Effect of tyrosine kinase inhibitor STI571 on the kinase activity ... - Nature

Oncogene (2003) 22, 660–664

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Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit receptors found in mast cell neoplasms Yael Zermati1,7, Paulo De Sepulveda2,7, Frederic Fe´ger1, Sebastion Le´tard2, Joelle Kersual1, Nathalie Caste´ran2, Guy Gorochov3, Michel Dy1, Antoni Ribadeau Dumas1, Karim Dorgham3, Christophe Parizot3, Yann Bieche5, Michel Vidaud5, Olivier Lortholary6, Michel Arock1, Olivier Hermine*,1,4 and Patrice Dubreuil*,2 1 CNRS UMR 8603, Hoˆpital Necker, Universite´ Rene´ Descartes, Paris, France; 2INSERM U 119, Marseille, France; 3Laboratoire d’ Immunologie Cellulaire et Tissulaire, Inserm U584, Pr Debre´, GHPS, Paris, France; 4Department of Adult Hematology, Hoˆpital Necker, Paris, France; 5Laboratoire de Geńe´tique Molećulaire, UPRES JE 2195, Faculte´ de Pharmacie, Universite´ Rene´ Descartes, Paris, France; 6Department of Internal Medicine and Infectious Diseases, Hoˆpital Avicennes, Bobigny, France

Systemic mastocytosis (SM) is a rare disease caused by an abnormal mast cell accumulation in various tissues. Two classes of constitutive activating c-kit mutations are found in SM. The most frequent class occurs in the catalytic pocket coding region with substitutions at codon 816 and the other in the intracellular juxtamembrane coding region. Therefore, kinase inhibitors that block mutated c-kit activity might be used as therapeutic agents in SM. Here, we show that STI571 inhibits both wild-type and juxtamembrane mutant c-kit kinase activity, but has no effect on the activity of the D816 V mutant. Accordingly, STI571 selectively decreases the survival of normal mast cell and of mast cell lines either with juxtamembrane c-kit mutations, but not that of tumoral mast cell from patient with SM or of mast cell lines with the D816 V mutation. Therefore, STI571 is not a good candidate to treat SM and specific kinase inhibitors should be designed to inhibit constitutive activating mutations at codon 816. Oncogene (2003) 22, 660–664. doi:10.1038/sj.onc.1206120 Keywords: mastocytosis; c-kit; STI571; kinase inhibitor

Introduction Systemic mastocytosis (SM) is a neoplastic disease caused by an abnormal mast cell (MC) migration, survival, proliferation and/or activation (Longley et al., 1995). Recent studies have implicated mutations that cause spontaneous activation of c-kit kinase receptors as a probable cause of the majority of SM (Longley et al., 1999). Two classes of c-kit mutations leading to its constitutive activation have been described. In most cases, these mutations consist of substitutions in codon *Correspondence: O Hermine, Department of Adult Hematology and CNRS UMR 8603, Hoˆpital Necker, 149-161 Rue de Se`vres, 75743 Paris Cedex 15, France; E-mail: [email protected] or P Dubreuil, INSERM U 119, Institut Paoli Calmettes, 27 Boulevard de leı¨ Roure, 13009 Marseille, France; E-mail: dubreuil@marseille. inserm.fr 7 Both authors contributed equally to this work Received 27 June 2002; revised 7 October 2002; accepted 9 October 2002

