Biology of Human Tumors

Protein Kinase C Epsilon Is a Key Regulator ofMitochondrial Redox Homeostasis in AcuteMyeloid LeukemiaDaniela Di Marcantonio1, Esteban Martinez1, Simone Sidoli2, Jessica Vadaketh1,3,Margaret Nieborowska-Skorska4, Anushk Gupta1,3, Jake M. Meadows1,Francesca Ferraro1, Elena Masselli5, Grant A. Challen6, Michael D. Milsom7,8,Claudia Scholl9, Stefan Fr€ohling9, Siddharth Balachandran1, Tomasz Skorski4,Benjamin A. Garcia2, Prisco Mirandola5, Giuliana Gobbi5, Ramiro Garzon10,Marco Vitale5,11, and Stephen M. Sykes1

Abstract

Purpose: The intracellular redox environment of acutemyeloidleukemia (AML) cells is often highly oxidized compared tohealthy hematopoietic progenitors and this is purported to con-tribute to disease pathogenesis. However, the redox regulatorsthat allow AML cell survival in this oxidized environment remainlargely unknown.

Experimental Design: Utilizing several chemical and geneti-cally-encoded redox sensing probes across multiple human andmouse models of AML, we evaluated the role of the serine/threonine kinase PKC-epsilon (PKCe) in intracellular redox biol-ogy, cell survival and disease progression.

Results: We show that RNA interference-mediated inhibitionof PKCe significantly reduces patient-derived AML cell survival aswell as disease onset in a genetically engineered mouse model(GEMM) of AML driven by MLL-AF9. We also show that PKCeinhibition induces multiple reactive oxygen species (ROS) and

that neutralization of mitochondrial ROS with chemical antiox-idants or co-expression of the mitochondrial ROS-bufferingenzymes SOD2 and CAT, mitigates the anti-leukemia effects ofPKCe inhibition. Moreover, direct inhibition of SOD2 increasesmitochondrial ROS and significantly impedes AML progressionin vivo. Furthermore, we report that PKCe over-expression protectsAML cells from otherwise-lethal doses of mitochondrial ROS-inducing agents. Proteomic analysis reveals that PKCe may con-trol mitochondrial ROS by controlling the expression of regula-tory proteins of redox homeostasis, electron transport chain flux,aswell as outermitochondrialmembranepotential and transport.

Conclusions: This study uncovers a previously unrecognizedrole for PKCe in supporting AML cell survival and disease pro-gression by regulating mitochondrial ROS biology and positionsmitochondrial redox regulators as potential therapeutic targets inAML. Clin Cancer Res; 24(3); 608–18. �2017 AACR.

IntroductionThe intracellular redox environment is largely influenced by

the production and clearance of reactive species such as reactiveoxygen species (ROS). ROS encompass a heterogeneous class ofsmall oxygen-containing reactive species that are produced byseveral intracellular processes such as aerobic glucose metab-olism and enzymatic reactions (1) and contribute to physio-logic functions such as innate and acquired immune defense.However, aberrantly elevated ROS levels, referred to as "oxi-dative stress," can cause DNA lesions, organelle dysfunction,and metabolic alterations that contribute to tumorigenesis (2).Furthermore, extremely high ROS levels can promote cell death(2). Therefore, intracellular ROS levels are tightly regulated byan array of enzymatic and nonenzymatic systems (1).

Several human cancers, including AML, display perturbationsin genes that encode regulators of intracellular ROS biology (3).For example, primary AML patient samples display decreasedexpression of the mitochondrial superoxide-neutralizing geneSOD2 and decreased glutathione metabolism (4). The activitiesof the ROS-generating enzymes NADPH-oxidases (NOX) are alsoelevated in primary human AML samples compared with healthy

1Fox Chase Cancer Center, Philadelphia, Pennsylvania. 2Penn Epigenetics Insti-tute, Department of Biochemistry and Biophysics, Perelman School of Medicine,University of Pennsylvania, Philadelphia, Pennsylvania. 3Immersion ScienceProgram, Fox Chase Cancer Center, Philadelphia, Pennsylvania. 4Departmentof Microbiology and Immunology, Fels Institute for Cancer Research andMolecular Biology, Temple University School of Medicine, Philadelphia, Penn-sylvania. 5Department of Medicine and Surgery (DiMeC), University of Parma,Parma, Italy. 6Division of Oncology, Department of Internal Medicine, Washing-ton University School of Medicine, Saint Louis, Missouri. 7Division of Experi-mental Hematology, German Cancer Research Center (DKFZ) Heidelberg,Germany. 8Heidelberg Institute for Stem Cell Technology and ExperimentalMedicine (HI-STEM), Heidelberg, Germany. 9Department of Translational Oncol-ogy, NCT Heidelberg, German Cancer Research Center (DKFZ), Heidelberg,Germany. 10Division of Hematology, The Ohio State University, Columbus, Ohio.11CoreLab, Parma University Hospital, Parma, Italy.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Stephen M. Sykes, Blood Cell Development & FunctionProgram, Fox Chase Cancer Center, Room R297, 333 Cottman Avenue. Phila-delphia, PA 19111. Phone: 215-728-3539; Fax: 215-728-2412; E-mail:Stephen.Sykes@fccc.edu

doi: 10.1158/1078-0432.CCR-17-2684

�2017 American Association for Cancer Research.

ClinicalCancerResearch

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controls and this is associated with increased steady-state levels ofintracellular superoxides (5). Moreover, the total antioxidantcapacity of leukemic cells from AML patients at initial diagnosisand relapse is decreased compared with healthy controls (6).Several of the oncogenic signaling molecules that are eithermutated (e.g., FLT3ITD, KRASG12D or BCR-ABL1; refs. 7–12) ordysregulated (e.g., c-MYC; ref. 13) in myeloid malignancies areknown to drive ROS production. Furthermore, small nucleotidepolymorphisms (SNP) of several redox-regulatory enzymes havebeen identified to be associated with myeloid neoplasia suscep-tibility and prognosis (14). Despite these studies as well as theemerging evidence that elevated ROS levels promote the prolif-eration and survival of solid cancers (15, 16), the underlyingmolecular mechanisms that govern ROS biology in AML remainunresolved.

