lack of constitutively active dna repair sensitizes ... · kamalakannan palanichamy1, disha patel1,...

Lack of Constitutively Active DNA RepairSensitizes Glioblastomas to Akt Inhibition andInduces Synthetic Lethality with RadiationTreatment in a p53-Dependent MannerKamalakannan Palanichamy1, Disha Patel1, John R. Jacob1, Kevin T. Litzenberg1,Nicolaus Gordon1, Kirstin Acus1, Shin-ei Noda2, and Arnab Chakravarti1

Abstract

Treatment refractory glioblastoma (GBM) remains a majorclinical problem globally, and targeted therapies in GBM havenot been promising to date. The Cancer Genome Atlas integrativeanalysis ofGBMreported the strikingfinding of genetic alterationsin the p53 and PI3K pathways in more than 80% of GBMs. Giventhe role of these pathways in making cell-fate decisions andresponding to genotoxic stress, we investigated the reliance ofthese two pathways in mediating radiation resistance. We select-ed a panel of GBM cell lines and glioma stem cells (GSC) withwild-type TP53 (p53-wt) and mutant TP53,mutations known tointerfere with p53 functionality (p53-mt). Cell lines were treatedwith a brain permeable inhibitor of P-Akt (ser473), phosphati-dylinositol ether lipid analogue (PIA), with andwithout radiationtreatment. Sensitivity to treatment was measured using Annexin-V/PI flow cytometry and Western blot analysis for the markers of

apoptotic signaling, alkaline COMET assay. All results were ver-ified in p53 isogenic cell lines. p53-mt cell lines were selectivelyradiosensitized by PIA. This radiosensitization effect corre-sponded with an increase in DNA damage and a decrease inDNA-PKcs levels. TP53 silencing in p53-wt cells showed a similarresponse as the p53-mt cells. In addition, the radiosensitizationeffects of Akt inhibition were not observed in normal humanastrocytes, suggesting that this treatment strategy could havelimited off-target effects. We demonstrate that the inhibition ofthe PI3K/Akt pathway by PIA radiosensitizes p53-mt cells byantagonizing DNA repair. In principle, this strategy could providea large therapeutic window for the treatment of TP53-mutanttumors. Mol Cancer Ther; 17(2); 336–46. �2017 AACR.

See all articles in this MCT Focus section, "DevelopmentalTherapeutics in Radiation Oncology."

IntroductionGlioblastomas (GBM) are among the most treatment-resis-

tant solid tumors, often recurring after resection, radio-, andchemotherapy treatment. There has been a considerable effortto identify therapeutics that radiosensitize GBMs because mostpatients will receive radiotherapy. However, identifying suchradiosensitive chemotherapeutic agents has been difficult dueto the complex molecular heterogeneity of GBMs that promotesredundant pro-growth and pro-survival pathways. To overcomethis obstacle, there is a need to devise therapeutic strategiestargeting these redundant treatment-resistant pathways thatpromote the intrinsic radioresistance of GBMs.

The Cancer Genome Atlas (TCGA) reported that the PI3K andp53 pathways are each altered in over 80% of GBMs (1). Altera-tions in the PI3K pathway result in the constitutive activation ofthe signaling node Akt. High levels of Akt activation frequentlyresults from the downregulation of the tumor-suppressor PTENphosphatase or increased activity of upstream receptor tyrosinekinases (RTK; refs. 2–4). In GBMs, the most frequently upregu-lated RTKs are the EGFR, insulin-like growth factor 1 receptor beta(IGF1Rb), and the platelet-derived growth factor receptor beta(PDGFRb). The increased activity of Akt had been correlated withpro-growth and pro-survival factor. Conversely, p53 activity issuppressed inmost GBMs. Decreased p53 activity predominantlystems from inactivating mutations or increased activity of the E3ubiquitin-protein ligase (MDM2), a negative regulator of p53.Suppression of p53 activity alters cell fate decisions, DNA damagerepair, apoptosis, and genetic stability. Given the high dysregula-tion of the PI3K and p53 pathways in GBMs and their role inresponding to cellular stress, we set out to determine the inter-dependence of these two pathways in responding to radiationtreatment.

In this study, we assess the radiosensitizing effect of inhibit-ing Akt activity in a panel of GBM cell lines with wild-type ormutant TP53. To inhibit Akt, we used the inhibitor phospha-tidylinositol ether lipid analogue (PIA), which binds to thesame region on the N-terminal Pleckstrin (PH) domain of Aktas PIP3 and blocks phosphorylation at serine 473 (5). Inhibi-tors competing for ATP-binding sites are common to many

1Department of Radiation Oncology, The Ohio State University College ofMedicine and Comprehensive Cancer Center, Columbus, Ohio. 2Department ofRadiation Oncology, Saitama Medical University International Medical Center,Saitama, Japan.

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

Corresponding Author: Kamalakannan Palanichamy, The Ohio State UniversityCollege of Medicine and Comprehensive Cancer Center, 410 West 12th Avenue,Room385E,WisemanHall, ColumbusOH43210. Phone: 614-685-4245; Fax: 614-292-5435; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0429

�2017 American Association for Cancer Research.

