The Src Related Tyrosine Kinase Lyn Mediates Proliferative Signals from Leukemia-Related Flt3 Mutants.

Abstract

Fms-like tyrosine kinase-3 (Flt3) is a hematopoietic growth factor receptor that regulates the survival and proliferation of myeloid and B-cell precursors. Mutations of Flt3 resulting in constitutive activation are the most common known molecular abnormalities in acute myeloid leukemia. Cells expressing activated Flt3 mutants show enhanced survival and proliferation, and leukemia-associated Flt3 mutations produced a fatal myeloproliferative disorder, in a mouse model (Kelly et al. Blood 2002). Nevertheless, the signaling pathways mediating Flt3 effects on proliferation are incompletely understood. Src kinases, such as Lyn, have also been linked to myeloproliferative disease, but the participation of Src-related kinases in Flt3 signaling has not been established. We therefore determined whether Src-family kinases were activated by Flt3 in human leukemic cell lines that express either wild-type or mutant Flt3. Flt3-ligand stimulation of THP1 cells, which express wild-type Flt3, significantly increased the phosphorylation of Lyn, the principal Src-family kinase found in these cells. In MV4;11 cells, which express a constitutively-active tandem duplication mutant of Flt3, Lyn phosphorylation is constitutively elevated. In both cases, a Lyn phosphorylation state specific antibody confirmed phosphorylation at tyrosine 397 of human Lyn; phosphorylation at this site is indicative of Lyn kinase activation. To determine whether the increase in Lyn phosphorylation seen in MV4;11 cells could be attributed specifically to effects of mutant Flt3, we examined Lyn phosphorylation in the murine leukemic Baf3 cells transduced with either wild-type or mutant human Flt3. Lyn phosphorylation and activation in Baf3 cells expressing the Flt3 mutant was significantly increased, compared to basal Lyn phosphorylation in Baf3 cells expressing wild-type Flt3 or vector alone. This result suggested that the constitutive Lyn phosphorylation seen in MV4;11 cells could be caused by mutant Flt3. In Baf3 cells expressing wild-type Flt3, Lyn phosphorylation was stimulated by Flt3-ligand treatment as expected. The Baf3 cell line is growth factor dependent, requiring IL-3 for survival and proliferation. However, as previously reported, expression of activated Flt3 mutants render the cells growth factor independent. We found that the Src-family specific inhibitors PP1 and PP2, but not the inactive analog PP3, inhibited the growth factor-independent proliferation of Baf3 cells expressing mutant Flt3. Moreover, anti-sense oligonucleotides that specifically reduced Lyn expression also inhibited proliferation of cells expressing Flt3 mutants. Previous studies identified Stat5 and Erk as mediators of the proliferation signal from mutant Flt3. We therefore examined the effects of Src kinase inhibitors on the activation of these signaling proteins by mutant Flt3. No change in Stat5 phosphorylation was detected, but inhibition of Lyn did inhibit Erk phosphorylation. These results suggest that Lyn is upstream from Erk in a Flt3 signaling pathway important for proliferation. Flt3 activation of Lyn also raises the possibility that Src kinase inhibitors could have therapeutic efficacy in Flt3-related acute leukemias.