NEK2 Promotes Aerobic Glycolysis in Multiple Myeloma Through Regulating Splicing of Pyruvate Kinase
Gu et al. Journal of Hematology & Oncology
NEK2 Promotes Aerobic Glycolysis in Multiple Myeloma Through Regulating Splicing of Pyruvate Kinase
Zhimin Gu 0
Jiliang Xia 0 1
Hongwei Xu 0
Ivana Frech 0
Guido Tricot 0
Fenghuang Zhan 0
0 Department of Medicine, Division of Hematology, Oncology and Blood and Marrow Transplantation and Holden Comprehensive Cancer Center, University of Iowa , 585 Newton Rd, 52242 Iowa City, IA , USA
1 Institute of Cancer Research, School of Basic Medical Sciences, Southern Medical University , Guangzhou , China
Background: Aerobic glycolysis, a hallmark of cancer, is characterized by increased metabolism of glucose and production of lactate in normaxia. Recently, pyruvate kinase M2 (PKM2) has been identified as a key player for regulating aerobic glycolysis and promoting tumor cell proliferation and survival. Methods: Tandem affinity purification followed up by mass spectrometry (TAP-MS) and co-immunoprecipitation (Co-IP) were used to study the interaction between NIMA (never in mitosis gene A)-related kinase 2 (NEK2) and heterogeneous nuclear ribonucleoproteins (hnRNP) A1/2. RNA immunoprecipitation (RIP) was performed to identify NEK2 binding to PKM pre-mRNA sequence. Chromatin-immunoprecipitation (ChIP)-PCR was performed to analyze a transcriptional regulation of NEK2 by c-Myc. Western blot and real-time PCR were executed to analyze the regulation of PKM2 by NEK2. Results: NEK2 regulates the alternative splicing of PKM immature RNA in multiple myeloma cells by interacting with hnRNPA1/2. RIP shows that NEK2 binds to the intronic sequence flanking exon 9 of PKM pre-mRNA. Knockdown of NEK2 decreases the ratio of PKM2/PKM1 and also other aerobic glycolysis genes including GLUT4, HK2, ENO1, LDHA, and MCT4. Myeloma patients with high expression of NEK2 and PKM2 have lower event-free survival and overall survival. Our data indicate that NEK2 is transcriptionally regulated by c-Myc in myeloma cells. Ectopic expression of NEK2 partially rescues growth inhibition and cell death induced by silenced c-Myc. Conclusions: Our studies demonstrate that NEK2 promotes aerobic glycolysis through regulating splicing of PKM and increasing the PKM2/PKM1 ratio in myeloma cells which contributes to its oncogenic activity.
NEK2; Pyruvate kinase; Multiple myeloma; Alternative splicing
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
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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
, breast cancer [15–18], pancreatic cancer [19, 20],
colorectal cancer [21, 22], bladder cancer , 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
. Additionally, inhibition of aerobic glycolysis was found
to contribute to melphalan treatment in myeloma .
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 . 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 . 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.
Gene expression profiling
The data of gene expression profile (GEP), which were
derived from NIH Gene Expression Omnibus (http://
www.ncbi.nlm.nih.gov/geo/), include 22 healthy subjects
(accession number GSE5900), 44 monoclonal
gammopathy of undetermined significance (MGUS) patients
(accession number GSE5900), 305 low-risk, and 46 high-risk
myeloma patients (accession number GSE2658).
Affymetrix U133Plus2.0 microarrays were used to analyze these
samples as previously described . Signal intensities
were preprocessed and normalized by GCOS1.1 software.
The expression and relationship between NEK2, c-Myc,
PKM2, and aerobic glycolysis relative genes were
investigated in these samples.
Human myeloma cell lines, ARP1, OPM2, and the B cell
line P493-6 (a gift from Dr. Thomas-Tikhonenko,
University of Pennsylvania) were cultured at 37 °C and 5% CO2
in RPMI 1640 (Gibco, Grand Island, NY) supplemented
with 10% heat inactivated fetal calf serum (Gibco, Grand
Island, NY) and 1% penicillin and streptomycin (Gibco,
Grand Island, NY).
