MX2008000894A - Compositions for treatment of systemic mastocytosis - Google Patents

Abstract

The present invention relates to the use of the combination of tyrosine phosphate inhibitors AMN107 and PKC412 for the preparation of a drug for the treatment of a mast cell-related proliferative disease. The present invention is also drawn to a combination treatment of a tyrosine phosphate inhibitor and a TK-inhibitor that is effective against a mast cell-related proliferative disease, including especially systemic mastocytosis (SM) including aggressive SM (ASM) and mast cell leukemia (MCL).

Description

COMPOSITIONS FOR THE TREATMENT OF SYSTEMIC MASTOCYTOSIS FIELD OF THE INVENTION The present invention relates to the use of tyrosine kinase inhibitors for the preparation of a medicament for the treatment of systemic mastocytosis. The present invention also relates to a method for treating systemic mastocytosis.

BACKGROUND OF THE INVENTION Systemic mastocytosis (SM) can be classified into indolent SM (little or no evidence of impaired organ function), aggressive SM (presence of impaired organ function), non-mast cell haematological disease associated with MS (SM- AHNMD) and mast cell leukemia. Clinical presentation in adult MS is heterogeneous and includes skin disease (usually urticaria pigmentosa), symptoms of release of mediators of mast cells (headache, blushing, dizziness, syncope, anaphylaxis, etc.), and direct organ damage or indirect (bone pain from lytic bone lesions, osteoporosis or bone fractures, hepatosplenomegaly, bone marrow involvement cytopenia). In addition, about 20% of patients with MS may exhibit significant and sometimes isolated blood eosinophilia (Tefferi and Pardanani 2004). In general, mast cell leukemia is a terminal disease with survival measured in months and no effective therapy to date. The natural history of indolent MS is much better with survival measured in decades and infrequent progression to aggressive SM and SM-AHNMD. The result in SM-AHNMD is determined by associated AHNMD and is significantly worse than SM without AHNMD. Both in indolent and aggressive MS without AHNMD, the content of eosinophils and mast cells of increased bone marrow, elevated serum alkaline phosphatase, anemia and hepatosplenomegaly, have been associated with poor prognosis (Tefferi and Pardanani 2004). Complete clinical and histological remission has been achieved in patients with MS with the fusion of gene FIP1 L1 -PDGFRa when treated with Gleevec® (Pardanani 2003a, Pardanani 2003b).

BRIEF DESCRIPTION OF THE INVENTION Several emerging treatment concepts for myeloid neoplasms are based on novel drugs that focus on critical tyrosine kinases (TK) or downstream signaling molecules.1"5 Systemic mastocytosis (SM) is a hematopoietic neoplasm that behaves like an indolent myeloproliferative disease in most patients, but may also present as an aggressive disease (aggressive MS is denoted in the present as "ASM") or even as a leukemia, for example, mast cell leukemia (denoted herein as "MCL"). 6"1 1 In patients with ASM and MCL, the response to conventional therapy is poor in most of the cases and the prognosis is serious.6"12 Therefore, a variety of attempts have been made to identify novel neoplastic mast cell (MC) therapy targets and to define new treatment strategies for these patients.9" 12 the majority of all patients with MS including those who are diagnosed as having ASM or MCL, the point mutation c-KIT somatic D816V (Asp816Val) is detectable in neoplastic cells (mast cells) .13"17 This point mutation is associated with independent phosphorylation of ligand and activation of KIT, and autonomous differentiation and growth of affected cells.17, 18 Based on this association with constitutive tyrosine kinase activity (TK), the variant mutated with D816V of KIT is a target at therapy ractivity.9"12'19 A variety of efforts have been made in recent years to identify suitable drugs that inhibit TK activity of KIT D816V.9" 12, 19"24 The TK inhibitor has been recently found imatinib (STI571) which is widely used in clinical hematology, counteracts the growth of neoplastic MCs that exhibit natural-type KIT (wt) or the variant mutated with F522C that occurs in a rare way in KIT.20"23, this drug is found to block the growth of neoplastic cells in patients having MS associated with clonal eosinophilia and a FIP1 L1 / PDGFRA fusion gene (SM with associated chronic eosinophilic leukemia, denoted herein as "SM-CEL"). 24"26 However, imatinib failed to inhibit the growth of neoplastic MCs harboring the c-KIT D816V20" 22 mutation, which points to the clear need for additional research for novel TK inhibitors that can block KIT D816V and thus the growth of MC neoplastic in SM.

The novel targeting drugs of TK PKC412 and AMN 107 counteract the TK activity of D816V-KIT and inhibit the growth of neoplastic human MC and Ba / Fe with inducible doxycycline expression of KIT-D816V, growth of primary neoplastic mast cells, and growth of the human MCL line HMC-1, which hosts this mutation of c-KIT: PKC412 is found a superior drug with IC50 values of 50-250 nm and with differences seen between HMC-1 cells that exhibit or lack KIT- D816V. In contrast, AMN 107 exhibits more potent effects in HMC-1 KIT-D816V-negative cells. The corresponding results are obtained with Ba / F3 cells that exhibit natural type KIT or mutated with D816V. The growth inhibitory effects of PKC412 and AMN 107 on HMC-1 are associated with induction of apoptosis and down-regulation of CD2 and CD63. It is found that PKC412 cooperates with AMN 107, imatinib and cladribine (ie, 2CdA), to produce growth inhibition in HMC-1. We also show that PKC412 has synergy with AMN107 and cladribine (2CdA) to produce growth inhibition in HMC-1 cells. Together, PKC412 and AMN 107 represent promising novel agents for targeted therapy of SM. This invention is directed to a combination treatment of PKC412 and AMN 1 07 which is effective against SM, especially SM associated with the oncogenic mutation of c-KIT D816V. This invention is also directed to a combination treatment of PKC412 and a TK inhibitor that is effective against MS, especially, systemic mastocytosis (SM) including aggressive MS (ASM) and mast cell leukemia (MCL). In a preferred embodiment, SM, ASM and MCL are associated with the oncogenic mutation of c-KIT, including especially D816V.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A-D is a representation of the effects of TK inhibitors on KIT phosphorylation in neoplastic cells. FIG. 1 A, B: phosphorylation of KIT in HMC-1 cells.1 (FIG.1A) and HMC-1.2 cells (exhibiting KIT D816V; FIG.1B) after incubation in control medium (Control), imatinib STI571 (1 μM), PKC412 (1 μM) or AMN 107 (1 μM) for 4 hours. FIG. 1 C, D: phosphorylation of KIT in Ton cells. Kit. Wt (FIG.1C) and Ton cells. Kit. D816V.27 (FIG: 1 D) after incubation in control medium (Control), imatinib (i.e., STI571) (1 μM), PKC412 (1 μM) or AMN 107 (1 μM) for 4 hours. Before exposure to medication, Ton cells. Kit. Wt and Ton. Kit. D816V.27 are maintained in doxycycline, 1 μg / ml for 24 hours to induce KIT expression: In the case of the Ton clone. Kit. Wt, cells are also exposed to SCF (100 ng / ml, 4 hours) to induce phosphorylation of KIT (p-KIT). In all cells, immunoprecipitation is conducted using the anti-KIT mAb SR-1. Western blotting was performed using anti-phospho-mAB 4G10 for detection of p-KIT and anti-KIT mAB 1 C1 for detection of total KIT protein. Figure 2A-D is a graphical representation of the effects of PKC412, AMN107 and imatinib on the proliferation of HMC-1 cells. FIG. 2A: Time-dependent effects of PKC412 on uptake of 3H-thymidine in HMC-1 cells .2. The HMC-1 .2 cells are incubated with control medium or PKC412 (200 nM) at 37 ° C) and 5% CO2 for various periods of time as indicated. After the incubation. The uptake of 3H-thymidine is analyzed. The results are expressed as control percentage (ie, the uptake of 3H-thymidine in control medium at each point in time) and represent the mean ± S. D. in triplicate. FIG. 2B-D: Dose-dependent effects of TK inhibitors on uptake of 3H-thymidine in HMC-1 cells. HMC-1 .1 (• - •) and HMC-1 .2 (B-B) cells are incubated in control medium in the absence (0) or presence of various concentrations of either PKC412 (FIG: 2B) , AMN 107 (FIG.2C) or imatinib (FIG 2D) at 37 ° C for 48 hours. After incubation, uptake of 3H-thymidine is measured. The results are expressed as percent control (0, 100%) and represent the mean ± S.D. of at least 3 independent experiments. Figure 3A-B is a graphical representation of the effects of PKC412, AMN 107 and imatinib on the uptake of H-thymidine in Ton cells. Kit. FIG.3A: Ton cells. Kitwt are kept in control medium (hollow bars) or are induced to express activated natural KIT when adding doxycycline (1 μg / ml) and SCF (filled bars). In both conditions, the cells are exposed either to control medium (Co) or various concentrations of PKC412, AMN 1 07 or imatinib (STI571), as indicated, for 48 hours (37 ° C, 5% CO2). Subsequently, uptake of 3H-thymidine is assessed as described in the text. The results are expressed as control percentage (Co) and represent the mean ± S.D. of three independent experiments.

FIG. 3B: Ton.Kit.D816V.27 cells are maintained in control medium (hollow bars) or are induced to express KIT D816V by adding doxycycline (1 μg / ml) (filled bars), and then exposed to ysa be medium control (Co) or various concentrations of PKC412, AMN 107 or imatinib (STI571), as indicated, for 48 hours (37 ° C, 5% CO2). Subsequently, uptake of 3H-thymidine is determined. The results are expressed as percent control (cells exposed to control medium, denoted herein as "Co") and represent the mean ± S.D. of three independent experiments. Figure 4 is a graphical representation of downregulation of PKC412 growth of primary neoplastic cells (mast cells) that exhibit D816V. Primary neoplastic bone marrow cells expressing KIT D816V are isolated from patients with latent systemic mastocytosis. The isolated cells are incubated in control medium (Co) or with various concentrations of PKC412, AMN 107 and imatinib as indicated. Cell growth is quantified by measuring uptake of 3H-thymidine. The results are expressed as a percentage of control (where Co equals 1 00%) and represent the average ± S.D. of tripilicated. In normal bm cells, no effect of PKC412 is seen (not shown). Figure 5A-F is a graphical representation of the effects of TK inhibitors on apoptosis of HMC-1 cells. HMC-1 cells (FIG 5A, C, E) and HMC-1 cells 2 (FIG 5B, D, F) are cultured in the absence (Co) or presence of various concentrations of PKC412 (FIG. 5A, B), AMN 107 (FIG.5C, D), or imatinib (FIG.5E, F) as indicated at 37 ° C for 24 hours. Subsequently, the percentages of apoptotic cells are quantified by light microscopy. The results represent the mean ± S.D. of three independent experiments. Figure 6A-D is a representation of electron microscopic examination of PKC412-induced apatoptosis in HMC-1 cells. HMC-1 .2 cells are incubated with control medium (FIG 6A), PKC412, 500 nM (FIG 6B) or PKC412, 900 nM (FIG 6C, D) at 37 ° C for 24 hours. Then, the cells are collected and analyzed for signs of ultrastructural apoptosis. While apoptotic cells are rarely seen in cultures maintained with control medium (FIG 6A), HMC-1 cells cultured in PKC412 (FIG 6B-D) frequently exhibited signs of apoptosis including cell shrinkage, membrane shirring , vacuolization and condensation of nuclear chromatin. Figure 7 is a representation of HMC-1 .2 cells exposed to control medium (Control), PKC412 (1 μM), AMN 107 (1 μM) or imatinib (1 μM) at 37 ° C for 24 hours. Then, the cells are examined for viability and apoptosis by staining with combined propidium (Pl) / Annexin V-FIT iodide. Figure 8A-H is a representation of apoptosis in HMC-1 cells evaluated by Tunnel assay. HMC-1 cells (FIG 8A-D) and HCM-1 cells 2 (FIG 8E-H) are incubated in control medium (FIG 8A, E), PKC412, 1 μM (FIG 8B) , F), AMN 107, 1 μM (FIG 8C, G), or imatinib, 1 μM (FIG 8D, H) at 37 ° C for 24 hours. Subsequently, the cells are collected and subjected to tunnel testing. As it is visible, PKC412 produced apoptosis in the majority of cells HMC-1 .1 and HMC-1 .2, whereas AMN107 and imatinib showed potent apoptosis-inducing effects only in HNC-1 cells (FIG 8C, D), but not in HMC-1 cells exhibiting KIT D816V (FIG: 8G, H). Figure 9A-B is a graphical representation of the effects of TK inhibitors on the expression of cell surface antigens in HMC-1 .2 cells. FIG. 9A: HMC-1 .2 cells are exposed to control medium (Co, hollow bars), PKC41 2, 1 μM (filled bars), AMN 107, 1 μM (shaded bars) or imatinib (ie STI571), μM (gray bars) at 37 ° C for 24 hours. The results show the percentage of control and represent the mean ± S.D. of 3 independent experiments. FIG. 9B: Dose-dependent effect of PKC412 on the expression of CD63 in HMC-1 .2 cells. Cells are incubated with various concentrations of PKC412 as indicated at 37 ° C for 24 hours. Subsequently, the cells are harvested and examined for DC63 expression by flow cytometry. The figure shows a normal result of an experiment. As is visible, the expression of CD63 decreased dependently on the dose of PKC412. Figure 10A-J is a graphic representation of the effects of synergistic drugs on growth of HMC-1 cells. HMC-1 .1 cells lacking KIT D816V (FIG.10A-F) and HMC-1 .2 cells exhibiting KIT D816V (FIG.10G-J) are incubated with control medium (0) or various combinations of medications (in fixed proportion) as indicated, at 37 ° C for 48 hours to determine cooperative antiproliferative effects. Fig. 10A, C, E, G, I: After incubation with simple medications (FIG: 10A: PKC412, B-B; AMN107, • - •; FIG. 10C: ST571, B-B; AMN107, • - •; FIG. 10E: ST571, B-B; PKC412, • - •; FIG. 10G: PKC412, B-B; AMN107, • - •; FIG. 101: PKC412, B-B; 2CdA, • - •) or drug combinations (A-A), the cells are analyzed for uptake of 3H-thymidine. The results show that the uptake of 3H-thymidine as control percentage (medium control, denoted as "0" or as "100%") and represent the mean ± S.D. of triplicates from a normal experiment (corresponding results are obtained in at least 2 other experiments for each drug combination). The images on the right (FIG 10B, D, F, H, J) show determined combination index values for each affected fraction according to the Chou and Talalay method39 using the calcusyn computation program. A combination index (Cl) value of 1.0 indicates an additive effect, a Cl greater than 1.0 indicates antagonism and a Cl of less than 1.0 indicates synergism. Figure 11 A-D is a representation of the effects of dasatinib on the phosphorylation of KIT in neoplastic cells. FIGS. 11A.B represent tyrosine phosphorylation of KIT in HMC-1.1 cells (FIG 11A) and HMC-1.2 cells (exhibiting KIT D816V) (FIG 11 B) after incubation in control medium or various concentrations of dasatinib for 4 hours. hours. FIGS. 11C.D represent KIT phosphorylation in Ton cells. Kit wt exposed to doxycycline (FIG 11C) and Ton cells. Kit D816V.27 (FIG 1 1 D) after incubation in control medium (0) or dasatinib (10"3 - 103 nM) for 4 hours Before exposure to drug, Ton cells Kit. wt and Ton cells Kit.D816V.27 were maintained in control medium (control), or in doxycycline for 24 hours to induce KIT expression.In the case of Ton.Kit.wt, the cells were also exposed to SCF (100 ng / ml, 4 hours) to induce KIT expression In the case of Ton Kit, the cells were also exposed to SCF (100 ng / ml, 4 hours) to induce KIT phosphorylation (p- KIT) In all cells, immunoprecipitation was conducted using anti-KIT mAB 1 C1.Western blotting was performed using anti-phospho-tir-mAb 4G 1 0 for detection of p-KIT and anti-KIT mAb 1 C1 for detection of total KIT protein (KIT).

