Original Article

Yonago Acta Medica 2017;60:09–15

DNA-PK Inhibition by NU7441 Enhances Chemosensitivity to Topoisomerase
Inhibitor in Non-Small Cell Lung Carcinoma Cells by Blocking DNA Damage Repair
Masaaki Yanai, Haruhiko Makino, Bingqiong Ping, Kenichi Takeda, Natsumi Tanaka, Tomohiro Sakamoto,
Kosuke Yamaguchi, Masahiro Kodani, Akira Yamasaki, Tadashi Igishi and Eiji Shimizu
Division of Medical Oncology and Molecular Respirology, Department of Multidisciplinary Internal Medicine, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504, Japan

ABSTRACT

cause of cancer-related death worldwide. Over half ofpatients with NSCLC have been advanced or metastatic
lesions at the time of diagnosis.1 Systemic chemotherapy
is the standard treatment for patients with advanced NSCLC. In many countries, including Japan, cytotoxic chemotherapeutic agents are used as the first-line treatment
for advanced NSCLC patients. However, these cytotoxic
chemotherapies have reached a therapeutic plateau andhave limited survival benefits for advanced NSCLC patients. In recent years, treatment with targeted agents has
markedly improved progression-free and overall survival
in patients with advanced NSCLC harboring EGFR mutation or ALK rearrangement.2, 3 However, only 20–30%
of NSCLC patients harbor targets available for molecular targeting agents and most acquire resistance against
molecular target agents within 1 year. Therefore, newagents and new combination chemotherapy regimens are
required to improve the outcomes of all advanced NSCLC patients.
DNA-dependent protein kinase, catalytic subunit(DNA-PKcs) is an essential component in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair.4 In response to DNA
DSBs, DNA-PKcs is rapidly recruited to damage sites
by the Ku70/Ku80 heterodimer and phosphorylated at
Thr2609 and Ser2056 clusters by ATM and itself, respectively.5, 6 The recruitment and phosphorylation of
DNA-PKcs contributes to the processing and direct ligation of broken DNA ends. Several studies demonstrated
increased protein levels of DNA-PKcs in NSCLC,7–9 andthe elevation of DNA-PKcs has been correlated with

BackgroundDNA double-strand breaks (DSBs) are
the most cytotoxic form of DNA damage and are induced by ionizing radiation and specific chemotherapeutic agents, such as topoisomerase inhibitors. Cancer cells
acquire resistance to such therapies by repairing DNA
DSBs. A major pathway for the repair of DNA DSBs is
non-homologous end-joining (NHEJ), which requires
DNA-dependent protein kinase (DNA-PK) activity. In
this study, we investigated the effect of NU7441, a synthetic small-molecule compound, as a specific inhibitorof DNA-PK on the chemosensitization of non-small celllung carcinoma (NSCLC) A549 cells.
Methods The combined effects of chemotherapeutic
agents and NU7441 were evaluated by isobologram
analysis using Cell Counting Kit-8. DNA DSBs wereassessed by immunofluorescence assay. Apoptosis was
examined by flow cytometry using an Annexin V apoptosis kit. Activation of DNA-PK was assayed by western
blotting.
Results The combination of NU7441 and topoisomerase inhibitors such as amrubicin and irinotecan had
a synergistic effect on cell proliferation in A549 cells.
NU7441 increased 53BP1 foci and apoptosis induced
by topoisomerase inhibitors and decreased phospho-DNA-dependent protein kinase, catalytic subunit(pDNA-PKcs) (S2056) protein expression caused by
topoisomerase inhibitors. Interestingly, mitotic inhibitors
such as pacritaxel did not cause the pDNA-PKcs (S2056)
protein expression and the combination of NU7441 andpacritaxel had an only additive effect.
Conclusion NU7441 inhibited the growth of NSCLC
cells and enhanced the chemosensitization to topoisomerase inhibitors by blocking DNA repair. A combination
of NU7441 and topoisomerase inhibitor may be a promising treatment for NSCLC.