Briefly, total proteins from myeloma cells were extracted
using Mammalian Cell Extraction Kit (K269–500,
Biovision, Milpitas, CA). Protein samples (20 μg/sample) were
separated using SDS-PAGE and transferred into the
nitrocellulose membrane via Bio-Rad Mini-Protean
electrotransfer system. The membranes were blocked with
5% non-fat dry milk in TRIS buffered saline (TBS)
containing 0.05% Tween-20 (TBST) prior to incubation
overnight at 4 °C with primary antibody including NEK2
(Santa Cruz, USA), FLAG (Sigma, USA), HA (C29F4),
PKM2 (D78A4), c-Myc (D84C12), hnRNPA1 (D21H11),
hnRNPA2 (A2A), and β-actin (D6A8) (Cell Signaling,
USA). Respective HRP-conjugated secondary antibodies
were added and protein signals were developed with the
use of the HRP substrate luminol reagent (Millipore,
CA). The developed images were obtained and analyzed
using ChemiDocTM XRS+System (Bio-Rad, USA).
Co-immunoprecipitation (Co-IP) was performed as
previously describe  with some modifications. Briefly,
total proteins from NEK2 overexpressing ARP1 cells
were extracted with IP lysis buffer (Thermo Scientific,
USA). HA antibodies (C29F4, Cell Signaling, USA) or
control immunoglobulin (IgG) (Cell Signaling, USA)
were added and incubated with cell lysate overnight at
4 °C. Then followed by protein A Dynabeads (Invitrogen,
USA) incubation for 2 h at 4 °C. The beads were washed
three times with TBST (Sigma, USA). The pulled-down
proteins were extracted and examined by Western
blotting as described above.
RNA immunoprecipitation (RIP) was carried out as
previously described  with some modifications. Briefly,
NEK2 overexpressing ARP1 cells were cross-linked with
1% formaldehyde (Covaris, USA) for 5 min at room
temperature and then followed by Covaris quenching
buffer (Covaris, USA) incubation for 5 min to stop the
cross-link. Cells were lysed by Covaris lysis buffer
(Covaris, USA) containing protease inhibitor cocktail
(Covaris, USA) and RNase inhibitor (Invitrogen, USA).
Nuclear pellets were collected and lysed through
sonication. Nuclear lysates were incubated with HA antibody
or control IgG conjugated protein A Dynabeads
(Invitrogen, USA) overnight at 4 °C followed by stringent
washing of bead pellets with final resuspension in TRIzol
(Invitrogen, USA). Co-precipitated RNAs were isolated
and RT-PCR was performed to determine the sequence
of EI9. EI9 forward and reverse primer sequences,
respectively, 5′-TGCATGCTTCCACAGGCATC-3′; EI9
reverse primer 5′-TGGGCTAACTTGTGAGAGGC-3′.
Cells (1 × 105) were spun down on glass slides and then
fixed with 4% paraformaldehyde solution (Affymetrix,
USA) for 15 min at room temperature. NEK2,
hnRNPA1, and hnRNPA2 antibodies were diluted in
TBS buffer with 0.1%, Triton 100, and 1% BSA. These
antibodies were dripped on glass slides and incubated
overnight at 4 °C. After 3 washes with TBST, secondary
antibodies coupled to Alexa-Fluor® 488 goat anti-rabbit
IgG(H+L) (Invitrogen, USA) or Alexa-Fluor® 594 goat
anti-mouse IgG(H+L) (Invitrogen, USA) were added and
incubated for 1 h at room temperature. Nuclei were
labeled with DAPI (Vector Laboratories, CA).
Fluorescence was observed under a fluorescence microscope.
Total RNA was extracted using RNeasy RNA isolation
kit (Qiagen, USA) according to the manufacturer’s
instructions. After digestion with RNase-free DNase
(Roche, USA), 200 μg of total RNA was retrotranscribed
using the 5×iScriptTM RT Supermix (Bio-Rad, USA).
