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Cancer Biology and Translational Studies

Mechanisms Behind Resistance to PI3K InhibitorTreatment Induced by the PIM KinaseJin H. Song1,2, Neha Singh2, Libia A. Luevano2, Sathish K.R. Padi2,Koichi Okumura3, Virginie Olive4, Stephen M. Black3,4, Noel A.Warfel1,2,David W. Goodrich5, and Andrew S. Kraft2,4

Abstract

Cancer resistance to PI3K inhibitor therapy can be in partmediated by increases in the PIM1 kinase. However, theexact mechanism by which PIM kinase promotes tumor cellresistance is unknown. Our study unveils the pivotal controlof redox signaling by PIM kinases as a driver of this resis-tance mechanism. PIM1 kinase functions to decrease cellu-lar ROS levels by enhancing nuclear factor erythroid2-related factor 2 (NRF2)/antioxidant response elementactivity. PIM prevents cell death induced by PI3K-AKT–inhibitory drugs through a noncanonical mechanism of

NRF2 ubiquitination and degradation and translationalcontrol of NRF2 protein levels through modulation ofeIF4B and mTORC1 activity. Importantly, PIM also controlsNAD(P)H production by increasing glucose flux through thepentose phosphate shunt decreasing ROS production, andthereby diminishing the cytotoxicity of PI3K-AKT inhibitors.Treatment with PIM kinase inhibitors reverses this resistancephenotype, making tumors increasingly susceptible tosmall-molecule therapeutics, which block the PI3K-AKTpathway. Mol Cancer Ther; 17(12); 2710–21. �2018 AACR.

IntroductionIntrinsic and acquired resistance to PI3K inhibitors has been

poorly understood, and recent studies in breast cancer identifiedthe PIM (Proviral Integration site for Moloney murine leukemicvirus) protein kinase as additional therapeutic targets that sensi-tize cancer cells to anticancer therapeutics that inhibit this path-way (1). These findings are highly relevant to prostate cancerwhere PIM is highly expressed and aberrations in the PI3Kpathway and deletion of PTEN are detected in nearly 70% ofpatients with metastatic castration-resistant prostate cancer(mCRPC; ref. 2). The prevalence of PI3K pathway activation inmCRPC cells makes this pathway an attractive target to inhibittumor growth. However, in clinical trials PI3K inhibitor therapyinduced a partial response in only 12% of patients whose tumorsharbor mutations in PTEN or PIK3C isoforms (3), suggesting apossible role for PIM kinase in blocking this pathway.

PIM kinases, a family comprised of PIM1, PIM2, and PIM3, areoncogenic serine/threonine kinases that are overexpressed inmultiple malignancies (4, 5) that promote tumor progression by

regulating cell-cycle progression, proliferation, and survival (6).PIM1 expression is elevated in approximately 50% of humanprostate cancer specimens, particularly in high Gleason grade andaggressive metastatic prostate cancer cases (5, 7). These proteinkinases share significantmechanistic similarities to theAKT familywhich might account for their ability to act as part of a resistancemechanism. For example, both PIM and AKT families are knownto regulate protein translation by affecting activity of mTORC1, anutrient/energy/redox sensor and regulator of protein synthesis,through an ability to modify identical proteins. PIM and AKToverlap in substrate modification; PIM phosphorylates TSC2,PRAS40, and eIF4B on serine (S) 406 (8).

Activation of the AKT pathway produces reactive oxygenspecies (ROS). Because excessive oxidative stress can lead tosenescence or cell death, proliferating cancer cells limit cellularROS levels by inducing an array of antioxidants and ROSscavengers (9). AKT-stimulated mitogenic and survival pro-grams (10) control ROS levels through nuclear factor erythroid2-related factor 2 (NRF2), a basic leucine zipper transcriptionfactor, that binds to antioxidant response elements (ARE) toactivate a battery of cytoprotective and antioxidant gene targetsincluding heme oxygenase (HMOX1), NAD(P)H quinonedehydrogenase (NQO1), glutamate-cysteine ligases [GCLC andGCLM; the first rate-limiting enzymes of glutathione (GSH)synthesis], glutathione peroxidases (GPX), and superoxide dis-mutases (SOD; ref. 11). The activated NRF2, in addition tocytoprotective function, also promotes metabolic reprogram-ming by activating metabolic genes (12). Recently, it wasreported that NRF2 promotes EGFR autocrine signaling tofacilitate eIF4F complex formation for cap-dependent transla-tion initiation in KRAS-mutant pancreatic adenocarcinomas(13). The activated NRF2 promotes the further activation ofthe PI3K-AKT pathway to accelerate aggressive cancer progres-sion. Thus, therapeutic agents targeting NRF2 signaling mayrender cellular vulnerability to PI3K-AKT–inhibitory drugs.

1Department of Cellular and Molecular Medicine, University of Arizona, Tucson,Arizona. 2University of Arizona Cancer Center, Tucson, Arizona. 3Department ofPhysiology, University of Arizona, Tucson, Arizona. 4Department of Medicine,University of Arizona, Tucson, Arizona. 5Department of Pharmacology andTherapeutics, Roswell Park Cancer Institute, New York, New York.

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

Corresponding Authors: Jin H. Song, University of Arizona, 1515 N. CampbellAvenue, Room 4999B at UACC, Tucson, AZ 85724. Phone: 520-626-9048; Fax:520-626-6898; E-mail: [email protected]; and Andrew S. Kraft,[email protected]

doi: 10.1158/1535-7163.MCT-18-0374

�2018 American Association for Cancer Research.

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Previously, we reported that PIM kinasemediates an importantadaptive survival response upon inhibition of AKT through feed-back activation of receptor tyrosine kinase signaling, leading tocancer cell resistance (14). Recently, a genome scale shRNAscreening identified the PIM kinase as conferring resistance tothe PI3K inhibitor alpelisib (BYL719; ref. 1). Concurrent treat-ment with a PIM inhibitor and PI3K inhibitor overcame thisresistance. Moreover, PIM1was identified as a pivotal therapeutictarget in triple-negative breast cancer (15, 16), inhibiting PIMsensitized these cells to chemotherapy.

