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1 Multiple defects sensitize p53-deficient head and neck cancer cells to the WEE1 kinase inhibition Ahmed Diab 1 , Michael Kao 2 , Keffy Kehrli 3,5 , Hee Yeon Kim 1 , Julia Sidorova 3,6, * and Eduardo Mendez 1,2,4,6 . 1 Clinical Research Division, Fred Hutchinson Cancer Research Center 2 Department of Otolaryngology: Head and Neck Surgery, University of Washington 3 Department of Pathology, University of Washington, Seattle, Washington, USA 4 Seattle Cancer Care Alliance, Seattle, WA 5 Present address: Genetics program, Stony Brook University, Stony Brook, New York, USA 6 Co-senior authors Running title: p53 loss compromises response to WEE1 inhibition * Corresponding Author: Julia Sidorova, Department of Pathology, P.O. Box 357705, 1959 NE Pacific St., Seattle, WA 98155; phone (206) 616-3189; [email protected] Keywords: head and neck cancer, WEE1, TP53, cell cycle, mitosis, replication stress, DNA damage. Abbreviations: HNSCC, head and neck squamous cell carcinoma. E. Méndez received commercial research grants and other commercial research support from AstraZeneca. The authors declare no other potential conflicts of interest on March 23, 2021. © 2019 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 24, 2019; DOI: 10.1158/1541-7786.MCR-18-0860

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Multiple defects sensitize p53-deficient head and neck cancer cells to the WEE1 kinase

inhibition

Ahmed Diab1, Michael Kao2, Keffy Kehrli3,5, Hee Yeon Kim1, Julia Sidorova 3,6, * and Eduardo

Mendez1,2,4,6.

1 Clinical Research Division, Fred Hutchinson Cancer Research Center

2 Department of Otolaryngology: Head and Neck Surgery, University of Washington

3 Department of Pathology, University of Washington, Seattle, Washington, USA

4 Seattle Cancer Care Alliance, Seattle, WA

5 Present address: Genetics program, Stony Brook University, Stony Brook, New York, USA

6 Co-senior authors

Running title: p53 loss compromises response to WEE1 inhibition

*Corresponding Author: Julia Sidorova, Department of Pathology, P.O. Box 357705, 1959 NE

Pacific St., Seattle, WA 98155; phone (206) 616-3189; [email protected]

Keywords: head and neck cancer, WEE1, TP53, cell cycle, mitosis, replication stress, DNA

damage.

Abbreviations: HNSCC, head and neck squamous cell carcinoma.

E. Méndez received commercial research grants and other commercial research support from

AstraZeneca. The authors declare no other potential conflicts of interest

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Abstract:

The p53 gene is the most commonly mutated gene in solid tumors but leveraging p53

status in therapy remains a challenge. Previously, we determined that p53 deficiency sensitizes

head and neck cancer cells to AZD1775, a WEE1 kinase inhibitor, and translated our findings

into a Phase I clinical trial. Here we investigate how p53 affects cellular responses to AZD1775

at the molecular level. We found that p53 modulates both replication stress and mitotic

deregulation triggered by WEE1 inhibition. Without p53, slowing of replication forks due to

replication stress is exacerbated. Abnormal, H2AX-positive mitoses become more common

and can proceed with damaged or under-replicated DNA. p53-deficient cells fail to properly

recover from WEE1 inhibition and exhibit fewer 53BP1 nuclear bodies despite evidence of

unresolved damage. A faulty G1/S checkpoint propagates this damage into the next division.

Together, these deficiencies can intensify damages in each consecutive cell cycle in the drug.

Implications: The data encourage the use of AZD1775 in combination with genotoxic modalities

against p53-deficient HNSCC.

Introduction

Resistance 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 association

between loss of the p53 tumor suppressor and the emergence of resistance. HNSCCs have a

very heterogeneous mutational landscape with few shared oncogenic mutations excepting the

p53 gene (TP53), where mutations were noted in up to 72% of tumors (TCGA Research

Network: http://cancergenome.nih.gov/." , (1)) Despite the central role of TP53 in HNSCC

carcinogenesis (2), to date no standard-of-care therapy leverages the tumor’s p53 status, albeit

preclinical work and clinical trials are bringing this goal closer (3-5). We have previously found

that inhibition of the cell cycle kinase WEE1 with a small molecule AZD1775 is significantly

more cytotoxic to p53-mutated than to p53 wt HNSCC cell lines (6). Also, we recently completed

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a phase I trial of AZD1775 in combination with CDDP and docetaxel in HNSCC, which showed

very promising results for patients with mutant or HPV-inactivated p53 (7). Our goal is to

understand how p53 deficiency sensitizes HNSCC cells to AZD1775 as a single agent or in

combination with genotoxic modalities.

WEE1 controls S phase and mitosis via inhibitory phosphorylation of cyclin-dependent

kinases CDK2 and CDK1, respectively. Upon DNA damage or replication blockage, the ATM –

CHK2 and/or ATR – CHK1 checkpoints block mitosis by acting on WEE1 and CDK1, thus

allowing cells to complete DNA replication and repair. Inhibiting WEE1 can compromise the

checkpoint, leading to forced mitosis and mitotic catastrophe (8-10). WEE1 inhibition also

overactivates CDK2 during S phase, inducing replication stress through excessive initiation of

replication and exhaustion of supplies of dNTPs, concomitant stalling of replication forks, and

breakage of nascent DNA (11-13). Upon WEE1 inhibition, hyperactivation of CDK1/2 also

suppresses RRM2 expression, exacerbating dNTP depletion (14), while precocious activation of

CDK1 and PLK1 in S phase causes cleavage of stalled replication forks by the prematurely

activated MUS81 endonuclease complex, MUS81/SLX4 (15). The cytotoxic effect of WEE1

inhibitor AZD1775 as a single agent is often attributed to induction of replication stress (16).

The prominence of mitotic and S-phase responses to AZD1775 and their relative

contributions to the drug’s cytotoxicity may differ depending on the cancer cells’ rewiring of the

cell cycle regulatory circuitry. Studies document different responses to AZD1775 in cell lines

derived from sarcomas, carcinomas, leukemias, and other cancers (17-21). In some studies S-

phase arrest followed by addition of AZD1775 promoted premature mitosis and cell death in the

absence of p53 (8-10). However, in a study by Guertin et al (22) induction of DNA damage in S-

phase, not premature mitosis, correlated with cytotoxicity of WEE1 inhibition in a panel of cell

lines, and this effect was not dependent on the p53 status. Similarly, Van Linden et al (18) noted

no sensitization of AML lines to AZD1775 upon p53 inactivation.

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Focusing on HNSCC cell models and isolating for p53-specific effects with an isogenic

cell line pair, we previously reported p53-independent replication stress and p53-dependent

unscheduled mitosis in an AZD1775-treated HNSCC cell line (23). Here, by following specific

subpopulations of cells through more than one cell cycle, we reveal novel and confirm known

p53-specific phenotypes in the response to WEE1 inhibition. Our results support the conclusion

that an interplay of replication stress and G1/S and G2/M checkpoint failures can explain

sensitivity of p53-deficient cells to AZD1775, and will help to optimize therapeutic window when

targeting p53-mutated HNSCC.

