PIM Kinase Inhibitors Kill Hypoxic Tumor Cells by Reducing...

Cancer Biology and Signal Transduction

PIM Kinase Inhibitors Kill Hypoxic TumorCells by Reducing Nrf2 Signaling and IncreasingReactive Oxygen SpeciesNoel A.Warfel1,2, Alva G. Sainz3, Jin H. Song1,2, and Andrew S. Kraft2,4

Abstract

Intratumoral hypoxia is a significant obstacle to the suc-cessful treatment of solid tumors, and it is highly correlatedwith metastasis, therapeutic resistance, and disease recurrencein cancer patients. As a result, there is an urgent need todevelop effective therapies that target hypoxic cells within thetumor microenvironment. The Proviral Integration site forMoloney murine leukemia virus (PIM) kinases represent aprosurvival pathway that is upregulated in response to hyp-oxia, in a HIF-1–independent manner. We demonstrate thatpharmacologic or genetic inhibition of PIM kinases is signif-icantly more toxic toward cancer cells in hypoxia as comparedwith normoxia. Xenograft studies confirm that PIM kinaseinhibitors impede tumor growth and selectively kill hypoxictumor cells in vivo. Experiments show that PIM kinases

enhance the ability of tumor cells to adapt to hypoxia-inducedoxidative stress by increasing the nuclear localization andactivity of nuclear factor-erythroid 2 p45-related factor 2(Nrf2), which functions to increase the expression of antiox-idant genes. Small molecule PIM kinase inhibitors preventNrf2 from accumulating in the nucleus, reducing the tran-scription of cytoprotective genes and leading to the build-upof intracellular reactive oxygen species (ROS) to toxic levels inhypoxic tumor cells. This toxic effect of PIM inhibitors can besuccessfully blocked by ROS scavengers, including N-acetylcystine and superoxide dismutase. Thus, inhibition of PIMkinases has the potential to oppose hypoxia-mediated thera-peutic resistance and induce cell death in the hypoxic tumormicroenvironment. Mol Cancer Ther; 15(7); 1637–47. �2016 AACR.

IntroductionA significant obstacle to the successful treatment of advanced

cancer is intratumoral hypoxia. Multiple clinical studies in pros-tate cancer have reported an association between hypoxia andpoor clinical outcome in patients with various types of cancerfollowing chemotherapy or surgery (1, 2). A study of 247 prostatecancer patients with localized intermediate-risk disease showedthat tumor hypoxia is associated with early biochemical relapseand local recurrence in the prostate gland (3). Similarly, theexpression of hypoxia-induced proteins (HIF1a and VEGF) pre-dicts treatment failure independent of tumor stage, Gleason score,and serum PSA levels (4). Therefore, elucidating the molecularand cellular mechanisms controlling tumor cell survival in hyp-oxia is critical to the development of effective therapeutic strat-egies to kill hypoxic tumor cells.

Cellular hypoxia is a state of reductive stress that is character-ized by the elevation of reducing equivalents (i.e., NADH andFADH2) that build up in themitochondria when insufficientO2 isavailable for reduction by the electron transport chain (ETC;ref. 5). Hypoxic cells rely largely on anaerobic glycolysis togenerate ATP, while the remaining low level of oxygen supportsthe production of ATP through the tricarboxylic acid cycle andETC. Electrons leaking from the mitochondrial ETC generate anexcess of reactive oxygen species (ROS) (i.e., oxidative stress;refs. 6, 7). Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)is a basic leucine zipper redox-sensitive transcriptional factor thatregulates the expression of cellular antioxidant and cytoprotectivegenes (8). During normoxia, Nrf2 is held in the cytoplasmby a cytoskeletal-associated inhibitory protein, Kelch-like ECH-associated protein 1 (KEAP1). KEAP1 promotes the degradationof Nrf2 via ubiquitination and rapid proteasomal degradation(9). In response to oxidative stress, thiol oxidation occurs in thehinge region of KEAP1, resulting in a conformational change andthe loss of Nrf2 binding (10). Nrf2 then accumulates and trans-locates into the nucleus where it binds to DNA-containing anti-oxidant responsive elements (ARE/EpRE), activating the tran-scription of critical antioxidant genes, including heme oxyge-nase-1 (HMOX1), NAD(P)H dehydrogenase, Quinone 1(NQO1), and Peroxiredoxin 1 (PRDX1; refs. 11, 12). Thus, theNrf2 signaling pathway represents the primary protectiveresponse against oxidative stress.

The Proviral Integration site for Moloney murine leukemiavirus (PIM) kinases are implicated in cell survival and metabo-lism, and their expression is enhanced and correlated with poorprognosis in several types of cancer (13). The PIM kinase family iscomposed of three different isoforms (PIM1, PIM2, and PIM3)

1Department ofCellularandMolecularMedicine,UniversityofArizona,Tucson, Arizona. 2University of Arizona Cancer Center, Tucson, Ari-zona. 3UROC-PREP program, University of Arizona College of Under-graduate Studies, Tucson, Arizona. 4Department of Medicine, Univer-sity of Arizona, Tucson, Arizona.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

N.A. Warfel and A.S. Kraft are co-senior authors of this article.

Corresponding Authors: Noel A. Warfel, University of Arizona, Rm 0985A,1515 N. Campbell Avenue, Tucson, AZ 85724. Phone: 520-626-7756; Fax: 520-626-6898; E-mail: [email protected]; and Andrew S. Kraft,[email protected]

doi: 10.1158/1535-7163.MCT-15-1018

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

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on November 21, 2018. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst May 16, 2016; DOI: 10.1158/1535-7163.MCT-15-1018

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that are highly conserved throughout evolution (13). In contrastto many prosurvival kinases, PIM isoforms do not possessregulatory domains, so their expression levels are directly corre-lated with activity (14). The overexpression of PIM kinases hasbeen observed in hematopoietic malignancies and prostate can-cer, and mutations in PIM kinases have been identified in lym-phoma (15, 16). Given the potential role of PIM in regulatingmalignant transformation, a number of newly developed pan-PIM kinase inhibitors are currently being tested in clinical trials(17), and these compounds have shown some success inmultiplemyeloma and hematopoietic cancers (18). It was previouslyreported that PIM1 expression is increased in response to chem-ically induced hypoxia and promotes resistance to chemotherapy(19, 20). However, the biologic role of PIM kinases in regulatingthe protective cellular response to hypoxia and the utility of smallmolecule PIM inhibitors as agents to target hypoxic tumor cellshas not been examined.