In this study, we have defined the potential mechanism under-lying the PIM1-mediated inhibition of PI3K-AKT inhibitor inboth tissue culture and mouse models harboring PI3K-AKT path-way aberrations.

Materials and MethodsChemicals

PIM447 (17) was provided by Novartis Oncology. Buparli-sib (BKM120) (18), AZD1208 (19), AZD5363 (20), andTorin1 (21) were purchased from AdooQ Bioscience.CM-H2DCF-DA (22) was purchased from Molecular Probes(Thermo Scientific). All other chemicals were purchased fromSigma-Aldrich.

Cell culture, stable cell lines, transfection, and CRISPR-Cas9The human prostate cancer cell lines LNCaP, PC-3, and

PC3-LN4 were cultured as previously described (23, 24).Mouse prostate epithelial cells were grown in DMEM mediumsupplemented with 2.5% charcoal-stripped FCS, 5 mg/mL ofinsulin/transferring/selenium, 10 mg/mL of bovine pituitaryextract, 10 mg/mL of epidermal growth factor, and 1 mg/mL ofcholera toxin as described previously (25). All cell lines weremaintained at 37�C in 5% CO2 and were authenticated byshort tandem repeat DNA profiling performed by the Univer-sity of Arizona Genetics Core Facility. The cell lines wereroutinely tested for mycoplasma and used for fewer than50 passages. Cells were transfected with RNAi or transducedwith lentiviruses as previously described (23, 26). eIF4BsgRNA CRISPR-Cas9 system was purchased from ABM Inc.Lentiviruses were produced in 293T cells coexpressing thepackaging vectors (psPAX2 and VSV-G), concentrated byultracentrifugation (20,000 g for 2 hours at 4�C), and resus-pended with culture media for an infection (48 hours). Alt-RCRISPR-Cas9 System is used for genome editing of the eIF4Bserine 406 site. A detailed description is provided in theSupplementary Methods.

Immunoblotting, immunofluorescence, and IHCFor immunoblotting, total lysates were prepared, resolved by

SDS-PAGE, and transferred to immobilon membranes. The fol-lowing primary antibodies were utilized from the indicatedvendors: PIM1, p-IRS1 (S1101), g-H2AX (S139), p-AKT (S473),AKT, p-p70S6K (T389), p70S6K, and pS6 (S240/244) were fromCell Signaling Technology; S6, NRF2, HMOX1, GCLC, GCLM,andPRDX3were fromSantaCruz Biotechnology; SOD2was fromAbcam; ACTIN, FLAG, HA, and ubiquitin antibodies were pur-chased from Sigma. IHC and immunofluorescence staining wereperformed as described in detail in the Supplementary Methods.Staining with H2DCF-DA followed by flow cytometry was aspreviously described (26).

Metabolic analysisLactate dehydrogenase, NADPH, and GSH production was

assessed using an Amplite Colorimetric L-Lactate Dehydrogenase(LDH)Assay Kit (AATBioquest), bioluminescent cell–basedNAD(P)/NAD(P)H assay, and GSH-Glo kits (Promega), respectively,and was carried out as per the manufacturer's instructions. Mea-surement of oxygen consumption rate (OCR) and extracellularacidification was performed using a Seahorse Bioscience XF24Extracellular Flux Analyzer. Following exposure of LNCaP to[U-13C] glucose, carbon flux through metabolic pathways wasexamined at the University of Michigan Regional ComprehensiveMetabolomics Resource Core (http://mrc2.umich.edu).

AnimalsFor xenograft studies, male SCIDmice (8 weeks old) were used

by approval of the Institutional Animal Care and Use Committeeat the University of Arizona and described in detail in theSupplementary Methods.

Prostate cancer patient-derived organoidsProstate cancer biopsieswere provided byUniversity of Arizona

Cancer Center Tissues Acquisition and Cellular/Molecular Anal-ysis Shared Resources, and the study was conducted under Uni-versity of Arizona Institutional Review Board approval. Prostatecancer patient-derived organoids were cultured based on previ-ously established procedures (27, 28) and are described in detailin the Supplementary Methods.

Statistical analysisStatistical significance was determined, when appropriate,

using unpaired, two-tailed Student t test. P values of less than0.05 were considered statistically significant.

ResultsPIM kinase and PI3K-AKT inhibitors synergize to suppresstumor growth

Proliferation of human prostate cancer cells, e.g., LNCaP andPC-3 lines that express constitutively active AKT, can be inhibitedby the pan-PI3K inhibitor buparlisib. However, when PIM1 isoverexpressed, these cells becomehighly resistant to this inhibitor(Fig. 1A and B). In PIM1-overexpressing LNCaP prostate cancercells (which contain a highly activated AKT pathway secondary todeletion of PTEN), simultaneously inhibiting both PI3K and thePIM kinase enhanced growth inhibition (Fig. 1C). PC3-LN4 cellsare a highly metastatic variant of PC-3 cells (29, 30) that haveincreased PIM transcript levels compared with other prostatecancer cell lines (Supplementary Fig. S1A), and are relativelyresistant to buparlisib (Fig. 1D and E). Synergistic inhibition ofsurvival and growth was demonstrated when PC3-LN4 cells weretreated with both PIM and PI3K/AKT inhibitors, PIM447 andbuparlisib, respectively (Supplementary Fig. S1B). This growthinhibition effect is not limited to these agents, as theAKT inhibitorAZD5363 and PIM inhibitor AZD1208 also abrogated prolifer-ationof these prostate cancer cells (Supplementary Fig. S1C–S1E).To determine whether these effects were observed in vivo, micewere xenografted with PC3-LN4 tumors and then treated withbuparlisib (10mg/kg) with or without PIM447 (30mg/kg) for 24days. The combination treatment significantly inhibited in vivogrowth, indicated by a decrease in tumor volume and weightcomparedwith animals treatedwith either buparlisib, PIM447, orvehicle control (Fig. 1F and G). The combination treatmentsignificantly reduced tumor cell proliferation compared with

Exploiting Redox Mechanism to Overcome Tumor Cell Resistance

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either agent alone (Supplementary Fig. S1F and S1G), evidencedby the marked decrease in Ki67 staining. Similar results wereobtained using theAKT inhibitor AZD5363 (40mg/kg) in place ofbuparlisib (Fig. 1H and I). These results suggest that tumor cellresistance to PI3K/AKT inhibitorswasmediated, at least in part, bythe PIM kinases.