PCR primers were purchased from Integrated DNA
Technologies (Coralville, IA). Real-time quantitative
PCRs (qPCR) were performed using iTaqTM Universal
SYBR® Green Supermix (Bio-Rad, USA). Fold changes
were calculated using the ΔΔCt method and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA as
reference. Primer sequences are listed in Table 1.
Glucose uptake and lactate production assay
Glucose uptake was detected by a glucose uptake assay
kit (Biovision, CA). Myeloma cells or P493-6 cells were
plated into a 96-well plate. Cells were washed 3 times
with PBS and then starved by preincubating with 100 μL
Krebs Ringer Phosphate Hepes (KRPH) buffer for
40 min. Cells were stimulated with or without insulin
(1 μM) for 20 min to activate glucose transporter, and
10 μL of 10 mM 2-deoxyglucose (2-DG) was added and
incubated for 20 min. Cells were lysed with 90 μL of
extraction buffer and then frozen/thawed once and heated
at 85 °C for 40 min. The cell lysate was neutralized by
adding 10 μL of neutralization buffer. The glucose
uptake was measured by the cellular fluorescence (Ex/Em
= 535/587 nm) in a microplate reader (BioTake, USA).
Lactate production was detected by a lactate assay kit
(Biovision, CA). Myeloma cells or P493-6 cells were
cultured in fresh phenol free RPMI1640 medium, and the
culture medium was collected at the indicated times.
Mix the culture medium with lactate assay buffer to
50 μL/well in a 96-well plate. Then 50 μL reaction buffer
was added to every well and incubated for 30 min at
room temperature. The lactate production was measured
by the absorbance (570 nm) in a microplate reader.
FITC-conjugated annexin V (eBioscience, USA) was
used to label apoptosis cells. Dead cells were labeled by
propidium iodide (PI) (eBioscience, USA). Staining
experiment was performed according to the product
instructions. Cells were then analyzed for apoptosis by
flow cytometry (FACS) using the Cell Quest software.
The results were analyzed using FlowJo software.
All data were analyzed using two-tailed Student’s t test
and expressed as mean ± SD between two groups. The
difference of gene expression in multiple groups was
analyzed by one-way ANOVA. A p value of 5% (*p < 0.05)
was considered significant. Event-free (ES) and overall
Table 1 Primer sequences for real-time PCR
PKM1 pyruvate kinase isozymes M1, PKM2 pyruvate kinase isozymes M2, GLUT4 glucose transporter type 4, ENO1 enolase 1, LDHA lactate dehydrogenase A, MCT4
monocarboxylate transporter 4, HK2 hexokinase 2, NEK2 NIMA-related kinase 2, GAPDH glyceraldehyde 3-phosphate dehydrogenase
survivals (OS) were presented by the Kaplan-Meier
curves, and the log-rank test was used to determine
significance between gene expression levels with patient
outcome. Significance was set at p < 0.05.
NEK2 interacts with hnRNPA1/2 in myeloma cells
NEK2 has been identified as an oncogenic protein which
promotes tumorigenesis, tumor progression, and drug
resistance. In this study, a TAP-MS analysis was
performed to identify NEK2 interacting proteins in
myeloma cells. To reduce nonspecific binding, plasmids
containing human NEK2-cDNA tagged with HA and
3xFLAGS were transfected into a human myeloma cell
line ARP1 by lentiviral delivery. Western blotting results
confirmed that NEK2 was overexpressed in myeloma
cells (Fig. 1a). NEK2 and its binding proteins were pulled
down using sequential HA and Flag antibodies
immunoprecipitation, and proteins bound to NEK2 were
identified by mass spectrometry. The TAP-MS analysis
showed that NEK2 binds at least to 67 proteins (data
not shown), and the major functional category is the
splicing factor family. We were particularly interested in
hnRNPA2 because it is the key regulator of PKM
premRNA alternative splicing. It is known that hnRNPA2
forms a heterodimer with hnRNPA1 to play its biological
function . To confirm the interaction between NEK2
and hnRNPA1/2, immunofluorescence and Co-IP
experiments were performed. Immunofluorescence images
showed that NEK2 is co-localized with hnRNPA1/2 in
the nucleus (Fig. 1b). As NEK2 protein was tagged with
HA, we used HA antibody to immunoprecipitate NEK2,
then detecting NEK2, hnRNPA1, and hnRNPA2 by
Western blotting. As shown in Fig. 1c, NEK2, hnRNPA1,
and hnRNPA2 were detected in HA immunoprecipitated
proteins but not in IgG control. These results indicate
that NEK2 binds to hnRNPA1 and hnRNPA2 proteins.
