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Therapeutics, Targets, and Chemical Biology
Therapeutic Targeting of the Warburg Effect inPancreatic Cancer
Relies on an Absence of p53FunctionN.V. Rajeshkumar1, Prasanta
Dutta2, Shinichi Yabuuchi1, Roeland F. de Wilde1,Gary V. Martinez2,
Anne Le1, Jurre J. Kamphorst3, Joshua D. Rabinowitz3, Sanjay K.
Jain4,Manuel Hidalgo5, Chi V. Dang6, Robert J. Gillies2, and
Anirban Maitra7
Abstract
The "Warburg effect" describes a peculiar metabolic featureof
many solid tumors, namely their increased glucose uptakeand high
glycolytic rates, which allow cancer cells to accumu-late building
blocks for the biosynthesis of macromolecules.During aerobic
glycolysis, pyruvate is preferentially meta-bolized to lactate by
the enzyme lactate dehydrogenase-A(LDH-A), suggesting a possible
vulnerability at this target forsmall-molecule inhibition in cancer
cells. In this study, we usedFX11, a small-molecule inhibitor of
LDH-A, to investigate thispossible vulnerability in a panel of 15
patient-derived mousexenograft (PDX) models of pancreatic cancer.
Unexpectedly,the p53 status of the PDX tumor determined the
response toFX11. Tumors harboring wild-type (WT) TP53 were
resistant toFX11. In contrast, tumors harboring mutant TP53
exhibitedincreased apoptosis, reduced proliferation indices, and
atten-
uated tumor growth when exposed to FX11. [18F]-FDG PET-CTscans
revealed a relative increase in glucose uptake in mutantTP53 versus
WT TP53 tumors, with FX11 administration down-regulating metabolic
activity only in mutant TP53 tumors.Through a noninvasive
quantitative assessment of lactate pro-duction, as determined by
13C magnetic resonance spectros-copy (MRS) of hyperpolarized
pyruvate, we confirmed thatFX11 administration inhibited
pyruvate-to-lactate conversiononly in mutant TP53 tumors, a feature
associated with reducedexpression of the TP53 target gene TIGAR,
which is known toregulate glycolysis. Taken together, our findings
highlight p53status in pancreatic cancer as a biomarker to predict
sensitivityto LDH-A inhibition, with regard to both real-time
noninvasiveimaging by 13C MRS as well as therapeutic response.
Cancer Res;75(16); 3355–64. �2015 AACR.
IntroductionPancreatic ductal adenocarcinoma (PDAC) is the
fourth most
common cause of cancer-related mortality in the United
States,with an alarming rise in incidence and a projection that it
willbecome the second most common cause of cancer deaths by2030
(1). The 5-year survival rate of patients with advancedPDAC is
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dehydrogenase-A (LDH-A) is involved in the conversion ofpyruvate
into lactate, utilizing NADH as a cofactor. By con-verting pyruvate
to lactate, LDH-A regenerates the NADþ need-ed to maintain
glycolysis and diverts pyruvate from beingconverted to acetyl-CoA
for oxidative phosphorylation (6).Aerobic glycolysis provides
bioenergetic intermediates andgenerates ATP, while simultaneously
suppressing excessivereactive oxygen species (ROS) production. The
lactate producedby cancer cells acidifies the extracellular
microenvironment,promoting invasion and metastases, reduced drug
efficacythrough ion tapping, and evading immune recognition (7–9).
The increase in glycolytic flux is a metabolic strategy oftumor
cells to ensure survival and growth in
nutrient-deprivedenvironments (10).
LDH-A is upregulated by various oncogenic transcriptionfactors,
such as hypoxia-inducible factor-1a (HIF1a) and c-Myc, in cancers
(11). Conversely, it has been documentedthat reduction of
fermentative glycolysis through LDH-Ablockade results in the
inhibition of tumor growth andmetastases in various preclinical
models, implicating LDH-A as a viable therapeutic target (12–17).
Blockade of LDH-Aactivity with the pharmacologic inhibitor FX11
attenuatestumor progression across various preclinical models,
includ-ing in PDAC cell lines (18). Given the expanding portfolio
ofpharmacologic inhibitors that target aberrant cancer metab-olism
(19, 20), it is imperative that molecular determinants
ofsensitivity and resistance to these inhibitors be identified,
andfurther, clinically feasible assays that can provide insights
intoin vivo response in real-time be developed. In this study,
wedemonstrate that PDAC tumors are responsive to FX11 treat-ment in
a TP53-dependent manner. We further demonstratethat 13C magnetic
resonance spectroscopy (MRS) using hyper-polarized pyruvate
provides a biochemically specific and clin-ically feasible approach
for predicting in vivo response to LDH-Ainhibition.
Materials and MethodsPatient-derived PDAC xenografts
All animal experiments were performed in accordance with
theGuidelines for the Care and Use of Laboratory Animals and
wereapproved by the Institutional Animal Care and Use Committeeof
Johns Hopkins University (Baltimore, MD) and the Universityof South
Florida (Tampa, FL). Male nu/nu athymicmice (Harlan)were used for
the study. Animals were maintained under patho-gen-free conditions
and a 12-hour light/12-hour dark cycle.Fresh PDAC specimens
resected from patients at the time ofsurgery were implanted
subcutaneously (s.c.) into the flanks of6-week-oldmice, as
previously described (21, 22).Grafted tumorswere subsequently
transplanted frommouse tomouse andmain-tained as a live PDX Bank in
Johns Hopkins University, Baltimoreas per the guidelines of
Institutional Review Board-approvedprotocol.