816 in the catalytic pocket coding region (CP). The second class, rarely observed in human SM, includes a single nucleotide substitution, frame deletion or insertion in the intracellular juxtamembrane coding region (JM). Currently, no treatment is available to reduce the MC burden and to cure SM (Worobec, 2000; Marone et al., 2001). STI571, a recently discovered inhibitor of abl and bcr-abl, also called Imatinib or Gleevec, has been used successfully to induce a complete hematological response in chronic myelogenous leukemia (CML) (Druker et al., 2001). In vitro data have suggested that STI571 is also able to block kinase activity of PDGF and c-kit receptors (Buchdunger et al., 2000). Since c-kit kinase activity is necessary in vitro (Valent et al., 1992) and in vivo (Kitamura et al., 1978) for the generation and survival of MC and because c-kit is spontaneously activated in SM, we tested in vitro the effect of STI571 on normal, tumoral MC lines and on MCs derived from patients with SM. STI571 inhibited the growth of the positive UT-7-bcrabl cell line at the pharmacological dose of 0.1–1 mm (Figure 1a, left panel). This inhibition was related to cell cycle arrest and massive apoptosis (>80% of cell death at 72 h, Figure 1a, right panel). To test the effect of STI571 on cell growth induced by c-kit stimulation, UT-7/SCF cells were grown in the presence of 100 ng/ml of SCF with or without increasing doses of STI571 (0.1– 10 mm). After 72 h, STI571 inhibited cell growth, with an IC50 ranging between 0.1 and 1 mm (Figure 1a) as a consequence of cell cycle arrest (data not shown) and apoptosis (Figure 1a). In contrast, in the presence of GM-CSF (2 ng/ml) (data not shown) or Epo (2UI/L) cell growth inhibition was observed only at a suprapharmacological dose of 10 mm, of STI571 (Figure 1a). These results demonstrated that STI571 exerts a specific effect on cell growth induced by the interaction between SCF and its membrane receptor c-kit. We then tested the hypothesis that STI571, by blocking the c-kit signaling, could also impair the survival of normal human mature MC whose generation from cord blood CD34-positive progenitors and survival in vitro are strictly dependent on SCF (80 ng/ml). In the presence of STI571 (1 mm), cell survival decreased significantly at

STI571 efficiency is dependent on the Kit mutation type found in mast cell Y Zermati et al

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Figure 1 STI57 l decreases cell proliferation of c-kit-dependent cell lines and survival of human normal mast cells (MC), but does not alter the survival of MC from bone marrow aspirates of patients with mastocytosis. (a) Increasing concentrations of STI571 (0–10 mm) (kindly provided by E Buchdunger (Novartis Pharma, Basel, Switzerland)) were added for 72 h to UT-7 cells (Hermine et al., 1992) (transfected with Bcr-Abl (UT-7-bcr-abl) (Issaad et al., 2000) cultured without growth factor (’), or to UT-7 cells cultured in the presence of Epo (2 U/ml) (UT-7/Epo) (O) or of SCF (100 ng/ml) (UT-7/SCF) () in RPMI medium (Gibco, BRL) supplemented with 1% glutamine and 1% penni-streptomycin. Cell proliferation was assessed by [3H]thymidine incorporation (left panel). Results are shown in percentage and 100% is the proliferation rate obtained in the absence of STI571. Percentage of apoptotic cells was analysed by flow cytometry after annexin V/PI staining (right panel) as previously described (Zermati et al., 2000). Each point represents the mean value from triplicate observations. (b) Highly purified (95%) normal MCs were obtained by long-term culture (>8 weeks) in serum-free medium (Stempro, Stem Cell Technology, France) in the presence of 80 ng/ml human recombinant SCF (kindly donated by Amgen, Thousand Oaks, CA, USA) and 50 ng/ml human recombinant IL-6 (a kind gift of Novartis Biotechnology, Basel, Switzerland) of CD34-positive cells purified by the use of an immunomagnetic affinity column (MACS, Milteny Biotech, France) from cord blood mononuclear cells. MCs were cultured for 72 h in the presence of SCF (100 ng/ml) with increasing doses of STI571 as indicated. Percentage of viable cells was estimated after blue trypan staining. (c) Clinical characteristics of the patients tested. Male (M), female (F). Skin (s), bone marrow (bm), bone (b), liver (l), gastrointestinal tract (gi). Normal tryptase level o15 mg/l. c-kit Mutations were detected by PleI (New England Biolabs, France) digestion or sequencing with an automatic sequencer ABI 377 (Perkin-Elmer, USA) of the RT–PCR products (primer sense) 5-GGATGACGAGTTGGCCCTAGA, reverse primer 5-GTAGAACTTAGAATCGACCGGCA, 35 cycles: 941C, 30 s; 551C, 40 s; 721C, 40 s) obtained from patient bone marrow total RNA extracted with RNABle (Eurobio, France) from 105 to 4 106 cells. (d) Bone marrow cells obtained from six patients with SM were depleted with erythrocytes by NH4CL treatment and then cultured in serum-free medium (BIT, StemCell Laboratories, France) (0.5 106 cells/ml) in the presence of SCF (100 ng/ml) for 7 days with (’) or without (&) STI571 (1 mm). The percentage of MC (number MC/ total bone marrow cells) was estimated after May–Gru¨nwald–Giemsa staining by counting with an inverted microscope ( 40 magnification). Each point represents the mean value from triplicate observations

