Gain-Of-Function Mutant P53 R273H Interacts with Replicating DNA and PARP1 in Breast Cancer

Home , MCM2, PARP1

Author Manuscript Published OnlineFirst on November 27, 2019; DOI: 10.1158/0008-5472.CAN-19-1036 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Gain-of-Function Mutant p53 R273H Interacts with Replicating DNA and PARP1 in Breast Cancer

Gu Xiao1, Devon Lundine1&2, George K. Annor1&2, Jorge Canar1, Viola Ellison1, Alla Polotskaia1, Patrick L. Donabedian3, Thomas Reiner3,4,5, Galina F. Khramtsova6, Olufunmilayo I. Olopade6, Alexander Mazo7, and Jill Bargonetti1,2,8

1The Department of Biological Sciences Hunter College, Belfer Building, City University of New York, New York;

2The Graduate Center Biology and Biochemistry Programs of City University of New York, New York;

3Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY;

4Department of Radiology, Weill Cornell Medical College, New York City, NY 10065;

5Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York City, NY 10065;

6Center for Clinical Cancer Genetics and Global Health and Section of Hematology and Oncology, Department of Medicine, University of Chicago, Chicago, IL, 60637;

7 Department of Biochemistry and Molecular Biology and Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107

8Department of Cell and Developmental Biology, Weill Cornell Medical College, New York City, NY 10021

*Address correspondence to: Jill Bargonetti Ph.D., Department of Biological Sciences, Belfer Building, Hunter College, 413 East 69th Street, New York, NY 10021; Tel. 212- 896-0465; Email:[email protected]

Running title: Mutant p53 and PARP association with Replicating DNA Keywords: Mutant p53; DNA replication; MCM; PARP1; TNBC The authors declare no potential conflicts of interest.

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 27, 2019; DOI: 10.1158/0008-5472.CAN-19-1036 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Over 80% of Triple Negative Breast Cancers (TNBC) express mutant p53 (mtp53)

and some contain oncogenic gain-of-function (GOF) p53. We previously reported that

GOF mtp53 R273H upregulates the chromatin association of Mini Chromosome

Maintenance (MCM) proteins MCM2-7 and Poly-ADP-Ribose Polymerase (PARP) and named this the mtp53-PARP-MCM axis. In this study we dissected the function and association between mtp53 and PARP using a number of different cell lines, patient- derived xenografts, tissue microarrays (TMAs), and The Cancer Genome Atlas (TCGA) database. Endogenous mtp53 R273H and exogenously expressed R273H and R248W bound to nascent EdU-labeled replicating DNA. Increased mtp53 R273H enhanced the association of mtp53 and PARP on replicating DNA. Blocking poly-ADP-ribose gylcohydrolase also enhanced this association. Moreover, mtp53 R273H expression enhanced overall MCM2 levels, promoted cell proliferation, and improved the synergistic cytotoxicity of treatment with the alkylating agent temozolomide in combination with the PARP inhibitor (PARPi) talazoparib. Staining of p53 and PARP1 in breast cancer TMAs and comparison with the TCGA database indicated a higher double-positive signal in basal-like breast cancer than in Luminal A or Luminal B subtypes. Higher PARP1 protein levels and poly-ADP-ribosylated proteins were

detected in mtp53 R273H than in wild-type p53-expressing patient-derived xenograft

samples. These results indicate that mtp53 R273H and PARP1 interact with replicating

DNA and should be considered as dual biomarkers for identifying breast cancers that

may respond to combination PARPi treatments.

Introduction:

Breast cancer is the leading cause of cancer related death in women worldwide1.

Biological markers used to categorize breast cancer types include estrogen receptor

(ER), progesterone receptor (PR), and human epidermal growth factor receptor 2

(HER2). Triple negative breast cancer (TNBC) patients lack these three targetable makers and have a higher recurrence that results in a higher mortality1. The Cancer

Genome Atlas (TCGA) found that in 80% of TNBC the p53 tumor suppressor gene

(TP53) is mutated2. The p53 protein is a transcription factor that regulates its downstream target genes that control cellular functions, including cell cycle arrest, apoptosis and cell senescence3. Wild-type p53 has DNA replication functions that help to protect the genome during S phase, by enhancing the processivity of replication forks4. The majority of missense mutations in p53 are located within the DNA-binding domain, including six frequent ‘hotspot’ amino acid codons (R175, G245, R248, R249,

R273 and R282)5. Some mutant p53 (mtp53) proteins have oncogenic gain-of-function

(GOF) that promote tumor growth, genomic instability, invasion, chemo resistance, and alter diverse proteome and metabolic pathways6. These GOF properties are known to share some properties while being distinct for others, and therefore it has been described that different GOF mtp53 proteins can have specific oncogenic properties3.

Therefore, it is of interest to study specific mtp53 variants for their generalized functions.

We previously determined that high levels of stable mtp53 R273H drive the association of DNA replication proteins MCM2-7 and PARP onto chromatin and improve the synergistic cytotoxicity of the PARP inhibitor talazoparib in combination with the DNA damaging agent temozolomide7-8. Single agent treatment in this context is not cytotoxic.

The wtp53 protein works at replication forks to increase processivity but very little

information exists on the direct interaction of R273H mtp53 with replicating DNA4. The

GOF mtp53 proteins R175H and R273H have been shown to enhance transcription of genes that activate cell cycle and DNA replication: up-regulating Cyclin A expression at early S phase and stabilizing replication forks by up-regulating Chk1 expression9. Many

GOF mtp53 proteins also co-opt chromatin pathways to drive cancer proliferation10. Both mtp53 R273H and R175H increase serine-threonine kinase Cell division cycle 7 (Cdc7) transcription and stimulate Cdc7 dependent DNA replication initiation11. Cdc7 phosphorylates the N-terminus of MCM2/4/6 proteins and recruits Cdc45, GINS and DNA polymerase to origins12. In addition, mtp53 R273H and

R248W, but not R175H, directly enhance DNA replication by bridging the interaction between TopBP1 and Treslin and colocalize with PCNA suggesting a direct replication association13.

PARP inhibitors are treatment options for a subset of TNBCs with a defective homologous recombination (HR) pathway due to mutations in BRCA1 or BRCA214-15. It is of broad interest to determine if high expression of GOF mtp53 and PARP also coordinate biologically on replicating DNA, and as dual staining markers, in a large subset of TNBC. Poly ADP-ribose polymerase (PARP) synthesizes a polymer (ADP- ribose polymer) (PAR). PARP1 has been shown to interact with DNA polymerase  and multiprotein DNA replication complexes16-17. In addition, PARP1 attracts DNA repair complexes to sites of DNA replication fork stalling or damage18. PARP1 regulates Chk1 protein kinase, Mre11 nuclease and RECQ helicase activities resulting in replication fork resection, degradation and restart18-20. Moreover, PARP1 is a sensor of unligated

Okazaki fragments during DNA replication and facilitates their repair by activating PAR synthesis on PARP during S phase21.

