The Dual PI3K/mTOR Inhibitor
NVP-BEZ235 Is a Potent Inhibitor
of ATM- and DNA-PKCs-Mediated
DNA Damage Responses1,2
Bipasha Mukherjee3, Nozomi Tomimatsu3,
Kaushik Amancherla3, Cristel V. Camacho,
Nandini Pichamoorthy and Sandeep Burma
Department of Radiation Oncology, University of Texas
Southwestern Medical Center, Dallas, TX, USA
Inhibitors of PI3K/Akt signaling are being actively developed for tumor therapy owing to the frequent mutational
and potent dual PI3K/mTOR inhibitor that is currently in phase 1/2 clinical trials for advanced solid tumors. Here, we
show that NVP-BEZ235 also potently inhibits ATM and DNA-PKcs, the two major kinases responding to ionizing
radiation (IR)–induced DNA double-strand breaks (DSBs). Consequently, NVP-BEZ235 blocks both nonhomologous
end joining and homologous recombination DNA repair pathways resulting in significant attenuation of DSB repair.
In addition, phosphorylation of ATM targets and implementation of the G2/M cell cycle checkpoint are also attenuated
by this drug. As a result, NVP-BEZ235 confers an extreme degree of radiosensitization and impairs DSB repair in a
panel of GBM cell linesirrespective oftheir Akt activation status. NVP-BEZ235alsosignificantly impairs DSBrepair in a
mouse tumor model thereby validating the efficacy of this drug as a DNA repair inhibitor in vivo. Our results, showing
that NVP-BEZ235 is a potent and novel inhibitor of ATM and DNA-PKcs, have important implications for the informed
radiosensitizer for GBMs in the clinic.
Neoplasia (2012) 14, 34–43
The phosphatidylinositol 3-kinase (PI3K)–Akt-mTORC1 pathway is
frequently activated in a variety of human cancers, including glioblas-
tomas (GBMs). Therefore, inhibitors of PI3K/Akt signaling are being
actively developed for tumor therapy (reviewed in Liu et al.  and
Garcia-Echeverria and Sellers ). Dual PI3K-mTOR inhibitors are
particularly effective in blocking Akt activation because they prevent
the feedback activation of PI3K signaling normally observed with
mTORC1 inhibitors, such as rapamycin. NVP-BEZ235 is a potent
dual PI3K-mTOR inhibitor  that has shown great efficacy in inhib-
iting tumor growth in preclinical mouse models [4–11] and is cur-
rently being evaluated in phase 1/2 clinical trials for advanced solid
tumors (colorectal, breast, non–small cell lung carcinoma, renal, and
sarcoma; reviewed in Garcia-Echeverria and Sellers ). The design
of these trials is based on the premise that this compound specifically
and mTOR (IC50= 20 nM) with little cross-reactivity toward other
kinases, as previously reported . However, mTOR belongs to the
PI3K-like kinase (PI3KK) family, which includes the two major ki-
nases that respond directly to DNA double-strand breaks (DSBs),
ATM and DNA-PKcs, both of which have catalytic domains highly
homologous to that of the PI3Ks [1,12]. Because DSBs are induced
during radiotherapy and commonly generated during chemotherapy,
it is particularly important to understand the effects of NVP-BEZ235
on these two kinases.
Anumber of recent reports identify an important role for the PI3K-
Akt pathway in radioresistance (reviewed in Mukherjee et al.  and
Address all correspondence to: Sandeep Burma, PhD, Division of Molecular Radiation
Biology, Department of Radiation Oncology, University of Texas Southwestern
Medical Center, 2201 Inwood Rd, NC7-214E, Dallas, TX 75390. E-mail: sandeep.
1S.B. is supported by grants from the National Institutes of Health (RO1 CA149461),
National Aeronautics and Space Administration (NNX10AE08G), and the Cancer
Prevention and Research Institute of Texas (RP100644).
2This article refers to supplementary materials, which are designated by Tables W1 and
W2 and Figures W1 to W6 and are available online at www.neoplasia.com.
3These authors equally contributed to this study.