In vivo efficacy of the LDH-A antagonist FX11 in
patient-derivedPDAC xenografts
Fifteen individual patient-derived PDAC xenografts were usedfor
the study. The mutational status of common driver genes inPDAC has
been previously described for these PDXs (23).
Briefly,subcutaneously established tumors were harvested at the
expo-nential growth phase, and were cut aseptically into cubes of 1
to
2 mm3. The tumor pieces were dipped in Matrigel and implantedon
both flanks of 6-week-old male nu/nu athymic mice (Harlan).When
cohorts of tumors reached approximately 200 mm3, mice(5 mice/group;
8–10 tumors in each arm) were randomlyassigned to (i) control
(vehicle) and (ii) FX11 (2.2 mg/kg),injected intraperitoneally,
once daily for 4 weeks. Tumors weremeasured twice per week using a
digital caliper, allowing discrim-inationof
sizemodifications>0.1mm. Individual tumor volumeswere calculated
using the formula: tumor volume V ¼ a � b2/2,where a being the
largest dimension of the tumor, b the smallest.
Effect of FX11 treatment on tumor cell proliferation
andapoptosis
Excised tumors from the vehicle and FX11 treatment
(harvestedfrom the 28 day efficacy study)werefixed in
10%neutral-bufferedformalin and processed into paraffin blocks.
Sections were depar-affinized in xylene and rehydrated in
graded-alcohol washes.Hematoxylin and eosin (H&E) staining was
performed usingstandard procedure. The assessment of cellular
proliferation wasconducted using an anti-MIB-1 (Ki-67) antibody
(clone K2,dilution 1:100, Ventana Medical Systems). IHC detection
ofapoptosis was performed using Terminal deoxynucleotidyl
trans-ferase-mediated dUTP nick end labeling (TUNEL) assay with
acommercial apoptosis detection kit (DeadEnd FluorometricTUNEL
System; Promega), according to the recommendationsof the
manufacturer. Stained sections were examined under lightmicroscope
and images were captured. Histograms for TUNELand Ki-67 staining
were generated by evaluating five high-powerfields (hpf) of tumor
section from two independent tumors pertreatment arms (24).
TP53-induced glycolysis and apoptosis regulator andp53 IHC
Anti-TP53–induced glycolysis and apoptosis regulator (TIGAR)and
anti-p53 antibodies (rabbit polyclonal to TIGAR, abcam,ab37910 and
rabbit polyclonal to p53, abcam, ab4060)were usedfor TIGAR and p53
IHC, respectively. The staining was performedas per manufacture's
protocol. Briefly, formalin-fixed, paraffin-embedded sections were
deparaffinized and subjected to heat-mediated antigen retrieval in
citrate buffer before blocking in 10%serum for 1 hour at room
temperature. The primary antibodieswere diluted at 1/400 (TIGAR)
and 1/100 (p53). The sectionswere incubated with the sample for 1
hour at room temperaturefor TIGAR and 12 hours at 4�C for p53. A
biotinylated goat anti-rabbit antibody was used as the secondary
antibody (25, 26).Stained sections were examined microscopically at
�40 magni-fication and images were captured. The neoplastic areas
wereexamined for staining and were graded according to the
preva-lence of both nuclear and cytoplasmic staining within the
tumor.We used a 0 to 3 scale for staining intensity: 0,
completelynegative; 1, weak staining; 2, moderate staining; 3,
strong staining(27). The staining was scored by evaluating sections
from twoseparate tumors each from P420, JH033, P286, JH024,
P253,P410, P194, and JH015.
Expression of human TIGAR transcripts in PDAC xenograftsBaseline
TIGAR expression was determined in frozen tumors of
two xenografts each with TP53 wild-type (WT) and
TP53-mutantstatus. Total RNA was extracted using RNeasy Mini Kit
(Qiagen),cDNA was synthesized with SuperScript First Strand
System(Invitrogen), and qRT-PCR for huTIGAR expression was
Rajeshkumar et al.
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conducted using FAST SYBR Green Master Mix (Applied Biosys-tems)
on a Step One Plus Real-Time PCR System (AppliedBiosystems). Human
PGK1 and murine b-actin were used ashousekeeping genes. The primer
sequences used for huTIGARare Forward 50-ATGGAATTTTGGAGAGAA-30
Reverse 50-CCATGGCCCTCAGCTCAC-30 (28). Relative expression of
themRNA was estimated using the 2�DDCt method.
