Lyn and Btk as novel therapeutic targets in ASM

Aggressive mast cell disorders are incurable neoplasms that usually are resistant against conventional antineoplastic drugs and have an unfavorable prognosis and a short survival. In most cases, activating mutations in the KIT proto-oncogene are detectable and are considered essential for disease-evolution. The KIT mutant D816V is most commonly found. However, this mutant and other KIT mutants are also detectable in patients with indolent mastocytosis who have a normal or near normal life expectancy. More recently 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 remission in patients with aggressive mast cell neoplasms. These observations suggest that other KIT-independent pro-oncogenic pathways and molecules may play a role in mast cell disease-progression. We have recently shown that neoplastic mast cells in aggressive mastocytosis display phosphorylated (p)Lyn and pBtk, and that the KIT D816V-targeting drug PKC412 downregulates the activation of KIT and of downstream signaling molecules without blocking Lyn- or Btk activation. Our preliminary data also suggest that a Btk-specific siRNA counteracts growth and survival of neoplastic mast cells. Based on these observations, it is tempting to speculate that the Lyn/Btk pathway of signaling and its downstream effector molecules play a role in disease- progression in mastocytosis. In the current project the hypothesis that the Lyn/Btk pathway plays an essential role in disease evolution/progression and thus represents a novel KIT-independent therapeutic target in mastocytosis, will be tested. The project will employ human and canine mast cell tumor models as well as Ba/F3 cells and human progenitor cells with inducible expression of KIT D816V. These cell line models have recently been established in the lab of the applicant. siRNAs as well as targeted drugs blocking Btk and/or Lyn (dasatinib, INNO-406, LFM- A13) will be used to establish a functional role for each target. Further aims of the project are to identify Lyn/Btk upstream target-molecules and to combine KIT-targeting and Lyn/Btk-targeting drugs to achieve synergistic effects on growth of neoplastic cells in advanced mast cell neoplasms. These experiments will be performed with HMC-1 cells expressing or lacking KIT D816V as well as primary neoplastic mast cells. To study a variety of different KIT mutants in primary cells and to establish an in vivo model, studies will also include neoplastic mast cells obtained from canine patients, where mast cell neoplasms are one of the most frequent tumors. It can be expected, that the 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 signaling pathways in neoplastic cells. Such novel information should ultimately lead to improved diagnosis of advanced mast cell neoplasms and to the development of novel more effective treatment concepts.

Systemic mastocytosis is a disease characterized by abnormal growth and accumulation of neoplastic mast cells in the skin and internal organs. The disease either behaves as a stable non-aggressive condition or as an aggressive tumor, sometimes even as mast cell leukemia. In all disease variants, neoplastic mast cells harbor the tumor-inducing mutation D816V in the KIT receptor gene. So far, however, little is known about additional factors and molecular lesions responsible for tumor progression or leukemic transformation. We have recently identified two KIT D816V-independent tumor-driving molecules in neoplastic mast cells, namely Lyn and Btk. These two molecules were found to be activated in mast cells in most patients with aggressive mastocytosis or mast cell leukemia, whereas in patients with non- aggressive disease, Lyn and Btk are usually expressed as non-activated molecules. During the course of the project, we were able to show that Lyn and Btk actively trigger growth and survival of neoplastic mast cells in advanced mastocytosis, and that those drugs that can de- activate one or both of these molecules, can also block the growth of neoplastic mast cells in these patients. Moreover, we found that KIT-blocking drugs and Lyn/Btk-blocking drugs act together to inhibit the growth of neoplastic mast cells in advanced mastocytosis. In a next step, we asked whether neoplastic mast cells also express other KIT-independent molecular drug targets. In these screening experiments, we found that neoplastic mast cells in man and dogs frequently express cell surface receptors that can serve as targets of antibody-based therapy, such as CD25, CD30, or CD52. However, these surface molecules are not regulated by Lyn or Btk. We also asked whether Lyn and Btk are expressed in abnormal (mutated) form in neoplastic mast cells in advanced mastocytosis. However, no activating mutations and no obvious structural defect of Lyn or Btk was found. In a next step, we asked whether neoplastic mast cells express activating mutations in other genes that would explain secondary activation of Lyn or Btk. However, no activating mutations in any potentially relevant primary genes examined were found. Finally, we asked whether neoplastic mast cells express a viral gene that would explain activation of Lyn and/or Btk. However, again, no viral antigens (EBV, CMV, HPV, HCV) were identified in neoplastic mast cells in advanced mastocytosis. We next asked whether growth factors, like IL-3, IL-4, IL-9, IL-10, or IL-13 can induce activation of Lyn or Btk in neoplastic mast cells, but no activating effects were seen. Together, activated Lyn and Btk are interesting new targets in aggressive mastocytosis and mast cell leukemia. Clinically, the most important observation was that carefully selected combinations of targeted drugs might be very helpful to better attack and finally eliminate neoplastic mast cells in these patients. This observation may have clinical implications and may lead to the preparation of clinical trials, which is an important issue, as aggressive mastocytosis and mast cell leukemia are incurable cancers with short survival times in humans and dogs.