14-3-3 Integrates Pro-Survival Signals in Hematopoietic Cells Transformed by Diverse Leukemogenic Tyrosine Kinases, and Represents a Common Target.

Constitutively activated tyrosine kinases associated with recurrent chromosomal abnormalities play an essential role in the pathogenesis and disease progression of a variety of hematopoietic malignancies. Selective tyrosine kinase inhibitors such as imatinib are effective in treating some forms of leukemia such as t(9;22) CML associated with expression of BCR-ABL fusion tyrosine kinase. However, they are not curative and clinical resistance may develop, prompting the design of alternate and/or complementary therapeutic strategies. To better understand the signaling properties of constitutively activated tyrosine kinases associated with different hematopoietic malignancies, we examined whether BCR-ABL, FLT3-ITD, NPM-ALK, TEL-PDGFbetaR, TEL-FGFR3 and ZNF198-FGFR1 activate the same set of signaling pathways. We found that they all activated AKT and MAPK signaling pathways. Activated AKT resulted in phosphorylation of FOXO3a at Thr-32 but not BAD at Ser-136, whereas activated MAPK led to phosphorylation of BAD at Ser-112. These phosphorylated residues subsequently sequestered the pro-apoptotic FOXO3a and BAD to 14-3-3, suggesting that 14-3-3 integrates pro-survival signals from AKT and MAPK pathways. We utilized a peptide-based 14-3-3 competitive antagonist, R18 to disrupt 14-3-3/ligand association. Expression of R18 effectively induced apoptosis in hematopoietic Ba/F3 cells transformed by these tyrosine kinases with significantly enhanced sensitivity compared to the control Ba/F3 cells. Moreover, doxycycline-induced expression of R18 significantly attenuated the disease latency and penetrance in mice induced by intravenous injection of representative ZNF198-FGFR1-transformed Ba/F3 cells. Co-immunoprecipitation experiments indicate that induced R18 expression disrupted interaction between 14-3-3 and FOXO3a, but not 14-3-3/BAD association. R18 induced apoptosis by rescuing the nuclear localization of FOXO3a and up-regulating FOXO3a transcription targets Bim and p27 in cells expressing ZNF198-FGFR1. Furthermore, fluorescent confocal microscopy revealed that expression of R18 generally resumed FOXO3a nuclear localization in cells transformed by the spectrum of diverse leukemogenic tyrosine kinases. Together, these data support a model that 14-3-3 functions as a general integrator of pro-survival signals in hematopoietic transformation induced by diverse leukemogenic fusion/mutant tyrosine kinases. Disrupting 14-3-3/ligand association may be a common and effective therapeutic strategy for hematopoietic neoplasms associated with these tyrosine kinases.