +/+ cells on both a mutant (W) and wildtype c-kit receptor background. Since SCF has been shown to be important for mast cell survival as well as proliferation, it is possible that differences in total numbers of mast cells at local injection sites between genotypes could be in part a result of differences in survival. If differences in total numbers of mast cells at local injection sites are secondary to increased mast cell survival, then the percent of BrdU positive cells (proliferating cells) would be equivalent between experimental groups despite differences in total numbers of mast cells.
  • Nfl +/- BMMCs of both W genotypes is compared to Nfl +/+ cells in two previously used methods (Nocka et al., 1990).
  • mast cells from each respective genotype are assayed in serum-free media in response to exogenous SCF only. Survival in this assay has been shown to correlate previously with c-kit receptor tyrosine kinase activity (Nocka et al., 1990).
  • the mast cell survival assay as previously established is conducted as follows: BMMCs from each genotype are deprived of growth factors for 24 hours and 3 x 10 5 cells are plated in 24 well dishes in serum- free RPMI containing 1% glutamine and 100 ng/ml of recombinant murine SCF in a total volume of 1 ml. The number of surviving cells are determined by trypan blue exclusion at 48, 72, and 96 hours of culture in a 37°C, 5% CO 2 , humidified incubator, and the number of apoptotic cells are calculated using a TUNEL assay as described in other studies (Haneline et al., 1998).
  • a second method of evaluating the intrinsic resistance of Nfl +/- cells independent of either growth factor or serum is to evaluate their survival in serum-free and growth factor-free conditioned media.
  • Cells are cultured as described above in 24 well dishes and evaluated for TUNEL positive cells 8, 16, 24, 36, and 48 hours after initiation of cultures and adjusted as indicated from preliminary studies. At least 4-5 independent experiments are conducted for each in vitro measure of apoptosis, and differences between genotypes will be examined using Student's T-test. Evaluation of Survival Advantage of Nfl +/- mast cells in vivo.
  • mice receive a daily subcutaneous injection of either vehicle or 100 ng recombinant murine SCF for 3 weeks analogous to previous studies by Galli et al. (Iemura et al., 1994).
  • Cohorts of mice (7-8 per group of each respective genotype) are treated as previously described above with vehicle or 100 ng/day of subcutaneous polyethylene glycol prepared SCF, and sacrificed at 1, 2, 4, and 7 days after daily injections of this long-acting SCF to quantitate total numbers of mast cells remaining at the injection site.
  • Galli has shown that increased numbers of mast cells at local injection sites quickly return to normal by 2-4 days after cessation of SCF subcutaneous treatment in wildtype mice (Iemura et al., 1994).
  • Sections are also prepared as described above, to quantitate total numbers of mast cells on days 1, 2, 4, and 7 after cessation of treatment. By comparing total numbers of mast cells on days 2, 4, and 7 post-treatment to total numbers of cells present on day 1 following the last injection of SCF, the proportionate decline in absolute numbers of cells is determined and compared between genotypes.
  • slides are prepared for evaluation of apoptosis using the TUNEL assay.
  • evaluation of apoptosis is conducted on sections stained with alcian blue and berberine sulfate, using characteristic mo ⁇ hologic indicators including condensed nuclear chromatin, pyknotic nuclei, and swelling or fusion of cytoplasmic mast cell granules.
  • SCF as a single stimulant can also induce degranulation of connective tissue derived mast cells found.
  • Nfl +1- and Nfl +/+ mast cells are pulse-treated with SCF alone in the absence of IgE sensitization, to evaluate the release of serotonin.
  • Nfl +/- mast cells will have greater serotonin release than wildtype cells in response to SCF and IgE alone or in combination.
  • coculture experiments of mast cells and fibroblasts in vitro. Since fibroblasts secrete growth factors such as SCF, the potential of Nfl deficient fibroblasts to alter the survival, proliferation and degranulation of Nfl +/- mast cells is determined in coculture experiments.
  • Example 5 The Biochemical Mechanisms Leading to Abnormal Cell Function in Nfl -/-. Nfl +/- and Nfl +/+ Mast Cells in Response to Stem Cell Factor Loss of neurofibromin leads to elevated levels of activated Ras which potentially results in the hyperactivation of multiple downstream effectors.
  • Ras- GTP binds and activates the serine/threonine kinase Raf-1 which leads to activation of the MEK and ERK kinases (Ras-Raf-ERK kinase pathway) (Marshall, 1996). Most prohferative stimuli in mammalian cells activate ERK kinases following growth factor stimulation.
  • Ras-GTP While Ras activation of the ERK kinase pathway is the best characterized biochemical pathway, Ras-GTP also binds and activates other downstream effectors via Raf independent mechanisms which are important for coordinating cellular responses to a wide variety of extracellular stimuli (White et al., 1995) ( Figure 8). Recently, activated Ras has been shown to bind and activate PI3-kinase (PI3K) (Marshall, 1996). Ras activation of PI3K leads to the activation of another MAP kinase family member, jun-N- terminal kinase (JNK), through a Raf-1 independent mechanism (White et al., 1995).