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kinases, therefore, choosing to use PIA allows for greaterspecificity when targeting Akt (6, 7). We validate the resultsfrom our panel of GBM cell lines in p53 isogenic cell lines.Moreover, we investigate the role of DNA damage repair in theobserved radiosensitizing effect of Akt inhibition in cell lineswith wild-type or mutant TP53. Overall, we demonstrate thatAkt inhibition by PIA before radiation treatment radiosensitizesGBM cells with mutant TP53.

Materials and MethodsStudy approval

This study was conducted in accordance with The Ohio StateUniversity Intuitional Review Boards for IRB (2009C0065 and2014C0115), IBC (2009R0169), and IACUC (2009A0127).

Cell cultureCommercially available cell lines U87, LN229, and LN18 were

purchased from the ATCC during 2015. Patient-derived cell linesOSU61, MGH8, VC3, and the GSC cell lines (OSU2GSC,OSU11GSC, and OSU20GSC) were authenticated by neuro-pathologists. The OSU cell lines were propagated during 2013to 2015,MGH8andVC3during 2007 to2008.Non-GSC cell lineswere maintained in Dulbecco's Modified Eagle's medium (Invi-trogen) supplemented with 10% FBS (Invitrogen) and 1% anti-biotic–antimycotic (Invitrogen). GSCs were maintained inDMEM-F12 (Invitrogen) supplemented with B-27 (Gibco), EGF(20 ng/mL; Thermo Fisher), bFGF (20 ng/mL) (Thermo Fisher),and 1% antibiotic–antimycotic (Invitrogen). Normal humanastrocytes (NHA)were obtained fromLonza andwere propagatedas per the manufacturer's protocol. All cells were cultured at 37�Cunder a gas phase of 95% air and 5% CO2 and were free fromMycoplasma contamination tested using a colorimetry-based assay(R&D systems) during the study period. All studies were con-ducted within 5 passages and were authenticated using STRprofiles obtained from genetics core at University of Arizona.

InhibitorsAkt Inhibitor II (PIA) was purchased from Calbiochem. MK-

2206 and PIK-75 were purchased from Selleckchem.

Lentiviral transductionU87 cells were plated at a density of 2.5� 105 cells per well in a

6-well plate. Media containing the lentivirus constructs for theNon-target or TP53 MISSION shRNA Lentiviral TransductionParticles (Sigma-Aldrich) were added at an MOI of 10. After 48hours, the spentmedia were replacedwith freshmedia containing10 mg/mL of puromycin (Sigma-Aldrich). Ten puromycin-resis-tant colonies were picked and each clone was expanded to assessthe extent of p53 knockdown using RT-QPCR at transcriptionallevel and Western blot at translational level.

Western analysisCells were harvested using RIPA buffer (Sigma) with 1% (v/v)

protease inhibitor cocktail (Sigma) and 1% (v/v) phosphataseinhibitor cocktail (Sigma). Samples were run on 10% SDS-PAGEgels and transferred to polyvinylidene difluoridemembranes. Themembranes were then incubated overnight with the followingprimary antibodies: EGFR, PDGFRb, IGF1R, phospho-MDM2,phospho-p44/42 (P-ERK), p44/42 (ERK), PI3K-p85, MGMT,phospho-PTEN, PTEN, phospho-BAD, BAD, XIAP, phospho-

mTOR, mTOR, ATM, phospho-Akt (Ser473), Akt, PARP, cleavedPARP, caspase-3, cleaved caspase-3, p38, phospho-p38, DNA-PKcs, Ku80, phospho-GSK3b, GSK3b, and g-H2AX (Cell Signal-ing Technology). b-actin was purchased from Sigma-Aldrich.

Reverse-transcriptase quantitative polymerase chain reactionTotal RNA from clonal cells was isolated using the RNaseEasy

spin columns (Qiagen) per the manufacturer's protocol. Forreverse-transcriptase reactions, first-strand cDNA was synthesizedusing Superscript reverse transcriptase (Invitrogen) per the man-ufacturer's protocol. TaqMan probes (Applied Biosystems) wereused to estimate the gene expression level of TP53 (Hs00153349_m1). GAPDH (Hs99999 905_m1) and RNA18S1 (Hs03928990_g1) were used as housekeeping genes.

Clonogenic survival assayCells were plated at 8,000, 4,000, 2,000, 1,000, 500, and 50

cells per well in petri dishes and radiated at 2 Gy fractions totaling10, 8, 6, 4, 2, and 0 Gy, respectively. The plates were incubated for10 to 14 days depending on colony size and cell type. Colonieswere stained with 0.1% methylene blue, dried, and counted.Colonies �50 cells were considered significant. The plating effi-ciency (PE) was calculated from the ratio of colonies formed overthe number of cells plated (Fig. 2B). Radiobiological effect wasquantified by computing radiation enhancement ratio (RER) at 2,4, and 6Gy using survival fraction (SF) of RT alone over SF ofPIAþRT (Fig. 2B) RER > 1 is indicative of radiosensitization.