Materials and Methods

Cell lines, vectors, and RNAi: Primary fibroblast cells (HFF4) were described previously (24).

Head and neck cancer cell lines UM-SCC-74a was from Dr. Carey at University of Michigan

(Ann Arbor, MI). Cells were used within one to three months after thawing, and tested for

mycoplasma contamination prior to cryopreservation or upon thawing. We used a pBabeHygro

retroviral vector expressing shRNA targeting p53 (25) (a gift from Dr. Kemp) to generate a

stable cell line with depleted p53 protein under hygromycin selection. siRNAs against p21

(CDKN1A) were from Qiagen (#SI00604898 and # SI00604905) and a non-targeting control

siRNA (#D-001810-01-05) was from Dharmacon.

Drugs and chemicals: AZD1775 was provided by AstraZeneca through a collaborative

agreement. CDDP (P4394) and Triapine (3-AP, SML0568) were purchased from Sigma-Aldrich.

EmbryoMax® Nucleosides (ES-008-D, EMD Millipore) were used at a final concentration of

1:25. 5-Iododeoxyuridine (IdU) and 5-chlorodeoxyuridine (CldU) were from Sigma-Aldrich and

used at 50uM from stock solutions of 2.5mM in PBS, and 10mM in PBS, respectively.

Antibodies: Antibodies used were to -H2AX (Ser139, JBW301 #05-636), p-HH3 (Ser10, 3H10,

#05-806) from EMD Millipore; to p-HH3 (Ser10, D2C8, #3377), p21 Waf1/Cip1 (12D1, #2947),

cleaved PARP (D214, #9541), and β-Actin-HRP (13E5, #5125) from Cell Signaling Technology;

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to P53BP1 (E-10, #sc-515841) and p53 (DO-1, #sc-126) from Santa Cruz, and to nucleolin (#

396400) from Life Technologies/Thermo Fisher. PE-conjugated Anti-Cleaved PARP antibody

(Asp214 #51-9007684) was from BD Pharmingen. Antibody to IdU/BrdU (B44, #347580) was

from BD Pharmingen and to CldU/BrdU (BU1/75 (ICR1), #OBT0030) from Bio-Rad / AbD

Serotec.

Flow cytometry: Cells were fixed and processed for flow cytometry as described previously

(24). Samples were run on FACS Canto II. FACS profiles were visualized using FACS Express

software (DeNovo).

DNA fiber assays on sorted cells (Sorted Microfluidics-assisted Replication Track Analysis or

SmaRTA): Approximately 5x106 cells were fixed in 2% formaldehyde in PBS for 10 min at 37°C

and stained with H2AX and histone H3S10P 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. Sorted cells were pelleted after supplementing PBS with

0.3% BSA, resuspended in Agarose plug buffer, embedded in agarose, and processed as

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). QIBC was performed as described in (28) with the

following modifications: images were captured using TissueFAXS, an automated slide scanner

(Zeiss AxioImager Z2 upright) microscope with a 20X objective. Automated image analysis for

QIBC utilized TissueQuest software.

TP53 signaling pathway PCR Array: The 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 uM CDDP for 24h. Cells were harvested and processed according to the

manufacturer's instructions.

Comet assays: DNA strand breaks were measured using a kit following manufacturer’s

instructions (Trevigen). For each experimental condition, “tail moments” (defined as the product

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of tail length and the fraction of total DNA in the tail) were determined for at least 500 nuclei

using ImageJ software (National Institutes of Health) with the Open Comet plugin. At least 2

independent experiments were scored.

Cell growth and viability assays: Cell proliferation after drug washout was conducted as

described previously (29). Alternatively, 103 cells were seeded into 96-well plates and were

subjected to the same treatment for 24 hours. After drug removal, media was replenished and

cells were allowed to grow for 96 hours before analysis with CellTiter-Glo (Promega), following

the protocol outlined by the manufacturer.

Synergy analysis for drug combinations: 103 cells were seeded into 96-well plates and treated

with serial dilutions of the respective drugs for 96 hours. Cell viability was assessed with

CellTiter-Glo (Promega) and the fraction affected was used to calculate the combination index (CI)

and isobologram analyses according to the median-effect method of Chou and Talalay (30)

using the CalcuSyn software (Biosoft, Ferguson, MO).

Statistical analyses: Un-paired t-tests were carried out in GraphPad Prism 7 software to analyze

in vitro data. All data were expressed as mean ± SD or (, ± SEM for large data sets) and P

values were indicated. SmaRTA data were analyzed in K-S tests using R studio software, and P

values and, in some cases, D statistics are shown.

Results

Cell cycle progression and DNA damage/replication stress response in WEE1 inhibitor-treated

HNSCC cells.

Phenotypes elicited by a therapeutically relevant dose of WEE1 inhibitor AZD1775 were

first demonstrated in the TP53 wild type HNSCC line UM-SCC-74a. Pulse-chase labeling of

cells with EdU and flow cytometry of EdU incorporation versus DNA content allows to see how

EdU-positive cells progress through the cell cycle for up to two consecutive S phases (Fig.1A,

S1 and S2 in Fig.1B). A majority of cells that were exposed to AZD1775 while in the S1,

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completed it and transited to G1 by 9.5 hrs after EdU pulse, similar to untreated cells (Figure

1B, C). The entry of the EdU+ population into the S2 and/or transit through it was delayed in

WEE1-inhibited cells. In order to follow cell cycle progression of AZD1775-treated cells past S1

more precisely, we consecutively labeled these cells with IdU and EdU as shown in Fig. 1C-E.

The dual-labeled cells should be the ones that remained in S1 phase for over 8 hrs. We

reasoned that these cells may be the most severely affected by AZD1775, and following their

progression will reveal the strongest response to AZD1775. Fig. 1D confirms the specificity of

dual staining. Without the drug, majority of IdU/EdU+ cells completed S1 and S2 phases, while

with the drug these cells slowed a delay traversing through S2 (Fig. 1E). Together, the data

suggest that response to WEE1 inhibition is heterogeneous and depends on the time in the

inhibitor, indicating an accumulation of damage over more than one cell cycle.

We next measured H2AX level as a function of DNA content and time in AZD1775.

H2AX accumulated over the course of 24 hrs, with subsets of cells remaining H2AX-negative

(Fig. 1F upper panels). We also saw late-onset accumulation of extra-high level of H2AX

(H2AX ++) in a subset of mid S phase cells (2-3% of all cells at 8.5hr and 10-14% of all cells at

24.5hr, orange profiles, Fig. 1F histograms). Appearance of H2AX ++ cells was not unique to

the UM-SCC-74A line, as it was also observed in human primary fibroblasts and normal oral

keratinocytes (Fig. S1A) as well as in another TP53 wild type HNSCC line, UM-SCC-81A

(Fig.S2E).

