vegfr2 mediated reprogramming of mitochondrial …cancer res; 78(3); 731–41. 2017 aacr....

Translational Science

VEGFR2–Mediated Reprogramming ofMitochondrial Metabolism Regulates theSensitivity of Acute Myeloid Leukemia toChemotherapySandrina N�obrega-Pereira1, Francisco Caiado1, Tania Carvalho1, Ines Matias1,Goncalo Graca2, Luís G. Goncalves2, Bruno Silva-Santos1,3, Haakan Norell1, andS�ergio Dias1,3

Abstract

Metabolic reprogramming is central to tumorigenesis, butwhether chemotherapy induces metabolic features promotingrecurrence remains unknown. We established a mouse xenograftmodel of human acute myeloid leukemia (AML) that enabledchemotherapy-induced regressions of established disease fol-lowed by lethal regrowth of more aggressive tumor cells. HumanAML cells from terminally ill mice treated with chemotherapy(chemoAML) had higher lipid content, increased lactate produc-tion and ATP levels, reduced expression of peroxisome prolifera-tor–activated receptor gamma coactivator 1a (PGC-1a), andfewer mitochondria than controls from untreated AML animals.These changes were linked to increased VEGFR2 signaling that

counteracted chemotherapy-driven cell death; blocking ofVEGFR2 sensitized chemoAML to chemotherapy (re-)treatmentand induced a mitochondrial biogenesis program withincreased mitochondrial mass and oxidative stress. According-ly, depletion of PGC-1a in chemoAML cells abolished suchinduction of mitochondrial metabolism and chemosensitiza-tion in response to VEGFR2 inhibition. Collectively, this revealsa mitochondrial metabolic vulnerability with potential thera-peutic applications against chemotherapy-resistant AML.

Significance: These findings reveal a mitochondrial metabolicvulnerability that might be exploited to kill chemotherapy-resistantacute myeloid leukemia cells. Cancer Res; 78(3); 731–41. �2017 AACR.

IntroductionMetabolic reprogramming is a hallmark of cancer and apart

from the well-knownWarburg effect of aerobic glycolysis, severalother metabolic adaptations have been described (1, 2). The useof alternative carbon sources and the establishment of metabolicinteractions within the tumor microenvironment are examples ofadaptation to secondary microenvironments and pharmacologictreatments (1–3). Tumors are further metabolically heteroge-neous, and several energetic programs may coexist (4). There isindeed compelling evidence across several tumor types thatquiescent "cancer stem cells" rely more exclusively on mitochon-drial respiration and oxidative phosphorylation (OXPHOS) forenergy production anddisplay impaired glycolytic capacity (5–8).

Conversely, suppression of mitochondrial genes was identified asa key metabolic signature of metastatic cancers and associatedwith the worst clinical outcome across several cancer types (9),and in particular, themitochondrial biogenesis factor peroxisomeproliferator–activated receptor gamma coactivator 1-alpha (PGC-1a) was found to play a suppressive role in melanoma andprostate cancer metastasis and to resensitize renal cell carcinomato cytotoxic therapies (10–12). These findings suggest that cancercells can undergo a multifaceted rewiring of cellular metabolismin order to support their biosynthetic needs and highlight newmetabolic vulnerabilities that can potentially be exploited toeradicate aggressive subsets of tumor cells.

Acute myeloid leukemia (AML) comprises a heterogeneousgroup of aggressive hematologic malignancies characterized bythe arrest of leukemic myeloblasts at an immature and selfrenew-ing stage of development. Conventional drug therapy for AMLhasremained unchanged over the last 40 years (a combination of thecell-cycle inhibitor cytosine arabinoside, Cytarabine, with ananthracycline derivative, likeDaunorubicin or Idarubicin; ref. 13).In spite of inducing initial AML remission in most patients,acquisition of therapy resistance results in frequent relapse,highlighting the need for therapies able to tackle recurrences.Several chemokine and growth factor signaling pathways, inparticular the VEGF signaling, regulate AML proliferation, surviv-al, and chemotherapy resistance (14–17). Key studies showed thatprofound changes in cellular metabolism are also criticallyinvolved in the progression and pathogenesis of AML, withincreased synthesis and consumption of lipids and enhanced

1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade deLisboa, Lisboa, Portugal. 2Instituto de Tecnologia Química e Biol�ogica, Avenidada Rep�ublica, Estac~ao Agron�omica Nacional, Oeiras, Portugal. 3Faculdade deMedicina, Universidade de Lisboa, Lisboa, Portugal.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: S�ergio Dias, Instituto de Medicina Molecular, Facul-dade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal. Phone:351-217999411; Fax: 351-217999412; E-mail: [email protected]; and HaakanNorell, [email protected]

doi: 10.1158/0008-5472.CAN-17-1166

�2017 American Association for Cancer Research.

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glycolytic flux associated with the consumption of fructose,representing important metabolic adaptations that affect leuke-mia cell survival, tumorigenic potential, and therapy resistance(6, 18–23). Additional studies highlighted an intimate relation-ship between metabolic changes and the differentiation status inAML, with the control of myeloid differentiation by pyrimidinebiosynthesis and the reliance of leukemic stem cells on OXPHOSfor energy metabolism (5, 6, 24). Despite these established rolesof altered metabolism for AML tumorigenesis and the severeimpact of chemotherapy resistance onpatient outcome, a putativelink between chemotherapy-induced AML phenotypes and met-abolic features that reduce chemosensitivity anddrive progressionof AML has not been established.

