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A new humanized in vivo model of KIT D816V+ advanced systemic mastocytosis monitored using a secreted luciferase
Systemic mastocytosis are rare neoplasms characterized by accumulation of mast cells in at least one internal organ. The majority of systemic mastocytosis patients carry KIT D816V mutation, which activates constitutively the KIT receptor. Patient with advanced forms of systemic mastocytosis, such as aggressive systemic mastocytosis or mast cell leukemia, are poorly treated to date. Unfortunately, the lack of in vivo models reflecting KIT D816V+ advanced disease hampers pathophysiological studies and preclinical development of new therapies for such patients. Here, we describe a new in vivo model of KIT D816V+ advanced systemic mastocytosis developed by transplantation of the human ROSAKIT D816V-Gluc mast cell line in NOD-SCID IL-2R gâ/â mice, using Gaussia princeps luciferase as a reporter. Intravenous injection of ROSAKIT D816V-Gluc cells led, in 4 weeks, to engraftment in all injected primary recipient mice. Engrafted cells were found at high levels in bone marrow, and at lower levels in spleen, liver and peripheral blood. Disease progression was easily monitored by repeated quantification of Gaussia princeps luciferase activity in peripheral blood. This quantification evidenced a linear relationship between the number of cells injected and the neoplastic mast cell burden in mice. Interestingly, the secondary transplantation of ROSAKIT D816V-Gluc cells increased their engraftment capability. To conclude, this new in vivo model mimics at the best the features of human KIT D816V+ advanced systemic mastocytosis. In addition, it is a unique and convenient tool to study the kinetics of the disease and the potential in vivo activity of new drugs targeting neoplastic mast cells
Identification of Bromodomain-Containing Protein 4 (BRD4) as a Novel Marker and Epigenetic Target in Systemic Mastocytosis and Mast Cell Leukemia
Les mastocytes humains (MC) sont des cellules tissulaires dâorigine hĂ©matopoĂŻĂ©tique impliquĂ©es dans une sĂ©rie de processus physiologiques et pathologiques. Les recherches sur les MC ont Ă©tĂ© entravĂ©es pendant longtemps en raison de l'accĂšs limitĂ© Ă des populations pures de ces cellules. Nous avons Ă©tabli une nouvelle lignĂ©e humaine de MC, ROSAKIT WT, dont les propriĂ©tĂ©s sont similaires Ă celles des MC primaires, constituant un nouvel outil pour la recherche sur les fonctions des MC humains, et permettant le criblage Ă haut dĂ©bit de thĂ©rapies anti-allergiques. Les MC sont impliquĂ©s dans les mastocytoses, oĂč ils sâaccumulent pathologiquement dans divers tissus. Bien que la plupart des cas de mastocytoses systĂ©miques (SM) sont chroniques et indolents, les patients atteints de SM avancĂ©e (SM agressive; ASM, et leucĂ©mie Ă mastocytes; MCL) ont un mauvais pronostic, car la plupart des thĂ©rapies disponibles ne sont pas curatives. Afin de mieux comprendre la physiopathologie des formes avancĂ©es de SM et pour trouver de nouvelles approches pour le traitement, nous avons profitĂ© de la disponibilitĂ© des cellules ROSAKIT WT pour Ă©tablir un nouveau sous-clone, la lignĂ©e cellulaire ROSAKIT D816V, reprĂ©sentant un Ă©quivalent des cellules nĂ©oplasiques sâaccumulant dans les SM. L'utilisation de cette lignĂ©e et de cellules des patients nous a permis dâidentifier BRD4 comme une nouvelle cible thĂ©rapeutique dans les ASM et les MCL. Nous avons dĂ©montrĂ© que les MC nĂ©oplasiques de patients avec ASM expriment des quantitĂ©s substantielles de BRD4. Fait intĂ©ressant, nous avons aussi dĂ©montrĂ© que les lignĂ©es cellulaires HMC-1 et ROSAKIT D816V expriment aussi BRD4, et que leur prolifĂ©ration est inhibĂ©e par un shRNA BRD4-spĂ©cifique. En outre, nous avons montrĂ© que le mĂ©dicament JQ1, inhibiteur de BRD4, induit une inhibition