Tracking development of H2AX by following EdU+ cells through the cycle (Fig. 1F,

middle panels and histograms), we saw that early S phase cells were H2AX-, and developed

H2AX signal as they reached mid S. A small subset of EdU+ cells was H2AX- as they reached

G2 and traversed into G1. H2AX level negatively correlated with the rate of progression

through S phase. 92% of H2AX++ population at 8.5 hr in AZD1775 were EdU+ cells in their S1

phase in AZD1775. At 24.5 hrs in AZD1775 there were more H2AX++ cells overall and only

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65% of them were EdU+ suggesting an accumulation of cells in the H2AX++ compartment over

time (Fig.1F and data not shown).

By labeling cells with EdU after 24 hrs in AZD1775, we found that H2AX++ cells

incorporated about 10 times less EdU than H2AX+ cells in S phase (Fig. 1G, histograms,

compare orange and black profiles). H2AX++ cells also expressed extra-high level of

CHK1S345P (Fig. S1B). Thus, these cells represent an S phase subpopulation with severely

inhibited DNA synthesis and highly upregulated replication stress response. Staining with an

antibody against CDK1/2Y15P confirmed WEE1 inhibition (Fig.S2A-D) and showed that at least

in some cell lines H2AX++ cells had a lower level of Y15 phosphorylation than H2AX+ cells,

potentially suggesting greater hyperactivity of CDK1/2 (Fig. S2D, E). However, this lower

staining for CDK1/2Y15P may be due to the fact that H2AX++ cells are exclusively in mid S

phase whereas H2AX+ cells can be in the late S/G2, and CDK1/2Y15P staining is normally

lower in mid S compared to G2 (Fig.S2D).

Depletion of p53 exacerbates the response to AZD1775.

We next depleted p53 in UM-SCC-74a background using shRNA (Fig. 2A). As expected,

p53 depletion sensitized cells to AZD1775 (Fig. 2B). Depletion of p53 caused greater

accumulation of H2AX+ and H2AX++ cells in AZD1775 (Fig. 2C,D). By immunofluorescence

(IF) in situ (Fig. 2E), H2AX++ expression level corresponded to extremely bright staining, either

pan-nuclear or localized to numerous foci. Alkaline comet assays indicated that ss- and dsDNA

breaks were elevated in AZD1775-treated p53 knockdown (kd) cells (Fig. 2F). Also, PARP1

cleavage (an apoptotic marker) was elevated in these cells (Fig. 2G,H).

Flow cytometric analyses of H2AX development and cell cycle progression showed that

AZD1775 sped up the traversal of cells out of S1 and through G2/M compared to untreated

controls (Fig.2J, e.g. compare 4 and 8hr time points with and without AZD1775). Compared to

controls, p53kd cells had a higher G2 fraction both with and without AZD1775. Also, more

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p53kd cells than controls entered their 2nd S phase (S2) since the start of the time course. The

HNSCC cell line PCI-15b line harboring a high-risk TP53 mutation (R273C) showed a similar

behavior (Fig.S3A, B).

Both p53kd cells and controls developed some H2AX upon entry into S2 (Fig. 2I,J).

Moderate expression of H2AX during S phase is not unusual for cancer cell lines. Most

importantly, AZD1775 treatment delayed entry of control cells into S2, while p53kd cells entered

S2 and expressed high level of H2AX upon entry, as indicated by the appearance of an EdU-

positive, H2AX + and ++ population at 24hrs in the drug (Fig. 2I). Lastly, pulse-labeling cells

with EdU after a prolonged treatment with AZD1775 showed that both p53kd and control cells

developed a mid-S phase population with severely depressed DNA synthesis (compare Fig. 2K

with Figs. 1G and S1A). Overall, the data suggest that p53-deficiency is associated with greater

replication-associated damage upon AZD1775 treatment. At least part of this phenotype can be

attributed to a failure of p53kd cells to activate the G1/S checkpoint and thus avoid an entry into

their second S phase in the presence of the drug.

AZD1775 increases prevalence of mitosis in p53kd cells.

WEE1 inhibition not only causes replication stress but also stimulates and in some cases

advances mitosis (10, 31). To explore this facet of WEE1 inhibition in more detail, we stained

cells for histone H3S10P modification as a marker of mitosis (Fig. 3). Correlation between the

level of H3S10P staining and mitotic condensation and alignment of chromosomes was verified

by IF (Fig. 3A). Of note, H3S10P level increased gradually in UM-SCC-74a cells and preceded

visible chromosome condensation, suggesting that only the highest level of H3S10

phosphorylation identifies mitosis.

High histone H3S10P-staining (HH3+) cells were more prevalent in p53kd cells

compared to controls (Fig. 3B). AZD1775 markedly increased HH3+ abundance, particularly in

p53-depleted cells (Fig. 3B, C). A minor fraction of HH3+ cells in the p53kd line also appeared

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to have < 4c DNA content (Fig 3B). Notably, in the control a vast majority of cells was either

H2AX+ or HH3+, whereas double-positive, HH3+/H2AX+ cells were detectable in p53kd cells

(Fig. 3D-G).

These differences were recapitulated in the p53-mutant PCI-15b cell line (Fig. S3C, D).

In particular, basal and AZD1775-induced percentage of HH3+ cells was higher in these p53

mutant cells than in a p53 wild type line, and a higher proportion of HH3+ cells was also H2AX-

positive. Furthermore, inactivation of p53 in UM-SCC-74a cells by expressing E6 protein of the

HPV16 virus (32) recapitulated AZD1775-associated phenotypes displayed by p53kd cells,

including suppression of growth, increased PARP1 cleavage, and elevated H2AX and

HH3+/H2AX+ cells (Fig.S4A-D). Thus, the data consistently show that induction of H2AX

expression and mitosis by WEE1 inhibition are more pronounced if p53 is altered, and second,

that p53 deficiency correlates with the presence of mitotic H2AX-positive cells. This feature

may mark a specific vulnerability of p53-defective cells to WEE1 inhibition and/or may serve as

a biomarker of WEE1i sensitivity.

The impact of p53 on the G2/M checkpoint can be conveyed through p21, a CDK

inhibitor inducible by p53 (33, 34). Indeed when we depleted p21 in p53 wild type cells, we

observed an increase in H2AX, HH3+, and HH3+/H2AX+(++) cells, which negatively

correlated with p21 level (Fig. 3H-J). p21 depletion also exacerbated reduction of p21 levels

observed in p53kd cells, and increased H2AX-positive mitoses, albeit less markedly than in

p53 wild type cells (Fig.3H-J).

Recovery from AZD1775 triggers additional H2AX induction in p53kd cells

We next asked whether recovery from AZD1775 was affected by p53 status. Cells were

incubated with AZD1775 and then allowed to recover for up to 24 hrs (Fig. 4A), and analyzed by

IF in situ and QIBC. p53kd cells had a higher percentage of H2AX-positive cells throughout the

recovery (Fig. 4B). Over 30% of p53kd cells scored H2AX-positive for 8 hrs after removal of

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AZD1775 versus 5% of control cells. Moreover, a persistently higher fraction of H2AX-positive

p53kd cells displayed aberrant, severely misshapen or fragmented nuclei, consistent with failed

segregation (Fig. 4C,D). However, this nuclear fragmentation was low overall, occurring in less

than 10% of H2AX-positive subpopulations throughout recovery.