PKCe is a serine/threonine kinase that is expressed in numer-ous tissues and supports tumorigenesis in many solid cancers(17). In normal hematopoietic development, PKCe influenceserythrocyte and megakaryocyte lineage commitments (18);however, in primary myelofibrosis, elevated PKCe expressionantagonizes megakaryocytic differentiation (19). Furthermore,downmodulation of PKCe expression is a key event duringphorbol ester–induced differentiation of primary human AMLsamples and increased PKCe expression protects AML cells fromTRAIL-induced apoptosis (20). However, the molecular mech-anism(s) by which PKCe influences AML cell fate has yet to beresolved.

Here, we show that PKCe inhibition obstructs disease progres-sion in a GEMM of AML driven by MLL-AF9 and impairs cellsurvival in multiple patient-derived AML samples. Furthermore,we find that PKCe inhibition results in increased steady-state levelsof multiple mitochondrial ROS and that chemical or geneticneutralization ofmitochondrial ROS counteracts the antileukemiaeffects of PKCe. Furthermore, we report that overexpression ofPKCe protects AML cells from otherwise lethal doses of mitochon-drial ROS–inducing agents. Finally, direct inhibition of the mito-chondrial ROS–neutralizing enzyme SOD2 phenocopies PKCeinhibition indicating that maintenance of mitochondrial ROShomeostasis is crucial to the maintenance of AML pathogenesis.

Materials and MethodsCell culture

Human AML cell lines were obtained from the ATCC and theGerman Collection ofMicroorganism and Cell Cultures (DMSZ).Cells were cultured in the recommended media conditions.Murine AML cells were cultured in cytokine-enriched media(CEM): RPMI1640 supplemented with 10% FBS and penicil-lin/streptomycin, 10 ng/mL mSCF (Peprotech), 6 ng/mL mIL3(Peprotech), and 5 ng/ml mIL6 (Peprotech).

Patient-derived AML samplesPatient-derived AML samples were obtained from The Ohio

State Comprehensive Cancer Center and used according to theapproved Institutional Review Board protocol 16-9037. Cellswere plated on irradiated monolayers of HS27 cells (21) andcultured in Stemspan (StemCell Technology) supplementedwith10% FBS, 100 ng/mL hSCF (Peprotech), 100ng/ml hFLT3 ligand(Peprotech), 20ng/ml hIL-3 (Peprotech), 20ng/mL hIL6 (Pepro-tech), 20 ng/mL G-CSF (Peprotech). Cells (5 � 105) were trans-duced with pLKO.1 GFP lentiviral shRNA vectors and evaluatedfor GFP expression every 3 days for 12 days after staining withhuman CD45 APC-Cy7 (BD Biosciences) and propidium iodide(PI) by flow cytometry.

Lentiviral transductionA total of 5 � 105 human AML cells were transduced for 30

hours with recombinant pLKO.1 lentiviruses coexpressing eitherGFP or a puromycin-resistant cassette with shRNAs from the TRCshRNA library (shRNA Pkce: TRCN0000022759; shRNA PKCe_1:TRCN0000000848 and shRNA PKCe_2: TRCN0000000846:shRNA SOD2: TRCN0000350349; shRNA Sod2_1:TRCN0000123392; shRNA Sod2_2: TRCN0000123390). Cellstransduced with recombinant lentiviruses expressing the puro-mycin-resistant cassette were initially selected with 2 mg/mLpuromycin for 48 or 72 hours (MOI ¼ 0.5–0.9) and thereaftermaintained in 0.5 mg/mL puromycin. To generate pLKO.1 GFPvectors, the puromycin-resistant cassette was replaced with eGFP.Cells transduced with lentiviruses expressing GFP (MOI ¼ 0.2–0.5) were purified by FACS at the indicated times followingtransduction.

Retroviral transductionA total of 5 � 105 human or murine AML cells were subjected

to spin transduction with recombinant retroviruses (MOI ¼0.2–0.3). TheMSCV.PKCe.GFP constructwas obtained by cloningmurine Pkce into the BglII and XhoI sites of the MSCV.IRES.GFPvector. Cells transducedwith vectors expressingGFPwere purifiedby FACS 96 hours after transduction. SF91-IRES-eGFP and SF91-SOD2/Catalase-IRES-eGFP vectors, were described previously(22) and provided by M. Milsom. SF91-Grx1-roGFP2, SF91-Mito-Grx1-roGFP2, SF91-roGFP2-Orp1, and SF91-Mito-roGFP2-Orp1 were described previously (23) and provided byDr. T. Dick (German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany).

Western blot analysisCell pellets were lysed and resolved on 4%–12% Bis Tris gels

(Thermo Fisher Scientific; Life Technologies). Following proteintransfer and blocking with 5% nonfat milk, blots were incubatedwith primary antibody overnight at 4�C. Secondary antibody was

Translational Relevance

Patient-derived AML cells often display significantlyhigher levels of intracellular ROS compared with theirnormal counterparts. Elevated ROS levels are often associ-ated with increased DNA damage, dysfunctional organellebiology, oncogenic signaling, and altered cellular metabo-lism and thus are purported to contribute to disease path-ogenesis and therapeutic responses in a variety of humantumor settings. However, excess ROS can also promotetumor cell death and therefore intracellular ROS are tightlymaintained by various regulatory mechanisms. Therefore,regulators of redox biology may provide opportunities fortherapeutic intervention. We have revealed that PKCe sup-presses mitochondrial ROS to support AML and that block-ing PKCe or enzymes that directly neutralize mitochondrialROS, such as SOD2, diminishes AML cell survival by induc-ing lethal ROS levels.