  • PI3K PI3-kinase
  • JNK jun-N- terminal kinase
  • PI3K activation of JNK requires the small G proteins Rac and Cdc42, but these pathways are cureently not well defined (Minden et al., 1995; Coso et al., 1995).
  • simultaneous activation of multiple downstream effectors by Ras-GTP underscores the importance of examining the coordinated response of these downstream pathways in specific cell types, particularly in primary cells where Ras is hyperactivated.
  • the biological importance of multiple effectors in Ras signaling has been recently illustrated using new Ras point mutants which bind and selectively activate only subsets of Ras effectors (White et al., 1995).
  • Nfl +/- and Nfl +/+ mast cell proliferation were used to evaluate the contribution of each pathway to the proliferation of each genotype to SCF.
  • pharmacologic inhibitors of two well characterized mitogenic pathways were used to evaluate the contribution of each pathway to the proliferation of each genotype to SCF.
  • 1 x 10 5 Nfl +/- and Nfl +/+ BMMCs were growth factor starved for 24 hours and then cultured in complete RPMI media containing 10% fetal calf serum with 100 ng/ml SCF, in the presence of either LY294002 or vehicle, in triplicate cultures for 48 hours.
  • BMMCs were lysed and ERK activity was measured in a kinase activity assay as previously described (Bollag et al., 1996). Autoradiography and quantitative densitometry of the phosphorylation of Elk- 1 fusion protein by ERK2 from lysates obtained from SCF stimulated BMMCs are shown. ERK2 activity in Nfl +/- mast cells was higher at baseline and 2 and 5 minutes after stimulation with SCF compared to wildtype cells. Data represents 1 of 3 independent experiments with similar results. Interestingly, preincubation of Nfl +/- and Nfl +1+ BMMCs with Ly294002 prior to stimulation significantly inhibited ERK activity in both genotypes at both time points tested.
  • Nfl +/+ and Nfl +/- BMMCs were growth factor starved for 24 hours, and cultured in complete RPMI media containing 10% fetal calf serum, 100 ng ml SCF, and either 50 ⁇ M PD98059 or vehicle.
  • PAK activity was determined using a kinase activity assay in Nfl +/- and Nfl +/+ cells after stimulation with a low concentration of SCF (10 ng/ml). This dose was selected because it is closer to the range (3-5 ng/ml) observed in vivo in normal animals as compared to traditional saturating cytokine concentrations used in in vitro studies.
  • PAK activity was greatly enhanced in Nfl +1- mast cells compared to wildtype cells at all time points tested.
  • PAKs may confer a growth advantage to Nfl +/- BMMCs following growth factor stimulation.
  • Nfl +/- and +/+ primary bone marrow derived mast cells following stimulation with stem cell factor.
  • Primary bone marrow derived mast cells (BMMCs) lines from Nfl +/- and +/+ mice are generated as previously described (Serve et al., 1995), and Nfl -I- mast cells are derived from primary dl3.5 gestation fetal liver cells. At least 3-4 independently derived primary mast cell lines of the respective genotypes are tested. Fetal liver and bone marrow derived mast cells behave similarly in culture (unpublished results).
  • BMMCs from each of the three genotypes are serum starved overnight to induce quiescence and stimulated with a range of concentrations (0, 3, 10, and 100 ng/ml) of SCF. Prior to stimulation, BMMCs are equalized for cell number and viability. Cells are stimulated for the following time course: 0, 15 seconds, 30 seconds, 1 minute, 5 minutes, and 15 minutes.
  • concentrations of SCF are tested because of recent studies linking the strength of the stimulus and subsequent activation of PI3K to cross- cascade activation of MAPkinase. Wennstrom et al.
  • PAKs Three iso forms of PAKs have been identified which include PAKl, PAK2, and PAK3. Most studies have characterized the ability of PAKl to mediate cross talk from the PI3K pathway by phosphorylating MEKl on serine 298. Thus, a specific antibody to PAKl is used for immunoprecipitation after cell lysis to test PAKl activity in these assays. Equal amounts of protein from each test group are used by performing a Bradford assay prior to immunoprecipitation. If differences are observed in PAK kinase activity between the three genotypes, total levels of PAK protein in BMMCs are detected to ensure that differences are not secondary to increased levels of protein in Nfl deficient cells.
  • PAK3 kinase was shown to specifically phosphorylate serine 338 on Raf-1 following PI3 kinase activation (King et al., 1998). Phosphorylation on this site enhanced Raf-1 kinase activity (King et al., 1998).
  • PAK3 may be another potential mediator of cross talk between the PI3 kinase pathway and the Ras-Raf-ERK pathway.
  • PAK3 activity is determined following stimulation with SCF.
  • PAK3 activity assays are performed to compare levels and time course of activation between the 3 genotypes.
  • PAK activation in any of the 3 experimental groups is dependent on PI3K activation, then preincubation of BMMCs with LY294009 prior to stimulation with SCF should inhibit PAK activation.
  • different isoforms of PAK utilize PI3K independent pathways for cross-cascade activation of kinases in the Ras-Raf-ERK pathway.
  • Rac or other downstream effectors
  • PI3 kinase may be simultaneously activated by Ras-GTP following SCF stimulation converging upon the activation of PAKs.
