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, ROSAKIT WT and MCPV-1) do not harbor KIT mutations, HMC-1 and ROSAKIT 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 ROSAKIT 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 ROSAKIT D816V cells in preclinical therapeutic research in mastocytosis.

The KIT and PDGFRA switch-control inhibitor DCC-2618 blocks growth and survival of multiple neoplastic cell types in advanced mastocytosis.

Systemic mastocytosis is a complex disease defined by abnormal growth and accumulation of neoplastic mast cells in various organs. Most patients exhibit a D816V-mutated variant of KIT, which confers resistance against imatinib. Clinical problems in systemic mastocytosis arise from mediator-related symptoms and/or organ destruction caused by malignant expansion of neoplastic mast cells and/or other myeloid cells in various organ systems. DCC-2618 is a spectrum-selective pan KIT and PDGFRA inhibitor which blocks KIT D816V and multiple other kinase targets relevant to systemic mastocytosis. We found that DCC-2618 inhibits the proliferation and survival of various human mast cell lines (HMC-1, ROSA, MCPV-1) as well as primary neoplastic mast cells obtained from patients with advanced systemic mastocytosis (IC50 <1 µM). Moreover, DCC-2618 decreased growth and survival of primary neoplastic eosinophils obtained from patients with systemic mastocytosis or eosinophilic leukemia, leukemic monocytes obtained from patients with chronic myelomonocytic leukemia with or without concomitant systemic mastocytosis, and blast cells obtained from patients with acute myeloid leukemia. Furthermore, DCC-2618 was found to suppress the proliferation of endothelial cells, suggesting additional drug effects on systemic mastocytosis-related angiogenesis. Finally, DCC-2618 was found to downregulate IgE-mediated histamine release from basophils and tryptase release from mast cells. Together, DCC-2618 inhibits growth, survival and activation of multiple cell types relevant to advanced systemic mastocytosis. Whether DCC-2618 is effective in vivo in patients with advanced systemic mastocytosis is currently under investigation in clinical trials.

A new human mast cell line expressing a functional IgE receptor converts to tumorigenic growth by KIT D816V transfection.

In 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.

Co-operating STAT5 and AKT signaling pathways in chronic myeloid leukemia and mastocytosis: possible new targets of therapy.

Chronic myeloid leukemia and systemic mastocytosis are myeloid neoplasms sharing a number of pathogenetic and clinical features. In both conditions, an aberrantly activated oncoprotein with tyrosine kinase activity, namely BCR-ABL1 in chronic myeloid leukemia, and mutant KIT, mostly KIT D816V, in systemic mastocytosis, is key to disease evolution. The appreciation of the role of such tyrosine kinases in these diseases has led to the development of improved therapies with tyrosine kinase-targeted inhibitors. However, most drugs, including new KIT D816V-blocking agents, have failed to achieve long-lasting remissions in advanced systemic mastocytosis, and there is a similar problem in chronic myeloid leukemia, where imatinib-resistant patients sometimes fail to achieve remission, even with second- or third-line BCR-ABL1 specific tyrosine kinase inhibitors. During disease progression, additional signaling pathways become activated in neoplastic cells, but most converge into major downstream networks. Among these, the AKT and STAT5 pathways appear most critical and may result in drug-resistant chronic myeloid leukemia and systemic mastocytosis. Inhibition of phosphorylation of these targets has proven their crucial role in disease-evolution in both malignancies. Together, these observations suggest that STAT5 and AKT are key drivers of oncogenesis in drug-resistant forms of the diseases, and that targeting STAT5 and AKT might be an interesting approach in these malignancies. The present article provides an overview of our current knowledge about the critical role of AKT and STAT5 in the pathophysiology of chronic myeloid leukemia and systemic mastocytosis and on their potential value as therapeutic targets in these neoplasms.

Th1-Polarized, Dengue Virus-Activated Human Mast Cells Induce Endothelial Transcriptional Activation and Permeability.