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incubated at room temperature for 1 hour and blots were devel-oped with ECL prime (GE Healthcare). The following antibodieswere used: anti-PKCe (catalog no.: 2683S: lot no.: 4) and anti-Catalase (catalog no.: 12980S, lot no.: 1) from Cell SignalingTechnology with the dilution 1:1,000, anti-SOD2 (catalog no.:ADI-SOD-111, lot no.: 08021202) from Enzo Life Sciences withthe dilution 1:1,000, anti-a-tubulin (catalog no.: T9026, lot no.:083M4847V) from Sigma-Aldrich with the dilution 1:5,000.Secondary antibodies anti-rabbit-HRP (catalog no.: 7074S, lotno.: 26) and anti-mouse-HRP (catalog no.: 7076S, lot no.: 31)were from Cell Signaling Technology with the dilution 1:4,000.

MTS assayCells were plated at the density of 1–2 � 105 cells/mL at either

72 or 96 hours posttransduction (day 0). At days 0, 2, and 4,100 mL of cell suspension was incubated with 20 mL of CellTiterAqueous One solution (Promega) for 90minutes. Plates were readat the wavelength of 495 nm. Culture media was used as blank.

Apoptosis assayCells were washed in PBS and stained with Annexin V and

Propidium Iodide (PI) or 7AAD according to the manufacturer'sinstruction (BD Biosciences). Cells were acquired and analyzedusing an LSRII flow cytometer (Beckton Dickinson). All the datawere analyzed using FlowJo software.

CellROX and MitoSOX StainingHuman AML cell lines were washed and incubated with PBSþ

5mmol/L ofCellROXDeepRed reagent (Life Technologies; catalogno.: C10422) at 37�C for 30 minutes, or with PBS þ 5 mmol/L ofMitoSOXRed reagent (Life Technologies; catalog no.:M36008) at37�C for 10 minutes. After incubation, cells were washed twotimes in PBS and stained with Annexin V-APC according to themanufacturer's instructions (BDBiosciences). Cellswere analyzedusing an LSRII flow cytometer (Beckton Dickinson).

roGFP analysisHuman AML cell lines stably expressing the roGFP2 probes

(27) were transduced with recombinant lentiviruses for 30 hours,then selected with 2 mg/mL puromycin for 72 hours and subse-quently analyzed by flow cytometry. RoGFP2 is excited at 400 nm(oxidized state) and 475–490 nm (reduced state) when fluores-cence emission ismonitored at 510nm.Diamide (DIA; 1mmol/L)or dithiothreitol (DTT; 1mmol/L) were used as controls to inducea complete oxidized or reduced state of the probes, respectively.Propidium iodide staining was performed to exclude dead cellsfrom the analysis.

Pro-oxidant treatmentHuman AML cells stably expressing PKCe, PKCe-targeting

shRNAs, or corresponding controls were seeded at the concen-tration of 2 � 105 cells/mL in 24-well plates and treated with theindicated concentrations of Antimycin A (AA; Sigma AldrichA8674) and Thenoyltrifluoroacetone (TTFA, Sigma AldrichT27006) for 24 hours and then analyzed by flow cytometry afterAnnexin V and 7AAD staining.

Bone marrow transplant leukemia modelAll animal studies conducted were approved by the IUCAC of

the Fox Chase Cancer Center. For recombinant viral transduction,

bonemarrow cells recovered from leukemiamicewere cultured inCEMmedia overnight. The next day, cells were counted and then5 � 105 cells were subjected to spin transduction with recombi-nant lentiviruses (MOI ¼ 0.4–0.6) or retroviruses (MOI ¼ 0.2–0.3) supplemented with polybrene (5 mg/mL) in 12-well non-adherent plates. Plates were centrifuged at 2,400 rpm at 30 �C for90 minutes. Transduced cells were then incubated overnight. Thefollowing day, viral supernatants were removed from cells andreplenished with fresh CEM. For in vivo survival assays, sortedGFPþ cells (1 � 106 cells/mouse) were transplanted into suble-thally irradiated (450 rad) syngenic recipient mice 48 hours post-transduction. For Western blot analysis and colony formationassays, transduced cells were subjected to FACS to isolate GFPþ

cells 48 hours post-transduction. For colony formation assays,500 purified GFPþ leukemia cells were cultured in 1 mL ofmethylcellulose supplemented with cytokines (M3434, StemCellTechnologies) for 5–7 days.

ResultsPKC« inhibition impairs in vitro and in vivo AML cell expansionand survival

To evaluate the functional role of PKCe expression in AMLbiology, we employed an shRNA approach in a panel of genet-ically distinct AML cell lines in vitro (OCI-AML3, THP-1, NOMO1,and U937). We identified two shRNA constructs, PKCe shRNA_1and PKCe shRNA_2, which target distinct regions of the PKCemRNA and effectively deplete PKCe protein levels (Supplemen-tary Fig. S1A). Each of these PKCe-targeting shRNAs significantlyreduced the expansion of the four AML cell lines compared withnontargeting shRNA controls (CTRL shRNA; Supplementary Fig.S1B). This reduction in cell growth was accompanied by a signif-icant increase in the percentage of Annexin Vþ cells (Supplemen-tary Fig. S1C), and CD11b expression (Supplementary Fig. S1Dand S1E) indicating that PKCe inhibition is detrimental to sur-vival and growth andmay induce differentiation of these AML celllines.

To assess the impact of PKCe inhibition in vivo, we utilized aGEMM of AML that is driven by the human leukemogenic fusionprotein MLL-AF9 (24). Mouse MLL-AF9 leukemia cells weretransduced with recombinant lentiviruses that coexpress GFP incombination with either control (CTRL shRNA) or murine Pkce-targeting shRNAs (Pkce shRNA). The transduced cells were sub-sequently purified by FACS to assess protein expression, cellgrowth in vitro and leukemia induction in vivo (SupplementaryFigs. S2A and S2B). Depletion of Pkce protein significantlyreduced the growth of mouse MLL-AF9 leukemia cells in cyto-kine-enriched liquid culture (Fig. 1A and B; Supplementary Fig.S2C–S2E). Furthermore, mice transplanted with FACS-purifiedmouse MLL-AF9 leukemia cells expressing Pkce shRNA exhibiteda significantly longer onset of disease compared with CTRLshRNA–expressing cells (Fig. 1C). Furthermore, depletion of Pkceprotein significantly reduced the colony-forming capacity (CFC)of mouse MLL-AF9 leukemia cells in cytokine-enriched methyl-cellulose (Fig. 1D).

