Unique Effects of KIT D816V in BaF3 Cells: Induction of Cluster Formation, Histamine Synthesis, and Early Mast Cell Differentiation Antigens

Abstract

Oncogenic tyrosine kinases (TK) usually convert growth factor-dependent cells to factor independence with autonomous proliferation. However, TK-driven neoplasms often are indolent and characterized by cell differentiation rather than proliferation. A prototype of an indolent TK-driven neoplasm is indolent systemic mastocytosis. We found that the D816V-mutated variant of KIT, a TK detectable in most patients with systemic mastocytosis, induces cluster formation and expression of several mast cell differentiation and adhesion Ags, including microphthalmia transcription factor, IL-4 receptor, histamine, CD63, and ICAM-1 in IL-3-dependent BaF3 cells. By contrast, wild-type KIT did not induce cluster formation or mast cell differentiation Ags. Additionally, KIT D816V, but not wild-type KIT, induced STAT5 activation in BaF3 cells. However, despite these intriguing effects, KIT D816V did not convert BaF3 cells to factor-independent proliferation. Correspondingly, BaF3 cells with conditional expression of KIT D816V did not form tumors in nude mice. Together, the biologic effects of KIT D816V in BaF3 cells match strikingly with the clinical course of indolent systemic mastocytosis and with our recently established transgenic mouse model, in which KIT D816V induces indolent mast cell accumulations but usually does not induce a malignant mast cell disease. Based on all these results, it is hypothesized that KIT D816V as a single hit may be sufficient to cause indolent systemic mastocytosis, whereas additional defects may be required to induce aggressive mast cell disorders.

Identification of Oncostatin M as a STAT5-Dependent Mediator of Bone Marrow Remodeling in KIT D816V-Positive Systemic Mastocytosis

“…Of note, wild-type KIT (in the presence of SCF) did not activate STAT5 15 or induce expression of OSM in Ba/F3 cells (Figure 4). To investigate the effects of wild-type KIT and SCF on expression of OSM in greater detail, CD34 ϩ cord blood cells were analyzed.…”

“…15 Induction of KIT D816V by addition of doxycycline up-regulated OSM promoter activity as measured by a luciferase reporter assay, and increased OSM mRNA and OSM protein levels in Ba/F3 cells ( Figure 4, A-C). The effects of KIT D816V on expression of OSM were more pronounced in the absence of IL-3 (approximately 100-fold up-regulation; Figure 4B) than in the presence of IL-3 (approximately 10-fold upregulation; Figure 4D), which is best explained by the previously described stimulatory effect of IL-3 on expression of OSM in Ba/F3 cells.…”

“…Ba/F3 cells with inducible expression of KIT D816V (Ton.Kit.D816V) or wild-type KIT (Ton.Kit.wt) 15 were grown in RPMI 1640 medium with 10% FCS and IL-3 (1 ng/mL) at 37°C and 5% CO 2 . (Ba/F3 is a murine, IL-3-dependent progenitor cell line.)…”

“…14,15 To identify the signal transduction pathways in- Figure 5A). In contrast, piceatannol, a compound targeting multiple targets including STAT5, 44 inhibited expression of OSM mRNA and OSM protein levels in a dose-dependent manner in KIT D816V ϩ HMC-1 cells (Figure 5, A and B), as well as in Ba/F3 cells inducibly expressing KIT D816V ( Figure 5C).…”

“…14 -16 In particular, various signal transduction pathways including the phosphoinositide 3-kinase (PI3-kinase)/Akt pathway, the mitogen-activated protein kinases (MAP kinases), and signal transducer and activator of transcription 5 (STAT5) have been shown to be activated by KIT D816V. 14,15 Several observations suggest that STAT5 may play a particular role in growth and survival of normal and neoplastic mast cells. Specifically, STAT5-deficient mice have a profound defect in mast cell development.…”