Received 26 October 2011; Revised 23 December 2011; Accepted 3 January 2012
Copyright © 2012 Neoplasia Press, Inc. All rights reserved 1522-8002/12/$25.00
Volume 14 Number 1January 2012pp. 34–43
Begg et al. ). Our laboratory and those of others have shown that
GBM-relevant cells and tumors with epidermal growth factor receptor
vIII (EGFRvIII) amplification or PTEN loss exhibit proficient DSB
repair, which confers radioresistance [15–19]. Because radiotherapy
plays a key role in the treatment of GBM, we investigated the poten-
tial utility of NVP-BEZ235 as a radiosensitizing agent against human
GBM lines. Surprisingly, we found that very low concentrations of
this drug conferred a high degree of radiosensitization that was signif-
icantly greater than that previously reported with PI3K/Akt inhibition
[15–19], and this correlated with attenuation of DSB repair. Detailed
experimentation revealed that NVP-BEZ235 potently inhibits ATM
and DNA-PKcs, thereby blocking both nonhomologous end joining
(NHEJ) and homologous recombination (HR), the two major path-
ways of DSB repair. In addition, phosphorylation of ATM targets and
implementation of the G2/M cell cycle checkpoint are also attenuated
by this drug. The consequence is profound radiosensitization at very
low concentrations of NVP-BEZ235 (100 nM). This radiosensitizing
effect is significantly more potent than that seen with much higher
concentrations (10 μm) of current inhibitors of DNA-PKcs  or
ATM  that are being optimized for clinical testing (reviewed in
Ding et al. ). These results have significant translational impor-
tance, both for the design of current clinical trials involving this drug
and for the potential use of this drug as a powerful radiosensitizer in
Materials and Methods
Cell Culture and Drug Treatment
The cell lines used in this study are U251, U118, LN18, T98G,
LN229, SF188, 1BR3, AT5, M059K, and M059J. The U87 glioma
line expressing EGFRvIII has been described before . All cell lines
were maintained in Dulbecco modified Eagle medium containing 10%
fetal bovine serum in a humidified 37°C incubator with 5% CO2. All
10-mM stocks were stored at −20°C. Cells were treated with drugs for
1 hour before irradiation.
Irradiation of Cells
Cells were irradiated with gamma rays from a137Cs source (JL
Shepherd and Associates, CA) at the indicated doses. Subcutaneous
tumors in mice were irradiated with an x-ray device (X-RAD 320,
Precision X-ray, North Branford, CT; 300 kV, 12 mA, 1.65 mm Al)
fitted with a specifically designed collimator providing a 1-cm-diameter
field size iso-dose exposure. For laser irradiation, cells were microirra-
diated with a pulsed nitrogen laser (Spectra-Physics, Newport Corpo-
ration, Santa Clara, CA; 365 nm, 10 Hz) with output set at 75% of the
maximum, as described .
Western Analyses and Immunofluorescence Staining
For Western blot analysis of ATM and DNA-PKcs, nuclear extracts
were prepared as described . For Western blot analysis of all other
proteins, whole-cell extracts were prepared as described . Western
blot analysis was carried out as described . Immunofluorescence
(IF) staining of cells and tumor sections was performed as described
. Antibodies used were as follows: anti–phospho-Akt(S473), anti-
Akt, anti–phospho-Chk2(T68), anti–phospho-p53(S15), and anti–
phospho-S6(S235/236) (Cell Signaling, Danvers, MA); anti-actin
and anti-ATM (Sigma); anti-Rad51, anti–cyclin A, anti-p53, and
anti-53BP1 (Santa Cruz, Biotechnology, Santa Cruz, CA); anti–phospho-
SMC1(S966), anti-SMC1, anti–phospho-KAP-1(S824), anti–KAP-1,
anti-Chk2, and anti-H2AX (Bethyl Laboratories, Montgomery, TX );
anti-γH2AX and pHistone-H3(S10) (Upstate, Billerica, MA); anti–
phospho-ATM(S1981) (GenScript, Piscataway, NJ); anti–phospho-
DNA-PKcs (S2056) (Abcam, Cambridge, MA); anti–DNA-PKcs
HRP-conjugated secondary antibodies (Biorad, Hercules, CA); and
Alexa488/568–conjugated secondary antibodies (Molecular Probes,
Grand Island, NY). Primary antibodies were incubated overnight at
4°C, and secondary antibodies were incubated for 1 hour at room tem-
perature for both IF and Western blot analysis. Antibody dilutions are
provided in Table W1.