  • Nfl +/- BMMCs Stimulation of Nfl +/- BMMCs with 10 ng/ml of SCF results in elevated levels of PAK activity as compared to wildtype mast cells.
  • a MEK inhibitor reduced the proliferation of Nfl +/- mast cells to wildtype levels, while addition of a MEK inhibitor to wildtype cells had a minimal effect on proliferation.
  • the effect of a dominant negative PAKl or PAK3 on the prohferative responses of Nfl -/-, Nfl +/-, or +/+ mast cells is determined.
  • Prohferative assays provide a sensitive measure of the biological effects of PAK activation in BMMCs.
  • MSCV-pac PAKl or PAK3 cDNAs
  • the retrovirus encodes the selectable marker gene Pac which renders cells resistant to puromycin.
  • Pac which renders cells resistant to puromycin.
  • transduced cells expressing the dominant negative PAKl or PAK3 are selected by adding puromycin to the cultures.
  • This backbone has been used with the same promotor sequences to express genes in murine fetal liver hematopoietic stem/progenitor cells for up to 20 months in vivo (Clapp et al., Blood. 86. 94 (1995)).
  • this virus was used to transduce the NFl GRD sequences into Nfl -I- mast cells
  • prohferative responses to SCF of BMMCs containing either a DN PAKl or a DN PAK3 from each genotype are compared to BMMCs from each experimental group transduced with the constructs encoding only the reporter genes.
  • Mast cells are growth factor starved overnight in complete mast cell media containing 10% FCS, 2% penicillin/streptomycin, 1% glutamine.
  • 3 x 10 5 viable BMMCs are counted using a trypan blue exclusion assay and plated in triplicate cultures in 24 well plates in complete mast cell media containing either 10, 25, 50, or 100 ng of SCF.
  • Mast cells are then placed in a humidified incubator at 37°C and 5% CO 2 and counted on days 1, 3, and 5 following incubation by trypan blue exclusion. Numbers of viable mast cells are expressed as a percentage of input cells to assess the prohferative response. Though 100 ng/ml is considered to be the maximal prohferative dose of SCF for BMMCs, the proliferation of mast cells in response to lower doses of SCF is tested to generate a dose response curve and to approximate more physiologic concentrations of SCF. Thymidine inco ⁇ oration assays are also performed to confirm any prohferative differences observed in the experimental.
  • PAKl or PAK3 is mediating cross-cascade activation of ERK via PI3K in Nfl -I- and Nfl +/- mast cells, then elevated levels of PAKl or PAK3 activity in Nfl deficient mast cells are found compared to wildtype cells following SCF stimulation. Similarly, increased phosphorylation on serine residue 298 on MEK in Nfl deficient cells is observed if PAKl activity is elevated, and an increase in phosphorylation of serine 338 on Raf-1 is found if PAK3 activity is elevated.
  • PAKl or PAK3 is mediating cross-cascade activation of ERK via PI3K to confer a prohferative advantage to Nfl +/- and Nfl -I- mast cells, then transduction of the dominant negative PAKl or PAK 3 cDNAs into Nfl deficient (Nfl -I- or Nfl +/-) cells results in a restoration of normal cytokine responsiveness to SCF in proliferation assays, similar to that observed in wildtype cells.
  • Nfl +/- mice were obtained from Tyler Jacks at the Massachusetts institute of Technology (Cambridge, MA) in a C57BL/6.129 background and backcrossed for 13 generations into the C57BL/6 strain. Wildtype C57BL/6 mice were obtained from Harlan Laboratories (Indianapolis, IN). These studies were conducted with a protocol approved by the Indiana University Laboratory Animal Research Center. The Nfl allele was genotyped as described in Bollag et al. (1996) and Zhang et al. (1998).
  • BMMCs bone marrow derived mast cell lines
  • FACS analysis revealed similar forward and side light scatter characteristics and the same percentage of c-kit + expression in BMMCs of both murine genotypes (data not shown).
  • the mast cell survival assay was done as follows: BMMCs from each genotype were deprived of growth factors for 24 hours and 3 x 10 5 cells were plated in 24-well dishes in serum-free RPMI containing 1% glutamine, 1.5% HEPES (BioWhittaker, Walkersville, MD), 2% penicillin/streptomycin (BioWhittaker, Walkersville, MD) and 100 ng/ml recombinant murine stem cell factor (rmSCF) (Peprotech) in a total volume of 1 ml.
  • rmSCF murine stem cell factor
  • LY294002 Sigma
  • PD98059 New England Biolabs
  • the number of surviving cells was determined by trypan blue exclusion at 24 hours of culture in a 37°C, 5% CO 2 , humidified incubator. Apoptosis was determined as described in Haneline et al. (1998) by examining DNA fragmentation using the TUNEL assay according to the manufacturer's protocol. Assays were performed in triplicate.
  • BMMCs were deprived of growth factors for 24 hours and 1 x 10 5 cells were plated in 96 well dishes in 200 ml of RPMI containing 1% glutamine, 10% fetal bovine serum (Hyclone Laboratories) and 100 ng/ml of rmSCF or no growth factors, as indicated, in a 37°C, 5% CO 2 , humidified incubator.