The site-specific DNA binding activity of GOF mtp53 proteins is lost but these mt p53 proteins maintain a tight association with nuclear chromatin3. Many transcription factors that bind DNA, such as transcription factors NF-Y, SREBP2, Sp1, ETS1 and

VDR or non-transcription factors, such as TopBP1, Pin1, MRE11 or PML, have been suggested as mtp53 modulators for the chromatin interaction6. Structural DNA recognition has been noted for mtp53 (G245S, R248W and R273H) and wild type p53 which have the ability to interact with quadruplex structures22. However as of yet, GOF mtp53 binding to a functionally-specific DNA structure, such as DNA replication forks has not been reported. In this study we found that mtp53 R273H and R248W directly associated with replicating DNA. In addition, we found a positive association between mtp53 and PARP in breast cancer tissue culture cells, patient derived xenograft samples, tissue microarrays, and The Cancer Genome Atlas database suggesting that the dual biomarker of high mtp53 and PARP can be beneficial for subtyping TNBCs that may benefit from combination PARPi treatments.

MATERIALS AND METHODS

Cell culture and patient derived xenograft (PDX) cell lines

Human breast cancer cell lines MDA-MB-468 and MCF7 were purchased from ATCC

(www.atcc.org). Cell lines were authenticated every six months and tested for

mycoplasma (Genetica DNA Laboratories). Cells were maintained at 5% CO2 in DMEM

(Invitrogen) with 50 U/ml penicillin, 50 μg/ml streptomycin (Mediatech) and

supplemented with 10% FBS (Gemini) in 37°C humidified incubator. MDA-MB-468 cells

generated with mir30 shRNA controls, inducible p53 knockdown, and R273H and

R248W overexpression were described previously23-24. To induce shRNA expression,

cells were treated with 8 μg/mL doxycycline for time periods indicated, and fresh

medium with doxycycline was supplemented every 48 hr. PDX lines were derived at

Washington University from patients with extensive stage breast cancer25.

2 × 106 viable cells/mouse were injected with a 1:1 mix of DMEM and Matrigel basement

membrane (Corning) in a final volume of 100 μL subcutaneously into female NSG mice

(NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ; The Jackson Laboratory). Mice were 6 to 8 weeks old at time of PDX injection/implantation. The animals were euthanized by CO2(g)

asphyxiation when tumor size reached to 1.0cm3 and tumors were removed and snap

frozen. Protein extraction from frozen tumor was previously described 26. Proteins were

extracted by homogenization of tissue with RIPA buffer (0.1% SDS, 1% IGEPAL NP-40,

0.5% Deoxycholate, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Tris-Cl pH 8.0,

1mM PMSF, 8.5 µg/ml Aprotinin, 2 µg/ml Leupeptin and phosphatase inhibitor cocktail).

RIPA buffer was added at a ratio of 0.4 ml per 0.1 g tissue (1:4 w/v). Incubation was

carried out on ice for 10 min and the sample was vortexed every 2 minutes. Additional

sonication of the lysate (3x on ice for 30 sec on/off at 98% amplitude) was carried out

after the incubation. Samples were centrifuged at 13,000 rpm for 10 min at 4°C. This

study was given IACUC approval from the Institutional Animal Care and Use Committee

of Memorial Sloan Kettering Cancer Center (MSKCC). CRISPR of the mtp53 R273H in

MDA-MB-468 cells was carried out using in-situ assembled sgRNA and Cas9 enzyme plus a eGFP-Puro plasmid for selection introduced by Nucleofector at 1700V/20ms/1

pulse.

Isolation of proteins on nascent DNA (iPOND) iPOND was performed as previously described27 with modifications. 1 × 108 cells were

plated for each condition 1 day before EdU incubation. Cells were incubated with 10 μM

EdU for 45 min. Cells were fixed with 10 ml 0.5% formaldehyde in PBS for 20 min and

quenched by adding 1 ml 1.25 M glycine. Cells were permeabilized with 0.25% Triton X-

100 in PBS for 30 min and subsequently underwent a click reaction. Click reaction was

2 mM copper sulfate, 10 µM biotin-azide, and 10 mM sodium ascorbate added to PBS

for 1.5 h at room temperature with rotation. Cells were incubated in RIPA buffer on ice

for 30 min, vortexing every 5 min. Additional sonication of lysate (18x on ice for 30 sec

on/off at 98% amplitude) was done after the incubation. Samples were centrifuged at

13,000 rpm for 30 min at 4°C. Biotin-EdU-labeled DNA was incubated with streptavidin-

agarose beads at 4°C for 20 h. The beads were washed with RIPA buffer 3x and

proteins bound to nascent DNA were eluted by incubating in 2× SDS Laemmli sample

buffer containing 0.2 M dithiothreitol (DTT) for 25 min at 95°C.

In situ Proximity Ligation Assay (PLA) and 5-Ethynyl-2´-deoxyuridine (EdU) PLA

Cells were seeded at 2 × 105 per well in a 12-well glass bottom plate (MatTek). After

removing media, cells were rinsed with ice-cold PBS 3x, fixed in 4% formaldehyde for

15 min and permeabilized in 0.5% Triton x-100 in PBS for 10 min at room temperature.

After washing cells 3x in PBS, PLA was carried out using Duolink in-situ red kit (Sigma-

Aldrich). Briefly, cells were incubated in blocking buffer for 30 min at 37 °C in a humidified chamber and then incubated with primary antibodies overnight at room temperature in a humidified chamber. The next day, cells were washed with Sigma buffers (Cat# DUO82049). First, Buffer A for 5 min 3x and incubated with secondary antibodies conjugated oligonucleotides (PLA probes MINUS and PLUS) for 60 min at

37 °C in a humidified chamber. This was followed by 5 min wash in Buffer A 2x. The ligation reaction was carried out at 37 °C for 30 min in a humidified chamber followed by

2x 2 min wash in Buffer A. Cells were then incubated with the amplification mix for

100 min at 37 °C in a darkened humidified chamber. After washing with 1× Buffer B for

10 min 2x and a 1 min wash with 0.01× buffer B, cells were mounted with mounting media containing 4′,6-diamidino-2-phenylindole (DAPI). PLA with EdU (SIRF) was performed as previously described28-29. Cells were incubated with 125 µM EdU in growth media for 15 min and fixed with 4% formaldehyde in PBS (pH 7.4) for 15 min at room temperature. For the thymidine chase experiment, EdU was removed and cells were washed 2x with media before addition of media with 100 µM thymidine before fixation. After fixation, cells were washed with PBS 2x for 5 min each. Cells were permeabilized in 0.5% Triton X-100 in PBS for 15 min at room temperature then washed in PBS 2x for 5 min each. Click reaction contained 2 mM copper sulfate, 10 µM biotin- azide, and 100 mM sodium ascorbate added to PBS and incubated at room temperature for 1 hr. Cells were washed with PBS for 5 min and blocked 1 hr at room temperature. Primary antibodies incubation was 4°C overnight in a humidified chamber.