Here, we investigate the role of PIM kinases in modulating thecellular and physiologic response to intratumoral hypoxia. Bothtissue culture and animal models demonstrate that hypoxiaincreases PIM1/2 expression, and in turn, PIM increases thenuclear localization of Nrf2, resulting in increased expression ofcytoprotective proteins that combat the production of hypoxia-induced ROS. As a result, small molecule PIM kinase inhibitorsdramatically enhance ROS levels in hypoxic tumor cells, account-ing for their selective toxicity toward this population.Ourfindingsdescribe a mechanistic role for PIM in controlling the amplitudeofNrf2 signaling and provide a foundation for the pharmacologicinhibition of PIM kinases as a novel strategy for treating hypoxictumors.

Materials and MethodsPlasmids and siRNA

HA-HIF-1a (21) and HA-HIF-1a (P402/564A) (22) con-structs were purchased from Addgene. ARE-Luc was kindgift from Dr. Donna Zhang (University of Arizona, Tuscon,AZ; ref. 23). The sequences of each siRNA are as follows: Pim1sense 50-GAUAUGGUGUGUGUGGAUA-30, Pim1 antisense50-UAUCUCCACACACCAUAUC-30; Pim2 sense 50-ACCUU-CUUCCCGACCCUCA-30, Pim2 antisense 50- UGAGGGUCGG-GAAGAAGGU-30; Pim3 sense 50-GCACGUGGUGAAG-GAGCGG-30, Pim3 antisense 50-CCGCUCCUUCACCAC-GUGC-30; control sense 50-UUCUCCGAACGUGUCACGU-30

and antisense 50- ACGUGACACGUUCGGAGAA-30. HIF-1aand HIF-2a siRNA were purchased from Santa CruzBiotechnology.

Reagents and antibodiesAZD1208 (17) and LGB321 (24)were provided byAstraZeneca

and Novartis, respectively. Cycloheximide, propidium iodide,and MG-132 were purchased from Sigma. CM-H2DCFDA waspurchased from Invitrogen. Antibodies to HIF-1a and HIF-2awere purchased from BD Transduction Laboratories and NovusBiologicals, respectively. PIM1/2/3, IRS1 (S1101), cleaved cas-pase-3, and actin antibodies were purchased from Cell SignalingTechnology. An anti-HAmAb was purchased from Covance. Nrf2antibodies for immunofluorescence and Western blotting werepurchased from Santa Cruz Biotechnology. The anti-pimonida-zole mAb (Hypoxyprobe-1) was purchased from Hypoxyprobe,Inc. All other materials and chemicals were reagent-grade.

Cell transfection and immunoblottingPC4-LN4 prostate cancer and HCT-116 colon cancer cell lines

weremaintained inRPMImedium (CellGro) containing 10%FBS(Hyclone). MB-MDA-231 andMB-MDA-231-ARE-Luc breast can-cer cells were maintained in DMEM containing 10% FBS. All celllines were maintained at 37�C in 5% CO2. All cell lines wereauthenticated by short tandem repeat DNA profiling performedby the University of Arizona Genetics Core facility-ArizonaResearch Laboratories Division of Biotechnology at theUniversityof Arizona. (http://uagc.arl.arizona.edu/). The cell lines were usedfor less than 50 passages, and they were routinely tested formycoplasma contamination. When indicated, cells were main-tained in a hypoxic environment (1% O2) using a hypoxiachamber (In vivo2 400, Ruskinn). Transient transfection of DNAand siRNAwas carried out using Lipofectamine 3000 transfectionreagent (Invitrogen), according to the manufacturer's protocol.Immunoblottingwas performed as described previously (25). Forcellular fractionation, nuclear and cytoplasmic extracts were iso-lated as described previously (26). Densitometric analysis wasperformed with the NIH ImageJ analysis software.

qRT-PCR analysisMessenger RNAwas isolated from cell lysates using the RNeasy

Mini Kit (Qiagen), and cDNA was synthesized from each sampleusing the RT2 first strand synthesis kit (Qiagen). Changes in geneexpression in response to hypoxia and PIM inhibition weremeasured as follows: qRT-PCR reactions were performed withequal amounts of starting material (500 ng RNA) using Bio-RadSsoAdvanced Universal SYBR Green Supermix, according to themanufacturer's protocol. Validatedprimer sets (QuantiTechprim-er assays; Qiagen) for each of the following genes were purchasedfor qRT-PCR analysis of gene expression: PIM1, PIM2, PIM3,HIF-1a, HIF-2a, ANGPTL4, VEGF, HMOX1, NQO1, and PRDX1.

Luciferase, ROS, and viability assaysFor luciferase assays, approximately 3 � 105 MD-MBA-231-

ARE-Luc were plated in triplicate into black 96-well plates andallowed to adhere prior to incubation in hypoxia and/or drugtreatment. Firefly luciferase was measured at the indicatedtimes/conditions using the luciferase assay system (Promega),according to the manufacturer's protocol. For ROS assays, PC3-LN4 cells were plated in 6-well plates and cultured in normoxiaor hypoxia � AZD1208 for the indicated times. Then, the cellswere washed with PBS and incubated with CM-H2DCFDA (1mmol/L in PBS), a general oxidative stress indicator. After 10minutes, the CM-H2DCFDA was removed and the cells werewashed once with PBS. The cells were immediately imagedusing a fluorescence microscope (EVOS, Invitrogen) or har-vested by trypsinization, centrifuged, and resuspended in PBSfor FACS analysis (FACSCanto II; Beckon Dickinson). To mea-sure viability, cells were stained with crystal violet or sub-G1

DNA content was measured by FACS analysis after stainingwith propidium iodide, according to previously describedmethods (27, 28).

ImmunofluorescenceTwenty-four hours prior to the start of the assay, 2 � 106 PC3-

LN4 cells were plated in 6-well plates containing microscopecoverslips. Then, the cells were placed in normoxia or hypoxiain the presence or absence of 3 mmol/L AZD1208 or sulforaphane

Warfel et al.

Mol Cancer Ther; 15(7) July 2016 Molecular Cancer Therapeutics1638




for 24 hours. Cells were fixed with 4%paraformaldehyde at roomtemperature for 10 minutes. Fixed cells were incubated for over-night with an Nrf2 antibody in PBS containing 5% normal goatserum. Coverslips were washed and incubated with AlexaFluor488-conjugated anti-rabbit secondary antibody for 45 minutes,followed byDAPI to stain the nuclei. Coverslips were washed andmounted on glass slides, and images were obtained by fluores-cence microscopy at 40� magnification. The images were cap-tured and minimal alterations were performed on the digitalimages using NIS Elements software (Nikon).