PIM1kinase induces tumor cell resistance to inhibitors of PI3K/AKT signaling by increasing NRF2 levels and stimulating theproduction of ROS scavengers

Small-molecule PIM inhibitors inactivate the NRF2 transcrip-tion factor, reducing expression of cytoprotective genes andleading to the buildup of ROS in hypoxia (31). To understand

the biochemical mechanism by which PIM kinases cause resis-tance to inhibitors of PI3K/AKT, the relationship between NRF2signaling and PIM1 kinase was explored. To minimize geneticalterations affecting NRF2 regulation, we utilized mouse epithe-lial prostate cancer (mPrEC) cells derived from spontaneousprostate adenocarcinomas that arose in a mouse modelTrp53loxP/loxP; Rb1loxP/loxP; PbCre4 (Supplementary Fig. S2A).When PIM1 was expressed in cells from these mice (mPrEC/PIM1), these cells established larger colonies in soft agar (Sup-plementary Fig. S2B) and had elevated levels of NRF2-ARE genesincluding HMOX1, NQO1, SOD2, and SOD3 (SupplementaryFig. S2C; GEO repository accession number, GSE118786).Because activated AKT was detected in mPrEC/PIM1 cells, these

Figure 1.

PIM inhibition overcomes resistance toPI3K-AKT inhibitors. A, Overexpressionof PIM1 in LNCaP and PC-3 cells usinglentivirus. EV was used as a control.B, Representative crystal violet staining.EV- and PIM1-expressing LNCaP andPC-3 cells were treated with buparlisibfor 72 hours at the doses indicated. C,Dose-response analysis of LNCaP/PIM1versus LNCaP/EV. LNCaP/PIM1 cellswere treated with 3 mmol/L PIM447 andwere simultaneously exposed to varyingdoses of buparlisib for 72 hours. Thedata shown are the mean ofmeasurements � the SD (n ¼ 4). IC50

of buparlisib (mmol/L) was 0.80 forLNCaP/EV, 1.75 for LNCaP/PIM1, and0.39 for LNCaP/PIM1 cells cotreatedwith PIM447. D, Colony focus formationvisualized by crystal violet staining.Representative images are shown.PC3-LN4 cells (100 cells) were seededand then incubated in the absence orpresence of 3 mmol/L PIM447 for 7 daysalong with buparlisib at the dosesindicated. E, Dose-response analysisof PC3-LN4 cells exposed to buparlisibfor 72 hours in the absence or presenceof 3 mmol/L PIM447. The data shown arethe mean of measurements � SD(n ¼ 4). IC50 of buparlisib (mmol/L) was1.25 for DMSO and 0.63 for PIM447, F,PC3-LN4 xenografts treated withbuparlisib, PIM447, or the combination.The average tumor volume � SEM(n ¼ 5) is plotted, and the statisticalcomparison versus vehicle-treatedcontrol is shown using a t test(� , P < 0.05). G, The results of tumortreatment with single- or dual-kinaseinhibitors. The average tumorweight � the SEM (� , P < 0.05). H,PC3-LN4 xenografts treated withAZD5363 (40 mg/kg), AZD1208 (30mg/kg), or the combination. Theaverage tumor volume � SEM (n ¼ 7) isplotted, and the statistical comparisonwith vehicle treated control is shownusing a t test (� , P < 0.05). I, The tumorswere excised from themice described inH at the end of treatment. The averageweight is shown (� , P < 0.05).

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cells were treated with buparlisib together with PIM447. Treat-ment blocked AKT and PIM activity, judged by inhibition ofphospho-AKT (S473) and phospho-IRS1 (S1101) expression,respectively, which resulted in inhibition of mTORC1 and NRF2activity (Fig. 2A); the effects of combination treatment furtherresulted in DNA damage asmeasured by themarked induction ofg-H2AX expression. Sensitivity of mPrEC cells to buparlisib wasmarkedly enhanced by cotreatment with PIM447 (Fig. 2B). Theseresults suggest that in prostate cell lines with few mutations,combining PI3K and PIM inhibitors may be useful in blockingtumor growth. To test activity of combination therapy to killprostate cancer from primary patient samples, the laboratoryhas used organoids cultured from patient biopsies of the prostateobtained fromprostatectomy specimens. These patient organoidshad activated AKT and increased PSA levels (Fig. 2C; Supplemen-tary Fig. S2D and S2E). qPCR analysis confirmed the presence ofPIM1, 2, and 3 transcripts in these organoids (Supplementary Fig.S2F). These organoids had detectible levels of the PIM substrate,phopho-IRS1 (S1101), indicative of PIM kinase activity (23).Organoid data resembled cell culture and animal models ascombination treatment of buparlisib and PIM447 resulted in asignificant reduction in organoid number and viability (Fig. 2D).

To examine whether NRF2 signaling induced by PIM1 kinaseplays a role in resistance of human prostate cancer cell to PI3Kinhibitor, a Doxycycline (Dox)-inducible vector was used toinduce PIM1overexpression in LNCaP cells; NRF2 protein expres-sion was markedly increased (Supplementary Fig. S2G) withoutsignificant increase ofNRF2mRNA. This increase inNRF2 proteininduced multiple downstream targets of NRF2, including ROSscavengers (HMOX1 and NQO1), an enzyme needed for GSHsynthesis (GCLM), and enzymes that modulate cellular NADPHlevels (i.e., ME1, IDH1, and G6PD; Supplementary Fig. S2H). Todemonstrate that the increase in ROS scavengers was secondary toPIM1-mediated NRF2 induction, and not a direct effect of ele-vating the PIM kinase, NRF2-targeted shRNAwas expressed in thehuman prostate cancer cells containing Dox-inducible PIM1.PIM1-mediated induction of HMOX1 andNQO1 expression wasabrogated bydepletionofNRF2 (Fig. 2E; Supplementary Fig. S2I).Treatment of LNCaP cells with buparlisib, a pan PI3K inhibitor,blocks AKTphosphorylation andmarkedly decreasesNRF2 levels,leading to reduction in ROS scavengers, NQO1, HMOX1, SOD2,and in the GCLM enzyme. In contrast, when the expression ofPIM1 was increased even in the presence of buparlisib treatmentand blocked AKT phosphorylation, there was no change in theROS scavenger protein levels (Fig. 2F). Over a broad range ofdoses, buparlisib failed to downregulate HMOX1 in PIM1-over-expressing prostate cancer cells (Supplementary Fig. S2J).