Apoptosis assayTo determine the number of apoptotic and necrotic cells,

Annexin V/PI assays were performed using an apoptosis detectionkit (Life Technologies). Briefly, cells were plated onto 6-well platesat a density of 2�105 cells perwell and treatedwithPIA twohoursbefore radiation treatment. After incubation, the cells were har-vested andwashed in cold PBS. For every 100mLof sample, 5mL ofFITCAnnexinV and1mLof 100mg/mLPIwere added and sampleswere incubated for 15 min at room temperature in the dark. Thecells were then analyzed using flow cytometry and FlowJosoftware.

Alkaline comet assaysAlkaline comet assays were performed per manufacturer's

instruction (Trevigen). Cells were trypsinized and suspended incold PBS at a concentration of 1.0 � 105 cells/mL. The cells werethen mixed with lowmelting agarose (Trevigen) at a ratio of 1:10(v/v) and immediately plated onto Cometslide (Trevigen). Alka-line electrophoresis was run at 21 V for 30 minutes in theCometAssay Electrophoresis System (Trevigen). Data were col-lected using a fluorescence microscope (Zeiss).

In vivo studiesThe glioblastoma xenograft model used has been outlined

previously and the sample size for the study was determinedon the basis of the power calculation from our previousexperiments (8).

Statistical analysisAll results were confirmed in at least three independent experi-

ments, and data from one representative experiment were shown.All quantitative data are presented as mean � SD. The statistical

TP53 Mutation and Radiosensitization

www.aacrjournals.org Mol Cancer Ther; 17(2) February 2018 337

analysis was performed using SAS 9.2 (SAS Institute) orGraphPadPrism 5. Student t tests were used for comparisons of means ofquantitative data between groups. P values <0.05were consideredsignificant.

ResultsBasal expression of PI3K pathway proteins and MGMT inGBM cell lines

We selected a panel of commercially available GBM cells,patient-derived GBM cells, and glioma stem cell (GSC), con-taining five cell lines with mutant TP53 (p53-mt) and four celllines with wild-type TP53 (p53-wt; Fig. 1A). MGH8 cells have awild-type p53 sequence, whereas U87, VC3, OSU2GSC, andOSU11GSC cells have a mutation in the proline-rich domainleading to the amino acid change at residue 72 from prolineto arginine. This P72R is a documented polymorphism andknown to exhibit wild-type p53 function (9). LN229 have amutation in the proline-rich domain P98L, this mutation hasbeen reported to have a partial wild-type functionality (10).LN18, OSU61, and OSU20GSC have mutations in the DNA-binding domain of p53. Because PTEN is also an importantregulator of Akt activity and apoptosis, we included the muta-tional status of PTEN to ensure observations based on the statusof p53 were independent of PTEN (Fig. 1A). We began bydetermining the basal expression levels of the key proteins andregulators of the PI3K pathway (Fig. 1B–D). Akt was phosphor-ylated at serine 473 (P-Akt) in all cell lines, indicating activation

of the PI3K pathway. U87, OSU61, and OSU20GSC showedrelatively higher levels of P-Akt, a likely result of low PTENexpression. Conversely, the high levels of P-PTEN and lowlevels of PI3K in OSU2GSC and MGH8 cells likely account forthe relatively low levels of P-Akt in these cell lines. All cell linesexpressed the RTKs EGFR, PDGFRb, and IGFR1b as well as thep53 inhibitor P-MDM2 (Ser166). In contrast to the GBM celllines, normal human astrocytes (NHA) did not express any ofthe RTKs, Akt, or P-MDM2. The selected cell lines recapitulatethe major genetic alterations of the PI3K and p53 pathways inGBM, and therefore were used to study the interdependent roleof these two pathways in conferring radiation sensitivity. Next,we determined the basal levels of O6-methylguanine-DNAmethyltransferase (MGMT), a predictive marker for responseto temozolomide þ radiotherapy (standard-of-care) in GBMs.The higher expression of MGMT in LN18, and OSU61 indicatedthat the MGMT promoter was likely unmethylated in these celllines (Fig. 1C). Importantly, the expression of MGMT did notcorrelate with the status of p53, meaning observations relatingto p53 will be independent of MGMT.

Mutational status of p53 determines the radiosensitizingeffect of PIA treatment

The activation of Akt one hour after radiation treatment wasobserved in all cell lines (Fig. 1E), independent of p53 status,supporting the role of Akt in mediating radioresistance. Next,we characterized the sensitivity of U87 and LN18 cells toPIA treatment alone. PIA treatment-induced apoptosis in a

Figure 1.

Mutational status of TP53 and PTEN, basal expression levels of proteins implicated in treatment resistance, Akt activation after RT and growth inhibitoryeffect of PIAþRT in LN18 and U87 cells. Sensitivity of GBM cell lines to radiation and PIA treatment alone. A, Mutational status of TP53 and PTEN.B–D, Western blotting was used to assess the key members of the PI3K–Akt signaling pathway and MGMT. E, The activation of Akt following radiationtreatment. F, The MTS cell proliferation assay was used to assess growth inhibition of the U87 and LN229 cell lines 24 hours after the indicated treatments(Two-tailed Student t test: � , P < 0.05). WT: Wild-type; (WT): Functionally wild-type; (PWT): Partially functional wild-type.