Material and MethodsIntrabone marrow xenotransplantation of human AML cellsinto immunodeficient Rag2�/�IL2rgchain

�/� mice, diseasemonitoring, and in vivo chemotherapeutic treatment

All animal experiments were conducted in accordance withstandard institutional animal care procedures and followedethical committee protocols. An orthotopic xenoengraftmentmodel of human AML was established by injection of 10,000human erythroleukemia (HEL; DSMZ), pretransduced with alentiviral vector encoding luciferase and GFP and enriched forGFPþ cells, into the BM (intratibial injection in the right tibia)of sublethally irradiated (250 Rad, 1 day before) immunode-ficient, 8- to 16-week-old Rag2�/�IL2rgchain

�/� mice (kindlyprovided by Dr. Barata; ref. 25) on oral antibiotics (Bactrim,2 days before). Tumor progression was assessed by flow cyto-metry–based enumeration of the number of tumor cells in theblood (longitudinally) and peripheral organs (terminal diseasestage). A predefined threshold of 100 circulating GFPþ cells/mLof PB was used to, on an individual basis, define the trigger(systemic disease detection) and thus assignment to receivingin vivo chemotherapy treatment or not (sham control) at similarsystemic disease stage/burden. The chemotherapy protocolapplied consisted of two cycles of a combination of dailyCytarabine (100 mg/kg, intraperitoneal injections on days 1 to5 from triggering) and doxorubicin (3 mg/kg, intraperitonealinjections on days 1 to 3 from triggering), both clinical gradereagents kindly provided by the pharmacy of Hospital de SantaMaria in Portugal.

Primary human AML and cell lines processing and in vitroculture

HEL, KG1, KG1a, GDM-1, MV-4-11, HL-60, OCI-AML3, AML-193, Kasumi-1, and U-937 human AML cell lines were purchasedfromDSMZbetween 2012 and 2016, tested as beingmycoplasmafree by using the PCR Mycoplasma Test Kit II (AppliChem), andauthenticated by examination of morphology and consistent invitro performance. For ex vivo isolation of untreatedAML andchemoAML from terminally illmice from thebonemarrow, femurswere flushed with 500 mL of PBS containing EDTA. The brain andsurroundingmeningeswere isolated, cut repeatedlywith a scalpel,and further disaggregated by passing through a 5 mL pipette. Inboth cases, cellswere strained (40mmmesh) to ahomogenous cellsuspension, centrifuged at 300 x g for 5 minutes, followed by RedCell Lysis using RCLB (Santa Cruz Biotechnology) for 10minutesin the dark at room temperature. The remaining cells werewashed with PBS and seeded in complete media. Ficoll density

centrifugationmedium (GEHealthcare Life Sciences) was used asper the manufacturer's recommendation to eliminate dead cellsand debris. All cell lines were cultured in RPMI 1640 media withL-glutamine, Gibco, supplementedwith 10%heat-inactivated FBS(Gibco) and 1x antibiotic–antimycotic (Gibco) at 200,000 cells/mL in a 37�C incubator containing 5% CO2, stored in liquidnitrogen and thawed and used within 15 passages. For primaryhuman AML samples, bone marrow samples of adult AMLpatientswere collected at theHematologyDepartment at InstitutoPortugues de Oncologia (IPO, Lisbon, Portugal) after informedconsent and Institutional Review Board approval (IPO), in accor-dance with the Declaration of Helsinki (see more in Supplemen-tary Information). For drug treatment, equal number of liveAML cells was treated with VEGFR2ki (VEGF receptor 2 kinaseinhibitor II, Calbiochem, IC50 ¼ 70 nmol/L, used at 70 nmol/L),SU5614 (Semaxanib, Calbiochem, IC50 ¼ 1.2 mmol/L, used at150 nmol/L), FASN inhibitor cerulenin (0.25 mg/mL; Sigma), orhuman VEGF (250 ng/mL; SIGMA) for 48 hours. For the in vitrochemotherapy protocol, AML cells were treated with VEGFR2ki,SU5614, cerulenin, or humanVEGF for 48hours (or 108hours forhAML samples) alone or in combination with chemotherapy(Cytarabine, 0.6 mmol/L; doxorubicin, 81 nmol/L) added duringthe last 24 hours or the last 84 hours for primary hAML samples(see more in Supplementary Information).

Flow cytometryGFPþ cells fromtheBMof terminally ill 1arymicewere isolated ex

vivo by FACS, using the same staining procedure as for the assess-ment of tumor progression and sorting on a BD FACS Aria III flowcytometer. For cell death in vitro, cells were washed in PBS andresuspended in 300 mL Annexin V–binding buffer (BD Bio-sciences), containing 5 mL of 7-aminoactinomycin D (7-AAD; BDPharmingen) and 5 mL Annexin V–PE (BD Biosciences), andincubated for 15 minutes at room temperature before analysis ina LSR Fortessa (BDBiosciences) flow cytometer. FormitochondrialROS, cells were incubated with 1 mmol/L MitoSOX (Life Technol-ogies) for 15 minutes at room temperature and analyzed in a BDFACS Aria III (BD Biosciences) flow cytometer. For lipid droplets,cells were stained with 1 mL Nile Red solution (1:2,000 in acetonefrom stock at 100 mg/mL; Sigma) for 15 minutes at room temper-ature, followed by similar type of analyses. For mitochondrialmass, cells were incubated with 2 nmol/L MitoTracker Deep Red(Molecular Probes) for 15 minutes at room temperature andanalyzed in LSR Fortessa flow cytometer. Mitochondrialmembrane potential was assessed using the TMRE MitochondrialMembrane Potential Assay Kit (Abcam) at 100 nmol/Lfor 20 minutes at 37�C in growth media, washed with PBS-0.2%BSA, and followed by similar type of analyses. FCCP (20 mmol/L)treatment was used for control of TMRE-specific staining. Datawere analyzed using the FlowJo software (LLC; see more at Sup-plementary Information).

Cell extracts and NMR spectroscopyThe same number of AML cells (100 to 110 million cells) were

pelleted by centrifugation and rinsedwith ice-cold PBS. Cells werelysed with ice-cold methanol at constant vortexing, followed bythe sequential addition of ice-cold chloroform and deionizedwater. After phase separation and solvent removal, the organicphase (lipidic fraction) was evaporated under nitrogen and sus-pended in deuterated chloroform, and the aqueous phase super-natants were freeze-dried and suspended in phosphate buffer

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(0.35 mol/L, pH 7.0 in 99.8% D2O containing 2 mmol/L NaN3)before NMR analysis. 1H NMR spectra were acquired at 25�C in aBruker Avance IIþ spectrometer, operating at a frequency of800.33 MHz, equipped with a 5 mm three-channel probe (TXI-ZH/C/N/-D; see more in Supplementary Information).