Corresponding author: Haruhiko Makino, MD, PhD
hmakino@med.tottori-u.ac.jp
Received 2016 December 1
Accepted 2016 December 21
Abbreviations: 53BP1, p53 binding protein 1; ALK, anaplastic
lymphoma kinase; AMR, amrubicin; CCK-8, Cell Counting Kit8; CI, combination index; CPT-11, irinotecan; DAPI, 4,6-diamidino-2-phenylindole; DNA-PK, DNA-dependent protein kinase;
DNA-PKcs, DNA-dependent protein kinase catalytic subunit;
DSB, double-strand break; EGFR, epidermal growth factor receptor; IC50, 50% inhibition of cell growth; NHEJ, non-homologous
end-joining; NSCLC, non-small cell lung carcinoma; PTX, pacritaxel

Key words

DNA-dependent protein kinase; non-homologous end-joining; non-small cell lung carcinoma;
NU7441; topoisomerase inhibitor
Non-small cell lung carcinoma (NSCLC) is one of themost commonly diagnosed cancers and the leading9

M. Yanai et al.

radioresistance in some advanced stage cancers.9 Therefore, targeting of DNA-PKcs is an attractive approachfor enhancing radio-sensitivity in NSCLC. Given its important role in the NHEJ repair pathway, different antiDNA-PKcs strategies have been explored to enhance thesensitivity to radiation or DNA damage-based agents. In
a series of small-molecule ATP-competitive inhibitorsof DNA-PKcs, NU7441 was found to be the most potent
and specific inhibitor of DNA-PKcs at a low concentration relative to other members of the PI3KK family (ATMand ATR).10 Several studies have shown that NU7441
can enhance the sensitivity to radiation in NSCLC.11
However, little is known about the potency of NU7441
in enhancing the sensitivity of cytotoxic chemotherapeutic agents.
Among cytotoxic anti-cancer drugs, topoisomerase inhibitors target DNA topoisomerases, which areessential nuclear enzymes that catalyze the breakage
and rejoining of DNA. There are two classes of DNA
topoisomerases, type I and type II, which alter the topology of single- and double-stranded DNA, respectively,
and are involved in genetic reactions including DNA
replication, transcription, and DNA repair.12 DNA topoisomerase inhibitors, such as irinotecan (CPT-11; topo
I inhibitor) and amrubicin (AMR, topo II inhibitor), areimportant in lung cancer chemotherapy.13, 14
In the present study, we evaluated the effect of
combining topoisomerase inhibitors, which cause DNA
damage, with DNA-PKcs inhibitors, which inhibit DNADSB repair by blocking the NHEJ pathway, on NSCLC.

NY) for 48 h. Next, the indicated drugs were added tothe culture media. After 72 h of drug treatment, 10%
CCK-8 solution (Dojindo, Kumamoto, Japan) was added
to each well and incubation was continued for an additional 3 h. The absorbance of each well was measured ata wavelength of 450 nm using a Sunrise scanning multiwell spectrometer (Tecan, Männedorf, Switzerland).
Each experiment was performed in triplicate for eachdrug concentration.
Assessment of combination effect

To qualitatively assess the combination effect of the indicated agents, isobologram analysis was conducted as described previously.15 The percentage of cell proliferation
was calculated as: [(mean absorbance of drug-treatedwells – mean absorbance of cell-free wells)/(mean absorbance of vehicle cells – mean absorbance of cellfree
wells)] × 100. We used the concentration resulting in
50% inhibition of cell growth (IC50) to evaluate dose-response interactions.
Apoptosis assay

Apoptosis of A549 cells was detected by the Annexin V
Apoptosis Detection Kit APC (eBioscience, San Diego,
CA). A549 cells were treated with or without NU7441
and with anticancer agents for 24 and 48 h. The cells
were harvested by trypsinization, collected in centrifuge tubes, and washed with ice-cold phosphate-buffered saline. Next, the cells were suspended in 500 μL
of binding buffer and incubated with Annexin V-APC
and propidium iodide or 4,6-diamidino-2-phenylindole(DAPI). The apoptotic population was assayed by flow
cytometry (LSRFortessa X-20; BD Biosciences, Franklin Lakes, NJ) and analyzed using FlowJo software(Treestar, San Carlos, CA).

MATERIALS AND METHODS
Chemicals and reagents

AMR (Dainippon Sumitomo Pharma, Tokyo, Japan),
CPT-11 (Daiichi Sankyo Pharma, Tokyo, Japan), and paclitaxel (PTX) (Bristol-Myers-Squibb, New York, NY)
were dissolved in distilled water and stored at –20 °C.
NU7441 (AdooQ Bioscience, Irvine, CA) was dissolved
in dimethyl sulfoxide and stored at –20 °C.