This is consistent with a recent study that NEK2
interacts and activates several splicing factors as a novel
splicing factor kinase . Based on these data, we
hypothesize that NEK2 may be involved in hnRNPA1/2
mediated pre-mRNA alternative splicing of PKM gene.
NEK2 regulates the PKM2/PKM1 complex in myeloma cells
The hnRNPA1/2 complex binds to the intronic
sequences flanking exon 9 of PKM pre-mRNA leading
to exon 9 exclusion and exon 10 inclusion [37, 38].
In cancer or embryonic cells, increased hnRNPA1/2
proteins by c-Myc or others promotes exon 10
splicing and inclusion resulting in generation of pyruvate
kinase isozyme type M2 (PKM2) . We have
confirmed that NEK2 binds with hnRNPA1/2 in myeloma
cells described above, we then determined whether
high NEK2 enhances its binding to the intronic
sequences flanking exon 9 of PKM pre-mRNA. The RIP
using HA-tag antibodies was performed to pull down
NEK2 binding RNA sequences, and real-time PCR
revealed that the intronic sequences flanking exon 9 of
PKM pre-mRNA was significantly enriched in the
NEK2 binding RNA compared with the IgG control
(Fig. 2a). We further examined whether NEK2
regulates alternative splicing of PKM pre-mRNA in NEK2
silencing myeloma cells. NEK2 expression and PKM2
expression showed a decrease after addition of
doxycycline by Western blotting in ARP1 and OPM2
myeloma cells (Fig. 2b). The expression of PKM1 and
PKM2 was measured by real-time PCR in myeloma
cells with or without knockdown of NEK2. Clearly,
inhibition of NEK2 upregulated PKM1 expression but
downregulated PKM2 (Fig. 2c). The ratio of PKM2/
PKM1 was significantly decreased in myeloma
NEK2silenced cells (Fig. 2c). Since NEK2 is also localized
Fig. 1 NEK2 interacts with hnRNPA1/2 proteins. a Western blots confirm that NEK2 cDNA conjugated with tags HA-3xFLAG are transfected into
the myeloma cell line ARP1. b Immunofluorescence analysis of ARP1 cells stained with NEK2 antibody (red), hnRNPA1/2 (green), and DAPI (blue).
c HA antibody was used to pull down NEK2, and its interacting proteins were analyzed by Western blotting. The lysates before IP were used as a
positive control and IgG pulled down proteins as a negative control
Fig. 2 High NEK2 increases the ratio of PKM2/PKM1. a RNA immunoprecipitation using anti-HA antibody to pull down NEK2 binding RNA in
ARP1 NEK2-HA OE cells. Real-time PCR was performed to test the enrichment of intronic sequence flanking exon 9 of PKM pre-mRNA. All values
were normalized by genomic GAPDH, and IgG was used as negative control, *p < 0.05. b Western blots were performed to test the levels of NEK2
and PKM2 in NEK2-shRNA ARP1 and OPM2 MM cells. c Real-time PCR analyses of the ratio of PKM2/PKM1 in NEK2 knocked down ARP1 and
OPM2 MM cells. Results of real-time PCR were normalized against GAPDH and presented means ± SD of triplicate determinations from an experiment
representative of three, *p < 0.05
in the nucleus, it is possible that NEK2 directly binds
to the PKM pre-mRNA and regulates its splicing. If
this is the case, we can prove it by pulling down
RNA sequences using anti-NEK2 antibodies and
determine if PKM pre-mRNA can be detected by PCR
in future studies.
