Natural Product Micheliolide (MCL) Irreversibly Activates Pyruvate Kinase M2 and Suppresses Leukemia

Article abstract of DOI:10.1021/acs.jmedchem.8b00241

Metabolic reprogramming of cancer cells is essential for tumorigenesis in which pyruvate kinase M2 (PKM2), the low activity isoform of pyruvate kinase, plays a critical role. Herein, we describe the identification of a nature-product-derived micheliolide (MCL) that selectively activates PKM2 through the covalent binding at residue cysteine424 (C424), which is not contained in PKM1. This interaction promotes more tetramer formation, inhibits the lysine433 (K433) acetylation, and influences the translocation of PKM2 into the nucleus. In addition, the pro-drug dimethylaminomicheliolide (DMAMCL) with similar properties as MCL significantly suppresses the growth of leukemia cells and tumorigenesis in a zebrafish xenograft model. Cell-based assay with knock down PKM2 expression verifies that the effects of MCL are dependent on PKM2 expression. DMAMCL is currently in clinical trials in Australia. Our discovery may provide a valuable pharmacological mechanism for clinical treatment and benefit the development of new anticancer agents.

Full text of DOI:10.1021/acs.jmedchem.8b00241

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Natural Product Micheliolide (MCL) Irreversibly Activates
Pyruvate Kinase M2 and Suppresses Leukemia
Jing Li, Shanshan Li, Jianshuang Guo, Qiuying Li, Jing Long, Cheng Ma, Yahui Ding, Chunli Yan,
Liangwei Li, Zhigang Wu, He Zhu, Keqin Kathy LI, Liuqing Wen, Quan Zhang, Qingqing Xue, Caili
Zhao, Ning Liu, Ivaylo Ivanov, Ming Luo, Rimo Xi, Haibo Long, Peng George Wang, and Yue Chen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00241 • Publication Date (Web): 11 Apr 2018
Downloaded from http://pubs.acs.org on April 11, 2018
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Natural Product Micheliolide (MCL) Irreversibly
Activates Pyruvate Kinase M2 and Suppresses
Leukemia
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Jing Li,† Shanshan Li,‡ Jianshuang Guo,† Qiuying Li,† Jing Long,† Cheng Ma,‡ Yahui Ding,†
Chunli Yan,‡ Liangwei Li,‡ Zhigang Wu,‡ He Zhu,‡ Keqin Kathy Li,‡Liuqing Wen,‡ Quan
Zhang,† Qingqing Xue,† Caili Zhao,† Ning Liu,† Ivaylo Ivanov,‡ Ming Luo,‡ Rimo Xi,† Haibo
Long,§ Peng George Wang,*,† ,‡ Yue Chen*, †
†State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key
Laboratory of Molecular Drug Research, Nankai University, Haihe Education Park, 38 Tongyan
‡
Road, Tianjin 300350, China. Center for Diagnostics and Therapeutics, Department of
§
Chemistry, Georgia State University, Atlanta, GA 30303, USA. Department of Nephrology,
Zhujiang Hospital, Southern Medical University, Guangzhou, 510280, China.
KEYWORDS: micheliolide, pyruvate kinase, activator, leukemia
ABSTRACT Metabolic reprogramming of cancer cells is essential for tumorigenesis, in which
pyruvate kinase M2 (PKM2), the low activity isoform of pyruvate kinase, plays a critical role.
Herein, we describe the identification of a nature-product-derived micheliolide (MCL) that
selectively activates PKM2 through the covalent binding at residue cysteine424 (C424), which is
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not contained in PKM1. This interaction promotes more tetramer formation, inhibits the
lysine433 (K433) acetylation, and influences the translocation of PKM2 into the nucleus. In
addition, the pro-drug dimethylaminomicheliolide (DMAMCL) with similar properties as MCL,
significantly suppresses the growth of leukemia cells and tumorigenesis in a zebrafish xenograft
model. Cell-based assay with knock down PKM2 expression verifies the effects of MCL are
dependent on PKM2 expression. DMAMCL is currently in clinical trials in Australia and shows
encouraging treatment results, our discovery may provide valuable pharmacological mechanism
for the clinical treatment and benefit the development of new anti-cancer agents.
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INTRODUCTION
Metabolic reprogramming towards aerobic glycolysis is a critical hallmark of cancers,
enabling cancer cells to obtain more biosynthetic materials for the growth and division of rapidly
proliferating cells.1 Pyruvate kinases, which catalyzes the conversion of phosphoenol-pyruvate
(PEP) to pyruvate at the final step of glycolysis, has been shown to be crucial in the regulation of
cancer metabolic reprogramming.2-3 Multiple lines of evidence have indicated that the M2
isoform of pyruvate kinase (PKM2) is upregulated in most cancer cells and tumors tested so far.1
In contrast to its splice variant PKM1, which is expressed in normal differentiated tissues as a
stable tetramer, PKM2 exists in equilibrium among monomer, dimer, and tetramer forms, which
was regulated by the allosteric binding of metabolic effectors or posttranslational modifications
(Figure 1A).2-4 These different catalytic and regulatory properties are all ascribe to 22 distinct
amino acids between PKM1 and PKM2 (Figure 1B).1 In cancer cells, more negative allosteric
modulations, including phosphor-tyrosine and numerous post-translational modifications, cause
the PKM2 tetramer to dissociate into a dimeric formation. Dimeric PKM2 is much less active,
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and its presence diverts the glycolytic flux to increased biomass production, contributing to
oncogenesis.5-6 In addition, dimeric PKM2 has the potential to translocate into the nucleus to act
as transcriptional regulators of genes that promote tumorigenesis and migration.5-8 Consequently,
much effort has focused on the discovery and development of small-molecule PKM2 activators
to promote tetramer formation or initiate its activity.9, 10 Herein, we describe a novel natural-
product-derived PKM2 activator that shows promising efficacy for leukemia treatment and has
entered clinical trials.