Cells were washed 3x with wash buffer A for 5 min each. Duolink In-Situ PLA probes anti–mouse plus and anti–rabbit minus were diluted 1:5 and incubated for 1 hr at 37°C.

Cells washed in Buffer A solution 3x for 5 min each. Ligation reaction was incubated at

37°C for 30 min. Cells were washed in buffer A 2x for 2 min each. Amplification reactions were incubated at 37°C for 100 min. Cells were washed in wash buffer B solution 3x for 10 min each and one time in 0.01× diluted wash Buffer B solution for 1 min. EdU was detected by incubation with FITC-conjugated anti-mouse antibody for 1 hr at room temperature and washed with PBS 3x for 5 min each and once in 0.01× diluted wash buffer B solution for 1 min. Cells were mounted with mounting media containing

DAPI. z-stack images were taken using Nikon A1 confocal microscope with 60x objective oil immersion, acquired by Nikon elements.

Quantification of mtp53 R273H/EdU foci

Images acquired through Nikon NIS Elements were processed using ImageJ.

Maximum intensity projections were generated by taking 2 representative z-stack slices for the determination of S-phase progression. Look-up tables for each resulting image were automatically applied by ImageJ to DAPI, EdU, and mtp53R273H/EdU channels.

For each cell line, a pipeline was constructed in CellProfiler for the identification of cells positive for EdU, followed by identification and quantification of foci per cell. S-phase progression (Early, Mid and Late) was manually scored according to EdU staining30.

Graphpad Prism 8 was used for statistical analysis. siRNA transfection plus Aphidicolin treatment

siRNA electroporation was performed with the Neon® Transfection System (MPK5000).

MDA-MB-468 cells were grown to 70 % confluence on 100mm tissue culture dishes and rinsed with PBS without Ca2+ and Mg2+. This was followed by addition of 1× Trypsin-

EDTA and incubated for 2 min at 37 °C. After neutralizing the reaction with supplemented DMEM plus 10% FBS, the cells were resuspended and counted by hemocytometer. Cells were pelleted by centrifugation at 1100rpm for 5 minutes and resuspended in Resuspension Buffer R (Neon 100 μl kit Invitrogen) to a final concentration of 1 × 107 cells ml−1. 1.5 × 106 cells were transferred to a sterile 1.5 ml microcentrifuge tube and mixed with 10 μl of 20 μM siRNA (Dharmacon; PARP, L-

006656-03-0005; Non-targeting Pool, D-001810-10-05). Electroporation was then carried out with 100 μl of cells plus siRNA at 1100 volts/30 milliseconds/2 pulses. Cells were seeded in 6-well cell culture plate with 2 ml of pre-warmed supplemented DMEM plus 10% FBS without antibiotics for 48hr and treated with 5 μM Aphidicolin for an additional 24hr.

Cell proliferation assay

20,000 cells/well were seeded in 6-well plates and grown for 1, 3 and 4 days, respectively. At each time point, cells were trypsinized and cells were counted by hemocytometer.

Co-Immunoprecipitation (IP) assays

Cells were lysed in NP-40 buffer (50mM Tris PH 8.0, 5mM EDTA, 150mM NaCl, 0.5%

NP40, 1mM PMSF, 8.5 µg/ml Aprotinin, 2 µg/ml Leupeptin and Phosphatase Inhibitor

Cocktail (Sigma-Aldrich) for 30 min at 4°C with rotation. After every 10 min a 21G1 ½

needle was used to homogenize the sample 10 times. Cell lysates were centrifuged at

13,000 rpm for 15min at 4°C. 1mg total protein were precleared with 1µg mouse IgG

(2.5 µl), 30 µl Protein A/G PLUS-agarose (Santa Cruz), balance with wash buffer

(50mM Tris 8.0, 5mM EDTA, 150mM NaCl, 1mM PMSF, 8.5 µg/ml Aprotinin, 2 µg/ml

Leupeptin and Phosphatase Inhibitor Cocktail (Sigma-Aldrich) to 1ml. Rotate for 30 min

at 4°C. Add 35ul anti-p53 DO1-AC antibody beads (Santa Cruz) or mouse IgG-AC

beads to precleared lysates and incubated at 4°C for 2 hr with rotation. Wash beads

with 1 ml wash buffer for 4 times for 4 min each at 4°C. Bound proteins were eluted by

incubating in 2× SDS Laemmli sample buffer containing 0.2 M dithiothreitol (DTT) at

95°C for 10 min and loaded onto SDS-PAGE.

Protein extraction

Cells were harvested at 1100 rpm for 5 min at 4°C on Sorvall benchtop centrifuge. Cells

were washed 3x with ice-cold PBS. Cells then were resuspended in RIPA buffer. The

cell suspension was incubated on ice for 30 min, vortexing every 5 min. Additional

sonication of lysate (3x on ice 30 sec on/off at 98% amplitude) was done after the

incubation. Samples were centrifuged at 13,000 rpm for 30 min at 4°C and supernatant

was collected.

Immunoblotting assay

Cell extracts were run on 10% SDS-PAGE followed by electrotransfer onto

Polyvinylidene fluoride membrane (GE). The membrane was blocked with 5% non-fat

milk (Biorad) in either 1X PBS-0.1% Tween-20 or 1X TBS-0.1% Tween-20 followed by incubation with primary antibody overnight at 4°C. The membrane was washed with either 1X PBS-0.1% Tween-20 or 1X TBS-0.1% Tween-20 and incubated with

secondary antibody for 1hr at room temperature. The signal was detected by chemiluminescence with Super Signal Kit (Pierce) and autoradiography with Hyblot CL films (Denville Scientific) or Typhoon FLA 7000 laser scanner (GE Healthcare).