In addition to MLL-AF9, we observed that PKCe supports thegrowth of mouse hematopoietic stem and progenitor cells(HSPC) by expressing alterations in genes commonly mutatedin AML. Briefly, leukemia cells coexpressing MLL-AF9 and aninternal tandem duplication (ITD) of the murine Flt3 gene(MLL-AF9;Flt3ITD) were transduced with lentiviruses expressing

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GFP in combinationwith eitherCTRLorPkce shRNAs.HSPCsnullfor the combination of Dnmt3a and Tet2 deletion (Dnmt3a�/�;Tet2�/�) as well as Dnmt3a�/�;Tet2�/� HSPCs coexpressinghuman FLT3-ITD and GFP (Dnmt3a�/�;Tet2�/�;FLT3ITD) weretransduced with lentiviruses expressing RFP in combination witheither CTRL or Pkce shRNAs. Following stable transduction, cellsfrom each condition were purified by FACS and plated separatelyin cytokine-enriched methycellulose. In all of the models ana-lyzed, shRNA-mediated inhibition of PKCe expression significant-ly hindered the CFC of leukemic cells compared with CTRLshRNA–expressing cells (Fig. 1E–G).

PKC« supports the survival of patient-derived AML samplesTo investigate the functional role of PKCe in patient-derived

AML samples, we employed an shRNA approach to inhibit theexpression of PKCe in 10 subtype diverse patient-derived AMLsamples (Supplementary Table S1; Supplementary Fig. S3A).Briefly, cryopreserved patient-derived samples were thawed andplated on the HS-27 supportive stroma in cytokine-enrichedmedia. After a short recovery period, cells were transduced withlentiviruses coexpressing GFP and either CTRL shRNA or PKCeshRNA_1 and subsequently replated on fresh HS-27 stroma cells.Three days after transduction, cells were assessed by flow cyto-metry to determine the percentage of live hCD45þ, GFPþ cells(represented as time point day 0). Cells were cocultured for anadditional 9 days and then assessed for the percentage of livehCD45þ, GFPþ cells (time point day 9). We then determined thefold change in the percentage of hCD45þ, GFPþ cells by dividingthe day 9 percentages of hCD45þ, GFPþ divided by the percen-tages of hCD45þ, GFPþ at day 0. From this analysis, we observedthat 8 of the 10 patient samples expressing PKCe-targeting

shRNAs displayed a significantly lower fold change in the per-centage of hCD45þ, GFPþ cells compared with those samplesexpressing CTRL shRNAs (Fig. 2; Supplementary Fig. S3B). Thesedata indicate that PKCe supports leukemia growth in a geneticallydiverse subset of patient-derived AML samples.

PKC« regulates the intracellular redox environment of AMLcells

Given the emerging recognition that ROS influences bothnormal and malignant HSPCs (4–6) and the previous connec-tions of PKCe to redox biology (25–28), we investigated therelationship between PKCe and ROS biology in AML cells. As aninitial assessment of whether shRNA-mediated inhibition ofPKCe impacted intracellular ROS levels, we stained CTRL andPKCe-shRNA expressing cells with the fluorogenic probe Cell-ROX, which detects multiple types of ROS. From this analysis, wefound that inhibition of PKCe resulted in an increase of CellROXstaining in OCI-AML3, NOMO1, and THP-1 cells (Fig. 3A; Sup-plementary Figs. S3C and S3D).

To determine the localization and further define the specifictypes of ROS regulated by PKCe, OCI-AML3 cells were engineeredto express the redox-sensitive roGFP2 protein genetically fusedwith glutaredoxin (Grx1) or the hydrogen peroxide (H2O2)-neutralizing yeast peroxidase Orp1. The Grx1-roGFP2 probemeasures the redox potential of GSH:GSSG redox couples, whereroGFP2-Orp1 probe measures changes in H2O2 levels. Bothprobes are expressed in the cytoplasm, however, we also engi-neered cells to express versions of each probe that are tagged withmitochondrial localization signals (Grx1-roGFP2–mito androGFP2-Orp1–mito) to evaluate changes in mitochondria ROSbiology (Supplementary Figs. S4A and S4B). roGFP2 has an

Figure 1.

PKCe inhibition impairs AML cell expansion and survival in vitro and in vivo.A,Western blot analysis of mouse MLL-AF9 leukemia cells transduced with CTRL or PkceshRNA. B, In vitro competitive growth curve of mouse MLL-AF9 cells transduced with either CTRL or Pkce shRNA GFP lentiviruses. %GFPþ cells were evaluatedevery two days by flow cytometry and normalized to fold change in %GFPþ at day 3 post-transduction, which represents day 0 in the figure (day 6 ¼ day 9post-transduction). C, Kaplan–Meier survival curve analysis of mice transplanted with mouse MLL-AF9 leukemia cells coexpressing GFP and either CTRL or PkceshRNAs (P ¼ 0.0014; n ¼ 7). MLL-AF9 (D) and MLL-AF9;Flt3ITD knock in (KI; E) cells transduced with lentiviruses coexpressing GFP with either CTRL orPkce shRNA were FACS-purified and plated in M3434. Dnmt3a�/�;Tet2�/� (F) and Dnmt3a�/�;Tet2�/�;FLT3ITD cells (G) transduced with lentiviruses coexpressingRFP with either CTRL or Pkce shRNA were FACS-purified and plated in M3434. D–G, Bar graph representing the number of colonies formed by mouseMLL-AF9 leukemia cells expressing CTRL or Pkce shRNAs in methylcellulose culture. Data are represented as the mean � SD of three technical replicates forB and D–G (��� , P � 0.001; ���, P � 0.0001).