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time- and concentration-dependent manner (Fig. 1F; Supple-mentary Fig. S1). In addition, cells treated with greater than5 mmol/L PIA showed a decreased colony formation ability(Supplementary Fig. S1). Interestingly, the colony formationwas completely inhibited in LN18 after radiation treatment,whereas LN229 cells had colonies following radiation treat-ment, which may be explained on the basis of the partial wild-type functionality of p53 LN229. These results agree with aprevious report that demonstrates PIA-induces a moderate levelof apoptosis in cancer cell lines (11).

To determine whether PIA would radiosensitize GBM cells, weadministered PIA 2 hours before radiation treatment. Cell lineswith p53-mut (LN18, LN229, OSU61, and OSU20GSC) wereradiosensitized by PIA, showing increased apoptosis anddecreased colony forming ability (Figs. 2A and B, 3A; Supple-mentary Fig. S2). Clonogenic survival assay on the OSU61 cellsand GSCs were not included because this cell line does notform colonies in a manner compatible with this assay. The celllines with p53-wt (U87, MGH8, VC3, and OSU2GSC) wereradioresistant to the additionof PIA, showingdecreased apoptosisand increased colony forming ability (Figs. 2A and B, 3A; Sup-plementary Fig. S3). The plating efficiency (PE) and radiationenhancement ratio (RER) calculated are provided as tablesaccompanying Fig. 2B. As radiation dose increases RER increasesin TP53-mt radiosensitive subset and decreases in the TP53-wtsubset. Even at higher concentrations of PIA (50 mmol/L)U87 and LN18 cells followed the same radioresistant and

radiosensitization trends, respectively (Supplementary Fig. S4).The radiosensitivity of these cell lines following Akt inhibitionwas independent of PTEN mutational status and MGMT expres-sion. Representativemicroscopic images of LN18 (radiosensitive)andMGH8 (radioresistant) provide a snapshot of this differentialradiosensitization (Fig. 2C). LN18 cells weremore sensitive to PIAand radiation treatment, as indicated by the appearance of round,floating dead cells. Therefore, on the basis of these findings, weconcluded that the radiosensitization of PIA might be dependenton the mutational status of p53.

Allosteric Akt inhibitor MK2206 radiosensitizes GBM cellsindependent of p53 status

To determine whether the radiosensitization effect of PIA waspathway or inhibitor specific, experiments were conductedusing the Akt inhibitor MK-2206. MK-2206 binds and inhibitsthe phosphorylation of Akt at threonine 308 and serine 473 in anon-ATP competitive manner (12). Administration of MK-2206 and radiation did not show any selective radiosensitizingeffects as seen with PIA (Supplementary Fig. S5). This differ-ential effect between PIA and MK2206 could be due to theinhibition of Ser473 by PIA and both Ser473 and Thr308 byMK2206. Reports have shown the substrate specificity of DNA-PKc and Akt (Ser473) phosphorylation that further supportsthis conclusion (13). Further studies are required to confirmthis DNA-PK–specific activity of Akt phosphorylation at Ser473and Thr308 residues.

Figure 2.

Apoptosis and clonogenic assay and microscopic images of GBM cells after treatment with PIAþRT. PIA and radiation treatment synergisticallyradiosensitize a subset of GBM cell lines. A, Annexin-V/PI apoptosis assay was used to determine the relative cell death of GBM cell lines and NHAs 24 hoursafter radiation treatment, PIA, and PIA þ radiation treatment (Two-tailed Student t test: �, P < 0.05). B, Clonogenic survival assay following radiationtreatment and PIA þ radiation treatment. The plot with the LN18 and LN229 cell lines represents the radiosensitive subset of cell lines, whereas the plotcontaining MGH8 and VC3 represents the radioresistant subset of cell lines. PE refers to plating efficiency, and RER refers to radiation enhancement ratio.C, Microscopic images of LN18 and VC3, 24 hours after radiation treatment and 5 mmol/L PIA.



PIA and radiation treatment induce increased apoptosis inp53-mt cell lines

Western blots were run to determine the molecular targets bywhich PIA � radiation treatments induce apoptosis. FollowingPIA and radiation treatments, the radiosensitive subset of cell lineshad relatively higher levels of the apoptotic markers cleavedcaspase-3 and cleaved PARP compared with the radioresistantcell lines (Fig. 3B). In addition, the radiosensitive cell linesshowed relatively lower levels of the antiapoptotic proteins X-linked inhibitor of apoptosis (XIAP), survivin, and phosphory-lated Bcl-2-associated death promoter (BAD; Fig. 3B). Thesealterations in apoptotic and antiapoptotic markers support theresults of the Annexin-V/PI assays (Fig. 2A; Supplementary Fig. S2and S3).

