MultipleDefectsSensitizep53-DeﬁcientHeadand Neck Cancer ... · slide scanner (Zeiss AxioImager Z2 upright) microscope with a 20 objective. Automated image analysis for QIBC utilized

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Multiple Defects Sensitize p53-Deficient Head andNeck Cancer Cells to the WEE1 Kinase InhibitionAhmed Diab1, Michael Kao2, Keffy Kehrli3, Hee Yeon Kim1, Julia Sidorova3, andEduardo Mendez1,2,4,†

Abstract

The p53 gene is the most commonly mutated gene insolid tumors, but leveraging p53 status in therapy remains achallenge. Previously, we determined that p53 deficiencysensitizes head and neck cancer cells to AZD1775, a WEE1kinase inhibitor, and translated our findings into a phase Iclinical trial. Here, we investigate how p53 affects cellularresponses to AZD1775 at the molecular level. We found thatp53 modulates both replication stress and mitotic deregu-lation triggered by WEE1 inhibition. Without p53, slowingof replication forks due to replication stress is exacerbated.Abnormal, gH2AX-positive mitoses become more common

and can proceed with damaged or underreplicated DNA.p53-deficient cells fail to properly recover from WEE1 inhi-bition and exhibit fewer 53BP1 nuclear bodies despiteevidence of unresolved damage. A faulty G1–S checkpointpropagates this damage into the next division. Together,these deficiencies can intensify damages in each consecutivecell cycle in the drug.

Implications: The data encourage the use of AZD1775 incombination with genotoxic modalities against p53-deficienthead and neck squamous cell carcinoma.

IntroductionResistance to genotoxic therapy is the main reason that

patients with head and neck squamous cell carcinoma(HNSCC) die of cancer, and evidence shows a strong associ-ation between loss of the p53 tumor suppressor and theemergence of resistance. HNSCCs have a very heterogeneousmutational landscape with few shared oncogenic mutationsexcepting the p53 gene (TP53), where mutations were noted inup to 72% of tumors (TCGA Research Network: http://cancergenome.nih.gov/; ref. 1). Despite the central role of TP53 inHNSCC carcinogenesis (2), to date no standard-of-care therapyleverages the tumor's p53 status, albeit preclinical work, andclinical trials are bringing this goal closer (3–5). We havepreviously found that inhibition of the cell-cycle kinase WEE1with a small-molecule AZD1775 is significantly more cytotoxicto p53-mutated than to p53 WT HNSCC cell lines (6). Also, we

recently completed a phase I trial of AZD1775 in combinationwith CDDP and docetaxel in HNSCC, which showed verypromising results for patients with mutant or HPV-inactivatedp53 (7). Our goal is to understand how p53 deficiency sensi-tizes HNSCC cells to AZD1775 as a single agent or in combi-nation with genotoxic modalities.

WEE1 controls S phase and mitosis via inhibitory phosphor-ylation of cyclin-dependent kinases CDK2 and CDK1, respective-ly. Upon DNA damage or replication blockage, the ATM–CHK2and/or ATR–CHK1 checkpoints block mitosis by acting onWEE1and CDK1, thus allowing cells to complete DNA replication andrepair. Inhibiting WEE1 can compromise the checkpoint, leadingto forced mitosis and mitotic catastrophe (8–10). WEE1 inhibi-tion also overactivates CDK2 during S phase, inducing replicationstress through excessive initiation of replication and exhaustion ofsupplies of dNTPs, concomitant stalling of replication forks, andbreakage of nascent DNA (11–13). Upon WEE1 inhibition,hyperactivation of CDK1/2 also suppresses RRM2 expression,exacerbating dNTP depletion (14), while precocious activationofCDK1andPLK1 in Sphase causes cleavage of stalled replicationforks by the prematurely activatedMUS81 endonuclease complexMUS81/SLX4 (15). The cytotoxic effect of the WEE1 inhibitorAZD1775 as a single agent is often attributed to induction ofreplication stress (16).

The prominence of mitotic and S-phase responses to AZD1775and their relative contributions to the drug's cytotoxicity maydiffer depending on the cancer cells' rewiring of the cell-cycleregulatory circuitry. Studies document different responses toAZD1775 in cell lines derived from sarcomas, carcinomas, leu-kemias, andother cancers (17–21). In some studies, S-phase arrestfollowed by the addition of AZD1775 promoted prematuremitosis and cell death in the absence of p53 (8–10). However,in a study by Guertin and colleagues (22), induction of DNAdamage in S phase, not premature mitosis, correlated with cyto-toxicity of WEE1 inhibition in a panel of cell lines, and this effectwas not dependent on the p53 status. Similarly, Van Linden and

1Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle,Washington. 2Department of Otolaryngology, Head and Neck Surgery, Univer-sity of Washington, Seattle, Washington. 3Department of Pathology, Universityof Washington, Seattle, Washington. 4Seattle Cancer Care Alliance, Seattle,Washington.

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

Current address for K. Kehrli: Genetics Program, Stony Brook University, StonyBrook, New York.

J. Sidorova and E. Mendez share senior authorship of this article.

†Deceased.

Corresponding Author: Julia Sidorova, University of Washington, P.O. Box357705, 1959 NE Pacific St., Seattle, WA 98195. Phone: 206-616-3189; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-18-0860

�2019 American Association for Cancer Research.

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colleagues (18) noted no sensitization of AML lines to AZD1775upon p53 inactivation.

Focusing on HNSCC cell models and isolating for p53-specificeffects with an isogenic cell line pair, we previously reported p53-independent replication stress and p53-dependent unscheduledmitosis in an AZD1775-treated HNSCC cell line (23). Here, byfollowing specific subpopulations of cells throughmore than onecell cycle, we reveal novel and confirm known p53-specific phe-notypes in the response to WEE1 inhibition. Our results supportthe conclusion that an interplay of replication stress andG1–S andG2–M checkpoint failures can explain sensitivity of p53-deficientcells to AZD1775, and will help to optimize therapeutic windowwhen targeting p53-mutated HNSCC.

Materials and MethodsCell lines, vectors, and RNAi

Primary fibroblast cells (HFF4) were described previously (24).Head and neck cancer cell lines UM-SCC-74a was from Dr. Careyat University ofMichigan (AnnArbor,MI). Cells were usedwithinone to 3 months after thawing and tested for Mycoplasma con-tamination prior to cryopreservation or upon thawing. We used apBabeHygro retroviral vector expressing shRNA targeting p53(ref. 25; a gift from Dr. Kemp) to generate a stable cell line withdepleted p53 protein under hygromycin selection. siRNAsagainst p21 (CDKN1A) were from Qiagen (#SI00604898 and# SI00604905), and a nontargeting control siRNA (#D-001810-01-05) was from Dharmacon.

Drugs and chemicalsAZD1775was provided byAstraZeneca through a collaborative

agreement. CDDP (P4394) and Triapine (3-AP, SML0568) werepurchased from Sigma-Aldrich. EmbryoMax Nucleosides (ES-008-D, EMD Millipore) were used at a final concentration of1:25. 5-Iododeoxyuridine (IdU) and 5-chlorodeoxyuridine(CldU) were from Sigma-Aldrich and used at 50 mmol/L fromstock solutions of 2.5 and 10 mmol/L in PBS, respectively.