Colony Formation Assays
treated with the indicated drugs, and irradiated 1 hour later with graded
was replaced with drug-free medium. Surviving colonies were stained
with crystal violet approximately 10 to 14 days later as described .
DSB Repair Assays
DSB repair rates were assessed by quantifying the rates of dissolu-
tion of 53BP1 foci after irradiation of cells with 1 Gy of gamma rays
as described . For quantifying Rad51 foci, cells were irradiated
with 6 Gy of gamma rays and coimmunostained with Rad51 and
cyclin A antibodies 3 hours later as described . The average numbers
of Rad51 foci for cyclin A–positive (S/G2) nuclei were determined after
scoring at least 50 nuclei.
HR and NHEJ Assays
In the HR assay, GFP expression was quantified (by flow cytometry)
in MCF7-DRGFP cells transfected with an I-SceI plasmid as described
. In the NHEJ assay, repair of site-specific I-SceI–induced breaks in
engineered 293T cells by NHEJ results in the conversion of a GFP
signal to RFP (quantified by flow cytometry) as described .
G2/M Checkpoint Assay
The G2/M checkpoint was evaluated by quantifying histone H3
phosphorylation by flow cytometry as described .
DNA-PKcs Kinase Assays
In vitro kinase assays were carried out with purified DNA-PKcs
as described . Sheared herring testis DNA (Clontech, Mountain
View, CA) was added to the DNA-PKcs kinase assays to stimulate ki-
Mouse Tumor Studies
Tumors were generated by subcutaneous injection of U87 cells
overexpressing EGFRvIII  into 6-week-old female Nu/Nu mice.
Once these tumors reached an average size of 150 mm3, mice were
treated with a single dose of 45 mg/kg NVP-BEZ235 by oral gavage
or with vehicle (NMP/polyethylene glycol 300; 10:9 vol/vol) as con-
trol. Two hours later, tumors were irradiated with 2 Gy of x-rays,
and tumorswere excisedattheindicatedtime points.All animalstudies
were performed under protocols approved by the Institutional Animal
Care and Use Committee of UT Southwestern Medical Center.
Neoplasia Vol. 14, No. 1, 2012NVP-BEZ235 Inhibits DNA RepairMukherjee et al.
Statistical significance was determined by a 2-tailed t test using
GraphPad Prism software (San Diego, CA; **P < .01, ***P < .001).
Error bars represent the SEM for all plots.
Because PI3K-Akt signaling has been shown to promote DSB repair
in GBM cells and tumors [15–19], we assessed whether NVP-
BEZ235 could radiosensitize human GBM cells by inhibiting DNA
repair. We chose a panel of six GBM lines, of which four (U251,
U118, LN18 and T98G) exhibit high levels of activation of the
PI3K-Akt pathway as evidenced by high levels of Akt phosphorylation
(Ser473), whereas two (SF188 and LN229) exhibit lower levels of
Akt activation (Figure 1A). Radiation survival was measured by the
colony formation assay. We chose a concentration of 100 nM NVP-
BEZ235 for colony survival assays because this was the highest dose
at which plating efficiency was largely unaffected for most cell lines
(Table W2). We observed significant attenuation of Akt signaling with
NVP-BEZ235 treatment at 100 nM, in accord with previous reports
[3,5,6,8,11] (Figure 1A). We tested the radiosensitizing potential
of NVP-BEZ235 (100 nM) compared with the established radiosensi-
tizers and DNA repair inhibitors KU55933 (ATM inhibitor, 10 μM)
 and NU7026 (DNA-PKcs inhibitor, 10 μM) . We found that
NVP-BEZ235 elicited a significantly greater degree of radiosensi-
tization compared with KU55933 or NU7026, and this was consis-
tent among all cell lines irrespective of their Akt activation status
(Figure 1B). Radiosensitization was drug dose dependent with a lesser
(Figure W1). As indicated by IF staining for 53BP1 foci 
(Figure 1C), all glioma lines could complete DSB repair by 24 hours
after irradiation. However, NVP-BEZ235–treated cells showed higher
numbers of unresolved foci at 24 hours (Figure 1D), which correlates
with the high degree of radiosensitization observed.