Subcellular Fractionation

Cells were harvested, and subcellular fractionation was performed using the Stillman protocol31. After removing the media, cells were rinsed with ice-cold PBS twice, scraped from plates, and pelleted by centrifugation at 1100 rpm for 5 min. Cell pellets were suspended in buffer A [10 mM Hepes, pH 7.9, 10 mMKCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% (vol/vol) glycerol, 1 mM DTT, 0.5 mM PMSF, 2 µg/mL leupeptin, 8.5

µg/mL aprotinin] with 0.1% Triton X-100. After 5 min incubation on ice, cells were spun down at 3600 rpm for 5 min at 4°C. The supernatant was spun down for an additional 5 min at 13,000 rpm at 4°C (cytoplasmic fraction). Pellets were washed two times with buffer A by centrifugation at 3600 rpm for 5 min at 4°C. Resuspended nuclear pellet in buffer B (3 mM EDTA, 0.2 mM EGTA, 0.5 mM PMSF, 2 µg/mL leupeptin, 8.5 µg/mL aprotinin) was incubated on ice for 30 min with vigorous vortexing every 5 min and spun down at 4000 rpm for 5 min at 4°C. The supernatant was nuclear soluble proteins, and the pellet, enriched in chromatin, was washed two times with buffer B, resuspended in buffer B, and sonicated 3x on ice for 30 sec on/off at 98% amplitude.

MTT

Cells were seeded at 1.25 × 105 cells per well in 12-well plates. MTT solution (5 mg mL−1 (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) was added to the

cells (10% v/v) and incubated at 37°C in 5% CO2 for 1 h. The cells were resuspended in

0.04 N hydrochloric acid diluted in isopropanol. Samples were incubated for 5 min at RT.

The absorbance was quantified by measuring the absorbance at 550 nm subtracted

from the absorbance at 620 nm.

Live cell imaging

Cells were seeded at 2 × 105 per well in a 12-well glass bottom plate one day before

imaging (MatTek, Ashland, MA, USA). Cells were incubated with 500nM PARPi-FL for

20 min at 37°C. PARPi-FL was removed and cells were incubated in fresh medium for

10 min, washed twice with PBS at room temperature, co-stain the nuclear DNA with 1

μg/ml Hoechst 33342 (ThermoFisher) in PBS for 5 min and z-stack images of stained

cells were taken by confocal microscopy using a Nikon A1 confocal microscope with

40x objective.

Tissue microarray p53 and PARP1 immunohistochemistry

Tissue microarrays were constructed from FFPE tumor samples (tumor and adjacent

normal). Cores were precisely arrayed into a new recipient paraffin block using an

automated arrayer (ATA-27, Beecher Instruments, Silver Spring, MD) with the method

described by Kononen et al32. Documentation for each tissue core’s location was done on a ready-made grid using the program Microsoft Excel. MCF-7 and Hela cells were purchased from ATCC. Cells were allowed to reach semi-confluence, then collected and fixed in 10% neutral formalin for 15 min. Cell blocks were prepared by embedding cells

in HistoGel and processing as a piece of tissue, then arrayed into a TMA block as

controls.

Immunohistochemistry and evaluation of p53 and PARP1

The 4-µm thick tissue microarray (TMA) tissue sections were deparaffinized in xylene

and rehydrated through a graded alcohol series to distilled water. Slides were cooled to

room temperature and washed in tris-buffered saline. Antigen retrieval was performed in

a vegetable steamer in Universal Decloaker target retrieval solution (BioCare Medical

cat. # UD1000 M), then slides were left to cool in solution for 15 min. Slides were then

incubated in Peroxidase 1 (BioCare Medical cat. # PX968) for 10 min, followed by

incubation for 15 min in Background Sniper (BioCare Medical cat. # BS966L)

Immunohistochemical assays were performed using the following antibodies: p53 (DO-1)

antibody (Santa Cruz Biotechnology, dilution 1:100) and PARP-1 (F-2) (Santa Cruz

Biotechnology, dilution 1:250). The BioCare Medical Polymer Detection Kit (cat.#

M3M530 L) was used as a detection system for the Anti-P53 MACH 3 Probe (10mins),

ready to use MACH 3 HRP-Polymer (10mins). Slides were treated for 5 min with 3–3'-

diaminobenzidine (DAB) chromogen (BioCare Medical cat. # BDB2004 H),

counterstained with hematoxylin, and cover slipped. For the immunohistochemical

analysis, Allred scores were determined semi-quantitatively by two observers as

described33. Scoring was based on intensity and percentage of positive stained cells.

Final scores (range 0-8) were converted to a scale from 0 to 3 (0-1 considered as negative-0; 2-3 considered as weakly positive-1; 4-5 considered as moderately positive

-2; 6-8 considered as highly positive-3. All discrepancies were resolved by a second examination by two observers simultaneously using a multi-head microscope.

Results

Mutant p53 R273H and PARP1 interact with replicating DNA

We previously identified that mtp53 R273H increases the chromatin association

of the DNA repair protein PARP and essential DNA replication protein complex Mini

Chromosome Maintenance (MCM2-7), and showed mtp53 (but not wtp53) directly

associates with MCM2 and MCM47-8. To investigate whether mtp53 plays a direct role in regulating DNA replication, here we examined the association of mtp53 R273H in MDA-

MB-468 breast cancer cells and PANC-1 pancreatic cancer cells on replicating DNA by

using the isolation of proteins on nascent DNA (iPOND) assay (Fig. 1A). In MDA-MB-

468 cells the influence of mtp53 R273H was compared in miR30 expressing control

MDA-MB-468 cells, miR30-based doxycycline-inducible shRNA isogenic knockdown in

MDA-MB-468.shp53 cells, and increased expression that was shRNA-resistant in MDA-

MB-468.shp53 cells (468.shp53+R273H)24. After thymidine analog 5-ethynyl-2′-

deoxyuridine (EdU) pulse labeling and covalent linkage to a biotin-azide using click

chemistry, proteins bound to newly replicated DNA were analyzed. We detected

significant mtp53 R273H on the replicating DNA in MDA-MB-468 shp53+R273H cells

(Fig. 1A MDA-MB-468). The exogenous expression of mtp53+R273H (Fig. 1A MDA-

MB-468 see input lane 4) increased the mtp53 R273H on replicating DNA (Fig. 1A

MDA-MB-468, compare lane 8 to lanes 6 and 7). Furthermore, PARP1 association on

replicating DNA was increased when mtp53 R273H was overexpressed (Fig. 1A MDA-

MB-468, compare lane 8 to lanes 6 and 7) and knockdown of mtp53 R273H reduced

the mtp53 R273H and PARP on replicating DNA (Fig. 1A MDA-MB-468, compare lanes

6 and 7). The increased exogenous expression of mtp53 R273H in MDA-MB-468 cells

also caused a slight increase in the chromatin association of histone H2B (Fig. S1). We also detected endogenous mtp53 R273H on replicating DNA in PANC-1 cells by iPOND