Metabolic and biochemical assaysFree-fatty acids were determined using the Free Fatty Acid

Quantification Colorimetric Assay Kit (Abcam) according to themanufacturer's instructions using fresh cultured cells with valuesnormalized to cell number. Lactate levels were determined usingthe Lactate Assay Kit (Sigma) and according to manufacturer'sinstructions and samples were deproteinized before assay mea-surement using AmiconUltracel 10k centrifugal filters (Milipore).ATP levels were determined according to the manufacturer'sinstructions by the ATP Determination Kit (Life Technologies),where cells where homogenized in RIPA buffer supplementedwith a protease inhibitor cocktail (Roche). In both cases, proteinwas quantified for normalization using the Bio-Rad Protein Assay(BIO-RAD). VEGF production was determined in AML culturesusing RPMI media supplemented with 2% FBS and quantifiedusing the human VEGF ELISA Kit (Calbiochem). All colorimetricand luminescent measurements were performed using the micro-plate reader TECAN Infinite M200.

Statistical analysisStatistical analyses using a two-tailed Student t test or Mann–

Whitney test were performed with Graph-Pad Prism 5.0 (Graph-Pad software), and one-way ANOVA followed by Tukey posttestformultiple comparisons, anddata are presented asmean� SDor� SEM, as indicated.

ResultsEstablishment of an immunodeficient mouse xenograft modelof human AML that enables relevant in vivo exposure tochemotherapy

To evaluate if in vivo chemotherapy treatment induced acqui-sition of adaptive metabolic and phenotypic features in humanAML, we established a Rag2�/�IL2rgchain

�/� mouse model usingintrabone marrow (IBM) injection of a GFPþLuciferaseþ–trans-duced version of the human erythroleukemia cell line HEL(Fig. 1A). Intratibial injection of 10,000 of these parentalAML cellscaused a highly reproducible disease development and spread (asobserved by luciferase and histologic detection of leukemic cells;Supplementary Fig. S1A and S1B) with 100%mortality (Fig. 1B).In vivo chemotherapy treatment (Cytarabine plus antracyclincombination), applied only after circulating tumor cell numbersin peripheral blood reached a predefined threshold, induceddisease regression in all analyzed compartments and translatedto significantly prolonged survival (Fig. 1A and B; SupplementaryFig. S1C and S1D). This notwithstanding, all mice died fromthe (re-)growing human tumor cells. AML cells from terminallyill mice treated (chemoAML) or not (untreatedAML) by chemother-apy were ex vivo isolated and retransplanted or phenotypicallycharacterized after re-establishment and expansion in vitro.When BM-derived human tumor cells were IBM injected into 2ary

preconditioned Rag2�/�IL2rgchain�/� recipients, chemoAML

showed enhanced aggressiveness (faster and greater disease bur-den) compared with untreatedAML, leading to inferior host sur-vival (Fig. 1C–E).

Chemotherapy-exposed AML cells exhibited distinctmetabolic features

To determine the chemotherapy-induced changes inmetabolicfeatures,weperformedametabolomic analysis of in vitro–expand-ed untreatedAML and chemoAML by 1H-Nuclear Magnetic Reso-nance spectroscopy (1H-NMR). Principal component analysis(PCA) of the intracellular metabolites enabled the differentiationof untreatedAML from chemoAML samples (SupplementaryFig. S1E). Specifically, the loadings from 1H-NMR analysisrevealed that chemoAML cells exhibited increased levels of pyru-vate (the endproduct of glycolysis), phosphocholine, and myo-inositol (soluble metabolites related to lipid metabolism; Sup-plementary Fig. S1F; Supplementary Table S1). The scores fromPCA corresponding to the lipid extracts did not enable thecomplete differentiation of untreatedAML from chemoAML sam-ples (Supplementary Fig. S1E). However, the univariate compar-ison of each variable from the spectral fingerprints revealed higherlevels of saturated fatty acid residues and a tendency for increasedtotal lipids (Supplementary Fig. S1G and S1G'; SupplementaryTable S1). Additional assays confirmed increased levels of lipiddroplets (Fig. 1F; Supplementary Fig. S1H) and long-chain free-fatty acids (Fig. 1G) in chemoAMLwithout significantly altering theexpression of enzymes frommainmetabolic pathways, includingglycolysis, TCA cycle, glutaminolysis, lipid metabolism, andOXPHOS (Supplementary Fig. S1I).

We next investigated if in vivo exposure to chemotherapyaffected the cellular bioenergetics of the AML cells. Indeed, che-moAML exhibited decreased mitochondrial DNA (mtDNA) con-tent (Fig. 1H) andmitochondrial mass (MitoTracker staining; Fig.1I), elevated intracellular lactate levels (Fig. 1J), and ATP produc-tion (Fig. 1K). Transmission electron microscopy (TEM) analysisconfirmed the reducednumber ofmitochondria in chemoAML andfurther revealed that morphologically these mitochondriaappeared swollen when compared with untreated conditions(Fig. 1L). Mitochondrial biogenesis is controlled by a networkof nuclear transcription factors and coactivators in which PGC-1aplays a dominant role. ChemoAML indeed had lower expression ofthe mitochondrial biogenesis factors PGC-1a and the nuclearrespiratory factor 1 (NRF1; Fig. 1M).

In order to validate these findings in an alternative human AMLmodel with decrease response to chemotherapy, we took advan-tage of the commercially available human AML cell line KG1 andits derived subline KG1a that has been reported to be resistant tochemotherapy-induced cytotoxicity (26). Similarly, comparedwith KG1, the KG1a subline showed reduced expression ofPGC-1a (Supplementary Fig. S2A). Together, these results indicatethat chemotherapy exposure of the AML cells in vivo droveemergence of distinct metabolic features, a particularity that isshared by other chemotherapy-resistant AML cells.

Chemotherapy-exposed AML cells displayed enhanceddependency on VEGFR2 signaling

Several chemokine and growth factor signaling pathwaysmod-ulate AML biology, and we have previously demonstrated thatVEGF/VEGFR2 signaling pathway plays a key role for both pro-gression and chemotherapy resistance (14, 16). We found thatchemoAML expressed higher levels ofCCR7, CXCR4, and VEGFR1,2, and 3 (Fig. 2A). In addition, chemoAML had increased totallevels of VEGFR2 protein, as determined by flow cytometryintracellular staining (Fig. 2B; Supplementary Fig. S2B) andincreased levels of VEGF-A production (Supplementary

VEGFR2 Affects Mitochondria and Chemosensitivity in AML

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Figure 1.