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Figure 2 Effect of STI571 on the growth of various leukemic MC lines and of Ba/F3 cell lines transfected with juxtamembrane and catalytic domain mutants of the c-kit receptor. Cell lines Ba/F3 kit, Ba/F3 kit G559 (V559G substitution), Ba/F3 kit 814 (D814 V), Ba/ F3 human kit 816 (816 V) and IC2KITV814 (D814 V), are derived from the murine IL-3-dependent Ba/F3 proB lymphoid and IC2 mast cells, respectively, and transfected with wild-type murine Kit and various mutated forms of murine or human Kit as indicated (Tsujimura et al., 1999). The FMA3 and P815 cell lines are mastocytoma cells expressing endogenous mutated forms of Kit, that is, frame deletion in the murine juxtamembrane coding region of the receptor-codons 573 to 579 and D814 V substitution, respectively Tsujimura et al., 1996a; Tsujimura et al., 1996b. The HMC-1 line expresses both mutations JM-V560G and CP-D816 V (Butterfield et al., 1988). (a) Proliferation of leukemic MC lines analysed by [3H]thymidine incorporation. Cells (1 104) were cultured without growth factor. Cell proliferation was assessed in the presence of increasing concentration of STI571 (0.1–10 mm). Results are shown in percentage where 100% is the proliferation obtained without addition of the drug. Data represent the averages of triplicates (mean7s.d.) and the result of a representative experiment is shown. (b) Anti-phosphotyrosine blots of immunoprecipitated mutated ckit receptors from FMA-3 and P815 cells. Phosphorylation status of receptors was analysed in the presence of increasing concentration of STI571 (0.1–10 mm). The upper panel shows the immunoblot with anti-PY. Blot represented in the upper panel was stripped and reprobed with rabbit anti-Kit immune-serum to illustrate the total c-kit proteins (medium panel), c-kit Receptors are indicated by arrows. Total cell lysates were probed with anti-GRB2 antibody to illustrate the amount of total proteins in the lysates (lower panel). Size markers are shown in kilodaltons. (c) Cell proliferation analysed by [3H]thymidine incorporation. Unstimulated or SCF-stimulated Ba/F3Kit or Ba/F3-derived cells (1 104) were cultured in the presence of increasing concentration of STI571 (0.1–10 mm). The procedure was the same as above in (a). (d) Antiphosphotyrosine blots of immunoprecipitated wild-type c-kit and mutated c-kit receptors from Ba/F3 and Ba/F3-derived cells. Lysates from unstimulated 5 106 Ba/F3 c-kit and Ba/F3-derived cells cultured in the presence of increasing concentration of STI571 (0.1–10 mm) were immunoprecipitated with a rabbit immune-serum directed against the murine c-Kit cytoplasmic domain. Immunoprecipitated proteins were run on SDS–polyacrylamide gel electrophoresis and immunobloted either with the 4G10 antiphosphotyrosine monoclonal antibody or with the rabbit immune-serum anti-murine c-Kit as described earlier (Beslu et al., 1996). Panel descriptions are the same as in (b)

72 h (70% inhibition, IC50 0.1–1 mm) (Figure 1b). These results suggest that STI571 inhibited the survival of human MC and thus could be a good candidate for inhibiting the abnormal accumulation of MC in human SM. For this reason, we have investigated the effect of STI571 on in vitro survival of tumoral MC from patients with SM that was diagnosed based on clinical and biological features, and on bone marrow and/or skin biopsies (Valent et al., 2001; Horny and Valent, 2001). Oncogene

The characteristics of the patients are summarized in Figure 1c. In all cases, bone marrow samples were significantly infiltrated by tumoral MC and the c-kit point mutation D816 V was present in RNA extracted from the unfractionated bone marrow cells (data not shown). At pharmacological concentrations of 0.1–1 mm, STI571 exerted a moderate overall inhibitory effect (o15% of inhibition) on bone marrow cells survival. In contrast, the absolute number of tumoral MCs remained