Figure 12A-F is a representation of the effects of dasatinib on the proliferation of HMC-1 cells and growth and clumping of BaF / 3 cells. FIG. 12A depicts time-dependent effects of dasatinib on uptake of 3H-thymidine in HMC-1 .1 (B-B) cells and HMC-1 .2 (• - •) cells. HMC-1 .1 cells were incubated with dasatinib at 10 nM and HMC-1 .2 cells with dasatinib at 1 μM, at 37 ° C and 5% CO2 for several periods as indicated. After incubation, uptake of 3H-thymidine was measured. The results are expressed as percent control (= uptake of 3H-thymidine in cells maintained in control medium) and represent the mean ± S.D. of 3 independent experiments. FIG. 12B depicts the dose-dependent effects of dasatinib on uptake of 3H-thymidine in HMC-1 .1 (B-B) cells and HMC-1.2 cells (• - •). The cells were incubated in control medium in the absence or presence of various concentrations of dasatinib at 37 ° C for 48 hours. After incubation, uptake of 3H-thymidine was measured. The reusable ones are expressed as percentage of control and they represent the mean ± S. D. of 3 independent experiments. (FIG.12C) Effects of dasatinib on the growth of Ton cells. Kit. Wt. The cells were either maintained in medium containing IL-3 before and during incubation with dasatinib (• - •) or preincubated with doxycycline (1 μg / ml) in the presence of I L-3 for 24 hours, and then incubated with various concentrations of dasatinib in medium containing doxycycline and SCF (100 ng / ml) without I L-3 for 48 hours at 37 ° C (B-B). After incubation, the cells were harvested and subjected to 3H-thymidine uptake experiments. The results are expressed as control porcnetaje and represent the mean ± S.D. of 3 independent experiments. (FIG: 12D) Effects of dasatinib on the growth of Ton cells. Kit.D816V. Cells were incubated in control medium (+ IL-3) and various concentrations of dasatinib (as indicated) in the absence (• - •) or presence (B-B) of doxycycline (1 μg / ml) for 48 hours (37 ° C). Subsequently, cell viability was determined by the trypan blue exclusion test. The results are expressed as percentage of viable cells (calculated from the percentage of trypan blue positive cells) compared to control (without dasatinib = 100%) and represent the mean ± S.D. of 3 independent experiments. (FIG 12E.F) Effects of dasatinib (FIG 12E) and AMN 107 (FIG: 12F) on cluster formation induced by D816V in Ton cells. Kit. D816V. The cells were incubated without doxycycline (co) or in doxycycline (1 μg / ml) in the absence or presence of various concentrations of dasatinib or AMN107 as indicated for 24 hours. After incubation, bunch numbers were counted under an inverted microscope. The results are expressed as percentage of cluster formation compared to the cells maintained in control medium (co) and doxycycline (= 100%) and represent the mean ± S.D. of 3 independent experiments. Figure 13 is a representation that dasatinib downregulates the growth of primary neoplastic cells in a patient with SM KIT D816V-positive with associated leukemia. The primary neoplastic bone marrow cells were isolated from a patient with ASM KIT D816V-positive with AML. The isolated cells were incubated in control medium or in various concentrations of dasatinib (• - •), PKC412 (B-B), AMN107 (A-A) or imatinib (T-T), as indicated. Cell growth was quantified by uptake of 3H-thymidine. The results are expressed as percentage of control (cells maintained in control medium = 100%) and represent the mean ± SD of triplicates. In normal bone marrow cells, no effects of the applied TK inhibitors (not shown) are seen. Figure 14A-C represents that dasatinib induces apoptosis in HMC-1 cells. (FIG.14A.B) Cells HMC-1 .1 (FIG.14A) and HMC-1 .2 cells (FIB.14B) were cultured in the absence (Co) or presence of various concentrations of dasatinib as indicated for 24 hours. hours. Subsequently, the percentages of apoptotic cells were quantified by light microscopy. The results represent the mean ± S.D. of three independent experiments. The asterisk indicates p <; 0.05. (FG: 14C) Electron microscopic examination of apopotosis induced by dasatinib in HMC-1 .1 ceand HMC-1 .2 ce HMC-1 cewere incubated with control medium or dasatinib (1 μM) at 37 ° C for 24 hours. Then, the cewere collected and analyzed for signs of ultrastructural apoptosis. While apoptotic cewere rarely seen in cultures maintained in control medium, the majority of HMC-1 cultured in dasatinib exhibited signs of apoptosis including cell shrinkage, membrane shirring, vacuolization and nuclear chromatin condensation. (FIG 14D.E) Apoptosis induced by dasatinib in HMC-1 ceevaluated by Tunnel assay. CeHMC-1 .1 (FIG.14D) and HMC-1 .2 ce(FIG.14E) were incubated in control medium, various concentrations of dasatinib (as indicated), or PKC412 (1 00 nM and 1 μM ) as indicated at 37 ° C for 24 hours. Subsequently, the cewere collected and subjected to tunnel testing. As it is visible, dasatinib produced dose-dependent apoptosis in ceHMC-1 .1 and HMC-1 .2. Figure 1 5A-F depicts the effects of dasatinib on the expression of cell surface antigens in HMC-1 ce (FIG.15A) HMC-1 ce1 and HMC-1 ce2 (FIG.1B) were exposed to control medium or various concentrations of dasatinib (as indicated) or PKC412 (1μM) at 37 ° C for 24 hours. After incubation, the cewere examined for expression of several CD antigens by flow cytometry using CD-specific mAbs. FIGS. 15C-D show the average fluorescence intensity levels (MFI) as control percentage (= 100%). The results represent the average ± S.D. of 3 independent experiments. The asterisk: p < 0.05. (FIG: 15E.F) Expression of CD63 in HMC-1.1 ce(FIG 15ED) and HMC-1.2 ce(FIG 15F) after incubation in control medium, various concentrations of dasatinib, or pKC412 (1 μM) at 37 ° C for 24 hours. Flow cytometry was performed with CD63 mAb CLB-gran12 (black line). The dotted line represents the control antibody matched with isotype. Figure 16A-D depicts synergistic drug effects on the growth of HMC-1 ce HMC-1.1 ce(FIG.16A-B) and HMC-1.2 ceexhibiting KIT D816V (FIG.16C-D) were incubated with single drugs or various drug combinations (fixed ratio) at 37 ° C for 48 hours before to determine the uptake of 3H-thymidine. (FIG 16A) HMC-1.1 were incubated with various concentrations of dasatinib (B-B) or PKC412 (• - •), or combinations of both drugs (A-A). (FIG: 16B) HMC-1.1 were incubated with various concentrations of dasatinib (B-B) or imatinib (• - •), or combinations of both drugs (A-A). (FIG: 16C) HMC-1.2 cewere incubated with various concentrations of dasatinib (B-B) or PKC412 (• - •), or combinations of both drugs (A-A). (FIG 16D) HMC-1.2 cewere incubated with various concentrations of dasatinib (B-B) or 2CdA (• - •), or combinations of both drugs (A-A). The results represent the mean ± SD of triplicates from a normal experiment. As assessed by the calcusyn program, drug interactions (FIG 16A-D) were found to be synergistic in nature. Figure 17 depicts HMC-1 .2 cethat were incubated with control medium or various concentrations of dasatinib as indicated at 37 ° C for 48 hours. Subsequently, uptake of 3H-thymidine was measured. The results are expressed as percentage of control (the cemaintained in control medium = 100%) and represent the mean ± S-D- of three independent experiments.

DETAILED DESCRIPTION The problem to be solved by the present invention is the use of a combination of PKC412 and AMN 1 07 in the treatment of systemic mastocytosis, especially SM associated with the oncogenic mutation c-KIT D816V. In the majority of all patients with systemic mastocytosis (SM), including aggressive SM and mast cell leukemia (MCL), the neoplastic cells express the oncogenic mutation c-Kit D816V. This mutation activates the tyrosine kinase (TK) of the KIT receptor, which thus represents an attractive target of therapy. However, most of the available TK inhibitors including STI571 (imatinib, Novartis Pharma AG), fail to block the TK activity of KIT D816V at pharmacological concentrations. We provide evidence that the novel targeting drugs of TK PKC412 and AMN 1 07 (Novartis) block TK activity of KT mutated with D816V and counteract the growth of Ba / F3 cells with doxycycline-induced expression of KT D816V as well as growth of the cell line of human mast cell leukemia HMC-1, which express this mutation of c-KIT. PKC41 2 is found as the most potent agent with IC50 values of 50-200 nM and no differences seen between HMC-1 cells, which exhibit or lack KT D816V. In contrast, AMN 107 exhibited potent effects only in the absence of KIT D816V in HMC-1 cells (IC50 5-10 nM compared to HMC-1 expressing KIT D816V: IC50 1 -5 μM). The corresponding results are obtained with Ba / F3 cells that exhibit the wild type or the mutated variant with KIT D816V. Subsequently, the effect of PKC412 on primary neoplastic MC obtained from the bone marrow of a patient with MS exhibiting KIT D816V is examined. In line with our cell line data, PKC412 independently inhibited the uptake of 3H-thymidine in neoplastic MC (IC50: 50 nM) in this patient, while no significant effect was found with AMN 107 (0.1 -3 μM) . The growth inhibitory effects of PKC412 and AMN 107 on HMC-1 cells are associated with inhibition of KIT TK in phosphoblot experiments, and with induction of apoptosis as assessed by conventional morphology and by electron microscopy. In addition, PKC412 is found to sub-regulate the expression of CD2 and CD63 (two cell surface antigens over-regulated in SM) in HMC-1 cells. In co-incubation experiments, it was found that PKC412 synergizes with AMN107, imatinib, and cladribine (2CdA) by producing growth inhibition in HMC-1 cells harboring KIT D816V as well as in HMC-1 cells lacking KT D816V. In summary, our data show that PKC412 and AMN107 alone and in combination counteract the growth of neoplastic mast cells expressing the mutated variant with KITD816V. Both drugs can therefore be considered as promising new agents for targeted therapy in patients with aggressive MS and MCL. Therefore, the present invention relates to the use of N - [(9S, 10R, 11R, 13R) -2,3,10,11,12,13-hexahydro-10-methoxy-9-methyl-1-oxo -T.IS-epoxy-IH.TH-diindoloIlm ^ .Sg ^ '^ M'-lmJpirrololS ^ -j] [1,7] benzodiazonin-11-yl] -N-methylbenzamide of formula (I) (hereinafter forward: "PKC412"): T •: "-% - ^ (0 in combination with a 4-methyl-3 - [[4- (3-pyridinyl) -2-pyrimidinyl] amino] -N- [5- (4-methyl-1 H-imidazol-1-yl) -3 - (trifluoromethyl) phenyl] benzamide of formula (II) (hereinafter: "AMN107"): or a pharmaceutically acceptable salt of either or both, for treatment of systemic mastocytosis. The abbreviations used herein preferably have within the context of this disclosure the following meanings, unless otherwise indicated: ASM aggressive systemic mastocytosis bm bone marrow cladribine 2-chlorodeoxyadenosine FCS fetal calf serum IFNa interferon-alpha IP immunoprecipitation MCL mast cell leukemia PBS phosphate buffered saline PE phycoerythrin recombinant human RT room temperature SCF stem cell factor SM systemic mastocytosis SSM latent systemic mastocytosis TK tyrosine kinase wt natural type The general terms used herein are preferably within the context of this description the following meanings, unless otherwise indicated: Where the plural form is used for compounds, salts and the like, this is taken to mean also a single compound, salt or the like. Any asymmetric carbon atom may be present in the (R), (S) or (R, S) configuration, preferably in the (R) or (S) The compounds can be present in this manner as mixtures of isomers or as pure isomers, preferably as pure enantiomer diastereomers. The invention also relates to possible tautomers of the compounds of formula I and formula I I. The salts are especially the pharmaceutically acceptable salts of compounds of formula I and formula I I. The compounds of formula I and formula II can be administered sequentially or concurrently. The compounds of formula I and formula I I may be combined in a single formulation or may be in separate formulations. Such salts are formed, for example, as acid addition salts, preferably with organic or inorganic acids, from compounds of formula I and / or formula I I with a basic nitrogen atom, especially pharmaceutically acceptable salts. Suitable inorganic acids are, for example, halogen acids, such as hydrochloric acid, sulfuric acid or phosphoric acid. Suitable organic acids are, for example, carboxylic, phosphonic, sulphonic or sulphamic acids, for example, acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid , pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, amino acids, such as glutamic acid or aspartic acid, maleic acid, hydroxymeleic acid, methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methano- or ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, acid 1, 5-naphthalene-disulfonic acid, dodecyl sulfuric acid, 2-, 3- or 4-methylbenzenesulfonic acid, Methylsulfuric acid, ethylsulfuric acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-proylsulfamic acid or other organic protonic acids, such as ascorbic acid. In the presence of negatively charged radicals, such as carboxy or sulfo, the salts can also be formed with bases, for example, metal or ammonium salts, such as alkali metal or alkaline earth metal salts, for example, sodium salts, potassium, magnesium or calcium, or ammonium salts with ammonia or suitable organic amines, such as tertiary monoamines, for example, triethylamine or tri (2-hydroxyethyl) amine, or heterocyclic bases, for example, N-ethyl-piperidine or N. N'-dimethylpiperazine. When a basic group and an acid group are present in the same molecule, a compound of formula I and / or formula I I can also form internal salts. For isolation or purification purposes, it is also possible to use pharmaceutically unacceptable salts, for example, picrates or perchlorates. For therapeutic use, only pharmaceutically acceptable salts or free compounds are used (where they are applicable in the form of pharmaceutical preparations), and accordingly, these are preferred. In view of the close relationship between the novel compounds in free form and those in the form of their salts, including those salts that can be used as intermediates, for example in the purification or identification of the novel compounds, any reference to free compounds hereinbefore and hereafter shall be understood as also referring to the corresponding salts, as appropriate and timely. In each case where the citations of patent applications or scientific publications are made, the subject matter of the final products, the pharmaceutical preparations and the claims are incorporated herein in the present application by reference to these publications. If any discrepancy appears between statements in an incorporated reference and the present description, then the statements in the present description will govern. The structure of the active agents identified by the code numbers, generic or commercial names, can be taken from the current edition of the standard compendium "The Merck Index" or from the databases, for example, Patents International (for example, IMS World Publications). The corresponding content thereof is incorporated herein by reference. It has now been surprisingly found that the combination of AMN 107 and PKC412 possesses therapeutic properties, which makes it particularly useful as an inhibitor of tyrosine kinase activity and especially for the treatment and prophylaxis of diseases induced by oncogenic KIT-D816V, such as a systemic mastocytosis. KIT-D816V, as used hereinbefore and below, is the designation of the mutation product of the c-Kit gene, wherein the nucleic acid encoding the aspartic acid at residue 816 of the KIT polypeptide is mutated to encode a valine . KIT-D816V also refers to the polypeptide product of the mutated oncogenic c-KIT gene. The present invention thus concerns the use of the combination of AMN 107 and PKC412 for the preparation of a medicament for the treatment of systemic mastocytosis induced by the oncogenic c-KIT D816V mutation, or other diseases associated with the oncogenic c-KIT D816V mutation or similar tyrosine kinase-activating mutations. Systemic mastocytosis (MS) includes indolent MS, aggressive MS, and non-mast cell haematological disease associated with MS and mast cell leukemia. The term "mastocytosis" as used herein, refers to systemic mastocytosis, eg, mastocytoma, and also canine mast cell neoplasms. Mastocytosis is a myeloproliferative disorder with limited treatment options and generally a poor prognosis. The pathogenesis of mastocytosis has been attributed to the constitutive activation of the KIT tyrosine kinase receptor. In a large majority of patients with mastocytosis, the deregulated KIT tyrosine kinase activity is due to a mutation within codon 816 of the protein (D816V), which also confers resistance to imatinib or imatinib mesylate, the latter being marketed as Gleevec® in the United States or Glivec® elsewhere, in vitro and in vivo. The mast cells play an important role as the primary effector cells in the allergic disorders mentioned herein. The IgE-mediated degranulation of mast cell antigen leads to the subsequent release of chemical mediators and multiple cytokines and leukotriene synthesis. Additionally, mast cells are involved in the pathogenesis of multiple sclerosis.