Modulation of ROS scavengers is important for buparlisib-mediated LNCaP tumor cell killing as evidenced by the fact thatforced NRF2 or HMOX1 expression conferred buparlisib resis-tance (Fig. 2G). Conversely, depletion of NRF2 in the resistantPIM1 prostate cancer lines rendered these cells susceptible tobuparlisib-induced killing (Fig. 2H). Depletion of HMOX1expression in the PIM1-overexpressing cells also restored cellsensitivity to buparlisib, as evidenced by cleavage of PARP-1, amarker of apoptosis, and markedly increased cell death (Fig. 2I).In PC3-LN4 cells, siRNA-mediated depletion of HMOX1marked-ly increased buparlisib-induced cell death as characterized bycaspase-3 and PARP-1 cleavage (Fig. 2J). Therefore, PIM1preventssmall-molecule PI3K inhibitor–induced cell death by regulatingthe levels of NRF2 and ROS scavenger proteins.

Redox regulation of GSH is a critical factor in PIM-mediatedresistance to PI3K-AKT inhibitor

Luminescence-based assays demonstrate that increased expres-sion of PIM1 blocks the ability of buparlisib treatment to decreasethe cellular levels of both NADPH and GSH (Fig. 3A). To furtherexamine the role of ROS in the regulation of cell death induced byPI3K inhibitors, LNCaP/PIM1 were treated with buthionine sul-foximine (BSO), an inhibitor of GSH synthesis. PIM1 transduc-tion made these cells less sensitive to BSO killing (Fig. 3B), incomparison with control cells (LNCaP-expressing empty vector,LNCaP/EV). To test whether the addition of BSO significantlysuppressed in vivo tumor growth, LNCaP/PIM1 cells were grownsubcutaneously in immunocompromisedmice, and animalsweretreated with BSO (400 mg/kg), buparlisib, or the combination.Combination treatmentwith these agents inhibited tumor growthsignificantly better than either agent alone (Fig. 3C). To documentROS stimulation by these agents, prostate cells were stainedwith H2DCF-DA. In LNCaP/EV, buparlisib increased ROSaccumulation, which was blocked by the addition of either theROS scavenger N-acetylcysteine (NAC) or Trolox (Fig. 3D). Incontrast, buparlisib alone did not increase ROS accumulation inLNCaP/PIM1 (Fig. 3E). Combined BSO/buparlisib treatment ofthese cells produced robust ROS accumulation, paralleling theresults of animal experiments. These data indicate that increasedexpression of PIM1 induces resistance to buparlisib in part bystimulating GSHproduction and suppressing ROS accumulation.

Given the ability of PIM1 to regulate NRF2 levels, potentiallycontrolling the activity of multiple genes which modulate energyproduction, the effect of PIM1 overexpression on the metabolismof prostate cancer cells was studied. LNCaP/EV or LNCaP/PIM1cells were labeled with 13C-glucose, and this labeling was chasedat 0, 1, and 3 hours. LNCaP/PIM1 had increased carbon fluxthrough both glycolysis and the pentose phosphate shunt. Spe-cifically, there was an increased rate of labeling of glucose6-phosphate (G6P)/fructose 6-phosphate (F6P) (combined),phosphoenol pyruvate (PEP), 2-phosphogluconate (2PG)/3-phosphogluconcate (3PG) (combined), 6-phosphogluconate(6PG), ribulose 5-phosphate (R5P)/xylulose 5-phosphate (X5P)(combined), and sedoheptulose-7-phosphate (S7P; Supplemen-tary Fig. S3). This increased flux enhanced the mass isotopeabundance of NAD(P)H and NADPþ (Fig. 4A). As predicted,13C abundance in GSHwas increased (Fig. 4B). The Seahorse assaywas then used to assess OCR and extracellular acidification rate(ECAR). Consistent with the increased flux through the glycolyticpathway, decreased OCR and increased ECAR were evident inLNCaP/PIM1 (Fig. 4C). Moreover, induction of PIM1 resulted inrestoration of LDH activity (which was decreased by buparlisib;ref. Fig. 4D). Change in mitochondrial energetics (i.e., OCR) wasinhibited by buparlisib treatment but was maintained in LNCaP/PIM1 cells (Fig. 4E). Thus, increased PIM1 appears to stimulateglucose carbon flux, increasing GSH synthesis, potentially throughthe pentose phosphate pathway, and NAD(P)H production.

Combined inhibition of PIM and AKT blocks the survival andproliferation of prostate cancer cells by inhibiting NRF2expression through the regulation of mTORC1 activity

Both PC-3 and PC3-LN4 cells with aberrant activation of AKTsignaling are relatively insensitive to either buparlisib or AZD5363,but buparlisib cotreatment with PIM447 or AZD1208 (two struc-turally different PIM inhibitors) blocked their proliferation (Sup-plementary Figs. S1 and S4A). Genetic inhibition of the AKT