de la croissance et une apoptose dose-dĂ©pendante dans les mĂȘmes cellules. De plus, nous avons dĂ©montrĂ© que JQ1 supprime Ă©galement la prolifĂ©ration des MC nĂ©oplasiques primaires de patients atteints dâASM ou de MCL Ă de faibles concentrations. Enfin, nous avons observĂ© que la midostaurine (PKC412) et lâacide rĂ©tinoĂŻque tout-trans (ATRA) coopĂšrent avec JQ1 pour induire des effets inhibiteurs synergiques sur lâinhibition de la survie des mĂȘmes cellules. En conclusion, nos rĂ©sultats reprĂ©sentent une avancĂ©e sur ce qui Ă©tait prĂ©cĂ©demment connu sur lâimplication de BRD4 dans les mastocytoses et nous ont permis d'identifier cette protĂ©ine comme cible thĂ©rapeutique prometteuse dans le traitement des formes avancĂ©es de SM.Human mast cells (MCs) are hematopoietic stem cell (HSC)-derived, tissue-resident, multifaceted cells involved in a myriad of physiological and pathological processes. Researches on MCs have been hampered for a long time, due to limited access to pure populations of these cells. We have established a new human MC line, ROSAKIT WT, whose properties are similar to those of primary HSC-derived MCs, providing a novel tool for research on human MC functions, and enabling the high-throughput screening of anti-allergic therapies. Among others, MCs are involved in a group of diseases termed mastocytosis, where they accumulate pathologically in various tissues. Although most cases of systemic mastocytosis (SM) are chronic with an indolent course, patients with advanced SM (aggressive SM; ASM, and mast cell leukemia; MCL) have a reduced life expectancy and a poor prognosis, since most of the therapies already available are not curative. In order to better understand the pathophysiology of advanced SM and to. find new approaches for treatment, we took advantage of the availability of the ROSAKIT WT cells to establish a new subclone, the ROSAKIT D816V cell line, representing a paradigm of the neoplastic cells accumulating in SMUsing these malignant cell line and patientsâ cells, we identified the epigenetic reader bromodomain-containing protein-4 (BRD4) as a novel drug target in ASM and MCL. Indeed, we demonstrated that neoplastic MCs from ASM patients expressed substantial amounts of BRD4. Interestingly, we then demonstrated that HMC-1 and ROSAKIT D816V cell lines express BRD4, and that their proliferation is inhibited by a BRD4-specific shRNA. Moreover, we showed that the BRD4-targeting drug JQ1 induced a dose-dependent growth inhibition and apoptosis in the same cells. In addition, we demonstrated that JQ1 suppressed also the proliferation of primary neoplastic MCs of patients with ASM or MCL at low concentrations. Finally, we reported that midostaurin (PKC412) and all-trans retinoic acid (ATRA) cooperated with JQ1 in producing synergistic inhibitory effects on the survival of HMC-1 and ROSA cells. Together, our data represent a significant advance over what was previously known on the involvement of BRD4 in mastocytosis and identify this epigenetic reader bromodomain-containing protein as a promising drug target in advanced S
Identification de BRD4 comme nouvelle cible thérapeutique dans le traitement des mastocytoses systémiques agressives (ASM) et des leucémies à mastocytes (MCL)
Human mast cells (MCs) are hematopoietic stem cell (HSC)-derived, tissue-resident, multifaceted cells involved in a myriad of physiological and pathological processes. Researches on MCs have been hampered for a long time, due to limited access to pure populations of these cells. We have established a new human MC line, ROSAKIT WT, whose properties are similar to those of primary HSC-derived MCs, providing a novel tool for research on human MC functions, and enabling the high-throughput screening of anti-allergic therapies. Among others, MCs are involved in a group of diseases termed mastocytosis, where they accumulate