Mast cell neoplasms occur in both humans and animals. In dogs, mast cell neoplasms are called mastocytomas and the disease is common, representing 7% -21% of canine tumors. A distinction must be drawn between human mastocytosis, which is usually transient or indolent and canine mast cell neoplasia, which behaves unpredictably and is frequently aggressive and metastatic. For example, human solitary mast cell tumors often do not metastasize; in contrast, 50% of canine mast cells behave in a malignant manner, as estimated by Hottedorf & Nielsen (1969) after the review of 46 published reports of tumors in 938 dogs. The involvement of KIT receptor in the pathogenesis of mastocytosis is suggested by the observation that several mutations resulting in costitutive activation of KIT have been detected in a variety of mast cell lines. For example, a point mutation in human c-KIT, which causes the substitution of Val by Asp816 in the phosphotransferase domain and receptor autoactivation, occurs in a long-term human mast cell leukemia line (HMC-1) and in the corresponding codon in two rodent mast cell lines. Moreover, this activating mutation has been identified in situ in some cases of human mastocytosis. Two other activating mutations have been found in the intracellular juxtamembrane region of KIT, ie, the Val560Gly substitution in the human HMC-1 mast cell line and suppression of seven amino acids (Thr573-His579) in a line of rodent mast cells called FMA3 . The present invention relates more particularly to the use of the combination of AMN 107 and PKC412 for the preparation of a medicament for the treatment of systemic mastocytosis. In another embodiment, the present invention provides a method of treating systemic mastocytosis comprising administering to a mammal in need of such treatment, a therapeutically effective amount of the combination of AMN 107 and PKC412 or pharmaceutically acceptable salts or prodrugs thereof.

Preferably, the present invention provides a method for treating mammals, especially humans, suffering from systemic mastocytosis comprising administering to a mammal in need of such treatment an inhibiting amount of KIT-D816V from the combination of AMN107 and PKC412 or pharmaceutically acceptable salts. thereof. In the present description, the term "treatment" includes both prophylactic or preventive treatment as well as disease suppressive or curative treatment, including the treatment of patients at risk of contracting the disease or suspected of having contracted the disease, as well as sick patients. This term also includes the treatment for the delay in the progression of the disease. The term "curative," as used herein, means efficacy for treating ongoing episodes involving systemic mastocytosis. The term "prophylactic" means the prevention of the onset or recurrence of diseases involving systemic mastocytosis. The term "delay of progression", as used in the present, means the administration of the active compound to patients who are in a pre-stage or early stage of the disease to be treated, in which the patients, for example , a pre-form of the corresponding disease is diagnosed or which patients are in a condition, for example, during a medical treatment or a condition resulting from an accident, under which a corresponding disease is likely to be developed. This unpredictable range of properties means that the use of the combination of AMN 107 and PKC412 are of particular interest for the manufacture of a medicament for the treatment of systemic mastocytosis. This effect may be especially relevant only for patients with systemic mastocytosis. To demonstrate that the combination of AMN 107 and PKC412 is particularly suitable for the treatment of systemic mastocytosis with good therapeutic margin and other advantages, clinical trials can be performed in a manner known to the skilled person. The precise dosage of the combination of AMN 1 07 and PKC412 to be employed to inhibit systemic mastocytosis depends on several factors including the host, the nature and severity of the condition being treated, the mode of administration. The combination of AMN107 and PKC412 can be administered either together or independently by any route, including orally, parenterally, for example, intraperitoneally, intravenously, intramuscularly, subcutaneously, intratumorally, or rectally or enterally. Preferably the combination of AMN 107 and PKC412 is administered orally, preferably at a daily dosage of 1 -300 mg / kg body weight or, for most higher primates, a daily dosage of 50-5000, preferably 500- 3000 mg. A preferred oral daily dosage is 1-75 mg / kg of body weight or, for most higher primates, a daily dosage of 10-2000 mg, administered as a single dose or divided into multiple doses, such as dosing twice daily. day. The precise dosage of PKC412 administered in combination with AMN 107 depends on several factors including the host, the nature and severity of the condition being treated, the mode of administration. However, in general, satisfactory results are achieved when PKC412 is administered parenterally, for example intraperitoneally, intravenously, intramuscularly, subcutaneously, intratumorally, or rectally, or enterally, for example, orally, preferably intravenously or, preferably orally , intravenous at a daily dosage of 0.1 to 10 mg / kg of body weight, preferably 1 to 5 mg / kg of body weight. In human trials, a total dose of 225 mg / day is very presumably the maximum tolerated dose (MTD). A preferred intravenous daily dosage is 0.1 to 10 mg / kg body weight or, for most higher primates, a daily dosage of 200-300 mg. A normal itnravenous dosage is 3 to 5 mg / kg, three to five times a week. Most preferably, PKC412 is administered orally, by dosage forms, such as microemulsions, soft gels or solid dispersions in dosages up to about 250 mg / day, in particular 225 mg / day, administered once, twice or three times a day. Usually, a small dose is administered initially and the dosage is gradually increased until the optimum dosage for the host under treatment is determined. The upper limit of dosage is that imposed by lateral effects and can be determined by assay for the host being treated. The combinations of AMN 107 and PKC41 2 may be combined, independently or together, with one or more pharmaceutically acceptable carriers and, optionally, one or more conventional pharmaceutical auxiliaries and administered enterally, for example, oral, in the form of tablets, capsules, tablets, etc. or parenterally, for example, intraperitoneally or intravenously, in the form of sterile injectable solutions or suspensions. The enteric and parenteral compositions can be prepared by conventional means. The combination of AMN 107 and PKC412 can be used alone or combined with at least one other pharmaceutically active compound for use in these pathologies. These active compounds can be combined in the same pharmaceutical preparation or in the form of combination preparations "set of parts" in the sense that the commingling partners can be dosed independently or by the use of different fixed combinations with distinguished amounts of the combination partners, that is, simultaneously or at different points in time. The parts of the set of parts can then be, for example, administered simultaneously or chronologically in stages, that is at different points in time and with equal or different time intervals for any part of the set of parts. Non-limiting examples of compounds, which may be cited for use in combination with the combination of AMN 107 and PKC412 are cytotoxic chemotherapeutic drugs, such as, cytosine arabinoside, daunorubicin, doxorubicin, cyclophosphamide, VP-16 or imatinib, etc. In addition, the combination of AMN 1 07 and PKC412 could be combined with other inhibitors of signal transduction or other drugs focused on oncogenes with the expectation that it would result in significant synergy.

The invention further pertains to the combination of AMN107 and PKC412 as described herein with imatinib for the treatment of the diseases and conditions described herein. The administration of such a combination can be affected at the same time, for example, in the form of a combined, fixed or faraceutical composition or composition, or sequentially or in appropriate stages. The administration of the combination of AMN 107 and PKC412 in a dosage form as described herein and of imatinib in its marketed form of GLEEVEC® in the US (GLIVEC® in Europe) and with the dosages provided for these dosage forms is currently preferred. The treatment of systemic mastocytosis with the above combination may be a so-called first-line treatment, that is, the treatment of a newly diagnosed disease without some preceding chemotherapy or the like, or it may also be a so-called second line treatment, ie , the treatment of the disease after a previous treatment with imatinib or the combination of AMN 1 07 and PKC412, depending on the severity or stage of the disease as well as the overall condition of the patient, etc. The efficacy of the combination of AMN 107 and PKC412 for the treatment of systemic mastocytosis is illustrated by the results of the following examples. These examples illustrate the invention without limiting its scope in any way.

EXAMPLES EXAMPLE 1: CLINICAL ICO STUDY The effect of compound (II) on c-KT transcription levels and c-kit mutation status in malignant cells taken from blood and / or bone marrow is evaluated. SM can result from altered kinase activity. SM associated with c-Kit K816V may also result from an activating mutation in the KIT gene. Q-RT-PCR for transcription of c-KIT D816V in baseline, cycle 1 day 15, cycle 1, 2, 3 day 28 and each subsequent third cycle, end of the study. Mutation analysis of c-kit: Three separate groups, each with the following patient populations: SM endpoints: response rates after 3 months of therapy.NSa EXAMPLE 2: COMBINATION OF AMN 107 and PKC412 In the current study, we show that the novel TK inhibitors PKC4125 and AMN10727 counteract the growth of neoplastic human Ba / F3 and MC cells that expire KIT D816V quite effectively. PKC412 seems to be the most potent compound in this regard. We also show that PKC412 and AMN 107 have synergy to produce growth inhibition in HMC-1 cells expressing or lacking KIT D816V. These data show that PKC412 and AMN107 may be novel promising drugs for the treatment of mastocytosis.

MATERIALS AND REAGENT METHODS The TK inhibitors imatinib (STI571), AMN 10727 and PKC4125 are obtained from Novartis Pharma AG (Basel, Switzerland). The stock solutions of AMN 107 and PKC412 are prepared by dissolving in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany). The recombinant human stem cell (SCF) factor (rH) is purchased from Strathmann Biotech (Nannover, Germany), RPMI 1640 medium and fetal calf serum (FCS) from PAA laboratories (Pasching, Austria), L-glutamine and medium from Dulbecco modified by Iscove (IMDM) from Gico Life Technologies (Gaithersburg, MD), 3H-thymidine from Amersham (Buckinghamshire, UK) and propidium iodide from Sigma (St. Louis, MO). Interferon alfa (IFNa is obtained from Roche (Basel, Switzerland), 2-chlorodeoxyadenosine (cladribine, denoted herein as "2CdA") by Janssen Cilag (Titusville, NJ) and rh interleukin-4 (I L-4) of Peprotech (Rocky Hill, NJ): Monoclonal antibodies (mAbs) labeled with phycoerythrin (PE), IVTO85 (CD2), WM15 (CD1 3), YB5, B8 (CD1 17), N6B6.2 (CD164) and 97A6 (CD203c) ) were purchased from Becton Dickinson (San José, CA) and mAB CIB-gran12 PEG-conjugated (CD63) to Immunotech (Marseille, France).

HMC-1 cells expressing or lacking C-KITD816V The human mast cell line HMC-128 generated from a patient with mast cell leukemia was kindly provided by Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN). Two subclones of HMC-1 are used, namely HMC-1 .1 which hosts the mutation of c-KIT V560G, but not the mutation of c-KIT D816V20, and a second subclone, HMC-1 .2, which houses both dec-KIT mutations, that is, V560G and D816V20. and a second subclone, HMC-1 .2, which hosts both mutations of c-KIT, ie, V560G and D816V20. HMC-1 cells are grown in IMDM supplemented with 10% FCS, L-glutamine, and antibiotics at 37 ° C and 5% CO2. The HMC-1 cells are re-thawed from an original stock every 4 to 8 weeks and are passed weekly. As control of "phenotypic stability", the HMC-1 cells are periodically verified by i) the presence of metachromatic granules, ii) expression of surface KIT, and iii) the submodulator effect of I L-4 (100 U / ml, 48 hours) in the expression of KIT.29 These control experiments are done before each set of experiments and only HMC-1 cells that exhibit all the characteristics of the original clone29 are used.

Ba / F3 cells with inducible expression of wt c-KIT or c-KIT D816V The generation of Ba / F3 cells with inducible doxycycline expression of wt c-KIT (Ton-Kit.wt) or c-KIT D816V is described in more detail elsewhere.30 Briefly, Ba / F3 cells expressing the reverse tet trativator31, 32 are co-transfected with the pTRE2 vector (Clontec, Palo Alto, CA) containing cDNA from c-KIT D816V (or wt cDNA). c-KIT, both kindly provided by Dr. JB Longley, Columbia University, New York, US) and pTK-Hyg (Clontech) by electroporation. After electroporation, stably transfected cells are selected by growing in hygromycin and cloned by limiting dilution. In the present study, the Ton.Kit subclone. D816V.27 is used in all experiments. These Ton cells. Kit.D816V exhibits a low growth rate on exposure to doxycycline30. As it is assessed by western blotting, immunocytochemistry, PCR and restriction fragment length polymorphism analysis (RFLP) 16, the expression of KIT D816V can be induced in Ton cells. Kit.D816V.27 within 12 hours by exposure to doxycycline (1 μg / ml) .30 Isolation of primary neoplastic mast cells MC from primary bone marrow (bm) are obtained from a female patient (54 years of age) with latent systemic mastocytosis (SSM), a subvariant other than MS characterized by involvement of multiple hematopoietic lineages and detection of c- KIT D816V in myeloid cells of MC lineage and not MC.34"36 For control purposes, bm obtained from a patient suffering from malignant lymphoma (without involvement of bm) who underwent phases, is analyzed and both patients gave informed consent Before the bm puncture, bm aspiration is obtained from the posterior iliac crest and collected in syringes containing preservative-free heparin.The cells are stratified on Ficoll to isolate mononuclear cells (MNC). MNC contain 5% MC in the patient with SSM and less than 1% MC in the control sample (normal bm.) Cell viability is> 90%. the mutation of c-KIT D816V in MNC of bm in the patient with SSM is confirmed by RT-PCR and RFLP analysis was performed as previously described.16 Analysis of KIT phosphorylation by western blotting Cells HMC-1 (106 / ml) and Ba / F3 cells (106 / ml) containing either wt KIT (Ton. Kit. Wt) or KIT D816V (Ton Kit. D816 V.27) are incubated with PKC412 (1 μM), AMN107 (1 μM), imatinib (1 μM), or control medium at 37 ° C for 4 hours. Before exposure to inhibitory drugs, Ton cells. Kit. Wt and Ton. Kit. D816V.27 are incubated with doxycycline (1 μg / ml) at 37 ° C for 24 hours to induce KIT expression. In case of Ton cells. Kit. Wt, phosphorylation of KIT is induced by adding rhSCF (100 ng / ml). Immunoprecipitation (IP) and western blotting are performed as previously described. 32 In brief, the cells are washed at 4 ° C and resuspended in RI PA buffer (1 ml of buffer per 108 cells) consisting of 50 mM Tris, 150 mM sodium chloride (NaCl), 1% nonidet P40 (NP-40 ), 0.25% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 1 mM ethylene diamine tetraacetic acid (EDTA), 1 mM sodium fluoride (NaF), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM orthovanadate of sodium (Na3VO4). After incubation in RIPA buffer supplemented with proteinase inhibitor cocktail (Roche) for 30 minutes at 4 ° C (vigorously shaken every 5 minutes), the lysates are centrifuged to remove the insoluble particles. For IP, lysates of 1 x 107 cells are incubated with anti-KIT SR137 antibody (kindly provided by Dr. V. Broudy, University of Washington, Seattle, WA) or with the anti-KIT 1 C138 antibody (kindly provided by Dr. H.-J. Bühring, University of Tubingen, Germany) and G Sepharose protein beads (Amersham) in IP buffer (50 mM Tris-CI, pH 7.4, 150 mM NaCl, 100 mM NaF, and 1% NP40 ) at 4 ° C during the night. Then, the beads are washed 3 times in IP buffer. The lysates and immunoprecipitates are then separated under reducing condicines by SDS 7.5% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Protran, Schleicher &Schuell, Keene, NH) in buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol at 4 ° C. The membranes are blocked for 1 hour in 5% blocking reagent (Roche) and then incubated with anti-KIT 1 C1 antibody or with monoclonal antibody 4G1 0 (Upstate Biotechnology, Lake Placid, NY) directed against tyrosine-phosphorylated proteins, at 4 ° C during the night. Antibody reactivity is made visible by sheep anti-mouse IgG antibody and Lumingen PS-3 detection reagent (both from Amersham) and a CL-Xposure film (Pierce Biotechnology, Rockford, I L).