AntibodiesAntibodies used were g-H2AX (Ser139, JBW301 #05-636),

p-HH3 (Ser10, 3H10, #05-806) from EMD Millipore; p-HH3(Ser10, D2C8, #3377), p21 Waf1/Cip1 (12D1, #2947), cleavedPARP (D214, #9541), and b-Actin-HRP (13E5, #5125) from CellSignaling Technology; P53BP1 (E-10, #sc-515841) and p53 (DO-1, #sc-126) from Santa Cruz; and nucleolin (#396400) from LifeTechnologies/Thermo Fisher. PE-conjugated anti-cleaved PARPantibody (Asp214 #51-9007684) was from BD Pharmingen.Antibody to IdU/BrdUrd (B44, #347580) was from BD Pharmin-gen and to CldU/BrdUrd [BU1/75 (ICR1), #OBT0030] from Bio-Rad/AbD Serotec.

Flow cytometryCells were fixed and processed for flow cytometry as described

previously (24). Sampleswere runonFACSCanto II. FACSprofileswere visualized using FACS Express software (DeNovo).

DNA fiber assays on sorted cells (Sorted Microfluidics-assistedReplication Track Analysis or SmaRTA)

Approximately 5 � 106 cells were fixed in 2% formaldehyde inPBS for 10 minutes at 37�C and stained with gH2AX and histoneH3S10P antibodies as described for flow cytometry. DNA content

was visualized by DAPI. Cells were sorted in PBS on Aria III sorter(BD Biosciences) to yield at least 50,000 cells per fraction. Sortedcells were pelleted after supplementing PBS with 0.3% BSA, resus-pended inagaroseplugbuffer, embedded inagarose, andprocessedas described previously for the maRTA procedure (24, 26, 27).

Immunofluorescence and quantitative image-based cytometry(QIBC)

Cells were fixed and stained as described previously (24). QIBCwas performed as described in ref. 28 with the following mod-ifications: images were captured using TissueFAXS, an automatedslide scanner (Zeiss AxioImager Z2 upright) microscope with a20� objective. Automated image analysis for QIBC utilizedTissueQuest software.

TP53 signaling pathway PCR arrayThe human p53 Signaling Pathway RT2 Profiler PCR array

(Qiagen) was used as described by the manufacturer. UM-SCC-74a cells (p53wt vs. shp53) were treated with 1 mmol/L CDDP for24 hours. Cells were harvested and processed according to themanufacturer's instructions.

Comet assaysDNA strand breaks were measured using a kit following the

manufacturer's instructions (Trevigen). For each experimentalcondition, "tail moments" (defined as the product of tail lengthand the fraction of total DNA in the tail) were determined for atleast 500 nuclei using ImageJ software (NIH) with the OpenComet plugin. At least 2 independent experiments were scored.

Cell growth and viability assaysCell proliferation after drug washout was conducted as

described previously (29). Alternatively, 103 cells were seededinto 96-well plates and were subjected to the same treatment for24 hours. After drug removal, media were replenished and cellswere allowed to grow for 96 hours before analysis with CellTiter-Glo (Promega), following the protocol outlined by themanufacturer.

Synergy analysis for drug combinationsA total of 103 cells were seeded into 96-well plates and treated

with serial dilutions of the respective drugs for 96 hours. Cellviability was assessed with CellTiter-Glo (Promega), and thefraction affected was used to calculate the combination index(CI) and isobologram analyses according to the median-effectmethod of Chou and Talalay (30) using the CalcuSyn software(Biosoft).

Statistical analysesUnpaired t tests were carried out in GraphPad Prism 7 software

to analyze in vitro data. All data were expressed as mean � SD (�SEM for large data sets), andP valueswere indicated. SmaRTAdatawere analyzed in Kolmogorov-Smirnov (K-S) tests using R studiosoftware, and P values and, in some cases, D statistics are shown.

ResultsCell-cycle progression and DNA-damage/replication stressresponse in WEE1 inhibitor-treated HNSCC cells

Phenotypes elicited by a therapeutically relevant dose of theWEE1 inhibitor AZD1775 were first demonstrated in the TP53

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wild-type HNSCC line UM-SCC-74a. Pulse-chase labeling of cellswith EdU and flow cytometry of EdU incorporation versus DNAcontent allows to see how EdU-positive cells progress through thecell cycle for up to two consecutive S phases (Fig. 1A; S1 and S2

in Fig. 1B). A majority of cells that were exposed to AZD1775while in the S1 completed it and transited to G1 by 9.5 hours afterEdU pulse, similar to untreated cells (Fig. 1B and C). The entry ofthe EdUþ population into the S2 and/or transit through it wasdelayed in WEE1-inhibited cells. In order to follow cell-cycleprogression of AZD1775-treated cells past S1 more precisely, weconsecutively labeled these cells with IdU and EdU as shownin Fig. 1C–E. The dual-labeled cells should be the ones thatremained in the S1 phase for over 8 hours. We reasoned thatthese cells may be the most severely affected by AZD1775, andfollowing their progression will reveal the strongest response toAZD1775. Figure 1D confirms the specificity of dual staining.Without the drug, a majority of IdU/EdUþ cells completed S1 andS2 phases, while with the drug these cells slowed a delay traversingthrough S2 (Fig. 1E). Together, the data suggest that response toWEE1 inhibition is heterogeneous and depends on the time in theinhibitor, indicating an accumulation of damage over more thanone cell cycle.

We next measured the gH2AX level as a function of DNAcontent and time in AZD1775. gH2AX accumulated over thecourse of 24 hours, with subsets of cells remaining gH2AX-negative (Fig. 1F, top). We also saw late-onset accumulation ofextra-high level of gH2AX (gH2AXþþ) in a subset of mid Sphase cells (2%–3% of all cells at 8.5 hours and 10%–14% ofall cells at 24.5 hours, orange profiles, Fig. 1F, histograms).Appearance of gH2AXþþ cells was not unique to the UM-SCC-74A line, as it was also observed in human primary fibroblastsand normal oral keratinocytes (Supplementary Fig. S1A) as wellas in another TP53 wild-type HNSCC line, UM-SCC-81A(Supplementary Fig. S2E).

Tracking development of gH2AX by following EdUþ cellsthrough the cycle (Fig. 1F, middle and histograms), we saw thatearly S phase cells were gH2AX� and developed gH2AX signal asthey reached mid S. A small subset of EdUþ cells was gH2AX� asthey reached G2 and traversed into G1. gH2AX level negativelycorrelatedwith the rate of progression through the S phase. Ninety-two percent of the gH2AXþþ population at 8.5 hours in AZD1775were EdUþ cells in their S1 phase in AZD1775. At 24.5 hours, inAZD1775 there weremore gH2AXþþ cells overall and only 65%ofthem were EdUþ, suggesting an accumulation of cells in thegH2AXþþ compartment over time (Fig. 1F and data not shown).