NEK2 promotes aerobic glycolysis in myeloma cells
PKM2 plays an important role in aerobic glycolysis. We
then tested whether NEK2 alters aerobic glycolysis via
regulating PKM2 expression. The expression of NEK2 and
aerobic glycolysis genes was examined in plasma cells
derived from 22 healthy subjects, 44 monoclonal
gammopathy of undetermined significance (MGUS) patients, 305
low- and 46 high-risk myeloma patients using gene
expression profiling (GEP). The expression of NEK2 and
glycolysis-enhancing genes, such as hexokinase 2 (HK2),
alpha-enolase (ENO1), and lactate dehydrogenase A
(LDHA), was significantly increased in high-risk myeloma
samples and positively correlated each other (Fig. 3a). We
then confirmed these gene expressions in NEK2 silenced
ARP1 and OPM2 myeloma cells by real-time PCR
(Fig. 3b). Consistently, the expression of HK2, ENO1,
LDHA, glucose transporter type 4 (Glut4), and
monocarboxylate transporter 4 (MCT4) was downregulated in
NEK2 silenced myeloma cells. To determine whether
NEK2 regulates aerobic glycolysis, we tested glucose
uptake and lactate production in NEK2 knockdown cells and
control cells at normoxia or hypoxia (1% oxygen)
conditions. As shown in Fig. 3c, d, both glucose uptake and
lactate production decreased in NEK2 knockdown ARP1 and
OPM2 myeloma cells compared to the control cells in
c-Myc transcriptionally regulates NEK2 expression
Although NEK2 expression is increased in various cancers,
the regulation of its expression remains unclear. It is known
that NEK2 is a potential target of c-Myc from chromatin
immunoprecipitation (ChIP) sequencing , and c-Myc
regulates pyruvate kinase mRNA splicing in cancer by
upregulation of hnRNPA1/2 and PBT . Given that both
NEK2 and c-Myc are involved in hnRNPA1/2
mediatedFig. 3 NEK2 regulates aerobic glycolysis in myeloma cells. a GEP analysis of NEK2, HK2, ENO1, and LDHA on plasma cells derived from normal
healthy donors (n = 22), MGUS patients (n = 44), low-, and high- (n = 305) risk MM patients (n = 46). b Real-time PCR was performed to test the
expression of GLUT4, NEK2, HK2, ENO1, and LDHA in NEK2 silenced ARP1 and OPM2 MM cell lines, *p < 0.001. c, d Glucose uptake and lactate
production were analyzed in NEK2 knocked down ARP1 MM cells cultured at normoxia (black column) or hypoxia (grey column), *p < 0.05
PKM splicing, we hypothesize that c-Myc induces PKM
splicing may depend on upregulation of NEK2, at least
partially. We compared the expression of NEK2, c-Myc and
PKM2 in plasma cells derived from healthy donors, MGUS
patients, low- and high-risk MM patients described above.
As shown in Fig. 4a, the expression of c-Myc, NEK2, and
PKM2 is positively correlated and increased significantly in
high-risk MM samples (p < 0.001 by one-way ANOVA). A
chromatin immunoprecipitation-qPCR (ChIP-qPCR) assay
was performed using anti-c-Myc antibodies to pull down
binding DNA in a human B cell line P493-6 that is stably
transfected with EREB2-5 and the construct c-Myc-tet .
Consistently, NEK2 promoter DNA sequences were
significantly enriched by c-Myc antibody pulling down in P493-6
cells without addition of doxycycline compared with
addition of doxycycline (Fig. 4b). Addition doxycycline in
P493-6 cells decreases c-Myc expression (Fig. 4d). NEK2
expression was significantly decreased at both
transcription and protein levels following inhibition of c-Myc
(Fig. 4c, d) further suggesting that c-Myc regulates the
expression of NEK2.