  • Cells were cultured for 48 hours, and tritiated thymidine (New Life Science Products, Inc.) was added to cultures 6 hours prior to harvest. Cells were harvested on glass fiber filters (Packard, Meriden, CT) and ⁇ emission was measured. Assays were performed in triplicate.
  • BMMCs were serum starved for 16-18 hours and 2.5 x 10 5 BMMCs were resuspended in 100 ⁇ l of serum free RPMI containing 1% glutamine and 0.5% BSA (Sigma). BMMCs were then loaded onto each transwell filter (8- ⁇ m pore filter Transwell, 24-well cluster; Costar, Boston, MA). Filters were then placed in wells containing 600 ⁇ l of serum free RPMI supplemented with 10 ng/ml rmSCF.
  • Chamber temperature was maintained at 37°C using a stage heater (Instec Instruments Inc., Boulder, CO). The chamber was allowed to equilibrate for 20 minutes to allow a stable gradient to form. Images were recorded digitally at 15-second intervals using a 40x LWD objective and a Spot RT cooled CCD camera and analyzed using Metamo ⁇ h software (Universal Imaging, Brandywine, PA). The centroid coordinates of cells at each timepoint were determined on calibrated images and used to calculate the speed and direction of migration.
  • F- Actin quantitation F-actin quantitation was performed as previously described (Roberts et al., 1999). Briefly, flow cytometry was used to quantitate the amount of filamentous actin per mast cell. Mast cells (10 6 ) were resuspended in 1 ml HBSS in polypropylene tubes and warmed at 37° for 3 minutes prior to addition of SCF or carrier. Cells were fixed after the specified time by addition of 10 volumes of PBS containing 4.6% formaldehyde and 0.1% BSA.
  • Coverslips were treated with block solution (wash buffer PBS with 1 mg/ml BSA, 0.5 mM MgCl 2 , 2.5 mM calcium chloride containing 50 mM ammonium chloride, 25 mM glycine, 25 mM L-lysine and 10% goat serum) for 30 minutes and then incubated in block solution containing 0.1 ⁇ g/ml rhodamine phalloidin (Molecular Probes, Eugene, OR) for 1 hour. Coverslips were washed briefly with wash buffer and mounted on 50% glycerol- PBS containing 100 mg/ml diamino-bicyclo-[2.2.2] octane (Sigma). Slides were imaged using a Zeiss LSM 510 laser scanning confocal microscope using a lOOx, 1.4 N.A. oil immersion objective.
  • block solution wash buffer PBS with 1 mg/ml BSA, 0.5 mM MgCl 2 , 2.5 mM calcium chloride containing
  • ERK and PAK kinase immunoprecipitations were carried out with an anti-ERK-2 (C-14) antibody and an anti-PAK(C-19) antibody (Santa Cruz), respectively.
  • Raf-1 and Mek kinase immunoprecipitations were carried out with an anti-Raf-1 (C-12) antibody (Santa-Cruz) and an anti-Mek-1 (C-18) antibody (Santa Cruz), respectively.
  • Akt kinase immunoprecipitations were carried out with an anti-Akt antibody (Cell Signaling Technology).
  • kinase immune complex assay was performed as previously described (Bollag et al., 1996a) and the following proteins were used as phosphorylation substrates: Elk-1 fusion protein (New England Biolabs) for JNK and ERK kinase, histone 2B (Roche) for Akt, myelin basic protein (Sigma) for PAK kinase and recombinant MEKl fused with GST at the N-terminus (Upstate Biotechnology) for Raf-1 kinase. For Mek, Raf-1, and ERK, kinase reactions were resolved on 10% SDS-P AGE gels (No vex).
  • kinase reactions were resolved on 4-20% gradient SDS-P AGE gels (Novex), and transfened to PVDF filters. Gels and PVDF filters were dried and subjected to autoradiography. Densitometry of individual bands was conducted using NTH Image software.
  • Ras. Cdc42 and Rac activation assays Bone marrow derived mast cells were deprived of serum and growth factors for 24 hours and stimulated at various amounts of time with 10 ng/ml rmSCF. Ras, Cdc42 and Rac activation was subsequently determined using Ras, Cdc42, and Rac activation assay kits (Upstate Biotechnology) according to the manufacturer's protocol and as previously described in Gille and Downward (1999).
  • BMMCs were lysed in nonionic lysis buffer as previously described (Bollag et al., 1996). Cleared lysates were normalized for protein content using the BCA assay (Pierce Chemical Co.) and proteins were immunoprecipitated with protein A sepharose beads (Amersham) coupled with polyclonal antibodies for Raf-1, Mek, PI-3 kinase pi lO ⁇ (H-219) (Santa Cruz), PI-3 kinase pi 10a (H-201) (Santa Cruz), or c-kit (C-19) (Santa Cruz) for 2 hours at 4°C.