FDG-PET imaging of PDAC xenografts[18F]-FDG-PET imaging was used
to determine the baseline
glucose uptake and the impact of FX11 therapy. Tumor-bearing
mice (N ¼ 5 each) from two PDX models - JH024 (TP53 WT)and JH015
(TP53 mutant) were used for the study. Tumor-bearing mice in JH024
and JH015 PDXs were treated withvehicle or FX11 (2.2 mg/Kg) i.p.
for 7 consecutive days. Micewere imaged for determining the tumor
glucose uptake by[18F]-FDG-PET before the commencement of vehicle
or FX11treatment (D1) and on day 7 of vehicle or FX11
treatment(D7). On the day of imaging, each mouse was injected
with250 mCi of [18F]-FDG via the tail vein and imaged 45
minutespostinjection using the Mosaic HP (Philips) Small Animal
PETimagers with 15 minutes static acquisition. PET images were
*****
*
Contr
olFX
110
2
4
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10
JH015Contr
olFX
110
2
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Rel
ativ
e tu
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gro
wth
(fol
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P253 Contr
olFX
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P194
TP53 WT xenografts TP53 MUT xenografts
–50
–40
–30
–20
–10
0
10
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P420 JH024 JH033 JH034 P286 P219 P198 P281 P374 P215 P410 P265
P194 JH015 P253
Tum
or g
row
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ompa
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to c
ontr
ol (%
)
Human pancreatic cancer xenografts
*****
*
A
B
Figure 1.In vivo antitumor effects of LDH-A inhibitor FX11 in
human pancreatic cancer xenografts. A, FX11 treatment delays tumor
progression, selectively in TP53-mutant(MUT) tumors. Tumors from
fifteen individual patient-derived pancreatic cancer xenografts
were implanted subcutaneously in athymic mice. Animals
withestablished tumors (�200 mm3) were injected with vehicle or
FX11 (2.2 mg/kg) i.p. for 4 weeks. Relative TGI was compared with
vehicle-treated mice on day 28.Tumors with TP53 WT tumors were
completely resistant to glycolytic inhibition, while a range of TGI
was observed with mutant TP53 PDXs. B, threePDXs (P194, JH015, and
P253) showed significant delay in tumor progression compared with
vehicle-treated mice. Points, mean� SEM.N¼ 8–10 tumors per group.�
, P ¼ 0.0478; �� , P ¼ 0.0011; ��� , P ¼ 0.0002 compared with
vehicle-treated mice.
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reconstructed and coregistered with CT images using Amiraversion
5.2.2 (Visage Imaging; ref. 29).
Pyruvate sample preparation and animal handlingSamples (�30mL)
of [1-13C] pyruvic acid (Isotec) containing 15
mmol/L of OX63 trityl radical and 1.5 mmol/L of the
gadoliniumchelate dotarem were polarized at 3.35T and 1.4K in the
Hyper-sense DNP polarizer (Oxford Instruments) for one hour.
Thehyperpolarized pyruvate sample was rapidly dissolved in 4 mL ofa
superheated alkaline buffer comprising 100 mg/L
ethylendiami-netetraacetic acid, 40mmol/L of TRIS buffer, 40mmol/L
ofNaOH,and 30 mmol/L of NaCl. This produces 80 mmol/L
concentrationof pyruvate with physiologic pH (7.4) for
administration toanimals. Hyperpolarized [1-13C] pyruvate solution
of 350 mL wasintravenously injected over a period of 12 to 15
seconds through acatheter placed in the jugular vein of the
tumor-bearing mice.
Representative PDXs that were sensitive (JH015, P253)
andresistant (JH024, JH033) to FX11 treatment were chosen for
thehyperpolarized 13C-MRS study and T2-weighted MRI measure-ments.
Mice with 500 mm3 subcutaneous tumors were selectedfor the imaging
study. The jugular vein catheter was surgicallyimplanted to
facilitate polarized substrate injections. The micewere treated
with vehicle or FX11 (2.2 mg/kg) i.p. for 7 consecu-tive days (N ¼
4 mice/group).
In vivo 13C MR spectroscopyAs preparation for the MRI
experiment, mice were infused
with isofluorane in a plastic anesthesia chamber with
scaveng-ing. Anesthetized mice were placed in a mouse-specific
holderwithin the MRI coil, outfitted with a mouse-specific nose
coneinhalant anesthesia and scavenging system for imaging.
Thissystem also contains a pad for respiration monitoring,
andendo-rectal fiber optic temperature monitoring device, a
heatedpad for maintaining core body temperature and leads
forelectrocardiography (ECG monitoring). In vivoMR experimentswere
performed on a 7T, 31-cm horizontal bore magnet (Agi-lent).
Multi-slice T2-weighted anatomic images were acquiredusing a
respiratory-gated spin-echo sequence with a TE ¼ 60 ms,TR ¼ 4000
ms, fat saturation, FOV 40 � 40 mm, echo trainlength ¼ 8, matrix
256 � 128, slice thickness ¼ 1 mm, 15 slices.Dynamic 13C MR spectra
were acquired utilizing a dual tuned1H - 13C volume coil (M2M, 35
mm). All spectra were acquiredusing slice-selective pulse with a
nominal flip angle of 9�, TR ¼1 second, 30,000 complex points, and
spectral width of 50 kHz.In vivo data acquisition started right
before the pyruvate injec-tion and collected single transient
spectra over a period of 150seconds from a 5-mm-thick tumor slice.