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Figure 1. MCL is a novel allosteric activator of PKM2. (A), Allosteric regulations of PKM2. (B),
Splice variant amino acids between PKM1 and PKM2. The 22 distinct amino acids are marked in
red for PKM1 and in blue for PKM2. (C), the mechanism of sustainable release of MCL by
DMAMCL under neutral conditions. (D), Inhibition profile of cancer cell lines by DMAMCL.
Eight cancer cell lines were incubated with different concentrations of DMAMCL for 48 h. Cell
proliferation was determined by MTT assay. Graphs depict mean ± SEM from 6 independent
experiments.
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Sesquiterpene lactones (SLs) are a class of natural products with varied biological and
pharmacological activities, including the common incorporation of trans- or cis-fused γ-lactone
containing an α-methylene group.11 Parthenolide (PTL), a SL isolated from feverfew (Tanacetum
parthenium L.) reportedly inhibits the proliferation of various human cancer cells in vitro.12, 13
However, PTL’s lack of stability under both acidic and basic conditions, coupled with poor
solubility, has severely limited its medicinal applications.14 In our previous work, we identified
micheliolide (MCL), a more stable guaianolide SL (GSL) (Figure 1C), as a new potent lead
compound for reducing the proportion of acute myelogenous leukemia (AML) cells and glioma
cells.15, 16 Most strikingly, dimethylaminomicheliolide (DMAMCL, commercial name: ACT001),
the pro-drug of MCL, can release MCL slowly under physiological conditions16 and was recently
approved for clinical trials in Australia (trial ID: ACTRN12616000228482). However, the
pharmacodynamic markers and cellular mechanism of DMAMCL remain unknown, making
their identification crucial for clinical treatment and designing more effective treatments.
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In this present work, we determined that MCL is a novel natural-product-derived, covalent,
and selective activator of PKM2 via binding to the conserved cysteine424 (C424) residue of
PKM2, but not PKM1. This binding induces the irreversible tetramerization of PKM2, which is
essential for high pyruvate kinase activity. Concomitantly, this allosteric regulation decreases
lysine433 (K433) acetylation of PKM2, and lowers PKM2 nucleus translocation, which is vital
for its non-metabolic function in cell proliferation and tumorigenesis. Combined with the
proliferation inhibition of several cancer cell lines and the repression of leukemia in vivo,
DMAMCL represents the first natural lead compound for the development of PKM2-targeted
therapeutic agents to enter clinical development.
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RESULTS AND DISCUSSION
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Micheliolide derivative DMAMCL inhibits multiple cancer cells in vitro.
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16
Our previous work has demonstrated that MCL is a potent inhibitor of AML cells
and
glioma cells.15 To increase the bioavailability of MCL, we also synthesized the dimethylamino
16
Michael adduct of MCL (DMAMCL),
which exhibited high stability, lower toxicity and
sustainable release of MCL, under physiological or neutral conditions (Figure 1C). Here, a wide
range of cancer cell lines, including drug-resistant and sensitive leukemia cell lines (HL-60,
K562, and KG-1a), a human pancreatic adenocarcinoma cell line (SW1990), and a human
neuroblastoma cell line (SH-SY5Y), were treated with DMAMCL, and assayed for the cell
viability by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazo- lium). Cell growth was
inhibited in all treated cancer cell lines (IC50: 3.9–16.2 µM; IC90: 16.4–54.14 µM) in a
concentration-dependent manner (Figure 1D and Figure S1). Consistent with our previous
studies, the leukemia cell lines were found to be more sensitive to DMAMCL, with an IC50 of
less than 12 µM.16
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Figure 2. MCL directly binds to PKM2. (A), Structures of MCL (1, active) and S-MCL (2,
inactive). (B), Michael addition mechanism of MCL and cysteine. (C), Strructure of Biotin-MCL
(3, active) and Biotin-S-MCL (4, inactive). (D), Silver staining of the pull
cell lysates were incubated with 3 and 4 at 4°C overnight. For the treatment of 3, a high
concentration of MCL was added to the co-incubation. The lysates were used for streptavidin
agarose pull-down assays, and the precipitates were resolved by SDS-PA E, followed by silver
ut developed by anti-
recombinant proteins
l-down fraction. HL60
-
G
staining. (E), Western blot with the same sample as in silver staining b
PKM2. (F), Western blot detection of the binding abilities of 3 and 4 withh
PKM1 and PKM2, respectively. rPKM1 and rPKM2 were incubated witth probe 3 or 4 at 4°C
overnight. The mixture was then subjected to Western blot with streptaviidin-HRP to detect the
biotin-conjugated complex (top). The same sample was developed wwith anti-PKM1/PKM2
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antibody (bottom) to show the sample amount. All experiments were repeated at least three times,
with similar results.