  • Immunoprecipitates were washed three times in lysis buffer, resuspended in sample buffer, boiled for 5 minutes, subjected to SDS- PAGE and transfened to nitrocellulose. The membranes were blocked in TBS- Tween containing 5% BSA overnight. Membranes were incubated for 1-3 hours at room temperature with following antibodies: anti-phospho-Mek 1/2 (1:1000) (New England Biolabs), anti-phosho-Raf-1 338 (1 :5000) (Upstate
  • Osmotic pumps were surgically placed under light avertin anesthesia. rmSCF or vehicle was released over 7 days at a rate of 0.5 ⁇ l/hr, and osmotic pumps were surgically removed on day 7 following sacrificed by cervical dislocation.
  • the dorsal skin was stained with a drop of India ink at the point of exit of rmSCF from the osmotic pump prior to removal of the pump. Three cm sections of skin marked with India ink were removed, fixed in buffered formalin and processed in paraffin-embedded sections.
  • Specimens were stained with hematoxylin-eosin to assess routine histology and with Giemsa stain to identify mast cells. Cutaneous mast cells were quantitated in a blinded fashion by counting 2 mm 2 sections in proximity to the India ink stain. Results
  • Nt7 +/- mast cells Stimulation of Nt7 +/- mast cells with stem cell factor.
  • SCF binding to its receptor, c-kit causes a rapid increase in p21 ras activity in primary BMMCs (Serve et al., 1995).
  • Nfl +/- and wildtype BMMCs were stimulated with SCF and assayed for changes in active p21 ras -GTP levels.
  • pi lO ⁇ is the predominantly expressed class ⁇ PI-3 kinase catalytic subunit isoform, and active p21 ras -GTP can directly bind to the p21 ras binding domain of pi lO ⁇ to increase PI-3 kinase activity (Luo-Kuo et al., 2000, and Vanhaesebroeck et al., 1997).
  • Nfl +/- and wildtype BMMCs were stimulated with SCF for 5 minutes and pi lO ⁇ was immunoprecipitated from lysates using a specific polyclonal antibody.
  • immune complexes were separated on SDS-P AGE gels and Western blotting was performed with a specific antibody for p21 ras .
  • Akt PI-3 kinase dependent and provides a sensitive measure for PI-3 kinase activity (Alessi and Cohen, 1998).
  • Nfl +/- BMMCs demonstrated greater basal and SCF- stimulated Akt activity compared to wildtype cells ( Figure 13C).
  • inhibitors of PI-3 kinase inhibited Akt activity in both mast cell genotypes ( Figure 13C) confirming that the p21 ras contribution to Akt activation is via activation of PI-3 kinase.
  • Proliferation of wildtype BMMCs in response to SCF is dependent on PI- 3 kinase activation and subsequent stimulation of downstream effectors including the protein kinase JNK (c-jun N-terminal kinase) and Akt (Lu-Kuo et al., 2000; Timokhina et al, 1998). While it was previously shown that Nfl +/- mast cells have increased proliferation in response to SCF, the biochemical mechanisms responsible for this phenotype remain unclear.
  • Nfl +/- BMMCs were tested whether SCF-induced hype ⁇ roliferation of Nfl +/- BMMCs is mediated through hyperactivation of PI-3 kinase.
  • DNA synthesis was measured in Nfl +/- and wildtype BMMCs when incubated with SCF or medium alone, in the presence or absence of LY294002.
  • Thymidine inco ⁇ oration was determined forty-eight hours after addition of SCF.
  • Nfl +/- BMMCs showed a 2-fold greater increase in DNA synthesis compared with wildtype cells, as previously observed (Figure 14B).
  • JNK activity in both mast cell genotypes was compared in the presence or absence of PI-3 kinase inhibitors following stimulation with SCF. Both wortmannin ( Figure 14C) and LY294002 (data not shown) inhibited JNK activity in both mast cell genotypes indicating that JNK activation is dependent on signals from PI-3 kinase.
  • Hype ⁇ roliferation of Nfl +/- Mast Cells is Independent on Crosscasade Activation of ERK from PI-3 Kinase. While hyperactivation of the PI-3 kinase - JNK pathway provides a simple model for the hype ⁇ roliferation of Nfl +/- BMMCs, other downstream effectors of PI-3K could contribute to the proliferation of Nfl +/- BMMCs. In other cell types, signals from PI-3K converge upon the p21 ras -Raf-Mek-ERK pathway to activate ERK and promote transcription of genes that control mitogenesis (Frost et al., 1997; Tang et al., 1999).
  • Nfl +/- BMMCs have elevated ERK activity relative to wildtype cells following stimulation with SCF, but the contribution of ERK activation to the proliferation of mast cells is unclear (Ingram et al., 2000).
  • DNA synthesis was measured in Nfl +/- and wildtype mast cells when incubated with SCF or medium alone, in the presence or absence of the MEK inhibitor PD98059, which blocked ERK activation in both genotypes (data not shown). Forty eight hours after the addition of SCF, thymidine inco ⁇ oration was determined.
  • Crosscascade Activation of ERK from PI-3 kinase Occurs at the Level of MEK and Raf-1 kinase in Nfl +/- and Wildtype Mast Cells.
  • crosscascade activation of ERK from PI-3 kinase occurs at the level of either MEK or Raf-1 kinase (Frost et al., 1998; Garrington et al, 1999; Tang, et al 1999).