The peak heights ofpyruvate and lactate resonance spectra were used
to calculaterelevant ratios (Lac/Pyr).
A
B
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JH024
Cont
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67 s
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pf
JH015
Cont
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Ki–
67 s
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i/hpf
P253
** **
*****
Figure 2.FX11 treatment induces apoptosis and inhibits tumor
cell proliferation selectively in tumors with mutant TP53 status.
IHC staining of TUNEL and Ki-67 in paraffin-embedded pancreatic
tumors. Tumors resected on day 28 from vehicle and FX11 treatment
groups (JH033, JH024, P253, and JH015) were used for
analysis.Formalin-fixed, paraffin-embedded sections (5 mm) were
stained for nuclear Ki-67 and TUNEL-positive tumor cells. A,
representative photomicrographs(�20) from TP53 WT tumors (JH033 and
JH024). No significant induction of apoptosis or inhibition of
Ki-67 was seen in FX11-treated tumors compared withvehicle
treatment. B, representative photomicrographs (�20) from mutant
TP53 tumors (P253 and JH015) showing that FX11 treatment
significantly inducedapoptosis and inhibited tumor cell
proliferation compared with vehicle treatment. Quantification of
TUNEL and Ki-67–positive tumor cells is shown in theright panel of
figures. The data representative of mean� SD were generated by
evaluating five different hpf from three different tumors per
group. �� , P < 0.01 and��� , P < 0.005 compared with
vehicle-treated mice.
Rajeshkumar et al.
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Statistical analysisStatistical analysiswas carried out by a
two-tailed unpaired t test
usingGraphPad Prism 5 software. SEMor SD is either representedin
the graphs or following the means of all measures, as stated
infigure legends. Statistical significance was considered at the P
<0.05 level.
ResultsPharmacologic LDH-A inhibition leads to tumor
growthinhibition selectively in TP53-mutant PDAC xenografts
We sought to determine the treatment effect of FX11, a
smallmolecule that can inhibit LDH-A, in a panel of 15
pancreaticcancer PDXs. A wide range of responses to 4 weeks of
FX11therapy was observed in these PDXs, with tumor growth
inhi-bition (TGI) ranging from approximately 40% to a few
PDXsreaching a marginally larger volume than the control group(Fig.
1A). Overall, three of the PDXs had significant TGI withFX11
monotherapy (P253, JH015, and P194; Fig. 1B). In thispanel, only
two PDXs were KRAS WT (P410 and P420), and nospecific pattern of
response to FX11 was noted based on KRASmutational status.
Similarly, SMAD4 mutational status did notyield any discernible
pattern of response (Supplementary TableS1). On contrary, when the
waterfall plot was segregated basedon TP53 status, nearly all PDXs
with any evident TGI (includingthe three tumors with significant
inhibition) were TP53mutant,while PDXs with minimal response or
marginal enhancement
in growth were uniformly TP53 WT. We assessed proliferation(by
Ki-67 nuclear labeling) and apoptosis (using TUNEL stain-ing) in
two TP53WT (JH033 and JH024) and two TP53mutant(P253 and JH015)
PDXs, and identified a significant reductionin proliferation, and a
significant increase in apoptosis post-FX11 therapy that was
restricted to the TP53-mutant xenografts(Fig. 2A and B). Overall,
these findings suggested that TP53status is likely to be a key
molecular determinant of response topharmacologic LDH-A
inhibition.
We also measured the expression of the p53 target TIGAR,whose
expression has been linked to decreased glycolytic conver-sion of
pyruvate to lactate. Using IHC, we found that TIGARexpression was
positively correlated with TP53WT status, and itsexpression
appeared diminished in TP53-mutant tumors (Fig. 3Aand B).
Quantification of the p53 and TIGAR staining intensity intumor
sections of TP53WTPDXs revealed a significant increase inp53 and
TIGAR expression compared with TP53-mutant PDXs(Fig. 3C). Baseline
TIGAR mRNA expression was also reduced inTP53-mutant tumors
compared with tumors with TP53 WTtumors, with a significant
downregulation in one of two PDXs(Supplementary Fig. S1).
FDG-PET imaging of PDAC xenograftsIn light of the differential
responses to LDH-A inhibition, we
interrogated the pattern of 18F-deoxy-glucose (FDG) uptake
atbaseline and following FX11 therapy in two PDXs, one with
A
B
TIG
AR
p53
P420 JH033
TP53 WT PDXs
P286
P253 P410TP53 MUT PDXs
TIG
AR
p53
P194
JH024
JH015
C
**
***
TP53
MUT
PDXs
TP53
WT P
DXs
0
1
2
3
4
p53
stai
ning
inte
nsity
(a.u
.)
TP53
MUT P
DXs
TP53
WT PD
Xs0
1
2
3
4
TIG
AR
sta
inin
g in
tens
ity (a
.u.)