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pathway by expression of PTEN in PC-3 cells downregulatedNRF2expression (Fig. 5A) and resulted in a mild inhibition of prostatecancer viability. However, this effect was blunted by PIM1 over-expression. Conversely, treatment with PIM447 enhanced PTEN-mediated suppression of prostate cancer viability even in PIM1-overexpressing cells (Fig. 5B). Treatment with the combination ofPIM447 and buparlisib increased the accumulation of ROS in thePC3-LN4 cells (Fig. 5CandD; Supplementary Fig. S4B).Consistentwith the induction of ROS, NRF2 protein expressionwasmarkedlyreduced by combination treatment (Fig. 5E). Addition of tert-Butylhydroperoxide further increased ROS and enhanced the cytotox-icity of buparlisib as well as PIM447 (Supplementary Fig. S4C). Inparallel, decreases of cellularGSH andNAD(P)H levels induced byPIM447were potentiated by buparlisib treatment (Fig. 5F). Forceddepletion of all three isoforms of PIMkinases with siRNAs directedat PIM1, 2, and 3 resulted in reduced GSH, NADPH, as well asHMOX1 levels (Supplementary Fig. S4D–S4F), validating thatthese changes were secondary to PIM inhibition. The increasedtoxicity ofROS in these cellswas reflected in inductionof g-H2AX, aDNA damage marker (Supplementary Fig. S4G). Thus, the com-binationof PIMandPI3K/AKT inhibitor killed prostate cancer cellsthrough increasing ROS levels.

Although buparlisib treatment of LNCaP cells effectively inhib-ited mTORC1 activity as judged by phosphorylation of p70S6K,an mTORC1 substrate, PIM1 overexpression abrogated themTORC1-inhibitory activity of buparlisib (Fig. 5G). When PC-3or PC3-LN4 cells were treated with buparlisib and PIM447,phosphorylation levels of p70S6K, S6, and 4E-BP1 protein weremore strongly inhibited compared with buparlisib alone. Togeth-er, these data validate that both the AKT/PI3K pathway and PIMcontrol mTORC1 signaling (Fig. 5H). In LNCaP cells, AZD5363,buparlisib, or Torin1 (an mTOR kinase inhibitor) all blockedmTORC1 signaling and decreased HMOX1 levels, but in PIM1-overexpressing cells, only Torin1 suppressed mTORC1 andHMOX1 expression (Supplementary Fig. S4H). The NQO1-AREluciferase vector measures the stimulation of transcription byNRF2. When PC3-LN4 cells expressing NQO1-ARE luciferasewere treated with Torin1, luciferase activity was inhibited to asimilar extent as PIM447 plus buparlisib (Supplementary Fig.S4I). PIM kinase in part regulates mRNA translation by phos-phorylating eIF4B on S406 (8). Using CRISPR-Cas9 technology, aknock in of serine to alanine (eIF4B S406A) mutation wasproduced in human prostate cancer line PC3-LN4. Similar to

PIM inhibitor treatment, blocking this eIF4B S406 phosphoryla-tion via a genetic approach markedly inhibited NRF2 andHMOX1 protein expression (Fig. 5I). To examine whethereIF4B is needed for PIM-mediated induction of NRF2 signaling,PIM1-inducible PC-3 cells were transduced with lentivirus ofCRISPR-Cas9 eIF4B (sgRNA eIF4B_2) and control vector. Com-pared with the increased expression of NRF2 and HMOX1 uponDox-stimulated PIM1 induction in control vector–expressingcells, increases of HMOX1 and NRF2 were blunted in eIF4B-deficient cells even with Dox stimulation (Fig. 5J and K), indi-cating critical role of eIF4B in PIM1 regulation of NRF2 signaling.Mouse embryonic fibroblasts (MEF) obtained frommice that aregenetically deleted in all PIM kinases (triple knockout; TKO) donot phosphorylate eIF4B. These results suggest that inhibition ofmTORC1 by PI3K and PIM inhibitors functions to block NRF2activation, and thus increase ROS production. It is noteworthythat when PIM triple-knockout MEF cells were engineered toexpress either PIM1, PIM2, or PIM3 (23, 26), expression of eachPIM isoform was capable of increasing NRF2 expression (Sup-plementary Fig. S4J), indicating the overlapping effects of theseisoforms on NRF2 signaling.

Because mTOR inhibition activates overall protein degrada-tion by the ubiquitin proteasome system (32), we exploredwhether increased expression of PIM changes NRF2 levels bypartially blocking ubiquitination and subsequent proteasome-mediated protein degradation. To test this possibility, LNCaPcells were cotransfected with PIM1, hemagglutinin (HA)-taggedNRF2, and Flag-tagged ubiquitin plasmids. NRF2 protein wasimmunoprecipitated using anti-HA agarose beads, and ubiqui-tylated NRF2 was further detected by immunoblot analysiswith an anti-Flag antibody. In LNCaP cells cotransfected withPIM1, a decrease in the level of ubiquitylated NRF2 protein wasdetected (Fig. 6A), indicating that PIM1 prevents NRF2 ubiqui-tination. To further examine the role of PIM1, prostate cancercells were cotransfected with HA-NRF2 and Flag-Ubiquitinfollowed by the treatment with PIM447 in the presence ofMG132, a compound that blocks the degradation of ubiqui-tylated NRF2. PIM447 treatment induced accumulation ofubiquitinated NRF2 protein (Fig. 6B). Reduction of NRF2protein levels by PIM447 was blocked by the application ofMG132 (Fig. 6C). MG132 also blocked the decrease in NRF2induced by genetic deletion of PIM (Supplementary Fig. S4K).The half-life of NRF2 protein was decreased by treatment with