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emission of 510nmaswell as excitation peaks at 400 and 490nm.Upon oxidation, the intensity of the 400-nm roGFP2 peakincreases while the amplitude of the 490-nm peak decreasesresulting in the ratio of peak intensities shifting toward 400 nm(23). OCI-AML3 cells were treated with either the oxidizing agentdiamide (DIA) or the reducing agent dithiothreitol (DTT) toestablish the oxidized and reduced gates (data not shown). Usingthese probes, we observed that the expression of PKCe shRNA_1or shRNA_2 resulted in a significantly higher percentage ofoxidized OCI-AML3 cells expressing either cytoplasmic or mito-chondrial Grx1-roGFP2, compared with control shRNAs. We alsoobserved that PKCe inhibition led to a significant increase in thepercentage of cells expressing oxidized roGFP2-Orp1 in the mito-chondria (Fig. 3B; Supplementary Fig. S4A and S4B).

The most well-defined ROS generated by mitochondria aresuperoxides, which are either neutralized by glutathione or con-verted to H2O2 by superoxide-dimutase (SOD) enzymes. Giventhat PKCe inhibition consistently altered mitochondrial levels ofglutathione and H2O2, we next examined how PKCe inhibitionimpacted mitochondrial superoxide levels using the fluorogenicprobe MitoSOX. From this analysis, we observed that shRNA-mediated inhibition of PKCe led to a significant increase in

MitoSOX staining in both human [OCI-AML3, Fig. 3C; Supple-mentary Figs. S5A (THP-1) and S5B] and mouse (MLL-AF9, Fig.3D) AML cells.We also observed that shRNA-mediated inhibitionof PKCe in patient-derived AML cells expressing MLL-AF9 alsodisplay elevated MitoSOX levels (Supplementary Fig. S5C). Col-lectively, these findings reveal that PKCe regulates steady-statelevels of mitochondrial ROS and possibly certain cytoplasmicROS in human and mouse AML cells.

Reducing mitochondrial ROS partially reverses theantileukemia effects of PKC« inhibition

To determine whether changes in the intracellular redox biol-ogy contribute to the antileukemia effects of PKCe inhibition, weassessed how various chemical antioxidants impacted the growthand survival of AML cells expressing CTRL and PKCe-targetingshRNAs. Twice daily treatment with either N-acetyl-L-cysteine(NAC) or glutathione was unable to reverse the antileukemiaeffects of PKCe inhibition in both human andmouse cells despitebeing able to effectively block the cytotoxicity of the glutathione-depleting pro-oxidant, Menadione (Supplementary Fig. S6A andS6B and data not shown). However, administration of eitherbutylated hydroxyanisole (BHA) or MitoTEMPO, both

Figure 2.

PKCe inhibition impedes patient-derived AML growth. Ex vivo growthcurve analysis of 10patient-derivedAMLsamples transduced with lentivirusescoexpressing GFP and CTRL, PKCeshRNA_1 and then analyzed by flowcytometry for the GFPþ, hCD45þ,PI� cells every 3 days for a total of12 days, post-transduction (P.T.). AMLsamples displayed maximal GFP meanfluorescence intensity (MFI) at threedays post-transduction and thusrepresents analysis point, day 0. Thedatapresented are expressed as the foldchange of GFPþ cells at day 9 (day 12post-transduction) versus day 0,relative to the mean of the CTRL shRNA(� , P� 0.05; �� , P� 0.01; ���, P� 0.001;���� , P < 0.0001). Data shownare the endpoint analyses (day 12post-transduction) and are representedas mean � SD of three technicalreplicates.

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compounds that neutralize mitochondrial ROS, was able tosignificantly reduce the cell death of human and mouse AMLcells mediated by PKCe inhibition (Fig. 4A and B; SupplementaryFig. S6C).

To examine how specific neutralization of mitochondrial ROSimpacts the antileukemia effects of PKCe inhibition, we engi-neered OCI-AML3 cells to constitutively coexpress SOD2 andCatalase (SOD2-Catalase), which neutralize mitochondrialsuperoxides and H2O2, respectively. Control and SOD2-Cata-lase–expressing OCI-AML3 cells were then transduced with CTRLor PKCe shRNAs and subsequently analyzed for MitoSOX levelsand cell death. SOD2-Catalase expression significantly blockedthe induction of MitoSOX staining mediated by PKCe inhibition(Supplementary Fig. S6D) and significantly reduced cell deathinduced by PKCe-targeting shRNAs (Fig. 4C). We also observedthat SOD2-Catalase expression restored the CFC of mouse MLL-AF9 leukemia cells expressing Pkce shRNA (Fig. 4D) furtherindicating that AML cells rely on PKCe tomaintainmitochondrialROS levels and survival.

Themitochondrial ROS–neutralizing enzyme, SOD2, supportsAML in vitro and in vivo

To examine how a specific induction of mitochondrial super-oxides impacts AML cell growth, we evaluated how shRNAs

directly targeting SOD2 impacted human AML cell biology (Sup-plementary Fig. S7A). Similar to PKCe inhibition, shRNA-medi-ated depletion of SOD2 led to increased steady-state levels ofMitoSOX and reduced AML cell growth and survival (Supple-mentary Fig. S7B–S7D). Furthermore, direct inhibition ofSOD2 inmouseMLL-AF9 leukemia cells (Fig. 5A) led to increasedlevels of MitoSOX staining (Fig. 5B), reduced CFC (Supplemen-tary Fig. S7E) and significantly delayed the time of disease onsetin vivo (Fig. 5C). These results show that similar to the antileu-kemia effects of PKCe inhibition, SOD2 inhibition leads to anaccumulation of mitochondrial ROS and diminishes AML cellgrowth and survival.

PKC« regulates the expression of proteins that regulatemitochondrial biology

To identify potential downstream effectors of PKCe in AML, weperformed nanoscale liquid chromatography coupled to tandemmass spectrometry (nanoLC/MS-MS) on OCI-AML3 cells expres-sing CTRL shRNA, PKCe shRNA_1, or PKCe shRNA_2. From thisanalysis, 3,192 peptides were captured and using a statisticalcutoff of P < 0.05, we found that 707 peptides were differentlyexpressed between CTRL shRNA and PKCe shRNA_1–expressingcells and 641 peptides were differently expressed between CTRLshRNA and PKCe shRNA_2–expressing cells. Furthermore, we

Figure 3.

