In the 1920s, Dr. Otto Heinrich Warburg observed that cancer cells uptake more glucose compared with normal tissues and metabolize glucose via glycolysis, a low efficient pathway for generating ATP, rather than mitochondrial oxidative phosphorylation, regardless of oxygen availability [
1‐
3]. This process is now known as “Warburg effect” or aerobic glycolysis. In the past decades, researches confirmed that aerobic glycolysis is the hallmark of cancer cells and important for their proliferation and survival [
4‐
9]. In addition to generating energy, aerobic glycolysis is involved in the biosynthesis of cancer cells. The intermediate of glycolysis is used as a carbon source for the generation of nucleic acids, phospholipids, fatty acids, cholesterol, and porphyrins [
1,
6,
8]. Aerobic glycolysis also affects tumor microenvironment. In cancer cells, glucose is metabolized to lactate through glycolysis, and then the lactate is released outside the cells by monocarboxylate transporters. The release of lactate results in environmental acidosis, which protects cancer cells against attack from the immune system [
1,
6,
8]. Additionally, aerobic glycolysis was found to affect the cells signaling of tumor cells through maintaining the appropriate balance of reactive oxygen species (ROS) and histone acetylation [
1,
6,
8]. The inhibition of Warburg effect deprives the generation of ATP, decreasing cancer cells growth and proliferation [
10,
11]. Thus, Warburg effect has received substantial attention as a novel therapeutic target in cancers including lung cancer [
12,
13], leukemia [
14], breast cancer [
15‐
18], pancreatic cancer [
19,
20], colorectal cancer [
21,
22], bladder cancer [
23], and multiple myeloma [
24,
25]. In multiple myeloma, dichloroacetate, which is an inhibitor of aerobic glycolysis, has been reported to increase myeloma cell sensitivity to bortezomib [
24]. Additionally, inhibition of aerobic glycolysis was found to contribute to melphalan treatment in myeloma [
25]. Pyruvate kinase (PK) is one of the key regulators of the Warburg effect that convert phosphoenolpyruvate (PEP) to pyruvate and generate one molecular of ATP [
26,
27]. PK family consists of four isoforms: liver‑type PK (PKL), red blood cell PK (PKR), and PK muscle isozyme M1 and M2 (PKM1 and PKM2, respectively) [
27,
28]. PKM1 and PKM2, produced by an alternative splicing of the primary RNA transcript of the PKM gene, play important roles on Warburg effect. PKM1 is constitutively activated and expressed in terminally differentiated tissues to promote oxidative phosphorylation, whereas PKM2 is highly expressed in embryonic and cancer cells, which is an allosteric isoform and exhibits a dimer with low affinity for PEP. Tetramer PKM2 exhibits highly catalytic activity leading to ATP synthesis and catabolic metabolism. In contrast, dimeric PKM2, which is the low active state of PKM2, accelerates glycolytic intermediates to enter the glycolysis, such as glycerol synthesis and the pentose phosphate pathway [
26‐
30]. Increased PKM2/PKM1 ratio has been reported in multiple cancers and has been closely associated with shorter overall survival in cancer patients [
31‐
36]. Understanding the regulation of PKM pre-mRNA alternative splicing is of great importance for developing cancer therapy. The splicing factors of heterogeneous nuclear ribonucleoproteins (hnRNP) A1/2 and polypyrimidine-tract binding (PTB) protein, which mediate c-Myc enhanced PKM2/PKM1, drive alternate splicing of PKM pre-mRNA by selectively inclusion of exon 10 and the exclusion of exon 9 [
37‐
39].
Never in mitosis (NIMA)-related kinase 2 (NEK2) is a serine/threonine kinase that promotes centrosome splitting and ensures correct chromosome segregation during the G2/M phase of the cell cycle [
40]. Former studies from our group and others have indicated that NEK2 promotes tumor cell proliferation, tumor progression, and drug resistance. High expression of NEK2 is associated with poor survival in various cancers [
41‐
44]. Naro et al. reported that NEK2 is localized at the splicing speckles and phosphorylates the oncogenic splicing factor SRSF1 [
45]. We recently performed a tandem affinity purification followed up by mass spectrometry (TAP-MS) analysis and identified that NEK2 binds to hnRNPA proteins in myeloma cells. Therefore, we hypothesize that NEK2 regulates alternative splicing of PKM2/PKM1 through interacting with hnRNPA proteins, leading to modulation of aerobic glycolysis in myeloma cells. In this study, we determine whether NEK2 increases PKM splicing and PKM2 expression resulting in high aerobic glycolysis in myeloma cells using engineered isogenic myeloma cell lines with over or lower expression of NEK2. We also explore whether NEK2 is a direct target of the transcription factor c-Myc. In summary, our studies show the first evidence that NEK2 plays a functional role in aerobic glycolysis and provide mechanistic insights how NEK2 promotes aerobic glycolysis in myeloma.