We also pulse-labeled cells with EdU 1 hr prior to release in order to mark replicating

cells and follow them through recovery. H2AX, EdU, and H3S10P signal intensities were

manually collected in digital images for specific cell categories, i.e. mitotic, premitotic, or

H2AX++ (Fig. S5A). This allowed us to define a range of signal intensities that was associated

with a particular category. In parallel, we also collected H2AX, H3S10P, and EdU signal

intensities for thousands of individual nuclei using automated image acquisition and analysis.

We determined the distributions of H2AX and histone H3S10P signal intensities in cells

that displayed none/low (termed EdU-) or high EdU incorporation when labeled just prior to

release from AZD1775 (Fig. 4E). EdU- cells correspond to mid S phase H2AX++ cells as well

as cells that were outside of S phase at the moment of labeling (Figs. 1G,H, 2K). These cells

most frequently displayed high levels of H2AX (signal intensity >125) consistent with H2AX++

status. The prevalence of these cells and their signal intensity declined over time in control but

less so in p53kd cells. The majority of EdU- cells were mitotic (H3S10P signal >75) in AZD1775

and before 4 hr of recovery, with subsequent wave of mitoses developing at 24 hrs of recovery

in both control and p53kd cells. Based on the overall EdU levels in the populations, between 12

and 24 hrs of recovery most of control and p53kd cells have divided once (Fig. S5A)

EdU-high signal is consistent with cells in early to mid S phase at the point of release

from AZD1775, and these cells were overwhelmingly H2AX-negative in both cell lines

(consistent with the data in Fig. 1F, G). Mitoses in these cells peaked at 8 to 12 hrs of recovery,

which was predictably later than mitoses in the EdU-negative cells. Interestingly however, in the

p53kd line, cells that were virtually H2AX-negative in AZD1775 began to develop H2AX signal

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at 24 hrs after removal of the drug. This development followed completion of mitosis by several

hours and thus is consistent with the entry of the EdU-high subpopulation into the next S phase.

An independent experiment confirmed that EdU-high population of p53kd cells increased its

H2AX level at 24hrs compared to the 0hrs after the drug, unlike the mock-depleted controls

(Fig. S5B). Overall, the findings suggest that early S phase cells that display virtually no H2AX

response in AZD1775 nevertheless incur some kind of damage. In p53-deficient background

this damage persists for hours after AZD1775 removal, and is revealed at the time point 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 recovering from AZD1775. While in AZD1775, neither cell line

had 53BP1 signal above background, consistent with other studies (35). At 24hrs of recovery, a

subset of control cells clearly displayed elevated 53BP1 signal (Fig. 4G, H). Remarkably, p53kd

cells displayed lower 53BP1 signal. In both cell lines the majority of 53BP1-positive cells did not

express H2AX, arguing against 53BP1 colocalization with DSBs. Thus, both control and p53-

deficient cells retain unresolved damage after mitosis, however, p53-deficient cells have an

altered response to it.

H2AX level in AZD1775-treated cells correlates with replication fork slowing

Our data suggest that H2AX++ cells represent a qualitatively distinct subpopulation with

severe replication stress (Figs. 1G, 2K and Fig. S1). WEE1 inhibition by AZD1775 is known to

cause replication fork slowing and stalling (12). We wanted to determine whether severe

replication stress in the H2AX++ population corresponded to the slowest forks. We were also

interested to see if p53 status affected fork response to AZD1775, and if H2AX++/HH3+ double

positive cells were quantitatively more affected than H2AX++/HH3- cells, reasoning that

premature mitosis of H2AX++/HH3+ cells may be stimulated by their complete inhibition of

replication, as suggested in (15).

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To answer these questions, we devised a combination of flow sorting and DNA fiber

analysis (Fig. S6). We sequentially labeled cells with CldU and IdU (with or without AZD1775) to

mark ongoing replication forks, immunostained these cells with antibodies against H2AX and

H3S10P, and flow-sorted them. H2AX-, + and ++ fractions were obtained for all cell lines. For

p53kd cells, the H2AX++ fraction was subdivided into HH3+ and HH3- populations (Fig. 5A).

DNA was isolated from these fractions and subjected to our DNA fiber stretching

protocol, maRTA (Fig. 5B) (27). AZD1775 treatment slowed fork progression in all cells, but as

we expected, fork progression rate negatively correlated with H2AX level (Fig. 5C, D),

confirming that H2AX++ cells experience the highest level of replication stress. In addition,

H2AX+ subpopulation of p53kd cells had slower forks compared to H2AX+ controls, and

overall, p53kd cells displayed a more pronounced reduction in fork progression in H2AX-

positive compared to H2AX-negative subpopulations (Fig. 5E). Interestingly, fork progression in

H2AX++/HH3+ cells was not slower but in fact faster than in H2AX++ cells (Fig. 5C, D). The

data suggest that, while p53 status affects the severity of replication stress at the replication fork

level, the p53kd-specific H2AX++/HH3+ cells do not exhibit higher replication stress than

H2AX++/HH3- cells. Thus, the level of replication stress/suppression of replication alone

cannot explain the prevalence of this double-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 an independent test, we asked if survival of p53kd

cells was improved by supplementation of the media with extra nucleosides. Such

supplementation is known to alleviate WEE1i-induced replication stress (12, 14). Indeed,

addition of nucleosides improved survival of the control but not p53kd UM-SCC-74a cells (Fig.

5F).

Synthetic lethality of AZD1775 in combination with CDDP or triapine

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Only subsets of p53kd populations exhibited extreme dysfunction or cytotoxicity when

exposed to the clinically safe doses of AZD1775 in the experiments above. In order to amplify

cytotoxic outcome, and given that AZD1775 is being tested in combination with chemotherapy in

clinical trials, we next explored the effects of combining AD1775 with other drugs.

Triapine (3-AP) is a potent inhibitor of ribonucleotide reductase (RNR) currently in

clinical trials. A phase II study of 3-AP in metastatic HNSCC noted that the drug was well

tolerated but was not effective as a single agent (36). We reasoned that 3-AP can exacerbate

AZD1775-induced replication stress. On the other hand, CDDP, a standard of care drug for

HNSCC, is a DNA crosslinker. In this case, the ability of AZD1775 to override the G2/M DNA

damage checkpoint induced by CDDP may enhance cell killing if the two drugs are combined.

3-AP and CDDP used at doses that had minimal to no effect on their own, suppressed

colony formation when combined with AZD1775 (Fig. 6A). Proliferation of AZD1775/3-AP-

treated cells was more affected in the p53kd line than in the control (Fig. 6B), and the drugs

showed weak (additive) interaction in p53kd cells and not in controls (isobologram analysis in

Table S1). The 3-AP dose that had no effect on H2AX and H3S10P as a mono-therapy,

dramatically enhanced H2AX expression and virtually arrested S phase both in p53kd and

control cells when combined with AZD1775 (Fig. 6C). While 3-AP/AZD1775 combination and

AZD1775 alone increased the prevalence of cells with low to intermediate H3S10P levels (Fig.