Figure 2.PIM1 upregulates the NRF2-HMOX1 antioxidant response to contribute to PI3K inhibitor resistance. A, Representative dose-response of mouse prostate epithelialcancer cells overexpressing PIM1 (mPrEC/PIM1) to buparlisib, PIM447 (3 mmol/L), or the combination (48-hour treatment). The data shown are the mean ofmeasurements � SD (n ¼ 4). IC50 of buparlisib (mmol/L) was 1.18 for DMSO and 0.35 for PIM447. B, Following 24-hour treatment with either buparlisib (varyingdoses), PIM447 (3 mmol/L), or the combination, mPrEC/PIM1 cells were lysed and immunoblotted with the indicated antibodies. C, Immunoblot analysis of AKT andPIM activation in prostate cancer patient-derived organoids. Total lysates of cell organoids from patients (identification number P6T2, P4T2, and P3T2) weresubjected to detection of p-AKT (S473), p-S6 (S240/244), and p-IRS1 (S1101). p-AKT/AKT ratio was calculated from densitometric analysis.D,Organoid cells (5,000cell density) derived from patient tissues (P6T2 and P4T2) were plated in matrigel in a 96-well plates and then treated with DMSO, 3 mmol/L PIM447, 1 mmol/Lbuparlisib, or the combination of PIM447 plus buparlisib. Organoid number was calculated on day 7 of respective drug treatment. The data shown are themean of measurements � SD (n ¼ 3). � , P < 0.05. E, Immunoblot analysis of the NRF2 and its target proteins in the Dox-inducible PIM1 LNCaP model. These cellsexpress shRNA scrambled control (shC) or shNRF2 are treatedwith 20 ng/mLDox (þDox) or PBS (-).F, Immunoblot analysis of the indicated proteins in stable PIM1-overexpressing LNCaP and control vector (EV)–expressing cells exposed to 1 mmol/L buparlisib for 6 hours. G, Representative buparlisib dose-responsecurves in NRF2- or HMOX1-overexpressing LNCaP cells. The data shown are the mean of measurements � SD (n ¼ 4). Immunoblots confirming NRF2 and HMOX1expression compared with pcDNA3 control vector are shown. H, Representative buparlisib dose-responses of LNCaP/PIM1 cells expressing shNRF2, as comparedwith the control shC. The data shown are the mean of measurements � SD (n ¼ 4). I, Immunoblots of HMOX1-depleted LNCaP/PIM1 cells exposed to1mmol/L buparlisib. Cell deathwasmeasuredby trypanblue uptake after treatmentwithDMSOorbuparlisib. The arrow indicates the cleavageproduct of PARP-1. Theaverage number of dead cells � SD is shown (n ¼ 3). J, Immunoblots of HMOX1-depleted PC3-LN4 cells exposed to 1 mmol/L buparlisib. The cleavagefragments of caspase-3 and PARP-1 are identified by arrows. Cell death is measured by trypan blue uptake in the cells treated with buparlisib or DMSO. Theaverage number of dead cells � SD is shown (n ¼ 3).






Figure 3.

Inhibition of GSH synthesis abrogates resistance to PI3K inhibitor. A, The cellular levels of GSH and NADPH after treatment with buparlisib. EV- and PIM1-expressingLNCaP cells were exposed to 1 mmol/L buparlisib for 48 hours. The data shown are the mean of measurements � SD (n ¼ 4). � , P < 0.05; n.s., not significant.B, Impact of BSO pretreatment on the sensitivity of LNCaP/PIM1 versus LNCaP/EV cells to 1 mmol/L buparlisib (72 hours). The data shown are the mean ofmeasurements� SD (n¼ 4). C, Tumor volume of implanted LNCaP/PIM1 cells treated with the indicated agents for varying periods of time. The average volume�SEM (n ¼ 6), and statistical comparison with the vehicle-treated control is shown (�, P < 0.05). The tumors shown were excised from the mice at the end oftreatment.D, Fluorescence imaging of ROS in LNCaP/EV cells treatedwith buparlisib for 16 hours in the presence of PBS, Trolox (1 mmol/L), or NAC (3mmol/L; scalebar, 20mm).QuantitationofDCFgreen intensity following treatmentwith 1mmol/L buparlisib. The averageof these values� the SD is shown.E,Fluorescence imagingofROS in LNCaP/PIM1 cells treatedwith 1mmol/Lbuparlisib for 16 hours in thepresenceor absence ofBSO (scale bar, 20mm). The averagevalueofROSdeterminedbyDCF green florescence intensity and SD of these measurements is shown.

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PIM447 compared with untreated PC3-LN4 control cells from32.8 to 21.9 minutes (Fig. 6D). This demonstrates the potentialability of PIM kinases to maintain NRF2 protein stability and itsfunction by controlling both the translation and degradationpathways that regulate the levels of NRF2. Therefore, PIM andPI3K-AKT inhibitors synergize to block NRF2 activity, augment-ing oxidative stress to suppress tumor growth (Fig. 6E).

DiscussionThe PIM protein kinases have been shown to confer resistance

to PI3K and AKT inhibitors in part by activating PI3K downstreameffectors in an AKT-independent manner (1, 14, 33). PIM inhi-bition enhances sensitivity to AKT inhibitors, and cancers withmutation in PIK3CA cells often develop increased levels of thePIM kinases (1). Treatment of tumors with agents that inhibit cellsurface tyrosine kinases, e.g., MET inhibitors, blocks AKT activa-tion, elevates PIM levels, and ultimately, induces drug resistance(34). Experiments reported here demonstrate the mechanism bywhich PIM functions to induce resistance to these agents, namely,(1) by increasing NRF2 levels and consequently augmenting thelevel of ROS scavengers and (2) by stimulating cellular metabo-lism to drive reduced GSH levels.

The NRF2 transcription factor is an important regulator ofcancer survival and proliferation. Redox homeostasis is essential

to maintain cellular equilibrium and the survival of cancer cellsthat have abnormal metabolism (12). Approximately 70% oftumors in biopsies from patients with metastatic prostate cancerharbor aberrant PI3K pathway signaling. This leads to AKT acti-vation, and in this tumor type, HMOX1 plays a pivotal role inantioxidant defense and the regulation of resistance to PI3Kinhibition. In prostate cancer cell lines harboring PTEN deletionor mutation, PI3K inhibitor treatment increases ROS levels whiledecreasing scavenger enzymes. Here, the importance of ROSgeneration as a means by which these compounds kill prostatecancerwas demonstrated by theobservation that the expressionofHMOX1blocked the cytotoxicity of the pan-PI3K inhibitor bupar-lisib, whereas the depletion of ROS scavengers enhanced theactivity of this compound. Importantly, even in buparlisib-treatedtumor cells, expression of PIM1 elevated NRF2 levels andincreased ROS scavengers, e.g., HMOX1, preventing PI3K inhib-itor killing of prostate cancer. The combination of a PIM and anAKT inhibitor markedly decreased NRF2 and increased ROSproduction. Importantly, further increasing ROS generation withtert-Butyl Hydroperoxide stimulated the death of these prostatecancer cells.