Furthermore, PIA� radiation treatment reduced the activationof mTOR and Erk in all cell lines, corresponding with decreasedcell proliferation (Supplementary Fig. S6). In contrast with aprevious report, we did not observe the off-target activation ofp38 after PIA treatment (7). However, we did observe an increasein p38 activity following PIA and radiation treatment in some celllines (Supplementary Fig. S6). We do not expect that the activa-tion of p38 is responsible for the observed radiosensitizationeffects as it is increased in both radiosensitive and radioresistantcell lines. In addition, the same report demonstrated that theinduction of apoptosis after PIA treatment was independent ofp38 activation. Since p73, like p53, is a tumor suppressor thatinduces cell-cycle arrest and participates in PTEN-induced apo-ptosis (14), we extended our investigation to determine the

reliance of p73 on PIA-induced radiosensitization. Most the celllines used in this study did not express p73. This is unsurprisinggiven the p73 gene is in the commonly deleted chromosomalregion 1p36.2-3. Furthermore, there was no change in PTENexpression following radiation or PIA� radiation treatment (Fig.3B). Therefore, we concluded that the alterations in apoptosisobserved between the radiosensitive and radioresistanceGBMcelllines were a result of Akt inhibition and the mutational status ofp53 in these cell lines.

Validation of PIA-mediated radiosensitization by geneticapproach

Five shRNA constructs were used to silence p53 expression inp53-intact U87 cells (Fig. 4A). U87 cells transduced with the p53shRNA construct 1673 (U87-p53KD) did not have enhanced celldeath following radiation treatment alone compared to the U87non-target control cells (U87-NT; Fig. 4B). However, in the PIA�radiation treatment arm, U87-p53KD cells experienced increasedcell death (Fig. 4B). In addition, the U87-p53KD cells showed adecreased colony forming ability compared to U87-NT cells(Fig. 4C). The increased PIA and radiation-induced cell death inU87-p53KD cells were validated using the U87-p53KD 427construct (Supplementary Fig. S7). Furthermore, U87-p53KDcells had an increased activation of the apoptotic markers cleavedcaspase-3 and cleaved PARP, decreased Erk activation, and limit-ed off-target p38 activation (Fig. 4D). These data support ourprevious findings that p53 may be a major determinant of radio-sensitization following PIA treatment.

Figure 3.

Apoptosis assay of GSCs and intracellular signaling cascades after PIAþRT in radiosensitive and radioresistant subsets of GBM cells. Regulation of apoptoticand antiapoptotic markers by PIA and radiation treatment. A, Annexin-V/PI apoptosis assay was used to assess the cell death of the OSU2GSC andOSU20GSC cell lines 24 hours after 6 Gy radiation, 5 mmol/L PIA, or 6 Gy radiation and 5 mmol/L PIA treatment. B, Apoptotic signaling cascades of theradiosensitive and radioresistant subsets of GBM cell lines 24 hours following radiation treatment, PIA, or radiation and PIA treatment. The numbers underthe blots represents the ratio of phosphorylated or cleaved protein to the total protein.

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Normal human astrocytes are not radiosensitized by PIANHAs treated with PIA had 17% cell death and no increase in

apoptotic or necrotic cell death following PIA and radiationtreatment (Fig. 4E). This result could be due to the presence offunctional p53 or the lower expression level of P-Akt in NHAs(Fig. 1B). Importantly, this suggests that normal tissue toxicitymay be negligible if PIA-induced Akt inhibition is used incombination with radiation treatment in vivo. This is importantbecause this could be a tumor-specific effect and provides alarge therapeutic window and rationale for targeting P-Aktusing PIA for the treatment of GBMs.

PIA and radiation treatment alters the DNA damageresponse in p53-mt cell lines

To determine the underlying cause of increased apoptosis inthe cell lines radiosensitized by PIA treatment, we investigatedthe role of the cellular DNA damage repair response. The kineticsof radiation-induced DNA double-strand break repair (DNA-DSBR) through the non-homologous end joining (NHEJ) path-way have an ATM-independent fast and ATM-dependent slowcomponent (15). Given that the expression of ATM followingPIA � radiation treatment did not show any trend specific to theradiosensitive or radioresistant cell lines (Supplementary Fig.S6), we directed our focus to the ATM-independent fast com-ponent of the NHEJ pathway. The fast component accounts forapproximately 85% of DSBR and occurs within the first 2 to 3hours following radiation treatment. The effect of PIA on dou-

ble-strand DNA breaks (DSB) was estimated using a single cellgel electrophoresis COMET assay. This assay provides a quali-tative way to compare DNA damage by observing tails offragmented DNA behind cell nuclei. COMET assays performedon the radiosensitive LN18 and OSU61 cell lines following PIAtreatment demonstrated a significant increase in DSBs in com-parison to the U87 radioresistant cell line (Fig. 5A and B).Furthermore, p53 silencing in the U87-p53KD cell line hadsignificantly higher amount of DSBs after PIA and radiationtreatment (Fig. 5C). We concluded that this increase in DSBsfollowing PIA � radiation treatment may account for theenhanced cytotoxicity and radiosensitization of the GBM celllines with functionally mutant or null p53.