(see Fig. 1A PANC-1 lane 4). To verify the association of mtp53 R273H on nascent

DNA by an additional method we carried out single-cell in situ proximity ligation assay

(PLA) to detect the direct protein interactions with newly replicated DNA (Fig. 1B MDA-

MB-468 cells on the left and PANC-1 cells on the right). EdU detection distinguishes between early, mid, and late S-phase replication structures by the area of nucleus showing the EdU incorporation in confocal microscopy28, 30. Replicating DNA was bound by mtp53 R273H in early, mid, and late S-phase cells in both the breast cancer and pancreatic cancer cells (Fig. 1B) after a 15 min EdU pulse and biotin-azide click chemistry as evident by the co-association with the EdU in the different replication patterns seen in confocal imaging sections30. With maximum projection images after a

15 min EdU pulse and biotin-azide click chemistry showing clear replication association of mtp53 R273H (Fig. 1C upper panel), and PARP (Fig. 1C lower panel) on newly replicated DNA as evident by red foci. This supports our earlier report that mtp53 directly associates with MCM2 and MCM48. In addition, we detected that another mtp53 hotspot mutant, R248W, when exogenously expressed was also associated with replicating DNA (Fig. S2). The observed PLA signal between biotinylated EdU and mtp53 R273H decreased after a 1-hour thymidine chase following the15 minute EdU pulse. Endogenous PAR is detected during S phase at sites of DNA replication21 and p53 is PARylated by PARP1 and this activates the interaction between PARP1 and wild-type p5334. To investigate how increased PARylation influenced replication associated mtp53 and endogenous PARP in the absence of DNA damage, we carried

out PLA with short incubation with the poly (ADP-ribose) glycohydrolase inhibitor

(PARGi) (Fig. 1D). Increased mtp53 R273H was found in association with newly

replicated DNA in PARGi treated cells versus vehicle treated cells. Moreover, increased

mtp53 R273H and PARP were co-associated in PARGi treated MDA-MB-468 and

PANC-1 cells (Fig. S3 A and B). We also observed an endogenous protein-protein

interaction between PARP and MCM2 and mtp53 R273H by co-Immunoprecipation with anti-PARP (Fig. S3 C). No mtp53 R273H was detected on replicating DNA when

Okazaki fragments formation was suppressed by emetine (Fig. S3D). Furthermore,

when the cells were blocked in the G2 phase of the cell cycle with nocodazole, the

chromatin interactions of mtp53 R273H, PARP and MCM2 were reduced (Fig. S3 E).

Collectively, the iPOND and PLA assays with pulse EdU labeling identified a direct

interaction of mtp53 R273H and PARP1 on replicating DNA in the MDA-MB-468 breast

cancer cells and PANC-1 pancreatic cancer cells.

Mutant p53 R273H, MCM2, and PARP Promote Cell Proliferation

An increased frequency of origin firing has been implicated in genomic

abnormalities35. Replication origins are licensed in the G1 phase by the binding of MCM

helicase complexes36. Phosphorylation of MCMs by Cdc7 promotes subsequent

assembly of replisome members and initiates DNA synthesis in the S phase12. The

MCM2 amino acid residue serine 108 is a Cdc7 phosphorylation site37. We examined

the levels of MCM2 over the cell in MDA-MB-468 control cells and a CRISPR mtp53

targeted clone that expressed extremely low levels of mtp53 R273H (Fig. 2A). The level

and phosphorylation of MCM2 varied depending on the cell cycle stage, and lower

expression levels of mtp53 R273H correlated with lower levels of MCM2 (Fig. 2A). This was consistent with our previous results for total MCM2 protein reduction when mtp53

R273H was depleted by shRNA knockdown methods8. Moreover, when we used siRNA targeting PARP (see Fig. 2B) we reduced the ability of the MDA-MB-468 cells to enter

S-phase even in the absence of changes in mtp53 recruitment.

To investigate whether mtp53 R273H increased cell proliferation, we performed the MTT and cell counting assay in MDA-MB-468 mir30 expressing, 468.shp53 knockdown and 468.shp53-R273H increased mtp53 expressing cells (Figs. 2C, D & E).

The mtp53 R273H protein was reduced in 468.shp53 cells and was higher in

468.shp53+R273H cells (Fig. S1). Mitochondrial activity was reduced by 28.4% in response to knockdown of mtp53 and a 33.5% increase was observed when mtp53

R273H was exogenously expressed. At day 4 in the growth curve a 33.4% reduction of cell number was observed when mtp53 was knocked down and a 17.6% increase in cell number was detected when mtp53 R273H was exogenously expressed. Our results demonstrate that mtp53 R273H increases MCM2 and induces cell proliferation, and that the level of PARP does not influence mtp53 levels but PARP is needed to increase progression into S-phase.

Overexpression of mutant p53 R273H increases PARP1 chromatin interaction and synergistic PARPi plus temozolomide treatment sensitivity

We carried out the PLA assay to identify if mtp53 R273H interacts with PARP1 on chromatin (Fig. 3A). The detection of PARP1 and mtp53 in close proximity showed an average (3.455±0.1546, n = 233) fluorescent foci per cell in MDA-MB-468 cells. In contrast, detection of PARP and wtp53 in MCF7 cells showed a lower average

(1.085±0.1305, n = 117) fluorescent foci per cell (Fig. 3B). A higher p53 protein level

was detected in MDA-MB-468 cells compared to MCF7 cells (Fig. 3C) and this could

account for the increased detectable interaction. The interaction of endogenous mtp53

R273H with PARP1 was also detected with the co-immunoprecipitation assay (Fig. 3D).

Importantly, in MDA-MB-468 cells the endogenous mtp53 R273H and MCM7 were also

detected in a protein-protein interaction (Fig. 3D).

We previously reported that while single agent treatment was not very cytotoxic

there was a synergistic activation of apoptosis in MDA-MB-468 cells treated with the

PARP inhibitor/trapping agent (PARPi) talazoparib (Tal) in combination with the DNA

damaging agent temozolomide (Temo)8. We hypothesized that overexpression of mtp53

R273H would further sensitize cells to the combination treatment. The cell viability was

slightly reduced in mtp53 exogenously expressing 468.shp53+R273H cells compared to

knockdown 468.shp53 cells (Fig. 3E). The increased synergistic sensitivity to the PARPi

(Tal) with DNA damaging agent (Temo) in mtp53 R273H overexpressing cells supports

the conclusion that the increased interaction of mtp53 and PARP sensitizes the cells.