In vivo chemotherapy treatment drives distinct metabolic processes in human AML cells. A, Representation of the procedures employed in the establishment of theorthotopic IBM xenotransplantation mouse model of human AML. 1, Sublethal-irradiated immunodeficient Rag2�/�IL2rgchain

�/� mice were IBM injected withparentalAML cells (GFPþLuciferaseþ human erythroleukemia HEL cells). 2, Longitudinal measurement of the number of tumor cells (GFPþ, mouseCD45�, 7AAD�);flow cytometry events/mL of peripheral blood (PB) for definition of animals receiving in vivo chemotherapy treatment or not (sham control) at similar systemicdisease burden.B, Flow cytometry quantification of tumor cells in the PBof 1ary HEL-xenotransplantedmice following chemotherapy (black columns) or shamcontrol(white columns) treatment cycles betweenweek 3/4 and 6/7. Tumor cells from terminally ill untreated (week 8: untreatedAML) and chemotherapy-treated (week 10:chemoAML) mice were isolated ex vivo. C–E, Flow cytometry quantification of tumor cells in the PB (C), BM, spleen, liver, and lung (D), and overall survival of 2ary

recipients (E), following injection of 10,000GFPþ cells isolated by FACS from the BMof untreated (untreatedAML) or chemotherapy-treated (chemoAML) terminally ill1ary mice. Each line corresponds to one animal. F, Flow cytometry quantification of lipid droplets (Nile Red staining) in untreatedAML and chemoAML (n ¼ 5).G, Quantification of free-fatty acids by a colorimetric assay in untreatedAML and chemoAML (n ¼ 5). H, mtDNA content assessed by qPCR analysis of the humanmitochondrial ND1 (mtND1) gene relative to the nuclear b2-microglobulin gene in DNA samples from untreatedAML and chemoAML (n ¼ 5). I, Flow cytometryquantification of MitoTracker Deep Red staining in untreatedAML and chemoAML (n ¼ 5). J, Lactate production in cell extracts from untreatedAML and chemoAML

(n¼ 5).K,ATP levels in cell extracts fromuntreatedAML and chemoAML (n¼ 5). L,Quantification of the number ofmitochondria per cell in TEM low-power field imagedsections (left; n ¼ 15 cells from three independent untreatedAML and chemoAML) and representative images (right; scale bars, low-power field 2 mm andhigh-power field 500 nm).M, qPCR analysis of the relative expression of the indicated genes in chemoAML relative to untreatedAML (n¼ 5). All primer sequences aredescribed in Supplementary Table S2. MFI, median fluorescent intensity. Data are presented as mean � SD (B and D) or SEM (F–M). Statistical significance wasdetermined by the Mann–Whitney test (B, D, and E) or two-tailed Student t test (F–M). � , P < 0.05; �� , P < 0.01; �� , P < 0.001.





Figure 2.

Chemotherapy-exposed AML cells displayed enhanced dependency on VEGFR2 signaling. A, qPCR analysis of the relative expression of the indicated genes inchemoAML relative to untreatedAML (n¼ 5/7). B, Flow cytometry analysis of VEGFR2 intracellular staining in untreatedAML and chemoAML (n¼ 7). C,Western blot forBcl-2 andBad proteins in untreatedAML and chemoAML under control or upon treatment with SU5614 or VEGFR2ki (representative images; uncropped images of blotsare shown in Supplementary Fig. S6A). b-Actin was used as a loading control. D, Flow cytometry cell death analysis of untreatedAML and chemoAML-treated withchemotherapy (chemo) alone or in combination with VEGFR2ki depicted as the quantification of Annexin Vþ; 7AADþ cells (left; n ¼ 5) in respect to basal andrepresentative flow cytometry plots (right), where fold (x) refers to increase over basal. E, Flow cytometry cell death analysis of untreatedAML treated withchemotherapy (chemo) alone or in combination with human VEGF depicted as the quantification of Annexin Vþ; 7AADþ cells (n ¼ 4) in respect to basal. F, Flowcytometry cell death analysis of KG1 and KG1a cell lines treated with chemotherapy (chemo) alone or in combination with VEGFR2ki depicted as the quantificationof Annexin Vþ; 7AADþ cells (n ¼ 6) in respect to basal. G, Flow cytometry analysis of primary human AML (hAML) samples depicted as the quantification ofAnnexin V�; 7AAD� (viable) cells in chemotherapy (chemo) alone or combinationwith VEGFR2ki (fold to chemo; n¼ 6). Due to the variability in cell viability at basallevels, the data are expressed as the fold of viable cells under combined treatment (VEGFR2kiþchemo) compared with chemotherapy alone. Data are presentedas mean � SEM or SD. F, Statistic significance was determined by the two-tailed Student t test, with the exception of D and F, where one-way ANOVAfollowed by Tukey posttest was used. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.





Fig. S2C). We detected increased proportion of Bcl2:Bad proteinlevels in chemoAML compared with untreatedAML at baseline (Fig.2C, control). Notably, treatment with cell-permeable VEGFR2kinase inhibitors (semaxanib, SU5614, and VEGFR2 tyrosinekinase inhibitor, VEGFR2ki) decreased the proportion of Bcl2:Bad in chemoAML (Fig. 2C; Supplementary Fig. S2D), suggestingthat chemotherapy-exposedAML cells rely on aVEGFR2 autocrineinternal signaling loop for survival.