PKCe regulates intracellular ROS biology in AML. A, OCI-AML3 cells stably expressing CTRL shRNA or PKCe shRNAs were stained with CellROX, 5 dayspost-transduction. Left, representative histogram plot of a single experiment; right, the average MFI of CellROX. Data are represented as the mean � SD ofthree technical replicates. B, OCI-AML3 cells were stably transduced with the indicated roGFP2 probes to evaluate glutathione or H2O2 redox potential incytoplasm or mitochondria. Cells were transduced with lentiviruses expressing CTRL shRNA or PKCe shRNA_1 or PKCe shRNA_2 and analyzed by flow cytometry5 days later. Bar graph represents the percentage of live (PI�) oxidized cells in the cytoplasm (Cyto) or mitochondria (Mito). Data represents the mean � SDof three independent experiments. C, OCI-AML3 cells were transduced with CTRL shRNA or PKCe shRNA and stained after 4 days with MitoSOX and Annexin V (toexclude dead cells). Left, representative histogram plot of a single experiment; right, shows the average MitoSOX MFI of live cells (Annexin V�) expressed asthe mean � SD of three independent experiments. D, MLL-AF9 leukemia cells coexpressing GFP and either CTRL or PKCe shRNAs were stained and evaluated forMitoSOX levels by flow cytometry, 5 days after transduction. Bar graph represents the MitoSOX MFI of live cells (Annexin V�) expressed as the mean � SDof three independent experiments (� , P � 0.05; �� , P � 0.01; ��� , P � 0.001; ���� , P < 0.0001).

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observed that 226 peptides were similarly differentially expressedbetween OCI-AML3 cells expressing CTRL shRNA versus PKCeshRNA_1 and CTRL shRNA versus PKCe shRNA_2. Integratedpathway analysis did not reveal any significant enrichment ofparticular molecular pathways or processes however, we didobserve that approximately 15% (34 proteins) of these differen-tially expressed proteins between CTRL shRNA and either PKCeshRNA_1 or PKCe shRNA_2 were related to mitochondrial biol-ogy. Aberrantmitochondrial ROS arise frommultiple disruptionsin mitochondrial biology such as reduced expression of antiox-idant systems, perturbations in the activities of the complexes thatregulate electron flux through the electron transport chain (ETC),as well as alterations in outer mitochondrial membrane (OMM)potential and transport. Many of the proteins whose expression isaltered by PKCe inhibition are components of ETC complexesinvolved in the regulation of OMM potential (UQCR10, ATPC1,ATP5H, COX6C, SDHAF2, and NDUFB10) or proteins involvedin mitochondrial membrane transport (VDAC1, VDAC3,TOMM22, SLC25A1, SLC25A11, and SLC25A12; Fig. 6A). Wealso observed that two antioxidant proteins, GSS and TXN, weresignificantly reduced by PKCe inhibition as well as a variety ofother antioxidant proteins that were reduced but not statisticallysignificant (Supplementary Fig. S7F).

PKC« protects AML cells against agents that promotemitochondrial dysfunction and induce superoxides

To assess the role PKCe in superoxide-induced oxidativestress, we examined how modulating PKCe expression impact-ed the survival of AML cells challenged with either of themitochondrial superoxide inducing agents, 2-thenoyltrifluor-oacetone (TTFA) or antimycin A (AA). To examine whetherincreased expression of PKCe is able to protect AML cells from

the cytotoxic effects of TTFA or AA, we generated OCI-AML3,THP-1, and mouse MLL-AF9–expressing AML cells that consti-tutively express PKCe (Supplementary Fig. S8A and data notshown). Constitutive PKCe expression did not impact OCI-AML3 growth or the CFC of mouse MLL-AF9 leukemia cells(Supplementary Fig. S8B and S�C). However, PKCe-expressingOCI-AML3 or THP-1 cells treated with TTFA or AA displayedsignificantly lower percentages of cell death compared withsimilarly treated control cells (Fig. 6B and C; SupplementaryFig. S8E and S8F). In addition, constitutive PKCe expressionsignificantly improved the CFC of mouse MLL-AF9 leukemiacells challenged with TTFA or AA compared with similarlytreated control cells (Fig. 6D). Of all the human AML cell lineswe tested, U937 cells were the least impacted by PKCe inhibi-tion, cell viability–wise (Supplementary Fig. S1B, bottom).Therefore, we evaluated whether PKCe inhibition renderedU937 cells more sensitive to TTFA and/or AA treatment. Fromthis analysis, we found that shRNA-mediated inhibition ofPKCe exacerbated the cytotoxic effects of TTFA and AA incomparison with PKCe inhibition or pro-oxidant treatmentalone (Supplementary Fig. S8G and S8H). Collectively, theseresults suggest that PKCe protects AML cells from agents thatperturb mitochondrial function and induce oxidative stress.

DiscussionPKCe has been implicated as an oncogenic kinase in several

human cancers including prostate, breast, colon, lung, and certainforms of squamous cell carcinoma (29–34). In AML, downmo-dulation of PKCe is necessary for the prodifferentiating actions ofphorbol esters (20, 35), insinuating that PKCe supports leukemiagrowth and expansion. However, the impact of blocking PKCe

Figure 4.