By labeling cellswith EdUafter 24hours inAZD1775,we foundthat gH2AXþþ cells incorporated about 10 times less EdU thangH2AXþ cells in S phase (Fig. 1G, histograms, compare orangeand black profiles). gH2AXþþ cells also expressed extra-high levelof CHK1S345P (Supplementary Fig. S1B). Thus, these cells rep-resent an S phase subpopulation with severely inhibited DNAsynthesis and highly upregulated replication stress response.Findings of gH2AX induction are summarized in Fig.1H.

Staining with an antibody against CDK1/2Y15P confirmedWEE1 inhibition (Supplementary Fig. S2A–S2D) and showedthat at least in some cell lines gH2AXþþ cells had a lower levelof Y15 phosphorylation than gH2AXþ cells, potentially suggest-ing greater hyperactivity of CDK1/2 (Supplementary Fig. S2D andS2E). However, this lower staining for CDK1/2Y15P may be dueto the fact that gH2AXþþ cells are exclusively in the mid S phase,whereas gH2AXþ cells can be in the late S–G2, and CDK1/2Y15P

staining is normally lower in mid S compared with G2 (Supple-mentary Fig. S2D).

Depletion of p53 exacerbates the response to AZD1775We next depleted p53 in UM-SCC-74a background using

shRNA (Fig. 2A). As expected, p53 depletion sensitized cells toAZD1775 (Fig. 2B). Depletion of p53 caused greater accumula-tion of gH2AXþ and gH2AXþþ cells in AZD1775 (Fig. 2C and D).By immunofluorescence (IF) in situ (Fig. 2E), gH2AXþþ expres-sion level corresponded to extremely bright staining, either pan-nuclear or localized to numerous foci. Alkaline comet assaysindicated that single- and double-strand DNA breaks (SSB andDSB) were elevated in AZD1775-treated p53 knockdown (kd)cells (Fig. 2F). Also, PARP1 cleavage (an apoptotic marker) waselevated in these cells (Fig. 2G and H).

Flow-cytometric analyses of gH2AXdevelopment and cell-cycleprogression showed that both p53kd cells and controls developedsome gH2AX upon entry into their second S phase (S2) (Fig. 2Iand J). Moderate expression of gH2AX during the S phase is notunusual for cancer cell lines. Most importantly, AZD1775 treat-ment delayed entry of control cells into S2, while p53kd cellsentered S2 and expressed high level of gH2AX upon entry, asindicated by the appearance of an EdU-positive, gH2AX þ, and þþ

population at 24 hours in the drug (Fig. 2I).Also of note, AZD1775 sped up the traversal of cells out of S1

and through G2–M compared with untreated controls (Fig. 2J,e.g., compare 4- and 8-hour time points with and withoutAZD1775). Compared with controls, p53kd cells had a higherG2 fraction both with and without AZD1775. The unrestrainedentry into S2 observed in p53kd cells was also displayed by theHNSCC cell line PCI-15b harboring a high-risk TP53 mutationR273C (Supplementary Fig. S3A and S3B).

Pulse-labeling cells with EdU after a prolonged treatment withAZD1775 showed that both p53kd and control cells developed amid-S phase population with severely depressed DNA synthesis(compare Fig. 2K with Fig. 1G; Supplementary Fig. S1A). Overall,the data suggest that p53 deficiency is associated with greaterreplication-associated damage upon AZD1775 treatment. At leastpart of this phenotype can be attributed to a failure of p53kd cellsto activate the G1–S checkpoint and thus avoid an entry into theirsecond S phase in the presence of the drug.

AZD1775 increases prevalence of mitosis in p53kd cellsWEE1 inhibition not only causes replication stress but also

stimulates and in some cases advances mitosis (10, 31). Toexplore this facet of WEE1 inhibition in more detail, we stainedcells for histone H3S10P modification as a marker of mitosis(Fig. 3). The correlation between the level of H3S10P staining andmitotic condensation and alignment of chromosomes was veri-fied by IF (Fig. 3A). Of note, H3S10P level increased gradually inUM-SCC-74a cells and preceded visible chromosome condensa-tion, suggesting that only the highest level of H3S10 phosphor-ylation identifies mitosis.

High histone H3S10P-staining (HH3þ) cells were more prev-alent in p53kd cells compared with controls (Fig. 3B). AZD1775markedly increased HH3þ abundance, particularly in p53-depleted cells (Fig. 3B and C). A minor fraction of HH3þ cellsin the p53kd line also appeared to have < 4c DNA content(Fig. 3B). Notably, in the control, a vast majority of cells wereeither gH2AXþ or HH3þ, whereas double-positive, HH3þ/gH2AXþ cells were detectable in p53kd cells (Fig. 3D–G).

p53 Loss Compromises Response to WEE1 Inhibition

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Figure 1.

Response to WEE1 inhibition by AZD1775 develops over consecutive cell cycles in the HNSCC cell line UM-SCC-74a. A, Experimental design. Cells were pulse-labeled with EdU for 30 minutes, then grown for up to 36 hours with or without 300 nmol/L AZD1775. B, Flow-cytometric analyses. Left, representativedensity plots of cells stained for EdU incorporation and DNA content. Populations in the first S, G1, G2, and the second S phases since the EdU pulse aremarked by arrows. Right, histograms of cell-cycle distribution of EdU-positive cells at the indicated times after the EdU pulse. C, Experimental design and adiagram of dual labeling: IdU is the first label and EdU is the second label. AZD1775 (400 nmol/L) was added after the first label where indicated. D, Anexample of immunofluorescent staining of dual-labeled, AZD1775-treated UM-SCC-74a cells harvested 15 hours after the second label. Scale bar, 40 mm. E,Histograms of cell-cycle distributions of dual-labeled cells at indicated times after the second label. F, Flow-cytometric analysis of cells treated as in A,harvested at indicated times of incubation with AZD1775, and immunostained for gH2AX expression, EdU incorporation, and DNA content. Top two rows aredensity plots of, respectively, gH2AX and EdU levels versus DNA content. The bottom row is histograms of cell-cycle distributions (by DNA content) of thefollowing subpopulations: EdU-positive/gH2AX-negative (green), EdU-positive/gH2AX-positive (purple), gH2AX-superpositive (orange). G, Histograms of cellcycle (top) and EdU level (bottom) distributions of cells from the indicated subpopulations. Cells were incubated with AZD1775 for 24 hours and pulse-labeledfor 30 minutes with EdU prior to harvest. H, A summary of findings presented in the figure. Red color intensity corresponds to the gH2AX level. Cells remaingH2AX-negative in early S phase, develop gH2AX signal as they progress through S, and at least some of them retain gH2AX staining in the next G1 and S. Asubset of cells develops ultra-high level of gH2AX associated with suppressed DNA synthesis.

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Figure 2.

