Induced pluripotent stem cells with KIT D816V mutation for modeling myeloproliferative diseases

Induced pluripotent stem cells (iPS cells) represent a particularly appealing tool for modeling of human diseases in vitro, such as myeloproliferative neoplasms. Myeloproliferative diseases are chronic malignant diseases of the myeloid lineage and are associated with mutations in key signaling pathways. In mastocytosis patients a point mutation in the stem cell factor (SCF) receptor tyrosine kinase KIT (KIT D816V) leads to a constitutive active form of the receptor and confers resistance against the tyrosine kinase inhibitor imatinib (Gleevec/Glivec). Thus, KIT D816V patients in the terminal phase of disease are left without effective therapy, rendering this a fatal disease. Here we generated iPS cells from KIT D816V patients to investigate the mechanisms underlying their pathology and to develop models for screening compounds on a patient-specific background. Patient-derived KIT D816V iPS cells and unmutated iPS cells were differentiated into hematopoietic progenitor cells. The progenitor cells obtained showed characteristic in vitro surface marker expression. Additionally, human embryonic stem cells (ES cells) without and with KIT D816V mutation, introduced by CRISPR/Cas9n technology, were used as isogenic pairs. In compound screens KIT D816V iPS cell-derived and ES cell-derived hematopoietic progenitors showed sensitivity to the tyrosine kinase inhibitor nintedanib. A contrary effect of nintedanib on the viability of a well-established human mast cell line was observed. In addition, the efficacy of nintedanib on the KIT downstream signaling was differing between KIT D816V iPS cell-derived progenitors and the human mast cell line, demonstrating the need of a reliable model for this disease. The patient specific KIT D816V iPS cells and KIT D816V hematopoietic progenitors derived thereof, overcome the limitation of cell numbers from primary patient samples and were used for the development of novel therapeutic strategies. Patient-specific KIT D816V iPS cell clones can be used as a valuable in vitro model for further investigations of this pathology and might give new insights into the disease pathophysiology

Nintedanib Targets KIT D816V Neoplastic Cells Derived from Induced Pluripotent Stem cells of Systemic Mastocytosis

The KIT D816V mutation is found in more than 80% of patients with systemic mastocytosis (SM) and is key to neoplastic mast cell (MC) expansion and accumulation in affected organs. KIT D816V therefore represents a prime therapeutic target for SM. Here we generated a panel of patient-specific KIT D816V induced pluripotent stem cells (iPSCs) from patients with aggressive SM (ASM) and mast cell leukemia (MCL) to develop a patient-specific SM disease model for mechanistic and drug discovery studies. KIT D816V iPSCs differentiated into neoplastic hematopoietic progenitor cells and MCs with patient-specific phenotypic features, thereby reflecting the heterogeneity of the disease. CRISPR/Cas9n-engineered KIT D816V human embryonic stem cells (ESCs), when differentiated into hematopoietic cells, recapitulated the phenotype observed for KIT D816V iPSC hematopoiesis. KIT D816V causes constitutive activation of the KIT tyrosine kinase receptor and we exploited our iPSCs and ESCs to investigate new tyrosine kinase inhibitors targeting KIT D816V. Our study identified nintedanib as a novel KIT D816V inhibitor. Nintedanib selectively reduced the viability of iPSC-derived KIT D816V hematopoietic progenitor cells and MCs in the nanomolar range. Nintedanib was also active on primary samples of KIT D816V SM patients. Molecular docking studies show that nintedanib binds to the ATP binding pocket of inactive KIT D816V. Our results suggest nintedanib as a new drug candidate for KIT D816V targeted therapy of advanced SM