Immunofluorescence staining

DNA-DSBs were assessed by quantifying the rates ofdissolution of p53 binding protein 1 (53BP1) foci as described. Immunofluorescence staining was performedin cells cultured in glass chamber slides (Lab-Tek II
Chamber Slide w/Cover RS Glass Slide Sterile; Nalge
Nunc). A549 cells were treated with or without NU7441,
and then treated with each anticancer agent for 1 h. At
the indicated times, cells were fixed with 4% PFA, permeabilized with 0.5% Triton X-100, and immunostainedwith 53BP1 primary antibody (diluted 1:1000; Cell
Signaling Technologies, Beverly, MA) and Alexa Fluor
488 goat anti-rabbit secondary antibody (diluted 1:2000;
Life Technologies, Carlsbad, CA). The nuclei of cells
were stained using blue fluorescent DAPI in Vectashield
mounting medium (Vector Laboratories, Burlingame,Cells

The lung adenocarcinoma A549 cell line was purchasedfrom the American Type Culture Collection (ATCC,
Manassas, VA). A549 cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum and antibiotics. These cells were grown in a humidified atmosphere of 5% CO2 and 95% air at 37 °C.
Cell Counting Kit-8 (CCK-8) assay

A CCK-8 assay was performed to evaluate cell proliferation inhibition. Cells (1000 per well) were cultured in
96-well flat-bottom multi-plates (Nalge Nunc, Penfield,
10Synergism of NU7441 and topoisomerase inhibitors in NSCLC

Data analysis

CA). Images were obtained with a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). The 53BP1
foci per nucleus were visually counted in the acquired
images.

The experimental data was analyzed by using Graphpad
Prism (Graphpad Software, San Diego, CA). The results
were expressed as the mean ± standard deviation. Two
independent samples were analyzed by the t-test.

Western blotting

RESULTS

Intracellular proteins from cells treated with AMR, CPT11, or PTX and with or without NU7441 at the indicatedconcentrations and time points were prepared using
NE-PER Nuclear and Cytoplasmic Extraction Reagents(Thermo Scientific, Waltham, MA). Proteins were measured using the Bio-Rad Protein assay reagent (Bio-Rad
Laboratories, Hercules, CA), and protein lysates containing 45 μg of total intracellular protein were subjected to
discontinuous SDS-polyacrylamide gel electrophoresis.
Proteins were electrotransferred to a polyvinylidene
fluoride membrane (GE Healthcare, Little Chalfont,UK) overnight at 4 °C at 45 V. Non-specific binding was
blocked by incubation with 5% bovine serum albumin
or non-fat milk in Tris-buffered saline containing 0.1%
Tween-20 for 2 h at room temperature. The following
primary antibodies were used: anti-DNA-PKcs (diluted
1:1000; Thermo Scientific), anti-phospho-DNA-PKcs(Serine2056) (diluted 1:5000; AbCam, Cambridge, MA),and anti-Lamin B (diluted 1:1000; AbCam) overnight
and washed twice with Tris-buffered saline containing
0.1% Tween-20. After washing, proteins were detectedby incubation with horseradish peroxidase-labeled secondary antibodies (GE Healthcare). Each protein was
detected using an enhanced chemiluminescence detection system (ECL prime) (GE Healthcare) and detectedusing ImageQuant LAS400 (GE Healthcare).

First, we assessed the IC50 concentrations of chemotherapeutic agents and NU7441 on A549 cells in the CCK8 assay at 72 h. The IC50 values of AMR, CPT-11, PTX,
and NU7441 on A549 cells were 3.1 μM, 27 μM, 7.5
nM, and 0.8 μM, respectively, and these results weresimilar to those of previous reports.16–18 We evaluated the
combination effects of NU7441 and chemotherapeutic
agents in A549 cells by isobologram analysis. Representative results of isobologram analysis are shown in Fig.
1. The combination of NU7441 with AMR and CPT11 synergistically inhibited cell growth, whereas only
additive effects were observed for the combination of
NU7441 and PTX. We conducted the analyses at least
three times and similar trends were obtained.
DNA DSB induced by chemotherapeutic agentsand combination effect of NU7441

