PKM2 Mediates Chronic Myeloid Leukemia Imatinib Resistance By Regulating Glycolysis Energy Metabolism

Background: Pyruvate kinase M2 (PKM2) plays an important role in malignant tumor metabolism, and provides tumor cells with enough energy and a lot of metabolic intermediate by mediating Warburg effect. Recently, studies indicate that PKM2 protein can mediate tumor chemoresistance through reprogramming cellular metabolism pathway and participate in chemotherapy resistance mechanisms of many tumor cells. Currently, the problem of imatinib resistance is still a difficult point in the treatment of chronic myeloid leukemia. This study is aimed at investigating the function of PKM2 protein in the metabolism of chronic myeloid leukemia cells and the relationship between PKM2 and drug resistance of chronic myeloid leukemia cells.

Methods: We selected the CML primary cells (the cells from new diagnosed patients and refractory patients respectively) and cell lines (KBM5 cells were served as drug-sensitive cells and KBM5 cell with T315I mutation (KBM5-STI) were served as drug-resistant cell) as research objects. We used a low dose PKM2 inhibitor and/or IM treat with CML cells, and detected cell proliferation by CCK-8, apoptosis by flow cytometry. The cell glycolysis level is detected by a real-time cell energy detector, and the results represent the level of glycolysis by the acidification rate. Western blot and RT-qPCR analyzed the protein and mRNA expression level of PKM2 in TKI-sensitive and -resistant primary cells and cell lines. After transfecting control or PKM2 siRNA into KBM5 and KBM5-STI cells for 72hrs, we extracted total RNA and proteins, and determined the mRNA and protein level of PKM2, and the protein level of mTOR、P13K/AKT.

Results:

1. PKM2 inhibitor can inhibit KBM5 and KBM5-STI cells growth with IC50 5uM, 6.8uM (P=0.001), respectively, and induce cell apoptosis.

2. Low dose PKM2 inhibitor combined with IM greatly induced KBM5 and KBM5-STI cell growth inhibition and cell death compare to using single drug.

3. PKM2 was higher expressed in the TKI-resistant CML primary cells and cell line KBM5-STI. In the meanwhile, we found that mTOR, HIF-1a were increased in TKI-resistant CML primary cells and cell line too.

4. PKM2 knockdown decreased the cell proliferation and growth, and increased the apoptosis of TKI-resistant cell line after treated with IM. In the mentime, PKM2 knockdown showed significantly lower glycolytic capacity than normal cell line with lower oxygen intake and lacitic acid production, and downregulating with mTOR and P13K/AKT protein expression.

Conclusion: Pyruvate kinase M2 (PKM2) acts as an important rate-limiting enzyme in the aerobic glycolytic pathway, and mediates abnormal metabolic pathways which promote tumor cell proliferation, invasion and drug resistance. Compared to the TKI-sensitive primary cell and cell line, PKM2 was increased in the TKI-resistant primary cell and cell line and related to glycolytic level. PKM2 inhibitor can inhibit CML cells growth, induce cell apoptosis, and combined with IM at a low dose can exhibited a synergistic anti-leukemia effect on TKI-resistant cells. Low dose PKM2 inhibitor combined with IM can enhance targeted killing to the CML cells, suggesting that PKM2 protein plays an important role in the formation of drug resistance. After interfering PKM2 protein, it showed a significant downregulated level of glycolysis in the cell line, and the decreased level of P13K/AKT/mTOR signaling pathway. This study demonstrates that PKM2 may be involved in the regulation of energy metabolism in leukemia cells and induce drug resistance.