Figure 3.TIGAR expression is elevated in human pancreatic PDX,
with WT TP53 compared with tumors with mutant TP53 status. A and B,
representative high power (�40)photomicrographs of p53 and TIGAR in
stained tumor sections from tumors with WT TP53 status (P420,
JH033, P286, and JH024; A) and tumors withmutant TP53 status (P253,
P410, P194, and JH015; B). Higher cytoplasmic expression of TIGAR
was detected in WT TP53 PDXs compared with mutant TP53 tumors.C,
quantification of the staining intensity. TIGAR immunostaining in
tumor sections of TP53 WT PDXs revealed a significant increase in
TIGAR expressioncompared with TP53 MUT PDXs. Bars represent
aggregate mean staining scores � SD of TP53 MUT and TP53WT PDXs.
��� , P ¼ 0.0001; �� , P ¼ 0.0016 comparedwith TP53 MUT PDXs. a.u.,
arbitrary units.
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WT TP53 (JH024) and the second with a TP53 mutant(JH015). The
FDG uptake rates varied between JH024 andJH015 tumors. At baseline
(D1), JH015 tumors had elevatedFDG uptake compared with JH024,
indicating greater relianceon glycolysis (Fig. 4A and B). Notably,
there were no altera-tions in tumor FDG uptake following 7 days of
FX11 therapyin JH024 (Fig. 4A). In contrast, the avid glucose
uptakeobserved in JH015 xenografts at baseline was
significantlyreduced following 7 days of FX11 therapy (Fig.
4B).
Hyperpolarized 13C MR spectroscopy confirms that changes
inlactate flux upon FX11 therapy are restricted to
TP53-mutantxenografts
The recent development of hyperpolarized MR
spectroscopicimagingmethods to probe the biochemical andmetabolic
profileof tumors using 13C-labeled pyruvate as the tracer, and
thenmonitor its intracellular conversion to various metabolites,
offersa unique opportunity to examine in vivo responses
tometabolism-targeted therapies. In order to assess the metabolic
conversion ofhyperpolarized pyruvate to lactate, two sets of PDXs
that wereeither sensitive to FX11 treatment (JH015 and P253, both
TP53mutant) or resistant to FX11 treatment (JH024 and JH033,
both
TP53WT), respectively, were chosen for hyperpolarized
13C-MRSstudy.
Representative dynamic 13C MRS spectra of JH033 tumor(FX11
resistant) captured before FX11 treatment and 7 days FX11treatment
are shown in Fig. 5A and B, respectively. Pyruvate andlactate peak
intensities over time are shown in Fig. 5E and F. FX11treatment was
ineffective in altering the conversion flux frompyruvate to lactate
in JH033. In contrast with the JH033 spectra,dynamic 13CMRS spectra
of P253 showed that pyruvate-to-lactateconversion was significantly
reduced after 7 days of FX11 treat-ment as compared with spectra
captured before FX11 treatment(Fig. 5C and D), and this was
confirmed in the pyruvate andlactate peak intensities over time
(Fig. 5G and H). Seven daysvehicle treatment did not alter the flux
of hyperpolarized pyruvateto lactate conversion in both JH033 and
P253 tumors (13C MRSspectra not shown).
The integrated lactate-to-pyruvate ratio (Lac/Pyr) canbe used
torepresent a drug therapy response marker in this study. The
Lac/Pyr flux ratio was calculated from the area under the curve
(asregarded a "Model-Free" approach) of themetabolic flux from
thedynamic scan (30). The Lac/Pyr flux ratios in response to
FX11treatments were significantly different between tumors
sensitive
FX11 Control
Ani
mal
#2
D1
Ani
mal
#1
D7 D7D1
JH015 (TP53 MUT)
FX11 Control
Ani
mal
#2
D1
Ani
mal
#1
D7D1D7
JH024 (TP53 WT)A B
*
Vehic
leFX
110
1
2
3
JH015Veh
icle
FX11
0
1
2
3
JH024
18F-
FDG
upt
ake
in tu
mor
(D7/
D1)
18F-
FDG
upt
ake
in tu
mor
(D7/
D1)
Figure 4.FX11 treatment significantly reduces 18F-FDG uptake in
tumor with mutant TP53 status. Subcutaneous PDXs including WT TP53
(JH024; A) and mutant TP53(JH015; B) were treated with vehicle or
FX11 (2.2 mg/Kg) i.p. for 7 consecutive days. On the day of imaging
(D1 and D7), mice were injected with 250 mCi of[18F]-FDG via the
tail vein and imaged 45 minutes postinjection. While FX11 treatment
did not significantly reduce the tumor 18F-FDG uptake in
micebearing JH024 (A), the treatment significantly reduced the
18F-FDG in mice bearing JH015 (B). Arrowheads, tumor location on
the mouse flank. Histogramsshown at the bottom panels of figure
represent the standard uptake values in tumors. Standard uptake
values in tumors were normalized by dividing the valueswith values
of 18F-FDG update in the liver of each animal at the time of image
acquisition. Points, mean � SEM. N ¼ 3–4 mice per group. � , P ¼
0.0446 comparedwith vehicle-treated mice.
Rajeshkumar et al.
Cancer Res; 75(16) August 15, 2015 Cancer Research3360
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-
(P253 and JH015) and resistant (JH024 and JH033) to
FX11treatment (Fig. 6A). The Lac/Pyr ratios increased with time in
theresistant (JH024 and JH033) tumors (Fig. 6A). In stark
contrast,the Lac/Pyr of both sensitive tumors (P253 and JH015)
showed astriking decrease in response to FX11 treatment (Fig. 6A).