Interestingly when more mtp53 R273H was exogenously expressed there was a

reduction in the amount of the DNA repair protein 53BP1 localized to the chromatin (Fig.

3F). It has been shown that the inhibition of PARP blocks 53BP1 foci38, and mtp53

R273H increasing this inhibition could be part of the improved killing mechanism in the

presence of high mtp53 R273H.

High levels of expression of PARP1 and mutant p53 are correlated in Breast

Cancer Patients and support the use of live imaging of PARP as a TNBC diagnostic

High expression levels of mtp53 correlate with poor TNBC patient outcomes39.

Analyses of the global proteome with genomic profiles generated through TCGA have provided insights into identifying new biomarkers. We examined the data sets from

TCGA Cell 201540 and asked whether there were correlations between the expression of p53 (using p53 positivity as an indicator of mutant p53 expression) and PARP1 proteins in different subtypes of breast cancer (Fig. 4A). A total of 817 breast tumor samples were profiled and 633 cases were also profiled by reverse-phase protein array

(RPPA). Estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status were clinically determined by immunohistochemistry40. At the protein expression level, p53 and PARP1 showed a strong correlation (R2:0.2796, p value: 2.2e-16) patients with all types of breast cancer

(n=672). Further analysis revealed that both p53 and PARP1 protein levels were significantly elevated in TNBC (red) while in estrogen receptor–positive (ER+) patients

(green) the p53 was low. We carried out Immunohistochemical (IHC) staining of p53 and PARP1 on tissue microarrays (TMAs) that contain samples from 101 patients diagnosed with intrinsic subtypes of breast cancer (Fig. 4B). The results were scored using a scale from 0 to 3: no staining (0), weak staining (1), moderate staining (2) and strong staining (3) (Fig. 4B left panel, zoomed in examples, full slide images in Fig. S4).

A higher average IHC score of p53 was present in basal-like breast cancer samples than Luminal A or Luminal B subtypes (Fig. 4B top panel). Among 20 basal-like tumor

samples, we found in the p53 high group (n=10) nuclear PARP1 intensity was higher than in the p53 low group (n=10) (Fig. 4B lower panel). Due to the small sample size of basal-like tumor samples, the protein intensity of p53 and PARP1 was not statistically significant. However, in 62 Luminal A tumor samples, the protein intensity of p53 and

PARP1 showed a statistically significant positive correlation (Pearson r: 0.2745, p value:

0.0308). Taken together these data indicate that high mtp53 expression often correlates with high PARP1 in different breast cancer subtypes.

PARPi-FL imaging agents are extremely promising for detecting oral cancers with high PARP expression41. We carried out live cell imaging with PARPi-FL and detected PARPi-FL stains TNBC cells MDA-MB-468 more intensely than ER+ cells

MCF7 and this staining is nuclear (Fig. 4C). Furthermore, we tested PARP1 protein level and PARylated proteins in patient-derived xenograft (PDX) samples to determine if mtp53 expression correlated with PARP detection (Fig. 4D). PDX models maintain the histological and molecular heterogeneity of the progenitor and can be used to test cytotoxic drug responsiveness42. We detected more PARP protein and significant higher levels of PARylated proteins in WHIM25 expressing mtp53 R273H than in

WHIM6, which expresses wtp53. In conclusion, our data provide strong evidence that the increased co-expression mtp53 and PARP proteins can be biomarkers for companion diagnostics to identify TNBCs that have elevated expression of mtp53 and

PARP thereby giving a potential proxy for cancers that are candidate for combined talazoparib and temozolomide treatment.

Discussion

DNA replication and repair are two essential biological processes that ensure

accurate duplication of the genome. We previously showed a co-association of mtp53

and MCM DNA replication proteins8. Here we demonstrated that mtp53 and PARP1 are

associated on replicating DNA in TNBC cells. Furthermore, mtp53 enhanced PARP1

association with nascent DNA in TNBC and enhanced cell proliferation. We observed a positive association of increased p53 expression and PARP1 in analysis of cancer samples from the TCGA and TMA analysis for all subtypes of breast cancer patients

suggesting that scoring mtp53 and PARP expression as a prognostic marker can act as

a proxy for potential TNBCs that would benefit from combination talazoparib-

temozolomide treatment. Our findings prompt us to propose a model (see Fig. 5A) in

which mtp53 (shown in green) associates with replicating DNA and recruits MCM

proteins (shown in purple) and PARP1 (shown in red) on stressed replicating DNA

allowing replication to proceed even in the presence of DNA damage, thereby

promoting tumorigenesis. Talazoparib has recently been granted FDA approval for

metastatic breast cancers with BRCA mutations43. We previously showed that mtp53

R273H increases the ability of talazoparib to trap PARP1 on the chromatin7-8. We

propose that mtp53 R273H expression in TNBC allows the PARPi agent talazoparib in

combination with temozolomide to induce cell death because of increased PARP

trapping (shown in yellow) (see Fig. 5B).

DNA replication is regulated by various protein complexes that regulate origin

licensing and firing (licensing and initiating factors), unwinding (helicases), and

relaxation (topoisomerases). GOF mtp53 increases DNA replication origin firing9 and mutant p53 transactivates Cdc711. In addition, mtp53 R273H has been shown to facilitate the interaction of TopBP1 with Treslin, which is induced by Cdk2 under normal conditions13. We previously identified that mtp53 directly interacts with MCM replication licensing factors in multiple breast cancer cell lines and mice tumors with the analogous human R175H knockin mutation (Trp53R172H/R172H)8. The overexpression of MCMs is strongly associated with shorter survival in breast cancer patients44. Upregulation of Cdc7 expression during mammary tumorigenesis is linked to accelerating cell cycle progression, arresting tumor differentiation, increasing genomic instability and reducing disease-free survival45. Cdc7-mediated phosphorylation triggers the assembly of the initiation complex to fire the replication origins12. Thus, regulation of Cdc7 kinase to preformed pre-RCs is a major determinant for replication timing control.