Depletion of p53 in UM-SCC-74a HNSCC cells modifies response to AZD1775.A, AWestern blot verifies knockdown (kd) of the p53 protein and functionaldeficiency in p53 as demonstrated by the inability of cells to induce p21 after ionizing radiation. B, Fold change in proliferation relative to the untreated controlsafter treatment with 300 nmol/L AZD1775 for 16 hours in UM-SCC-74a and the primary fibroblast line HFF4. C, Flow-cytometric analysis of gH2AX response inmock and p53kd cells after 24 hours of 300 nmol/L AZD1775. D, Relative enrichment of gH2AX-positive cells in p53kd cells compared with the mock-depletedcontrol (n¼ 3). E, Examples of immunofluorescent staining for gH2AX in AZD1775-treated p53kd cells. F, Alkaline comet assays in p53kd versus control cellstreated 300 nmol/L AZD1775 for 16 hours (n¼ 3). G, Flow-cytometric analysis of cleaved PARP1. Fraction of cleaved PARP1 was assessed by gating cells relativeto untreated controls and averaging the results (n¼ 9). H, AWestern blot of cleaved PARP1 levels in cells treated with indicated doses of AZD1775 for 17 hours. I,Flow-cytometric analysis of EdU incorporation versus gH2AX expression. Black: all-negative cells; green: gH2AX-positive and superpositive (if any); blue: EdU-positive. Cells were labeled with EdU for 30 minutes and incubated for 24 hours with or without 300 nmol/L AZD1775. J, Histograms of cell-cycle distributions ofEdU-positive cells pulse-labeled with EdU for 30minutes and incubated with or without 300 nmol/L AZD1775 for the indicated times. EdU-positive cells thatwere gH2AX-negative (red) or gH2AX-positive (and superpositive, if any, blue) are plotted separately. K, Flow-cytometric analyses of EdU incorporation of cellsincubated with AZD1775 for 24 hours with EdU labeling for 30minutes prior to harvest. Low EdU-incorporating cells appearing upon AZD1775 treatment aremarked by arrows.






These differences were recapitulated in the p53-mutant PCI-15b cell line (Supplementary Fig. S3C, S3D, and S3E). In partic-ular, basal and AZD1775-induced percentage of HH3þ cells washigher in these p53-mutant cells than in a p53 wild-type line, anda higher proportion of HH3þ cells was also gH2AX-positive.Furthermore, inactivation of p53 in UM-SCC-74a cells by expres-sing E6 protein of the HPV16 virus (32) recapitulated AZD1775-associated phenotypes displayed by p53kd cells, including sup-pression of growth, increased PARP1 cleavage, and elevatedgH2AX and HH3þ/gH2AXþ cells (Supplementary Fig. S4A–S4D). Thus, the data consistently show that induction of gH2AXexpression andmitosis byWEE1 inhibition aremore pronounced

if p53 is altered, and second, that p53 deficiency correlates withthe presence of mitotic gH2AX-positive cells. This feature maymark a specific vulnerability of p53-defective cells to WEE1inhibition and/or may serve as a biomarker of WEE1i sensitivity.

The impact of p53 on the G2–M checkpoint can be conveyedthrough p21, a CDK inhibitor inducible by p53 (33, 34). Indeedwhen we depleted p21 in p53 wild-type cells, we observed anincrease in gH2AX, HH3þ, and HH3þ/gH2AXþ(þþ) cells, whichnegatively correlated with the p21 level (Figs. 3H–J). p21 deple-tion also exacerbated reduction of p21 levels observed in p53kdcells and increased gH2AX-positive mitoses, albeit less markedlythan in p53 wild-type cells (Figs. 3H–J).

Figure 3.

Depletion of p53 in UM-SCC-74a cells increases prevalence of mitotic cells and leads to abnormal mitoses with high expression of gH2AX. A,Immunofluorescent staining for histone H3 phosphorylated on S10 (H3S10P) as a function of premitotic and mitotic stages. B, Flow-cytometricanalysis of the H3S10P level versus DNA content. Cells were incubated with 500 nmol/L AZD1775 for 8 hours. C, Relative enrichment of highhistone H3S10P (HH3þ) cells in p53kd cells compared with the mock-depleted control (n ¼ 3). D, Examples of immunofluorescent staining ofAZD1775-treated mock and p53kd cells incubated with 300 nmol/L AZD1775 for 24 hours. Red: gH2AX; green: H3S10P. An arrow marks a cellstaining positive for both markers. E, An example of an abnormal mitotic figure positive for both gH2AX and H3S10P in p53kd, AZD1775-treatedcells. F, QIBC of mean fluorescent signals of gH2AX and H3S10P per nucleus in cells treated with 300 nmol/L AZD1775 for 24 hours. pB, n ¼4,062; p53kd, n ¼ 2,328. Dashed frames indicate expected positions of the gH2AXþ/HH3þ subpopulation, and cells positive for both markers aremarked by an arrow. G, Fractions of gH2AX-positive cells among the histone H3S10-positive, mitotic cells in p53kd cells and mock-depletedcontrol. Treatment regimens were 300 nmol/L AZD1775/10 hours or 500 nmol/L AZD1775/8 hours. Cells were analyzed by flow cytometry(n ¼ 5). H, A Western blot of siRNA-mediated depletion of p21 (CDKN1A) in control and p53kd cells. nc is nontargeting siRNA control. I, Levelsof gH2AXþ/þþ and HH3þ cells relative to untreated control cells transfected with nontargeting siRNA, as measured by flow cytometry (n ¼ 2).AZD1775 treatment was for 8 hours at 300 nmol/L. J, Fractions of gH2AX-positive cells among the histone H3S10-positive, mitotic cells in p53kdand control cells (n ¼ 2).

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Recovery from AZD1775 triggers additional gH2AX inductionin p53kd cells

Wenext askedwhether recovery fromAZD1775was affected byp53 status. Cells were incubated with AZD1775 and then allowedto recover for up to 24 hours (Fig. 4A), and analyzed by IF in situandQIBC. p53kd cells had a higher percentage of gH2AX-positivecells throughout the recovery (Fig. 4B). Over 30% of p53kd cellsscored gH2AX-positive for 8 hours after removal of AZD1775versus 5%of control cells. Moreover, a persistently higher fractionof gH2AX-positive p53kd cells displayed aberrant, severely mis-shapen or fragmented nuclei, consistent with failed segregation(Figs. 4C and D). However, this nuclear fragmentation was lowoverall, occurring in less than 10%of gH2AX-positive subpopula-tions throughout recovery.

We also pulse-labeled cells with EdU 1 hour prior to release inorder to mark replicating cells and follow them through recovery.gH2AX, EdU, and H3S10P signal intensities were manually col-lected in digital images for specific cell categories, i.e., mitotic,premitotic, or gH2AXþþ (Supplementary Fig. S5A). This allowedus to define a range of signal intensities that was associated with aparticular category. In parallel, we also collected gH2AX,H3S10P,and EdU signal intensities for thousands of individual nucleiusing automated image acquisition and analysis.