Elevated GSH biosynthesis is known to be required for PI3K/AKT-driven resistance to oxidative stress, initiation of tumorspheroids, and anchorage-independent growth (35). The addi-tion of buparlisib to prostate cancer treatment loweredGSH levels

Figure 4.

PIM1 overexpression alters metabolicactivity. A and B, Flux analysis ofLNCaP/PIM1 versus LNCaP/EV cellstreated with 13C-glucose at the timesindicated. The bars demonstrate therelative average abundance� the SD ofdiffering mass species. C, Differences ofOCR and ECAR of LNCaP/EV andLNCaP/PIM1 cells treated withbuparlisib (1 mmol/L) or vehicle (DMSO).The average � SD is shown (n ¼ 5).Statistical comparison with EV controlwas determined by t test (� ,P<0.05).D,LDH levels in LNCaP/PIM1 or LNCaP/EVtreatedwith varying doses of buparlisib.E, Representative OCR data afterbuparlisib treatment. LNCaP/PIM1 orLNCaP/EV cells were exposed to 1mmol/L buparlisib or DMSO for 16 hoursand then subjected to Seahorseanalysis. Oligomycin (2.5 mmol/L), FCCP(2.5 mmol/L), antimycin A (1 mmol/L),and rotenone (1 mmol/L) weresequentially added.






along with lowering NAD(P)H, a molecule essential for keepingGSH reduced and inducing oxidative stress. In contrast to bupar-lisib, PIM1 overexpression enhanced GSH synthesis, abrogatingthe inhibitory effects of buparlisib. Recent studies have shown

that NRF2 regulates genes that are involved in anabolic metab-olism. Shown here, in prostate cancer cells, PIM1 facilitatedglucose flux into substrates for GSH production and the recyclingprocess. Genes involved in GSH synthesis were not induced by

Figure 5.

NRF2 activity is inhibited by both PIM1and PI3K/AKT inhibitors via inhibitionof the mTORC1 pathway. A, PTEN wasinduced in PC-3 cells by varying dosesof Dox, and immunoblottingperformed for varying proteins usingactin as a control. B, Representativeimages of crystal violet–stained PTEN-inducible cells overexpressing PIM1treated with or without Dox and eitherDMSO or 3 mmol/L PIM447 for 7 days.C and D, Following treatment ofPC3-LN4 cells with buparlisib (1 mmol/L) with or without PIM447 (3 mmol/L),ROS was imaged by quantitation ofDCF green intensity (C). The averageDCF values � SD are shown. PC3-LN4cells treated with these inhibitorsindividually or in combination werelabeled with DCF and Hoechst 33342to measure ROS and document nuclei(Nuc; D). E, Immunoblot analysis ofNRF2 expression. LNCaP andPC3-LN4cells were treated with buparlisib(1 mmol/L), PIM447 (3 mmol/L), or thecombination for 24 hours. F, Followingtreatments in E, in PC3-LN4 cells, GSHand NADPH levels were measuredusing the procedures outlined inMaterials and Methods. G,Immunoblots of LNCaP/EV andLNCaP/PIM1 cells following 24-hourtreatment with buparlisib (1 mmol/L)or DMSO. H, PC3-LN4 cells weretreatedwith varying does of buparlisibwith or without PIM447 for 24 hours,followed by immunoblotting with theantibodies specified. I, Immunoblotsof wild-type or eIF4B S406A-mutantPC3-LN4 cells treated with PIM447(1 mmol/L, 72 hours). PC3-LN4 eIF4BS406A knock-in cells were generatedusing CRISPR-Cas9 gene editing.J, Immunoblots of eIF4B, NRF2, andHMOX1 expression in LNCaP cells afterlentiviral transduction of eIF4BCRISPR-Cas9 carrying three differentsgRNA target sequences. Vectorcontrol was the scrambled sgRNACRISPR lentivector.K, Immunoblots ofeIF4B, NRF2, andHMOX1 expression inPIM1-inducible PC-3 (TRIPZ-PIM1) cellstransduced with eIF4B CRISPR-Cas9(carrying sgRNA eIF4B_2) orscrambled sgRNA CRISPR lentivector(Vector). After 72-hour transduction,Dox at the indicated dose was addedfor 48 hours to induce PIM1overexpression.

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Figure 6.

NRF2 ubiquitination and protein stability is regulated by PIM1 kinase.A, Immunoblot analysis of ubiquitinated NRF2 protein. LNCaP cells were transfected with Flag-Ub, HA-NRF2, andPIM1 as indicated. After 48hours, these transfectantswere exposed to 10mmol/LMG132 for 4 hours. NRF2was immunoprecipitatedwith an anti-HAantibody and was subjected to immunoblot analysis with an anti-Flag antibody used for detection of ubiquitylated NRF2. An aliquot of the total lysate wasevaluated for immunoblot analysis. B, Immunoblot analysis of endogenous NRF2 ubiquitination. Following exposure to MG132 total extracts from LNCaP, DU145,PC-3, and PC3-LN4 cells were immunoprecipitated with an anti-NRF2 antibody. Immunoprecipitated NRF2 was subjected to immunoblot analysis withan anti-ubiquitin (anti-Ub) antibody for detection of endogenous ubiquitylated NRF2. An aliquot of the total cell lysate was used for immunoblot analysis. C,Immunoblot analysis of NRF2 accumulation after MG132 treatment. PC3-LN4 cells were treated with 3 mmol/L PIM447 for 24 hours in the presence or absence of 10mmol/LMG132.D,Thehalf-life ofNRF2 inPC3-LN4cells following treatmentwithDMSOor 3mmol/L PIM447. After 24-hour treatment, cellswere exposed to 10mg/mLcycloheximide for the indicated times. Total lysates were subjected to immunoblot analysis. NRF2 and actin levels were determined by densitometry, and thelevel of NRF2 relative to that of actin was plotted as a function of time to determine the decrease in NRF2 protein. E, Schematic outline of the resistancemechanism induced by PIM kinase. Expression of the PIM protein kinase induces tumor resistance to PI3K inhibitors. PIM promotes NRF2 synthesis throughphosphorylation of eIF4B (S406) and increases NRF2 activity by inhibiting ubiquitination-mediated proteasome degradation. PIMmodulates glycolytic metabolismto favor a decrease in ROS accumulation by producing NADPH and GSH. Genetic depletion of ROS scavenger proteins overcomes PIM-mediated drug resistance.Combining PIM and PI3K inhibitors promotes cancer death by inhibition of NRF2 signaling.