  • Nfl +/- and wildtype BMMCs were stimulated with SCF in the presence or absence of a PI-3 kinase inhibitor and measured MEK activity.
  • Nfl +/- BMMCs demonstrated a greater increase in MEK activity from baseline compared to wildtype cells and MEK activity was inhibited in the presence of PI-3 kinase inhibitors in both mast cell genotypes (Figure 15 A).
  • Raf-1 functions as the immediate upstream effector of MEK by phosphorylating Ser217/221 (Alessi et al., 1994)
  • Nfl +/- BMMCs had increased phosphorylation of Mek Ser217/221 which conelates with increased Raf-1 activity in these cells (Figure 15C).
  • phosphorylation of these residues was also inhibited by a PI-3K inhibitor in both mast cell genotypes ( Figure 15C).
  • p21 -activated kinase can mediate crosstalk between PI-3 kinase and the p21 ⁇ -Raf-MEK-ERK pathway by phosphorylating Ser298 on MEKl and Ser338 on Raf-1 (Frost et al., 1997; King et al., 1998). Phosphorylation of Ser298 increases MEKl binding to Raf-1 to enhance the kinase activity of the complex and phosphorylation of Ser338 is critical for Raf- 1 activation in a number of experimental systems (Chaudhary et al., 2000; Frost et al., 1997; King et al., 1998).
  • PAK activity was measured in both mast cell genotypes in response to SCF. While both mast cell genotypes demonstrated PAK activation after stimulation with SCF, Nfl +/- BMMCs had dramatically increased basal and SCF stimulated PAK activities compared with wildtype cells ( Figure 16 A). In addition, hyperactivation of PAK in Nfl +/- BMMCs conelated with increased phosphorylation of Ser298 on MEKl and Ser338 on Raf-1 ( Figure 16B).
  • PAK has been shown to be located either upstream or downstream of PI-3 kinase in different cell types (Chaudhary et al., 2000; Sun et al., 2000; Yablonski et al., 1998), the PAK activity assay was repeated in the presence or absence of a PI-3K inhibitor.
  • wortmannin inhibited both PAK activation (data not shown) and phosphorylation of Ser298 on MEKl and Ser338 on Raf-1 following stimulation with SCF (Figure 16B).
  • PAK interacts specifically with GTP bound forms of its direct upstream effectors, the small Rho GTPases Rac and Cdc42.
  • the catalytic activity of PAK is regulated by binding of Rac-GTP and Cdc42-GTP to the highly conserved N- terminus, known as the CRIB binding domain (Bagrodia and Cerione, 1999; Daniels and Bokoch, 1999).
  • Cdc42-GTP and Rac-GTP levels were compared in Nfl +/- and wildtype BMMCs after stimulation with SCF.
  • Nfl +/- BMMCs had higher basal and SCF stimulated levels of both active Cdc42- GTP and Rac-GTP compared with wildtype cells ( Figures 16C-D).
  • FIGs 16C-D shows that other effectors may mediate crosstalk between PI-3 kinase and ERK in both mast cell genotypes, these data offer evidence that hyperactivation of PAK may directly contribute to elevated ERK activity and enhanced proliferation in Nfl +/- mast cells.
  • Nfl +/- mast cells Given increases in Cdc42/Rac-GTP levels and PAK activity in Nfl +/- mast cells, F-actin content was compared in Nfl +/- and wildtype BMMCs following stimulation with SCF. After stimulation, both mast cell genotypes were stained with phalloidin to quantitate levels of F-actin by fluorescence cytometry and fluorescence microscopy. In both assays, Nfl +/- BMMCs displayed increased F-actin compared with wildtype cells ( Figures 17A-B).
  • Nfl +/- BMMCs had greater F- actin content at baseline compared to F-actin levels obtained in wildtype cells stimulated with SCF ( Figures 17A-B).
  • increased F-actin content in Nfl +/- BMMCs is consistent with hyperactivation of proteins known to regulate the actin cytoskeleton.
  • these experiments were repeated in the presence of PI-3 kinase inhibitors. Preincubation of both mast cell genotypes with either LY294002 or wortmannin inhibited increases in F-actin content after stimulation with SCF (data not shown).
  • ADF/cofilin is a class of small actin binding proteins that promote depolymerization of actin by increasing the off rate for actin monomers at the pointed end (slow growing end) and by promoting filament fragmentation (Moon and Drubin, 1995; Theriot,
  • ADF/cofilin is phosphorylated by LIM-kinase.
  • the ability of cofilin to disassemble actin filaments is reduced following its phosphorylation by LIM- kinase resulting in increased actin content (Arber et al., 1998; Yang et al., 1998).
  • Nfl +/- mast cells have increased PAK activity and F-actin content, the phosphorylation of cofilin was compared in both mast cell genotypes after stimulation with SCF by Western blot.
  • Nfl +/- BMMCs had higher levels of phosphorylated cofilin at both baseline and after stimulation with SCF compared to wildtype cells (Figure 17C) consistent with hyperactivation of a signaling cascade which controls F-actin polymerization.
  • Nfl +/- Mice Have Greater Accumulation of Mast Cells in Response to Local Cutaneous Administration of Stem Cell Factor.