Measurement of 3H-thymidine uptake To determine the effects of growth inhibitory drug, HMC-1 cells and Ba / F3 cells containing either wt KIT (Ton. Kit. Wt) or KIT D816V (Ton.Kit.D816 V. 27) activated with SCF are incubated with various concentrations of PKC412 (100 pM - 10 μM), AMN 107 (1 nM - 100 μM), or imatinib (3 nM - 300 μM) in 96 well culture dishes (PTT, Trasadingen, Switzerland) at 37 ° C in 5% CO2 for 48 hours. In ongoing experiments, HMC-1 cells are exposed to PKC412 (300 nM) for several periods (1, 12, 24, 36, and 48 hours). In selected experiments, HMC-1 cells (both subclones) are incubated with various concentrations of IFNa (0.1-500,000 U / ml) or 2CdA (0.005-10 pg / ml). The primary cells (bm cells from a patient with SSM and control bm cells) are cultured in the presence or absence of inhibitors (PKC412, 50-500 nM; AMN107, 100 nM-30 μM; imatinib, 1 μM) for 48 hours. After incubation, 1 μCi of 3 H-thymidine is added to each well and maintained for 12 hours (37 ° C). Then, the cells are harvested on filter membranes (Packard Bioscience, Meriden, CT) in a Filtermate 196 (Packard Bioscience) harvester. The filters are air dried and the bound radioactivity is counted in a ß counter (Top-Count NXT, Packard Bioscience). In a separate set of experiments, we determined the effects of drug combinations (additives against synergists) on the growth of neoplastic MCs. For this purpose, HMC-1 cells (both sublcones) are exposed to various drug combinations (PKC412, AMN 107, imatinib, IFNa, 2CdA) at a fixed ratio of drug concentrations. The drug interaction (additive versus synergistic) is determined by calculating the combination index values using a commercially available program (Calcusyn, Biosoft, Ferguson, MO) .39 All experiments are performed in triplicate.

Evaluation of apoptosis by conventional morphology and electron microscopy The effects of TK inhibitors on apoptosis in HMC-1 cells are analyzed by morphological examination, flow cytometry and electron microscopy. In normal experiments, HMC-1 cells are incubated with various concentrations of PKC412 (500 nM - 1 μM), AMN 107 (50 nM - 10 μM), imatinib (50 nM - 10 μM) or control medium in culture dishes of 6 cavities (PTT) in IMDM medium containing 10% FCS at 37 ° C for 24 hours. The percentage of apoptotic cells is quantified in cytospin preparations stained with Wright-Giemsa. Apoptosis is defined according to conventional cytomorphological criteria (cell shrinkage, condensation of the chromatatin structure) .40 To confirm apoptosis in HMC-1 cells, electron microscopy is performed as described 41, 42 using HMC-1 cells (both subclones ) exposed to PKC412 (500 nM, 900 nM, or 1 μM), AMN107 (1 μM), imatinib (1 μM), or medium control medium in 25 ml plastic culture flasks (PTT) for 24 hours. After incubation, the cells are washed and fixed in 2% paraformaldehyde, 2.5% glutaraldehyde and 0.025% CaCl2 buffered in 0.1 mol / l sodium cacodylate buffer (pH 7.4) at room temperature (RT) for 60 minutes . Then, the cells are washed three times in 0.1 mol / l sodium cacodylate buffer, suspended in 2% agar and centrifuged. The pellets are post-fixed with 1.3% OsO4 (buffered at 0.66 mol / l collidine) and stained "en bloc" in 2% uranyl acetate and sodium maleate buffer (pH 4.4) for 2 hours at RT . Then, the pellets are rinsed, dehydrated in series of alcohol, and embedded in EPON 812. Ultra-thin sections (85 nM) are cut and placed in gold grids. The sections are contrasted in uranyl acetate and lead citrate and viewed in a transmission electron microscope JEOL 1200 EX I I (JEOL, Tokyo, Japan). The presence of apoptotic cells is determined using conventional morphological criteria (see above).

Evaluation of apoptosis by Tunnel assay and flow cytometry To confirm apoptosis in HMC-1 cells exposed to PKC412 (1 μM), AMN107 (1 μM), or imatinib (1 μM) for 24 hours, a Tunnel assay (for its acronym in English Terminal transferase-mediated dUTP-fluorescence Nick End-Labeling (nickel end labeling) dUTP-fluorescence-mediated terminal transferase in situ)) that applied as previously described.43, 44 In brief, cells are first washed in phosphate-buffered saline (PBS) and fixed in 1% formaldehyde at pH 7.4 to 0 ° C for 1 5 minutes. Then, the cells are treated with 70% ethanol (ice-cold) for 1 hour, washed in PBS and incubated in terminal transferase reaction solution containing CoCl2, deoxy-nucleotidyl-exotransferase DNA, and biotin-16-2'-deoxy- uridin-5'-triphosphate (prepared according to the manufacturer's instructions Boehringer Mannheim, Germany) at 37 ° C for 10 minutes. After incubation, the cells are washed and then incubated with Streptavidin fluorescein (Boehringer Mannheim) (10 μg / ml) at 37 ° C for 20 minutes. The HMC-1 cells are then washed and analyzed with a Nikon Microphot-FXA fluorescence microscope (Tokyo, Japan). For cytometric flow determination of apoptosis and cell viability, staining with annexinV / propidium iodide is performed. For this purpose, HMC-1 cells are exposed to PKC412 (0.5, 1, and 2.5 μM), AMN1 07 (0.5, 1, and 2.5 μM), imatinib (0.5, 1, and 2.5 μM), or control medium at 37 ° C for 24 hours. Subsequently, the cells are washed in PBS and then incubated with annexinV-APC (Alexis Biochemicals, Lausen, Switzerland) in binder buffer containing HEPES (10 mM, pH 7.4), NaCl (140 mM), and CaCl2 (2.5 mM). Subsequently, propidium iodide (1 μg / ml) is added. The cells are then washed and analyzed by flow cytometry in a FACSCalibur (Becton Dickinson).

Evaluation of expression of surface antigens linked to activation in HMC-1 cells The expression of cell surface antigens in HMC-1 cells carrying KIT D816V (HMC-1 .2 cells) is determined by flow cytometry after short-term culture. term (for 24 hours) in control medium or medium supplemented with TK inhibitors (PKC412, 1 μM, AMN 107, 1 μM, imatinib, 1 μM). In selected experiments, various concentrations of PKC412 (50, 100, 250, 500, and 1000 nM) are applied. After incubation with medicaments, the HMC-1 cells are washed and subjected to simple color flow cytometry using PE-conjugated antibodies against MC antigens known to be overexpressed in neoplastic MCs in SM (compared to normal MCs) and / o are expressed at an early stage of mastopoiesis (CD2, CD13, CD63, CDI 17, CD164, CD203c) .45,47 Flow cytometry is performed on a FACSan (Becton Dickinson) as previously described.29 Statistical analysis To determine the significance in differences between proliferation rates, apoptosis and levels of surface expression after exposure of HMC-1 cells to inhibitors, the student's t test for dependent samples is applied. The results are considered statistically significant when p is > 0.05.

Results Effects of PKC412 and AMN 107 on TK activity of KIT mutated with D816V As assessed by IP and western blotting, PKC412 (1 μM) decreased phosphorylation of KIT in HMC-1 cells.1 (exhibiting the mutation of c-1). KIT V560G, but not the mutation of c-KIT D816V), as well as in HMC-1 .2 cells harboring V560G-mutated as well as the variant mutated with D816V of KIT (Figure 1 A and 1 B). The novel TK AMN 107 inhibitor (1 μM) strongly reduced KIT phosphorylation in HMC-1.1 cells, but showed only a weak effect on KIT phosphorylation in HMC-1 .2 cells at 1 μM. Similarly, imatinib (1 μM) reduced KIT phosphorylation in HMC-1 .1 cells, but did not inhibit KIT phosphorylation in HMC-1 .2 cells (Figure 1 A and 1 B). In a next step, we examined the effects of TK inhibitors on Ba / F3 cells exhibiting wt KIT (Ton Kit.wt) or KIT D816V (Ton.Kit.D816 V.27) after exposure to doxycycline. In Ton cells. Kit. Wt, KIT appeared to be phosphorylated in the presence (but not in the absence) of SCF, whereas KIT was found constitutively phosphorylated in Ton cells. Kit.D816V.27 (Figure 1 C). As is visible in Figure 1 C, the 3 inhibitors of TK (PKC412, AMN107, imatinib, each 1 μM) decreased the independent phosphorylation of SCF of KIT in Ton cells. Kit. Wt. In contrast, only PKC412, and to a lesser extent AMN 107, decreased the independent phosphorylation of SCF of KIT in Ton cells. Kit.D816 V-27. Imatinib (1 μM) showed no detectable effect on KIT phosphorylation in these cells (Figure ID). These data show that KC412 is a novel potent inhibitor of the TK activity of wt KIT, KIT V560G, and KIT D816V, and that AMN 107 is a novel potent inhibitor of the TK activity of wt KIT and KIT V560G, and an inhibitor weak (auto) phosphorylation of KIT D816V.

Effects of TK inhibitors on 3H-thymidine uptake in HMC-1 cells In the ongoing experiments, the maximal inhibitory effects of PKC412, AMN 107, and imatinib on the growth of HMC-1 .1 cells and HMC-1 cells. 2 are seen after 36-48 hours. Figure 2A shows the time-dependent effect of PCK412 (300 nM) on the growth of HMC-1 cells.2 As shown in Figure 2B and 2C, PKC412 and AMN 107 are found to counteract 3H-thymidine uptake in HMC-1 .1 cells and HMC-1 .2 cells in a dose-dependent manner. Interestingly, the IC50 for the effects of PKC412 on these two subclones appeared to be in the same range (50-250 nM) (Figure 2B). In contrast, the IC 50 values for the effects of AMN 107 on proliferation are significantly higher in HMC-1 .2 cells (1 -5 μM) compared to that found in HMC-1 .1 cells (3-10 nM) F (Figure 2C). As expected, imatinib is only effective at pharmacologically relevant concentrations in HMC-1 cells.1 (IC50: 10-30 nM); whereas the non-significant antiproliferative effects of imatinib on HMC-1 .2 cells are seen (Figure 2D) confirming previous data.20"22 An interesting observation is that AMN 107 is the most potent compound (on a molar basis), when it is compared to the inhibitory effects of growth of the three drugs on HMC-1 .1 cells that exhibit the mutation of c-KIT V560G (but not the mutation of c-KIT D816V) (Figure 2B-D).

Effects of KIT inhibitors on growth of Ba / F3 cells expressing wt KIT or KIT D816V As shown in Figure 3A, all 3 TK inhibitors are found to counteract the SCF-dependent growth of Ton cells. Kit. Wt exposed to doxycycline (expressing KIT) in a dose-dependent manner with IC50 values of 3-30 nM for PKC412, 30-300 nM for AMN107 and 3-30 nM for imatinib. In contrast, in Ton cells. Kit. D816V, only PKC41 2 (IC50: 100-300 nM), and to a lesser degree AMN 1 07 (IC50: 1 -3 μM) are found to inhibit the incorporation of H-thymidine, while no significant effect is obtained with imatinib over the dose range tested (Figure 3B). None of the three inhibitors used are found to counteract the growth of Ton cells. Kit. Wt or Ton cells. Kit.D816V.27 in the absence of doxycycline, that is, in the absence of KIT (Figure 3A and 3B). In additional control experiments, neither doxycycline (1 μg / ml), nor the TK inhibitors (imatinib, PKC412, AMN 107), showed growth inhibitory effects on control (untransfected) Ba / F3 cells (not shown).

PKC412 and AMN107 counteract the growth of primary neoplastic cells (mast cells) expressing KIT D816V to reconfirm the anti-proliferative effects of PKC412 and AMN 107 in systemic mastocytosis, we examined the response of MCs derived from primary neoplastic bone marrow (bm) in a patient with latent MS, a special subvariant of MS, in which the majority of myeloid cells (MC as well as lineage cells do not of MC) exhibit KIT D816V. In fact, although MC purity is only 4%, most of the myeloid cells in this patient exhibited KIT D816V. In these neoplastic bm cells, PKC412 and AMN 107 are found to inhibit the spontaneous uptake of 3H-thymidine in a dose-dependent manner, while no significant effect is seen with imatinib (1 μM) (Figure 4). In the control sample (normal bm, without haematological disease), PCK412 showed no effect on uptake of 3H-thymidine (not shown).