The DNA fiber assay showed that activated oncogenes induce an increase in the fraction of terminated forks and fork asymmetry, along with a decrease in replication fork speed and increased origin firing35. Oncogenes induce the firing of novel replication origins, unlike the constitutive origins, which are intragenic and give rise to replication forks that are prone to collapse46. The collapse of forks initiating from intragenic, oncogene-induced origins can be attributed to replication-transcription conflicts, which is increased in cells with shortened G146. Remarkably, genome wide isolation and sequencing of Okazaki fragments (OK-Seq) revealed the comprehensive landscape of human genome replication47. Replication initiation zones are mostly non- transcribed, enriched in open chromatin and with replication fork progression

significantly co-oriented with transcription initiation47. More work is required to elucidate the role of GOF mtp53 on replicating DNA.

PARP inhibitors impair the enzymatic activity of the ADP-ribose addition to substrates, which in turn suppresses the function of PARP in base excision repair, single-strand break repair and DNA damage sensing48. Many PARP inhibitors also cause the protein to be trapped on chromatin thus causing more DNA damage and killing of cells via the formation of toxic DNA-PARP complexes49. PARP inhibitors are used on familial and sporadic breast and ovarian cancers with bi-allelic mutations in the

HR repair genes BRCA1, BRCA2, or PALB249. The inhibition of PARP1 function in rapidly dividing cancer cells results in an accumulation of single-strand breaks leading to broken replication forks that are essentially double strand breaks (DSBs)50. These

DSBs that normally would be repaired by HR are not repaired in BRCA1 deficient cells.

In this study, we demonstrated that PARP1 associated with mtp53 R273H on chromatin and enhanced PARylation also increased this association. Furthermore, higher levels of

PARylation were detected in a R273H mtp53 expressing breast cancer PDX model than in a wtp53 expressing PDX model. As PARP activity is associated with the levels of

PARylation, the detection of low levels of PAR may indicate an intrinsic low activity of

PARP with a limited PARP inhibitor toxicity in tissue that bears wtp53. Figure 4A shows a positive correlation between p53 and PARP in TNBC samples (see red dots).

Interestingly ER+ breast cancer samples show a positive correlation for some, but not all, of the patients examined (see green dots). High levels of p53 are associated with stabilized mtp53 and this is found more often in TNBC than in ER+ breast cancer. A possible molecular mechanism for the stabilization of PARP and mtp53 in the samples

could be due to mtp53 being stabilized during replication stress resulting in PARP

activation and therefore increased recruitment. Replication stress may be a key

facilitator of the activation of pathways that result in stabilization of both mtp53 and

PARP. However there remains the possibility that mtp53 changes the transcription of

PARP. Our data demonstrate the complexity of the crosstalk between mtp53 R273H

and PARP and suggest that in cancer cells the increased levels of PARP and mtp53

can be used as a proxy for cell survival in the presence of replication stress. It will be

interesting to determine if talazoparib in combination with the DNA damaging agent

temozolomide suppresses organoid growth in 3-D culture and possibly xenograft

(and/or PDX) growth in vivo. Our results suggest that the detection of co-expression of

mtp53 (which is mutated in more than 80% of TNBC) and high expression of PARP may

be a good combined biomarker to precisely identify the cancer patient populations that

will benefit from combined PARPi talazoparib with temozolomide treatment.

Acknowledgements

Grants to JB from the Breast Cancer Research Foundation (BCRF-18-011, and

BCRF-17-011) supported this work. This work was also supported by National Institutes of Health grants R01 CA204441 and P30 CA008748. Members of the Bargonetti lab are acknowledged for helpful discussion. We thank Dr Weigang Qiu for providing TCGA data analysis and Dr Sheryl Roberts for providing PARPi-FL. Thanks to Jeffery Luo for

help with PLA analysis and Ayden Ackerman for creative assistance with the art.

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Figure Legends:

Fig. 1 Mutant p53 R273H Interacts with Newly Replicated DNA in Association with PARP.

A) iPOND analysis of newly replicated DNA association with mtp53 R273H and PARP in MDA-MB-468.vector, MDA-MB-468.shp53 cells and mtp53R273H overexpression MDA-MB-468.shp53+R273H cells (left panel) and PANC-1 cells (right panel). 1 × 108 cells were labeled with 10 μM EdU for 45 min. The protein-DNA complexes were cross- linked, nascent DNA was conjugated to biotin using click chemistry, and protein-DNA complexes were purified. The eluted proteins were analyzed using western blot. A sample that did not include biotin-azide was used as a negative control. Representative of two independent experiments. B) Single cell in situ proximity ligation assay (PLA) with pulse EdU labeling showed mtp53 R273H associated with newly replicated DNA throughout S-phase. Cells were labeled with 125 µM EdU for 15 min. The protein-DNA complexes were cross-linked, nascent DNA was conjugated to biotin using click chemistry. Representative maximum intensity projection images of 2 central slices for S- phase progression of MDA-MB-468 (upper left panel) and PANC-1 (upper right panel) are shown; thickness: 2m. PLA of mtp53 R273H/EdU (red) was performed using anti- biotin and anti-p53 antibodies. Anti-biotin IF (green) indicates cells undergoing DNA synthesis. DNA was counterstained with DAPI (blue). Analysis of mtp53/EdU foci per nuclei by CellProfiler and S-phase progression for each nucleus was manually grouped into Early, Mid, or Late S-phase using GraphPad Prism 8. Statistical analysis for MDA- MB-468 and PANC-1: * represents a p-value ≤ 0.05, ** represents a p-value ≤ 0.01, **** represents a p-value ≤ 0.0001, ns= not significant. The p-value was determined by 2- tailed student t-test. MDA-MB-468: Early: n=86, Mid: n=49, Late: n=69; PANC-1: Early: n=51, Mid: n=23, Late: n=44. Representative of three independent experiments with two technical replicates each. C) The z-stack maximum intensity projection images showed mtp53 R273H and PARP associated with newly replicated DNA in MDA-MB-468 cells. Cells were labeled with 125 µM EdU for 15 min with or without 100 µM thymidine chases for 60 min. PLA with EdU (red) was performed as in Fig. 1B. Three independent experiments were performed. D) PLA with or without 20 min incubation of 10 µM poly(ADP-ribose) glycohydrolase inhibitor (PARGi) and 10 µM EdU labeling. Maximum intensity projection images are shown. Representative of two technical replicates. Fig. 2 Mutant p53 R273H, MCM2, and PARP Promote Cell Proliferation. A) Protein levels of phosphorylated MCM2 at serine 108, MCM2 and mtp53 in MDA-MB-468 and 468 CRISPR-C13 cells were determined by Western blot analysis. Cells were synchronized in G1/S phase by 5 µM Aphidicolin treatment for 24hr and then released into fresh media. 200 nM Nocodazole treated cells for 24 hr were used to arrest cells in G2/M phase. 10 µg of whole cell extract was loaded on 10% SDS-PAGE gel. Representative of four independent experiments for reduced mtp53 R273H protein levels and two experiments for cell cycle stages. B) Protein levels of mtp53, PARP, MCM2, Actin and Lamin A were determined by Western blot analysis with or without PARP knockdown by siRNA in addition to in the presence or absence of 5 µM

