ALBERT

Description: 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. Disclosures: No relevant conflicts of interest to declare.

Description: 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. Disclosures Kirito: Novartis Pharma KK: Honoraria.

Description: The endoplasmic reticulum (ER) is a cytoplasmic organelle required for proper protein folding and sorting. When protein load exceeds the capacity of the ER, the accumulation of the misfolded proteins triggers a quality control response known as the unfolded protein response (UPR). Recent studies have revealed that increased activation of the UPR is one of the hallmarks of hematological malignancies. In addition, excess UPR is associated with pro-fibrotic conditions, i.e., pulmonary fibrosis, renal fibrosis and kidney fibrosis.Lysyl-oxidase (LOX) is an enzyme that regulates the crosslinking of extracellular matrix proteins, such as collagen, and enhances a fibrotic phenotype. Recent studies revealed that the genes of LOX family members are upregulated in myeloproliferative neoplasms (MPN). Furthermore, megakaryocytes and platelets derived from MPN patients had increased LOX protein concentrations in comparison with healthy subjects. It is also reported that the UPR is one of the important regulators of LOX. Based on this knowledge, we speculated that the enhanced UPR might be involved in the elevation of LOX in JAK2V617F harboring MPN and may contribute to the development of myelofibrosis. To study this hypothesis, we initially confirmed that JAK2V617F-positive cell lines(HEL and SET-2) show enhanced activation of UPR, including phosphorylation of eIF2-alpha, nuclear localization of ATF6 and XBP1s and upregulation of glucose response protein 78 (GRP78). We also confirmed the presence of elevated levels of LOX in these cells. Treatment of the cells with a chemical chaperone, namely, tauroursodeoxycholic acid (TUDCA), suppressed UPR and decreased expression of LOX, suggesting that UPR is responsible for elevation of LOX in MPN cells. To analyze whether JAK2V617F is responsible for the enhanced UPR and expression of LOX in MPN cells, we treated the cells with JAK1/JAK2 inhibitor ruxolitinib. Unexpectedly, ruxolitinib did not block UPR nor expression of LOX. Previously, we reported that metformin, which is a member of the biguanide family, inhibited growth of MPN cells through activation of AMPK and PP2A (Kawashima et al. Exp.Hematol, 2016). Furthermore, a number of studies demonstrated that metformin suppressed UPR in several cancer cells through activation of AMPK. Therefore, we investigated whether metformin could block UPR and LOX levels in these cells. As expected, metformin reduced the phosphorylation levels of eIF2alpha and nuclear localization of XBP1 and ATF6. Expression of GRP78 was also blocked by metformin. These results clearly indicated that metformin suppressed UPR in MPN cells. Metformin also blocked the activation of the molecules required for proteins synthesis including p70S6kinase, mTOR and 4E-PB1. These results indicated that suggested that metformin blocked aberrant UPR through inhibition of enhanced protein synthesis. Importantly, we also confirmed that LOX expression was also suppressed by metformin in MPN cells.Our observations indicated that the UPR is enhanced in MPN cells irrespective of JAK2V617F, and metformin could block this process leading to suppression of LOX levels. Combination of ruxolitinib with metformin might be a new and attractive method to suppress myelofibrosis in MPN. Disclosures Kirito: Novartis Pharma KK: Honoraria.