Phosphatidylinositol 3'-kinase is required for growth of mast cells expressing the kit catalytic domain mutant.
The Kit receptor tyrosine kinase is critical for the growth and development of hematopoietic cells, germ cells, and the interstitial cells of Cajal. Gain-of-function mutations in codon 816 of the catalytic domain of human Kit [codon 814 of murine Kit (mKit)] are found in patients with mastocytosis, leukemia, and germ cell tumors. There are no drugs that inhibit the activity of Kit catalytic domain mutants to a greater extent than wild-type Kit. The objective of this study was to understand the biochemical mechanisms mediating mast cell transformation by this Kit mutant to identify molecular targets for pharmacological intervention. To this end, we examined signaling pathways activated in the murine mast cell line IC2 infected with either wild-type (IC2-mKit) or mutant mKit (IC2-mKit(D814Y)). In this study, we show that mKit(D814Y) is constitutively phosphorylated on tyrosine 719, and this likely results in constitutive association with activated phosphatidylinositol 3'-kinase (PI3K). In vitro growth of IC2-mKit(D814Y) cells is more sensitive to inhibition of PI3K than SCF-induced growth of IC2-mKit cells. s.c. injection of IC2-mKit(D814Y) in syngeneic mice results in mast cell tumors. To determine whether inhibition of PI3K could reduce mKit(D814Y)-mediated tumorigenicity, mice were treated with 1.5 mg/kg wortmannin three times a week. Five weeks after injection of tumor cells, a 75% reduction in tumor weight was observed when wortmannin treatments were initiated 2 days after inoculation with tumor cells. A 66% reduction occurred when treatment was initiated 2 weeks after inoculation. Treatment with wortmannin increased necrosis in the tumors, and this was associated with apoptosis. Interestingly, there was no effect on tumor vasculature. Thus, PI3K is required for survival and growth of the IC2-mKit(D814Y) mast cell line both in vitro and in vivo. These findings may provide insight into designing strategies for treatment of mastocytosis and other diseases associated with mutations in the Kit catalytic domain.
Shivakrupa
R
R
Basic Research Laboratory, Center for Cancer Research, Building 567, Room 226, National Cancer Institute-Frederick, Frederick, MD 21702, USA. krupas@ncifcrf.gov
Bernstein
Alan
A
Watring
Nicole
N
Linnekin
Diana
D
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002471:Cell Transformation, Neoplastic / Q000378:metabolism* / Q000473:pathology; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D008407:Mast Cells / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D012515:Mast-Cell Sarcoma / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D051379:Mice; D008811:Mice, Inbred DBA; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D019089:Stem Cell Factor / Q000494:pharmacology; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019009:Proto-Oncogene Proteins c-kit; D019089:Stem Cell Factor; D000077191:Wortmannin