6C right panels), corresponding to premitotic cells (as before, see Fig. 4), only in p53kd cells did

these drugs markedly induced premature mitosis (Fig. 6C, note the HH3+ high /<4c DNA

content cell population marked by an arrow).

3-AP/AZD1775 combination dramatically increased single- and double-strand breaks

(SSBs and DSBs) in the DNA of p53kd cells (Fig. 6D). It is unlikely that these breaks were

associated only with cells undergoing premature mitosis, as the latter comprised only a minor

fraction (2.2% on average) of the entire population. Addition of extra nucleosides completely

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suppressed these breaks in the control and largely suppressed them in p53kd cells (Fig. 6E), as

well as improved survival of control but not p53kd cells (Fig. 6F), in line with the findings in Fig.

5F.

We next looked into the effect of the CDDP/AZD1775 combination (Fig. 7). The

combination had a more severe growth-suppressive effect and triggered a greater accumulation

of SSBs and DSBs in p53kd cells compared to controls (Fig.7A, B). Isobolograms of the

combination showed a synergistic interaction for p53kd cells and no interaction for controls

(Table S2). AZD1775 and CDDP synergized in inducing H2AX (Fig.7C), and suppressed

mitosis in the control but not in p53kd cells (HH3+cells in Fig.7C). In order to visualize cell cycle

distribution in the CDDP/AZD1775-treated cells, the isogenic pair was treated with the drug

combinations and then pulse-labeled with EdU prior to harvest (Fig.7D). p53kd cells had a far

greater accumulation of S and G2 cells in CDDP that the control, but in both cases addition of

AZD1775 together with CDDP overrode it. However, unlike in the control, the combination

triggered appearance of cells that had S phase-like DNA content but failed to incorporate EdU

(about 10% of total population).

A greater effect of CDDP on accumulation of cells in S and G2 in p53kd line prompted

us to measure contribution of p53 to the activation of the G2/M checkpoint regulators. We

profiled expression of p53 pathway genes w/out CDDP treatment in the control and p53kd UM-

SCC-74A cells (Fig. 7E). p53kd cells showed a markedly higher expression of S and G2/M

checkpoint regulators (WEE1, CHK1/2, CDC25A), DNA damage response (BRCA1/2) and

replication (PCNA, MLH1, MSH2, CDK2, CCNE1) genes upon treatment with CDDP (right

panel, Fig 7E), which agrees with a more pronounced accumulation of these cells in S/G2

(Fig.7D). As expected, p53kd cells failed to activate pro-apoptotic genes (BAX, FAS, CASP9,

etc., left panel, Fig. 7E). This confirms a greater G2/M engagement at the transcriptional level

upon genotoxic stimuli specific to p53 depletion, and is in agreement with the inability of p53kd

cells to escape from the CDDP- and AZD1775-vulnerable S and G2 phases by arresting in the

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G1 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 that enhance replication stress or

inflict DNA damage in the setting of WEE1 inhibition can enhance cytotoxic outcome in p53-

deficient cancer cells in positive correlation with, respectively, premature or forced mitosis.

Discussion

Differential effects of p53 deficiency

WEE1 inhibition by AZD1775 elicits both p53-dependent and independent phenotypes,

some of which develop over a course of more than one cell cycle. For instance, S phase-

associated DNA damage triggered by WEE1 inhibition (as revealed by H2AX expression), is

more prevalent in the second S phase of the time course of incubation with the clinically

relevant dose of the drug. This suggests a carryover of a lesion or a particular cellular state from

one cell cycle to the next. Interestingly, a recent study showed that WEE1 inhibition can result in

an elevated CDK1 activity persisting throughout the G1 phase, which may affect DNA repair and

the G1/S transition (37). Consistent with their weakened G1/S checkpoint, p53kd (Fig. 2) and

mutant (R273C, Fig.S3) cells are more likely to enter their second S phase in AZD1775 than

wild type controls, and thus exhibit more damage as a population (Figs. 1, 2, S3).

In addition to this expected difference, we observed three more differential phenotypes.

First, p53kd cells continue to experience effects of AZD1775 after its removal. This is most

obvious in the subpopulation of cells that has not had a chance to develop H2AX while in

AZD1775 (Fig.4F-H, Fig.S5). This subpopulation undergoes mitosis and enters the next G1 on a

similar schedule in the wild type and p53-deficient lines, however, only in the latter it

subsequently upregulates H2AX. Interestingly, at this time p53BP1 bodies are detected in

control but less so in p53kd cells. It is possible that some type of DNA damage persists or

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becomes detectable after AZD1775 but it is not properly responded to by p53-deficient cells.

Alternatively, the type of damage that persists in p53kd cells is invisible to 53BP1. While the

latter can’t be ruled out, comet assays suggest that both in control and p53kd cells the carryover

damage may be derived from single strand breaks and gaps (visible to 53BP1); and these are in

fact more prevalent in AZD1775-treated p53kd cells (Fig. 2). p53BP1 bodies have been

implicated as one of the contributors to the G1/S checkpoint activation by p53 wt cells (38).

However, to our knowledge virtually no evidence (with one exception, (39)) thus far points at

p53 as a factor contributing to the formation of the 53BP1 bodies despite the original finding of

association between 53BP1 and p53 (40). Thus our finding may suggest a previously

undetected interplay between p53 and 53BP1 in regulating the G1/S checkpoint in the aftermath

of WEE1 inhibition in HNSCC.

WEE1 inhibition is known to slow replication fork progression (12). By performing DNA

fiber analyses on subpopulations of cells, we found another differential phenotype of AZD1775-

treated p53kd cells: while exhibiting the same H2AX response as controls, they had a more

severe replication fork slowing compared to their respective H2AX-negative baseline (Fig.5).

This novel observation can imply that on a cell by cell basis H2AX response to replication

stress is actually dampened by the knockdown of p53, and/or that p53 facilitates fork

progression under stress. A stimulatory role of p53 in stressed fork progression was found by

some studies (41, 42) but not others (43). Resistance to AZD1775-induced replication stress at

a replication fork level likely involves a RAD18--TLS polymerase kappa-dependent tolerance

and a RAD51-dependent fork protection pathways (13). We propose that p53 may regulate a

choice between these pathways both directly (42, 43) and in a transcription-mediated manner

(44).