Aphidicolin treatment for 24hr. 20 µg of cytosolic or chromatin protein was loaded on a 10% SDS-PAGE. FACS data for the siRNA treated samples are shown. Representative of two independent siRNA PARP experiments. C) Mtp53 protein levels in MDA-MB-468 vector, shp53 and shp53+R273H cells with or without shRNA induction were determined by Western blot analysis prior to MTT and cell proliferation assays. Mtp53 depletion was achieved by growing cells in 8 μg/ml doxycycline for 7 days. 50 µg of whole cell extract was loaded on 10% SDS-PAGE gel. Two independent experiments were performed. D) MTT assay showed enhancement of mitochondrial activity when mtp53+R273H was overexpressed. Cells were seeded at 1.25 × 105 cells per well in 12- well plates with or without mtp53 depletion or mtp53 overexpression. Absorbance was quantified after 48hr. Four independent experiments were performed with 2 technical replicates each. * represents p-value ≤ 0.05, ** represents p-value ≤ 0.01, *** represents p-value ≤ 0.001. The p-value was determined by 2-tailed Student t-test. E) Cell proliferation assay was carried out in MDA-468.shp53 cells and mtp53 R273H overexpression MDA-468.shp53+R273H cells by cell counts. 2x105 cells/well were seeded in 6-well plate and grown 1, 3, and 4 days, respectively. At each time point, cell number was determined by hemocytometer. Three independent experiments were performed with 2 technical replicates.

Fig. 3 Mutant p53 R273H Associates with PARP and MCM Proteins and Increases Synergistic PARPi Plus Temozolomide Treatment Sensitivity.

A) Analysis of p53/PARP1 complexes (red) by immunofluorescence microscopy in combination with in situ proximity ligation assay (PLA) in MDA-MB-468 and MCF7 cells. DNA was counterstained with DAPI (blue). The z-stack maximum intensity projection images are shown. Two independent experiments were performed. B) Analysis of fluorescent foci per cell by CellProfiler software and demonstrated by scatter plot using Prism7 GraphPad. **** represents p-value ≤ 0.0001. The p-value was determined by 2- tailed Student t-test. Two independent experiments were performed. C) p53 protein level in MCF7 and MDA-MB-468 cells. Two independent experiments were performed. D) Co-Immunoprecipitation (Co-IP) of PARP and MCM7 with mtp53 in MDA-MB-468 cells. Two independent experiments were performed. E) MTT assay showed reduction of mitochondrial activity after combination treatment 1mM temozolomide plus 10 µM talazoparib for 24hr in cells with mtp53 R273H overexpression compared to cells with endogenous mtp53. Cells were seeded at 1.25 × 105 cells per well in 12-well plates and attached overnight. Cells were treated with either 10 µM talazoparib or 1mM temozolomide, or both for 24 hrs. The absorbance was quantified by measuring the absorbance at 550 nm subtracted from the absorbance at 620 nm. All MTT data are represented as mitochondrial dehydrogenase activity as percentage of a dimethyl sulfoxide (DMSO) vehicle-treated control. Three independent experiments were performed with two technical replicates. F) Chromatin protein levels of mtp53, 53BP1 and Fibrillarin were determined by Western blot analysis with or without combination treatment Temo plus Tal in MDA-MB-468 mtp53 R273H overexpression compared to

cells with endogenous mtp53. 50 µg of chromatin protein were loaded on 10% SDS- PAGE gel. Two independent experiments were performed.

Fig. 4 Mutant p53 and PARP1 Expression Are Correlated in Breast Cancer Patients and Live Imaging of PARP as Diagnostic Platforms. A) Analysis of protein expression correlation of p53 and PARP1 in breast invasive carcinoma samples from The Cancer Genome Atlas (TCGA) database40. A total of 817 breast tumor samples were profiled and 633 cases were also profiled by reverse-phase protein array (RPPA). ER, PR and HER2 status was clinically determined by immunohistochemistry. Scatter plots showed p53 and PARP1 protein expression correlation analysis by protein level (z-score) determined by RPPA. Red: TNBC; Green: estrogen receptor–positive (ER+) patients. B) Left panel: Examples of manually scored p53 and PARP1 staining intensity categories: no staining (0), weak staining (1), moderate staining (2) and strong staining (3) at magnification 200x. Right top panel: dot plot diagram showed immunohistochemical (IHC) staining of p53 score in tissue microarray (TMA) from Basal-like, Luminal A and Luminal B subtype of breast cancer. Right lower panel: Dot plot diagram showed immunohistochemical (IHC) staining of PARP1 score in tissue microarray (TMA) from Basel-like and Luminal A subtype of breast cancer grouped by p53 level. C) Live cell imaging of MDA-468 and MCF7 cells PARPi-FL (Green). Cells were imaged by confocal microscopy after 20 min incubation of 500 nM of PARPi-FL. Hoechst staining (blue) used to visualize nuclei. Two independent experiments were performed. D) Protein levels of p53, PARP1 and PAR in breast cancer PDX model WHIM6 and WHIM25 were determined by western blot analysis. Whole cell proteins were extracted from NSG mice xenograft tumors and 50ug of protein was loaded on 10% SDS-PAGE gel. Three technical replicates were performed.

Fig. 5 Model for Mutant R273H p53, PARP1 and MCM2-7 on Replicating DNA Promoting Tumorigenesis versus Cell Death. The coordination of mtp53 R273H, PARP, and MCM2-7 on replicating DNA implies a role in tumorigenesis. A) MCM2-7 (shown in purple), mtp53 R273H (shown in green), and PARP (shown in red) interact with replicating DNA (i), when damage occurs, they help facilitate aberrant repair (ii), allowing cells to survive, promoting tumorigenesis (iii). B) When high PARP and mtp53 expressing cells (i) are treated with talazoparib (shown in yellow) in combination with temozolomide, PARP is trapped on replicating chromatin, this increases unrepaired DNA damage (shown in brick) (ii), promoting cell death (iii).

Gain-of-Function Mutant p53 R273H Interacts with Replicating DNA and PARP1 in Breast Cancer

Gu Xiao, Devon Lundine, George K Annor, et al.

Cancer Res Published OnlineFirst November 27, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-1036

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2019/11/27/0008-5472.CAN-19-1036.DC1

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