Thetumor growth curves of representative tumors resistant
(JH033)and sensitive (P253) to FX11 treatment are shown in Fig. 6B
andC. We also evaluated the unidirectional conversion rate
constants(kp ¼ pyruvate-to-lactate) using modified Bloch's equation
ofkinetic model (31). Consistent with the above results, the
con-version rate constantly (kp) decreased in FX11-sensitive
tumorsand increased in FX11-resistant tumors (Table 1).
DiscussionPersistent aerobic glycolysis is a key metabolic
dependency in
tumorigenesis. LDH-A is a key regulator of glycolysis and playsa
critical role in tumor maintenance (6). LDH-A is
frequentlyupregulated in aggressive cancers, and expression levels
fre-quently correlate with poor prognosis (32, 33).
Furthermore,knockdown of LDHA compromises the tumorigenic
potentialof malignant cells (6). LDH-A expression is significantly
ele-vated in pancreatic cancer compared with paired normal
tissues(12, 34). Although forced expression of LDH-A enhances
theproliferation of pancreatic cancer cells, knocking down ofLDHA
transcript expression inhibited cell growth and inducedapoptosis
(12). Silencing LDHA expression also inhibitedgrowth of pancreatic
cancer cells in vivo, suggesting LDH-A apotential target for
pancreatic cancer therapy (12). Of note,
systemic inhibition of LDH-A may not produce side effects
inhumans, since hereditary LDH-A deficiency does not provokeany
symptoms under baseline circumstances (35).
The advent of metabolism-targeted agents has garneredinterest in
identifying genetic determinants of responses tothese agents in
cancer cells, especially for therapies that arenot geared directly
against specific activating events like IDH1mutations. In this
study, we examined the therapeutic efficacyof a small-molecule
LDH-A inhibitor across a panel of PDACPDXs. Human pancreatic cancer
xenografts used in the presentstudy are annexed with desmoplastic
stroma (SupplementaryFig. S2), a well-characterized feature of
primary tumors andmetastatic lesions of human PDAC (36). Masson's
trichromestaining and H&E histology from TP53 WT and
TP53-mutanttumors revealed that tumors were enriched with fibrotic
stro-ma, irrespective of their TP53 status (Supplementary Fig.
S2).We identify that the sensitivity to LDH-A inhibition is
depen-dent on p53 status of the tumor, TP53-mutant tumors
dem-onstrating sensitivity, and TP53 WT tumors
demonstratingrefractoriness to FX11 treatment. The basis for
resistance toglycolytic inhibition could be the reduced dependence
onglucose as an elemental fuel, as TP53 wild-type PDXs hadminimal
FDG uptake. Of note, expression of TIGAR was higherin PDXs with WT
TP53 function, whereas TP53 mutationsresulted in reduced TIGAR
levels. TIGAR is a p53-inducibleprotein that functions to lower
glycolytic flux and reducescancer cell sensitivity to reactive
oxygen species associatedapoptosis (37–39). In TP53 WT cells, TIGAR
expression func-tions to decrease the levels of
fructose-2-6-biphosphate, which
1008060402000
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150
Sign
al in
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ity (a
.u.)
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Pyruvate Lactate
1008060402000
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200
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al in
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ity (a
.u.)
Time (s)
Pyruvate Lactate
D7 FX11 treatmentPrior to FX11 treatment
Lactate Pyruvate PyruvateLactate
JH033 (TP53 WT)
Lactate Pyruvate Lactate Pyruvate
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Sign
al in
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.u.)
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Pyruvate Lactate
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.u.)
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Pyruvate Lactate
BA C D
E F G H
P253 (TP53 MUT)
D7 FX11 treatmentPrior to FX11 treatment
185 180 175 170 165 ppm 185 180 175 170 165 ppm 180 175 170 165
ppm185 180 175 170 165 ppm
Figure 5.Pyruvate-to-lactate conversion flux varies in PDXs,
which were resistant and sensitive to FX11 treatment.
Representative dynamic 13C MR spectra acquired fromFX11-resistant
JH033 (TP53 WT) and FX11-sensitive P253 tumor, a TP53-mutant PDX.
Spectra captured before D7 FX11 treatment of JH033 and P253
areshown in A–D, respectively. Sequential spectra were acquired
from a 5-mm tumor slice over 150 seconds after i.v. injection of
hyperpolarized [1-13C] pyruvate asdescribed in Materials and
Methods. Tumor lactate and pyruvate peak intensities with time
after i.v. injection of hyperpolarized 13C pyruvate are shownbefore
FX11 treatment (E and G) and D7 FX11 treatment (F and H) in JH033
and P253, respectively. Representative anatomical T2-weighted axial
MR imagescontaining slice ROI (red box) are shown in inset.
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-
suppresses glycolysis by diverting glucose-6-phosphate into
thepentose phosphate pathway. The correlation of TIGAR expres-sion
with p53 status in pancreatic cancer PDXs could explainone
potential mechanism for the reduced sensitivity to LDH-Ainhibition
in p53 WT, high TIGAR cases, by reducing glycolyticdependence.