In vivo studiesMale NOD/SCID mice (Charles River Laboratories) were

housed and maintained in accordance with the InstitutionalAnimal Care and Use Committee and state and federal guidelinesfor the humane treatment and care of laboratory animals. PC3-LN4 cells were injected subcutaneously into the rear flanks ofmice, at a density of 2� 106 cells per injection in PBS/Matrigel (v:v) in 200 mL total volume. Once tumors reached a volume ofapproximately 250mm3, the mice were separated into groups fortreatment with vehicle (30% propylene glycol, 5% Tween-80,65% of 5% dextrose in water, pH 4–5) or AZD1208 (30 mg/kg/day) by oral gavage once daily for 14 days. Tumor volumes weremonitored over time by caliper measurement. At the end of thestudy, tumors were fixed, embedded in paraffin, and sectioned forstaining with hematoxylin and eosin (H&E) or antibodies specificfor cleaved caspase-3 (CC3) and hypoxyprobe-1. The percentageof CC3-positive cells was calculated using NIH ImageJ (4 fieldswere counted on 3 separate tumors from the Vehicle and AZDgroups).

Statistical analysisAll Western blots shown are representative of at least three

independent experiments. Differences between independentgroups were determined by the Student's t test and linear regres-sion analysis. Two-way ANOVAwas used to analyze differences insurvival between groups with two independent variables (i.e.,normoxia vs. hypoxia). Three-way repeated measured ANOVAwas used to analyze experiments with three independent variables(i.e., time, oxygen concentration, and drug treatment). The Pvalues were adjusted using Tukey adjustment. The data are pre-sented as the mean � SE, and a P < 0.05 was considered to bestatistically significant.

ResultsHypoxia sensitizes prostate cancer cells to small molecule PIMinhibitors

The PIM Ser/Thr protein kinases promote cell survival throughpleotropic mechanisms, and their role in hypoxic tumor cells hasnot been defined. It was previously reported that the expression ofPIM1 is increased in hypoxia (14), but whether other PIM iso-forms are also induced is unknown. The expression of PIMisoforms was examined in prostate (PC3-LN4), colon (HCT116),and breast (MD-MBA-231) cancer cell lines. In response to hyp-oxia (6hours at 1.0%O2), the protein levels of all PIM1andPIM2,but not PIM3, were significantly increased across all cell linesexamined (Fig. 1A, Western blot analysis). PIM mRNA levelsremained relatively stable, suggesting that hypoxia regulates PIMat the protein level (Fig. 1A, bar graph). Next, the sensitivity ofcancer cells to PIM kinase inhibitors was examined in hypoxia

compared with normoxia. PC3-LN4 prostate cancer cells weretreated for 72 hours in normoxia or hypoxia with a dose range ofAZD1208, a pan-PIM kinase inhibitor that has been used inpreclinical (17, 29) and clinical trials (NCT01489722 andNCT01588548). Strikingly, the IC50 of AZD1208 was nearly anorder of magnitude lower in PC3-LN4 cells cultured in hypoxiacomparedwith normoxia (IC50¼ 0.2� 0.1 vs. 16.8� 0.2 mmol/L,respectively; Fig. 1B). Similar results were obtained usingHCT116and MD-MBA-231 cells, demonstrating that the selective toxicityof PIM inhibitors toward hypoxic cancer cells is not specific to aparticular cell line or type of cancer (Supplementary Fig. S1).Moreover, PARP cleavage, which is indicative of apoptosis, waspresent selectively in hypoxic cells after 48 hours of AZD1208treatment (Fig. 1C). To demonstrate that this drug inhibitedPIM activity in these cells, phosphorylation of IRS1 (S1101), ahighly sensitive PIM substrate, was measured (30). Phosphor-ylation of this protein was inhibited in both normoxia andhypoxia following AZD1208 treatment (Fig. 1C). Propidiumiodide staining revealed that hypoxia alone did not significant-ly impact cell viability (approximately 11% apoptosis at 72hours). However, AZD1208 treatment caused a significant,time-dependent increase in the sub-G1 population, whereasthis compound did not induce apoptotic cell death in nor-moxia. To confirm that the sensitivity of hypoxic cells toAZD1208 could be attributed to PIM inhibition and not off-target effects of the drug, siRNA was used to knockdown eachPIM isoform and viability was measured after 48 hours. Inhypoxia, knockdown of PIM1 and PIM2 significantly reducedviability, whereas the loss of PIM isoforms was not toxic innormoxia (Fig. 1E, bar graph). In addition, we observed thatknockdown of PIM1 and PIM2 reduced the protein levels ofHIF-1a and HIF-2a (Fig. 1E, Western blot analysis). These datademonstrate that pharmacologic or genetic inhibition ofPIM1/2 selectively kills hypoxic cancer cells.

PIM inhibitors kill hypoxic cancer cells in aHIF-1–independent manner

Because HIF-1 is widely considered the master regulator of thecellular response to hypoxia and knockdown of PIM1/2 reducedthe protein levels of HIF-a isoforms, we asked whether the HIF-1signaling pathway is essential for PIM inhibitor–mediated toxic-ity. First, we examined the effect of PIM inhibition on theexpression of HIF-a isoforms in hypoxia. A panel of cancer celllines was incubated in hypoxia � AZD1208 for 6 hours, andWestern blotting used to monitor HIF-a protein. In all the celllines tested, AZD1208 significantly reduced HIF-1a and HIF-2aprotein levels (Fig. 2A). To determine whether PIM inhibitors killhypoxic cells in a HIF-dependent manner, PC3-LN4 cells weretransfected with siRNA to knockdown HIF-1a, HIF-2a, or thecombination of both, effectively eliminating HIF-1 signaling.These cells were then incubated in hypoxia for 72 hours in thepresence or absence of AZD1208. Knockdown ofHIF-1a, HIF-2a,or both did not significantly reduce PC3-LN4 cell viability after 72hours in hypoxia, providing evidence that HIF-1 signaling is notessential for this cell line to survive in hypoxia (Fig. 2B, compareDMSO-treated cells). Furthermore, crystal violet staining revealedthat AZD1208 reduced cell viability to a similar extent in celllacking HIF-a isoforms, indicating that PIM inhibitors are toxictoward hypoxic cells even in the absence of HIF-1 signaling(Fig. 2B, compare DMSO vs. AZD). Similar results were observedusing HCT116 and MD-MBA-231 cells (Supplementary Fig. S2).

PIM Inhibitors Selectively Kill Hypoxic Tumor Cells

www.aacrjournals.org Mol Cancer Ther; 15(7) July 2016 1639




In addition, the protein levels of PIM1 and PIM2were unchangedin response to knockdown of HIF-1a isoforms, demonstratingthat PIM levels are not regulated by the HIF-1 pathway in hypoxia(Fig. 2B, Western blot analysis).