To evaluate DNA damage caused by chemotherapeutic
agents and the additional effect of NU7441, we subjected A549 cells exposed to chemotherapeutic agents withor without NU7441 to immunofluorescence staining
for 53BP1 (green), a DNA damage response factor.19 As
shown in Fig. 2, we observed 53BP1 foci in the nuclei ofA549 cells exposed to AMR and CPT-11. Furthermore,

0

0

1

AMR

NU 7441

1

NU 7441

1

NU 7441

1

Combination effects of NU7441 and chemotherapeutic agents

0

0

1

C P T -1 1

0

0

1

PTX

Fig. 1. Combination effects of NU7441 and chemotherapeutic agents in A549 cells.
A549 cells were treated with a combination of NU7441 and AMR, CPT-11, or PTX for 72 h. The combination effects were evaluated by
Figure 1. 	
isobologram analysis. The envelopes of additivity are defined by three isoeffect lines constructed from the dose-response curves of thesingle agents. The concentration resulting in 50% cell growth inhibition (IC50) of NU7441, AMR, CPT-11, or PTX alone is expressed as
one on the ordinate and the abscissa. The plotted data points show the relative values of the concentrations resulting in the IC50. AMR,
amrubicin; CPT-11, irinotecan; IC50, 50% inhibition of cell growth; PTX, paclitaxel.

11

M. Yanai et al.

A

B
100

100

80

80

C o n tro l
AMR 1 h100

% > 10 fo ci% > 10 fo ci

CPT 1 h

60

U

N

–

+

+1 0

–

U

U

N

% > 10 fo ci

Fig. 2. DNA DSBs in A549 cells exposed to chemotherapeutic agents in the presence and absence of DNA-PK inhibitor (NU7441).
A549 cells were treated with the IC50 dose of AMR (3 μM), CPT-11 (25 μM), or PTX (7.5 nM) for 1 h with or without NU7441 (10 μM,
1 h prior to chemotherapeutic agent exposure) and samples were immunostained with anti-53BP1 antibodies (green) and DAPI (blue) at
C o n tro l
4 0 and 24 h (B) after chemotherapeutic agent exposure.
1 h (A)
Representative images for each condition are shown. Percentages of cells
C P T -1 1 2 4 hwith > 10 53BP1 foci treated as in A (C) and B (D). Dateareexpressed
as the mean ± SE (n = 3). *P < 0.05. 53BP1, p53 binding protein
PTX 24 h
1; AMR, amrubicin; CPT-11, irinotecan; DAPI, 4,6-diamidino-2-phenylindole; DNA-PK, DNA-dependent protein kinase; DSB, dou30
ble-strand
break; IC50, 50% inhibition of cell growth; ns, not significant; NU, NU7441; PTX, pacritaxel.
ns20

CPT-11 (P < 0.05, Fig. 2D). In contrast, the rate of cellswith > 10 53BP1 foci did not increase following combination treatment with PTX and NU7441. Unfortunately,
the cells at 24 h after exposure of AMR were difficult toevaluate because of large changes in cell morphology.

the number of cells with 53BP1 foci increased under
combination treatment with NU74441. In contrast, thenumber of 53BP1 foci in the nuclei of A549 cells exposed to PTX was similar to the control condition anddid not increase following the addition of NU7441. To
quantify DNA DSB, we counted and graphed the number of cells with > 10 53BP1 foci. Immediately after
drug exposure, the rate of cells with > 10 53BP1 focitreated with a combination of AMR and NU7441 washigher than that of cells treated with only AMR (P < 0.05,
Fig. 2B). At 24 h after drug exposure, the rate of cellswith > 10 53BP1 foci treated with a combination of CPT11 and NU7441 was higher than that treated with only
ns

10

–

U

U
+

N

N

–

+

N
U

N
U

+

U

N

N

U

–

0

Apoptotic effects of NU7441 and chemotherapeutic agentsTo investigate the combination effect of NU7441 andchemotherapeutic agents on cell apoptosis, we assayed
the cells treated with or without NU7441 and with
AMR, CPT-11, or PTX for 24 or 48 h using an Annexin
V apoptosis kit. At 24 h after drug exposure (Fig. 3A),
12

