Nucleophosmin Mutant Promotes Aerobic Glycolysis In Acute Myeloid Leukemia By Stabilizing PKM2

Objective The dysregulated metabolism is a fundamental property of cancer cells for increased growth and proliferation. Oncogenic mutations can directly change cellular metabolism and promote aerobic glycolysis in a cell-intrinsic manner, priming cells for malignancy. Mutations involving NPM1 gene are the most frequently molecular alteration in acute myeloid leukemia(AML) with cytogenetically normal(CN-AML). NPM1 mutant has an important role in leukemia pathogenesis, treatment and prognosis. However, the function of NPM1 mutation in AML aerobic glycolysis is still obscure. Here, we explored the influence of NPM1 mutant in AML aerobic glycolysis and the underlying the related molecular mechanism.Method To explore the influence of NPM1 mutation in AML aerobic glycolysis, the expression plasmid pEGFPC1-NPM1-mA carrying NPM1 mutation A(NPM1-mA) was transfected into THP-1 cells. Then, the cells with stable expression of NPM1-mA protein(NPM1-mA group) were constructed. Meanwhile, the lentivirus harboring RNA interference sequence targeting the NPM1 gene was used to infect OCI/AML3 cells(sh-NPM1 group). And the cells with stable knockdown of NPM1-mA protein were constructed. Quantitative real time PCR(q RT-PCR) and Western blot were performed to verify the NPM1-mA mRNA and protein expression. Glucose Assay Kit and Lactate Assay Kit were used to detect the glucose content and lactate content in cell culture medium, calculated the glucose consumption and lactate production. 2?, 7?-Dichlorofluorescin Diacetate(DCF-DA) was used to detect cell reactive oxygen species(ROS). Immunoprecipitation(IP) tandem mass spectrometry(MS) in OCI/AML3 led to the discovery of glycolysis-related proteins which interacted with NPM1-mA. And the interaction between glycolysis-related proteins and NPM1-mA were confirmed by Co-IP. Furthermore, to investigate the function of the glycolysis-related proteins in aerobic glycolysis of AML with NPM1-mA mutant, we had regulated the protein expression of the glycolysis-related proteins in leukemia cells and detected the glycolysis level. In addition, cell proliferation and colony forming ability were assayed through CCK-8 and colony formation.Results We had successfully established the THP-1 cells with NPM1-mA expression, and the glycolysis level was elevated in NPM1-mA group(p<0.05). Meanwhile, the glycolysis level was decreased in knockdown NPM1-mA OCI/AML3 cells(p<0.05). We found the connection between NPM1-mA and PFKL/ PKM2. Then, we confirmed the interaction of PFKL and PKM2 with NPM1-mA by Co-IP. The results verified that NPM1-mA could interact with PKM2. Moreover, NPM1-mA and PKM2 also co-located in cytoplasm assayed by IF. Besides, the overexpression of NPM1-mA resulted in the PKM2 protein expression increased and the interference of NPM1-mA decreased the PKM2 protein expression. And the protein stability experiment showed that the PKM2 protein stability impaired when NPM1-mA knockdown. Next, we had successfully established the OCI/AML3 cells with PKM2 knockdown. The glycolysis level was decreased in sh-PKM2 OCI/AML3 cells(p<0.05), and the rescue Flag-PKM2 in sh-NPM1 OCI/AML3 cells elevated the glycolysis level(p<0.05). Finally, the cell proliferation and colony forming ability were inhibited in sh-PKM2 OCI/AML3 cells(p<0.05), but increased in Flag-PKM2 group(p<0.05).Conclusion NPM1-mA played an important role in leukemia cells’ aerobic glycolysis phenotype. Cytoplasm NPM1 mutant could interact with the glycolysis rate-limiting enzyme PKM2 and enhance the PKM2 protein stability. And PKM2 promoted the development and progression of leukemia cells by intensive aerobic glycolysis level. Thus, suppression of the aerobic glycolysis by targeting PKM2 could become a strategy for clinical therapy of leukemia with NPM1 mutant.