To further establish that the ability of PIM inhibitors to killcells was independent of their effect on the HIF-1 pathway, weutilized a HIF-1a mutant in which Pro402 and Pro564 weremutated to alanine (P/A). This construct is unable to behydroxylated by prolyl hydroxylase domain-containing pro-teins (PHD1, 2, 3), making it highly stable. To determinewhether PIM inhibitors could reduce the expression of thismutant, PC3-LN4 cells were transfected with HA-tagged WTHIF-1a or the P/A mutant, placed in hypoxia � AZD1208 for 6hours, and exogenous HIF-1a expression monitored using ananti-HA antibody. WT HIF-1a was reduced by AZD1208, butthe P/A mutant was unchanged, indicating that PIM inhibitorslikely reduce HIF-1a levels by interfering with the canonicaldegradation pathway (Fig. 2C, Western blot analysis). Impor-tantly, AZD1208 significantly reduced the transcriptional activ-ity of WT HIF-1 but not the P/A mutant, as determined by theexpression of HIF-1 target genes, Angiopoietin-Like 4(ANGPTL4) and VEGF (Fig. 2C). To determine whether aHIF-1 construct that is refractory to AZD1208-mediated down-regulation could block the toxic effect of this drug in hypoxia,PC3-LN4 cells were transfected with WT HIF-1a or the P/Amutant, which is not degraded in response to AZD1208 treat-ment. The next day, the cells were treated with DMSO orAZD1208 and incubated in hypoxia for 48 hours. AZD1208

reduced the viability of prostate cancer cells overexpressing theP/A mutant to the same extent as cells expressing the vector andWT HIF-1 (Fig. 2D). Taken together, these data demonstratethat PIM inhibitors kill hypoxic cancer cells through a mech-anism that is unrelated to their ability to reduce HIF-1 activity.

PIM inhibitors are toxic toward hypoxic cells in aROS-dependent manner

Similar to the metabolic phenotype associated with hypoxia,the loss of PIM kinases was recently shown to alter the cellularredox state by reducing the levels of metabolic intermediates inthe glycolytic and pentose phosphate pathways, increasing theproduction of reactive oxygen species (ROS; ref. 31). Treatmentwith PIM inhibitors could therefore increase the ROS levelsresulting from hypoxic stress, leading to tumor cell death. Toassess ROS production, PC3-LN4 cells were placed in normoxia orhypoxia � AZD1208 for 24 hours. After incubation for theindicated time, cells were loaded with a chloromethyl derivativeof H2DCFDA (DCF), an intracellular dye that indicates ROSlevels, and harvested for FACS analysis or imaged under a fluo-rescence microscope. At 24 hours, ROS levels were significantlyincreased in hypoxia compared with normoxia (Fig. 3A, compareDMSO). Strikingly, two structurally unrelated pan-PIM kinaseinhibitors, LGB321 and AZD1208 (Supplementary Fig. S3),caused a further increase in ROS levels, especially in hypoxia(7-fold increase compared with hypoxia alone). AZD1208increased ROS levels by 2-fold in normoxia, whereas LGB321hadno effect, indicating that PIMactivity is particularly important

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Hypoxia increases PIM protein levels and sensitizes prostate cancer cells to AZD1208. A, PC3-LN4, HCT116, and MD-MBA-231 cells were cultured in normoxia orhypoxia, and protein and mRNA were harvested to assess the expression of PIM isoforms. B, PC3-LN4 cells were treated with a dose range of AZD1208 innormoxia or hypoxia for 72 hours, and cell viability was measured by crystal violet staining (� , P < 0.05; normoxia vs. hypoxia). C, in parallel, cells were cultured innormoxia or hypoxia � AZD1208 (3 mmol/L) for 48 hours, and protein levels were monitored by Western blotting. D, PC3-LN4 cells were treated withDMSOorAZD1208 (3mmol/L) for the indicated times, and sampleswere stainedwith propidium iodide for FACSanalysis (� ,P<0.05; comparedwith normoxia at eachtime point). E, siRNAs targeting PIM1, PIM2, and PIM3 were transfected into PC3-LN4 cells. The cells were incubated for 48 hours � AZD1208 (3 mmol/L), andviability was measured at 48 hours (� , P < 0.05; n.s., not significant). N, normoxia; H, hypoxia.

Warfel et al.





for controlling ROS levels in the context of hypoxia (Fig. 3A, bargraph). If AZD1208-induced ROS production accounts for thedecrease in cell viability in hypoxic prostate cancer cells, ROSscavengers, N-acetyl cysteine (NAC) and polyethylene glycol-superoxide dismutase (PEG-SOD), should reduce intracellularROS and rescue viability in AZD1208-treated cells. To evaluatethis possibility, PC3-LN4 cells were treated with AZD1208, NAC,PEG-SOD, or the indicated combinations for 48 hours in hypoxia,and viabilitywasmeasured by crystal violet staining.DCF stainingconfirmed that both NAC and PEG-SOD significantly reducedROS levels in AZD1208-treated cells in hypoxia (Fig. 3B). Asexpected, AZD1208 significantly reduced cell viability (Fig. 3C;compare bars 1 and 2). However, NAC and PEG-SOD largelyrescued AZD1208-induced cell death, restoring viability toapproximately 85% (Fig. 3C; compare bar 2 to bars 5 and 6).These data show that PIM inhibition selectively kills cancer cells ina ROS-dependentmanner.Moreover, NAC and PEG-SODdid notrescue the AZD1208-mediated downregulation of HIF-a expres-sion, providing further evidence that PIM inhibitors kill hypoxiccells through amechanism that is distinct fromany effect onHIF-1(Fig. 3C, Western blot analysis).

PIM kinase activity regulates the cellular antioxidant responseby controlling NRF2 signaling

A major mechanism in the cellular defense against oxidativestress is activation of the Nrf2-antioxidant response elementsignaling pathway, which controls the expression of genes thateliminate ROS and enhance the cellular antioxidant capacity. To

determine whether PIM kinases alter Nrf2 signaling, the effect ofAZD1208 on Nrf2 DNA–binding capacity and its transcriptionalactivity was measured. First, PC3-LN4 cells were incubated for 24hours in normoxia or hypoxia� AZD1208. Nuclear extracts wereisolated and the relative amount of Nrf2 binding to a specificdsDNA sequence containing the Nrf2 response element wasmeasured by a colorimetric assay. No change was observedfollowingAZD1208 treatment innormoxia, but therewas approx-imately a 40%decrease inNrf2DNAbinding following AZD1208treatment of hypoxic cells (Fig. 4A). This result indicates that lessNrf2 is present in the nucleus to bind the promoters of target genesin the absence of PIM activity. Next, Nrf2 transcriptional activitywas measured using a breast cancer cell line (MD-MBA-231)stably expressing a luciferase reporter construct fused to theARE of hNQO1 (ARE-Luc). Luciferase expression in these cellsis directly correlated with Nrf2 transcriptional activity (23).MD-MBA-231-ARE-Luc cells were incubated in normoxia orhypoxia for 24 hours in the presence or absence of AZD1208,LGB321, or a known inducer of Nrf2 signaling, sulforaphane(SULF). In normoxia, neither PIM inhibitor significantly alteredluciferase expression, while there was a 3-fold increase inresponse to SULF. In contrast, both PIM inhibitors significantlyreduced luciferase expression in hypoxia, demonstrating thatPIM inhibition reduced the transcriptional activity of Nrf2 inhypoxia (Fig. 4B). In agreement with previous reports, weobserved that hypoxia increased the expression of several Nrf2target genes (NQO1, HMOX2, and PRDX1; ref. 7). Treatmentwith AZD1208 reduced the hypoxia-mediated increase in Nrf2

