A gain-of-function mutant JAK2 has been demonstrated to play a causal role in myeloproliferative neoplasms (MPN) development. The mutant JAK2, typically a substitution of position 617 valine (V) to phenylalanine (F) (hereinafter called JAK2V617F), activates STAT, Akt, and ERK pathways, which subsequently promotes cell proliferation. Although these pathways are well documented, their downstream effectors in MPN are still poorly understood.
FOXO3A, a subfamily of Forkhead transcription factors, paradoxically regulates cell death and survival in a variety of cells. We and others have previously shown that an oncogene product Bcr-Abl tyrosine kinase suppresses FOXO3A activity by phosphorylation and subsequently represses cell death in chronic myelogenous leukemia (CML), proposing a potential therapeutic strategy for CML treatment. Thus, defining FOXO3A function and uncovering its regulation in MPN are important for the understanding of this disease and the development of a novel therapeutic strategy.
To demonstrate this, we employed a model cell line Ba/F3/EpoR that requires IL-3 for survival but becomes independent of the cytokine when JAK2V617F is expressed. When JAK2V617F-expressing Ba/F3/EpoR cells were treated with a JAK2 inhibitor, FOXO3A phosphorylation diminished in a dose-dependent manner. The trend of diminution paralleled to the reduction of phospho-STAT5, a major JAK2V617F substrate, suggesting that FOXO3A phosphorylation is regulated by JAK2V617F activity. Removal of JAK2 inhibitor from the media induced a massive FOXO3A phosphorylation within 20 minutes in JAK2V617F-expressing but not in the wild-type JAK2-expressing Ba/F3/EpoR cells. This observation indicates that at least in this model system, JAK2V617F positively modulates the phosphorylation of FOXO3A.
To further investigate the function of FOXO3A in JAK2V617F-expressing Ba/F3/EpoR cells, we knocked down FOXO3A expression by shRNA. When treated with a JAK2 inhibitor to induce apoptotic cell death, FOXO3A-knocked down cells were more resistant to the inhibitor than the control cells. This strongly suggests that when JAK2V617F activity is blocked and subsequently FOXO3A is dephosphorylated, FOXO3A induces apoptosis in JAK2V617F-expressing cells.
Finally, we elucidated that the inhibition of JAK2V617F activity results in a reduction of FOXO3A phosphorylation and the induction of apoptosis in CD34-positive cells obtained from MPN patients. Taken together, we propose a model that shows JAK2V617F induces FOXO3A phosphorylation and subsequently blocks FOXO3A activity to repress apoptosis in JAK2V617F positive MPN cells.
Kobayashi:Sysmex Corporation: Employment. Komatsu:Sysmex Corporation: Research Funding.
Asterisk with author names denotes non-ASH members.