PIM1whenNRF2was depleted, suggesting that PIM regulation ofNRF2 is coordinating the high GSH content in PIM1-overexpres-sing tumor cells. Synthetic lethal interaction between NRF2 lossand mutant KRAS was detected in pancreatic ductal adenocarci-nomas (13). Cells that lack PIM kinase or are treated with a PIMinhibitor were not tolerant of mutant KRAS expression due tolethal ROS accumulation (26), again suggesting that PIM kinasesare important regulators of cellular redox signaling. In agreementwith other reports (13, 35), AKT inhibitor efficacy was markedlyenhanced by cotreatment with the GSH synthesis inhibitor BSO.Here, it is shown that tumor suppression is enhanced by com-bining BSO and buparlisib. BSO resensitized PIM1-overexpres-sing tumors to buparlisib both in vitro and in vivo. This suggeststhat the maintenance of GSH is important to the mechanism bywhich PIM protects cells from death. Clearly, the regulation ofredox homeostasis by PIM1 overlaps with the action of AKT andcontrols cellular homeostasis.

PIM1 regulates translation by phosphorylating eIF4B, a pro-tein associated with mRNA unwinding, on S406 (8). Shownhere, mutation of the PIM phosphorylation site in eIF4B(S406A) markedly decreases NRF2 protein levels, suggestinga mechanism by which PIM controls NRF2 translation. NRF2exists in complex with KEAP1 via direct protein–protein inter-actions between the KEAP1 Kelch domain and the ETGE andDLG motifs on the Neh2 domain of NRF2 (36). We furthershow that PIM kinases appear to partially block the ubiquitina-tion of NRF2, enhancing its half-life, accounting in part for theincreased levels of NRF2 when PIM is expressed. However, themechanism by which PIM regulates NRF2 degradation is notclear. Although there is variability in the level of KEAP1 inprostate cancer cell lines, experiments did not find that PIM1regulated the levels of NRF2 by modifying either the interactionof NRF2 with KEAP1 or the degradation of KEAP1. NRF2degradation could be regulated in these cells by phosphoryla-tion on the Neh6 domain of NRF2. Phosphorylation on thissite by GSK3 can be recognized by b-TrCP, which acts as areceptor for the SKP1-CUL1-RBX1/ROC1 ubiquitin ligase com-plex (37) and leads to the degradation of NRF2 in a KEAP1-independent fashion. In human lung cancer A549 cells, inhi-bition of the PI3K/AKT pathway markedly reduced endogenousNRF2 protein through this mechanism (38).

The mechanism by which PIM regulates NRF2 can havebroader impact for molecularly targeted therapy in variedtumor types. Targeting the PI3K signaling pathway has resultedin the development of novel PI3K inhibitors including bupar-lisib, pictilisib, ZSTK474, dactolisib, apitolisib, and omipali-sib. A PI3K-a–selective inhibitor alpelisib is currently in clin-ical development for breast cancer therapy in a phase IIIclinical trial (NCT02437318) and phase II study for treatment

of head and neck squamous cell carcinoma (NCT02051751).In 2014, the p110d-specific inhibitor idelalisib was approvedin the United States and Europe as the first-in-class PI3Kinhibitor for use in the treatment of chronic lymphocyticleukemia and follicular lymphoma. As well, copanlisib withactivity against the PI3K-a and -d isoforms was recentlyapproved by the FDA for treatment of patients with relapsedfollicular lymphoma. However, in prostate cancer althoughmutations activate the PI3K pathway, inhibitors targeting thispathway have not shown activity. Our findings in prostatecancer models that combining PI3K inhibitors with a PIMinhibitor enhances the induction of cell death by markedlyincreasing ROS generation may provide molecular basis toovercome the limited efficacy of PI3K-targeted therapies. Thetherapeutic vulnerabilities caused by redox regulation in can-cer suggest the use of PIM inhibitors as an additional treatmentmodality.

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

Authors' ContributionsConception and design: J.H. Song, N.A. Warfel, A.S. KraftDevelopment of methodology: J.H. Song, L.A. Luevano, K. Okumura, V. Olive,S.M. BlackAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.H. Song, N. Singh, L.A. Luevano, S.M. Black,D.W. GoodrichAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.H. Song, N. Singh, L.A. Luevano, A.S. KraftWriting, review, and/or revision of the manuscript: J.H. Song, N. Singh,S.K.R. Padi, K. Okumura, V. Olive, N.A. Warfel, D.W. GoodrichAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.H. Song, N. Singh, L.A. Luevano, S.K.R. Padi,A.S. KraftStudy supervision: J.H. Song, A.S. KraftOther (development of key experimental models): D.W. Goodrich

AcknowledgmentsThis research was supported by University of Arizona Cancer Center support

grant NIH P30CA023074, NIH award R01CA173200, DOD award W81XWH-12-1-0560 (to A.S. Kraft), and American Lung Association Award LCD-504131(to N.A. Warfel). The authors thank Novartis Oncology for providing PIM447.TheUACCShared Resources provided support for histologic and tissue staining,microarray, and flow cytometry analysis.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 10, 2018; revised May 27, 2018; accepted August 30, 2018;published first September 6, 2018.

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2018;17:2710-2721. Published OnlineFirst September 6, 2018.Mol Cancer Ther Jin H. Song, Neha Singh, Libia A. Luevano, et al. Induced by the PIM KinaseMechanisms Behind Resistance to PI3K Inhibitor Treatment

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