The understanding of cancer metabolism has deep roots
inmolecular biology and genetics but the ability to detect
metab-olism as it occurs in tumors without a biopsy is largely
limited toPET imaging of enhanced glucose uptake into tumors using
18F-fluorodeoxyglucose. The recently discovered hyperpolarized
MRIusing dynamic nuclear polarization technology has the
potentialof providing real-time images ofmetabolic processes in
tumors atunprecedented levels of sensitivity for monitoring
therapyresponses (40–43). Hyperpolarized 13C magnetic
resonanceallows rapid and noninvasive monitoring of dynamic
pathway-specificmetabolic and physiologic processes. Hyperpolarized
13CMRS provides a unique ability to noninvasively assess
metabolic
fluxes and downstream product(s) in real time, which has
beenutilized for tumor diagnosis, to stage the extent of disease,
and tomonitor the response to therapy.
Transmembrane transport of pyruvate, lactate, and ketonebodies
is mediated by monocarboxylate transporters -1 and 4(MCT-1 and
MCT-4; ref. 44). It has been documented thatlactate produced by
tumor cells can be taken up by stromal cellsto regenerate pyruvate
that either can be extruded to refuel thecancer cell or can be used
for oxidative phosphorylation (45).HIF1a stimulates the conversion
of glucose to pyruvate andlactate by upregulating glucose
transporter isoform 1 (GLUT1),hexokinase, and LDH-A, as well as the
lactate-extruding enzymeMCT-4 (46). A recent 13C MRS study
confirmed the modulatoryeffect of LDH-A, lactate, and MCT isoforms
in hyperpolarizedpyruvate to hyperpolarized lactate conversion in
cancer cell lines(47). Results obtained in our 13CMRS experiments
demonstrateda decrease in pyruvate to lactate conversion following
FX11treatment, in JH015 and P253, both TP53-mutant PDXs. In
order
A
B C
0
100
200
300
400
500
600
700
292522181511841Time (days)
% G
row
th (m
ean
± SE
M)
Control FX11
JH033
0
100
200
300
400
500
600
700
800
292522181511841Time (days)
% G
row
th (m
ean
± SE
M)
Control FX11
P253
**
0
1
2
3
4
86420
Lac.
/Pyr
. (a.
u.)
Time (days)
JH033 (resistant to FX11)JH024 (resistant to FX11)JH015
(sensitive to FX11)P253 (sensitive to FX11)
Figure 6.FX11 treatment reduces the lactate/pyruvate ratio,
selectively in PDXs sensitive to FX11 treatment. A, Lac/Pyr ratio
was reduced with FX11 treatment in PDXssensitive to FX11 (P253 and
JH015). However, the ratio was increased in PDXs resistant to FX11
treatment (JH024 and JH033). B and C, tumor growth
curvesrepresentative of tumors that were resistant (JH033) and
sensitive (P253) to FX11 treatment. N ¼ 8–10 tumors in each group.
Errors bars representstandard error of mean � SEM. �� , P ¼ 0.0011
compared with vehicle-treated mice.
Rajeshkumar et al.
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-
to characterize the contribution of tumor lactate pool,
LDH-A,HIF-1a, and MCT isoforms (MCT-1 and MCT-4) in
attenuatinghyperpolarized pyruvate to lactate production, we
conductedbaseline gene expression analysis andmeasuring the tumor
lactatepool. Baseline tumor lactate, LDH-A, HIF-1a, MCT-1,
andMCT-4gene expression were not significantly different in
tumorsresponded to FX11 treatment as compared with tumors
resistantto FX11 treatment (data not shown).
We measured the differences in pyruvate metabolism in
pan-creatic cancer xenografts, which were sensitive or resistant
toFX11 treatment. Multiple injections of hyperpolarized pyruvatein
the same animal enabled us to measure the flux changes
anddifferential kinetics in pyruvate-to-lactate conversion within
anindividual tumor over time. Of note, we were able to
gaugedifferences in pharmacodynamics of pyruvate to lactate
conver-sion in real time at a point even before reduction in
tumorvolumes was observed in FX11-sensitive tumors. Our
studiesdemonstrated the feasibility of using 13C hyperpolarized
meta-bolic imaging as a noninvasive biomarker for
monitoringresponse to metabolic therapy. The capability of MRS to
nonin-vasively obtain metabolic information is a valuable asset in
themetabolic profiling of tumors, and can provide molecular
signa-tures of specific biologic processes as a complementary
imagingmodality of FDG-PET.
The small number of fifteen xenografts studied and the lack
ofdetailed investigation of factors that might also influence
depen-dence on glycolytic metabolism, including but not limited
tointratumoral pH,O2 tension, interstitial pressure, tumor size,
andgrowth fraction, are limitations to this study. Even
thoughwehavenot conducted in vitro studies in support of the impact
of mutantp53 in targeting Warburg effect, previous report indicated
thattumor-associated mutant p53 stimulates the Warburg effect
incultured cells and demonstrated that targeting altered
glucosemetabolism could be a feasible therapeutic strategy for
tumorcarrying mutant p53 (48). Additional studies are needed to
fullyunderstand the mechanistic link in supportive of the influence
ofmutant p53 in deciding the sensitivity of LDH-A–targeted
ther-apy, or whether the phenomenon is demonstrable only in thein
vivo milieu. Our data show that pharmacologic inhibition ofLDH-A
may have utility in the treatment of tumors by reducingtumor growth
of TP53-mutant tumors.
