PMC

Schematic diagram describes the potential biological role of PKM2 in NPM1-mutated leukemic cells.

High PKM2 expression is associated with poor clinical outcome in patients with NPM1-mutated leukemia. A-B The survival outcome was showed by OS and EFS according to the levels of PKM2 in NPM1-mutated leukemia patients with the log-rank test applied for comparison.

High expression of PKM2 is regulated by PTBP1 in leukemic cells. A-B Basal mRNA and protein levels of PTBP1, PKM2 and PKM1 were detected by real-time PCR and western blot. C-D The mRNA and protein expression of PTBP1 and PKM2/PKM1 after PTBP1 knockdown were detected in OCI-AML3 cells. E-H The mRNA and protein expression of PTBP1, PKM2/PKM1 were detected after PTBP1 overexpression in THP-1 and NB4 cells. The data are expressed as the mean ± SD (n = 3). *p < 0.05.

PKM2 increases the phosphorylation of Beclin-1 in leukemic cells. A The gene expression of Beclin-1 was determined by real-time PCR in OCI-AML3 cells following PKM2 knockdown. B The phosphorylation levels of Beclin-1 were assessed by western blot in PKM2-silenced OCI-AML3 cells. C-D The expression of p-Beclin-1^Thr119, Beclin-1, and PKM2 proteins were determined by western blot in THP-1 and NB4 cells after PKM2 overexpression. E-F The protein expression was assessed from the THP-1 and NB4 cells transfected with the plasmids expressing PKM2 WT or PKM2 K367M. The data are expressed as the mean ± SD (n = 3). *p < 0.05.

PKM2 promotes cell survival via autophagic activation. A Cell proliferation was assessed by colony formation in PKM2-silenced OCI-AML3 cells. B Cell apoptosis was evaluated by flow cytometry analysis in PKM2-silenced OCI-AML3 cells. C Cell viability was detected in the PKM2-silenced OCI-AML3 cells and then treated with rapamycin for indicated times by CCK-8 assay. D Cell viability was detected by CCK-8 in THP-1 cells and then treated with 3-MA for indicated times by CCK-8 assay. E Cell viability was detected by CCK-8 in NB4 cells. F Cell proliferation was revealed by CCK-8 assay in PKM2-silenced OCI-AML3 cells and then treated with Tat-Beclin-1. The data are expressed as the mean ± SD (n = 3). *p < 0.05.

PKM2 mediates autophagic activation in leukemic cells. A Autophagy markers (LC3 II and p62) were detected by real-time PCR assay after silenced PKM2 and then treated with autophagy activator rapamycin (5 μM) for 6 hrs in the different groups. B LC3 II and p62 protein levels were detected by western blot, the bands were quantified and data were shown as mean ± SD of three independent experiments. C LC3 II and p62 mRNA were detected by real-time PCR after enforced PKM2 and treated with autophagy inhibitor 3-MA (2 mM) in THP-1 cells. D LC3 II and p62 protein levels were detected by western blot, the bands were quantified and data were shown as mean ± SD of three independent experiments. E-F LC3 II and p62 mRNA and protein were detected in NB4 cells with different treatments. The data are expressed as the mean ± SD (n = 3). *p < 0.05. n.s represents no significance.

PKM2 highly expresses in leukemia with NPM1 mutation. A PKM2 mRNA expression of AML samples was analyzed using Oncomine (https://www.oncomine.org/resource/login.html). B The mRNA expression of PKM2 was detected by real-time PCR in five indicated myeloid leukemia cell lines. C PKM2 protein expression was detected by western blot in leukemia cell lines. D RNA-seq mRNA expression data from the TCGA database were used to compare PKM2 expression between AML patients with (n = 22) and without NPM1 mutation (n = 25). (E) Dot plot showing the relative expression levels of PKM2 in 30 AML patients, consisting of 14 cases with NPM1 mutation and 16 cases with no NPM1 mutation. The data are expressed as the mean ± SD (n = 3). *p < 0.05.