To determine the clinical relevance of aerobic glycolysis
signaling, Kaplan-Meier analyses of event-frees (EFS) and
overall survivals (OS) were performed on 351 newly
diagnosed myeloma patients. Myeloma patients with high
PKM2 expression had shortened EFS (p = 0.040) and OS
(p = 0.007), which is similar to NEK2 (EFS p = 0.001; OS p
= 0.002) (Fig. 4e). To further investigate whether NEK2 has
a synergistic or additive effects in patient outcome, the 351
myeloma patients were classified into 4 groups including
low-NEK2/low-PKM2, low-NEK2/high-PKM2, low-PKM2/
high-NEK2, and high-NEK2/high-PKM2; and
KaplanMeier analyses showed clearly that the high NEK2/PKM2
group had the worst outcome in both EFS and OS (Fig. 4e).
Fig. 4 NEK2 is a transcriptional target of c-Myc. a Dot-plots present the expression of NEK2, c-Myc, and PKM2 in plasma cells of GEP derived from
normal donors (n = 22), MGUS patients (n = 44), low- (n = 305), and high- (n = 46) risk MM patients (p < 0.001). b ChIP PCR detected binding of
c-Myc to the promoter of Nek2 in P493-6 cells. IgG antibodies were used as negative control. c Real-time PCR shows the expression of NEK2 in
P493-6 cells after silencing c-Myc. d Western blots show protein expression of NEK2, c-Myc, and β-actin in P493-6 cells with knocking down
c-Myc. e Kaplan-Meier analyses of event-free survival (top panels) and overall survival (bottom panels) among MM patients with different expression
levels of NEK2 and PKM2
NEK2 contributes to c-Myc regulated aerobic glycolysis
and cell proliferation
hnRNPA1/2 were found to play an important role in c-Myc
regulated aerobic glycolysis. Similar to hnRNPA1/2, the
expression of NEK2 was regulated by c-Myc at transcription
level in myeloma cells. We hypothesize that NEK2 plays a
role in c-Myc-mediated aerobic glycolysis. To determine
whether NEK2 is involved in c-Myc-mediated aerobic
glycolysis, we examined the alternative splicing of PKM and
aerobic glycolysis in NEK2 overexpressed P493-6. In
P4936 cells c-Myc expression was inhibited by addition of
doxycycline. As shown in Fig. 5a, c-Myc was significantly
downregulated upon addition of doxycycline leading to decrease
PKM2 expression. However, the decreased expression of
PKM2 was rescued in P493-6 cells overexpressed NEK2
and the ratio of PKM2/PKM1 decreased more than two
folds in P493-6 cells with low c-Myc expression, while the
expression ratio of PKM2/PKM1 changed slightly in
P4936 cells silenced c-Myc and overexpressed NEK2 (Fig. 5b).
Consistently, with the expression alteration of PKM2 and
PKM1, both glucose uptake and lactate production were
significantly decreased in P493-6 cells with low c-Myc
expression. However, P493-6 cells overexpressed NEK2
showed high glucose uptake and lactate production
regardless of c-Myc alteration (Fig. 5c). These results indicate that
NEK2 can partially neutralize downregulation of
c-Mycmediated decrease of the PKM2/PKM1 ratio and aerobic
glycolysis. To further determine the functional role of
NEK2 in c-Myc regulated aerobic glycolysis, we evaluated
cell proliferation and cell viability in NEK2 OE and EV
Fig. 5 NEK2 mediates c-Myc-regulated aerobic glycolysis. a P493-6 cells with or without NEK2-OE were treated with Dox to inhibit c-Myc expression.