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MCL specifically targets PKM2, not PKM1.
To investigate the functional target of MCL (1) that is responsible for its anti-cancer activity,
we prepared biotin-conjugated probes for affinity purification. Our preliminary structure-activity
relationship (SAR) study revealed that when the 11,13-double bond of MCL was reduced to a
single bond to produce S-MCL (2) (Figure 2A), the inhibitory activity of MCL against leukemia
cells was completely abolished, even at a higher concentration (10 μM) .16 These data indicate
that the 11,13-double bond of MCL (1) is crucial for its anti-cancer activity, most likely acting as
a reactive Michael acceptor and forming a covalent bond with the cysteine residues of its target
protein (Figure 2B). In contrast, MCL with slight modification to its C-region largely
maintained its activity against AML cells at a level comparable to that of MCL.16 Thus, we
incorporated a biotin tag in the C-domain of MCL (1) and its inactive analogue S-MCL (2) to
yield a positive probe (3, Biotin-MCL) and a negative probe (4, Biotin-S-MCL) respectively
(Figure 2C and Scheme S1).
Next, HL60 cell lysates were incubated with positive (3) and negative (4) probes, and the
mixtures were separately pulled down with streptavidin-coated agarose beads, followed by gel
electrophoresis and silver staining. A single band with a molecular mass of ~60 kDa was clearly
precipitated by 3 but not by 4 (Figure 2D). In addition, this band could be suppressed by a high
concentration of MCL. Peptide mass fingerprinting data analysis identified the MCL-bound
protein was PKM2 (Figure S2). Using immunoblotting, we also observed the presence of PKM2
in the precipitates (Figure 2E). To verify the selectivity of this binding, we expressed
recombinant PKM2 (rPKM2) and PKM1 (rPKM1) in vitro and incubated them with positive (3)
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and negative (4) probes respectively. The positive probe (3) effectively bound to rPKM2 but not
rPKM1 (Figure 2F), further supporting the notion that PKM2, not PKM1, is the direct target of
MCL.
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Figure 3. MCL targets C424 of PKM2 and activates its pyruvate kinase activity. (A), MS/MS
analysis of the C424-containing tryptic peptide for rPKM2 incubated without (top) and with
(bottom) MCL for 60 min at RT. C in red color represents the cysteine bound by MCL. (B),
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Recombinant WT PKM2 and its mutant (C424S) incubated with biotin-MCL for 60 min at RT,
followed by SDS-PAGE (bottom) and Western blot to detect biotin (top). (C), Recombinant
PKM2 proteins were incubated with MCL, TEPP-46 at 4 µM for 50 min at RT, and their
pyruvate kinase activity was monitored over 40 min. (D), Determination of Kobs for the
interaction of PKM2 (0.25 µM) with biotin-MCL (20 µM) for different periods of time, as
described in the Supplementary Methods. (E), Plotting the Kobs values for the binding of PKM2
as a function of MCL at different concentrations.
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C424 of PKM2 is critical for binding to MCL.
The 11,13-double bond of MCL (1) is a reactive Michael acceptor, we speculated that some
conserved cysteine residues in PKM2 are the binding sites of MCL. To identify the specific
residues modified by MCL, we incubated the rPKM2 protein with or without MCL. After the
reaction, the rPKM2 protein was digested with trypsin and then analyzed by LC-MS/MS. The
mass of the Cys424-containing peptide CCSGAIIVLTK was measured as 1220.626 Da in the
absence of MCL and 1411.745 Da in the presence of MCL. The calculated mass shift of 191.120
Da is consistent with the addition of one molecular unit of MCL. MS/MS analyses of both
unmodified and modified C424-containing peptides provided a partial series of y-ion fragments
corresponding to the peptide sequence. Both MS/MS spectra had the same mass from y2 to y9,
whereas the mass shifted by 191.120 Da for the C424-containing fragment (from y10) in the
spectra of the modified peptide (Figure 3A). Interestingly, C424 is located at the C-C interface
of two dimers, which can affect subunit interaction, enzymatic activity, or both.17 To confirm
that C424 residue was the only modified site of MCL, rPKM2 and mutant rPKM2-C424S were
further expressed and incubated with biotin-MCL (3) or inactive biotin-MCL (4) and detected by
western blot. Contrast to rPKM2-C424, only rPKM2 incubated with biotin-MCL (3) produced a
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signal with anti-biotin, supporting our conclusion that MCL selectively binds to PKM2 at C424
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site (Figure 3B).
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MCL activates the pyruvate kinase activity of PKM2.