was significantly greater than what has been previously observed due to
inactivation of PI3K-Akt signaling [15–19], we hypothesized that this
compound might be inhibiting other kinases in addition to PI3K and
mTOR. The most likely candidates, given the striking inhibition of
DSB repair, are the DSB-responsive kinases, ATM and DNA-PKcs,
whose catalytic domains are highly homologous to that of PI3K and
mTOR [1,12]. We therefore examined the effects of NVP-BEZ235
in wild-type (1BR3) and ATM-deficient (AT5) human fibroblasts
 as well as in DNA-PKcs–proficient (MO59K) and –deficient
(MO59J) human glioma lines . We found that NVP-BEZ235
could sensitize 1BR3 cells to IR, and the degree of sensitization was
significantly greater than that seen with KU55933 or NU7026
(Figure 2A). As expected, NU7026 and KU55933 treatments resulted
in attenuated DSB repair, consistent with the role of DNA-PKcs in
NHEJ  and the role of ATM in promoting HR  and hetero-
chromatic DSB repair  (Figure 2B). Strikingly, NVP-BEZ235
treatment resulted in a repair defect that was much more severe than
that seen with either NU7026 or KU55933 and affected both “early”
and “late” phases of DSB repair , with almost 70% of breaks
remaining unrepaired at 24 hours after irradiation.
These data suggest that the profound radiosensitization conferred
by NVP-BEZ235 is due to the inhibition of more than one DNA
repair pathway. Indeed, we found that NVP-BEZ235 could further
radiosensitize ATM-null  and DNA-PKcs-null  cell lines, in-
dicating that the radiosensitizing effect of the drug was not due to its
effect on just one PI3KK i.e., either ATM or DNA-PKcs (Figure 2,
C and D). Similarly, the DSB repair defects of both lines could be
further exacerbated after NVP-BEZ235 treatment (Figure 2, C and
D), clearly indicating that this compound can block multiple PI3KK
Among the PI3KK family members, ATM plays a central role in
the mammalian DNA damage response (DDR), triggering cell cycle
arrest and promoting DSB repair . To examine if NVP-BEZ235
attenuates the activation of ATM, we irradiated 1BR3 cells and ana-
lyzed the autophosphorylation of ATM at Ser1981 by Western blot
analysis . We found that NVP-BEZ235 attenuated IR-induced
activation of ATM, similar to the specific ATM inhibitor KU55933
(Figure 3A, top panel). Moreover, autophosphorylation of ATM at
the sites of micro-laser-induced DSBs  was reduced by NVP-
BEZ235 pretreatment (Figure 3A, bottom panel). We irradiated
analysis with phospho-specificantibodies asdescribed , assessed the
phosphorylation status of the following key ATM substrates: Chk2
(Thr68), SMC1 (Ser966), p53 (Ser15), KAP-1 (Ser824), and H2AX
(Ser139). Phosphorylation of all of these ATM substrates was attenu-
ated (to varying extents) by pretreatment with NVP-BEZ235, similar
to that seen with KU55933 (Figure 3B). We validated the biologic
significance of this inhibition by examining the G2/M cell cycle check-
point in 1BR3 cells as described . The G2/M block manifests as a
decrease in M-phase cells at 2 hours after irradiation, and this was
attenuated by NVP-BEZ235 to the same extent seen with KU55933
(Figure 3C).Inaddition tocheckpointsignaling,ATMplays animpor-
tant role in DSB repair by HR in S/G2phases . Consistent with
inhibition of HR, we found that IR-induced Rad51 foci formation was
attenuated in 1BR3 cells pretreated with NVP-BEZ235 similar to that
seen with KU555933 treatment (Figure 3D, top panel). Abrogation of
HR by NVP-BEZ235 was also seen in a GFP-based assay, which mea-
sures reconstitution of a GFP gene by HR after the induction of DSBs
by I-SceI  (Figure 3D, bottom panel). These results indicate that
NVP-BEZ235 inhibits ATM activation, ATM-mediated phosphorylation
events, cell cycle checkpoints, and HR.
