Therapeutic Targeting of the Warburg Effect in Pancreatic ......patients underscores the urgent need to identify novel thera-peutic targets that exploit the underlying mechanistic

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

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.

Cancer Res; 75(16) August 15, 2015 Cancer Research3356

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Published OnlineFirst June 25, 2015; DOI: 10.1158/0008-5472.CAN-15-0108

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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

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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.

LDH-A Inhibition in Pancreatic Cancer

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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).

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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.

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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

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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.






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

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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.

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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

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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.






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

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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.





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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Table 1. Apparent rate constants kP (pyruvate-to-lactate conversion) of tumorsfrom FX11 pretreatment and D7 FX11 treatment

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Rajeshkumar et al.




2015;75:3355-3364. Published OnlineFirst June 25, 2015.Cancer Res N.V. Rajeshkumar, Prasanta Dutta, Shinichi Yabuuchi, et al. Relies on an Absence of p53 FunctionTherapeutic Targeting of the Warburg Effect in Pancreatic Cancer

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Therapeutic Targeting of the Warburg Effect in Pancreatic ......patients underscores the urgent need to identify novel thera-peutic targets that exploit the underlying mechanistic