Studies on the normal and oncogenic signaling networks from the hematopoietic growth factor receptors c-Kit and Flt3

Sammanfattning: The closely related receptor tyrosine kinases c-Kit and Flt3 are mainly expressed in hematopoietic cells and are involved in driving cell proliferation and promoting survival in response to growth factor stimulation. Several gain-of-function mutations of both c-Kit and Flt3 have been found in malignancies like acute myeloid leukemia, gastrointestinal stroma tumors and small cell lung carcinomas, and the constitutively activated receptors are very potent oncogenes. We have studied the detailed signal transduction pathways downstream of c-Kit and Flt3, in order to understand the exact mechanism by which processes such as cell growth and differentiation are regulated. We looked into the negative regulation of RTKs by focusing on the E3 ubiquitin ligase Cbl and its interaction with c-Kit, and found that this interaction was dependent on Src family kinases to target the receptor for lysosomal degradation. Src is found to be involved in the development of a variety of tumors and this mechanism is crucial for a balanced signaling. In Flt3, we identified several novel phosphorylation sites involved in Src binding and activation as well as the binding of the adaptor proteins Grb2/Gab2. Upon Y-to-F point mutations of these residues, we investigated the downstream signaling pathways affected and the biological outcomes of this. Moreover, we compared phosphorylation and signaling patterns between the wild-type and the mutated versions of Flt3. The most common mutation in Flt3, an internal tandem duplication (ITD) in the juxtamembrane region of the receptor, is found in about 30% of patients with AML and is correlated to a worse prognosis. The ITD receptor activates the downstream signaling protein Stat5, an important mediator of survival, whereas the wild-type Flt3 does not. We found that this activation of Stat5 to some extent is induced by the function of Gab2 and its recruitment to the Flt3 receptor, and that this as an important pathway for cell viability and proliferation. By using phospho-specific Flt3 antibodies produced in-house, we compared the site-specific phosphorylation and kinetics of wild-type Flt3 with the two most common mutants in AML; D853Y and the ITD. We found both quantitative and qualitative differences, indicating that the oncogenic receptors act through specific signaling pathways, which provides potential therapeutic targets.