To determine the affect of MCL on PKM2 activity, we measured the pyruvate kinase activity
of rPKM2 proteins in the presence or absence of MCL with TEPP-46 as positive control. The
results demonstrated that MCL effectively promoted the pyruvate kinase activity of rPKM2 with
half-maximum activating concentration (AC50) at 6 nM, which was better than TEPP-46 (AC50
= 92 nM) (Figure 3C; Figure S3). The potent activation of MCL may be due to the covalent
binding of MCL, which irreversibly activates PKM2 with cumulative effect. However, the action
mode of TEPP-46 is reversible and dependent on the available of FBP.9
To test if MCL binds to PKM2 through an irreversible covalent modification, we next
assessed this binding at certain time points for several biotin-MCL concentrations. The profile of
PKM2 binding with biotin-MCL at various concentrations revealed a time-dependent saturation
(Figure 3D), consistent with an irreversible modification mechanism.18 The data were fit to
determine the observed rate constants for binding (Kobs) at each concentration. Plotting the Kobs
values as a function of the biotin-MCL (3) concentration revealed a saturation curve (Figure 3E).
These data support a two-step reaction for the activation of PKM2 by MCL (1) that involves the
non-covalent reversible binding of PKM2 to biotin-MCL (initial binding step, Ki), which places
the moderately reactive electrophile of MCL (1) close to a specific nucleophile on the protein,
followed by a second step that results in a specific covalent linkage (kact). Based on this model,
the kact value for PKM2 binding with biotin-MCL (3) was calculated to be 0.031 min-1 (Figure
3E), suggesting a high specific reactivity, and the Ki value was 0.05 µM (Figure 3E), indicating
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a high affinity. Overall, the efficiency of the binding of MCL to PKM2 was high (kact / Ki
=1.03*104 M-1s-1), demonstrating that MCL (1) is a potent irreversible activator of PKM2.
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MCL promotes increased tetramer formation of PKM2 in vitro or in cells.
Tetrameric PKM2 is the only form with high pyruvate kinase activity, we next evaluated
whether the activation of MCL is accompanied by the tetramerization of PKM2. Size-exclusion
chromatography analysis and fraction detection by SDS-PAGE were performed on rPKM2 and
MCL-treated rPKM2. Chromatography of rPKM2 indicated that rPKM2 contained a mixed
population of configurations (i.e., monomers, dimers and tetramers), with a tendency to
tetramerize at higher concentrations (Figure S4A). Thus, a low concentration of rPKM2 (0.3
mg/mL), with a higher population of monomers, was used to detect the allosteric effect of MCL.
As expected, the incubation of rPKM2 with 10µM MCL resulted in a significant shift towards a
tetrameric configuration (Figure S4B, C).
Large existing phosphotyrosine (pTyr) proteins in cancer cells can bind PKM2 and catalyze
the release of FBP from the enzyme.19 To verify the same promotion effect of MCL on
endogenous PKM2, we cultured leukemia HL60 cells treated with DMSO, MCL, pervanadate (a
tyrosine phosphatase inhibitor, which can increase the amount of pTyr proteins) and pervanadate
combined with MCL. The cellular proteins including endogenous PKM2 were then extracted,
normalized, and analyzed by the same chromatographic method. According to tetramer and
monomer formation of PKM2, different fractions were collected and detected by western blot
with anti-PKM2 antibody. Compared to DMSO treatment, PKM2 with only pervanadate
treatment appeared primarily as a dimer. In contrast, MCL treatment increased tetramer
formation of PKM2, regardless of the presence or absence of pTyr. These data indicate that MCL
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acts as a potent tetramer inducer of PKM2 and cannot be competed-out by pTyr, even after
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treating the cells with pervanadate (Figure 4A).
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Figure 4. MCL promotes PKM2 tetramer formation and prevents AcK433 modification and
nucleus translocation. (A), PKM2 subunit association was determined by western blot using anti-
PKM2 (Cell signaling technology, 4053) of the chromatographic fractions of HL60 cells treated
with MCL, pervanadate, and MCL combined pervanadate, lysed hypotonically and analyzed by
size-exclusion chromatography. Mr, relative molecular weight. (B), Effect of MCL on the
AcK433 of PKM2. HL60 cells were preincubated with MCL (0, 2.5, 5 and 10 µM) for 20h,
followed by detection of endogenous PKM2 and AcK433 PKM2 by western blot. (C),
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Subcellular localization of total PKM2 and MCL treated PKM2 was examined by
immunofluorescence microscopy in HL60 cells. HL60 cells were treated with MCL (5 µM) or
DMSO for 20 h and reacted with anti-PKM2. After overnight incubation, the second antibody
Alexa Fluor 647 conjugated goat anti-rabbit IgG (red) for PKM2 and Hoechst 33342 (blue) for
nucleus were added to image.
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MCL decreases the K433 acetylation of PKM2 and reduces its nuclear translocation.