PKC412 and AMN107 induce apoptosis in HMC-1 cells To explore the underlying mechanisms of the growth inhibitory effects of PKC412 and AMN 107 on neoplastic human MCs exhibiting or lacking KIT D816V, we analyzed morphological and biochemical signs of apoptosis in HMC-1 cells. 1 and HMC-1 .2 cells after drug exposure. In these experiments, PKC412 was found to induce apotosis in both subclones of HMC-1 in a dose-dependent manner (Figure 5A and 5B). AMN 1 07 was also found to induce apoptosis in subclones HMC-1 in a dose-dependent manner, but the effect of this compound is much more pronounced in HMC-1 cells.1 (Figure 5C) compared to that found in HMC-1 cells. 1.2 (Figure 5D). Similarly, imatinib was found to produce apoptosis in HMC-1 .1 cells (Figure 5E), but showed no effect on HMC-1 .2 cells (Figure 5F). The effects that induce apoptosis of the drugs in HMC-1 cells could be confirmed by electron microscopy. Again, all drugs (each 1 μM) are found to induce apoptosis in HMC-1 cells.1, whereas in HMC-1 cells.2 only PKC412 and to a lesser extent AMN 107 are found to produce apoptosis in HMC-1 .2 cells. Figure 6 shows the apoptosis-inducing effect of PKC412 (1 μM, 24 hours) on HMC-1 cells.2 As is visible, many of the HMC-1 cells exposed to PKC412 (Figure 6B-D) exhibited normal ultrastructural signs of apoptosis compared with cells maintained in control medium (Figure 6A). Finally, we are able to demonstrate the apoptosis-inducing effects of PKC412 and AMN 107 on HMC-1 cells by staining combined with annexinV / propidium iodide and flow cytometry (Figure 7) as well as in a Tunnel assay (Figure 8). In both trials, PKC412 (1 μM) and to a lesser extent AMN107 (1 μM) are found to induce apoptosis in HMC-1 .2, whereas imatinib showed no effects (Figures 7 and 8E-H). In contrast, in the HMC-1 .1 cells, the 3 compounds are found to induce apoptosis as assessed by Tunnel assay (Figure 8A-D). These data provide evidence that the growth inhibitory effects of PKC412 and AMN 107 on HMC-1 cells are associated with induction of apoptosis.

PKC412 downregulates the expression of SM-related cell surface antigens and bound to activation in HMC-1 cells Several cell surface antigens that are normally (over) expressed in neoplastic MCs in SM, may play a role in growth, activation or distribution of neoplastic cells.45,46 Some of these molecules can be directly over-regulated by the K8 mutant variant with D816V.30 Therefore, we do not ask whether PKC412, AMN 107 or imatinib would influence the expression of cell surface antigens in HMC-1 .2 cells. The unstimulated HMC-1 .2 cells are found to express LFA-2 (CD2), aminopeptidase-N (CD13), CD63, KIT (CD1 17); CD164 and E-NPP3 (CD203c) confirming the previous data.45"47 Incubation of HMC-1 .2 cells with PKC412 resulted in a significant decrease in the expression of CD2, CD63 and CD164 (p <0.05) (Figure 9A In contrast, no significant effect of PKC412 on the expression of CD1 3 or CD203c is seen (Figure 9A). In case of KIT, a slight decrease in expression in HMC-1 cells.2 is found on exposure to PKC412 ( as well as on exposure to AMN 107 or imatinib), but the effect is not significant (p> 0.05) (Figure 9A) The effects of PKC412 on the expression of CD2 and CD63 are found to be dose dependent Figure 9B shows the effects of various concentrations of PKC412 on the expression of CD63 in HMC-1 cells.2 In contrast to PKC412, no significant effect of AMN 107 or imatinib on the expression of CD antigens in HMC-1 .2 cells is seen ( Figure 9A).

PKC412 cooperates with other conventional and directed drugs to produce growth inhibition in HMC-1 cells. As assessed by 3H-thymidine incorporation, PKC412 is found to cooperate with AMN 107 to produce growth inhibition in HMC-1 cells. HMC-1 .2 cells (Figure 10, Table 1). In case of HMC-1 .1 cells, the drug interaction is found to be clearly synergistic, whereas in HMC-1 .2 cells, the interactions are additive rather than synergistic (Figure 10; Table 1 ). In addition, PKC412 and 2CdA, a drug used successfully to treat aggressive mastocytosis, are found to inhibit the growth of HMC-1 cells.1 in a synergistic manner, and the same synergistic effect is seen with PKC41 2 and imatinib (Table 1) . However, no clear synergistic effect of PKC412 and 2CdA on growth of HMC-1 .2 cells is seen. In addition, AMN 107 and imatinib produced synergistic inhibitory effects only in HMC-1 .1 cells (Figure 10), but not in HMC-1 .2 cells carrying KIT D816V (Table 1). No synergistic or additive effect on the growth of HMC-1 cells is seen when combining PKC412 and interferon alpha (IFNa) or AMN 107 and IFNa (Table 1). A summary of drug interactions is shown in Table 1.

Table 1 Drug interactions in HMC-1 .1 cells and HMC-1 cells .2 As shown in Table 1, the effects of various drug combinations on growth of HMC-1 cells.1 (white squares, upper right) and HMC-1 .2 cells (lower left gray squares) are determined by incorporation assay of 3H-thymidine. Each drug combination is tested in at least three independent experiments. The drugs are applied at a fixed rate and the resulting effects (and the type of drug interaction) were determined by calcusyn computation program. Rating: +, synergistic growth inhibitory effect; +/-, additive effect; -, less than the additive (antagonist) effect. N .t. , not tested.

Discussion The mutation of somatic c-KIT D816V is a gene defect that leads to constitutive activation of the TK domain of the KIT receptor, which is critically involved in the growth of MC (neoplastic) and thus in the pathogenesis of SM .13"17 Therefore, recent attempts have focused on the identification and development of pharmacological compounds that can inhibit the TK activity of the mutated variant with KIT D81 V and thereforecan inhibit the growth of neoplastic MC in patients with SM.9"12 We describe here that the inhibitor of TK PKC412, and to a lesser degree AMN 107, inhibit the TK activity of KIT-D816V as well as growth of neoplastic human MC carrying this In addition, we show that both drugs cooperate with each other, as well as with other targeted and conventional drugs to produce growth inhibition in neoplastic MCs, PKC412 is a novel staurosporine-related inhibitor of PKC and several TKs including KDR, PDGFRA, FLT3, and KIT.5 In the current study, we show that PKC412 counteracts the growth of neoplastic human MC and Ba / F3 cells by expressing the mutated variant with KIT D816V. With respect to Ba / F3 cells, our Data are in line with the results of Growney et al.48 Interestingly, the effective dose range for Ba / F3 cells is found to be the same as that found in c HMC-1 cells ported two KIT D816V. Another interesting observation is that the IC50 for the effects of PKC412 on the two subclones of HMC-1 (expressing or lacking KIT D816V) appeared to be in the same range. Finally, we are able to confirm the growth inhibitory effects of PKC412 for primary neoplastic human (mast cell) cells expressing KIT D816V. Because the mutation of c-KIT D816V is detectable in a majority of patients with SM independent of the disease subtype, 13"17 these data are of considerable importance.In fact, PKC412 appears to be the first TK inhibitor to counteract reportedly the growth of human MCs carrying KIT D816V in the same way as MC expressing KIT wt It should also be noted in this regard, that the inhibitory effects of PKC412 on KIT D816V positive cells clearly exceed the antiproliferative activities of AMN107 and imatinib Based on these observations, PKC412 appears to be a novel targeted drug to be considered for use in clinical trials in patients with MS (aggressive) or MCL. Recent data suggest that AMN 107 is a very potent inhibitor of the activity of TK BCR / ABL.27 It has also been described that AMN107 inhibits the TK activity of natural type KIT.27 In the present study, we found that AMN 107 exerts potent it is effects on HMC-1 cells carrying the mutation of c-KIT V560G, but exhibits only weak effects in HMC-1 cells that house both KIT V560G and KIT D816V. Similarly, AMN 107 showed only weak effects on the growth of Ba / F3 cells expressing the mutated variant of D (16V of KIT.) These data suggest that the mutation of c-KIT D816V, but not the mutation of c-KIT V560G , confers relative resistance against AMN107, although AMN 107 still retains inhibitory effects on HMC-1 KIT D816V-positive cells, compared with imatinib.The impressive antiproliferative effects of AMN 107 on V560G-positive cells, also suggest that this compound can be a drug attractive candidate candidate for gastrointestinal stromal cell tu (GISTs), in which mutations in codon 560 of c-KIT have been recently reported.49 A variety of pharmacological inhibitors that direct TK activity of pro-oncogenic molecules have been recently developed in clinical hematology.5, 12, 19, 27 The inhibitory effects of growth of these TK inhibitors in neoplastic cells (which express the appropriate objective) are usually associated with loss of TK activity and with consecutive apoptosis. In the present study, we are able to demonstrate that the growth inhibitory effects of PKC412 on neoplastic human MC (HMC-1) are associated with the inhibition of KIT (mutated) TK as well as with apoptosis. In fact, we are able to show that PKC412 induces apoptosis in HMC-1 .1 cells (expressing KIT V560G but not KIT D816V), as well as in HMC-1 cells.1 (expressing KIT V560G and KIT D816V). The apoptosis inducing effect of PKC412 is demonstrable by light and electronic microscopy, as well as by flow cytometry and in a tunnel test. As expected, AMN 107 and imatinib showed significant apoptosis-inducing effects in HMC-1 .1 cells, but did not exhibit significant effects in HMC-1 cells.2. A variety of surface antigens are normally (over) expressed in neoplastic human MCs. Similarly, in contrast to normal MCs, neoplastic MCs in patients with MS express CD2 and CD25.45,46 In addition, the levels of CD63 and CD203c expressed in neoplastic MCs in SM are more compared to normal MCs. In several cases, such as CD63, the mutated variant with D816V of KIT can directly lead to enhanced surface expression.30 Therefore, we are interested in knowing whether the K8 D816V-mutated approach in HMC-1 cells by PKC412 is associated with a decrease in the expression of surface CD antigens "related to SM". The results of our experiments show that PKC412 downregulates the expression of CD2, CD63 and CD164 in HMC-1 .2 cells by displaying KIT D816V. A slight but insignificant effect of PKC412 (as well as of AMN 107) on the expression of KIT is also seen. An interesting observation is that AMN 107 failed to suppress the expression of CD2 and CD63 in HMC-1 cells.2. This is probably due to the weaker effect of this compound on the TK activity of KIT D816V when compared to the effect of PKC412. A variety of recent data suggest that the treatment of myeloid neoplasms with TK inhibitors as simple agents may be insufficient to control the disease for prolonged periods. This has been documented for the use of imatinib in CML50,51 (advanced), and may also apply for patients with ASM or MCL.52 In the latter patients, this is a particular problem because the D816V mutation confers a primary resistance ( relative) of KIT against imatinib and, to a lesser degree, relative resistance against AN 107. To overcome resistance, a variety of different pharmacological strategies can be envisioned. One possibility is to apply combinations of medications. Therefore, we are interested in learning if PKC412 and AMN 1 07 would exhibit synergistic antiproliferative effects on HMC-1 .1 and HMC-1 .2 cells. In fact, our data show that PKC412 cooperates with imatinib and AMN107 to produce growth inhibition in both clones of HMC-1. Additionally, PKC412 and 2CdA, a drug that has been described for counteracting the growth of neoplastic MC in vivo in patients with MS (aggressive), showed cooperative inhibitory effects on the growth of HMC-1 cells. and HMC-1 .2. However, interstingly, drug interactions are found to be synergistic only in HMC-1 .1 cells, but not in HMC-1 cells.2. This can be explained by the relatively weak (AMN107) or absent (imatinib) effects of co-applied drugs on KIT's TK activity and thus, the growth of HMC-1 .2 cells carrying D816V as compared to the effects much more pronounced of the same drugs in HMC-1 cells. No effect of cooperative medication is seen when IFNa is combined with AMN 107 or PKC412. Any drug combination consisting of PKC412 and other (targeted) medications that are of clinical value in patients with ASM or MCL remains unknown. Thus, until now, only a few agents with documented antiproliferative effects on neoplastic MC in vivo in patients with MS have been presented, and none of these drugs produces durable complete remissions in patients with ASM or MCL. The notion that PKC412 is a novel very potent inhibitor of neoplastic human MC growth bearing the D816V-mutated KIT variant is of particular interest in this regard.

In summary, we show that PKC412 and AMN 1 07 are promising new medicines focused on KIT natural type and mutated variants of KIT in SM. While each of the two drugs may exhibit a distinct pharmacological profile with unique effects on mutated KIT variants, a very effective and promising approach may be to combine both drugs with each other or with the clinically established 2CdA drug to treat patients with ASM or MCL in the future.

References of Example 1 1. Reilly JT. Class II I receptor tyrosine kinases: role in leukaemogenesis. Br J Haematol. 2002; 1 16: 744-757. 2. Deininger MW, Druker BJ. Specific targeted therapy of chronic myelogenous leukemia with imatinib. Pharmacol Rev. 2003 55: 401-423. 3. Pardanani A, Tefferi A. Imatinib targets other than bcr / abl and their clinical relevance in myeloid disorders. Blood. 2004; 1 04: 1 931 -1 939. 4. Shah NP, Tran C, Lee FY, Chen P, Norris D, Sawyers CL. Overriding imatinib resistance with a novel ABL kinase inhibitor. (Science, 2004; 305: 399-401. 5. Fabbro D, Ruetz S, Bodis S, et al. PKC412 - a protein kinase inhibitor with a broad therapeutic potential. Anticancer Drug Des. 2000; 15: 1 7- 28. 6. Lennert K, Parwaresch MR. Mast cells and mast cell neoplasia: a review. Histopathology 1979; 3: 349-365. 7. Metcalfe DD. Classification and diagnosis of mastocytosis: current status. J Invest Dermatol 1991; 96: 2S-4S. 8. Valent P. Biology, classification and treatment of human mastocytosis. Wien Klin Wschr. nineteen ninety six; 108: 385-397. 9. Valent P, Akin C, Sperr WR, et al. Diagnosis and treatment of systemic mastocytosis: state of the art. Br J Haematol 2003; 122: 695-717. 10. Akin C, Metcalfe DD. Systemic mastocytosis. Annu Rev Med. 2004; 55: 41 9-32. eleven . Tefferi A, Pardanani A. Clinical, genetic, and therapeutic insights into systemic mast cell disease. Curr Opin Hematol. 2004; 1: 58-64. 12. Valent P, Ghannadan M, Akin C, et al. On the way to targeted therapy of mast cell neoplasms: identification of molecular targets in neoplastic mast cells and evaluation of arising treatment concepts. Eur J Clin Invest. 2004; 34: 41 -52. 13. Nagata H, Worobec AS, Oh CK, et al. Identification of a point mutation in the catalytic domain of the protooncogene c-kit in blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder. Proc Nati Acad Sci (USA). 1995; 92: 10560-1 0564. 14. Longley BJ, Tyrrell L, Lu SZ, et al. Somatic c-kit activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm. Nat Genet nineteen ninety six; 12: 312-314.