Lastly, we observed that p53kd and p53 R273C mutant cells were more likely than the

wild type to undergo mitosis in AZD1775 despite an ongoing DNA damage response (Fig. 3 and

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S3). This forced mitosis manifested as increased prevalence of HH3+ cells, and moreover, it

was associated with the appearance of a unique subset of H2AX++/HH3+ cells, as well as a

higher percentage of micronuclei and abnormal (lobed, broken) nuclei, which are typically

associated with mitotic catastrophe. We previously reported (23) that a small fraction of

H2AX++/HH3+ p53-deficient cells displays less than 4c DNA content, i.e. enters mitosis with

under-replicated DNA, and in this study we demonstrate that this fraction of cells in mitotic

catastrophe can be increased by interfering with DNA replication during AZD1775 treatment

(Fig. 6). Similar findings have been recorded by other labs (10, 17, 20, 35), consistent with the

notion that p53 dysfunction weakens the G2/M checkpoint. This impact of p53 on the G2/M

checkpoint can be conveyed through p21, whose mitotic role has come into focus recently (33,

34, 45). Our data (Fig. 3) and (10), indeed suggest that p21 contributes to preventing the G2/M

checkpoint override by AZD1775. However, p53 – p21 axis is unlikely the only route by which

p53 may regulate the onset of mitosis. p53 may also inhibit transcription of Aurora A, a kinase

required for mitosis (46), or of FBW7, a negative regulator of Aurora B, another mitotic kinase

(47).

p53-dependent mitotic deregulation has been reported predominantly in cells derived

from carcinomas of the breast, colon, and head and neck, and is associated with p53 knock

down, 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 activities of different p53 mutants in an otherwise isogenic context of specific cancer

types as they respond to cell cycle deregulation.

Interaction between replication stress and mitotic deregulation, and the insights into combination

treatments with AZD1775

Our results are consistent with the notion that replication stress and mitotic deregulation

are independent variables in the response to AZD1775, and that replication stress is not

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sufficient to confer p53 sensitivity to AZD1775 in HNSCC. First, HH3-positive and HH3-negative

p53kd cells exhibited similar levels of replication stress as measured by their H2AX expression

and the extent of their replication fork slowing (Fig. 5), which suggests that a property other than

the severity of replication stress determined whether these cells underwent mitosis. Second,

relieving replication stress by supplementation of extra nucleosides in the media did not improve

survival of p53kd cells (in contrast to controls), while reducing such telltale symptom of

replication stress as single strand breaks in DNA (Fig. 5, 6). The ability of nucleosides to

improve replication fork progression and suppress phenotypes of AZD1775-induced replication

stress is well documented (12, 35).

At the same time, it is clear that replication stress can be manipulated to work together

with mitotic deregulation in p53kd cells in order to boost cytotoxicity. For example, combining

AZD1775 with a relatively novel, therapeutically relevant replication blocker triapine (3-AP),

increased premature mitosis of under-replicated DNA and additively suppressed survival of

p53kd HNSCC cells (Fig. 6).

A standard of care chemotherapy for HNSCC, CDDP represents another approach to

boosting cell killing by AZD1775. CDDP does not prevent complete replication of the genome

but slows it through engagement of the S phase checkpoint. CDDP induces cell death or

senescence, and successful repair of CDDP damage heavily relies on homologous

recombination in S and G2 phases of the cell cycle, and thus depends on a functional G2/M

checkpoint (49). In agreement with this, we observed that when treated with CDDP, p53kd cells

(incapable of G1 arrest) showed a greater induction of S and G2/M damage checkpoints than

controls (Fig. 7). AZD1775 overrode this response, with concomitant induction of mitosis and

PARP1 cleavage indicative of apoptosis. Together, these molecular phenotypes explain

synergistic interaction between CDDP and AZD1775 that we observed in p53kd HNSCC.

Our results are similar though not identical to the findings of Osman et al (48). For

example, p53-defective cells were not hypersensitive to AZD1775 alone in the PCI-13

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background used by the authors, while we detected sensitization of the UM-SCC74a cells by

p53 depletion or inactivation. The variability is likely an example of cell line heterogeneity,

nevertheless, taken together the results point to a promising therapeutic potential of combining

the standard of care CDDP with WEE1 inhibition in p53-defective HNSCC.

Intra-population heterogeneity of responses to AZD1775 – a p53-independent phenotype

We observed significant heterogeneity in the H2AX response of cells to AZD1775 and

demonstrated that it is associated with major functional differences among the cells. In

particular, we detected a pronounced negative correlation between cellular H2AX levels and

rates of replication fork progression, which brings up the question why subsets of cells

experience higher or lower replication stress in AZD1775. Since H2AX-negative cells in

AZD1775 typically belonged to early S phase, we hypothesize that their H2AX-negative state is

linked to an intrinsically low level of CDK1 at this point of the cycle. Alternatively, a critical

stress-signaling lesion may be slow to accumulate in these cells.

Extreme overactivation of CDK1 (and possibly CDK2) may also help explain why a

subset of H2AX-positive cells (H2AX++) in mid to late S phase go on to manifest extremely

high replication stress (Figs. 1, 2, 5, S1, S2). Indeed, a H2AX-positive subpopulation with

similar properties was previously observed upon overexpression of CDK1/2-activating

phosphatase, CDC25A (50). If so, it will be of interest to understand how and why some cells

within a population suffer a more severe overactivation of CDK1/2 than others, and find ways to

utilize this knowledge in cancer therapy.

In summary, our high-resolution analysis of head and neck carcinoma cells’ complex

response to WEE1 inhibition has highlighted both the known and the previously uncharacterized

deficiencies associated with p53 inactivation, and outlined specific directions for further inquiry

and for therapeutic exploitation.

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Acknowledgements

This work was supported by the NIH/NCI grant R01 CA215647 to E.M. and J.S., Seattle

Translational Tumor Research (STTR) programmatic investment grant to A.D., and University of

Washington Royalty Research Fund pilot grant to J.S. This research was also supported by the

Cellular Imaging and Therapeutic Manufacturing Shared Resources of the Fred

Hutch/University of Washington Cancer Consortium (P30 CA015704). E. Méndez received

commercial research grants and other commercial research support from AstraZeneca. The

authors declare no other potential competing financial interests.

Author contributions

A.D.: Conceptualization, Investigation, Methodology, Validation, Visualization, Writing – review

& editing.

M.K.: Conceptualization, Investigation, Methodology, Validation.

K.K.: Investigation, Methodology.

H.-Y. K: Investigation.

J.S.: Conceptualization, Investigation, Methodology, Supervision, Resources, Visualization,

Writing – original draft, Writing – review & editing, Funding acquisition.

E.M.: Conceptualization, Supervision, Resources, Funding acquisition, Project administration,

Writing – original draft.