Recently, Lv et al. reported that upon the stimulation of mitogenic and oncogenic signals,
PKM2 was acetylated by p300 acetyltransferase at lysine433 (K433), which inactivats PKM2 by
interfering with FBP binding and promotes the nuclear accumulation.20 Dimer PKM2, when
located to the nucleus, can function as a protein kinase and transcriptional coactivator to trigger
the expression of genes that promote tumoregenesis and migration.8, 20, 21 To explore the
intracellular effect of MCL on PKM2, we incubated HL60 leukemia cells with DMSO or MCL,
then extracted the cellular protein to detect the AcK433 modification of PKM2. Western blot
developed with anti-AcK433-PKM2 indicated that AcK433 decreased with MCL treatment in a
dose-dependent manner (Figure 4B, bottom). Meanwhile, western blot developed with anti-
PKM2 revealed that cellular PKM2 was present in comparable amounts among the different
samples (Figure 4B, top). These data indicate that the MCL treatment has no effect on the potein
level of PKM2, but reduces the acetylation at K433, contributing to the tetramerization of PKM2
indirectly. Interestingly, immune-fluorescence analysis revealed that MCL treatment at 5 µM
(note: most cells died at 10 µM, presumably as a result of nuclear damage) for 12 h resulted in
retardation of the nuclear accumulation of PKM2 in HL60 cells (Figure 4C). Western blot
analysis of sub-cellular fraction also confirmed the decrease of PKM2 in nuclear retardation
(Figure S5). These results demonstrate that MCL is an effective PKM2 modulator, which
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inhibits both acetylation modification and nuclear translocation in cells, further suppresses the
transcriptional regulation in tumorigenesis.
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Figure 5. Activator MCL promotes PKM2 tetramer formation (PDB ID: 11ZJH and 1T5A, 1ZJH
is the apo tetrameric PKM2 structure. 1T5A is the FBP bound tetrameric PKM2 structure). (A),
Computed B-factors mapped onto the PKM2 models highlights confoormational differences.
Coloring corresponds to computed B-factor values from high (red) to low (blue). (B), Computed
free energy of AA’ and CC’ interface. Per residue binding energies coomputed from the 1D
MM/GBSA decomposition. Residues are colored according to the value off the binding energy in
kcal/mol from red (positive) to blue (negative). The bound MCL are showwn as magenta spheres.
The large (AA’) and small (CC’) interfaces between monomers are pressented as dashed lines.
Only one monomer is shown.
Structural analysis of the binding mode between MCL and PKM2.
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To better understand the structure and regulatory properties of MCL binding to PKM2, we
docked MCL into the human X-ray structure of PKM2 (PDB ID: 1T5A) using the docking
package GOLD 5.1.22 Each PKM2 monomer is composed of four domains: the N-terminal, A-,
B- and C-domains (Figure S6A, B, C), and can form oligomeric assemblies (dimers, tetramers
and, to a lesser extent, higher order oligomers). In order to mimic covalent binding, we defined
two linking atoms, one from the MCL ligand (C13, Figure 1C) and the other from Cys424 (S),
and forced them to occupy the same steric volume.
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In the dimer formation, the subunit interface is formed by the adjacent C-domains (C-C' dimer
interface, Figure S6A). In the tetramer formation, an additional interface is introduced between
the neighboring A-domains (A-A' tetramer interface, Figure S6A). We firstly docked MCL
moiety among the A-, C- and N-terminal domains, and then performed extensive molecular
dynamics (MD) simulations and molecular mechanics/generalized Born surface area continuum
solvation (MM/GBSA) calculations to assess the stability of the tetramer or dimer with or
without MCL binding. The relative flexibilities of the MCL-bound and MCL-free complexes
were determined from the computed per residue B-factors. These values were calculated over the
last 40 ns of the MD trajectories, and subsequently mapped onto the PKM2 structures. Of the
four PKM2 domains (A-, B-, C- and N-terminus), the B-domain experienced larger fluctuations
during MD, while the A- and C- domains were less mobile, as these domains were involved in
dimer and tetramer formation (Figure 5A). Moreover, in the MCL-bound state, the A domain
exhibited considerably less flexibility, and the N-terminal domain induced tetramer formation,
which indicated that MCL binding can significantly stabilize the A-A' tetramer interface (Figure
5A). As for the interfacial binding energies of A-A' (tetramer formation) and C-C' (dimer
formation), C-C' binding energies were little fluctuated, but A-A' binding energies were greatly
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enhanced by the addition of MCL, and the binding free energy ΔGb increased from −253.8
kcal•mol–1 (PKM2) to −322.3 kcal•mol–1 (PKM2•MCL complex) (Figure 5B). To identify the
structural features responsible for stabilizing the A-A’ and C-C’ interfaces, we decomposed ΔGb
into per residue contributions (1D MM/GBSA decomposition) Residue contributions were color
mapped onto the structures of the respective interfaces (Figure S6D, E). In addition, ΔGnp (or
the buried surface area, BSA) correlated well with the overall binding energies, indicating that
MCL promoted tetramer formation by increasing the BSA at the tetramer interface. Overall,
these data suggest that new interactions at A-A' interface with MCL binding appear to be directly
responsible for the observed increase in binding free energy and stabilization of the PKM2
tetramers. In contrast, TEPP-46 was found in the A-A' interface and interacts with the
surrounding residues Asp354, Ala388, Tyr390 and Leu394 through polar and van der Waals
interactions. The binding residues and interaction energy of TEPP-46 are quite different from
that of MCL.