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PIM inhibitors selectively kill hypoxic cancer cells in a HIF-1–independent manner. A, PC3-LN4, HCT116, and MDA-MB-231 cells were placed in hypoxia�AZD1208 for6 hours, and protein was collected to assess the expression of HIF-a isoforms. B, PC3-LN4 cells were transfected with the indicated siRNAs and incubated inhypoxia�AZD1208 (3mmol/L) and viabilitywasmeasured by crystal violet; lysateswere collected in parallel forWestern blotting. C, PC3-LN4 cells were transfectedwith WT HIF-1 or the HIF-1 (P/A) mutant and incubated in hypoxia for 6 hours � AZD1208 and protein and mRNA were collected for analysis. D, in parallel,transfected cells were incubated in hypoxia � AZD1208 (3 mmol/L) for 48 hours. � , P < 0.05; n.s., not significant.






target genes to basal levels. This result suggests that PIMexpression is necessary for the full activation of Nrf2 in hypoxia(Fig. 4C). Moreover, we demonstrate that PIM1 overexpressionwas sufficient to increase the expression of Nrf2 target genes innormoxia (Fig. 4D).

PIM regulates Nrf2 activity by controlling its cellularlocalization

Nrf2 activation is associated with its nuclear translocation. Inresponse to oxidative stress, Nrf2 escapes its interaction withKEAP1 in the cytoplasm and enters the nucleus to enhance the

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PIM inhibitors kill hypoxic tumor cells by increasing ROS. A, PC3-LN4 cells were cultured in normoxia or hypoxia for 24 hours � LGB-321 (1 mmol/L) or AZD1208(3 mmol/L). DCF staining was imaged using a fluorescence microscope and quantified by flow cytometry (histograms). The geometric mean fluorescenceintensity (MFI) was calculated from three independent experiments. B, PC3-LN4 cells were placed in normoxia or hypoxia and treated with AZD1208 alone or incombination with NAC (5 mmol/L) or PEG-SOD (150 U/mL) for 24 hours prior to DCF staining. C, PC3-LN4 cells were treated with AZD1208, NAC, PEG-SOD,or the indicated combinations for 72 hours in hypoxia, and viability was assessed by crystal violet staining; in parallel, lysates were collected at 24 hours toassess HIF-a protein levels. � , P < 0.05.

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PIM regulates Nrf2 activity. A, PC3-LN4cells were incubated in normoxia orhypoxia � AZD1208 (3 mmol/L) for24 hours. Nuclear lysates were isolatedand Nrf2 DNA binding was measured.B, MD-MBA-231-ARE-Luc cells wereincubated in normoxia or hypoxia �AZD1208 (3 mmol/L), LGB321(1 mmol/L), or sulforaphane (15 mmol/L)for 24 hours, and luciferase expressionwas measured. C, PC3-LN4 cells wereplaced in normoxia or hypoxia �AZD1208 for 24 hours, and mRNA wascollected and gene expressioncompared with normoxia wasmeasured by qRT-PCR (� , P < 0.05compared with normoxia; þ, P < 0.05compared with hypoxia). D, mRNA andprotein were collected from PC3 cellsstably expressing Vector or PIM1, andgene expression was measuredby qRT-PCR. � , P < 0.05.

Warfel et al.





transcription of antioxidant genes (12). Therefore, PIM inhibitorsmight blunt Nrf2 activity by reducing its nuclear accumulation.To test this hypothesis, we performed immunofluorescencestaining to monitor the cellular localization of endogenousNrf2 in response to hypoxia and PIM inhibition. Similar toprevious reports, endogenous Nrf2 was evenly distributedthroughout the cell at basal conditions (Fig. 5A) (32, 33). After24 hours in hypoxia, Nrf2 accumulated in the nucleus of thecell, and treatment with a chemical activator of Nrf2, sulfo-raphane, further increased its nuclear localization (Fig. 5A,compare normoxia to DMSO and SULF in hypoxia). In con-trast, treatment with AZD1208 blocked the hypoxia-mediatedaccumulation of Nrf2 in the nucleus, as demonstrated by adecrease in nuclear localization compared with the DMSO- orSULF-treated cells in hypoxia. Interestingly, bright punctatestaining of Nrf2 was observed throughout the cytoplasm andperinuclear regions of the cell following AZD1208 treatment,suggesting that a majority of the protein is sequestered outsidethe nucleus and may be forming aggregates in the cytoplasm(Fig. 5A, compare AZD with DMSO).

To confirm that PIM kinase activity alters the cellular local-ization of Nrf2 and quantitatively compare the amount of Nrf2in the cytoplasm and nucleus, biochemical fractionation wasperformed. PC3-LN4 cells were treated with DMSO, AZD1208,or SULF for 24 hours in normoxia or hypoxia, and the cytosolicand nuclear fractions were isolated as described in the Materialsand Methods section. Equal amounts of nuclear and cyto-plasmic protein were loaded onto SDS-PAGE gels, and Westernblotting was used to determine the amount of Nrf2 in eachcellular compartment. The nuclear to cytoplasmic ratio wasdetermined using densitometry. To verify that we effectivelyisolated the indicated cellular fractionations, we probed forLamin A, a protein specifically found in the nucleus, as acontrol. Nrf2 was relatively evenly distributed between the

cytosol and nucleus in normoxic conditions, with a slightlyhigher amount of protein in the cytoplasm (Fig. 5B, normoxiaratio ¼ 0.6). As expected, Nrf2 exposure to hypoxia as well astreatment with sulforaphane caused a majority of Nrf2 totranslocate to the nucleus (Fig. 5B, hypoxia DMSO ratio ¼2.0 and SULF ratio ¼ 4.4). In contrast, Nrf2 levels increased inthe cytosol following AZD1208 treatment, demonstrating thatPIM inhibition retains this protein in the cytoplasm (Fig. 5B,AZD ratio ¼ 0.3). These data are consistent with the results ofour immunofluorescence staining. Consistent with a role forPIM kinases in affecting the nuclear translocation of Nrf2, PIM1expression caused the opposite effect; Nrf2 was accumulated inthe nuclear fraction in the PIM1-overexpressing cell line com-pared with the vector control (Fig. 5C, VEC vs. PIM ratio ¼ 0.6and 2.1, respectively). Together, these data demonstrate thatPIM kinases control Nrf2 activity by regulating its cellularlocalization.