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40. Iwabuchi K, Bartel PL, Li B, Marraccino R, Fields S. Two cellular proteins that bind to wild-type but not mutant p53. Proceedings of the National Academy of Sciences of the United States of America. 1994 Jun 21;91(13):6098-102. 41. Yeo CQ, Alexander I, Lin Z, Lim S, Aning OA, Kumar R, et al. p53 Maintains Genomic Stability by Preventing Interference between Transcription and Replication. Cell Rep. 2016 Apr 5;15(1):132-46. 42. Roy S, Tomaszowski KH, Luzwick JW, Park S, Li J, Murphy M, et al. p53 orchestrates DNA replication restart homeostasis by suppressing mutagenic RAD52 and POLtheta pathways. Elife. 2018 Jan 15;7. 43. Hampp S, Kiessling T, Buechle K, Mansilla SF, Thomale J, Rall M, et al. DNA damage tolerance pathway involving DNA polymerase iota and the tumor suppressor p53 regulates DNA replication fork progression. Proceedings of the National Academy of Sciences of the United States of America. 2016 Jul 26;113(30):E4311-9. 44. Lerner LK, Francisco G, Soltys DT, Rocha CR, Quinet A, Vessoni AT, et al. Predominant role of DNA polymerase eta and p53-dependent translesion synthesis in the survival of ultraviolet-irradiated human cells. Nucleic Acids Res. 2017 Feb 17;45(3):1270-80. 45. Kreis NN, Louwen F, Yuan J. Less understood issues: p21Cip1 in mitosis and its therapeutic potential. Oncogene. 2015 05/26/online;34:1758. 46. Wu CC, Yang TY, Yu CT, Phan L, Ivan C, Sood AK, et al. p53 negatively regulates Aurora A via both transcriptional and posttranslational regulation. Cell Cycle. 2012 Sep 15;11(18):3433-42. 47. Wang Y, Zhou BP. FBW7-Aurora B-p53 feedback loop regulates mitosis and cell growth. Cell Cycle. 2012 Nov 15;11(22):4113-4. 48. Osman AA, Monroe MM, Ortega Alves MV, Patel AA, Katsonis P, Fitzgerald AL, et al. Wee-1 Kinase Inhibition Overcomes Cisplatin Resistance Associated with High-Risk TP53 Mutations in Head and Neck Cancer through Mitotic Arrest Followed by Senescence. Molecular Cancer Therapeutics. 2015 February 1, 2015;14(2):608-19. 49. Dasari S, Tchounwou PB. Cisplatin in cancer therapy: molecular mechanisms of action. European journal of pharmacology. 2014 Oct 5;740:364-78. 50. Neelsen KJ, Zanini IM, Herrador R, Lopes M. Oncogenes induce genotoxic stress by mitotic processing of unusual replication intermediates. J Cell Biol. 2013 Mar 18;200(6):699-708.

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Figure Legends

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 min, then grown for up to 36 hrs with or without 300nM AZD1775. B) Flow cytometric

analyses. Left panels: representative density 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

are marked by arrows. Right panels: histograms of cell cycle distribution of EdU-positive cells at

the indicated times after the EdU pulse. C) Experimental design and a diagram of dual labeling:

IdU is 1st label and EdU is 2nd label. 400nM AZD1775 were added after the 1st label where

indicated. D) An example of immunofluorescent staining of dual-labeled, AZD1775-treated UM-

SCC-74a cells harvested 15 hrs after the 2nd label. Scale bar, 40m. E) Histograms of cell cycle

distributions of dual-labeled cells at indicated times after the 2nd label. F) Flow cytometric

analysis of cells treated as in (A), harvested at indicated times of incubation with AZD1775, and

immunostained for H2AX expression, EdU incorporation, and DNA content. Top two rows are

density plots of, respectively,H2AX and EdU levels versus DNA content. The bottom row is

histograms of cell cycle distributions (by DNA content) of the following subpopulations: EdU-

positive/ H2AX-negative (green), EdU-positive/H2AX-positive (purple), H2AX-superpositive

(orange). G) Histograms of cell cycle (top panel) and EdU level (bottom panel) distributions of

cells from the indicated subpopulations. Cells were incubated with AZD1775 for 24 hrs and

pulse-labeled for 30 min with EdU prior to harvest. H) A summary of findings presented in the

Figure. Red color intensity corresponds to the H2AX level. Cells remain H2AX-negative in

early S phase, develop H2AX signal as they progress through S, and at least some of them

retain H2AX staining in the next G1 and S. A subset of cells develops ultra-high level of H2AX

associated with suppressed DNA synthesis.

FIGURE 2. Depletion of p53 in UM-SCC-74a HNSCC cells modifies response to AZD1775.

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A) A Western blot verifies knockdown (kd) of the p53 protein and functional deficiency in p53 as

demonstrated by the inability of cells to induce p21 after ionizing radiation. B) Fold change in

proliferation relative to the untreated controls after treatment with 300nM AZD1775 for 16 hrs in

UM-SCC-74a and the primary fibroblast line HFF4. C) Flow cytometric analysis of H2AX

response in mock- and p53kd cells after 24hrs of 300nM AZD1775. D) Relative enrichment of

H2AX-positive cells in p53kd cells compared to the mock-depleted control (n=3). E) Examples

of immunofluorescent staining for H2AX in AZD1775-treated p53kd cells. F) Alkaline comet

assays in p53kd versus control cells treated 300 nM AZD1775 for 16 hrs (n=3). G) Flow

cytometric analysis of cleaved PARP1. Fraction of cleaved PARP1 was assessed by gating

cells relative to untreated controls and averaging the results (n=9). H) A Western blot of cleaved

PARP1 levels in cells treated with indicated doses of AZD1775 for 17hrs. I) Flow cytometric

analysis of EdU incorporation versus H2AX expression. Black: all-negative cells, green:

H2AX-positive and superpositive (if any), blue: EdU-positive. Cells were labeled with EdU for

30 min and incubated for 24hrs with or without 300nM AZD1775. J) Histograms of cell cycle

distributions of EdU-positive cells pulse-labeled with EdU for 30 min and incubated with or

without 300nM AZD1775 for the indicated times. EdU-positive cells that were H2AX-negative

(red) or H2AX-positive (and superpositive, if any, blue) are plotted separately. K) Flow

cytometric analyses of EdU incorporation of cells incubated with AZD1775 for 24 hrs with EdU

labeling for 30 min prior to harvest. Low EdU-incorporating cells appearing upon AZD1775

treatment are marked by arrows.

FIGURE 3. Depletion of p53 in UM-SCC-74a cells increases prevalence of mitotic cells and

leads to abnormal mitoses with high expression of H2AX. A) Immunofluorescent staining for

histone H3 phosphorylated on S10 (H3S10P) as a function of pre-mitotic and mitotic stages. B)

Flow cytometric analysis of H3S10P level versus DNA content. Cells were incubated with

500nM AZD1775 for 8 hrs. C) Relative enrichment of high histone H3S10P (HH3+) cells in

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p53kd cells compared to the mock-depleted control (n=3). D) Examples of immunofluorescent

staining of AZD1775-treated mock- and p53kd cells incubated with 300nM AZD1775 for 24 hrs.

Red: H2AX, green: H3S10P. An arrow marks a cell staining positive for both markers. E) An

example of an abnormal mitotic figure positive for both H2AX and H3S10P in p53kd, AZD1775-

treated cells. F) Quantitative image-based cytometry (QIBC) of mean fluorescent signals

ofH2AX and H3S10P per nucleus in cells treated with 300nM AZD1775 for 24 hrs. pB,

n=4062; p53kd, n=2328. Dashed frames indicate expected positions of H2AX+/HH3+

subpopulation, and cells positive for both markers are marked by an arrow. G) Fractions of

H2AX-positive cells among the histone H3S10-positive, mitotic cells in p53kd cells and mock-

depleted control. Treatment regimens were 300nM AZD1775/10 hrs or 500nM AZD1775/8hrs.