Apart from ATM, the other PI3KK family member that responds
directly to DSBs is DNA-PKs, a key enzyme in the NHEJ pathway
of DSB repair . To investigate whether NVP-BEZ235 also inhibits
the activation of DNA-PKcs, we irradiated 1BR3 cells and examined
DNA-PKcs autophosphorylation at Ser2056 by Western blot analysis
. We found that NVP-BEZ235 attenuated IR-induced activation
of DNA-PKcs, similar to the specific DNA-PKcs inhibitor NU7026
(Figure 4A, top panel). Similarly, autophosphorylation of DNA-PKcs
at the sites of micro-laser-induced DSBs  was impaired on NVP-
NVP-BEZ235 can directly block DNA-PKcs kinase activity, we carried
out in vitro kinase assays with purified DNA-PKcs using GST-p53
(1-393) as a substrate . DNA-PKcs efficiently phosphorylated p53
at Ser15 in vitro, and this was attenuated by NVP-BEZ235, indicating
that NVP-BEZ235 can directly block DNA-PKcs, similar to NU7026
of DSB repair , we used a GFP to RFP-conversion assay to inves-
tigate if NVP-BEZ235 might block NHEJ in addition to attenuating
HR . We found that NVP-BEZ235 could potently block NHEJ
(no RFP signal after transfection with an I-SceI–expressing plasmid)
(Figure 4C). These results clearly indicate that NVP-BEZ235 potently
NVP-BEZ235 Inhibits DNA Repair Mukherjee et al.Neoplasia Vol. 14, No. 1, 2012
Figure 1. The dual PI3K/mTOR inhibitor NVP-BEZ235 potently impairs DSB repair and confers significant radiosensitization in a panel of
human GBM lines. (A) Inhibition of PI3K/Akt signaling in mock-irradiated or irradiated human GBM lines after treatment with 100 nM
NVP-BEZ235 was analyzed by Western blot analysis with α–phospho-Akt (Ser473) antibody. Cells were irradiated (10 Gy) after 1 hour of
drug treatment and harvested at 30 minutes after IR. (B) Radiation survival of GBM lines after treatment with the indicated concentra-
tions of NVP-BEZ235 (PI3K/mTOR inhibitor), KU55933 (ATM inhibitor), NU7026 (DNA-PKcs inhibitor), or DMSO (solvent) was quantified
by colony formation assays. The fraction of surviving colonies (y axis) was plotted against corresponding radiation dose (x axis). (C) GBM
cells were irradiated with 1 Gy of gamma rays after a 1-hour period of drug treatment. Cells were immunostained for 53BP1 foci (green)
at 0.5 and 24 hours after IR. Nuclei were stained with DAPI (blue). Representative pictures are shown for DMSO- or NVP-BEZ235–treated
U251 cells. (D) 53BP1 foci were scored at 0.5 and 24 hours after IR (average of 50 nuclei), and after subtracting background (number of
foci in mock-irradiated nuclei), the average foci per nucleus was plotted against the indicated times.
Neoplasia Vol. 14, No. 1, 2012 NVP-BEZ235 Inhibits DNA RepairMukherjee et al.
blocks both ATM and DNA-PKcs, resulting in a DSB repair defect
that is more striking than that seen on the inhibition of ATM or
DNA-PKcs alone. Indeed, blocking both ATM and DNA-PKcs by
combining KU55933 and NU7026 resulted in greater numbers of
unrepaired DSBs, similar to that seen with NVP-BEZ235 alone, both
in 1BR3 cells (Figure 4D) as well as in the panel of GBM cell lines
(Figure W2). Given the potential cross-talk between ATM and
DNA-PKcs , we investigated whether NVP-BEZ235 could atten-
uate IR-induced ATM activation in DNA-PKcs-null M059J cells and
DNA-PKcs activation in ATM-null AT5 cells. We found inhibition of
kinase activation in both cell lines, demonstrating that this drug can
independently block either kinase (Figure W3). We also examined
ATM and DNA-PKcs activation in the panel of glioma lines that were
radiosensitized by NVP-BEZ235 (Figure 1B) and observed inhibition
of both ATM (Figure W4) and DNA-PKcs (Figure W5) to varying
extents. Taken together, these data implicate the impairment of both
HR and NHEJ repair pathways, due to inhibition of both ATM and
DNA-PKcs, as the underlying mechanism behind the profound radio-
sensitization conferred by NVP-BEZ235.