AZD1208 reduces tumor growth and selectively kills hypoxictumor cells in vivo

To investigate the effect of PIM inhibitors on hypoxic tumorcells in vivo, PC3-LN4 cells were injected subcutaneously intoeach flank of 4–6 week old, male NOD-SCID mice. The tumorswere allowed to grow to an average size of 250 mm3, whenregions of intratumoral hypoxia have formed. Then, the micewere randomly segregated into two groups (n ¼ 5 mice/10tumors) that received vehicle or AZD1208 (30 mg/kg/day).Tumor volume was measured every other day by caliper mea-surement for 14 days. Forty-five minutes prior to sacrifice, themice were injected with pimonidazole (i.p.), a marker ofhypoxia. Mice were sacrificed by cervical dislocation to avoidany ischemic artifact resulting from CO2-induced asphyxiation,and the tumors and mouse tissue were harvested for biochem-ical and histologic examination. Treatment with AZD1208

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PIM kinase activity regulates the cellular localization of Nrf2. A, PC3-LN4 cells were incubated in normoxia or hypoxia � AZD1208 (3 mmol/L) or sulforaphane(15 mmol/L) for 24 hours. Cells were fixed, and Nrf2 localization was monitored by immunofluorescence. B, PC3-LN4 cells were incubated in normoxia orhypoxia � AZD1208 (3 mmol/L) or sulforaphane (15 mmol/L) for 24 hours. Nrf2 levels were monitored in nuclear and cytoplasmic extracts by immunoblotting.C, PC3 cells stably expressing Vector or PIM1 were harvested, and Nrf2 levels were monitored by immunoblotting. � , P < 0.05.






significantly reduced tumor volume, especially at later timepoints, and the average tumor weight at the end of the studywas markedly lower in the AZD1208-treated cohort (Fig. 6Aand B, respectively). To account for intrinsic differences intumor size (i.e., larger tumors are more hypoxic), tumors withapproximately the same volume and weight were selected forbiochemical analysis at the time of harvest. These tumors wereimmediately frozen in liquid nitrogen and stored at �80�Cuntil lysis and subsequent Western blot analysis. Confirmingthat AZD1208 effectively inhibited PIM kinases in vivo, IRS1(S1101) phosphorylation was abolished in AZD1208-treatedmice (Fig. 6C). Moreover, the protein levels of both HIF-1a andHIF-2a were significantly reduced in the tumors of AZD1208-treated mice (Fig. 6C). To further investigate the effect of PIMinhibition on survival in normoxic versus hypoxic regions ofthe tumor, serial sections of vehicle and AZD1208-treatedtumors were stained with hypoxyprobe-1 (HP1; anti-pimoni-dazole), a marker of hypoxia, and CC3, a marker of apoptosis.CC3 staining was more widespread in PIM inhibitor–treatedtumors, and the number of apoptotic cells was significantlyincreased in regions of hypoxia compared with normoxia,demonstrating that hypoxic cells are preferentially killed byAZD1208 in vivo (Fig. 6D and E). In combination with our cellculture studies, these findings demonstrate the potential value

of small molecule PIM kinase inhibitors as hypoxia-targetingagents for use in solid tumors.

DiscussionA majority of the research on tumor hypoxia has focused on

HIF-1, a master regulator of the transcriptional response to lowoxygen. In contrast, the identification of HIF-1–independentsignaling pathways that regulate survival in hypoxia has beenelusive. Here, we establish and characterize one such pathwayand show that the PIM kinases are critical for survival in thehypoxic tumor microenvironment (Fig. 7). Our experimentsdemonstrate that PIM1 and PIM2 are induced in response tohypoxia, independent of HIF-1–mediated transcription (Fig.1A). We find that hypoxia markedly increases the sensitivity ofcancer cells to genetic or pharmacologic inhibition of PIM1/2,indicating that these enzymes play an important role in cellularadaptation and survival in hypoxia (Fig. 1B–D). Importantly, ina prostate cancer xenograft model treated with AZD1208, weobserved a dramatic increase in apoptosis specifically in hyp-oxic regions (Fig. 6), validating the utility of small moleculePIM kinase inhibitors as hypoxia-targeting agents in vivo. Inter-estingly, AZD1208 reduced the protein levels of HIF-1a andHIF-2a (Figs. 2A and 6C), suggesting that PIM kinase activity

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AZD1208 selectively kills hypoxic cells in vivo. A, tumor volumes were monitored by caliper measurements (line graph). B, at the end of the study, tumors from eachgroup were harvested and weighed (bar graph). C, size-matched tumors from each cohort were homogenized in lysis buffer and subjected to Westernblottingwith the indicated antibodies. D, tumors fromeach cohortwere immunostainedwith hypoxyprobe-1 (HP1) andCC3. Dotted lines delineate regions of hypoxiain serial sections. E, percent CC3þ area was determined as described in the Materials and Methods section. � , P < 0.05.

Warfel et al.





also influences HIF-1 signaling in hypoxia. However, the abilityof AZD1208 to kill hypoxic cells was not altered by siRNAknockdown of HIF-a isoforms, and the overexpression of aconstitutively active HIF-1a that could not be downregulated inresponse to AZD1208 did not rescue the toxic effect of PIMinhibition (Fig. 2B and C). These data demonstrate that thetoxicity of PIM inhibitors toward hypoxic cells is independentof the HIF-1 signaling pathway.

One mechanism by which hypoxia damages normal cells andtissue is through increased ROS production. As a result, tumorcells must buffer this excess ROS to maintain nontoxic levels inthe context of acute and/or chronic hypoxia. Our data suggestthat a critical function of PIM kinases in hypoxia is to protecttumor cells against hypoxia-induced ROS production. A recentstudy reported that TKO MEFs (lacking all three PIM isoforms)have decreased expression of several key ROS scavenger pro-teins, including superoxide dismutase 2 (SOD2) and Peroxir-edoxin-3 (Prdx3), resulting in increased basal ROS levels (31).We observed that hypoxia increased ROS levels in prostatecancer cells (Fig. 3A). Strikingly, treatment with chemicallydistinct PIM inhibitors increased ROS to a much greater extentin hypoxia compared with normoxia, leading to cell death (Fig.3A). Thus, in the presence of a PIM inhibitor, hypoxic cancercells are no longer able to maintain ROS at physiologic levels,resulting in apoptosis due to excessive free radicals. These datasupport a model where hypoxia increases mitochondrial ROSgeneration, and to combat the toxic effects of excess ROS,hypoxic cancer cells increase PIM protein levels. This enzymethen functions to increase the expression of detoxifying pro-teins to reduce the accumulation of free radicals and supportsurvival. This mechanism may explain the selective toxicity ofPIM inhibitors toward hypoxic tumor cells.