In order to investigate if VEGFR2 signaling modulated theresponse of the AML cells to chemotherapy, we tested inductionof cell death in vitro. UntreatedAML and chemoAML showed nodifference in the proliferation rate or survival at baseline (Fig. 2D,right, basal; Supplementary Fig. S2E and S2F). However,chemoAML showed a decreased sensitivity to chemotherapy-induced cell death in vitro (Fig. 2D, chemo). Importantly,the response of chemoAML to chemotherapy was restored whencells were treated in combination with a VEGFR2–specifickinase inhibitor (VEGFR2ki), almost doubling the cell deathcompared with retreatment with chemotherapy alone (Fig. 2D,VEGFR2kiþ chemo), whereas treatment with the inhibitor aloneproduced onlyminor effects (Fig. 2D, VEGFR2ki). Treatment withthe alternative less-potent VEGFR2 inhibitor SU5614 also pro-duced a slight increment in cell death response to chemotherapyin chemoAML cells (Supplementary Fig. S2G, SU5614 þ chemo).Conversely, the response of untreatedAML to chemotherapydecreased when cells were treated in combination with humanVEGF (Fig. 2E, VEGF þ chemo), resembling the lower sensitivityof chemoAML to chemotherapy-induced cell death (see Fig. 2D,chemo). We further confirmed these findings in the human AMLcell line KG1 and the less-chemotherapy responsive KG1a sublinethat displays increased expression of VEGFR2 in basal conditions(Supplementary Fig. S2A). KG1a showed adecreased sensitivity tochemotherapy-induced cell death in vitro (Fig. 2F, chemo) thatwas restored (to the level of KG1 response to chemotherapyalone) when cells were treated in combination with VEGFR2ki(Fig. 2F, VEGFR2ki þ chemo). Moreover, an increase cell deathresponse was also observed in several other human AML cell lineswhen VEGFR2 inhibitor was combined with chemotherapy (Sup-plementary Fig. S2H). Importantly, these findings were furthervalidated in primary human AML patient's samples, which dis-play decreased cell viability when chemotherapy treatment wascombined with VEGFR2ki (Fig. 2G, VEGFR2ki þ chemo).

Lipid synthesis and consumption play a pivotal role in AMLprogression and therapy resistance (18, 21). Interestingly, treat-mentwith the FASN inhibitor cerulenin produced no alteration inthe cell death response of chemoAML to chemotherapy butenhanced it in untreatedAML (Supplementary Fig. S2I), suggestingthat chemotherapy-exposed AML cells rely less on de novo fattyacid synthesis for the response to chemotherapy. Overall, theseresults suggest that VEGFR2 signaling modulates the cell deathresponse of AML cells to chemotherapy treatment in vitro.

VEGFR2 signaling regulates mitochondrial biogenesis andmetabolism in AML

We next investigated if VEGFR2 signaling modulated theresponse of AML cells to chemotherapy by inducing metabolicchanges. Treatment of chemoAML (and generally also untreate-dAML) with VEGFR2ki increased their mtDNA content (Fig. 3A;Supplementary Fig. S3A for SU5614), mitochondrial mass (Mito-Tracker staining, Fig. 3B; Supplementary Fig. S3B for SU5614),and mitochondrial transmembrane potential (TMRE

staining, Fig. 3C; Supplementary Fig. S3C for SU5614). TEManalysis confirmed the numerical increase in mitochondria uponVEGFR2 blockade (Fig. 3D; Supplementary Fig. S3D for SU5614),with VEGFR2ki–treated chemoAMLmitochondriamorphological-ly resembling untreatedAML. In contrast, treatment of untreate-dAML with VEGF led to the concomitant reduction of the mito-chondrial mass (MitoTracker staining; Supplementary Fig. S3E)and mitochondrial transmembrane potential (TMRE staining;Supplementary Fig. S3F). We further confirmed these findings inthe human AML cell line KG1a that, compared with KG1, showeddecreased mitochondrial mass (MitoTracker staining; Fig. 3E,control) and mitochondrial transmembrane potential (TMREstaining, Supplementary Fig. S3G, control), whereas those weremarkedly increased upon VEGFR2ki treatment (Fig. 3E; Supple-mentary Fig. S3G). Moreover, this was additionally validated inhuman primary AML patient samples with VEGFR2ki treatment,leading to increase mitochondrial mass (MitoTracker staining,Fig. 3F; Supplementary Fig. S3H) and mtDNA content (Supple-mentary Fig. S3I). Treatment of chemoAML with VEGFR2kiincreased the expression of several mitochondrial biogenesisfactors, including PGC-1a, PGC-1b, and the estrogen-relatedreceptor a (ERRa), a transcription factor through which PGC-1a function is transduced (Fig. 3G; Supplementary Fig. S3J forSU5614). An increased expression of mitochondrial biogenesisfactors upon VEGFR2ki treatment was also observed in KG1 andmore robustly in KG1a cells (displayed in Supplementary Fig.S2A) and in human primary AML patient samples (Supplemen-tary Fig. S3K). Interestingly, treatment of chemoAML with VEGFR2inhibitors did not change the content of intracellular lipid dro-plets, lactate, and ATP levels (Supplementary Fig. S3L–S3N).

We reasoned that elevated mitochondrial metabolism inducedbyVEGFR2blockage inAMLcellswould likely be accompaniedbyan increase in reactive oxygen species (ROS) production that canlead to detrimental oxidative stress. Treatment with VEGFR2kiproduced a slight increase in the levels of mitochondria-derivedROS (MitoSOX staining; Fig. 3H) and 8-hydroxyguanosine(8oxoG), an oxidized derivative of guanosine that is commonlyused as a biomarker of oxidative stress (Fig. 3I) only in untrea-tedAML; whereas treatment with the less potent VEGFR2 inhibitorSU5614 was more effective in increasing mitochondrial-derivedROS (Supplementary Fig. S3O) and oxidative stress markers(Supplementary Fig. S3P). Together, these results suggest thatblocking VEGFR2 signaling increases mitochondrial biogenesis,function, and, to some extent, mitochondrial-derived ROS for-mation and oxidative stress in AML, providing a rationale for theincreased sensitivity to chemotherapy mediated by VEGFR2 inhi-bition in AML cells.