Following radiation-induced DSBs, the fast component ofthe NHEJ pathways begins when the Ku70/80 heterodimer(Ku) binds the ends of DSBs and recruits DNA-dependentprotein kinase catalytic subunits (DNA-PKc) that facilitateDNA repair. We found that PIA and radiation treatment ofthe radiosensitive GBM cell lines decreased DNA-PKcs expres-sion in a time-dependent manner (Fig. 5D). Of note, compar-ing DNA-PKc expression levels after PIA þ radiation treatmentin LN18 and LN229 cells show a moderate decrease in DNA-PKc levels in LN229 cells compared to LN18 cells, likely due tothe partial functionality of p53 (Fig. 5D). In contrast, theradioresistant GBM cell lines had increased DNA-PKcs expres-sion following PIA and radiation treatment (Fig. 5E). U87-p53KD cells also had lowered expression of DNA-PKcs

Figure 4.

PIA radiosensitizes U87-TP53 KD cells, NHA cells exhibit radioprotection with PIA, clonogenic survival assay, and intracellular signaling cascade of isogenicU87-TP53 cells with PIAþRT. p53 silencing recapitulates radiosensitization effect of PIA in U87 cells. A, The protein and mRNA level of p53 in each ofthe p53 isogenic cell lines. B, Annexin-V/PI apoptosis assay measuring the cell death of U87-NT and U87-p53KD-1673 24 hours after radiation, PIA, orradiation and PIA treatment. C, Clonogenic survival assay of the p53 isogenic cell lines after either radiation treatment alone or radiation treatmentand 5 mmol/L PIA. D, Apoptotic signaling cascades of the U87-NT and U87-p53KD-1673 cell lines 24 hours after 6 Gy radiation, 25 mmol/L PIA, or 6 Gyradiation and 25 mmol/L PIA treatment. E, Annexin-V/PI apoptosis assay was used to assess the cell death of NHAs 24 hours after radiation, PIA, orradiation, and PIA treatment.



following PIA and radiation treatment compared with theU87-NT cells (Fig. 5E). Furthermore, OSU2GSC (TP53-wt;radioresistant) exhibited an increased DNA-PKc expressionlevels after PIAþRT and OSU20GSC (TP53-mt; radiosensitive)exhibited a decrease in DNA-PKc expression levels afterPIAþRT (Fig. 5D and E). The modest change in GSK3b acti-vation indicates limited off-target effects of PIA (Supplemen-tary Fig. S8). In addition, minor changes in Ku80 suggest thatthe altered expression of DNA-PKcs are the driving factorbehind differential DSBR. From these data, we concluded thatthe increased DNA damage in GBM cell lines with functionallymutant or null p53 likely results from the downregulation ofDNA-PKcs.

In vivo studies conducted using U87-NT and U87-p53KDcells in NOD-SCID mice

Because of the higher intracranial tumor take rate, we usedimmunodeficient NOD-SCID mice. About half a million cellswere intracranially implanted. The mice bearing U87-NT andU87-p53KD cells survived for 52 � 5 days and 29 � 5 days,respectively (Fig. 5F). On the basis of hematoxylin and eosinstaining of coronal sections, it appears that silencing p53 leadsto more aggressive tumors with an enhanced tumor growthkinetics and characteristics. We did not pursue in vivo studiesfurther due to the growth kinetic alterations in the p53isogenic cells, and owing to the loss of DNAPKc in NOD-SCID mice, and high sensitivity of these mice to radiationtreatment.

PIK-75 � PIA treatment radiosensitizes p53 intact GBMcell lines

Next, we investigated whether the dependence of p53-wt cellson NHEJ repair can be abrogated using the DNA-PKcs inhibitorPIK-75 (16). We determined the IC50 value of PIK-75 to be 0.5mmol/L (Fig. 6A). Treating the p53-intact U87 and MGH8 celllines with PIK-75 � PIA radiosensitized these cell lines (Fig. 6Band C). In addition, we observed a higher susceptibility of U87 tothe PIK-75 inhibitor in combination with radiation and PIAtreatment. There appeared to be no colony formation at anyradiation dose when U87 cells were treated with PIK-75 � PIA(Fig. 6C). These data further support our hypothesis that PIA-induces radiosensitivity in cell lines with p53-mut by alteringthe expression of DNA-PKcs. In addition, this provides a thera-peutic option for p53-wt GBM tumors.

Proof-of-concept from the previous studyA previous study (7) demonstrated that several commercially

available lung and breast cancer cell lines had differential sensi-tivity to a panel of PIA inhibitors depending on the basal level ofactivated Akt in each cell line. They reported that PIAs increasedapoptosis 20- to 30-fold in cancer cell lines with high levels ofendogenous Akt activity but only 4- to 5-fold in cancer celllines with low levels of Akt activity. All cell lines that showedhigher sensitivity to PIA analogs were p53 mutated. We obtainedsequencing data for H1703, H1155, and MB486 that had 1,009,4,091, and 689mutations, respectively. Among the cell lines usedin our study, sequencing data for p53-mt cells LN18 and LN229

Figure 5.