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Figure 6. The anticancer effects of DMAMCL are dependent on PKM2. (A), Western blot
detection of the expression of PKM1, PKM2 and PKM in the absence of endogenous PKM2，
which was knocked down with short hairpin RNA. (B), Cell-based detection of pyruvate activity
of HL60 cells with shPKM2 and shControl. (C), Cell viability detection of pyruvate activity of
HL60 cells with shPKM2 and shControl. (D), PKM2 depletion desensitizes cancer cells to MCL
treatment. (E), Representative fluorescent images of HL60 cell xenografted zebrafishes with
treatment of DMAMCL, cytarabine or negative control. Approximately 300 HL60 cells were
injected into the yolk sac of embryos. After injections, xenografted embryos were treated with a
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series of doses of DMAMCL (1, 3, 10 µg/mL), cytarabine at 200 µg/mL as a positive drug
control, and saline as a negative control. After 3 days incubation, the number of embryos
exhibiting cancer cell dissemination was counted by microscopic observation (15 larvae / group).
Data represent the average ± SD of three independent assays. * = P < 0.05. ** = P < 0.01.
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Anticancer activity of MCL is dependent on the expression of PKM2.
To investigate the influence of PKM2 on the MCL-mediated inhibition of leukemia cell
viability, we knock down the expression of PKM2 in HL60 cells by lentiviral delivery of specific
short hairpin RNA for PKM2 (shPKM2) or mock (shConrol). Western blot showed that shPKM2
inhibited the expression of PKM2 but not PKM1 (Figure 6A), indicating the specificity of
shPKM2. Concomitantly, stable knockdown of PKM2 resulted in decreased pyruvate kinase
activity of PKM2 (69% of shControl) and reduced cell viability (83% of shControl) (Figure 6B,
C). These data were consistent to the results of Goldberg et al., in which cell viability was
drastically reduced among 10 cancer cell lines with the treatment of shPKM2.23 Then shPKM2
and shControl cells were treated with MCL or DMSO for 24 h and assayed the cell viability.
Results indicate that the shPKM2 cells are less sensitive to the inhibitory effect of MCL than
shControl cells (Figure 6D), implying the effect of MCL on cancer cell viability is mainly
dependent on PKM2.
Evaluation of the inhibitive effect of DMAMCL in xenograft zebrafish.
To assess the anti-cancer effects of DMAMCL, xenograft zebrafishes were established via
injection of human leukemia HL60 cells in zebrafish embryos. HL60 cells were first stained with
cell tracker CM-Dil (red fluorescence color) and then transplanted into zebrafish embryos at 2
days post fertilization (2dpf). Following transplantation, nine injected zebrafish groups were
treated with drugs at different dosages over the course of 3 days. Compared with the control
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group, 80% zebrafish died when the dosage of DMAMCL exceeded 20 µg/mL, whereas the
survival rate approached 100% at a concentration of 10 µg/mL or lower. Notably, with dosages
between 1–10 µg/mL, DMAMCL produced a dose-dependent decrease in cell dissemination
(Figure 6E) with respect to both proliferation and migration. Results of this study indicated that
optimal inhibition of dissemination was observed with DMAMCL treatment at a dose of 10
µg/mL, with proliferation and migration inhibition rates of 56.44% (P<0.01) and 58.10%
(P<0.01), which exceeded rates following cytarabine treatment (200 µg/mL) of 43.28% (P<0.01)
and 44.04% (P<0.01) (Table S1 and Table S2).
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These data demonstrate that the activation of PKM2 by MCL results in significant suppression
of tumor growth in vivo, indicating DMAMCL acts as a novel PKM2 activator with promising
potential for anticancer therapeutics.
CONCLUSIONS
Targeting tumor-specific metabolic pathways is a novel strategy for cancer therapy.1 Recent
studies have identified that PKM2 is abundantly expressed in AML cell lines and primary AML
patient samples.24-26 Combined with the up-regulation in other cancer cells, PKM2 has been
identified as a promising potential target for cancer therapy.3 However, PKM2 knockdown in
HCT116 and RKO colon cancer cells demonstrated that the growth of xenograft tumors is
unaffected in vivo.27 Moreover, PKM2 knockdown accelerates tumor formation in a Brcal-loss-
driven mice model of breast cancer.5 In contrast, the genetic replacement of PKM2 with active
PKM1 decreased the proliferation of cancer cells in vitro and suppressed the formation of cancer
cell xenograft tumors in mice.1, 9 Thus, low pyruvate activity caused by inactive state of PKM2
(dimer) is the key for the proliferation of cancer cells within tumors.5 Fixation PKM2 in its
active tetrameric form by activators is anticipated to be a novel treatment for cancer therapy.3,9,10
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MCL, a natural guaianolide sesquiterpene lactone which discovered in michelia compressa
and michelia champaca plants, has been shown to exert potent anti-cancer properties on AML
and glioma cells.15, 16 The present study demonstrated that DMAMCL inhibited several cancer
cells growth in vitro, and repressed tumor growth in leukemia HL60 xenografted zebrafish. To
determine the specific target of MCL, we analyzed the SAR of MCL and synthesized both a
positive (3) and negative (4) probe. Using this pair of probes, we found that MCL selectively
targeted PKM2 but not PKM1 in HL-60 cells. Through LC-MS/MS analysis and a PKM2
activity assay, we found that MCL selectively binds to the conserved C424 residue of PKM2,
promoting tetramer formation, and elevating pyruvate activity. These promising results indicate
that activation of PKM2 via natural product compound might be a useful anticancer strategy in
the clinic.