PGC-1a–mediated mitochondrial metabolism is criticalfor the chemotherapy sensitization provided by VEGFR2inhibition in AML

To evaluate if PGC-1a and mitochondrial biogenesis weredirectly required for the sensitization to chemotherapy thatVEGFR2 inhibition provided in AML, we silenced PGC-1a usinggene expression knockdown (PGC-1a shRNA). Compared withscramble (SC) shRNA, we detected significantly less PGC-1atranscript in PGC-1a shRNA untreatedAML, and the reducedexpression of PGC-1a evident already at baseline in chemoAML

was further decreased (Supplementary Fig. S4A, left). Chemother-apy-exposed AML cells had reduced levels of PGC-1a protein(Supplementary Fig. S4B), and a reduction inPGC-1aproteinwas





also observed in AML cells transduced with PGC-1a shRNA(Supplementary Fig. S4B). Critically, the induction of PGC-1atranscript achieved upon VEGFR2 inhibition in the SC shRNA

lineages was abolished in PGC-1a–depleted AML (Supplemen-tary Fig. S4A, left). Interestingly, silencing PGC-1adid not alter theexpression of PGC-1b, andmoreover, VEGFR2 inhibition was still

Figure 3.

VEGFR2 signaling regulatesmitochondrial metabolism in AML.A,mtDNA content assessed by qPCR analysis of the humanmitochondrial ND1 (mtND1) gene relativeto the nuclear b2-microglobulin gene in DNA samples from untreatedAML and chemoAML alone (control) or treated with VEGFR2ki (n ¼ 5). B, Flow cytometryquantification of MitoTracker Deep Red staining in untreatedAML and chemoAML alone (control) or treated with VEGFR2ki (n ¼ 5; representative histograms inSupplementary Fig. S3H). C, Flow cytometry quantification of mitochondrial membrane potential (Dcm, TMRE staining) in untreatedAML and chemoAML

alone (control) or treatedwith VEGFR2ki (n¼ 5/6; representative histograms in Supplementary Fig. S3H).D,Quantification of the number ofmitochondria per cell inTEM-imaged sections (2 mm) in untreatedAML and chemoAML alone (control) or treated with VEGFR2ki (left, n¼ 9/14 cells from two independent untreatedAML andchemoAML) and representative images (right; scale bar, 500 nm). E, Flow cytometry quantification of MitoTracker Deep Red staining in KG1 and KG1a alone(control) or treated with VEGFR2ki (n ¼ 6/8). F, Flow cytometry quantification of MitoTracker Deep Red staining in primary human AML (hAML) samples alone(control) or treatedwithVEGFR2ki (n¼ 7; representative histograms in Supplementary Fig. S3H).G,qPCR analysis of the relative expression of the indicated genes inuntreatedAML and chemoAML alone (control) or treated with VEGFR2ki (n ¼ 5). H, Flow cytometry quantification of mitochondrial ROS (MitoSOX staining) inuntreatedAML and chemoAML alone (control) or treated with VEGFR2ki (n ¼ 5/6; representative histograms in Supplementary Fig. S3H). I, Flow cytometryquantification of 8oxoG in untreatedAML and chemoAML alone (control) or treated with VEGFR2ki (n ¼ 5; representative histograms in Supplementary Fig. S3H).Data are presented as mean � SEM or SD. E, Statistical significance was determined by one-way ANOVA, followed by Tukey posttest. � , P < 0.05; �� , P < 0.01;�� , P < 0.001.





Figure 4.

Regulation of PGC-1a andmitochondrial biogenesis underlies the chemotherapy-sensitization provided byVEGFR2 inhibition in AML.A,mtDNA content assessed byqPCR analysis of the human mitochondrial ND1 (mtND1) gene relative to the nuclear b2-microglobulin gene in DNA samples from untreatedAML and chemoAML

transduced with scramble (SC) shRNA or PGC-1a shRNA alone (control) or treated with VEGFR2i (n ¼ 5/6). Efficiency of PGC-1a shRNA knockdown wasevaluated in Supplementary Figs. S4A, S4B, and S6B. B, Flow cytometry quantification of Mitotracker Deep Red staining in untreatedAML and chemoAML transducedwith scramble shRNA or PGC-1a shRNA alone (control) or treated with VEGFR2ki (n¼ 5/6; representative flow cytometry histograms in Supplementary Fig. S4D).C, Flow cytometry quantification of mitochondrial membrane potential (Dcm, TMRE staining) in untreatedAML and chemoAML transduced with scrambleshRNA or PGC-1a shRNA alone (control) or treated with VEGFR2ki (n ¼ 5/6). D, Flow cytometry quantification of Mitotracker Deep Red staining in KG1 and KG1atransducedwith scramble shRNA or PGC-1a shRNA alone (control) or treatedwith VEGFR2ki (n¼ 5/8). E–E', Flow cytometry cell death analysis of untreatedAML andchemoAML transduced with scramble shRNA or PGC-1a shRNA treated with chemotherapy (chemo) alone or in combination with VEGFR2ki depicted asthe quantification of Annexin Vþ; 7AADþ cells (n ¼ 5/8; E) in respect to basal and representative flow cytometry plots (E'), where fold (x) refers to increase overchemo. F, Flow cytometry cell death analysis of KG1 and KG1a transduced with scramble shRNA or PGC-1a shRNA treated with chemotherapy (chemo) incombination with VEGFR2ki depicted as the quantification of Annexin Vþ; 7AADþ cells (n ¼ 5/8). G, ATP levels in cell extracts from untreatedAML and chemoAML

transduced with scramble shRNA or PGC-1a shRNA alone (control) or treated with VEGFR2ki (n ¼ 5/6). H, Flow cytometry quantification of mitochondrialROS (MitoSOX staining) in untreatedAML and chemoAML transduced with scramble shRNA or PGC-1a shRNA alone (control) or treated with VEGFR2ki (n ¼ 5/7).Data are presented as mean � SEM or SD. D and F, Statistical significance was determined by one-way ANOVA, followed by Tukey posttest. � ,#, P < 0.05;�� ,##, P < 0.01; �� , P < 0.001. Hashtag symbol indicates statistical significance to the corresponding chemo condition.





capable of inducing PGC-1b expression in PGC-1a–depletedchemoAML cells (Supplementary Fig. S4A, right). PGC-1a deple-tion resulted in suppressed mitochondrial biogenesis and func-tion in both untreatedAML and chemoAML, as assessed by reducedmtDNA content (Fig. 4A control), mitochondrial mass (Mito-Tracker staining, Fig. 4B control), and mitochondrial transmem-brane potential (TMRE staining, Fig. 4C). Treatment withVEGFR2ki was no longer capable of inducing the mitochondrialbiogenesis program in PGC-1a–depleted AML cells (Fig. 4A–C;Supplementary Fig. S4C–S4E for SU5614), suggesting that PGC-1a is fundamental for the induction of mitochondrial biogenesisand function elicited upon VEGFR2 blockage in AML cells. Sim-ilarly, PGC-1a depletion in KG1 and its derived subline KG1a(Supplementary Fig. S4F) also led to the reduction of mitochon-drial mass (MitoTracker staining, Fig. 4D control) and mtDNAcontent (Supplementary Fig. S4G), and treatment with VEGFR2kiwas no longer capable of inducing the mitochondrial biogenesisprogram inPGC-1a–depleted KG1a cells (Fig. 4D; SupplementaryFig. S4G).

