CD30 as a Novel Target in Advanced Mastocytosis

Aggressive mast cell disorders are incurable neoplasms with a poor prognosis and a short survival time. In most instances, patients are resistant to conventional cytostatic drugs. The activating KIT mutation D816V is detectable in most patients, and is considered essential for disease-evolution. However, the KIT D816V mutant is also detectable in patients with indolent mastocytosis who have a normal or near normal life expectancy. Moreover, it has been reported that KIT D816V per se is not a fully transforming oncoprotein, and that KIT D816V-targeting drugs alone are unable to produce long-lasting remissions in patients with aggressive mast cell disease. These observations suggest that other, KIT-independent, pro-oncogenic pathways and molecules play a role in mast cell disease-progression. We have recently shown that neoplastic mast cells in aggressive mastocytosis express CD30, a molecule that is otherwise expressed in (viral) transformed advanced lymphomas, Hodgkins Disease, and few other rare tumors, but is usually not expressed in other myeloid neoplasms. These observations suggest that CD30 is associated with, or even triggers, malignant transformation in systemic mastocytosis. We have recently established neoplastic mast cell lines through lentiviral-mediated gene transfer, and found that several of these mast cell lines also express CD30. Moreover, we have co-transfected KIT D816V into these lines and established a xenotransplant model using NSG mice to analyze the repopulation-capacity of primary patient-derived neoplastic stem cells and of established CD30+ mast cell lines. In the current project the hypothesis that CD30 plays an essential role in disease evolution and/or progression and/or represents a potential therapeutic target in mastocytosis, will be tested. Specifically, we will try to identify factors regulating the expression of CD30 in neoplastic mast cells, define the functional role of this surface antigen, and ask whether targeting of CD30 by iRNA or by specific pharmacologic compounds results in growth inhibition and apoptosis. The project will employ human and canine mast cell tumor models as well as human progenitor cell lines with stable or inducible expression of CD30 and/or KIT D816V. In addition, primary neoplastic mast cells and CD34+/CD38- stem cells, derived from patients with indolent and aggressive mastocytosis, will be employed to carry out the project. It can be expected, that results obtained in this project will significantly improve our knowledge about the pathogenesis of mast cell neoplasms, about factors and defects underlying disease-progression, and about possibilities to interfere with malignant cell growth through disruption of critical targets and target pathways in neoplastic cells. Such novel information should also lead to improved diagnosis of advanced mast cell neoplasms and could provide the basis for the development of novel pharmacological concepts with the ultimate aim to improved therapy in advanced mast cell disorders.

Advanced systemic mastocytosis (SM), including aggressive SM (ASM) and mast cell leukemia (MCL) is a myeloid neoplasm with poor prognosis. Neoplastic mast cells (MC) in ASM and MCL usually express the activating KIT mutation D816V. However, this mutant is also detectable in patients with indolent SM (ISM) who have a normal life expectancy. These observations suggest that other, KIT-independent pathways play a role in disease progression in advanced SM. Recently, we were able to show that neoplastic MC in SM aberrantly express the Ki-1 antigen (CD30). CD30 is a molecule that is otherwise only expressed in Hodgkins lymphoma and a few other rare tumors but is usually not expressed in other myeloid neoplasms. Therefore, our hypothesis was that CD30 is associated with, or even triggers, malignant transformation in systemic mastocytosis. The aim of the present project was to explore whether CD30 can serve as a therapeutic target in ASM and MCL. As assessed by flow cytometry, CD30 was found to be expressed on neoplastic human MC in 3 of 25 patients (12%) with ISM, 4 of 7 (57%) with ASM, and 4 of 7 (57%) with MCL. CD30 was also detectable on the surface of the HMC-1.1 and MCPV-1.1 human MC lines and the canine MC cell lines NI-1 and C2. In a next step we confirmed CD30 expression at the mRNA level by qPCR in CD30+ HMC-1.1 cells, MCPV-1.1 cells, NI-1 cells, and C2 cells as well as in primary CD30+ neoplastic MC. When examining the regulation of CD30 in neoplastic MC, we were able to show that the MEK inhibitors PD032509 and RDEA119 downregulate expression of cell surface CD30 and CD30 mRNA levels in HMC-1.1 cells and MCPV-1.1 cells. In a next step we were interested to learn whether targeting of CD30 by specific pharmacologic compounds results in growth inhibition of CD30+ neoplastic MC. Brentuximab-vedotin, a CD30 antibody-drug conjugate was found to inhibit the proliferation and to induce apoptosis in CD30+ human and canine neoplastic MC lines and primary neoplastic MC obtained from patients with ASM. We also examined the effects of brentuximab-vedotin on engraftment of MCPV-1.1 cells in NSG mice. Preincubation of MCPV-1.1 cells with brentuximab-vedotin resulted in a slightly reduced in vivo engraftment in NSG mice. Next, we examined the effects of a combination of Brentuximab-vedotin and the KIT-targeting drugs PKC412 and masitinib. Both combinations produced synergistic growth inhibition in human and canine CD30+ neoplastic MC lines. All in all, our data suggest that CD30 may serve as a novel therapeutic target in SM. However, drug combinations may be required to exert major and long-lasting growth-inhibitory effects on neoplastic cells in these patients.