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Interestingly, recent advances reported that various stimuli and post-translational
modifications can trigger PKM2 translocation into nucleus.17, 20, 28, 29 Once accumulation in
nucleus, PKM2 functions as co-activator or protein kinase to promote tumor growth or
proliferation.7, 8, 21, 30, 31 In this study, we found that MCL inhibited the K433 acetylation of
PKM2 but not the protein level. Meanwhile, the nuclear PKM2 was reduced concomitantly,
which will block its signaling role for proliferation. To our knowledge, MCL is the first known
or reported covalently-bound, natural-product-derived PKM2 modulator, which interfere the role
of PKM2 both in cytoplasm and nucleus.
PKM2 integrates metabolic inputs and nuclear signaling in tumorigenesis, and has been
investigated as a novel target for cancer therapy. Data reported in this study argue that MCL, the
active drug derived from DMAMCL, enhances PKM2 activity by stably promoting the more
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active tetrameric formation and reduces its nuclear translocation, which will unveil the
mechanism of DMAMCL and inform clinical trials for cancer treatment.
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EXPERIMENTAL SECTION
Reagents and General Synthetic Procedures.
Reagents were purchased at the highest commercial quality and used without further
purification, unless otherwise stated. NMR spectra were recorded with a 400 MHz (1H: 400 MHz;
13C: 100 MHz) spectrometer and referenced to the solvent peak for CDCl3. Synthesis and
chemical confirmation of micheliolide (MCL), S-MCL, DMAMCL, biotin-S-MCL and biotin-
MCL are described in SI Materials and Methods. The purity of these compounds was
confirmed to be ≥95% by analytical HPLC with an ODS-C18 column (4.6 × 150 mm, 5 μm)
eluted at 1 mL/min with Milli-Q water and CH3CN. Antibodies and other reagents used in this
study were purchased directly from the vendors listed in SI Materials and Methods.
Cell lines and culture.
HL-60, KG-1a, K562, HL-60/ADR, KG-1a/ADR, K562/ADR, SW1990, SH-SY5Y were
purchased from ATCC and authenticated by short tandem repeat testing. All the cell lines were
maintained in appropriate medium as the manufacturer suggested. HL-60, KG-1a, K562, HL-
60/ADR, KG-1a/ADR, K562/ADR were cultured in RPMI 1640 medium (Sigma-Aldrich, St.
Louis, MO, USA) supplemented with 12% fetal bovine serum (FBS; Gibco BRL, Gaithersburg,
ML, USA), penicillin (100 IU/mL) and streptomycin (100 µg/mL) in a humidified incubator at
37°C and 5% CO2 / 95% air. Prior to each experiment, HL-60/ADR, KG-1a/ADR, K562/ADR
cells were treated with 1 µmol/ml ADR (Sigma-Aldrich, St. Louis, MO, USA) for 10–28 days
and then cultured for 10 days without ADR exposure. SW1990, SH-SY5Y were cultured in
DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine
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serum (FBS; Gibco BRL, Gaithersburg, ML, USA), penicillin (100 IU/mL) and streptomycin
(100 µg/mL). All the cell lines were tested for mycoplasma contamination and were found to be
negative.
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For experiments, cells were treated with compounds for different concentrations with 0.1%
dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) as solvent control. Where
indicated, 100 μM pervanadate was added 10 min before cell lysis.
Plasmids, shRNA virus production, and infection.
Expression plasmids for human PKM1 and PKM2, pET28a-PKM1 and pET28a-PKM2
plasmids were donated by Dr. Zhiren Liu’s group. To get cysteine424 mutated PKM2 plasmid,
site-directed mutant was constructed using QuikChange II XL Site-Directed Mutagenesis Kit
(Stratagene, USA) and primers (Forward: 5'-attatggccccactg gagcacttgaaggagg-3'; Reverse: 5'-
cctccttcaagtgctccagtgggg ccataat -3'). After confirming a 100% identities match by DNA
sequencing (Invitrogen, Shanghai, China), the E. coli strain BL21 was transformed with
constructs for next step of proteins purification.
The shRNAs targeting the sequence of human PKM2 (corresponding to exon 10 of the human
PKM) and corresponding negative control (NC) were designed as previous reported.23 The
shRNA for PKM2 was 5’-CCATAATCGTCCT CACCAA-3’; the shRNA for negative control
was 5’-CUUACGCUGAGUACUUCGA-3’. The recombinant lentivirus containing shPKM2 or
shControl was packaged using hU6-MCS-Ubiquitin-EGFP-IRES-puromycin lenti-viral vector
provided by Shanghai GeneChem (China). HL60 was infected by adding the lentiviral particles
to the culture with 5 μg/mL polybrene. For generating stable cell lines, infected cells were
selected with 1 μg/mL puromycin (Sigma) for 72 h.