Neutralization of ROS partially reverses the antileukemia effects of PKCe inhibition. A and B, OCI-AML3 cells were transduced with CTRL shRNA, PKCe shRNA_1, orPKCe shRNA_2 and then administered 25 mmol/L BHA (A) or 100 nmol/L MitoTEMPO (B). Ninety-six hours post-transduction, cells from each condition wereassessed for Annexin V staining by flow cytometry. Data are represented as the mean � SD of two independent experiments. C, OCI-AML3 cells stably transducedwith retroviruses coexpressing SOD2-IRES-Catalase (SOD2-Catalase) and GFP or control vector (CTRL) were transduced with CTRL shRNA, PKCe shRNA_1or PKCe shRNA_2. Four daysafter transduction, cellswere analyzed for cell death evaluatedaspercent ofAnnexinVþ cells byflowcytometry. Data are represented asthe mean � SD of three independent experiments for each panel (� , P � 0.05; �� , P � 0.01). D, SOD2-Catalase or CTRL-expressing mouse MLL-AF9 weretransduced with CTRL shRNA or Pkce shRNA, selected with puromycin and grown in methylcellulose. After 5 days of culture, colonies were enumerated and datarepresents the mean � SD of two independent experiments (� , P � 0.05; �� , P � 0.01; ��� , P � 0.001; ���� , P < 0.0001).

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expression on AML growth and survival has not been compre-hensively investigated.

Here, we demonstrate that shRNA-mediated reduction of PKCesignificantly reduces the survival of human AML cells in vitro andsignificantly impedes disease progression in a GEMM of AMLdriven byMLL-AF9. In addition, inhibition of PKCe reduced the invitro growth properties of multiple, genetically diverse patient-derived AML samples confirming that the proleukemia roles ofPKCe is not restricted to a particular genetic subtype of AML.

At the molecular level, we have discovered that PKCe is a keyregulator of intracellular redox homeostasis in several mouse andhuman AMLmodels. Using multiple ROS detection strategies wehave found that shRNA-mediated inhibition of PKCe increasesthe steady-state levels of multiple ROS, including several mito-chondrial ROS. On the basis of these observations, we postulatedthat increased/excess production of mitochondrial ROS antago-nizes leukemia cell viability and that management of mitochon-drial ROS levels is a key proleukemia function of PKCe. We havemade three central observations that support these hypotheses.First, chemical antioxidants that specifically neutralize mitochon-drial ROS, such as BHA (36) and MitoTEMPO (37) are able tosignificantly and consistently blunt cell death mediated by PKCeinhibition, whereas indiscriminant antioxidants such as NAC andglutathione are not. Moreover, reconstitution of mitochondrialROS-neutralizing enzymes SOD2 and Catalase, partially reverses

mitochondrial superoxide induction and cell death mediated byPKCe inhibition. Second, similar to PKCe inhibition, shRNA-mediated inhibition of SOD2 increased mitochondrial ROS,limited disease progression in vivo and suppressed the growth ofpatient-derived AML samples. Third, elevated expression of PKCeis able to largely protect AML cells from otherwise toxic doses ofagents that drive mitochondrial ROS production. Moreover,impeding PKCe expression rendered leukemia cellsmore sensitiveto these pro-oxidants. Collectively, these results support thatcertain sub-types of AML rely on PKCe for proper managementof mitochondrial ROS biology and survival.

Our observations that the anti-leukemia effects of PKCeinhibition could not by fully rescued by SOD2-Catalase expres-sion, MitoTEMPO or BHA suggest that PKCe may regulateadditional molecular processes to support AML cell survival.Our proteomic analysis shows that, in addition to multiple keyROS-buffering enzymes, such as TXN and GSS, several proteinsrelated to mitochondrial biology and function are impacted byPKCe inhibition. Therefore, it remains possible that the increasein mitochondrial ROS mediated by PKCe inhibition is due tomitochondrial dysfunction either in addition to or in place ofthe observed decrease in ROS-regulating enzymes. Consistentwith this idea, several studies have shown that the mitochon-drial redox state of AML cells is directly related to their met-abolic needs. Specifically, compared to bulk AML cells, leuke-mia-initiating cells from multiple AML patients display a loweroxidized redox environment that is associated with lower ratesof oxidative phosphorylation (OXPHOS; ref. 38). Furthermore,cytarabine-treated AML cells display high levels of mitochon-drial ROS and OXPHOS and this altered mitochondrial statemay contribute to cytarabine resistance (39). Also, multipleAML patients display increased mitochondrial mass comparedwith healthy HSPCs and as a result are more sensitive toOXPHOS-induced oxidative stress (40).

Although our results indicate that a key proleukemia functionof PKCe is to manage mitochondrial ROS biology, they do notexclude the possibility that PKCe regulates additional redox-related (that are mitochondrial-independent) and/or redox-inde-pendent mechanisms to support AML cell survival. For example,our proteomic analysis shows that PKCe inhibition, although notstatistically significant, impacts multiple redox-regulatory sys-tems. For example, protein levels of both PRDX2 and PRDX4,which have been implicated as growth suppressors in AML andacute promyelocytic leukemia (APL), respectively, increase uponPKCe inhibition (41, 42). PKCe inhibition also altered the expres-sion of various glutathione-regulatory components and Pei andcolleagues (4) have shown that pharmacologic inhibition ofglutathione metabolic enzymes, such as GPX1 and GCLC, antag-onize primitive human leukemia cell survival. In fact, the rela-tionship of PKCe and redox biology varies among distinct bio-logical settings. PKCe activation in neuronal and cardiac tissuescorrelates with the induction of ROS mediated by ischemia,hypoxia or pro-oxidants such as buthionine sulfoximine (BSO)or AA. However, other studies have shown that PKCe activationpromotes ROS generation in smooth muscle and immortalizedepithelial cells and that hepatocytes void of PKCe displayenhanced stress-induced ROS formation (28, 43). These divergentobservationsmay result from distinct tissue- or ROS-specific rolesof PKCe; however, in the context of AML, our results establish thatPKCe works to suppress mitochondrial ROS and possibly othertypes of ROS.

Figure 5.

SOD2 inhibition phenocopies the antileukemic effects of PKCe inhibition. A,Mouse MLL-AF9 leukemia cells were transduced with CTRL shRNA or SOD2shRNA, sorted 48 hours later and analyzed for SOD2 expression bywestern blot.B, MitoSOX staining of mouse MLL-AF9 leukemia cells evaluated by flowcytometry 5 days after transduction with CRTL, Sod2 shRNA_1, or Sod2shRNA_2 (the bar graph shows the MitoSOX MFI of live cells (Annexin V�; CtrlshRNA vs. shRNA sod2_1 and _2; � , P�0.05). Data represents themean� SD ofthree technical replicates. C, Kaplan–Meier survival curve analysis of micetransplanted with mouse MLL-AF9 leukemia cells expressing either CTRL orSod2 shRNA_2 (P ¼ 0.0042; n ¼ 7).