Dengue virus (DENV), an arbovirus, strongly activates mast cells (MCs), which are key immune cells for pathogen immune surveillance. In animal models, MCs promote clearance of local peripheral DENV infections but, conversely, also promote pathological vascular leakage when widely activated during systemic DENV infection. Since DENV is a human pathogen, we sought to ascertain whether a similar phenomenon could occur in humans by characterizing the products released by human MCs (huMCs) upon direct (antibody-independent) DENV exposure, using the phenotypically mature huMC line, ROSA. DENV did not productively infect huMCs but prompted huMC release of proteases and eicosanoids and induced a Th1-polarized transcriptional profile. In co-culture and trans-well systems, huMC products activated human microvascular endothelial cells, involving transcription of vasoactive mediators and increased monolayer permeability. This permeability was blocked by MC-stabilizing drugs, or limited by drugs targeting certain MC products. Thus, MC stabilizers are a viable strategy to limit MC-promoted vascular leakage during DENV infection in humans.

In vitro and in vivo efficacy of an anti-CD203c conjugated antibody (AGS-16C3F) in mouse models of advanced systemic mastocytosis.

Antibody-drug conjugates (ADCs) are a new class of therapeutics that use antibodies to deliver potent cytotoxic drugs selectively to cancer cells. CD203c, an ecto-nucleotide pyrophosphatase-phosphodiesterase 3, is overexpressed on neoplastic mast cells (MCs) in systemic mastocytosis (SM), thus representing a promising target for antibody-mediated therapy. In this study, we have found that human neoplastic MC lines (ROSAKIT D816V and ROSAKIT D816V-Gluc), which express high levels of CD203c, are highly and specifically sensitive to the antiproliferative effects of an ADC against CD203c (AGS-16C3F). In these cell lines, AGS-16C3F induced cell apoptosis at very low concentrations. To characterize the effects of AGS-16C3F on leukemia progression in vivo, ROSAKIT D816V-Gluc NOD-SCID γ mouse models of advanced SM (AdvSM) were treated with AGS-16C3F or an ADC control for 2 weeks. Whereas AGS-16C3F had no apparent toxicity in xenotransplanted mice, in vivo neoplastic MC burden significantly decreased in both hematopoietic and nonhematopoietic organs. Furthermore, animals treated with AGS-16C3F had prolonged survival compared with the animals treated with control ADC, and AGS-16C3F efficiently prevented disease relapse. In conclusion, these preclinical studies identified CD203c as a novel therapeutic target on neoplastic MCs, and AGS-16C3F as a promising ADC for the treatment of patients with AdvSM.

Tyrosine Kinase Inhibition in Mastocytosis: KIT and Beyond KIT.

Mastocytosis is a group of rare disorders characterized by abnormal accumulation of mast cells in one or several organs. Mastocytosis can be seen at any age; but, in adults, the disease is usually systemic and chronic. Patients with indolent systemic mastocytosis (SM) are usually treated symptomatically, but cytoreductive treatments are needed in more advanced SM. In most patients with SM, an activating KIT D816V mutation is found. Thus, patients with advanced SM benefit from treatment with KIT-targeting tyrosine kinase inhibitors. However, none of these drugs are curative; new targeted drugs or combinations are still needed to improve patients' outcome.

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 γ-/- 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.

Molecular defects in mastocytosis: KIT and beyond KIT.

In all variants of mastocytosis, activating KIT mutations are frequently found. In adults, neoplastic mast cells (MCs) cells show the KIT mutation D816V, whereas in children, MCs invading the skin are frequently positive for non-KIT D816V mutations. The clinical course and prognosis of the disease vary among patients with systemic mastocytosis (SM). Additional KIT-independent molecular defects might cause progression. Additional oncogenic lesions have recently been identified in advanced SM. In advanced SM the presence of additional genetic lesions or altered signaling worsening the prognosis might lead to the use of alternative therapies such as combined antisignaling targeted treatments or stem cell transplantation.