STAT3 activation is required for cell transformation and survival mediated by Asp⁸¹⁶ mutant c-Kit

Activating mutations of c-kit at codon 816 (Asp816) have been described in some forms of nuilignancies, including acute myeloid leukemia (AML), mastocytosis and germ cell tumors. We have identified Asp8 mutations of c-kit from a revenant of the cytokinedependent AML cell line, MO7e (D816H), and de novo childhood AML (D816N). Following transduction of the mutant c-kit, MO7e cells cause leukemia in a murine xenograft model and acquire cytokine-independent survival, as well as increased resistance to apoptosis in response to chemotherapeutic drugs. To determine the molecular mechanisms responsible for the functional effects mediated by Asp⁸¹⁶ mutations of c-kit, we evaluated the role of STAT (Signal Transducer and Activator of Transcription) activation in mutant c-Kit bearing cells. Constitutive activation of Stat 1 and StatS is observed in both leukemia cells that contain mutant c-Kit and the human embryonic kidney cell line, 293, when transfected with mutant c-kit. Transfection of dominant negative Stat3, but not Stall, inhibits anchorage-independent growth of 293 cells induced by D816H mutant c-Kit. Expression of dominant negative Stat3 also significantly suppresses cytokine independent survival of MO7e cells transduced with mutant c-kit cDNAs. By constructing and expressing the cDNA that encodes a constitutively activated Stat3, we demonstrate that activated Stat3 by itself is able to restore the mutant receptor's transforming ability in 293 cells and partially reconstitute cytokine independent survival in MO7e cells. Expression of constitutive activated Stat3 has no protective effect on MO7e cells from drug-induced apoptosis. These results indicate that activation of Stat3 by Asp⁸¹⁶ mutant c-Kit is sufficient for malignant transformation and is partially required for the survival and growth of leukemia cells. Other signal transduction pathways activated by mutant c-Kit may be involved in conferring drug resistance in leukemia cells.