. Longley BJ, Metcalfe DD, Tharp M, Wang X, Tyrrell L, Lu S-Z, et al. Activating and dominant inactivating c-kit catalytic domain mutations in distinct forms of human mastocytosis. Proc Nati Acad Sci (USA). 1999; 96: 1609-1614. 16. Fritsche-Polanz R, Jordán JH, Feix A, et al. Mutation analysis of C-KIT in patients with myelodysplastic syndromes without mastocytosis and cases of systemic mastocytosis. Br J Haematol. 2001; 1 13: 357-364. 17. Feger F, Ribadeau Dumas A, Leriche L, Valent P, Arock M: Kit and c-kit mutations in mastocytosis: a short overview with special reference to novel molecular and diagnostic concepts. Int Arch Allergy Immunol. 2002; 1 27: 1 10-1 14. 18. Furitsu T, Tsujimura T, Tone T, et al. Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of the c-kit product. J Clin Invest. 1993; 92: 1 736-1744. 19. Tefferi A, Pardanani A. Systemic mastocytosis: current concepts and treatment advances. Curr Hematol Rep. 2004; 3: 197-202. 20. Akin C, Brockow K, D'Ambrosio C, et al. Effects of tyrosine kinase inhibitor STI571 on human mast cells bearing wild-type or mutated forms of c-kit. Exp Hematol 2003; 31: 686-692. twenty-one . Ma Y, Zeng S, Metcalfe DD, et al. The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory type mutations. Blood 2002; 99: 1741 -1 744. 22. Frost MJ, Ferrao PT, Hughes TP, Ashman LK. Juxtamembrane mutant V560GKit is more sensitive to Imatinib (STI571) compared with wild-type c-kit whereas the kinase domain mutant D816VK is resistant. Mol Cancer Ther. 2002; 1: 1 1 1 5- 1 124. 23. Akin C, Fumo G, Yavuz AS, Lipsky PE, Neckers L, Metcalfe DD. A novel form of mastocytosis associated with a transmembrane c-kit mutation and response to imatinib. Blood. 2004; 1 03: 3222-3225. 24. Pardanani A, Ketterling RP, Brockman SR, FIynn HC, Paternoster SF, Shearer BM, Reeder TL, Li CY, Cross NC, Cools J, Gilliland DG, Dewald GW, Tefferi A. CHIC2 deletion , a surrogate for FI P ILI -PDGFRA fusion, occurs in systemic mastocytosis associated with eosinophilia and predicts response to imatinib mesylate therapy. Blood. 2003; 102: 3093-3096.

. Pardanani A, Elliott M, Reeder T, Li CY, Baxter EJ, Cross NC, Tefferi A. Imatinib for systemic mast-cell disease. Lancet. 2003; 362: 535-536. 26. Pardanani A, Tefferi A. Imatinib targets other than bcr / abl and their clinical relevance in myeloid disorders. Blood. 2004; 1 04: 1 931 -1 939. 27. Weisberg E, Manley PW, Breitenstein W, et al. Characterization of AMN 107, a selective inhibitor of native and mutant Bcr-Abl. Cancer Cell. 2005; 7: 129-141. 28. Butterfield JH, Weiler D, Dewald G, Gleich GJ. Establishment of an immature mast cell line from a patient with mast cell leukemia. Leuk Res. 1988; 12: 345-355. 29. Sillaber C, Strobl H, Bevec D, et al. IL-4 regulates c-kit proto-oncogene product expression in human mast and myeloid progenitor cells. J Immunol. 1991; 147: 4224-4228. 30. Mayerhofer M, Gleixner K, Aichberger K, et al. c-kit gene mutation D816V as a single hit explains numerous features and the pathology of indolent systemic mastocytosis, submitted manuscript. 31 Daley GQ, Baltimore D. Transformation of an interleukin 3-dependent hematopoietic cell line by the chronic myelogenous leukemia-specific P210bcr / abl protein. Proc Nati Acad Sci (USA). 1 988; 85: 9312-9316. 32. Sillaber C, Gesbert F, Frank DA, Sattler M, Griffin JD. STAT5 activation contributes to growth and viability in Bcr / Abl-transformed cells. Blood. 2000; 95: 21 18-2125. 33. Valent P, Horny H-P, Escribano L, et al. Diagnostic criteria and classification of mastocytosis: a consensus proposal. Conference report of "Year 2000 Working Conference on Mastocytosis". Leuk Res. 2001; 25: 603-625. 34. Valent P, Horny H-P, Li CY, et al. Mastocytosis (Mast cell disease). World Health Organization (WHO) Classification of Tumors. Pathology & Genetics Tumours of Haematopoietic and Lymphoid Tissues, eds: Jaffe ES, Harris NL, Stein H, Vardiman JW. 2001; 1: 291-302. 35. Yavuz AS, Lipsky PE, Yavuz S, Metcalfe DD, Akin C. Evidence for the involvement of a hematopoietic progenitor cell in systemic mastocytosis from single-cell analysis of mutations in the c-kit gene. Blood. 2002; 100: 661 -665. 36. Valent P, Akin C, Sperr WR, Horny HP, Metcalfe DD. Smouldering mastocytosis: a novel subtype of systemic mastocytosis with slow progression. Int Arch Allergy Immunol. 2002; 1 27: 137- 1 39. 37. Broudy VC, Lin N, Zsebo KM, et al. Isolation and characterization of a monoclonal antibody that recognizes the human c-kit receptor. Blood. 1992; 79: 338-346. 38. Bühring HJ, Ashman LK, Gattei V, Kniep B, Larregina A, Pinto A, Valent P, van den Oord J. Stem-cell receptor factor (p145 (c-kit)) summary report (CD1 1 7). in Leucocyte Typing V. White Cell Differentiation Antigens, eds: Schlossmann SF, Boumsell L, Gilks W, et al. Vol 2. pp 1 882- 1 888. Oxford University Press. 1995. 39. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22: 27-55. 40. Van Cruchten S, Van Den Broeck W. Morphological and biochemical aspects of apoptosis, oncosis and necrosis. Anat Histol Embryol 2002; 31: 214-223. 41 Schedle A, Samorapoompichit P, Füreder W, et al. Metal ion-induced toxic histamine relase from human basophils and mast cells. J Biomed Mater Res. 1 998; 39: 560-567. 42. Samorapoompichit P, Kiener HP, Schernthaner GH, et al. Detection of tryptase in cytoplasmic granules of basophils in patients with chronic myeloid leukemia and other myeloid neoplasms. Blood. 2001 ¡98: 2580-2583. 43. Gorczyca W, Gong J, Darzynkiewicz Z. Detection of strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidal transferase and nick translation assays. Cancer Res. 1993; 53: 1945-1951. 44. Walker PR, Carson C, Leblanc J, Sikorska M. Labeling DNA damage with terminal transferase. Applicability, specificity, and limitations. Methods Mol Biol. 2002; 203: 3-19. 45. Escribano L, Diaz-Agustin B, Bellas C, et al. Utility of flow cytometric analysis of mast cells in the diagnosis and classification of adult mastocytosis. Leuk Res. 2001; 25: 563-570. 46. Valent P, Schernthaner GH, Sperr WR, et al. Variable expression of activation-linked surface antigens on human mast cells in health and disease. Immunol Rev. 2001; 179: 74-81. 47. Ghannadan M, Hauswirth AW, Schernthaner GH, et al. Detection of novel CD antigens on the surface of human mast cells and basophils. Int Arch Allergy Immunol. 2002; 127: 299-307. 48. Growney JD, Clark JJ, Adelsperger J, et al. Activation mutations of human-KIT resistant to imatinib are sensitive to the tyrosine kinase inhibitor PKC412. Blood. 2005, in the press. 49. Corless CL, Fletcher JA, Heinrich MC. Biology of gastrointestinal stromal tumors. J Clin Oncol. 2004; 22: 3813-3825. 50. Cowan-Jacob SW, Guez V, Fendrich G, et al. Imatinib (STI571) resistance in chronic myelogenous leukemia: molecular basis of the underlying mechanisms and potential strategies for treatment. Mini Rev Med Chem. 2004; 4: 285-299. 51 Weisberg E, Griffin JD. Resistance to imatinib (Glivec): update on clinical mechanisms. Drug Resist Updat. 2003; 6: 231 -238. 52. Gotlib J, Berube C, Rúan J, et al. PKC412, inhibitor of the KIT tyrosine kinase, demonstrates efficacy in mast cell leukemia with the D816V KIT mutation. Blood. 2003; 102: 919a (abst).

EXAMPLE 2: COMBINATION OF DASATIN I B AND PKC412 In the majority of patients with systemic mastocytosis (SM) including aggressive SM and mast cell leukemia (MCL), the neplasmic cells exhibit the D816V-mutated KIT variant. KIT-D816V exhibits constitutive trosine kinase (TK) activity and has been implicated in malignant cell growth. Therefore, several attempts have been made to identify KIT-D816V focusing drugs. We found that the TK inhibitor dasatinib (BMS-354825) counteracts the TK activity of wild type KIT (wt) and KIT-D816V in Ba / F3 cells with KIT expression inducible with doxycycline. In addition, dasatinib is shown to inhibit cluster formation induced by KIT D816V and viability in Ba / F3 cells as well as cell growth of HMC-1 .1 (KIT-D816V-negative) and HMC-1 .2 cells (KIT-D816V- positive). The effects of dasatinib are dose-dependent, with IC50 values 100-1,000 times higher in those harboring KIT-D816V compared to cells lacking KIT-D816V. The inhibitory effects of dasatinib on HMC-1 cells are found to be associated with apoptosis and a decrease in expression of CD2 and CD63. In addition, dasatinib is found to cooperate with PKC412, AMN 107, imatinib and 2CdA to produce growth inhibition. In HMC-1 .1 cells, all drug interactions applied are found synergistic. In contrast, in HMC-1 .2, only the combinations "dasatinib + PKC412" and "dasatinib + 2CdA" produce synergistic effects. These drug combinations can thus represent an interesting pharmacological approach for the treatment of aggressive MS or MCL.

Introduction The receptor tyrosine kinases, such as platelet-derived growth factor receptor (PDGFR) or stem cell factor receptor (SCFR, KIT), are frequently deregulated and show constitutive tyrosine kinase (TK) activity in patients with nesms. Hematopoietic agents.1"5 These molecules thus represent attractive targets for drug therapy.In fact, during the past few years, several emerging treatment concepts have been based on novel drugs that focus on critical TK in nestic myeloid cells.1" 5 Systemic mastocytosis (SM) is a myeloid nesm characterized by abnormal growth and accumulation of nestic mast cells (MC) in one or more organs. Variants of indolent as well as aggressive MS have been described.6"9 In patients with aggressive MS (ASM) and those suffering from the leukemic variant of MS, ie, mast cell leukemia (MCL), the response to conventional medications is poor and the prognosis is severe.6"12 Therefore, a variety of attempts have been made to identify new therapeutic targets in nestic MCs and to develop respective treatment concepts.9" 12 In the majority of patients suffering from MS including those with ASM or MCL, the mutation of KIT D816V is detectable.13 '17 This mutation is associated with KIT ligand-independent phosphorylation, as well as autonomous cell growth.17, 18 Based on this notion, the D861 variant V- The mutated KIT has been recognized as a primary therapy objective.9, 12, 19 Thus, a variety of efforts have been made to identify TK inhibitors that counteract KIT-D816V phosphorylation and growth. of MC nestic.9'12, 19"24 Imatinib (STI571), a potent inhibitor of BCR / ABL, has recently been described as counteracting growth of nestic MCs exhibiting wild-type KIT (wt) or the mutated F522C-variant that is rare It occurs in KIT.20"23 Furthermore, this medication was found to blunt nestic cell growth in patients who have chronic eosinophilic leukemia with FIP1 L1 / PDGRA fusion gene with or without co-existing SM.24'26 However , imatinib failed to inhibit the growth of nestic MCs that house KIT D816V.20"22 More recently, we and others have shown that PKC41227 counteracts the TK activity of KIT-D816V and thereby downregulates the growth of neoplastic MCs.28"30 It has also been described that the novel TK inhibitor AMN 10731 counteracts the growth of neoplastic cells that exhibit KIT-D816V at relatively high drug concentrations.30,32 However, most of these compounds may not produce durable complete remission in patients with ASM or MCL, at least as simple agents. these drugs act only on MCs exhibiting wt KIT, but do not inhibit growth of MCs harboring KIT-D816V.Therefore, it is important to additionally look for novel TK inhibitors targeting KIT and examine effects of cooperative drugs. Recently, we have shown that PKC412 and AMN 107 produce cooperative growth inhibitory effects in HMC-1 cells. However, while this drug combination produced synergistic inhibitory effects in HMC-1 cells lacking KIT-D816V, no synergistic effect was observed in HMC-1 .2 cells expressing KIT-D816V.30 Other drug combinations also fail in exert synergistic inhibitory effects on mast cells that exhibit KIT-D816V.30 Dasatinib (BMS-354825) is a novel inhibitor of src kinases and of several TK inhibitors including KIT.33, 34 It has also been reported that dasatinib inhibits KIT- phosphorylation D816V and the growth of neoplastic MCs.34, 35 In the current study, we show that dasatinib blocks several of the disease-related functions dependent on KIT-D816V in neoplastic cells including survival and cluster formation, as well as expression of CD2 and CD63. In addition, our data show that dasatinib synergizes with PKC412 as well as 2CdA to produce growth inhibition in HMC-1 cells.2. To our knowledge, this is the first combination of TK inhibitors described for acting synergistically on MCs that house KIT-D816V. Our data also suggest that dasatinib alone or in combination with other medications may be a promising agent for the treatment of patients with ASM or MCL.

Materials and methods Reagents Dasatinib (BMS-354825) 33 was provided by Bristol-Myers Squibb (New Brunswick, NJ), and imatinib (STI571), AMN 107.31 and PKC41227 by Novartis Pharma AG (Basel, Switzerland). The stock solutions of dasatinib, AMN107 and PKC412 were prepared by dissolving in dimethyl slufluoxide (DMSO) (Merck, Darmstadt, Germany). The mdre (recombinant human (rh) cell factor (SCF) was purchased from Strathmann Biotech (Hannover, Germany), RPMI 1640 medium and fetal calf serum (FCS) at PAA Laboratories (Pasching, Austria), L-glutamine and medium from modified Dulbecco from Iscove (IMDM) from Gibco Life Technologies (Gaithersburg, MD), 3H-thymidine from Amersham (Buckinghamshire, UK), 2-chloro-deoxyadenosine (cladribine = 2CdA) from Sigma (St. Louis, MO), and rh interleukin-4 (IL-4) from Peprotech (Rocky Hill, NJ): PE-labeled monoclonal antibodies (mAbs) RPA-2.10 (CD2), WM15 (CD13), YB5.B8 (CDI 17), and N6B6. 2 (CD164) as well as MOPC-21 (mlgGI) and G1 55-1 78 (mlgG2a) were purchased from Becton Dickinson (San Jose, CA), and the PE-conjugated mAb CLB-granl2 (CD63) from Immunotech (Marseille, France) The PE-labeled mAb VIM5 (CD87) was kindly provided by Dr. Otto Majdic (Institute of Immunology, Medical University of Vienna, Austria).

HMC-1 cells expressing or lacking KIT D816V The human mast cell line HMC-126 generated from a patient with MCL was kindly provided by Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN). Two subclones of HMC-1 were used, namely HMC-1 .1 which hosts the KIT V560G mutation but not KIT D816V, 20 and a second subclone, HMC-1 .2, which hosts both KIT mutations, i.e. HMC-1 V560G and D81 6V.20 cells were cultured in IMDM supplemented with 10% FCS, L-glutamine, alpha-thioglycerol (Sigma) and antibiotics at 37 ° C and 5% CO2. The cells were re-frozen from unstocked every 4-8 weeks and passed weekly. The HMC-1 cells were checked periodically for i) the presence of metachromatic granules, ii) expression of KIT, and iii) the sub-modulator effect of IL-4 (100 U / ml, 48 hours) on KIT expression. 37 Ba / F3 cells with inducible expression of wt KIT or KIT D816V The generation of Ba / F3 cells with inducible expression with doxillin from wt c-KIT (TonKit.wt) or c-KIT D816V has been previously described.30, 38 In Briefly, Ba / F3 cells expressing the reverse transactivator tet-39,40 were co-transfected with the pTRE2 vector (Clontech, Palo Alto, CA) containing KIT D816V cDNA (or wt KIT cDNA, both kindly sent by Dr. JB Longley, Columbia University, New York, USA) and pTK-Hyg (Clontech) by electroporation. The stably transfected cells were selected by culturing in hygromycin and cloned by imitant I dilution. In this study, the Ton subclone. Kit. D816 V.2738 was used in all experiments. The expression of KIT-D816V can be induced in these cells (within 12 hours) by exposure to doxycycline (1 μg / ml) .38 Isolation of primary neoplastic cells The bone marrow (bm) primarisa cells were obtained from a patient with ASM KIT D816V-positive ASM and associated AML and a patient with normal bm. The bm aspirate samples were collected in syringes containing conservative free heprin. The cells were stratified on Ficoll to isolate mononuclear cells (MNC). Cell viability was > 90% in both cases. In the patient with ASM-AML, isolated MNC was found to contain > 90% blast cells. Both patients gave written informed consent before bm puncture or blood donation.