–
U

U

N

N

–

+

U

U

–
U
N

–

+

U

U

N

+
U
N

–

+

U
N

N

–

0

+

U

U

N

10

U

N

N

U
N

N

U

+

–

0

ns

N

N

–

U

10

ns

N

N

+

U

+

U

N

N

N

N

–

+

U

N

U

+

U

N

20

+

N

–
U

+

U

U

N

ns

30

U

N

–

U

+

U

N

ns

PTX 24 h

–

+

U

–

U

N

ns

NNU
U–
+

–

U

N

20

C P T -1 1 2 4 h

N

N

–

+

U

N

C o n tro l

C o n tro l

U

U

–

U

N

30

0

N

0

–
+
U
U
N
N

N

+

U

N

ns

40

% > 10 fo ci% > 10 fo ci

ns

20

0

C P T -1 1 2 4 h

20

PTX 1 h

ns

+

U

N

40

30

CPT 1 h

40

–

U

N

% > 10 fo ci

–

D

60

+

U

N

PTX 24 h

ns

U

ns

N

ns

40

40

N

80

–

U

U

N

60

% > 10 fo ci% > 10 fo ci

0

ns

0

AMR 1 hns

C P T 12 0 h
PTX 1 h20

C o n tro lns40

AMR 1 h
ns

20

100

ns
60

C o n tro l

40

80

C

PTX 1 hns

Synergism of NU7441 and topoisomerase inhibitors in NSCLC

	
  A	
  	
  24	
  h

C o n tro l*

100

AM R 24 h

C o n tro l*

C P T -1 1 4 8 h

C P T -1 1 2 4 h

40

ns

N
U

N
U
–
N
U
+

N
U
–
N
U
+

N
U
–
N
U
+

N
U

–
N
U
+

0

–

–

–

N
U
+

20

N
U

0

ns

N
U
+

10

60

N
U

ns

ns
20

PTX 48 h
80

NU+

PTX 24 h30

A p o p to sis rate (%)

A p o p to s is ra te (%)

40

	
  B	
  	
  48	
  h

ns

Fig. 3. Apoptotic effect of combinations of NU7441 and chemotherapeutic agents.
A549 cells were treated with IC50 dose of AMR (3 μM), CPT-11 (25 μM), or PTX (7.5 nM) for 24 and 48 h with or without NU7441 (2.5μM, 1 h prior to chemotherapeutic agent exposure). Samples were assayed with Annexin V. Bar graphs representing the percentage ofapoptosis cells at 24 h (A) and 48 h (B). Date are expressed as the mean ± SE (n = 3). *P < 0.05. AMR, amrubicin; CPT-11, irinotecan;
IC50, 50% inhibition of cell growth; ns, not significant; NU, NU7441; PTX, pacritaxel.

	
  A	
  

	
  B	
  	
  

Fig. 4. Activation of DNA-PKcs induced by chemotherapeutic agents and effect of DNA-PK inhibitor (NU7441).
(A) A549 cells were pre-treated with or without a DNA-PKcs inhibitor (NU7441, 10 μM) and treated with 1/10 IC50 dose or IC50 dose ofPTX (0.75 nM, 7.5 nM), CPT-11 (2.5 μM, 25 μM), or AMR (0.3 μM, 3 μM) for 1 h. Activation of DNA-PKcs was assayed by western
blotting with antibodies as indicated. (B) A549 cells were pre-treated with or without NU7441 (10 μM) and treated with IC50 dose of PTX
Figure 4.
(7.5 nM) or CPT-11 (25 μM). Activation of DNA-PKcs was assayed at 1, 12, or 24 h after 1-h treatment with each agent by western blotting using the indicated antibodies. AMR, amrubicin; CPT-11, irinotecan; DNA-PKcs, DNA-dependent protein kinase catalytic subunit;
IC50, 50% inhibition of cell growth; pDNA-PKcs, phospho-DNA-dependent protein kinase catalytic subunit; PTX, paclitaxel.

