Novel Targets in Leukemic Stem Cells in SM-AHN

Advanced mast cell neoplasms are complex diseases in which mast cells expand in number in an uncontrolled manner. Despite novel, better therapies, the prognosis in these patients remains poor. Whereas most patients have the D816V mutation in the KIT gene, which results in uncontrolled growth of neoplastic mast cells, there are also mutations in other genes that are critically involved in disease evolution and progression in these patients. These additional mutations are more often present when the patients is suffering from an additional associated, hematologic (non-mast cell) neoplasm (AHN). Both, mastocytosis, and mastocytosis-associated AHN, are derived from neoplastic stem cells (NSC). In the past few years, we have identified and characterized these NSC in patients with aggressive systemic mastocytosis with or without an AHN as well as in patients with mast cell leukemia, the deadliest form of the disease. We have also shown that NSC in all these advanced mast cell neoplasms reside in a cell fraction defined by expression of CD34 and lack of CD38. In patients with AHN, AHN-specific NSC may be identified. With regard to therapy, the problem is that due the many NSC populations and molecular drivers involved, the disease cannot be brought under control using only a single therapeutic target. Finding suitable targets in all relevant NSC subsets in patients with advanced mastocytosis, including those who suffer from an AHN, would enable development of more effective targeted therapies that might even eliminate most or all disease-propagating NSC without harming normal stem cells. This could lead to more effective therapies exhibiting less drug-induced side effects. It may even lead to cure by eliminating all NSC subsets, including AHN-specific NSC which are often a source of relapsing disease. The objective of this study is to characterize AHN-related NSC and to identify possible target profiles and mechanisms of drug resistance in these cells. To achieve our objective, we will apply genetic analyses such as single cell DNA sequencing, RNA sequencing, and clonogenic assays. In addition, once we have identified suitable targets expressed in NSC, we will explore the effects of various targeted drugs and drug combinations on growth of neoplastic cells in cell cultures and in a xenotransplantation mouse model. We will also employ stem cell lines to confirm individual targets and to test the efficacy of NSC-targeting drugs. Target validation will be performed via gene knockout experiments using shRNA or CRISPR-Cas9. Our project should lead to a better understanding of target expression profiles in NSC in advanced mast cell diseases and associated hematologic neoplasms. In addition, uncovering factors underlying NSC resistance, and possibilities to interfere with relevant oncogenic machineries in these cells, will provide a useful basis for development of novel NSC-eradicating, and thereby curative, therapies.