The study was approved by a local institutional review board and was conducted in accordance with the Helsinki declaration.

KIT phosphorylation analysis by western blotting HMC-1 cells (106 / ml) and Ton cells. Kit D816V.27 (106 / ml) containing either wt KIT (Ton Kit.wt) or KIT D816V (Ton Kit.D816 V.27), incubated with dasatinib (1 pM, 1 nM, 10 nM, 100 nM, 1 μM), PKC412 (1 μM), AMN107 (1 μM), imatinib (1 μM), or control medium at 37 ° C for 4 hours. Before exposure to inhibitory drugs, Ton cells. Kit. Wt and Ton cells. Kit. D816V.27 were incubated with doxycycline (1 μg / ml) at 37 ° C for 24 hours to induce KIT expression. In the case of Ton cells. Kit. Wt, KIT phosphorylation was indicated by adding rhSCF (100 ng / ml). Immunoprecipitation (IP) and Western blotting were performed as describe.30'40 Briefly, cells were washed at 4 ° C and resuspended in RIPA buffer (1 ml of buffer per 108 cells) containing 50 mM Tris, 150 mM NaCl, 1% Nonidet P40 (NP-40), 0.25% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 1 mM ethylene diamine-tetraacetic acid (EDTA), 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride and 1 mM Na3VO4. After incubation in RIPA buffer supplemented with proteinase inhibitor cocktail (Roche) for 30 minutes at 4 ° C, the lysates were centrifuged. For IP, lysates from 107 cells were incubated with anti-KIT 1 C1 antibody (kindly provided by Dr. HJ. Bühring, University of Tubingen, Germany) 43 and protein G beads Sepharose (Amersham) in IP buffer (50 mM Tris -CI, pH 7.4, 150 mM NaCl, 100 mM NaF, and 1% NP-40) at 4 ° C overnight. The beads were then washed 3 times in IP buffer. The lysates and immunoprecipitates were separated under reducing conditions by SDS 7.5% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Protran, Schleicher &; Schuell, Keene, NH) in buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol at 4 ° C. The membranes were bocketed for 1 hour in 5% blocking reagent (Roche) and then incubated with anti-KIT 1 C1 antibody or with mAB anti-phospho-4G10 protein (Upstate Biotechnology, Lake Placid, NY) at 4 ° C during the night. Antibody reactivity was made visible by sheep anti-mouse GG antibody and Lumingen PS-3 detection reagent (both from Amersham), with CL-Xposure film (Pierce Biotechnology, Rockford, IL).

Evaluation of drug effects on growth and function of Ton cells. Lit. D816V.27 The Ton cells. Kit. D816V.27 were co-incubated with doxycycline (1 μg / ml) and various concentrations of dasatinib or AMN 107 at 37 ° C for 24-48 hours. Cell viability was determined by trypan blue exclusion. Cluster formation was analyzed using an inverted microscope. Previous studies have shown that the expression of KIT-D816V in Ton.Kit. D816V.27 is associated with significant cluster formation, and that PKC412, but not imatinib, downregulates clustering in Ton cells. Kit.D816V.27.38 In the present study, the effects of dasatinib (1 pM - 1 μM) and AMN 1 07 on clustering of Ton cells. Kit. D816V.27 were analyzed. For control purposes, the effects of PKC412 and imatinib were also examined. Cluster formation was determined by light microscopy (counted as a cluster by high energy field = HPF) and expressed as a percentage of control (= doxycycline alone without drugs = 1 00%). All the experiments were performed in triplicate.

Measurement of 3H-thymidine uptake To determine the effects of growth inhibitory drugs, HMC-1 cells were incubated with various concentrations of dasatinib (1 00 fM - 10 μM), PKC412 (100 pM - 10 μM), AMN 107 ( 1 nM-100 μM), or imatinib (3 nM-300 μM) in 96-well culture plates (TPP, Trasadingen, Switzerland) at 37 ° C for 48 hours. In the ongoing experiments, HMC-1 cells were exposed to dasatinib (HMC-1 .1: 10 nM; HMC-1 .2: 1 μM) for 12, 24, 36, or 48 hours. In selected experiments, HMC-1 cells were incubated with various concentrations of 2CdA (0.005-10 μg / ml). Primary cells (bm cells from a patient with ASM-AML; control bm cells) were cultured in control medium, dasatinib (100 pM - 10 μM), PKC412 (100 pM - 10 μM), AMN 107 (100 pM - 10 μM), or imatinib (100 pM - 1 0 μM) for 48 hours. After incubation, After incubation, 1 μCi 3 H-thymidine (37 ° C, 12 hours) was added. The cells were then harvested on filter membranes (Packard Bioscience, Meriden, CT) on a Filtermate 196 harvester (Packard Bioscience). The filters were air dried and the bound radioactivity was counted in a ß-counter counter (Top-Count NXT, Packard Bioscience). To determine the effects of potential additive or synergistic drugs on cell growth, HMC-1 cells (both subclones) were exposed to various drug combinations (dasatinib, PKC412, AMN107, imatinib, 2CdA) at a fixed ratio of drug concentrations. Drug interactions (additive, synergistic) were determined by calculating the combination index (Cl) values using the calcusyn computation program (Calcusyn, Biosoft, Ferguson, MO) .44 A Cl value of 1 indicates an additive effect, while Cl values below 1 indicate synergism of drug effects. All the experiments were performed in triplicate.

Evaluation of apoptosis by conventional morphology and electron microscopy The effects of TK inhibitors on apoptosis were analyzed by morphological examination, flow cytometry and electron microscopy. In normal experiments, HMC-1 cells were incubated with various concentrations of dasainib (1 pM - 1 μM) or control medium in 6-well culture plates (TPP) in IMDM containing 10% FCS at 37 ° C for 24 hours . The percentage of apoptotic cells was quantified in cytoliro preparations stained with Wright-Giemsa. Apoptosis was defined according to conventional cytomorphological criteria.45 To confirm apoptosis in HMC-1 cells, electron microscopy was performed as described46,47 using HMC-1 cells (both subclones) exposed to dasatinib (1 pM, 1 nM, 1 0 nM, 100 nM, 1 μM), PKC412 (1 μM), or control medium for 24 hours. After the incubation, the cells were washed and fixed in 2% paraformaldehyde, 2.5% glutaraldehyde and 0.025% CaCl2 buffered in 0.1 mol / l sodium cacodylate buffer (pH 7.4) for 1 hour. The cells were then washed, suspended in 2% agar and centrifuged. The pellets were post-fixed with 1.3% OsO4 (buffered at 0.66 mol / l collidine) and stained "en bloc" in 2% uranyl acetate and sodium maleate buffer (pH 4.4) for 2 hours. The pellets were then rinsed, dehydrated in series of alcohol and embedded in EPON 812. Ultra-thin sections were cut and placed in gold grids. The sections were contrasted in uranyl acetate and lead citrate and viewed in a JEOL 1200 EX II transmission electron microscope (JEOL, Tokyo, Japan).

Evaluation of apoptosis by Tunnel assay To confirm apoptosis in HMC-1 cells after exposure to dasatinib (1 pM, 1 nM, 10 nM, 100 nM, 1 μM) or PKC412 (100 nM, 1 μM), performed a tunnel assay for in situ Terminal transferase-mediated dUTP-fluorescence Nick End-Labeling (final labeling of nickel from dUTP-fluorescence-mediated terminal transferase in situ) using "In Situ Cell Death Detection Kit Fluorescein" (Fluorescein in situ cell death detection set) (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. In berve, the cells were placed in cytospin, fixed in 4% paraformaldehyde in PBS at pH 7.4 at RT for 60 minutes, washed and incubated in the terminal transferase reaction solution containing CoCl2, terminal deoxynucleotidyltransferase, and labeled dUTP. with fluorescein for 60 minutes at 37 ° C. The cells were then washed and analyzed with a Nikon Eclipse E 800 fluorescence microscope (Tokyo, Japan).

Evaluation of expression of surface antigens linked to activation in HMC-1 cells The expression of cell surface antigens in HMC-1 cells (both subclones) was determined by flow cytometry after culture in control medium or medium supplemented with inhibitors of TK (dasatinib, 1 pM - 5 μM; PKC412, 1 μM) at 37 ° C for 24 hours. After incubation with medicaments, the cells were washed and subjected to simple color flow cytometry using antibodies conjugated with PE against various MC differentiation antigens including determinants known to be expressed in an aberrant (selective) manner expressed in neoplastic MCs.48"51 The markers analyzed were CD2, CD1 3, CD63, CD87, CD1 17 and CD164 Flow cytometry was performed in a FACScan (Becton Dickinson) as described.30, 37.51 Statistical analysis To determine the significance of differences between proliferation rates, apoptosis and surface expression levels after exposure of HMC-1 cells to inhibitors, the student's t-test for dependent samples was applied.The results were considered statistically significant when p was <0.05.

Results Effects of dasatinib on TK activity of KIT-D816V As assessed by IP and western blotting, dasatinib (1 nM - 1 μM) decreased phosphorylation of KIT in HMC-1 cells.1 (expressing KIT-V560G but not KIT D816V) (Figure 1 1 A). In HMC-1 .2 cells harboring both mutations (KIT-V560G and KIT-D816V), dasatinib decreased phosphorylation of KIT to 1 μM, but did not counteract phosphorylation of KIT at lower concentrations (Figure 1 1 B). We next examined the effects of dasatinib on Ba / F3 cells expressing either wt KIT (Ton Kit.wt) or KIT D816V (Ton.Kit.D816V.27) after exposure to doxycycline. In Ton cells. Kit. Wt, KIT appeared to be phosphorylated in the presence of SCF, whereas KIT was found constitutively phosphorylated in Ton cells. Kit. D816V.27. As is visible in Figure 1C, dasatinib (10 nM-1 μM) decreased the SCF-induced phosphorylation of KIT in Ton cells. Kit. Wt. In contrast, in Ton cells. Kit.D816V.27 (expressing KIT-D816V after exposure to doxycycline), dasatinib decreased phosphorylation of KIT to 0.1 and 1 μM, but failed to decrease KIT- phosphorylation at lower concentrations (Figure 1 1 D).

Effects of TK inhibitors on uptake of 3H-thymidine in HMC-1 cells In the current experiments, the maximal inhibitory effects of dasatinib on the growth of HMC-1 .1 cells and HMC-1 .2 cells were seen after 36-48 hours. Figure 12A showed the time-dependent effects of dasatinib (10 nM for HMC-1 .1, 1 μM cells for HMC-1 .2 cells) on the growth of these cells. As shown in Figure 12B, dasatinib was found to counteract uptake of 3H-thymidine in HMC-1 .1 cells and HMC-1 .2 cells in a dose-dependent manner. Interestingly, the IC50 for the effects of dasatinib in HMC-1 .2 cells (200-500 nM) was considered to be higher compared to the IC50 values obtained for HMC-1 cells (1 NM) (Figure 12B; Figure 1 7). However, dasatinib was found to inhibit the growth of HMC-1 .2 cells much more effectively on a molar basis compared to imatinib (tested in parallel). Table 2 shows a summary of the IC50 values obtained for the effects of TK inhibitors applied on HMC-1 .1 cells and HMC-1 .2 cells. With respect to the effects of imatinib, AMN 1 07 and PKC412, these data confirmed the previous results.30 Table 2 Effects of focused drugs (IC50) on uptake of 3H-thymidine in HMC-1 cells Effects of TK inhibitors on the growth of Ba / F3 cells expressing wt KIT or KIT D816V (Ton.Kit.D816V.27) It was found that dasatinib counteracts the SCF-dependent growth of Ton cells. Kit. Wt exposed to doxycycline (expressing KIT) in a dose-dependent manner (IC50: 1 nM - 10 nM) (Figure 12C). In Ton cells. Kit.D816V.27, dasatinib was also found to inhibit cell growth and viability (Figure 1 2D). Dasatinib did not counteract the growth of Ton cells. Kit. Wt in the absence of doxycycline, that is, in the absence of KIT (Figure 12C). Similarly, dasatinib did not produce greater growth inhibitory effects on Ton cells. Kit. D816 V.27 in the absence of KIT D816V (- doxycycline) (Figure 1 2D). In addition, in additional control experiments, doxycycline (1 μg / ml) showed no growth inhibitory effects on untransfected Ba / F3 cells (not shown).

Effects of dasatinib and AMN107 on cluster formation dependent on KIT-D816V in Ton cells. Kit.D816V.27 We have previously shown that KIT-D186V induces not only mast cell differentiation, but also cluster formation in Ba / F3 cells, which is of particular interest because the formation of mast cell clusters is a primary finding and Major disease criterion in SM.38 It has also been reported that PKC412 upregulates cluster formation induced by doxycycline / KIT-D816V in Ton cells. Kit. D816 V.27 cells.38 In the present study, we found that dasatinib (100 nM - 1 μM) and to a lesser degree, AMN 107 (200 nM - 1 μM), counteract the formation of clusters dependent on KIT-D816V in Ba / F3 cells in a dose-dependent manner (Figure 12E and 12F).

Dasatinib counteracts the growth of primary neoplastic cells in a patient with SM KIT D816V-positive with associated AML To confirm effects of anti-proliferative drugs of dasatinib in SM, examine primary neoplastic cells in a patient with ASM KIT D816V-positive associated with AML. In this patient, dasatinib (IC50: 0.3-1.0 nM) as well as PKC412 (IC50: 10-30 nM) were found to inhibit the spontaneous growth (uptake of 3H-thymidine) of leukemic cells in a dose-dependent manner. AMN 1 07 also showed a growth inhibitory effect (100-300 nM); whereas imatinib (IC50> 1.0 μM) did not counteract the growth of neoplastic cells in this patient (Figure 13). In the control sample, ie, in normal bm cells, neither dasatinib nor the other inhibitors tested showed an effect on uptake of 3H-thymidine (not shown).