This study is the first to demonstrate that PIM kinasesinfluence the cellular antioxidant defense system via enhancingthe activity (Fig. 4) and cellular localization (Fig. 5) of Nrf2.Combined with the fact that PIM kinases are upregulated inhypoxia, this finding poises PIM kinases as key players inregulating the cellular cytoprotective response to low oxygen,

which is prevalent in solid tumors. Nrf2 expression levels areknown to influence both tumor progression and therapeuticefficacy. For example, Nrf2 has been shown to be elevated inlung, breast, and prostate cancers, and its expression is corre-lated with poor prognosis (34, 35). Moreover, Nrf2 overexpres-sion is associated with chemoresistance in many cancers,including colon, breast, and prostate cancers (36). Severalmechanisms have been proposed to explain the activation ofNrf2 in cancer, such as mutations that disrupt KEAP1 bindingand upregulation via amplification of oncogenic proteins,including Myc and Kras (37). We demonstrate that the over-expression of PIM kinases, which is frequently observed incancer, is sufficient to activate Nrf2. These data provide a newmechanism by which PIM kinases promote therapeutic resis-tance, independent of their role in regulating cell-cycle pro-gression and survival. Mechanistically, PIM kinases promotethe nuclear translocation of Nrf2 (Fig. 5). Thus, by enhancingPIM expression in response to hypoxia, cancer cells increaseNrf2 activity, allowing for more rapid and robust detoxificationof the cell and increased survival in response to the harsh tumormicroenvironment and/or therapeutic interventions. Previousreports have described that Nrf2 can be directly phosphorylatedby PKC (38) and MAPKs (39), leading to dissociation fromKEAP1, accumulation in the nucleus, and resistance to therapy;although, the importance of these phosphorylation events forNrf2 activity remains unclear. Further studies are warranted todetermine whether PIM kinases directly phosphorylate Nrf2 oract through an indirect mechanism to promote its accumula-tion in the nucleus.

In summary, we have identified a novel mechanism bywhich PIM kinases regulate Nrf2 activity and tumor cell sur-vival in hypoxia (Fig. 7). Inhibiting PIM in solid tumors hasthe unique advantage of opposing the oncogenic propertiesof hypoxia by selectively killing hypoxic tumor cells. Thesefindings provide new insights into pathways regulating thecellular response to oxidative stress and HIF-1–independentmechanisms that are essential for survival in hypoxia. Thebiochemical and in vivo results presented here support the

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PIM kinase inhibitors selectively targethypoxic cancer cells by reducing Nrf2activity and increasing ROS to toxiclevels. Model depicting the role of PIMkinases in controlling cell Nrf2 signalingand survival in hypoxia. A, hypoxiaupregulates PIM and ROS levels. In turn,PIM promotes the nuclear translocationof Nrf2, leading to the transcription/translation of cytoprotectivegenes thatrecued ROS levels and allow for cellsurvival. B, pharmacologic inhibition ofPIM blocks the nuclear translocation ofNrf2, leading to a significant increase inROS levels that ultimately kills hypoxiccancer cells. Thus, PIM inhibitionrepresents an attractive strategy tooppose hypoxia-mediated therapeuticresistance.






development of PIM kinase inhibitors as a novel therapeuticstrategy to increase ROS-mediated killing specifically inhypoxic tumor cells.

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

Authors' ContributionsConception and design: N.A. Warfel, A.S. KraftDevelopment of methodology: N.A. Warfel, A.G. Sainz, A.S. KraftAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N.A. Warfel, A.G. SainzAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N.A. Warfel, A.G. Sainz, J.H. SongWriting, review, and/or revision of the manuscript: N.A. Warfel, A.S. KraftAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N.A. Warfel, J.H. Song, A.S. KraftStudy supervision: N.A. Warfel

AcknowledgmentsThe authors thank Dr. Donna Zhang for providing ARE-Luc and MD-MBA-

231 (ARE-Luc) cells and Dr. Ningfei An at the University of Chicago for hertechnical assistance with in vivo experiments. The authors appreciate the supportof AstraZeneca and Novartis for making the PIM inhibitors available for thesestudies.

Grant SupportThis work was supported by University of Arizona Cancer Center support

grant P30CA023074, NIH award R01CA173200, and DOD award W81XWH-12-1-0560 (to A.S. Kraft).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 18, 2016; revised April 29, 2016; accepted May 2, 2016;published OnlineFirst May 16, 2016.

References1. Turaka A, Buyyounouski MK, Hanlon AL, Horwitz EM, Greenberg RE,

Movsas B. Hypoxic prostate/muscle PO2 ratio predicts for outcome inpatientswith localized prostate cancer: long-term results. Int J RadiatOncolBiol Phys 2012;82:e433–9.

2. Dekervel J, Hompes D, van Malenstein H, Popovic D, Sagaert X, DeMoor B, et al. Hypoxia-driven gene expression is an independentprognostic factor in stage II and III colon cancer patients. Clin CancerRes 2014;20:2159–68.

3. Milosevic M, Warde P, Menard C, Chung P, Toi A, Ishkanian A, et al.Tumor hypoxia predicts biochemical failure following radiotherapyfor clinically localized prostate cancer. Clin Cancer Res 2012;18:2108–14.

4. Vergis R, Corbishley CM, Norman AR, Bartlett J, Jhavar S, Borre M, et al.Intrinsic markers of tumour hypoxia and angiogenesis in localised prostatecancer and outcome of radical treatment: a retrospective analysis of tworandomised radiotherapy trials and one surgical cohort study. LancetOncol 2008;9:342–51.

5. Dawson TL, GoresGJ,NieminenAL,HermanB, Lemasters JJ.Mitochondriaas a source of reactive oxygen species during reductive stress in rat hepa-tocytes. Am J Physiol 1993;264:C961–7.

6. Chi AY, Waypa GB, Mungai PT, Schumacker PT. Prolonged hypoxiaincreases ROS signaling and RhoA activation in pulmonary arterysmooth muscle and endothelial cells. Antioxid Redox Signal 2010;12:603–10.

7. Guzy RD, Hoyos B, Robin E, Chen H, Liu L, Mansfield KD, et al. Mito-chondrial complex III is required for hypoxia-induced ROSproduction andcellular oxygen sensing. Cell Metab 2005;1:401–8.

8. Hybertson BM, Gao B, Bose SK, McCord JM. Oxidative stress in health anddisease: the therapeutic potential of Nrf2 activation. Mol Aspects Med2011;32:234–46.

9. McMahon M, Itoh K, Yamamoto M, Hayes JD. Keap1-dependent protea-somal degradation of transcription factor Nrf2 contributes to the negativeregulation of antioxidant response element-driven gene expression. J BiolChem 2003;278:21592–600.

10. ZhangDD.Mechanistic studies of theNrf2-Keap1 signaling pathway. DrugMetab Rev 2006;38:769–89.

11. Nguyen T, Sherratt PJ, Pickett CB. Regulatorymechanisms controlling geneexpression mediated by the antioxidant response element. Annu RevPharmacol Toxicol 2003;43:233–60.