pathologically in various tissues. Although most cases of systemic mastocytosis (SM) are chronic with an indolent course, patients with advanced SM (aggressive SM; ASM, and mast cell leukemia; MCL) have a reduced life expectancy and a poor prognosis, since most of the therapies already available are not curative. In order to better understand the pathophysiology of advanced SM and to. find new approaches for treatment, we took advantage of the availability of the ROSAKIT WT cells to establish a new subclone, the ROSAKIT D816V cell line, representing a paradigm of the neoplastic cells accumulating in SMUsing these malignant cell line and patientsâ cells, we identified the epigenetic reader bromodomain-containing protein-4 (BRD4) as a novel drug target in ASM and MCL. Indeed, we demonstrated that neoplastic MCs from ASM patients expressed substantial amounts of BRD4. Interestingly, we then demonstrated that HMC-1 and ROSAKIT D816V cell lines express BRD4, and that their proliferation is inhibited by a BRD4-specific shRNA. Moreover, we showed that the BRD4-targeting drug JQ1 induced a dose-dependent growth inhibition and apoptosis in the same cells. In addition, we demonstrated that JQ1 suppressed also the proliferation of primary neoplastic MCs of patients with ASM or MCL at low concentrations. Finally, we reported that midostaurin (PKC412) and all-trans retinoic acid (ATRA) cooperated with JQ1 in producing synergistic inhibitory effects on the survival of HMC-1 and ROSA cells. Together, our data represent a significant advance over what was previously known on the involvement of BRD4 in mastocytosis and identify this epigenetic reader bromodomain-containing protein as a promising drug target in advanced SMLes mastocytes humains (MC) sont des cellules tissulaires dâorigine hĂ©matopoĂŻĂ©tique impliquĂ©es dans une sĂ©rie de processus physiologiques et pathologiques. Les recherches sur les MC ont Ă©tĂ© entravĂ©es pendant longtemps en raison de l'accĂšs limitĂ© Ă des populations pures de ces cellules. Nous avons Ă©tabli une nouvelle lignĂ©e humaine de MC, ROSAKIT WT, dont les propriĂ©tĂ©s sont similaires Ă celles des MC primaires, constituant un nouvel outil pour la recherche sur les fonctions des MC humains, et permettant le criblage Ă haut dĂ©bit de thĂ©rapies anti-allergiques. Les MC sont impliquĂ©s dans les mastocytoses, oĂč ils sâaccumulent pathologiquement dans divers tissus. Bien que la plupart des cas de mastocytoses systĂ©miques (SM) sont chroniques et indolents, les patients atteints de SM avancĂ©e (SM agressive; ASM, et leucĂ©mie Ă mastocytes; MCL) ont un mauvais pronostic, car la plupart des thĂ©rapies disponibles ne sont pas curatives. Afin de mieux comprendre la physiopathologie des formes avancĂ©es de SM et pour trouver de nouvelles approches pour le traitement, nous avons profitĂ© de la disponibilitĂ© des cellules ROSAKIT WT pour Ă©tablir un nouveau sous-clone, la lignĂ©e cellulaire ROSAKIT D816V, reprĂ©sentant un Ă©quivalent des cellules nĂ©oplasiques sâaccumulant dans les SM. L'utilisation de cette lignĂ©e et de cellules des patients nous a permis dâidentifier BRD4 comme une nouvelle cible thĂ©rapeutique dans les ASM et les MCL. Nous avons dĂ©montrĂ© que les MC nĂ©oplasiques de patients avec ASM expriment des quantitĂ©s substantielles de BRD4. Fait intĂ©ressant, nous avons aussi dĂ©montrĂ© que les lignĂ©es cellulaires HMC-1 et ROSAKIT D816V expriment aussi BRD4, et que leur prolifĂ©ration est inhibĂ©e par un shRNA BRD4-spĂ©cifique. En outre, nous avons montrĂ© que le mĂ©dicament JQ1, inhibiteur de BRD4, induit une inhibition de la croissance et une apoptose dose-dĂ©pendante dans les mĂȘmes cellules. De plus, nous avons dĂ©montrĂ© que JQ1 supprime Ă©galement la prolifĂ©ration des MC nĂ©oplasiques primaires de patients atteints dâASM ou de MCL Ă de faibles concentrations. Enfin, nous avons observĂ© que la midostaurine (PKC412) et lâacide rĂ©tinoĂŻque tout-trans (ATRA) coopĂšrent avec JQ1 pour induire des effets inhibiteurs synergiques sur lâinhibition de la survie des mĂȘmes cellules. En conclusion, nos rĂ©sultats reprĂ©sentent une avancĂ©e sur ce qui Ă©tait prĂ©cĂ©demment connu sur lâimplication de BRD4 dans les mastocytoses et nous ont permis d'identifier cette protĂ©ine comme cible thĂ©rapeutique prometteuse dans le traitement des formes avancĂ©es de SM