Dasatinib induces apoptosis in HMC-1 cells To explore the mechanism underlying the growth inhibitory effect of dasatinib, we analyzed morphological and biochemical signs of apoptosis in HMC-1 cells after drug exposure. As assessed by light microscopy, dasatinib was found to induce apoptosis (i.e., increase the number of apoptotic cells) in both subclones of HMC-1 (Figure 14A and 14B). PKC412 was applied as a control and was also found to induce apoptosis in both HMC-1 subclones, while imatinib was found to produce apoptosis in HMC-1 cells.1 but showed no effects on HMC-1 cells.2 (not shown) ). Dasatinib apoptosis-inducing effect in HMC-1 cells was confirmed by electron microscopy. In fact, dasatinib induced apoptosis in both MC-1 .1 H and 1-M HMC-1 .2 cells (Figure 14C). Finally, we were able to demonstrate the apoptosis inducing effect of dasatinib in HMC-1 cells in a Tunnel assay (Figure 14D and 14E). In this assay, dasatinib was found to induce apoptosis in HMC-1 .1 cells between 1 and 1,000 nM; and induces apoptosis in both cell lines (Figure 14D and 14E). In contrast, imiatinib (1 μM) induced apoptosis only in HMC-1 cells.1 but showed no effects in HMC-1 cells.2 (not shown) confirming previous data.30 Dasatinib downregulates the expression of cell surface antigens linked to activation in HMC-1 cells Several cell surface antigens such as CD2 or CD63 are (over) normally expressed in neoplastic MCs in SM.48"50 Interestingly, some of these molecules can be expressed in neoplastic MCs in a manner dependent on KIT D816V38 and / or are expressed in an early stage of development of human mast cells.51 Therefore, we investigated whether dasatinib would influence the expression of these surface antigens in cells HMC-1. Unstimulated HMC-1 .1 cells were found to express CD1 3, CD63, CD87, CD1 17, and CD164, and HMC-1 .2 cells expressed CD2, CD13, CD63, CD87, CD17, and CD164, confirming data previos.30,38, 50 Incubation of HMC-1 .1 cells with dasatinib resulted in a significant decrease in the expression of CD13, CD63, CD87, and CD1 17 (p <; 0.05) (Figure 15C). In HMC-1 .2 cells, dasatinib significantly decreased the expression of CD2, CD63, and CD87 (p < 0.05), but did not lead to a significant decrease in the expression of CD13, CD1 17, or CD164 (Figure 15D). The down-regulation effects of dasatinib in flow cytometry experiments are exemplified by CD63 in Figures 15E (HMC-1 .1) and Figure 1 5D (HMC-1 .2).

Dasatinib cooperates with other TK inhibitors and with 2CdA to produce growth inhibition in HMC-1 cells. As assessed by 3 H-thymidine incorporation, dasatinib was found to cooperate with PKC412, AMN 107, imatinib, and 2CdA 81 Discussion In patients with MS, independent factor-independent growth and MC accumulation are characteristic features common to all disease variants. The somatic KIT mutation D816V is a defect related to SM considered responsible for the constitutive activation of KIT and autonomous cell growth.13"17 Therefore, recent attempts have been made to identify pharmacological compounds that inhibit KIT's TK activity -D816V, and thus, the growth / accumulation of neoplastic cells.9"12 We describe that the novel TK inhibitor sasatinib blocks the TK activity of KIT-D816V as well as several functions related to KIT D816V-dependent disease in neoplastic cells. . In addition, we show that dasatinib synergizes with PKC412 as well as other targeted and conventional drugs to produce growth inhibition in neoplastic MCs. Dasatinib, also known as BMS-3548925, is a novel TK inhibitor that exerts profound effects on several TKs including BCR / ABL and KIT, and also exhibits considerable activity against several src.33 kinases "35 Based on its activity" directed to TK ", dasatinib has been recently reported as an antineoplastic agent that can inhibit the growth of neoplastic cells in several myeloid neoplasms.33" 35 In the present study, we found that dasatinib counteracts the TK activity of the SM-related oncoprotein KIT-D816V and inhibits the in vitro growth of human MCs harboring this KIT mutation, which confirms previous publications.34,35 to cause growth inhibition in HMC-1 cells (Table 3, Figure 16). In HMC-1 .1 cells, all drug interactions tested were found to be synergistic in nature (Figure 16A and 16B). In contrast, in the HMC-1 .2 cells, only the combinations "dasatinib and PKC412" and "dasatinib and 2CdA" produced a clear synergism (Figure 16C and 16D), while other drug combinations showed more additive growth inhibitory effects. that synergists about cell growth (Table 3). As shown in Table 3, the effects of cooperative drugs on the growth of cells HMC-1 .2 (upper panels, gray) and HMC-1 cells .1 (lower panels, dark gray) were determined by measuring the uptake of 3H-thymidine. The effects of cooperative medications were calculated using the calcusyn counting program.

Table 3 Evaluation of the effects of synergistic drugs on the growth of HMC-1 cells Drug Interactions: +, synergistic effects; ±, additive effects; -, antagonistic effects.

In addition, we found that dasatinib counteracts the formation of clusters dependent on KIT-D816V in Ba / F3 cells as well as the expression of CD2 and CD63 in HMC-1 cells. In this way, dasatinib blocks several functions dependent on KIT-D816V and related to SM in neoplastic MCs. With respect to growth inhibition, an interesting observation was that the effect of dasatinib on wt KIT or KIT G560V was more pronounced compared to that seen with KIT D816V. A similar observation has recently been made with AMN 107 and imatinib.30 However, while the D816V KIT mutation contains almost complete resistance against imatinib, the other two inhibitors of TK, ie, AMN 107 and dasatinib, retain considerable activity. against KIT D816V, with lower IC50 values obtained by dasatinib compared to AMN 107 on a molar basis, which can be explained by different drug-target interactions or by the fact that dasatinib not only counteracts the activity of KIT TK but also other various potential objectives, such as, src kinases. An interesting observation was that the inhibitory effects of dasatinib growth in HMC-1 .2 cells occur at pharmacological concentrations, confirming previous publications.34, 35 In many cases, TK inhibitors act as growth inhibitors by blocking cell growth dependent on TK with consecutive apoptosis.30, 35 Similarly, in the case of dasatinib, we were able to show that the inhibition of HMC-1 cell growth is associated with loss of TK activity and is accompanied by signs of apotosis. The inducing effects of dasatinib apoptosis were demonstrated by light and electron microscopy, as well as in a tunnel assay. As expected, dasatinib showed more potent apoptosis-inducing effects on HMC-1 .1 cells than in HMC-1 .2, which is in line with recently published results.35 A key feature and major WHO criterion in SM is training of clusters of MCs in visceral organs.41, 42 We have recently shown that KIT D816V induces not only mast cell differentiation, but also cluster formation in Ba / F3.38 cells. Thus, the formation of MC clusters can be a step initial and very important in the pathogenesis of SM. In the present study, we were able to show that dasatinib and AMN107 counteract cluster formation induced by KIT D816V in Ba / F3 cells. This observation provides additional evidence for the specific action and effectiveness of these medications. Several cell surface membrane antigens are (over) normally expressed in neoplastic MCs.48"50 Similarly, in contrast to normal MC, neoplastic MCs in SMs express CD2 and CD25.48'50 Moreover, several cell surface molecules such as, CD63, are overexpressed in neoplastic MCs compared to normal MCs.49 In some cases (such as CD63), the expression of CD molecules may be dependent on KIT-D816V.38 Therefore, we wonder if the KIT D816V approach by dasatinib would be associated with a decrease in the expression of these CD antigens.The results of our study show that dasatinib downregulates the expression of CD2, CD63 and CD87 in HMC-1 .2 cells (exhibiting KIT D816V), while no inhibition Significant expression of CD13, CD1 17 = KIT or CD164 was found.In contrast, in HMC-1 .1 cells, it was also found that dasatinib downregulates CD1 3 and KIT expression.An explanation for this discrepancy It would be the different sensitivity (IC50) of the two subclones of HMC-1 for dasatinib. An alternative possibility would be that in HMC-1 .2, CD13 and KIT cells are generally not susceptible to drug-induced modulation. This hypothesis would be supported by the observation that CD13 and KIT were also expressed at the same levels after incubation with PKC412, although the IC50 values for this compound are identical in the two subclones of HMC-1. A variety of recent data suggest that the treatment of myeloid neoplasms with TK inhibitors as a single agent may not be sufficient to control the disease for a prolonged period. This has been documented for imatinib and advanced CML51, 52 and may also apply for patients with ASM or MCL29. Thus, in many of these patients, drug resistance is found. To overcome resistance, a variety of pharmacological strategies can be provided. A reasonable approximation may be to apply drug combinations. In a previous study, we found that PKC412, AMN 107 and 2CdA exhibit potent cooperative drug effects in HMC-1 cells. 30 However, while synergistic effects are seen with most drug combinations in HMC-1 cells. lacking KIT D816V, no synergistic drug interaction (but only additive) is seen in HMC-1 .2 cells that house KIT D816V. Therefore, we were highly interested in learning if dasatinib, which exhibits potent effects on mast cells carrying KIT D816V as a single agent, would produce synergistic effects in these cells when combined with other potent inhibitors of KIT D816V. In fact, our results show that dasatinib and PKC412 as well as dasatinib and 2CdA, a drug used to treat ASM and MCL54, inhibit the growth of HMC-1 .2 cells in a synergistic manner. To our knowledge, this is the first combination of TK inhibitors that produce a synergistic effect on the growth of neoplastic MCs carrying KIT D816V. In summary, we show that dasatinib and PKC412 are highly promising targeted drugs for the treatment of ASM and MCI. Based on our data, it seems reasonable to consider the application of combinations of these medications or combinations between these medications and 2CdA to improve therapy in patients with ASM or MCL.

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Claims (35)

1. CLAIMS 1 . A method for treating or preventing systemic mastocytosis, which comprises administering a compound of formula (I): in combination with a compound of formula (II): or a pharmaceutically acceptable salt thereof, wherein the combination treats or prevents a proliferative disease related to mast cells.
2. 2. The method according to claim 2, wherein the systemic mastocytosis has resistance to imatinib.
3. 3. The method according to claim 1, wherein the systemic mastocytosis is associated with the oncogenic mutation KIT-D816V.
4. 4. The use of a compound of formula (I): in combination with a compound of formula (II): or a pharmaceutically acceptable salt thereof, for the preparation of a pharmaceutical composition for the treatment of systemic mastocytosis.
5. 5. The use according to claim 4 for the treatment of systemic mastocytosis associated with the oncogenic mutation KIT-D816V.
6. 6. A method for treating mammals suffering from systemic mastocytosis comprising administering to a mammal in need of such treatment, an inhibiting amount of KIT activity of a compound of formula (I): in combination with a compound of formula (II): or a pharmaceutically acceptable salt thereof.
7. 7. A pharmaceutical preparation for the treatment of systemic mastocytosis associated with KIT-D816V, comprising a compound of formula (I): in combination with a compound of formula (II): or a pharmaceutically acceptable salt thereof.
8. 8. A method for treating mammals, including man, that suffer from systemic mastocytosis, which comprises administering to a mammal in need of such treatment a compound of formula (I): ) in combination with a compound of formula (II): or a pharmaceutically acceptable salt thereof.
9. 9. A method for treating or preventing systemic mastocytosis, which comprises administering to a compound of formula (I): in combination with a thymidine kinase inhibitor (TK); or a pharmaceutically acceptable salt thereof.
10. 10. A method for treating or preventing systemic mastocystosis, which comprises administering a compound of formula (I): in combination with a thymidine kinase inhibitor (TK), or a pharmaceutically acceptable salt thereof, wherein the treatment treats or prevents a proliferative disease related to mast cells. eleven .
11. The method according to claim 10, wherein the mast cell-related proliferative disease is selected from systemic mastocytosis, aggressive systemic mastocytosis and mast cell leukemia.
12. The method according to claim 10, wherein the mast cell-related proliferative disease has resistance to imatinib.
13. 13. The method according to claim 10 for the treatment of a mast cell-related proliferative disease associated with wild type KIT or with oncogenic mutation KIT G560V or KIT-D816V.
14. The method of claim 10, wherein the TK inhibitor is selected from dasatinib and 2CdA.
15. 15. The use of a compound of formula (I): in combination with a thymidine kinase inhibitor (TK), or a pharmaceutically acceptable salt thereof, for the preparation of a pharmaceutical composition for the treatment of a mast cell-related proliferative disease.
16. 16. The use according to claim 15, wherein the proliferative disease related to mast cells is selected from systemic mastocytosis, aggressive systemic mastocytosis and mast cell leukemia.
17. 17. The use according to claim 15, wherein the mast cell-related proliferative disease has resistance to imatinib.
18. 18. The use according to claim 15 for the treatment of a mast cell-related proliferative disease associated with wild type KIT or with an oncogenic mutation KIT G560V or KIT-D816V.
19. The use of claim 15, wherein the TK inhibitor is selected from dasatinib and 2CdA.
20. 20. A method for treating mammals suffering from a mast cell-related proliferative disease, comprising administering to a mammal in need of such treatment, an inhibiting amount of KIT activity of a compound of formula (I): in combination with a thymidine kinase inhibitor (TK), or a pharmaceutically acceptable salt thereof. twenty-one .
21. The method according to claim 20, wherein the mast cell-related proliferative disease is selected from systemic mastocytosis, aggressive systemic mastocytosis and mast cell leukemia.
22. 22. The method according to claim 20, wherein the mast cell-related proliferative disease has imatinib resistance.
23. 23. The method according to claim 20 for the treatment of a mast cell-related proliferative disease associated with wild-type KIT or with an oncogenic mutation KIT G560V or KIT-D816V.
24. The method according to claim 20, wherein the TK inhibitor is selected from dasatinib and 2CdA.
25. 25. A pharmaceutical preparation for the treatment of mast cell-related proliferative disease associated with wild-type or mutant KIT, comprising a compound of formula (I): in combination with a thymidine kinase inhibitor (TK), or a pharmaceutically acceptable salt thereof.
26. 26. The method according to claim 25, wherein the mast cell-related proliferative disease is selected from systemic mastocytosis, aggressive systemic mastocytosis and mast cell leukemia.
27. 27. The method according to claim 25, wherein the mast cell-related proliferative disease has resistance to imatinib.
28. 28. The method according to claim 25 for the treatment of a mast cell-related proliferative disease associated with wild-type KIT or with an oncogenic mutation of KIT 6560V or KIT-D816V.
29. 29. The method of claim 25, wherein the TK inhibitor is selected from dasatinib and 2CdA.
30. 30. A method for treating mammals, including man, that suffer from mast cell-related proliferative disease associated with wild type KIT or with an oncogenic mutation of KIT G560V or KIT-D816V, which comprises administering to a mammal in need of such treatment, a compound of formula (I): in combination with a thymidine kinase inhibitor (TK), or a pharmaceutically acceptable salt thereof. 15
31. 31. A method for treating mammals, including man, suffering from systemic mastocytosis, which comprises administering a compound of formula (I): in combination with a thymidine kinase inhibitor (TK); or a pharmaceutically acceptable salt thereof.
32. 32. The method according to any of claim 30 or claim 31, wherein the mast cell-related proliferative disease is selected from systemic mastocytosis, aggressive systemic mastocytosis and mast cell leukemia.
33. 33. The method according to any of claim 30 or claim 31, wherein the mast cell-related proliferative disease has imatinib resistance.
34. 34. The use according to any of claim 30 or claim 31, for the treatment of a mast cell-related proliferative disease associated with wild type KIT or with an oncogenic mutation of KIT G560V or KIT-D81 6V.
35. 35. The method according to any of claim 30 or claim 31, wherein the TK inhibitor is selected from dasatinib and 2CdA.