Cell-based pyruvate kinase activity assay.
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HL60 or HL60-shPKM2 leukemia cells were plated at 2×105 cells per well (6-well plate) in
RPMI 1640 media plus 12% FBS. 100 μM pervanadate was added 10 minutes prior to cell lysis.
9 Then, cells were lysed on ice with NP-40 buffer containing 2 mM DTT and protease inhibitors
about 30 minutes and clarified by centrifugation at 14,000 g for 5 minutes. The PK activity in
lysates was determined by Pyruvate Kinase Activity Assay (Sigma, MAK072-1KT). 32
In vivo tumor models.
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All animal studies were conducted according to protocols approved by the Animal Ethics
Committee of Nankai University, in accordance with ‘Principles of Laboratory Animal Care and
Use Programs’. Zebrafish (albino) were obtained from the Zebrafish International Resource
Center (Eugene, Oregon, USA) and housed in an automated fish housing system (Hunter
Biotechnology Inc, China) at 28°C. Care and treatment of zebrafish were conducted in
accordance with guidelines approved by the Association for Assessment and Accreditation of
Laboratory Animal Care (AAALAC).
Statistical analysis.
Kaplan-Meier survival analysis (SPSS 10.0; SPSS, Inc.) was used to compare drug-treated
versus control animals. An unpaired Student’s t-test was used to evaluate the difference between
two different treatments, where a P-value of less than 0.05 was considered statistically significant.
Statistical analyses were performed using PRISM software (Irvine, CA, USA).
SUPPORTING INFORMATION
Supplementary figures and table, methods for chemical synthesis, MTT assay for DMAMCL,
pull-down of biotin-MCL bound proteins, molecular modeling, western blot, Immunofluore-
scence analysis, preparation of recombinant proteins, gel filtration of cell fraction, recombinant
PKM2 activity assay, protein identification by LC/MS/MS, kinetic determination of PKM2-MCL
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interaction, and anti-cancer evaluation in HL60 cells xenografted zebrafish. These materials are
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available free of charge via the Internet at http://pubs.acs.org.
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Molecular formula strings (CSV).
PDB ID CODES.
Docked model of 1 into the FBP bound tetrameric PKM2 structure (PDB ID: 1T5A) and apo
tetrameric PKM2 structure (PDB ID: 1ZJH) were shown in Figure 5 and Figure S5. Authors
will release the atomic coordinates upon article publication.
ABBREVIATIONS
MCL, micheliolide; S-MCL, single bond MCL; Biotin-MCL, biotin conjugated micheliolide;
Biotin-S-MCL, biotin conjugated single bond micheliolide; PKM2, pyruvate kinase M2; PKM1,
pyruvate kinase M1; PEP, phosphoenol-pyruvate; SLs, sesquiterpene lactones; PTL, parthenolide;
GSL, guaianolide sesquiterpene lactone; AML, acute myelogenous leukemia; DMAMCL,
dimethylaminomicheliolide; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazo- lium; SAR,
structure-activity relationship; rPKM1, recombinant PKM1; rPKM2, recombinant PKM2; AC50,
half-maximum activating concentration; pTyr, phosphotyrosine; MD, molecular dynamics; Kobs,
the observed rate constants for binding; Ki,, the apparent dissociation constant for the initial
PKM2: biotin-MCL complex; MM/GBSA, molecular mechanics/generalized Born surface area
continuum salvation; shPKM2, specific short hairpin RNA for gene PKM2; shConrol, specific
short hairpin RNA mock; 2dpf, 2 days post fertilization; AAALAC, Assessment and
Accreditation of Laboratory Animal Care; PME, the particle mesh Ewald; MM/GBSA, the
generalized Born and surface area continuum salvation; IPTG, isopropyl β-D-1-
thiogalactopyranoside.
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AUTHOR INFORMATION
Corresponding Authors
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*Yue Chen: yuechen@nankai.edu.cn
*Peng G. Wang: pwang11@gsu.edu
Funding Sources
This work was supported, in part, by grants from the National Science Fund for Distinguished
Young Scholars of China (NO. 81625021), the National Institutes of Health (U01GM116263),
National Institutes of Health (GM110387), the Natural Science Foundation of China (NO.
31000371)，the International Science & Technology Cooperation Program of China (NO.
2015DFR40460), the Natural Science Foundation of Tianjin (15JCYBJC29000). Computational
resources were provided in part by a National Science Foundation XSEDE allocation
(CHE110042) and by the National Energy Research Scientific Computing Center (NERSC)
supported by the U.S.Department of Energy Office of Science (DE-AC02-05CH11231).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENT
We thank Qunying Lei of for kindly provided antibody for K433 acetylation of PKM2. We
also thank Jenny Yang, Zhiren Liu and Binghe Wang of Georgia State University for their help in
instruction of experiments.
REFERENCES
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1. Christofk, H. R.; Vander Heiden, M. G.; Harris, M. H.; Ramanathan, A.; Gerszten, R. E.; Wei,
R.; Fleming, M. D.; Schreiber, S. L.; Cantley, L. C. The M2 splice isoform of pyruvate kinase is
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