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stable after 7 days of culture under this treatment (Figure 1d). Thus, in contrast to normal MC, tumoral MCs were insensitive to the inhibitory activity of STI571. This result suggested that, in vivo, tumoral MC either developed secondary genetic events rendering them c-kit mutation independent, or that mutated c-kit kinase activity was not inhibited by STI571. To further explore this mechanism of resistance, we then tested the effect of STI571 on the human and murine leukemic MC lines HMC-1, IC2KITV814, P815 and FMA-3 (Figure 2a). STI571 exhibited an inhibitory effect on the proliferation of the FMA-3 cell line, but not of the three other cell lines (IC50 0.1 mm) (Figure 2a). Interestingly, this effect was correlated with the inhibition of ckit phosphorylation, which was observed only in the FMA-3 cells, but not in P815 (Figure 2b), HMC-1 and IC2KITV814 (data not shown). All the STI571-resistant MC lines or tumoral MC bear the D816 V mutation or the equivalent murine D814 V mutations (Figures 1d and 2a and data not shown). In contrast, the STI571-sensitive FMA-3 cell line possesses a juxtamembrane mutation of c-kit (Tsujimura et al., 1996a, b) This finding suggested that different effects of STI571 might be related to the type of c-kit mutation that is present. To prove this theory, we investigated the effect of STI571 on IL-3-dependent Ba/ F3 cell lines transfected with wild-type c-kit, murine CPD814 V or human CP-D816 V, or JM-G559 c-kit mutants. STI571 exerted contrasting effects depending on the type of c-kit receptor mutation. Cell proliferation of both wild-type (upon SCF stimulation at 80 ng/ml) and juxtamembrane mutant c-kit–transfected cell lines was inhibited with pharmacological doses of STI571 (IC50 0.1–1 mm) (Figure 2c). This effect was reversed in the presence of IL-3, ruling out a nonspecific toxicity of STI571. In contrast, no inhibition was observed in the Ba/F3 cells transfected with the CP-D814 V or CPD816 V c-kit mutants (Figure 2c). As shown in Figure 2d, the effects of STI571 on cell proliferation were correlated with its effect on c-kit phosphorylation. Indeed, at the pharmacological concentration of 1 mm, phosphorylation of wild-type (upon SCF stimulation) and juxtamembrane mutant c-kit receptors (G559) were inhibited by STI571, whereas distal mutants D814 V and D816 V were not, even at a higher dose of 10 mm of STI571.

In this report, we demonstrate that STI571 exerts an inhibitory effect on the kinase activity of only wild-type and juxtamembrane c-kit mutants, but not on catalytic domain mutant. Since in human SM virtually all mutations of c-kit are found in the catalytic domain (Longley et al., 1999), STI571 is unlikely to be effective in vivo to treat SM. In contrast, STI571 could be used in some cases of mastocytosis and particularly in urticaria pigmentosum without systemic involvement which, in most cases, do not exhibit c-kit mutations (Longley et al., 1999). Therefore, a molecular classification of SM based on the group of c-kit mutations may be more appropriate to treat patients if a kinase inhibitor strategy is used (Longley and Metcalfe 2000). Furthermore, our findings explain at the molecular level the efficacy of STI571 in gastrointestinal stromal cell tumors that exhibit juxtamembrane c-kit mutations (Joensuu et al., 2001; Tuveson et al., 2001). The understanding of the molecular mechanisms of the resistance of tumoral MC to STIS71 underlines the need to design new kinase inhibitors. These inhibitors should block constitutive kinase activity caused by mutations in the c-kit catalytic domain, particularly in SM and in other hematological malignancies such as acute myeloid leukemia (AML) (Beghini et al., 2000) and NK lymphoma (Hongyo et al., 2000). In addition, our findings may have important implications for the rational design and screening of new kinase inhibitors, and might be applicable to other malignancies in which various classes of constitutively activating mutations of kinase receptors are found such as those recently described in AML with mutant forms of Flt-3 (Kottaridis et al., 2001; Yamamoto et al., 2001). Acknowledgements This work was supported by grants from the Association Française pour les Initiatives de Recherche sur le Mastocyte et les Mastocytoses (AFIRMM), the Association pour la Recherche sur le Cancer (ARC) and the Ligue Nationale Contre le Cancer (LNCC). We are indebted to Dr A Turhan (IGR, Villejuif, France) who kindly provided the UT-7-bcr-abl cell line, to Dr Y Kanakura (Tokyo, Japan) who kindly provided FMA-3, Dr K Hashimoto for P815, Ba/F3 Kit G559 and IC2KitV814 cell lines, and to Dr Valent for kindly providing the HMCI cell line (Vienna, Austria). We thank A Tu and D Chez for their help in editing this manuscript.

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