To investigate if PGC-1a–mediated mitochondrial biogenesiswas required for the chemosensitization provided by VEGFR2inhibition, we exposed SC and PGC-1a shRNA manipulatedcultures of untreatedAML and chemoAML to chemotherapy in vitro(as above). None of the lines presented difference in viability atbaseline (Supplementary Fig. S4H). Moreover, depletion of PGC-1a did not affect the response of untreatedAML or chemoAML tochemotherapy in vitro, with chemoAML displaying decreased celldeath response to chemotherapy compared with untreatedAML

regardless of the shRNA conditions (Fig. 4E and E', chemo).Strikingly, and in great contrast to the SC shRNA conditions,blocking VEGFR2 signaling did not increase the response of PGC-1a–depleted AML cells to chemotherapy (Fig. 4E and E',VEGFR2ki þchemo), suggesting that the PGC-1a–induced mito-chondrial biogenesis program was critically required for thechemosensitizationmediated by VEGFR2 inhibition in AML cells.Similarly, PGC-1a–depleted KG1 and KG1a cells present lowercell death response to the combined treatment of chemotherapyand VEGFR2 blockade compared with the corresponding SCcondition (Fig. 4F, VEGFR2kiþchemo).

To get mechanistic insights into how PGC-1a and mitochon-drial function confer the chemosensitization mediated byVEGFR2 inhibition, we evaluated the energetic metabolismand ROS production in the shRNA-manipulated lines. PGC-1a depletion led to a dramatic reduction in ATP productionexclusively in chemoAML, under both control and VEGFR2inhibitor–treated conditions (Fig. 4G; Supplementary Fig.S4I for SU5614), with no major alterations detected uponshRNA manipulation of untreatedAML. In contrast, PGC-1adepletion abolished the increase in mitochondrial ROS drivenby VEGFR2 inhibition in untreatedAML (Fig. 4H; SupplementaryFig. S4J for SU5614), but had limited effect in chemoAML whereVEGFR2ki treatment in fact induced a small increase in Mito-SOX in the PGC-1a–depleted chemoAML (Fig. 4H). The mito-chondrial energetic metabolism was thus severely more affectedupon PGC-1a depletion in AML cells previously exposed tochemotherapy in vivo.

We next analyzed RNA sequencing data from over 160 AMLpatients (GSE12417_U133A) using the PROGgeneV2 PrognosticDatabase platform, which revealed that patients with relativelylow expression of PGC-1a had significantly worse overall survivalthan patients whose tumors expressed relatively high levels of

PGC-1a (Supplementary Fig. S5), indicating that low expressionof PGC-1a is an adverse clinical risk factor in AML.

Overall, these results suggest that VEGFR2 signaling decreasesthe response of AML cells to chemotherapy by repressing PGC-1a–mediated mitochondrial biogenesis, revealing a new meta-bolic vulnerability that potentially can be exploited to overcometreatment resistance in AML.

DiscussionTaken together, our data indicate that in vivo chemotherapy

exposure generated AML cells with adapted metabolic properties,including different content of soluble metabolites (pyruvate,phosphocholine, andmyo-inositol), higher lipid levels, increasedlactate and ATP production, reduced number of mitochondria,and lower expression of mitochondrial biogenesis factors includ-ing PGC-1a. The metabolic signature of chemotherapy-exposedAML cells is to the same extent compatible with an increasedreliance on glycolysis and decreased dependence on mitochon-drialmetabolism for bioenergetics. Suppression ofmitochondrialgenes was recently identified as a key metabolic signature ofmetastatic cancers and associated with worse clinical outcome(9), suggesting the adoption of a common metabolic landscapeby aggressive (metastatic and therapy-resistant) cancer cell popu-lations across several tumor types.

Existing therapeutic mouse xenograft models of human AML,in particular those using NSG mice, have been limited by thetoxicity induced by chemotherapy regimens (27). Specifically, theSCID mutation prevents exposure to really clinically relevantformulations and doses of chemotherapy in vivo. To achievesufficient in vivo treatment effects of chemotherapy on the phys-iology of the surviving tumor cells, we thus employed theRag2�/�IL2rgchain

�/� mouse model, compensating the relativelylower level of engraftment in this mouse strain with irradiationpreconditioning IBM injection of the aggressive human AML cellline HEL (Fig. 1; Supplementary Fig. S1). AML cells from micetreated with chemotherapy also exhibited increased expression ofseveral chemokine and growth factor receptors, which are well-known regulators of leukemia cell viability but less implicated inthe control of metabolic plasticity in AML. VEGF receptor expres-sion in acute leukemia is a prognostic factor that correlates withdisease progression and patient overall survival (28, 29). Inaddition, we have shown that VEGF/VEGFR2 signaling regulatesleukemia cell survival by both internal and external autocrineloops, with induction of prosurvival pathways and inhibition ofapoptosis downstream of VEGFR2 internal signaling (14, 16, 17).Here, we show that VEGFR2 signaling modulates the response ofAML cells (in several AMLmodels; including in vivo selected uponexposure to chemotherapy cell lines, established chemotherapy-resistant cell lines and in primary AML patient's samples) tochemotherapy by maintaining a low mitochondrial mass andbiogenesis. Accordingly, VEGFR2 inhibition restored mitochon-drial biogenesis and function (with no significant impact on ATPproduction), decreased the Bcl-2:Bad protein ratio, and, impor-tantly, increased the response of chemoAML cells to chemotherapyretreatment.