We determined the distributions of gH2AX and histoneH3S10P signal intensities in cells that displayed none/low(termed EdU�) or high EdU incorporation when labeled justprior to release fromAZD1775 (Fig. 4E). EdU� cells correspond tomid S phase gH2AXþþ cells as well as cells that were outside of theSphase at themoment of labeling (Figs. 1GandH; 2K). These cellsmost frequently displayed high levels of gH2AX (signal intensity>125) consistent with gH2AXþþ status. The prevalence of thesecells and their signal intensity declined over time in control butless so in p53kd cells. The majority of EdU� cells were mitotic(H3S10P signal >75) in AZD1775 and before 4 hours of recovery,with subsequent wave of mitoses developing at 24 hours ofrecovery in both control and p53kd cells. Based on the overallEdU levels in the populations, between 12 and 24 hours ofrecovery most of control and p53kd cells have divided once(Supplementary Fig. S5A)

EdU-high signal is consistent with cells in the early to mid Sphase at the point of release from AZD1775, and these cells wereoverwhelmingly gH2AX-negative in both cell lines (consistentwith the data in Fig. 1F andG).Mitoses in these cells peaked at 8 to12 hours of recovery, which was predictably later than mitoses inthe EdU-negative cells. Interestingly, however, in the p53kd line,cells that were virtually gH2AX-negative in AZD1775 began todevelop gH2AX signal at 24 hours after removal of the drug. Thisdevelopment followed completion of mitosis by several hoursand thus is consistent with the entry of the EdU-high subpopu-lation into the next S phase. An independent experiment con-firmed that EdU-high population of p53kd cells increased itsgH2AX level at 24 hours comparedwith the 0 hours after the drug,unlike the mock-depleted controls (Supplementary Fig. S5B).Overall, the findings suggest that early S phase cells that displayvirtually no gH2AX response inAZD1775nevertheless incur somekind of damage. In p53-deficient background, this damage per-sists for hours after AZD1775 removal and is revealed at the timepoint that is consistent with the entry into the next cell cycle.

We further addressed this by visualizing 53BP1 nuclear bodies(i.e., large, bright foci) in control and p53-deficient cells recov-ering from AZD1775. While in AZD1775, neither cell line had

53BP1 signal above background, consistent with other stud-ies (35). At 24 hours of recovery, a subset of control cells clearlydisplayed elevated 53BP1 signal (Fig. 4G and H). Remarkably,p53kd cells displayed lower 53BP1 signal. In both cell lines, themajority of 53BP1-positive cells did not express gH2AX, arguingagainst 53BP1 colocalization with DSBs. Thus, both control andp53-deficient cells retain unresolved damage after mitosis; how-ever, p53-deficient cells have an altered response to it.

gH2AX level in AZD1775-treated cells correlates withreplication fork slowing

Our data suggest that gH2AXþþ cells represent a qualitativelydistinct subpopulation with severe replication stress (Figs. 1G, 2Kand Supplementary Fig. S1). WEE1 inhibition by AZD1775 isknown to cause replication fork slowing and stalling (12). Wewanted to determine whether severe replication stress in thegH2AXþþ population corresponded to the slowest forks. Wewerealso interested to see if p53 status affected fork response toAZD1775, and if gH2AXþþ/HH3þ double-positive cells werequantitatively more affected than gH2AXþþ/HH3� cells, reason-ing that premature mitosis of gH2AXþþ/HH3þ cells may bestimulated by their complete inhibition of replication, as sug-gested in ref. 15.

To answer these questions, we devised a combination of flowsorting and DNA fiber analysis (Supplementary Fig. S6). Wesequentially labeled cells with CldU and IdU (with or withoutAZD1775) to mark ongoing replication forks, immunostainedthese cells with antibodies against gH2AX andH3S10P, and flow-sorted them. gH2AX�, þ, and þþ fractions were obtained for allcell lines. For p53kd cells, the gH2AXþþ fraction was subdividedinto HH3þ and HH3� populations (Fig. 5A).

DNA was isolated from these fractions and subjected to ourDNAfiber-stretching protocol,maRTA (Fig. 5B; ref. 27). AZD1775treatment slowed fork progression in all cells, but, as we expected,fork progression rate negatively correlated with the gH2AX level(Figs. 5C and D), confirming that gH2AXþþ cells experience thehighest level of replication stress. In addition, the gH2AXþ sub-population of p53kd cells had slower forks compared withgH2AXþ controls, and, overall, p53kd cells displayed a morepronounced reduction in fork progression in gH2AX-positivecompared with gH2AX-negative subpopulations (Fig. 5E). Inter-estingly, fork progression in gH2AXþþ/HH3þ cells was not slowerbut in fact faster than in gH2AXþþ cells (Fig. 5C and D). The datasuggest that, while p53 status affects the severity of replicationstress at the replication fork level, the p53kd-specific gH2AXþþ/HH3þ cells do not exhibit higher replication stress thangH2AXþþ/HH3� cells. Thus, the level of replication stress/sup-pression of replication alone cannot explain the prevalence of thisdouble-positive subpopulation in p53kd cells.

The above data suggest that replication stress may be necessary,but it is not sufficient to induce a p53-specific survival defect. As anindependent test, we asked if survival of p53kd cellswas improvedby supplementation of the media with extra nucleosides. Suchsupplementation is known to alleviate WEE1i-induced replica-tion stress (12, 14). Indeed, the addition of nucleosides improvedsurvival of the control but not p53kd UM-SCC-74a cells (Fig. 5F).

Synthetic lethality of AZD1775 in combination with CDDP ortriapine

Only subsets of p53kd populations exhibited extreme dysfunc-tion or cytotoxicity when exposed to the clinically safe doses of






Figure 4.

p53kd UM-SCC-74a cells recovering from AZD1775 retain more gH2AX but fail to form 53BP1 nuclear bodies. A, Experimental design. Cells were treated with 300nmol/L AZD1775 for 24 hours, then allowed to recover for up to 24 hours. In some experiments, cells were also labeled with EdU for 30 minutes between 22.5 and23 hours of the drug treatment. Cells were immunostained for gH2AX, H3S10P, and where indicated, EdU, and analyzed by QIBC. B, Relative enrichment ofgH2AX-positive fraction in p53kd cells compared with controls (n¼ 2). Mean nuclear gH2AX signal was scored in at least 2,000 and up to 16,000 cells in eachsample. Cells were considered gH2AX-positive if their mean gH2AX signal was >1/2 MAX of the population distribution, and the percentage of these cells wascalculated for each sample, expressed as fold enrichment over the control at time point 0 hours recovery in each experiment, and averaged. Both gH2AXþþ andgH2AXþ cells are included in this metric. C, Examples of immunofluorescent staining of p53kd, AZD1775-treated cells. The nucleus marked by an arrow displaysaberrant morphology. D, Relative enrichment of cells with aberrant nuclear morphology among gH2AX-positive p53kd cells compared with controls measured inthe same experiments as in B. Aberrant and normal gH2AX-positive nuclei were scored manually in digital images collected using a scanner microscope. A totalof 120–600 nuclei were analyzed per sample. Percentage of aberrant among gH2AX-positive nuclei was calculated for each sample, expressed as fold enrichmentover the control at time point 0 hours recovery in each experiment, and averaged. E, F,QIBC analysis of an experiment performed as in A. Mean nuclear EdU,gH2AX, and H3S10P signals were measured in 5,000–23,000 cells for each cell line/time point, and the data were subsetted based on EdU signal values. An EdU-low/negative subset has EdU values within the first quintile of a data set. EdU-high cells have EdU values >1/2 MAX of the data set. In E, gH2AX (top panels) andH3S10P (bottom panels) values in EdU-negative subsets of cells are plotted as a function of recovery time. In F, these same values are plotted for EdU-highsubsets. Numbers above plots are percentages of gH2AX-positive and HH3þ cells at each time point. See Supplementary Fig. S4 for more on selection andvalidation of EdU, gH2AXþ, and HH3þ value cutoffs. Diagrams on the right denote the inferred cell-cycle position of EdU-negative and EdU-high cells at the timeof EdU pulse labeling. G,QIBC analysis of AZD1775-treated cells recovering from the drug for 24 hours. Mean nuclear 53BP1 and gH2AX signals were measured inapproximately 5,700 each of control and p53kd cells. Elevated 53BP1 signal in controls compared with p53kd cells is marked with a bracket. H, Averagepopulation 53BP1 signal intensities were derived from QIBCmeasurements (n¼ 3). Cells were treated with AZD1775 as in A and released from the drug for 24hours.