AMR significantly increased apoptosis following addition of NU7441. At 48 h after drug exposure (Fig. 3B),
CPT clearly increased apoptosis when NU7441 was also
added. In contrast, PTX induced apoptosis, but there
was no combination effect with NU7441. Unfortunately,
apoptosis at 48 h after exposure of AMR was difficult toevaluate because of the high rate of cell death.

to PTX, CPT-11, and AMR by western blotting. The
nuclear proteins were extracted using NE-PER nuclear
and cytoplasmic extraction reagents. After 1-h exposure,
AMR clearly activated DNA-PKcs, while PTX and
CPT-11 only slightly activated DNA-PKcs (Fig. 4A).
After long-term incubation (12 and 24 h), following 1-h
drug exposure, CPT-11 clearly activated DNA-PKcs overtime, while PTX did not show this activation effect (Fig.
4B). These results suggest that the timing and intensityof phosphorylation of DNA-PKcs depends on the chemotherapeutic agent used. In addition, the activation of
DNA-PKcs induced by chemotherapeutic agents was
suppressed by NU7441.

Activation of DNA-PKcs induced by chemotherapeutic agents and suppression effect by NU7441

To assess the activation of DNA-PKcs induced by chemotherapeutic agents in NSCLC cells at different concentrations and times, we detected pDNA-PKcs (S2056)
protein expression in the nuclei of A549 cells exposed13

M. Yanai et al.

DISCUSSION
DNA-PKcs is an essential component of the NHEJ pathway and activation of DNA-PKcs is required for DNA
DSB repair.20 Inhibiting phosphorylation of DNA-PKcs
blocks DNA damage repair and leads to cell apoptosis.
NU7441, a potent and specific DNA-PKcs inhibitor,
has been predicted to enhance the therapeutic effect of
inducing DNA DSB. Previously, several studies showedthat NU7441 has potential for enhancing the radio-sensitivity in different tumors, including NSCLC,11 coloncancer,21 breast cancer,22 and prostate cancer,23 and chemo-sensitivity of etoposide (topoisomerase II inhibitor)
in colon cancer.21 The goal of this study was to determine the best combination drug for use with NU7441 toenhance the anti-proliferative effect on NSCLCs.
In this study, a DNA-PKcs inhibitor showed promise
in combination pharmacological medicine with specificchemotherapeutic agents against lung cancer. The DNAPKcs inhibitor had a synergistic anti-proliferative effecton the A549 lung cancer cell line when applied with
topoisomerase inhibitors. Interestingly, the inhibitor
only had an additive effect with the mitotic inhibitor.
Topoisomerases have been examined for use in thetreatment of certain diseases. Among them, eukaryotic
topoisomerases I and II are targets of anti-cancer drugs
that act to inhibit these enzymes by blocking the reactionthat reseals breaks in the DNA.24 Topoisomerase I inhibitors, including CPT-11, induce single-strand breaks in
DNA by blocking the dissociation of topoisomerase andDNA, leading to replication-mediated DNA damage.
Topoisomerase II inhibitors, including AMR, can act byeither stabilizing topoisomerase II-DNA complexes thatare easily cleaved or interfering with the catalytic activity of the enzyme, both resulting in DSBs. In contrast,
mitotic inhibitors, including PTX, bind to tubulin andinhibit its polymerization into microtubules, resulting
in tumors and leading to apoptosis without involvementwith DSBs.25, 26
Phosphorylation of DNA-PKcs occurs at the S2056
site,5 which provides a marker for assessing the effectof drugs on DNA-PKcs activity. In this study, we foundthat topoisomerase inhibitor induced DNA-PKcs phosphorylation of A549. Topoisomerase II inhibitor AMR
immediately phosphorylated DNA-PKcs, whereas topoisomerase I inhibitor CPT-11 only minimally induced
DNA-PKcs phosphorylation immediately after exposure
to drugs and gradually phosphorylated DNA-PKcs overtime. This delay in DNA-PKcs phosphorylation may berelated to the time required to shift from single-strand
breaks to DSBs. NU7441 clearly inhibited DNA-PKcs
phosphorylation caused by both AMR and CPT-11.
In this study, we assessed the degree of DNA DSBs