Western blots show the protein expression of c-Myc, NEK2, and PKM2. b Real-time PCR shows the relative expression of PKM1 and PKM2 in P493-6 cells
with altered expression of c-Myc and NEK2. c Glucose uptake and lactate production were evaluated in P493-6 cells with altered expression of c-Myc
and NEK2. d Cell growth was analyzed in P493-6 cells with altered expression of c-Myc and NEK2 by trypan blue staining (*p < 0.05). e Flow cytometry
analysis of apoptosis in P393-6 cells with silencing of c-Myc in the presense or absence of NEK2 overexpression using FITC-conjugated annexinV/PI
staining. Apoptotic cells were annexinV positive. Representative pictures of FCM were shown with quantification of percentage of cells with apoptosis.
Results from 3 independent experiments were shown. f Cell viability was analyzed in P493-6 cells with altered expression of c-Myc and NEK2 using
trypan blue staining, *p < 0.05
P493-6 cells after silencing of c-Myc. Notably, NEK2 OE
P493-6 cells grow faster than EV cells in the presence or
absence of c-Myc (Fig. 5d). Silence of c-Myc induced
significantly P493-6 cell apoptosis (EV NEK2-OE = result in
45.80 ± 0.43%, 25.90 ± 0.58%; p < 0.05) (Fig. 5e). Moreover,
P493-6 cells overexpressing NEK2 cells showed higher
viability than those control cells (Fig. 5f ). These results
demonstrated that NEK2 is involved in c-Myc-regulated aerobic
glycolysis which promotes cancer cell proliferation.
Almost one century ago, Dr. Warburg observed that
cancer cells, unlike normal cells, rely on glycolysis to
generate the energy needed for cellular processes rather
than mitochondrial respiration despite of oxygen
available . Recently, some factors have been found to
regulate Warburg effect including tumor microenvironment,
stabilization of hypoxia inducible factor 1 (HIF1),
oncogenic activation and/or tumor suppressor genes’
inhibition, mitochondrial dysfunction, glutamine metabolism,
and post-translational modifications . Our data from
this study indicate that NEK2 plays an important role
via regulating aerobic glycolysis resulting in MM cell
proliferation. Reprogramming of energy metabolism is
one of the eight hallmarks acquired during the multistep
development of human tumors [51, 52]. Genomic
instability, which causes genetic diversity, underlies these
hallmarks [51, 52]. We have demonstrated that high
NEK2 expression induces chromosomal instability and
cancer cell proliferation . In this study, we have
shown that NEK2 binds and interacts with hnRNPA1
and hnRNPA2, which control pyruvate kinase mRNA
splicing in cancer cells, and increases PKM2 expression
and PKM2/PKM1 ratio in myeloma cells. The complex
of hnRNPA1, hnRNPA2, and PTB binds to intronic
sequences flanking exon 9 (contained in PKM1) and
suppresses its splicing and activates exon 10 splicing of
PKM (contained in PKM2), resulting in upregulation of
PKM2 expression and downregulation of PKM1 .
Our RNA immunoprecipitation showed that NEK2
binds to intronic sequences flanking exon 9 of PKM
premRNA. Overexpression of NEK2 upregulates the
expression of PMK2 while decreases PKM1 expression leading
to increased PKM2/PKM1 ratio compared to control
cells. Our data demonstrate that knockdown of NEK2 in
myeloma cells decreased expression of PKM2 and the
ratio of PKM2/PKM1. Knockdown of NEK2 also altered
expression of critical genes involved in glycolysis under
normoxia and/or hypoxia. The glucose uptake and
lactate production were also impaired when NEK2 was
knocked down. Because PKM2 is an essential enzyme
for regulation of aerobic glycolysis in cancer cells, we
further determine that NEK2 expression is increased in
high-risk patients and positively correlates with aerobic
glycolysis genes including HK2, ENO1, and LDHA. The
subsequent assays show both glucose uptake and lactate
Fig. 6 Schematic model of NEK2-mediated aerobic glycolysis through splicing of PKM1/2. c-Myc enhances the transcription of NEK2 and hnRNPA1/2,
then NEK2 and hnRNPA1/2 complex bind to the intronic sequences flanking exon 9 of PKM pre-mRNA to out splicing exon 10 result in elevated
expression of PKM2 and increased aerobic glycolysis
production decrease in NEK2 silenced myeloma cells.