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AZD1775 in the experiments above. In order to amplify cytotoxicoutcome, and given that AZD1775 is being tested in combinationwith chemotherapy in clinical trials, wenext explored the effects ofcombining AD1775 with other drugs.

Triapine (3-AP) is a potent inhibitor of ribonucleotidereductase (RNR) currently in clinical trials. A phase II studyof 3-AP in metastatic HNSCC noted that the drug was well

tolerated but was not effective as a single agent (36). Wereasoned that 3-AP can exacerbate AZD1775-induced replica-tion stress. On the other hand, CDDP, a standard-of-care drugfor HNSCC, is a DNA cross-linker. In this case, the ability ofAZD1775 to override the G2–M DNA-damage checkpointinduced by CDDP may enhance cell killing if the two drugsare combined.

Figure 5.

AZD1775-treated UM-SCC-74a cells expressing different levels of gH2AX experience different degrees of replication fork slowing.A, Experimental design. Cellsincubated with 300 nmol/L AZD1775 and no-drug controls were labeled with consecutive 30minutes pulses of CldU and IdU prior to harvest, thenimmunostained for gH2AX and H3S10P, and sorted into subpopulations prior to DNA isolation and stretching. The gH2AXþþ/HH3þ fraction was available only inp53kd cells. B, Examples of replication tracks of ongoing forks in p53kd cells. Extremely short CldU and IdU segments in forks in treated samples prompted us tomeasure total (Cþ I) lengths for each ongoing fork, as shown below the images. Two representative images for each condition were compiled to showmoretracks. C,Ongoing fork track length distributions measured in the indicated gH2AX (white) and gH2AX/H3S10P (gray) subpopulations. Numbers of tracksanalyzed for each sample are shown below the graph.D,Ongoing fork track length distributions derived from an independent experiment with p53kd cells.Designations as in C. E, A summary of differences in ongoing fork track lengths between gH2AX-negative and -positive subpopulations in p53kd cells andcontrols, derived from two independent experiments. The differences were expressed as a D statistic, i.e., the maximal difference between two cumulativedistributions. D statistic values were calculated in K-S tests comparing each of the gH2AX-positive populations to the gH2AX-negative baseline for each cell line(AZD1775-treated). Differences with the D statistic of 0.12 and above were significant. F, Change in proliferation relative to the untreated controls after treatmentwith 300 nmol/L AZD1775 for 16 hours in the presence or absence of nucleosides in the media (n¼ 8).






The 3-AP andCDDPused at doses that hadminimal to no effecton their own suppressed colony formation when combined withAZD1775 (Fig. 6A). Proliferation of AZD1775/3-AP–treated cellswas more affected in the p53kd line than in the control (Fig. 6B),and the drugs showed weak (additive) interaction in p53kd cellsand not in controls (isobologram analysis in SupplementaryTable S1). The 3-AP dose that had no effect on gH2AX andH3S10P as amonotherapy dramatically enhanced gH2AX expres-sion and virtually arrested S phase both in p53kd and control cellswhen combined with AZD1775 (Fig. 6C). While 3-AP/AZD1775combination and AZD1775 alone increased the prevalence ofcells with low to intermediate H3S10P levels (Fig. 6C, right),corresponding to premitotic cells (as before, see Fig. 4), only inp53kd cells did these drugs markedly induce premature mitosis(Fig. 6C, note the HH3þ high/<4c DNA content cell populationmarked by an arrow).

The 3-AP/AZD1775 combination dramatically increased SSBsand DSBs in the DNA of p53kd cells (Fig. 6D). It is unlikely thatthese breaks were associated only with cells undergoing prema-ture mitosis, as the latter comprised only a minor fraction (2.2%on average) of the entire population. The addition of extranucleosides completely suppressed these breaks in the controland largely suppressed them in p53kd cells (Fig. 6E), as well asimproved survival of control but not p53kd cells (Fig. 6F), in linewith the findings in Fig. 5F.

We next looked into the effect of the CDDP/AZD1775 com-bination (Fig. 7). The combination had a more severe growth-suppressive effect and triggered a greater accumulation of SSBsand DSBs in p53kd cells compared with controls (Fig. 7A and B).Isobolograms of the combination showed a synergistic interac-tion for p53kd cells and no interaction for controls (Supplemen-tary Table S2). AZD1775 and CDDP synergized in inducinggH2AX (Fig. 7C) and suppressed mitosis in the control but notin p53kd cells (HH3þcells in Fig. 7C). In order to visualize cell-cycle distribution in the CDDP/AZD1775-treated cells, the iso-genic pair was treatedwith the drug combinations and then pulse-labeled with EdU prior to harvest (Fig. 7D). p53kd cells had a fargreater accumulation of S and G2 cells in CDDP than the control,but in both cases addition of AZD1775 together with CDDPoverrode it. However, unlike in the control, the combinationtriggered appearance of cells that had S phase-like DNA contentbut failed to incorporate EdU (about 10% of total population).

A greater effect of CDDPon accumulation of cells in S andG2 inthe p53kd line prompted us tomeasure contribution of p53 to theactivation of the G2–Mcheckpoint regulators. We profiled expres-sionof p53pathway geneswithorwithoutCDDP treatment in thecontrol and p53kd UM-SCC-74A cells (Fig. 7E). p53kd cellsshowed a markedly higher expression of S and G2–M checkpointregulators (WEE1, CHK1/2, CDC25A), DNA-damage response(BRCA1/2), and replication (PCNA, MLH1, MSH2, CDK2,CCNE1) genes upon treatment with CDDP (right, Fig. 7E), whichagrees with a more pronounced accumulation of these cells in S–G2 (Fig. 7D). As expected, p53kd cells failed to activate proapop-totic genes (BAX, FAS, CASP9, etc.; left, Fig. 7E). This confirms agreater G2–M engagement at the transcriptional level upon gen-otoxic stimuli specific to p53 depletion and is in agreement withthe inability of p53kd cells to escape from the CDDP- andAZD1775-vulnerable S andG2 phases by arresting in theG1 phase(Fig. 7D). Consistent with this, only in p53kd cells did CDDP/AZD1775 combo clearly enhance apoptotic PARP1 cleavage(Fig. 7F).