Finally, to examine the effect of NVP-BEZ235 on DSB repair in
tumors, we generated subcutaneous tumors in Nu/Nu mice using
U87 cells overexpressing EGFRvIII . We first confirmed that
Figure 2. NVP-BEZ235 impairs both “early” and “late” phases of DSB repair and can exacerbate the repair defects of ATM- and DNA-
PKcs-null cells. (A) Radiation survival of wild-type 1BR3 fibroblasts on treatment with the indicated concentrations of NVP-BEZ235,
KU55933, NU7026, or DMSO was quantified by colony formation assays. The fraction of surviving colonies (y axis) was plotted against
the corresponding radiation dose (x axis). Inhibition of PI3K/Akt signaling after NVP-BEZ235 treatment for 1 hour or 16 hours was ana-
lyzed by Western blot analysis with α–phospho-Akt (Ser473) antibody. (B) DSB repair kinetics of 1BR3 cells irradiated with 1 Gy of gamma
rays after a 1-hour period of drug treatment. Cells were immunostained for 53BP1 foci (green), and nuclei were stained with DAPI (blue).
Representative pictures are shown for DMSO- or NVP-BEZ235–treated 1BR3 cells. 53BP1 foci were scored at the indicated times after IR,
and percentage foci remaining was plotted against time. (C) Radiation sensitivity and DSB repair kinetics of ATM-deficient AT5 cells on
treatment with NVP-BEZ235. (D) Radiation sensitivity and DSB repair kinetics of DNA-PKcs–proficient (M059K) and –deficient (M059J)
cells on treatment with NVP-BEZ235.
NVP-BEZ235 Inhibits DNA RepairMukherjee et al.Neoplasia Vol. 14, No. 1, 2012
Figure 3. NVP-BEZ235 impairs ATM activation, phosphorylation of ATM substrates, G2/M cell cycle checkpoint, and HR repair. (A) Top
panel. 1BR3 cells were pretreated with the indicated concentrations of NVP-BEZ235 or KU55933 and irradiated with 10 Gy of gamma
rays. Autophosphorylation of ATM was assayed after 30 minutes by Western blot analysis with a α–phospho-ATM (Ser1981) antibody.
Bottom panel. 1BR3 cells, pretreated with DMSO or NVP-BEZ235, were laser microirradiated and co-IF stained with α–phospho-ATM
(Ser1981) antibody (red) and α-53BP1 antibody (green) after 30 minutes. (B) 1BR3 cells were pretreated for 1 hour with the indicated
concentrations of NVP-BEZ235 or KU55933 and irradiated with 10 Gy of gamma rays. Phosphorylation of key DDR proteins was assayed
after 30 minutes by Western blot analysis with phospho-specific antibodies as indicated. (C) IR-induced G2/M checkpoint in 1BR3 cells
treated with NVP-BEZ235 or KU55933 was analyzed by dual-parameter flow cytometry. Representative distributions show staining for DNA
content (x axis) and for histone H3 phosphorylation (y axis); cells in M phase are demarcated with blue circles. Percent cells in M-phase in
mock-irradiated and irradiated (4 Gy) cells are plotted. The G2/M checkpoint manifests as a decrease in mitotic cells at 2 hours after irradi-
ation in DMSO-treated cells; this decrease is not seen in NVP-BEZ235– or KU55933-treated cells. (D) Top panel. Representative image of
gamma-irradiated 1BR3 cells costained with α–cyclin A antibody (red) and α-Rad51 antibody (green) after 3 hours. Nuclei are stained with
DAPI (blue). Average numbers of Rad51 foci for cyclin A–positive (S/G2) nuclei at 3 hours after irradiation are plotted for 1BR cells pretreated
with NVP-BEZ235 or KU55933. Bottom panel. HR was measured by quantifying GFP expression (by flow cytometry) in MCF7-DRGFP cells
transfected with an I-SceI plasmid in the presence of NVP-BEZ235 or KU55933 as indicated. Plots show percentages of drug-treated cells
expressing GFP relative to DMSO-treated cells. GFP expression after transfection with a control plasmid with full-length GFP was quantified
to ensure that transfection efficiencies were comparable between the different drug treatments.
Neoplasia Vol. 14, No. 1, 2012 NVP-BEZ235 Inhibits DNA Repair Mukherjee et al.