The clinical data for survival analyses indicate that
myeloma patients with high NEK2 and PKM2 had the
shortened survival. Together, NEK2 promotes aerobic glycolysis
through activating pyruvate kinase mRNA splicing in
myeloma cells. NEK2 is a lethal target of c-Myc , we defined
that c-Myc directly binds to the NEK2 promoter sequence
and regulate its expression. We and others showed that
cMyc induced neoplastic tumor cells undergo high aerobic
glycolysis in accordance with Warburg effect . This
effect demonstrates that most cancer cells take up large
amount of glucose and convert them into lactic acid for
generation of energy in the presence of oxygen but reduce
rate of pyruvate oxidation. c-Myc is one of the most
frequently deregulated oncogenes in human malignancies
especially B cell lymphomas and multiple myeloma [54–56].
c-Myc increases PKM2 expression which regulates
chromosome segregation and cell cycle G1/S transition as
well as aerobic glycolysis in tumor cells . In light with
these studies, our ChIP-PCR confirmed that c-Myc directly
binds to the promoter of NEK2 in c-Myc overexpressing
P493-6 cells. Inhibition of c-Myc in P493-6 cells decreases
the expression of NEK2 and PKM2. Given that NEK2
regulates PKM2 expression and that the expression and activity
of NEK2 and PKM2 are controlled by c-Myc, NEK2 might
be involved in c-Myc regulated aerobic glycolysis. This was
supported by evidence that the PKM2/PKM1 ratio and
aerobic glycolysis were significantly decreased in P493-6 cells
by knocked down c-Myc, while overexpression of NEK2
blocked this decrease. Furthermore, knockdown of
c-Mycinduced cell death and cell growth arrest can be rescued by
overexpression of NEK2. We conclude that NEK2 is a
novel c-Myc target for regulation of PKM splicing and
aerobic glycolysis in myeloma. In general, our data shows the
first evidence that NEK2 promotes aerobic glycolysis and
provides mechanistic insights into how NEK2 regulates
aerobic glycolysis in MM. Our study not only uncovers a new
function of NEK2 but also contributes to study aerobic
glycolysis mechanism in cancer. Previous studies have
demonstrated that NEK2 promotes drug resistance in
multiple myeloma , it is very likely that enhanced aerobic
glycolysis by NEK2 may contribute to its function in drug
resistance. We also speculate that targeting aerobic
glycolysis may overcome NEK2 induced drug resistance in
In this study, we characterize NEK2 as a new positive
regulator of aerobic glycolysis through regulating PKM
pre-mRNA splicing. NEK2 is a direct target of the
transcription factor c-Myc and is involved in c-Myc-induced
aerobic glycolysis. We demonstrate that NEK2 may
interact with hnRNPA1 and hnRNPA2 proteins to
regulate PKM splicing and aerobic glycolysis (Fig. 6).
ChIP-qPCR: Chromatin immunoprecipitation-QPCR; Co-IP:
Coimmunoprecipitation; Dox: Doxycycline; EFS: Kaplan-Meier analyses of
event-frees; GEP: Gene expression profile; OS: Overall survivals; RIP: RNA
immunoprecipitation; shRNA: Small hairpin RNA; TAP-MS: Tandem affinity
purification plus mass spectrometry
This work was also supported by NIH grants R01CA152105 (F.Z.), the Multiple
Myeloma Research Foundation (F.Z.), the International Myeloma Foundation
(F.Z.), the America Society of Hematology (ASH) Bridge (F.Z.), the Leukemia &
Lymphoma Society TRP (6094-12), and institutional start-up funds from the
Department of Internal Medicine, Carver College of Medicine, University of
Iowa (F.Z. and G.T.).
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