Overall, the data indicate that combination treatments thatenhance replication stress or inflict DNA damage in the setting ofWEE1 inhibition can enhance cytotoxic outcome in p53-deficientcancer cells in positive correlationwith, respectively, premature orforced mitosis.

DiscussionDifferential effects of p53 deficiency

WEE1 inhibition by AZD1775 elicits both p53-dependent and-independent phenotypes, someofwhichdevelopover a course ofmore than one cell cycle. For instance, S phase–associated DNAdamage triggered by WEE1 inhibition (as revealed by gH2AXexpression) is more prevalent in the second S phase of the timecourse of incubation with the clinically relevant dose of the drug.This suggests a carryover of a lesion or a particular cellular statefromone cell cycle to the next. Interestingly, a recent study showedthat WEE1 inhibition can result in an elevated CDK1 activitypersisting throughout the G1 phase, whichmay affect DNA-repairand theG1–S transition (37). Consistent with their weakenedG1–

S checkpoint, p53kd (Fig. 2) andmutant (R273C; SupplementaryFig. S3) cells are more likely to enter their second S phase inAZD1775 than wild-type controls, and thus exhibit more damageas a population (Figs. 1 and 2; Supplementary Fig. S3).

In addition to this expected difference, we observed three moredifferential phenotypes. First, p53kd cells continue to experienceeffects of AZD1775 after its removal. This is most obvious in thesubpopulation of cells that has not had a chance to developgH2AX while in AZD1775 (Fig. 4F–H; Supplementary Fig. S5).This subpopulation undergoesmitosis and enters the next G1 on asimilar schedule in the wild-type and p53-deficient lines; how-ever, only in the latter it subsequently upregulates gH2AX. Inter-estingly, at this time, p53BP1 bodies are detected in control butless so in p53kd cells. It is possible that some type ofDNAdamagepersists or becomes detectable after AZD1775 but it is not prop-erly responded to by p53-deficient cells. Alternatively, the type ofdamage that persists in p53kd cells is invisible to 53BP1.While thelatter cannot be ruled out, comet assays suggest that both incontrol and p53kd cells the carryover damage may be derivedfrom SSBs and gaps (visible to 53BP1); and these are in fact moreprevalent inAZD1775-treatedp53kd cells (Fig. 2). p53BP1bodieshave been implicated as one of the contributors to the G1–Scheckpoint activation by p53 WT cells (38). However, to ourknowledge, virtually no evidence (with one exception; ref. 39)thus far points at p53 as a factor contributing to the formation ofthe 53BP1 bodies despite the original finding of associationbetween 53BP1 and p53 (40). Thus, our finding may suggest apreviously undetected interplay between p53 and 53BP1 inregulating the G1–S checkpoint in the aftermath of WEE1 inhi-bition in HNSCC.

WEE1 inhibition is known to slow replication fork progres-sion (12). By performing DNA fiber analyses on subpopulationsof cells, we found another differential phenotype of AZD1775-treated p53kd cells: while exhibiting the same gH2AX response ascontrols, they had a more severe replication fork slowing com-pared with their respective gH2AX-negative baseline (Fig. 5). Thisnovel observation can imply that on a cell-by-cell basis, gH2AXresponse to replication stress is actually dampened by the knock-down of p53, and/or that p53 facilitates fork progression understress. A stimulatory role of p53 in stressed fork progression wasfound by some studies (41, 42) but not others (43). Resistance to

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AZD1775-induced replication stress at a replication fork levellikely involves a RAD18–TLS polymerase kappa-dependent tol-erance pathway and a RAD51-dependent fork protection path-way (13). We propose that p53 may regulate a choice betweenthese pathways both directly (42, 43) and in a transcription-mediated manner (44).

Lastly, we observed that p53kd and p53 R273C-mutant cellswere more likely than the wild-type to undergo mitosis inAZD1775 despite an ongoing DNA-damage response (Fig. 3;Supplementary Fig. S3). This forced mitosis manifested asincreased prevalence of HH3þ cells, and moreover, it was asso-ciatedwith the appearance of a unique subset of gH2AXþþ/HH3þ

cells, as well as a higher percentage of micronuclei and abnormal

(lobed, broken)nuclei, which are typically associatedwithmitoticcatastrophe. We previously reported (23) that a small fraction ofgH2AXþþ/HH3þ p53-deficient cells displays less than 4c DNAcontent, i.e., enters mitosis with underreplicated DNA, and in thisstudy, we demonstrate that this fraction of cells in mitotic catas-trophe can be increased by interfering with DNA replicationduring AZD1775 treatment (Fig. 6). Similar findings have beenrecorded by other labs (10, 17, 20, 35), consistent with the notionthat p53 dysfunction weakens the G2–M checkpoint. This impactof p53 on the G2–M checkpoint can be conveyed through p21,whose mitotic role has come into focus recently (33, 34, 45). Ourdata (Fig. 3) and (10), indeed, suggest that p21 contributes topreventing theG2–Mcheckpoint override by AZD1775.However,

Figure 6.

Cotreatment with AZD1775 and triapine (3-AP) leads to mitosis with underreplicated DNA in a p53kd UM-SCC-74a cells. A, Colony formation of cells treated withthe indicated drug combinations. B, Change in proliferation relative to the untreated controls after treatment with 300 nmol/L AZD1775 with or without300 nmol/L 3-AP for 16 hours (n¼ 6). C, Flow-cytometric analyses of cells stained for gH2AX (top), H3S10P (bottom), and DNA content. Cells were treated with300 nmol/L 3-AP and/or 300 nmol/L AZD1775 for 16 hours. HH3þ cells with less than 4N DNA content are marked with a black arrow. D,Alkaline comet assaysperformed on cells treated with AZD1775 and/or 300 nmol/L 3-AP for 16 hours (n¼ 3). E, As in D, except nucleosides were added to the media during treatments(n¼ 3). F, Change in proliferation relative to the untreated controls after treatment with 300 nmol/LAZD1775 and 300 nmol/L 3-AP for 16 hours in the presenceor absence of nucleosides in culture media (n¼ 6).






the p53–p21 axis is unlikely the only route by which p53 mayregulate the onset ofmitosis. p53may also inhibit transcription ofAurora A, a kinase required for mitosis (46), or of FBW7, anegative regulator of Aurora B, another mitotic kinase (47).

p53-dependent mitotic deregulation has been reported pre-dominantly in cells derived from carcinomas of the breast, colon,and head and neck and is associated with p53 knockdown, TP53-null mutations, and, interestingly, only a subset of cancer-associated TP53 missense mutations (10, 20, 22, 35, 48). This

exposes a need for a better understanding of molecular activitiesof different p53 mutants in an otherwise isogenic context ofspecific cancer types as they respond to cell-cycle deregulation.

Interaction between replication stress and mitoticderegulation, and the insights into combination treatmentswith AZD1775

Our results are consistent with the notion that replication stressand mitotic deregulation are independent variables in the

Figure 7.