  • Local cutaneous injection of SCF into wildtype mice results in a dramatic accumulation of mast cells at the site of injection (Iemura et al., 1994; Tsai et al., 1991).
  • Local expansion of mast cells is dependent on mast cell proliferation, survival, and chemotaxis in response to SCF in this experimental model (Tsai et al., 1991).
  • Nfl+/- mice also had a greater percentage of degranulating mast cells at this dose (28% vs 8%) compared to wildtype mice at the site of SCF release.
  • Neurofibromatosis type I is a complex genetic disease that affects multiple cell lineages. Alterations in p21 ras activity have been linked with LOH in tumors generated from individuals with NFl and Nfl knockout mice, consistent with the classification of NFl as a tumor suppressor gene (Bollag et al., 1996; DeClue et al., 1992; Largaespada et al., 1996). However, the frequent nonmalignant manifestations found in patients with NFl infer a gene dosage effect. Consistent with this hypothesis is the slow and self-limited growth of most neurofibromas, combined with the observation that genetic analysis of many neurofibromas fail to detect LOH.
  • Nfl +/- mast cells have a 2-fold increase in each of these cellular functions in response to SCF as compared with wildtype cells in vitro.
  • these functions were dramatically reduced with PI-3 kinase inhibitors, confirming the importance of this pathway in Nfl+/- cells.
  • placement of micro- osmotic pumps containing SCF into the skin of Nfl +/- mice resulted in a greater accumulation of mast cells at the site of insertion compared to wildtype mice.
  • p21 ras -GTP can specifically bind the pi 10a and the pi lO ⁇ catalytic subunits of Class I A PI-3 kinase to increase protein kinase activity (Khwaja et al., 1997; Kodaki et al., 1994; Rodriguez- Viciana et al., 1996; Rodriguez- Viciana et al., 1997; Vanhaesebroeck et al., 1999).
  • these pi 10 catalytic subunits have been shown to control cellular functions necessary for accumulation of inflammatory cells at the site of stimulus (Vanhaesebroeck et al., 1999).
  • blocking antibodies directed at the pi 10a subunit inhibited the proliferation and survival of these cells in response to colony stimulating factor and antibodies raised against the pi lO ⁇ subunit inhibited migration and F-actin polymerization in response to the same stimulus
  • PAK can phosphorylate LIM- kinase which results in increased phosphorylation of the actin regulatory protein, cofilin.
  • cofilin When cofilin is phosphorylated, its F-actin depolymerizing activity is inhibited leading to the accumulation of actin filaments (Moon and Drubin, 1995; Theriot, 1997).
  • Nfl +/- mast cells displayed increased endogenous PAK activity and elevated levels of phosphorylated cofilin both at baseline and in response to SCF when compared to wildtype cells.
  • phosphorylation levels of cofilin observed in Nfl +/- mast cells were consistent with elevated F-actin content.
  • Nfl +/- mast cells elicit increased ERK activation compared to wildtype mast cells after stimulation with SCF. While hyperactivation of PI-3 kinase provides a simple model for the increased proliferation of Nfl +/- mast cells, unexpectedly it was found that activation of ERK is completely dependent on signals from PI-3 kinase in both mast cell genotypes and is linked to the hype ⁇ roliferative phenotype of Nfl +/- mast cells.
  • PI-3 kinase inhibitors completely blocked the activation of ERK in both mast cell genotypes linking ERK to PI-3 kinase.
  • physiological relevance of crosstalk between these pathways is shown by the action of a specific MEK inhibitor which reduced the SCF-stimulated proliferation of both mast cell genotypes. Since a MEK inhibitor reduces proliferation of Nfl +/- mast cells to wildtype levels, hyperactivation of ERK from PI-3 kinase confers a distinct growth advantage to these cells. Given the importance of this finding to the prohferative phenotype of Nfl +/- mast cells, PAK was focused on as a biochemical mediator of this crosstalk.
  • PAKl and PAKl are different isoforms of PAK. Different isoforms of PAK have been shown to increase signaling through the Raf-MEK-ERK pathway by phosphorylating specific residues on Raf and MEK (Chaudhary et al., 2000; Frost et al., 1997; Sun et al., 2000). Specifically, activated PAKl can phosphorylate MEKl at Ser298 to enhance the stable association between Raf-1 and MEKl (Frost et al., 1997). PAKl and
  • PAK3 can also phosphorylated Ser338 in the Raf-1 catalytic domain essential for activation of Raf-1 kinase (Chaudhary et al., 2000; King et al., 1998; Sun et al., 2000).
  • PAK has been positioned downstream of PI-3 kinase (Chaudhary et al., 2000; Sun et al., 2000).
  • Nfl +/- mast cells had higher Raf-1 and MEK activity compared with wildtype cells. The present study data suggests that PAK contributes to these differences in activation for two reasons.
  • Bourne HR, Sanders DA, McCormick F The GTPase superfamily: conserved structure and molecular mechanism. Nature. 349, 117-127 (1991).
  • Brillion G The NFl gene in myelopoiesis and childhood myelodysplastic syndromes. N. Engl. J. Med.. 330. 637-638 (1994).