NVP-BEZ235 could inhibit Akt activation and block DSB repair in
U87-EGFRvIII cells in culture (Figure W6). Next, tumor-bearing
mice were treated with a single dose of 45 mg/kg NVP-BEZ235 or
with vehicle as control. Tumors were mock irradiated or irradiated
(2 Gy of x-rays) 2 hours later, collected at 0.5 and 24 hours after
IR, and sectioned for IF. Tumors from NVP-BEZ235–treated mice
exhibited a marked reduction in the phosphorylation of Akt (Ser473)
and abrogation of phosphorylation of an mTOR substrate, the ribo-
somal protein S6 (Ser235/236), thereby confirming intratumoral
delivery of the drug and consequent inhibition of the PI3K-Akt-
mTOR pathway  (Figure 5A). Irradiated NVP-BEZ235– or vehicle-
treated tumor sections were IF stained for 53BP1 foci as described .
Vehicle-treated tumors were able to completely repair radiation-induced
DSBs by 24 hours after IR. Interestingly, NVP-BEZ235–treated tumors
exhibited higher levels of unresolved 53BP1 foci at 24 hours after IR,
indicating attenuation of DSB repair (Figure 5, B and C). These results
NVP-BEZ235 is also valid in a tumor setting.
Figure 4. NVP-BEZ235 impairs DNA-PKcs activation and NHEJ. (A) Top panel. 1BR3 cells were pretreated with NVP-BEZ235 or NU7026
and irradiated with 10 Gy of gamma rays. Autophosphorylation of DNA-PKcs was assayed after 30 minutes by Western blot analysis with
a α–phospho-DNA-PKcs (Ser2056) antibody. Bottom panel. 1BR3 cells, pretreated with DMSO or NVP-BEZ235, were laser microirradiated
and co-IF stained with α–phospho-DNA-PKcs (Ser2056) antibody (green) and α-γH2AX antibody (red) after 30 minutes. (B) In vitro kinase
assays were carried out with purified DNA-PKcs incubated with recombinant GST-p53 (1-393) in the presence of sheared herring testis
DNA and ATP. Increasing concentrations of NVP-BEZ235 or NU7026 were added to the reaction mixture as indicated. Phosphorylation of
p53 by DNA-PKcs was assessed by Western blot analysis with α–phospho-p53 (Ser15) antibody. (C) The NHEJ assay was initiated by the
transfection of an I-SceI plasmid into engineered 293T cells harboring a GFP gene (flanked by I-SceI sites) followed by a RFP gene. A
switch from GFP to RFP expression indicates ligation of I-SceI–generated breaks by NHEJ. Representative distributions show RFP +ve
cells (red circles) and GFP +ve cells (green circles) for different drug treatments; percentages of RFP +ve cells are indicated and are plotted
for cells treated with increasing concentrations of NVP-BEZ235 or NU7026. (D) 1BR3 cells were irradiated with 1 Gy of gamma rays after
a 1-hourperiod of treatment with NVP-BEZ235, KU55933, or NU7026 alone or with a combination of KU55933 and NU7026, asindicated. To
quantify residual (unrepaired) DSBs, cells were immunostained for 53BP1 foci at 24 hours after IR. Residual 53BP1 foci in irradiated cells
were scored (average of 50 nuclei), and after subtracting background (number of foci in mock-irradiated nuclei), average foci per nucleus
were plotted against the indicated treatment conditions.
NVP-BEZ235 Inhibits DNA RepairMukherjee et al.Neoplasia Vol. 14, No. 1, 2012
Using multiple approaches, we find that NVP-BEZ235, a drug already
in clinical trials, can inhibit IR-induced activation of ATM and DNA-
PKcs, the two major kinases responding to DSBs. This results in inhi-
bition of DSB repair, attenuation of cell cycle arrest, and profound
radio sensitization. In the original report describing the compound,
the authors examined the effects of NVP-BEZ235 only on doxorubicin-
has no cross-reactivity toward these two kinases . However, we ob-
serve attenuation of ATM (Ser1981) and DNA-PKcs (Ser2056) auto-
phosphorylations at low concentrations (100 nM) of NVP-BEZ235.
Differences in cell lines, phospho-specific antibodies, DNA-damaging
agents, and duration of incubation with drugs probably underlie these
discrepancies. It is also important to point out that, as DNA-PKcs is
phosphorylated at Thr2609 by ATM, Ser2056 autophosphorylation
might be a better indicator of DNA-PKcs activity in vivo . Regardless
of the extent of inhibition of ATM and DNA-PKcs autophosphoryla-
tion, our results clearly show that a low concentration of NVP-BEZ235
is sufficient to block key DDR events triggered by ATM and DNA-
PKcs — phosphorylation of DDR proteins, implementation of cell
cycle checkpoints, and repair of IR-induced DSBs by HR and NHEJ.