Preclinical human models and emerging therapeutics for advanced systemic mastocytosis
Mastocytosis is a term used to denote a group of rare diseases characterized by an abnormal accumulation of neoplastic mast cells in various tissues and organs. In most patients with systemic mastocytosis, the neoplastic cells carry activating mutations in; KIT; Progress in mastocytosis research has long been hindered by the lack of suitable; in vitro; models, such as permanent human mast cell lines. In fact, only a few human mast cell lines are available to date: HMC-1, LAD1/2, LUVA, ROSA and MCPV-1. The HMC-1 and LAD1/2 cell lines were derived from patients with mast cell leukemia. By contrast, the more recently established LUVA, ROSA and MCPV-1 cell lines were derived from CD34; +; cells of non-mastocytosis donors. While some of these cell lines (LAD1/2, LUVA, ROSA; KIT WT; and MCPV-1) do not harbor; KIT; mutations, HMC-1 and ROSA; KIT D816V; cells exhibit activating; KIT; mutations found in mastocytosis and have thus been used to study disease pathogenesis. In addition, these cell lines are increasingly employed to validate new therapeutic targets and to screen for effects of new targeted drugs. Recently, the ROSA; KIT D816V; subclone has been successfully used to generate a unique; in vivo; model of advanced mastocytosis by injection into immunocompromised mice. Such a model may allow; in vivo; validation of data obtained; in vitro; with targeted drugs directed against mastocytosis. In this review, we discuss the major characteristics of all available human mast cell lines, with particular emphasis on the use of HMC-1 and ROSA; KIT D816V; cells in preclinical therapeutic research in mastocytosis
In vitro and in vivo efficacy of an anti-CD203c conjugated antibody (AGS-16C3F) in mouse models of advanced systemic mastocytosis
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A new human mast cell line expressing a functional IgE receptor converts to tumorigenic growth by KIT D816V transfection
International audienceIn systemic mastocytosis (SM), clinical problems arise from factor-independent proliferation of mast cells (MCs) and the increased release of mediators by MCs, but no human cell line model for studying MC activation in the context of SM is available. We have created a stable stem cell factor (SCF)-dependent human MC line, ROSA(KIT WT), expressing a fully functional immunoglobulin E (IgE) receptor. Transfection with KIT D816V converted ROSA(KIT WT) cells into an SCF-independent clone, ROSA(KIT D816V), which produced a mastocytosis-like disease in NSG mice. Although several signaling pathways were activated, ROSA(KIT D816V) did not exhibit an increased, but did exhibit a decreased responsiveness to IgE-dependent stimuli. Moreover, NSG mice bearing ROSA(KIT D816V)-derived tumors did not show mediator-related symptoms, and KIT D816V-positive MCs obtained from patients with SM did not show increased IgE-dependent histamine release or CD63 upregulation. Our data show that KIT D816V is a disease-propagating oncoprotein, but it does not activate MCs to release proinflammatory mediators, which may explain why mediator-related symptoms in SM occur preferentially in the context of a coexisting allergy. ROSA(KIT D816V) may provide a valuable tool for studying the pathogenesis of mastocytosis and should facilitate the development of novel drugs for treating SM patients