Comet assay, DNA damage and repair signaling cascades, and in vivo studies of GBM cells. PIA and radiation induce increased DNA damage and decreasedDNA-PKcs in the radiosensitive subset of GBM cell lines. A–C, Alkaline comet assays measuring the relative number of double-strand DNA breaksfollowing 6 Gy radiation, 10 mmol/L PIA, or 6 Gy radiation and 10 mmol/L PIA treatment. D and E,Western blotting was used to track the changes in DNA-PKcs,Ku80, and g-H2AX following 5 mmol/L PIA and 6 Gy radiation treatment at the indicated times. F, Kaplan–Meier survival curves of NOD-SCID miceintracranially injected with U87-p53 isogenic cells and H and E staining of coronal sections.

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had about 949 and 563 mutations, respectively. We used thesesequencing data to find out whether there was an association ofany other mutations in all these cells lines in addition to p53 andapoptosis. By comparing the mutational profiles of p53-mt celllines with high sensitivity to PIAs, we found that only the TP53and PCLO genes were commonly mutated. In contrast, the radio-resistant cell lines from our study shared no common mutationswith the p53-wt cell lines which exhibited a decreased cytotoxicityto PIAs (Fig. 6D). Of note, U87 (radioresistant and p53-wt) alsohas the PCLOmutation, making TP53 the only commonly sharedmutation unique to the radiosensitive group of cell lines andthose cell lines with high susceptibility to PIAs. This furthersupports the correlation between the PI3K and p53 pathways inregulating survival in cancer cells.

DiscussionThrough this work, we aimed to clarify the interdependence

of the PI3K and p53 pathways in conferring resistance toionizing radiation in GBM cells. Inhibition of Akt by PIA wouldappear to be an effective radiosensitizer for GBMs, as summa-rized in the schematic in Fig. 6E. PIA is cell-permeable, revers-ible, and inhibits the activation of Akt with minimal off-targeteffects on PDK-1 or other kinases downstream of Ras, such asMAPKs (7). Because PIA is a lipid analog, it should have theability to cross the blood-brain barrier, a major obstacle to thedevelopment of chemotherapeutics for gliomas. The efficacyand activity of PIA analogues have been validated in twodifferent mouse models, proving that biologically effectivedoses of PIAs can be administered in vivo (17, 18). Furthermore,

NHAs were not radiosensitized by PIA, suggesting that thistreatment strategy may spare neighboring tissues from radia-tion induced-toxicity. Therapeutic targeting of several PI3K/Aktpathway members has been increasingly investigated and someof these inhibitors have made it to clinical trials (17, 19, 20).Some have tried to modulate the PI3K/Akt pathways by target-ing upstream molecules such as Ras, which has been found tobe activated in many tumor types (21–24). Others have tried totarget the PI3K/Akt pathway by using PI3K inhibitors, such aswortmannin and LY294002, or mTOR inhibitors, such asrapamycin (5, 25). These inhibitors have been shown to reducecancer cell growth in vitro and in vivo but have had limitedsuccess in clinical settings as single agents. Specifically, rapa-mycin's ability to induce Akt activity through upstream feed-back loops has reduced its clinical antitumor ability (26).Furthermore, a number of feedback loops that activate Akthave been identified in tumor cells, making it more likely thattargeting Akt directly may be a useful future cancer therapyapproach (27). A preclinical pharmacology report correlatingthe activity of an Akt inhibitor to the genetic background oftumor cells supports the potential for using Akt inhibitors forpersonalized medicine based on genetic status (28). However,there have been conflicting reports from previous studies eval-uating the use of P-Akt inhibitors as single agents or radio-sensitizers for the treatment of glioma (29–32).

Previous reports demonstrate that Akt inhibitors inducecell death in cancer cell lines based on their basal level ofP-Akt (4–7, 17, 19, 33, 34). In our study, PIA treatment inducedapoptosis to a moderate level in p53-wt cell lines and to agreater extent in p53-mt cell lines. However, when combining

Figure 6.

Radiosensitization rescue studies using DNA-PKc inhibitor in TP53-wt GBM cells, clonogenic survival assay, bioinformatics analysis, and graphical abstract.DNA-PKcs inhibition and radiation treatment radiosensitizes GBM cells with intact p53. A, U87 and MGH8 cell lines were administered PIK-75 at theconcentrations indicated and the DNA-PKcs, P-Akt, and Akt levels were determined and used to calculate the IC50 value for PIK-75. B–C, Clonogenic assay ofU87 and MGH8 cell lines following 6 Gy RT, PIA þ RT, PIK-75 þRT, or PIAþPIK-75þRT. D, Venn diagrams showing the number of overlapping mutations in thep53 mutant and p53 wild-type cell lines. E, Graphical summary of the role of p53 mutational status on the radiosensitization effect of PIA in GBM.



