Metformin Inhibits JAK2V617F Activity By Activating AMPK and PP2A Containing B56 Alpha Subunits in MPN Cells

Metformin, which is a member of the biguanide family, suppresses the growth of a variety of malignant hematological cells. It is widely accepted that metformin inhibits the growth of these cells primarily by suppressing the mTOR pathway. In contrast, we previously found that metformin directly suppresses the activation of JAK2V617F in HEL and SET-2 cells. In the present study, we investigated the precise molecular mechanisms through which metformin inhibits the auto-phosphorylation of JAK2V617F. Initially, we speculated that AMPK, which is one of the main downstream molecules activated by metformin, might be involved in the process. Compound C, an inhibitor for AMPK, resumed the JAK2 activity in cells treated with metformin. For the next step, we introduced siRNA against an α subunit of AMPK and analyzed the phosphorylation levels of JAK2V617F. We found that knock-down of AMPK enhanced the basal phosphorylation levels of JAK2V617F but that metformin-induced JAK2 inhibition was only partially diminished. These results suggested that although AMPK was responsible for the metformin-induced JAK2V617F inhibition, other mechanisms are also required to achieve the full inhibition of JAK2V617F activity.Recently, several groups have revealed that PP2A-activationg drugs suppress the activity of JAK2V617F. In addition, recent studies have demonstrated that metformin activates PP2A. These reports inspired us to investigate the possibility that PP2A might be responsible for the metformin-mediated suppression of JAK2V617F. PP2A consists of three different subunits: A, B and C. The A subunit works as a scaffold, and the C subunit has catalytic activity. The B subunit contains a variety of members, and the diversity of the B units determines the substrate specificity. Initially, we analyzed whether metformin activates PP2A in JAK2V617F-positive cells. We found that metformin decreases the phosphorylation of the C-subunit of PP2A at Tyr 308, which negatively regulates PP2A function. For further study, we prepared the A subunit of PP2A  knock-down cells using siRNA. We expected that the knock-down of PP2A would restore the JAK2V617F activity; instead, JAK2V617F was suppressed in these cells. To explain the discrepancy, we focused on the B subunits and hypothesized that only a specific type of PP2A complex is involved in the de-phosphorylation of JAK2V617F. To this end, we introduced a panel of siRNA targeting different B subunits and analyzed the effects on JAK2V617F activity. Knock-down of the B56α  subunit resulted in the activation of JAK2V617F. In contrast, the JAK2V617F phosphorylation levels were decreased in the B56γ knock-down cells. Interestingly, we found that B56γ knock-down cells have enhanced AMPK activity. These results indicate that among the PP2A complexes, B56α containing complex works as negative regulator for JAK2 but that the B56γ subunit-containing complex may positively regulate JAK2 by inhibiting AMPK.In conclusion, both AMPK and PP2A-containing B56α subunits are involved in the metformin-induced JAK2V617F inhibition. In contrast, the B56γ subunit-containing PP2A may diminish metformin-induced JAK2 inhibition via the negative regulation of AMPK. These results suggest that the specific inhibition of the B56γ subunit containing PP2A may enhance the anti-leukemic action of metformin.