using 53BP1 foci as a marker of DNA DSB.19 AMR and
CPT-11 caused DNA DSBs, whereas PTX caused few
DNA DSBs. The combined use of NU7441 induced agreater number of DNA DSBs. In addition, NU7441
in combination with topoisomerase inhibitors induced
apoptosis by more than the use of topoisomerase inhibitors alone.
This is the first study to demonstrate that the DNAPKcs inhibitor Nu7441 has a synergistic effect with
topoisomerase inhibitors, but not with a mitotic inhibitor, on NSCLC cells. Recently, several clinical studies
revealed that DNA-PKcs expression is significantly elevated in advanced disease and that an elevation in activated DNA-PKcs predicts the response to treatment andsurvival.27, 28 Further, DNA-PKcs inhibitors are currently
in phase I trials for advanced solid tumors and hematologic malignancies (NCT01280487, NCT01421524, and
NCT01682473) and the efficacy of DNA-PKcs inhibitors
has been demonstrated in clinical trials.29 Based on our
results, further studies, including preclinical studies andclinical trials, are necessary to clarify the efficacy of thiscombination therapy with NU7441 and topoisomerase
inhibitors in NSCLC.
In conclusion, NU7441 blocks DNA damage repair
induced by topoisomerase inhibitors in NCSLC cells andsubsequently leads to their apoptosis. Therefore, our results suggest that combination therapy of topoisomerase
inhibitors and NU7441 is a potential therapeutic option
for NCSLC.
Acknowledgments: This work was supported by JSPS KAKENHI
Grant Number 26830075.
The authors declare no conflict of interest.

REFERENCES

1 Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA
Cancer J Clin. 2015;65:5-29. PMID: 25559415.
2 Maemondo M, Inoue A, Kobayashi K, Sugawara S,
Oizumi S, Isobe H, et al. Gefitinib or chemotherapy for nonsmall-cell lung cancer with mutated EGFR. N Engl J Med.
2010;362:2380-8. PMID: 20573926.
3 Shaw AT, Yeap BY, Solomon BJ, Riely GJ, Gainor J,
Engelman JA, et al. Effect of crizotinib on overall survival inpatients with advanced non-small-cell lung cancer harbouring
ALK gene rearrangement: a retrospective analysis. Lancet
Oncol. 2011;12:1004-12. PMID: 21933749.
4 Chan DW, Chen BP, Prithivirajsingh S, Kurimasa A,
Story MD, Qin J, et al. Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks. Genes Dev. 2002;16:23338. PMID: 12231622.
5 Chen BP, Chan DW, Kobayashi J, Burma S, Asaithamby A,
Morotomi-Yano K, et al. Cell cycle dependence of DNA-dependent protein kinase phosphorylation in response to DNA
double strand breaks. J Biol Chem. 2005;280:14709-15.

14

Synergism of NU7441 and topoisomerase inhibitors in NSCLCPMID: 15677476.
6 Chen BP, Uematsu N, Kobayashi J, Lerenthal Y, Krempler A,
Yajima H, et al. Ataxia telangiectasia mutated (ATM) is essential for DNA-PKcs phosphorylations at the Thr-2609 cluster
upon DNA double strand break. J Biol Chem. 2007;282:65827. PMID: 17189255.
7 Hu S, Qu Y, Xu X, Xu Q, Geng J, Xu J. Nuclear survivin andits relationship to DNA damage repair genes in non-small
cell lung cancer investigated using tissue array. PloS One.
2013;8:e74161. PMID: 24066112.
8 Hsu FM, Zhang S, Chen BP. Role of DNA-dependent protein
kinase catalytic subunit in cancer development and treatment.
Transl Cancer Res. 2012;1:22-34. PMID: 22943041.
9 Xing J, Wu X, Vaporciyan AA, Spitz MR, Gu J. Prognostic
significance of ataxia-telangiectasia mutated, DNA-dependent protein kinase catalytic subunit, and Ku heterodimeric
regulatory complex 86-kD subunit expression in patients with
nonsmall cell lung cancer. Cancer. 2008;112:2756-64. PMID:
18457328.
10 Lea hy JJ, Golding BT, Gr iffin R J, Ha rdcastle IR,
Richardson C, Rigoreau L, et al. Identification of a highly
potent and selective DNA-dependent protein kinase (DNAPK) inhibitor (NU7441) by screening of chromenone libraries.
Bioorg Med Chem Lett. 2004;14:6083-7. PMID: 15546735.
11 Yu L, Shang ZF, Hsu FM, Zhang Z, Tumati V, Lin YF, et al.
NSCLC cells demonstrate differential mode of cell death inresponse to the combined treatment of radiation and a DNAPKcs inhibitor. Oncotarget. 2015;6:3848-60. PMID: 25714019.
12 Wang JC. DNA topoisomerases. Annu Rev Biochem.
1985;54:665-97. PMID: 2992360
13 Masuda N, Kudoh S, Fukuoka M. Irinotecan (CPT-11): pharmacology and clinical applications. Crit Rev Oncol Hematol.
1996;24:3-26. PMID: 8869796.
14 Nakao M, Oguri T, Suzuki T, Kunii E, Tomita Y, Iwashima
Y, et al. Amrubicin monotherapy for elderly patients withpreviously treated lung cancer. Intern Med. 2010;49:1857-62.
PMID: 20823645.
15 Steel GG, Peckham MJ. Exploitable mechanisms in combined
radiotherapy-chemotherapy: the concept of additivity. Int J
Radiat Oncol Biol Phys. 1979;5:85-91. PMID: 422420.
16 Ito S, Igishi T, Takata M, Ueda Y, Matsumoto S, Kodani M,
et al. Synergistic cell growth inhibition by the combination ofamrubicin and Akt-suppressing agents in K-ras mutation-harboring lung adenocarcinoma cells: implication of EGFR tyrosine kinase inhibitors. Int J Oncol. 2014;44:685-92. PMID:
24399305.
17 Kojima A, Hackett NR, Ohwada A, Crystal RG. In vivo hu-