Cotreatment with AZD1775 andCDDP (CDDP) leads to forcedmitosis in p53kd UM-SCC-74a cells.A, Change in proliferation relativeto the untreated controls aftertreatment with 300 nmol/L CDDPwith or without 100 nmol/LAZD1775 for 16 hours (n¼ 5). B,Alkaline comet assays performedon cells treated with AZD1775 and/or 300 nmol/L CDDP for 16 hours(n¼ 3). C, Flow-cytometricanalyses of cells stained for gH2AX(top), H3S10P (bottom), and DNAcontent. Cells were treated with 1mmol/L CDDP and/or 300 nmol/LAZD1775 for 16 hours. HH3þ cellsseen despite an ongoing DNA-damage response in p53kd cells aremarked with a black arrow. D, Cell-cycle distribution of cells incubatedwith the indicated doses of CDDPand/or 300 nmol/L AZD1775 for 24hours and pulse-labeled with EdUfor 30minutes prior to harvest.EdU-negative (orange) and-positive (blue) populations aregraphed separately to distinguishG1, G2, and S phase cells. E,A heatmap of the p53 Signaling PathwayRT2 Profiler PCR array analysis ofcells treated with no drug or with1 mmol/L CDDP for 24 hours. F, Aquantitation of flow-cytometricanalyses of cleaved PARP1 inuntreated cells and cells treatedwith 300 nmol/L AZD1775 and 1mmol/L CDDP combination for16 hours (n¼ 3).

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response to AZD1775, and that replication stress is not sufficientto confer p53 sensitivity to AZD1775 in HNSCC. First, HH3-positive and HH3-negative p53kd cells exhibited similar levels ofreplication stress as measured by their gH2AX expression and theextent of their replication fork slowing (Fig. 5), which suggeststhat a property other than the severity of replication stress deter-mined whether these cells underwent mitosis. Second, relievingreplication stress by supplementation of extra nucleosides in themedia did not improve survival of p53kd cells (in contrast tocontrols), while reducing such telltale symptom of replicationstress as SSBs in DNA (Figs. 5 and 6). The ability of nucleosides toimprove replication fork progression and suppress phenotypes ofAZD1775-induced replication stress is well documented (12, 35).

At the same time, it is clear that replication stress can bemanipulated towork together withmitotic deregulation in p53kdcells in order to boost cytotoxicity. For example, combiningAZD1775 with a relatively novel, therapeutically relevant repli-cation blocker triapine (3-AP), increased premature mitosis ofunderreplicatedDNAand additively suppressed survival of p53kdHNSCC cells (Fig. 6).

A standard-of-care chemotherapy for HNSCC, CDDP repre-sents another approach to boosting cell killing by AZD1775.CDDP does not prevent complete replication of the genome butslows it through engagement of the S phase checkpoint. CDDPinduces cell death or senescence, and successful repair of CDDPdamage heavily relies onhomologous recombination in the S andG2phases of the cell cycle and thus depends on a functionalG2–Mcheckpoint (49). In agreement with this, we observed that whentreated with CDDP, p53kd cells (incapable of G1 arrest) showed agreater induction of S and G2–M damage checkpoints than con-trols (Fig. 7). AZD1775 overrode this response, with concomitantinduction of mitosis and PARP1 cleavage indicative of apoptosis.Together, these molecular phenotypes explain synergistic inter-action between CDDP and AZD1775 that we observed in p53kdHNSCC.

Our results are similar, though not identical to the findings ofOsman and colleagues (48). For example, p53-defective cells werenot hypersensitive to AZD1775 alone in the PCI-13 backgroundused by the authors, while we detected sensitization of the UM-SCC74a cells by p53 depletion or inactivation. The variability islikely an example of cell line heterogeneity; nevertheless, takentogether the results point to a promising therapeutic potential ofcombining the standard-of-care CDDP with WEE1 inhibition inp53-defective HNSCC.

Intrapopulationheterogeneity of responses toAZD1775:Ap53-independent phenotype

We observed significant heterogeneity in the gH2AX responseof cells to AZD1775 and demonstrated that it is associated withmajor functional differences among the cells. In particular, wedetected a pronounced negative correlation between cellulargH2AX levels and rates of replication fork progression, which

brings up the question why subsets of cells experience higher orlower replication stress in AZD1775. Because gH2AX-negativecells in AZD1775 typically belonged to early S phase, we hypoth-esize that their gH2AX-negative state is linked to an intrinsicallylow level of CDK1 at this point of the cycle. Alternatively, a criticalstress-signaling lesion may be slow to accumulate in these cells.

ExtremeoveractivationofCDK1 (andpossiblyCDK2)may alsohelp explain why a subset of gH2AX-positive cells (gH2AXþþ) inmid to late S phase go on to manifest extremely high replicationstress (Figs. 1, 2, and 5; Supplementary Figs. S1 and S2). Indeed, agH2AX-positive subpopulation with similar properties was pre-viously observed upon overexpression of CDK1/2-activatingphosphatase, CDC25A (50). If so, it will be of interest to under-stand how and why some cells within a population suffer a moresevere overactivation of CDK1/2 than others, and find ways toutilize this knowledge in cancer therapy.

In summary, our high-resolution analysis of head and neckcarcinoma cells' complex response to WEE1 inhibition hashighlighted both the known and the previously uncharacter-ized deficiencies associated with p53 inactivation, and outlinedspecific directions for further inquiry and for therapeuticexploitation.

Disclosure of Potential Conflicts of InterestE. Mendez received commercial research grants and other commercial

research support from AstraZeneca. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: A. Diab, M. Kao, K. Kehrli, J. Sidorova, E. MendezDevelopment of methodology: A. Diab, M. Kao, J. Sidorova, E. MendezAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Diab, M. Kao, K. Kehrli, H.Y. Kim, J. SidorovaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Diab, M. Kao, J. Sidorova, E. MendezWriting, review, and/or revision of the manuscript: A. Diab, J. Sidorova,E. MendezAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H.Y. KimStudy supervision: J. Sidorova, E. Mendez

AcknowledgmentsThis workwas supported by theNIH/NCI grant R01 CA215647 to E.Mendez

and J. Sidorova, Seattle Translational Tumor Research (STTR) programmaticinvestment grant to A. Diab, and University of Washington Royalty ResearchFund pilot grant to J. Sidorova. This research was also supported by the CellularImaging and Therapeutic Manufacturing Shared Resources of the Fred Hutch/University of Washington Cancer Consortium (P30 CA015704).

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 August 12, 2018; revised November 27, 2018; accepted January 17,2019; published first January 24, 2019.

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Diab et al.




2019;17:1115-1128. Published OnlineFirst January 24, 2019.Mol Cancer Res Ahmed Diab, Michael Kao, Keffy Kehrli, et al. Cells to the WEE1 Kinase InhibitionMultiple Defects Sensitize p53-Deficient Head and Neck Cancer

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MultipleDefectsSensitizep53-DeﬁcientHeadand Neck Cancer ... · slide scanner (Zeiss AxioImager Z2 upright) microscope with a 20 objective. Automated image analysis for QIBC utilized