In summary, we observe that FX11, a LDH-A
small-moleculeantagonist, attenuated in vivo tumor growth, induced
apoptosisand reduced tumor cell proliferation, specifically in
pancreaticcancer PDXs with mutant TP53. Analysis of FDG PET-CT
scans ofanimals demonstrate that PDXs with mutant TP53 tumors
haverelatively high glucose uptake compared with tumors with
TP53
WT status. In addition, using hyperpolarized 13C magnetic
reso-nance spectroscopy, we demonstrate that FX11 treatment
inhib-ited thepyruvate to lactate conversion only in
tumorswithmutantTP53 status. Currently, we do not understand the
mechanisticbasis for the resistance of WT TP53 pancreatic cancer
PDXs toFX11, particularly regarding the lack of an effect on the in
vivoconversion of pyruvate to lactate. Furthermore, it is notable
thathigh concentrations of FX11 could have off-target effects
beyondinhibition of LDH-A. Nonetheless, our collective studies are
thefirst to document a link between tumor p53 status and
LDH-Ainhibitor sensitivity. Our work also suggests that the
elevatedglucose metabolism in TP53-mutant tumors can be exploited
forthe preferential targeting of these tumors by LDH-A
inhibitortherapy. The effectiveness of the therapy can be monitored
by invivometabolic imaging and can be readily applied to humans
formonitoring tumor response to therapy.
Disclosure of Potential Conflicts of InterestNo potential
conflicts of interest were disclosed.
Authors' ContributionsConception and design: N.V. Rajeshkumar,
P. Dutta, A. Le, M. Hidalgo,C.V. Dang, R.J. Gillies, A.
MaitraDevelopment of methodology: N.V. Rajeshkumar, P. Dutta, G.V.
Martinez,A. Le, R.J. Gillies, A. MaitraAcquisition of data
(provided animals, acquired and managed patients,provided
facilities, etc.): N.V. Rajeshkumar, P. Dutta, S. Yabuuchi, R.F.
deWilde, G.V. Martinez, J.J. Kamphorst, J.D. Rabinowitz, S.K. Jain,
M. Hidalgo,R.J. GilliesAnalysis and interpretation of data (e.g.,
statistical analysis, biostatistics,computational analysis): N.V.
Rajeshkumar, P. Dutta, S. Yabuuchi, R.F. deWilde, J.J. Kamphorst,
S.K. Jain, M. Hidalgo, C.V. Dang, R.J. Gillies, A. MaitraWriting,
review, and/or revision of the manuscript: N.V. Rajeshkumar,P.
Dutta, S. Yabuuchi, R.F. de Wilde, G.V. Martinez, A. Le, J.J.
Kamphorst,J.D. Rabinowitz, S.K. Jain, M. Hidalgo, C.V. Dang, R.J.
Gillies, A. MaitraAdministrative, technical, or material support
(i.e., reporting or organizingdata, constructing databases): N.V.
Rajeshkumar, R.F. de WildeStudy supervision: N.V. Rajeshkumar
AcknowledgmentsThe authors thankElizabethDeOliveira,
JohnsHopkinsUniversity for expert
help in animal experiments and Fatima Al-Shahrour and H�ector
Tejero, CNIO,Spanish National Cancer Research Center for assistance
in gene expression dataanalysis.
Grant SupportThisworkwas supported by funding
fromaStandUpToCancerDreamTeam
Translational Research Grant SU2C-AACR DT0509 (N.V. Rajeshkumar
andC.V. Dang), by P30CA016520, R01CA051497, R01CA057341 (C.V.
Dang),R01CA113669 (A. Maitra), R01CA163591 (J.D. Rabinowitz), and
the WayneHuizinga Trust at Moffitt Cancer Center, R01 CA077575-14
(R.J. Gillies). StandUp To Cancer is a program of the Entertainment
Industry Foundation admin-istered by the American Association for
Cancer Research.
The costs of publication of this articlewere defrayed inpart by
the payment ofpage charges. This article must therefore be hereby
marked advertisement inaccordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Received January 14, 2015; revised May 8, 2015; accepted May 27,
2015;published OnlineFirst June 25, 2015.
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2015;75:3355-3364. Published OnlineFirst June 25, 2015.Cancer
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Relies on an Absence of p53 FunctionTherapeutic Targeting of the
Warburg Effect in Pancreatic Cancer
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Published OnlineFirst June 25, 2015; DOI:
10.1158/0008-5472.CAN-15-0108
http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-15-0108http://cancerres.aacrjournals.org/content/suppl/2015/06/25/0008-5472.CAN-15-0108.DC1http://cancerres.aacrjournals.org/content/75/16/3355.full#ref-list-1http://cancerres.aacrjournals.org/content/75/16/3355.full#related-urlshttp://cancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://cancerres.aacrjournals.org/content/75/16/3355http://cancerres.aacrjournals.org/
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