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Several studies suggest that tumor cells, including leukemiacells, maintain high levels of ROS to drive cell growth and survivaland therefore targeting redox regulatorsmaybe a viable anticancertherapeutic strategy (2, 34, 44). In our models, reducing steady-state levels of mitochondrial superoxides through the combinedover-expression of SOD2 and Catalase does not impede colonyformation suggesting that elevated mitochondrial ROS are not acentral driver of AML cell growth in this model. However, increas-ing mitochondrial ROS, by inhibiting PKCe or SOD2 or byadministering chemical ROS-inducing agents diminishes AMLcell survival. Collectively, these results suggest that strategies forincreasing, rather than decreasing,mitochondrial ROSmay carry asignificant therapeutic potential. Consistent with this concept, theefficacy of arsenic trioxide, which is commonly used to treat APL,works primarily by inducing ROS (45). Moreover, high doses of

vitaminC,whichhavebeenpreviously shown to induce toxic ROSlevels in certain types of cancer cells (46),were recently reported toselectively eliminate AML cells carrying TET2 (47, 48) or IDH (49)mutations. However, it should be noted that the proposed mech-anismof actionof VitaminC inAML is to activate other TET familymembers (47, 48).

The intracellular redox environment of AML cells is oftendistinct from their normal counterparts. Therefore, identifyingand defining the molecular regulators of redox biology, such asPKCe and SOD2, may provide key insights into the etiology andpathogenesis of AMLaswell as possibly contribute to thedesignofmore effective antileukemia therapies. However, AML encom-passes a wide variety of genetic subtypes and individual tumorsoften display complex clonal heterogeneity (50) and it remainsunclear which genetic subtypes are susceptible to redox

Figure 6.

PKCe protects AML cells against agents that induce mitochondrial dysfunction and mitochondrial ROS-oxidative stress. A, Heatmap analysis displaying theexpression of mitochondrial-regulatory proteins that were significantly differentially expressed between CTRL shRNA versus PKCe shRNA_1 or versus PKCeshRNA_2 transduced OCI-AML3 cells. B and C, OCI-AML3 cells were transduced with retroviral vectors that constitutively express PKCe and GFP (PKCe) or justGFP-expressing control (Ctrl) viruses. Four days after transduction, GFPþ cells were isolated by FACS and treated with 100 mmol/L of AA (B) or TTFA (C).The percentage of Annexin Vþ cells was evaluated 24 hours after treatment by flow cytometry. Data are represented as the mean � SD of three independentexperiments. D, Ctrl and PKCe-expressing mouse MLL-AF9 leukemia cells were generated as described in B and C. GFPþ cells were isolated by FACS andseeded in methylcellulose with vehicle, 100 mmol/L AA or 200 mmol/L TTFA. The bar graph shows the number of colonies formed in methylcellulose after 5 daysof culture. The data represent the mean � SD of three technical replicates. (�� , P � 0.01; ��� , P � 0.001).

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imbalances. Our loss-of-function studies in patient-derived AMLcells show that not all AML samples rely on PKCe for growth andsurvival. Thus, it is possible that certain AML cells utilize PKCe-and/or SOD2-independent mechanisms to regulate mitochon-drial superoxide biology or that certain genetic subtypes or clonesare insensitive to increases in mitochondrial superoxide levels.Therefore, future studies defining the genetic subtypes that aresensitive to changes in redox homeostasis or PKCe/SOD2 inhi-bition as well as the role of PKCe/SOD2 in healthy HSPC biologywill be needed to fully gauge the therapeutic potential of targetingthese pathways in AML.

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

Authors' ContributionsConception and design: D.Di Marcantonio, E. Masselli, M.D. Milsom,G. Gobbi, S.M. SykesDevelopment of methodology: J. Michael Meadows, G. Gobbi, S.M. SykesAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):D.DiMarcantonio, E. Martinez, J. Vadaketh, A. Gupta,J. Michael Meadows, G.A. Challen, B. Garcia, R. Garzon, S.M. SykesAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D.Di Marcantonio, J. Vadaketh, A. Gupta, J. MichaelMeadows, F. Ferraro, M.D. Milsom, S. Froehling, P. Mirandola, G. Gobbi,S.M. Sykes

Writing, review, and/or revision of the manuscript: D.Di Marcantonio,E. Masselli, M.D. Milsom, C. Scholl, S. Froehling, S. Balachandran, T. Skorski,R. Garzon, M. Vitale, S.M. SykesAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):M. Nieborowska-Skorska, T. Skorski, S.M. SykesStudy supervision: P. Mirandola, M. Vitale, S.M. SykesOther (responsible for the design, execution and interpretation of massspectrometry studies): S. SidoliOther (advised in research design): C. Scholl

AcknowledgmentsThis work was supported by the NIH Grant R00 CA158461, the ASH Junior

Scholar Award, W.W. Smith and Bob and Jeanne Brennan (to S.M. Sykes); theRotary Foundation, Grant GG1414529 and the Board of Directors of Fox ChaseCancer Center Fellowship (to D. Di Marcantonio); CURE supplement(CA06927; to J. Vadaketh and A. Gupta); Jeanne E. and Robert F. OzolsUndergraduate Summer Research Fellowship, Fox Chase Cancer Center(to J. Michael Meadows); NIH grant P01CA196539, DOD grant W81XWH-113-1-0426 and the Leukemia and LymphomaSocietyDr. Robert Arceci ScholarAward (to B. Garcia), The Dietmar Hopp Stiftung (to M.D. Milsom), the NIHGrant 1R01DK102428 (to G.A. Challen).

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 September 13, 2017; revised October 15, 2017; accepted November6, 2017; published OnlineFirst November 10, 2017.

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