12. Kobayashi M, Yamamoto M. Molecular mechanisms activating the Nrf2-Keap1 pathway of antioxidant gene regulation. Antioxid Redox Signal2005;7:385–94.

13. Nawijn MC, Alendar A, Berns A. For better or for worse: the role of Pimoncogenes in tumorigenesis. Nat Rev Cancer 2011;11:23–34.

14. Qian KC, Wang L, Hickey ER, Studts J, Barringer K, Peng C, et al.Structural basis of constitutive activity and a unique nucleotidebinding mode of human Pim-1 kinase. J Biol Chem 2005;280:6130–7.

15. Liu HT,WangN,Wang X, Li SL. Overexpression of Pim-1 is associated withpoor prognosis in patients with esophageal squamous cell carcinoma.J Surg Oncol 2010;102:683–8.

16. Dhanasekaran SM, Barrette TR, GhoshD, Shah R, Varambally S, Kurachi K,et al. Delineation of prognostic biomarkers in prostate cancer. Nature2001;412:822–6.

17. Keeton EK, McEachern K, Dillman KS, Palakurthi S, Cao Y, Grondine MR,et al. AZD1208, a potent and selective pan-Pim kinase inhibitor, demon-strates efficacy in preclinical models of acute myeloid leukemia. Blood2014;123:905–13.

18. Lu J, Zavorotinskaya T, Dai Y, Niu XH, Castillo J, Sim J, et al. Pim2 isrequired for maintaining multiple myeloma cell growth through modu-lating TSC2 phosphorylation. Blood 2013;122:1610–20.

19. Chen J, KobayashiM, Darmanin S, Qiao Y, Gully C, Zhao R, et al. Hypoxia-mediated up-regulation of Pim-1 contributes to solid tumor formation.Am J Pathol 2009;175:400–11.

20. Chen J, Kobayashi M, Darmanin S, Qiao Y, Gully C, Zhao R, et al. Pim-1plays a pivotal role in hypoxia-induced chemoresistance. Oncogene2009;28:2581–92.

21. Kondo K, Klco J, Nakamura E, LechpammerM, Kaelin WG Jr. Inhibition ofHIF is necessary for tumor suppression by the von Hippel-Lindau protein.Cancer Cell 2002;1:237–46.

22. Yan Q, Bartz S, Mao M, Li L, Kaelin WG Jr. The hypoxia-induciblefactor 2alpha N-terminal and C-terminal transactivation domainscooperate to promote renal tumorigenesis in vivo. Mol Cell Biol2007;27:2092–102.

23. Du Y, Villeneuve NF, Wang XJ, Sun Z, Chen W, Li J, et al. Oridoninconfers protection against arsenic-induced toxicity through activation ofthe Nrf2-mediated defensive response. Environ Health Perspect2008;116:1154–61.

24. Garcia PD, Langowski JL, Wang Y, Chen M, Castillo J, Fanton C, et al. Pan-PIM kinase inhibition provides a novel therapy for treating hematologiccancers. Clin Cancer Res 2014;20:1834–45.

25. Warfel NA, Kraft AS. PIMkinase (and Akt) biology and signaling in tumors.Pharmacol Ther 2015;151:41–9.

26. Warfel NA, Niederst M, Stevens MW, Brennan PM, Frame MC,Newton AC. Mislocalization of the E3 ligase, beta-transducinrepeat-containing protein 1 (beta-TrCP1), in glioblastoma uncouplesnegative feedback between the pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) and Akt. J Biol Chem2011;286:19777–88.

27. FlickDA,GiffordGE. Comparison of in vitro cell cytotoxic assays for tumornecrosis factor. J Immunol Methods 1984;68:167–75.

28. Warfel NA, Dolloff NG, Dicker DT, Malysz J, El-Deiry WS. CDK1 stabilizesHIF-1alpha via direct phosphorylation of Ser668 to promote tumorgrowth. Cell Cycle 2013;12:3689–701.

29. Kirschner AN, Wang J, van der Meer R, Anderson PD, Franco-CoronelOE, Kushner MH, et al. PIM kinase inhibitor AZD1208 for treatment

Warfel et al.





of MYC-driven prostate cancer. J Natl Cancer Inst 2015;107: pii:dju407.

30. Song JH, Padi SK, Luevano LA, Minden MD, DeAngelo DJ, Hardiman G,et al. Insulin receptor substrate 1 is a substrate of the Pim protein kinases.Oncotarget. 2016 Mar 4. [Epub ahead of print].

31. Song JH, AnN, Chatterjee S, Kistner-Griffin E,Mahajan S, Mehrotra S, et al.Deletion of Pim kinases elevates the cellular levels of reactive oxygenspecies and sensitizes to K-Ras-induced cell killing. Oncogene 2015;34:3728–36.

32. Velichkova M, Hasson T. Keap1 regulates the oxidation-sensitive shuttlingof Nrf2 into and out of the nucleus via a Crm1-dependent nuclear exportmechanism. Mol Cell Biol 2005;25:4501–13.

33. Jain AK, Bloom DA, Jaiswal AK. Nuclear import and export signals incontrol of Nrf2. J Biol Chem 2005;280:29158–68.

34. Zhang P, Singh A, Yegnasubramanian S, Esopi D, Kombairaju P,Bodas M, et al. Loss of Kelch-like ECH-associated protein 1function in prostate cancer cells causes chemoresistance and radio-

resistance and promotes tumor growth. Mol Cancer Ther 2010;9:336–46.

35. Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO, et al.Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer.PLoS Med 2006;3:e420.

36. Wang XJ, Sun Z, Villeneuve NF, Zhang S, Zhao F, Li Y, et al. Nrf2 enhancesresistance of cancer cells to chemotherapeutic drugs, the dark side of Nrf2.Carcinogenesis 2008;29:1235–43.

37. DeNicolaGM,Karreth FA,HumptonTJ,GopinathanA,WeiC, FreseK, et al.Oncogene-induced Nrf2 transcription promotes ROS detoxification andtumorigenesis. Nature 2011;475:106–9.

38. Huang HC, Nguyen T, Pickett CB. Phosphorylation of Nrf2 at Ser-40 byprotein kinase C regulates antioxidant response element-mediated tran-scription. J Biol Chem 2002;277:42769–74.

39. Sun Z, Huang Z, Zhang DD. Phosphorylation of Nrf2 at multiple sites byMAP kinases has a limited contribution inmodulating theNrf2-dependentantioxidant response. PLoS One 2009;4:e6588.






2016;15:1637-1647. Published OnlineFirst May 16, 2016.Mol Cancer Ther Noel A. Warfel, Alva G. Sainz, Jin H. Song, et al. Signaling and Increasing Reactive Oxygen SpeciesPIM Kinase Inhibitors Kill Hypoxic Tumor Cells by Reducing Nrf2

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