  • Clapp DW, Williams DA The use of umbilical cord blood as a cellular source for conection of genetic diseases affecting the hematopoietic system. Stem Cells. 13, 613-621 (1995c).
  • Gutkind JS The small GTP-binding proteins Racl and Cdc42 regulate the activity of the JNK SAPK signaling pathway.
  • CeU CeU, 81, 1137-1146 (1995).
  • Coussens LM Raymond WW, Bergers G, Laig- Webster M, Behrendtsen
  • Galli S, Tsai M, Wershil B The c-kit receptor, stem cell factor, and mast cells. American Journal of Pathology. 142. 965-974 (1993). Galli SJ: New concepts about the mast cell. N. Engl. J. Med.. 328. 257- 265 (1993).
  • Hall A The Cellular Functions of Small GTP -Binding Proteins. Science. 249. 635-640 (1990). Hall, A: Signal Transduction through Small GTPases-A Tale of Two
  • Hawley R, Lieu F, Fong A, Hawley T Versatile retroviral vectors for potential use in gene therapy. Gene Therapy. I, 136-138 (1994).
  • Huson SM Upadhyaya M. Neurofibromatosis 1 : Clinical management and genetic counseling. In The Neurofibromatoses: A pathogenic and clinical overview.
  • Husan SM Hughes R.A. (eds), Chapman and Hall, London, Chapman and Hall, pp 355381 (1994).
  • Type 1 neuro fibromatosis selective expression of extracellular matrix genes by Schwann cells, perineurial cells, and fibroblasts in mixed cultures. J. Clin.Invest. 54, 253-261.
  • Lu-Kuo J, Austen K, Katz H Post-transcriptional stabilization by interleukin- 1 beta of interleukin-6 mRNA induced by c-kit ligand and interleukin- 10 in mouse bone manow-derived mast cells. The Journal of Biological Chemistry. 271. 22169-22174 (1996). Lu-Kuo, J. M., Fruman, D. A., Joyal, D. M., Cantley, L. C, and Katz, H.
  • the c-kit receptor ligand functions as a mast cell chemoattractant. Blood 79, 958-963.
  • the ADF/cofilin proteins stimulus- responsive modulators of actin dynamics. Mol. Biol. Cell 6, 1423-1431.
  • Nakahata T, Ogawa M Identification in culture of a class of hemopoietic colony- forming units with extensive capability to self-renew and generate multipotential hemopoietic colonies. Proc. Natl. Acad. Sci. USA. 79, 3843-3847 (1982).
  • Riccardi VM Neurofibromatosis: Phenotype, natural history and pathogenesis (ed 2nd). Baltimore, The Johns Hopkins University Press, 1992.
  • McCormick F Loss of the normal NE7 allele from the bone marrow of children with type 1 neurofibromatosis and malignant myeloid disorders. ⁇ . ⁇ ngl. J. Med.. 330. 597-601 (1994).
  • Tay Y Neurofibromatosis 1 and Piebaldism: A case report. Dermatology. 197. 401-402 (1998). Theriot, J. A. (1997). Accelerating on a treadmill: ADF/cofilin promotes rapid actin filament turnover in the dynamic cytoskeleton. J. Cell Biol. 136, 1165-1168. Timokhina, I., Kissel, H., Stella, G., and Besmer, P. (1998). Kit signaling through PI 3-kinase and Src kinase pathways: an essential role for Racl and JNK activation in mast cell proliferation. EMBO J. 17, 6250-6262.
  • Type 1 neurofibromatosis gene identification of a large transcript disrupted in three NFl patients. Science 249, 181-186.

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Abstract

La présente invention concerne une méthode pour identifier les inhibiteurs de Raf, MEK, protéine ERK1, protéine ERK2, phosphatidylinositol triphosphate kinase (P13K), protéine PAK2, protéine PAK3, Rac, CDC42, protéine PAK1, protéine kinase B (PKB), Akt, de c-protéine JNK, ou du couplage parasite entre P13-K et le trajet Ras-Raf-MEK-ERK, utilisés pour traiter les troubles tels que la neurofibromatose de type 1, ou au moins un de ses signes cliniques.
PCT/US2000/030924 1999-11-12 2000-11-10 Methodes de detection d'agents utiles pour inhiber la neurofibromatose de type 1 (nf1) Ceased WO2001034201A2 (fr)

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EP00975626A EP1227846A2 (fr) 1999-11-12 2000-11-10 Methodes de detection d'agents utiles pour inhiber la neurofibromatose de type 1 (nf1)
JP2001536198A JP2003513939A (ja) 1999-11-12 2000-11-10 神経繊維腫症タイプ１（ｎｆ１）の阻害方法
AU13647/01A AU1364701A (en) 1999-11-12 2000-11-10 Methods to detect agents useful to inhibit neurofibromatosis type 1 (nf1)
CA002388343A CA2388343A1 (fr) 1999-11-12 2000-11-10 Methodes de detection d'agents utiles pour inhiber la neurofibromatose de type 1 (nf1)

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US7118854B2 (en) * 2001-03-30 2006-10-10 Rigel Pharmaceuticals, Inc. PAK2: modulators of lymphocyte activation
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