Cells were analyzed by flow cytometry (n=5). H) A Western blot of siRNA-mediated depletion of

p21 (CDKN1A) in control and p53kd cells. nc is non-targeting siRNA control. I) Levels of

H2AX+/++ and HH3+ cells relative to untreated control cells transfected with non-targeting

siRNA, as measured by flow cytometry (n=2). AZD1775 treatment was for 8 hrs at 300nM. J)

Fractions of H2AX-positive cells among the histone H3S10-positive, mitotic cells in p53kd and

control cells (n=2).

FIGURE 4. p53kd UM-SCC-74a cells recovering from AZD1775 retain more H2AX but fail to

form 53BP1 nuclear bodies. A) Experimental design. Cells were treated with 300nM AZD1775

for 24 hrs, then allowed to recover for up to 24 hrs. In some experiments cells were also labeled

with EdU for 30 min between 22.5 and 23 hrs of the drug treatment. Cells were immunostained

for H2AX, H3S10P, and where indicated, EdU. and analyzed by QIBC. B) Relative enrichment

of H2AX-positive fraction in p53kd cells compared to controls (n=2). Mean nuclear H2AX

signal was scored in at least 2,000 and up to 16,000 cells in each sample. Cells were

considered H2AX-positive if their mean H2AX signal was >1/2MAX of the population

distribution, and the percentage of these cells was calculated for each sample, expressed as

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fold enrichment over the control at time point 0 hrs recovery in each experiment, and averaged.

Both H2AX++ and H2AX+ cells are included in this metric. C) Examples of immunofluorescent

staining of p53kd, AZD1775-treated cells. The nucleus marked by an arrow displays aberrant

morphology. Red: H2AX, blue: EdU. D) Relative enrichment of cells with aberrant nuclear

morphology among H2AX-positive p53kd cells compared to controls measured in the same

experiments as in (B). Aberrant and normal H2AX-positive nuclei were scored manually in

digital images collected using a scanner microscope. 120-600 nuclei were analyzed per sample.

Percentage of aberrant among H2AX-positive nuclei was calculated for each sample,

expressed as fold enrichment over the control at time point 0 hrs recovery in each experiment,

and averaged. E, F) QIBC analysis of an experiment performed as in (A). Mean nuclear EdU,

H2AX, 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 1st quintile of a dataset. EdU-high cells have EdU values >1/2MAX of the

dataset. In (E), H2AX (red panels) and H3S10P (green 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-

high subsets. Numbers above scatterplots are percentages of H2AX-positive and HH3+ cells at

each time point. See Fig.S4 for more on selection and validation of EdU, H2AX+, and HH3+

value cutoffs. Diagrams on the right denote the inferred cell cycle position of EdU-negative and

EdU-high cells at the time of EdU pulse-labeling. G) QIBC analysis of AZD1775-treated cells

recovering from the drug for 24 hrs. Mean nuclear 53BP1 and H2AX signals were measured in

approx. 5,700 each of control and p53kd cells. Elevated 53BP1 signal in controls compared to

p53kd cells is marked with a bracket. H) Average population 53BP1 signal intensities were

derived from QIBC measurements (n=3). Cells were treated with AZD1775 as in (A) and

released from the drug for 24 hrs.

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FIGURE 5. AZD1775-treated UM-SCC-74a cells expressing different levels of H2AX

experience different degrees of replication fork slowing. A) Experimental design. Cells incubated

with 300nM AZD1775 and no-drug controls were labeled with consecutive 30 min pulses of

CldU and IdU prior to harvest, then immunostained for H2AX and H3S10P, and sorted into

subpopulations prior to DNA isolation and stretching. The H2AX++/HH3+ fraction was available

only in p53kd 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 to measure total (C+I)

lengths for each ongoing fork, as shown below the images. 2 representative images for each

condition were compiled to show more tracks. C) Ongoing fork track length distributions

measured in the indicated H2AX (white) and H2AX/H3S10P (gray) subpopulations. Numbers

of tracks analyzed 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 H2AX-negative and positive

subpopulations in p53kd cells and controls, derived from two independent experiments. The

differences were expressed as a D statistic, i.e. the maximal difference between two cumulative

distributions. D statistic values were calculated in K-S tests comparing each of the H2AX-

positive populations to the H2AX-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 treatment with 300nM AZD1775 for 16 hrs in the

presence or absence of nucleosides in the media (n=8).

FIGURE 6. Co-treatment with AZD1775 and triapine (3-AP) leads to mitosis with under-

replicated DNA in a p53kd UM-SCC-74a cells. A) Colony formation of cells treated with the

indicated drug combinations. B) Change in proliferation relative to the untreated controls after

treatment with 300nM AZD1775 with or without 300nM 3-AP for 16 hrs (n=6). C) Flow

cytometric analyses of cells stained for H2AX (upper panels), H3S10P (lower panels), and

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DNA content. Cells were treated with 300 nM 3-AP and/or 300nM AZD1775 for 16 hrs. HH3+

cells with less than 4N DNA content are marked with a black arrow. D) Alkaline comet assays

performed on cells treated with AZD1775 and/or 300 nM 3-AP for 16 hrs (n=3). E) As in (D),

except nucleosides were added to the media during treatments (n=3). F) Change in proliferation

relative to the untreated controls after treatment with 300nMAZD1775 and 300nM 3-AP for 16

hrs in the presence or absence of nucleosides in culture media (n=6).

FIGURE 7. Co-treatment with AZD1775 and CDDP (CDDP) leads to forced mitosis in p53kd

UM-SCC-74a cells. A) Change in proliferation relative to the untreated controls after treatment

with 300nM CDDP with or without 100nM AZD1775 for 16 hrs (n=5). B) Alkaline comet assays

performed on cells treated with AZD1775 and/or 300nM CDDP for 16 hrs (n=3). C) Flow

cytometric analyses of cells stained for H2AX (upper panels), H3S10P (lower panels), and

DNA content. Cells were treated with 1uM CDDP and/or 300nM AZD1775 for 16 hrs. HH3+

cells seen despite an ongoing DNA damage response in p53kd cells are marked with a black

arrow. D) Cell cycle distribution of cells incubated with the indicated doses of CDDP and/or

300nM AZD1775 for 24 hrs and pulse-labeled with EdU for 30 min prior to harvest. EdU-

negative (orange) and positive (blue) populations are graphed separately to distinguish G1, G2

and S phase cells. E) A heatmap of the p53 Signaling Pathway RT2 Profiler PCR array analysis

of cells treated with no drug or with 1uM CDDP for 24hrs. F) A quantitation of flow cytometric

analyses of cleaved PARP1 in untreated cells and cells treated with 300 nM AZD1775 and 1uM

CDDP combination for 16 hrs (n=3).

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Published OnlineFirst January 24, 2019.Mol Cancer Res   Ahmed Diab, Micheal Kao, Keffy Kehrli, et al.   cells to the WEE1 kinase inhibitionMultiple defects sensitize p53-deficient head and neck cancer

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