PIA with radiation treatment, only cell lines with mutant p53were radiosensitized by PIA. This observation was independentof PTENmutational status as well as basal Akt activation. A casereport on the molecular profiles of a glioma patient whosurvived for 20 years described that the tumor was PTENpositive (wild-type) and negative for P-Akt, giving support tothe notion that this combination may have a favorable prog-nostic value (35). Therefore, the radiosensitization with PIArequires abrogation of "normal" p53 function/activity. Ofcourse, we cannot completely exclude the possibility of othersignaling molecules in modulating this effect. To account forthis, at least in part, we have evaluated the expression levels ofkey off-target signaling proteins involved in the PI3K, apopto-tic, and DNA-DSBR pathways. Furthermore, we found that thePIA-induced radiosensitization of the GBM cell lines testedwere independent of other regulatory proteins in the p53pathway, such as Murine double minute 2 (Mdm2) andCDKN2A. Mdm2 is a ubiquitin ligase that tags p53 for protea-some-mediated degradation. The level of P-Mdm2 expressionhas been reported to influence the extent to which radiationinduces p53-dependent apoptosis (36). Basal P-Mdm2 levelswere low in the GBM cell lines tested in this study (Fig. 1A) anddid not correlate with either the radiosensitive or radioresistantgroup of cell lines following PIA treatment. CDKN2A, whichencodes the MDM2 inhibitor p14ARF, was deleted in all celllines used in this study. This supports our conclusion that p53mutations that interfere with p53 wild-type functionalityinduce the radiosensitization effect of PIA.

The cell lines radiosensitized by PIA showed high levels ofapoptosis that correlated with increased DNA damage anddecreased levels of DNA-PKcs. There is a well-established linkbetween Akt and DNA-PKcs, the major effector of processingand repair in the fast component of the NHEJ pathway. Aktbinds to DNA-PKcs and is involved in facilitating binding toDNA damage sites and mediating the trans/auto-phosphoryla-tion of DNA-PKcs, ensuring their release from DNA for furtherligation of damaged ends (13, 37–41). The inhibition of Akt orDNA-PKcs is reported to induce high levels of DSBs andapoptosis after radiation treatment (21, 42–47). In contrastwith Akt, the role of p53 in mediating radiation resistance inGBMs remains unclear. Interestingly, the inhibition of Akt byPIA was only sufficient to impair the DSBR and decrease DNA-PKcs expression in cell lines with functionally mutant or nullp53. With regards to p53 status, we could only find one reporton the pharmacological inhibition of p53-sensitizing GBM cellsto BCNU and TMZ (48) and another report relating mutation ofp53 and chemosensitivity in malignant gliomas (49). In spiteof this, the link between DNA-PKcs and p53 is supported by anearlier finding that the defect in apoptosis in p53-deficient cellsis rescued by the inactivation of the DNA-PK holoenzyme (50).This suggests that p53 may play a regulatory role in the DNA-PKcs–dependent response to DNA damage. Clinical studiesattempting to seek a correlation between p53 status and radio-sensitivity have provided mixed results (49) and constitute an

area of improvement. On the basis of our results, we concludethat the inhibition of Akt in cell lines with functionally mutantor null p53 sensitizes GBM cells to radiation treatment byaltering the DSBR. The main function of wild-type p53 is itstumor-suppressor activity, whereas mutant p53 acquire severalfunctions to promote cancer phenotypes. Some of the p53mutation confer loss of function and others that confer achange of function. The distinct functional classes of TP53variants in cancers can lead to different consequences due toits regulation by an array of genetic and epigenetic alterationsthat occur in cancers which is beyond the scope of this study.However, in this study, the TP53 gene was sequenced in all thecell lines used and the interpretation of results were limited tothe mutations described thereof and cannot be applied to allother TP53 disruptive or non-disruptive mutations that werenot included. The findings of the study form a foundation forpersonalizing GBM therapies based on p53 status and supportsthe fact that, when using targeted therapies, treatment failure isprimarily due to the compensatory effect of complicated geneand protein networks that allow cancer cells to evade death. Asthe paradigm of cancer treatment moves toward personalizedcare, using genetic aberrations in an individual patient's tumorto select treatments that maximize efficacy will be key forproducing better treatment outcomes.

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

Authors' ContributionsConception and design: K. Palanichamy, S.-ei Noda, A. ChakravartiDevelopment of methodology: K. Palanichamy, A. ChakravartiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Palanichamy, D. Patel, J.R. Jacob, K.T. Litzenberg,N. Gordon, S.-ei NodaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Palanichamy, D. Patel, J.R. Jacob, N. Gordon,A. ChakravartiWriting, review, and/or revision of the manuscript: K. Palanichamy, D. Patel,J.R. Jacob, K.T. Litzenberg, A. ChakravartiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. ChakravartiStudy supervision: K. Palanichamy, K. Acus, A. Chakravarti

AcknowledgmentsNIH/NCI1RC2CA148190, RO1CA108633, and 1RO1CA188228 (to A.

Chakravarti), and The Ohio State University Comprehensive CancerCenter and College of Medicine (to K. Palanichamy and A. Chakravarti).

We thank theOSU-CCC core facilities and all the patients enrolled in our IRBfor making this possible. We thank Ananya Kamalakannan for her help withgraphical abstract.

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 May 11, 2017; revised June 27, 2017; accepted August 9, 2017;published OnlineFirst August 24, 2017.

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