Metformin Exerts Anti-Leukemic Effects Via Direct Inhibition Of Oncogenic Kinase Activity In Leukemia Cells Derived From Myeloproliferative Neoplasms

Metformin, a biguanide family member of a group of anti-hyperglycemic reagents, is one of the most widely used anti-diabetic drugs. Recent studies have revealed that metformin can block growth and induce apoptosis in several leukemia cells types. These studies also demonstrated that AMP-activated protein kinase (AMPK), a sensor for cellular energy status, is an important effector that mediates the anti-leukemic effects of metformin. Activation of AMPK leads to the inhibition of p70S6 kinase(p70S6K), an important regulator of protein synthesis, which consequently causes cell death. In contrast to these findings, other studies reported that metformin exerts anti-leukemic effects independent of AMPK. To clarify this discrepancy and determine the anti-leukemic function of metformin, we analyzed the molecular events induced by metformin with a series of leukemic cell lines: JAK2V617F positive cells (SET2), BCR-ABL expressing cells (KU812), and Flt-3-ITD mutant cells (PL21 and MV4-11). Treatment with metformin inhibited growth and induced apoptosis in all of the tested leukemic cell lines. However, metformin exerted differing effects on AMPK activity. In the Flt-3-ITD positive cells, metformin clearly induced the phosphorylation of AMPK at Thr172, which is required for the activation of the enzyme. Concomitant with this finding, the inhibition of p70S6K and activation of ACC (acetyl-CoA carboxylase), both of which are important downstream mediators of AMPK, were also observed in these cell lines. However, metformin did not enhance AMPK activity in either the SET-2 or KU812 cell lines. Furthermore, neither inhibition of p70S6K nor activation of ACC was induced by metformin in these cells. These results suggested that metformin exerts anti-leukemic action independent of AMPK in these cells. To investigate the molecular mechanisms of the AMPK-independent action of metformin, we investigated whether metformin affects any signal transduction pathways in these cells. We discovered that metformin inhibited the tyrosine phosphorylation of JAK2V61F and BCR-ABL in a dose and time dependent manner in SET2 and KU812 cells, respectively. This inhibitory function was confirmed with the HEL and K562 cells lines, which express JAK2V617F and BCR-ABL, respectively. Metformin also prevented the phosphorylation of targets downstream of JAK2V617F and BCR-ABL, including STAT5, ERK and AKT, in these cells. Compared to JAK2V617F and BCR-ABL, the phosphorylation of Flt-3-ITD was not inhibited by metformin, and downstream signal molecules were instead activated by metformin in Flt-3-ITD expressing cells. It is well known that both JAK2V617F and BCR-ABL induce overproduction of reactive oxygen species, and anti-oxidants (e.g., N-acetyl-L-cysteine) block the action of both oncogenic proteins. Interestingly, recent studies revealed that metformin modulates the intracellular oxidative status by inhibiting mitochondrial electron transport complex I. Together with our observations, this suggests that metformin might block the activity of JAK2V617F and BCR-ABL by modulating ROS levels. In conclusion, we elucidated a new anti-leukemic action of metformin: direct inhibition of oncogenic tyrosine kinases. These observations also provide a potential new treatment strategy for myeloproliferative neoplasms.