Shp2^E76K mutant confers cytokine-independent survival of TF-1 myeloid cells by up-regulating Bcl-X_L

Shp2 has been known to mediate growth factor-stimulated cell proliferation, but its role in cell survival is less clear. Gainof-function Shp2 mutants such as Shp2^E76K are associated with myeloid leukemias. We found that Shp2^E76K could transform cytokine-dependent human TF-1 myeloid cells into cytokine independence and further characterized the Shp2 ^E76K-induced cell survival mechanism in this study. Expression of Shp2^E76K suppressed the cytokine withdrawal-induced intrinsic/mitochondrial apoptosis pathway, which is controlled by the Bcl-2 family proteins. Analysis of Bcl-2 family proteins showed that Bcl-X_L and Mcl-1 were up-regulated in Shp2^E76K-transformed TF-1 (TF-1/Shp2^E76K) cells. Knockdown of Bcl-X_L but not Mcl-1 with short hairpin RNAs prevented Shp2^E76K-induced cytokineindependent survival. Roscovitine, which down-regulated Mcl-1, also did not prevent cytokine-independent survival of TF-1/Shp2^E76K cells, whereas the Bcl-X_L inhibitor HA14-1 did. Ras and mitogen-activated protein kinases Erk1 and Erk2 (Erk1/2) were constitutively activated in TF-1/Shp2^E76K cells, whereas little active Akt was detected under cytokine-free conditions. Shp2^E76K-induced Bcl-X_L expression was suppressed by Mek inhibitors and by a dominant-negative Mek1 mutant but not by the phosphoinositide 3-phosphate inhibitor LY294002 and the Akt inhibitor API-2. Inhibition of Erk1/2 blocked cytokine-independent survival of TF-1/Shp2^E76K cells, whereas inhibition of Akt had a minimal effect on cytokine-independent survival of TF-1/Shp2^E76K cells. These results show that Shp2^E76K can evoke constitutive Erk1/2 activation in TF-1 cells. Furthermore, Shp2^E76K induces cytokine-independent survival of TF-1 cells by a novel mechanism involving up-regulation of Bcl-X_L through the Erk1/2 pathway.