man carboxylesterase cDNA gene transfer to activate the prodrug CPT-11 for local treatment of solid tumors. J Clin Invest.
1998;101:1789-96. PMID: 9541511.
18 Zhao M, Lei C, Yang Y, Bu X, Ma H, Gong H, et al. Abraxane, the Nanoparticle Formulation of Paclitaxel Can Induce Drug Resistance by Up-Regulation of P-gp. PloS One.
2015;10:e0131429. PMID: 26182353.
19 Takai H, Smogorzewska A, de Lange T. DNA damage foci at
dysfunctional telomeres. Curr Biol. 2003;13:1549-56. PMID:
12956959.
20 Davis AJ, Chen DJ. DNA double strand break repair via
non-homologous end-joining. Transl Cancer Res. 2013;2:13043. PMID: 24000320.
21 Zhao Y, Thomas HD, Batey MA, Cowell IG, Richardson
CJ, Griffin RJ, et al. Preclinical evaluation of a potent novelDNA-dependent protein kinase inhibitor NU7441. Cancer
Res. 2006;66:5354-62. PMID: 16707462.
22 Ciszewski WM, Tavecchio M, Dastych J, Curtin NJ. DNAPK inhibition by NU7441 sensitizes breast cancer cells toionizing radiation and doxorubicin. Breast Cancer Res Treat.
2014;143:47-55. PMID: 24292814.
23 Shaheen FS, Znojek P, Fisher A, Webster M, Plummer R,
Gaughan L, et al. Targeting the DNA double strand breakrepair machinery in prostate cancer. PloS One. 2011;6:e20311.
PMID: 21629734.
24 Wang JC. Cellular roles of DNA topoisomerases: a molecularperspective. Nat Rev Mol Cell Biol. 2002;3:430-40. PMID:
12042765.
25 Diaz JF, Menendez M, Andreu JM. Thermodynamics
of ligand-induced assembly of tubulin. Biochemistry.
1993;32:10067-77. PMID: 8104479.
26 Rao S, Horwitz SB, Ringel I. Direct photoaffinity labeling of
tubulin with taxol. J Natl Cancer Inst. 1992;84:785-8. PMID:
1349352.
27 Goodwin JF, Kothari V, Drake JM, Zhao S, Dylgjeri E, Dean
JL, et al. DNA-PKcs-Mediated Transcriptional Regulation
Drives Prostate Cancer Progression and Metastasis. Cancer
Cell. 2015;28:97-113. PMID: 26175416.
28 Cornell L, Munck JM, Alsinet C, Villanueva A, Ogle L,
Willoughby CE, et al. DNA-PK-A candidate driver of hepatocarcinogenesis and tissue biomarker that predicts responseto treatment and survival. Clin Cancer Res. 2015;21:925-33.
PMID: 25480831.
29 Thijssen R, Ter Burg J, Garrick B, van Bochove GG, Brown
JR, Fernandes SM, et al. Dual TORK/DNA-PK inhibition
blocks critical signaling pathways in chronic lymphocytic leukemia. Blood. 2016;128:574-83. PMID: 27235137.

15