KIT D816V-induced Cytokines in Systemic Mastocytosis

Systemic mastocytosis (SM) is a stem cell disease characterized by accumulation of mast cell progenitors and the presence of the KIT D816V point mutation. A pathologic key feature in SM is the altered bone marrow (BM) microarchitecture with increased angiogenesis, sometimes BM fibrosis, thickening of bony trabeculae, or/and osteosclerosis. So far, little is known about microenvironment modulating cytokines in SM and the role the KIT D816V oncoprotein might play in their expression. Moreover, the role of KIT D816V-dependent cytokines as autocrine or intracrine growth regulators in neoplastic (stem) cells has not been investigated yet. We have recently found that KIT D816V strongly promotes the expression of oncostatin M (OSM), a cytokine modulating growth of BM fibroblasts and endothelial cells as well as maturation and survival of hematopoietic progenitors. The aims of this project are a) to study the effects of KIT D816V on expression of BM microenvironment- modulating cytokines, b) to characterize underlying signal transduction pathways, and c) to investigate the functional significance of these mediators for growth of neoplastic mast cells and stem cell-niche interactions. To address these aims, lentiviral-mediated expression of KIT D816V in human growth factor-dependent cell lines (TF-1 and Mo7e) and primary CD34+ cells has been established in our lab. In addition, Ba/F3 cell lines with doxycycline-inducible expression of KIT D816V have been generated and will be used in this project. Data obtained in these experiments will be confirmed in primary neoplastic cells and neoplastic CD34+/CD38-/Lin- stem cells. The functional role of KIT D816V-dependent cytokines will be investigated using cultured endothelial cells and BM fibroblasts. To investigate the role of cytokines in growth of leukemic (stem) cells, a combination of overexpression and knockdown studies using lentiviral-mediated gene delivery will be applied. In vivo data will be generated in NSG-hu-SCFm mice xenotransplanted with human CD34+/CD38-/Lin- stem cells and in BM transplantation experiments after lentiviral transduction of mouse BM with KIT D816V and/or shRNAs targeting KIT D816V-dependent cytokines. Expression of cytokines in the BM of patients will be correlated with clinical and histopathological features, follow-up parameters, and with prognosis. Results obtained from the current project are expected to contribute to our knowledge about KIT D816V- dependent expression of angiogenic factors and the role these cytokines play in BM remodeling, stem cell-niche interactions, and growth of neoplastic (stem) cells. In addition, new molecular targets will be identified in the signaling- and cytokine networks underlying angiogenesis in the BM, which should provide a valuable basis for the development of new treatment concepts.

Systemic mastocytosis is an incurable orphan disease with an incidence of approximately one in 10 000. Like leukaemia, mastocytosis is a hematologic neoplasm affecting the bone marrow and also other organs including intestine, liver and spleen by infiltration with malignant mast cells. The majority of patients harbours the somatic gene mutation KIT D816V. The clinical course of mastocytosis is highly variable with an average life expectancy of five years in aggressive forms of systemic mastocytosis. Within the FWF funded project, cytokines have been identified as major factors for disease progression. Cytokines affect the microenvironment in the bone marrow and promote the formation of new vessels and the pathologic propagation of connective tissue. This complex interplay of neoplastic mast cells and cells of the bone marrow stroma substantially contributes to the progression of the disease to aggressive forms. In particular, the cytokine CCL-2 is increased in the serum of patients with systemic mastocytosis. CCL-2 is a major mediator of inflammation and promotes the formation of vessels in solid tumours. Analysis of clinical data indicated a significant correlation of high CCL-2 serum level with advanced disease and shorter overall survival. Thus, measurement of cytokine levels improves the individual assessment of prognosis for patients with mastocytosis. In addition, novel methods for detection and quantification of the KIT D816V mutation burden in blood and bone morrow samples have been developed within the project. This optimized molecular diagnostic methods also contribute to the improved estimation of prognosis and are crucial for monitoring of response to therapy in systemic mastocytosis.Apart from theses diagnostic applications, results of the project have also promising implications for novel therapeutic approaches in mastocytosis. Thus, we have found that CCL-2 was essential for proliferation of mast cell tumours and targeting of CCL-2 showed efficacy both in vitro and in vivo. In particular, neutralizing antibodies against CCL-2 are currently evaluated in clinical trials for patients with breast or prostate cancer in whom elevated levels of CCL-2 had also been described. Our data indicate that this approach might also be effective in systemic mastocytosis and suggest targeting of CCL-2 as novel therapeutic strategy to be evaluated in combination with established treatment regiments.