Increased mitochondrial metabolism is believed to represent aliability to tumor cells, due to the role of mitochondria inapoptosis and generation of ROS (30), and a low ROS and highBcl-2 expression state has been described for human quiescentleukemia stem cells (6). Moreover, recently Ara-C–resistant AML





cells have been shown to exhibit metabolic features and genesignatures consistent with a high OXPHOS status (31). However,for chemotherapy (combined treatment of Cytarabine and doxo-rubicin)-exposed AML cells, we demonstrate that mitochondrialmetabolism is required for the sensitization to chemotherapyprovided by VEGFR2 inhibition in the absence of overt changes inmitochondrial ROS production and oxidative stress markers,similarly to what has been described for the suppressive functionof PGC-1a in prostate cancer metastasis (10). These findingssupport thenotion that VEGFR2 signalingdecreases the sensitivityof AML cells to chemotherapy by reprogramming mitochondrialbioenergetic and apoptotic functions, resulting in overallimproved tumor cell viability.

Autocrine VEGF signaling has been shown to regulate anabolicmetabolism and survival of endothelial cells (32). Moreover, thedevelopment of resistance to antiangiogenic multikinase inhibi-tors is accompanied by an increase reliance on mitochondrialrespiration necessary for solid tumor survival (33). These findingssuggest that VEGFR2 signaling–mediated repressionofmitochon-drial metabolismmay be a common strategy in cancer, leading todifferential tumorigenic outcomes according to the specific bioe-nergetic and biosynthetic requirements. In our studies, VEGFR2inhibition does not significantly alter other metabolic features ofchemoAML, such as lipid droplets content or lactate production.We detected no difference in the activity of FASN, the rate-limitingenzyme in de novo fatty acid synthesis, in chemoAML, suggestingthat increased fatty acid content in chemoAML cells is most likelydue to extracellular uptake, rendering chemoAML not susceptibleto sensitization to chemotherapy provided by inhibition of denovo fatty acid synthesis (Supplementary Fig. S2I; ref. 34). Leu-kemic stem cells have been shown to evade chemotherapy bymetabolic adaption to an adipose tissue niche in a CD36-depen-dent manner (35); however, the contribution of leukemia–BMniche metabolic interactions has not been evaluated in this work.

VEGFR2 inhibition elicited a transcriptional response in che-motherapy-exposed AML cells, resulting in increased expressionof PGC-1a, PGC-1b, ERRa, and NRF1 (Fig. 3G; SupplementaryFig. S2A and S3K), which are overall involved in oxidative metab-olism and mitochondrial biogenesis (36). PGC-1a has beenreported to have pro- and antitumorigenic functions in varioustumors (7, 10–12, 37, 38). Importantly, in a clinical AML data-base, low levels of PGC-1a expression were associated with worstoverall patient survival, and in chemotherapy-exposed AML cells,PGC-1adepletion abolished the capacity of VEGFR2 inhibition tosensitize AML cells to chemotherapy, revealing that PGC-1arepresents a suppressive axis for the VEGFR2 signaling–mediatedincreased viability of AML cells.

There is an increasing appreciation of the importance of target-ing mitochondrial metabolism for cancer therapy (39). In par-ticular, recent studies suggest that a PGC-1a/ERRa-mediated

metabolic program can be exploited to overcome therapy resis-tance in breast cancer (40, 41). Due to the implication of VEGFR2signaling in several biological processes (includinghematopoiesisand angiogenesis), it will be important to determine if PGC-1aactivation is a viable therapeutic modality to increase the apo-ptotic response of less-sensitive AML cells to conventionalchemotherapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S. N�obrega-Pereira, F. Caiado, H. Norell, S. DiasDevelopment of methodology: S. N�obrega-Pereira, F. Caíado, I. Matias,B. Silva-Santos, H. NorellAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. N�obrega-Pereira, F. Caiado, T. Carvalho, I. Matias,G. Graca, B. Silva-Santos, H. NorellAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. N�obrega-Pereira, F. Caiado, G. Graca, L.G.Goncalves, S. DiasWriting, review, and/or revision of the manuscript: S. N�obrega-Pereira,F. Caiado, G. Graca, L.G. Goncalves, H. Norell, S. DiasAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. N�obrega-Pereira, S. DiasStudy supervision: S. N�obrega-Pereira, H. Norell, S. Dias

AcknowledgmentsThe authors thank Dr. J. Serpa for performing FASN activity assays; Dr. Nuno

BarbosaMorais and Bernardo Carvalho Pereira de Almeida for performing RNAsequencing databases analysis; Dr. Vanessa Morais for TEM analysis and criticalreading of the article; Andreia Pinto for TEM technical assistance; and InesBarbosa and Maria Gomes da Silva for collection of primary human AMLsamples at Hematology Department, Instituto Portugues de Oncologia deFrancisco Gentil, Lisboa. The study was also supported by the following:Histology and Comparative Pathology Laboratory, Flow Cytometry, Bioima-ging and Rodent facilities at the Instituto de Medicina Molecular; InstitutoGulbenkian de Ciencia Electron Microscopy Facility for usage of the Hitachi H-7650microscope; CERMAX (Centro de RessonanciaMagn�etica Ant�onio Xavier)for NMR data acquisition. This study was supported by the research grants FCTPTDC/BIM-ONC/1242/2012 (to H. Norell), Associac~ao Laco (to S. Dias), andAPCL-SEMAPA 2014 (to H. Norell), Portugal. S. N�obrega-Pereira, F. Caiado, G.Graca, L.G. Goncalves, and H. Norell are recipients of individual FCT postdoc-toral fellowships (SFRH/BPD/91159/2012, SFRH/BPD/91344/2012, SFRH/BPD/93752/2013, SFRH/BPD/111100/2015, and SFRH/BPD/112968/2015,respectively). The costs of publication of this article were funded by LISBOA-01-0145-FEDER-007391, cofunded by FEDER through POR Lisboa 2020 Pro-grama Operacional Regional de Lisboa, from PORTUGAL 2020.

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 April 26, 2017; revised September 14, 2017; accepted November 30,2017; published OnlineFirst December 11, 2017.

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