Novel Diagnostic Markers and Targets in Activated Mast Cells

About 30% of the general population is suffering from clinical symptoms related to mast cell activation, either in the context of an IgE-dependent allergy, a primary mast cell disease (mastocytosis) or a mast cell activation syndrome (MCAS). However, little is known about the mechanisms of mast cell activation in MCAS and only a few diagnostic markers specific for MCAS have been identified to date. Moreover, effective therapies are lacking, especially for patients who are suffering from both, an IgE-dependent allergy and a primary mast cell disease. We have recently established diagnostic criteria for MCAS, thereby providing the basis for an in-depth analysis of markers, mechanisms and targets involved in these conditions. In addition, our group has identified several drugs that can interfere with mast cell activation in primary and secondary forms of MCAS. However, most of these drugs are anti-cancer agents that may produce substantial side effects. The aims of our project are to identify new specific markers and therapeutic targets in MCAS and to validate these markers and targets in patients with MCAS associated with IgE-dependent allergies, mastocytosis, or with both conditions. In a first step, the molecular mechanisms contributing to mast cell activation and mast cell expansion in mastocytosis and IgE-dependent allergies will be explored, with special focus on novel diagnostic markers and therapeutic targets. We will also explore critical target pathways and possibilities to interfere with mast cell growth and activation in these patients. Potential diagnostic markers will be validated by testing larger series of patient samples. Proteomics- based studies will reveal critical targets of drugs applied in cancer patients and should thereby assist in the selection and development of new more specific kinase blockers. One good example is the demonstration that BTK is a major dasatinib target mediating mast cell activation and that more specific and less toxic BTK blockers, like ibrutinib, can suppress histamine secretion and mast cell activation in vitro in the same way as dasatinib. Other drugs, like midostaurin, can inhibit both, the growth and activation of mast cells and therefore may be preferable drugs when patients are suffering from both, mastocytosis and severe mediator-related symptoms (primary/clonal MCAS). Again, however, midostaurin is an anti-cancer agent and may therefore not be suitable for all patients with a primary mast cell disease. Therefore, we will also examine the effects of drug combinations consisting of agents that are more specific and less toxic and can inhibit either mast cell activation or mast cell expansion in these patients. Drug partners will be selected based on specific target profiles and safety aspects, following the principles of personalized medicine. In vitro studies will be performed with several human cells line models established in our lab as well as with primary normal and neoplastic cells obtained from patients with mastocytosis and/or MCAS. The long-term aim of our project is to establish more effective, personalized, medicine approaches for patients with MCAS who also suffer from an underlying mastocytosis or an IgE-dependent allergy, or both.

Several different mechanisms work together to activate mast cells (MC) in patients with IgE-dependent allergies and those suffering from mastocytosis. In severe cases, a MC activation syndrome (MCAS) may be diagnosed. However, little is known about mechanisms and molecules triggering MC activation in these patients. The aims in the current project were to identify specific markers and mechanisms linked to MC activation in these disorders, and to develop diagnostic criteria and prognostic tools for MCAS. Additionally, the project aimed to explore new strategies to block MC activation and expansion using targeted drugs. During the course of our project, we were able to update and refine diagnostic criteria for MCAS together with an international (EU/US-based) expert consortium. In addition, we established a global classification of MC-related disorders, including variants of mastocytosis and of MCAS, as well as hereditary alpha tryptasemia (HT). IgE-dependent allergies were identified as most relevant co-morbidities triggering MC activation in patients with systemic mastocytosis (SM) and MCAS. To discover new, specific markers of MC and their activation, we analyzed the expression and function of various CD antigens on normal and neoplastic MC and basophils (BA). This led to the identification of several markers on MC and/or BA, including JAM1 (CD321), Tetherin (CD317), Siglec-6 (CD327), Siglec-7 (CD328), and CLEC4A (CD367). We also found that primary BA and skin MC express DPPIV (CD26), and that in patients with SM, MC express CD25, CD26, and CD123. Siglec-6 was identified as a cell-specific antigen that is highly expressed on normal and neoplastic BA and MC, but not on other myeloid cells in the blood or bone marrow, except for some monocytes. We also found that expression of Siglec-6 on BA and MC is influenced by cytokines. In fact, interleukin-3 (IL-3) was found to promote Siglec-6 expression on BA, and stem cell factor (SCF) increased the expression of Siglec-6 on tissue MC. Unexpectedly, IgE-dependent activation resulted in internalization of Siglec-6 in IL-3-primed BA whereas in tissue MC, IgE-dependent activation augmented SCF-induced upregulation of Siglec-6. Finally, we screened for drugs that can suppress MC growth and/or activation. We found that the HDAC inhibitors panobinostat and quisinostat are highly effective in suppressing growth of neoplastic MC (IC50: <10 nM). Both drugs also worked in combination with midostaurin and avapritinib to inhibit growth of neoplastic MC. Avapritinib was found to suppress growth and survival of various MC lines and primary neoplastic MC. In addition, avapritinib suppressed IgE-dependent histamine release in MC and BA obtained from patients with SM. The discoveries made in this project may pave the way for new therapeutic approaches to treat MC disorders and related conditions.