Moreover, we show that NVP-BEZ235 inhibits purified DNA-PKcs
in vitro indicating that the observed effects of NVP-BEZ235 are likely
due to a direct blockage of the kinase activity of this protein.
Cross-inhibition of ATM and DNA-PKcs by a dual PI3K-mTOR
inhibitor is not surprising because these kinases have homologous
catalytic domains [1,12]. In accord with our results, a previous report
demonstrated that NVP-BEZ235 could radiosensitize non–small cell
lung carcinoma cells expressing oncogenic K-RAS and this correlated
with higher levels of IR-induced DNA breaks, although the underly-
ing mechanism(s) were not investigated . Also, an earlier report
demonstrated inhibition of DNA-PKcs kinase activity by NVP-
BEZ235 in vitro, although the radiosensitizing effect of this drug
was not tested in this study . At the time this article was in prep-
aration, NVP-BEZ235 was shown to induce senescence in irradiated
cancer cells by inhibiting DNA-PKcs thereby supporting our data
showing radiosensitization by this drug due to inhibition of both
ATM and DNA-PKcs . Moreover, NVP-BEZ235 was recently
identified in a cell-based screen as a potent ATR inhibitor . Like
ATM and DNA-PKcs, ATR also belongs to the PI3KK family and
responds to ssDNA generated by the stalling of replication forks or
bytheresection of DSBs . Our findings, incombination with these
reports, unequivocally demonstrate that NVP-BEZ235 is a potent in-
hibitor of all members of the PI3KK family that respond to IR-induced
DNA breaks, either directly (ATM and DNA-PKcs) or indirectly
(ATR), resulting in profound radiosensitization at low concentrations
of the drug.
Figure 5. NVP-BEZ235 impairs the repair of IR-induced DNA dam-
age in tumors. (A) Nu/Nu mice bearing subcutaneous tumors
(U87-EGFRvIII) were treated with NVP-BEZ235 (45 mg/kg) or vehicle
Tumors were collected at 0.5 or 24 hours after IR and sectioned for
IF staining. Tumor sections were stained for phosphorylation of Akt
(Ser473) and ribosomal protein S6 (Ser235/236) to confirm intra-
tumoral drug delivery and consequent inhibition of the PI3K-Akt-
mTOR pathway. (B) Tumor sections were IF stained for 53BP1 foci
(green) to visualize radiation-induced DSBs (at 0.5 hours) and resid-
ual breaks (at 24 hours). Representative pictures are shown for NVP-
BEZ235– or vehicle-treated tumors. (C) 53BP1 foci were scored at
0.5 and 24 hours after IR (average of 50 nuclei), and after subtracting
background (average number of foci in mock-irradiated tumors),
average foci per nucleus were plotted against the indicated times.
Please note residual 53BP1 foci in NVP-BEZ235–treated tumors,
confirming that the impairment of DSB repair by NVP-BEZ235 is also
valid in a tumor setting.
Neoplasia Vol. 14, No. 1, 2012 NVP-BEZ235 Inhibits DNA RepairMukherjee et al.
Our study not only shows that NVP-BEZ235 is a novel inhibitor
of both ATM and DNA-PKcs but also establishes that this drug is
significantly more potent than specific inhibitors of ATM and DNA-
PKcs that are being optimized for use as radiosensitizers in the clinic
. A very high degree of radiosensitization was seen with 100-fold
lower concentrations of NVP-BEZ235 (100 nM) relative to KU55933
(10 μM) or NU7026 (10 μM). Indeed, radiosensitization by NVP-
BEZ235 was greater than the higher degree of sensitization achieved
by combining both ATM and DNA-PKcs inhibitors (data not shown),
indicating that radiosensitization by NVP-BEZ235 is not just due to
combined inhibition of both DSB-responsive kinases. Thus, the ex-
treme radiosensitivity conferred by NVP-BEZ235 is clearly a combina-
torial effect of disabling multiple pathways impinging on 1) DSB repair
(due to DNA-PKcs and ATM inhibition), 2) cell cycle checkpoints
(due to ATM and ATR inhibition), and 3) cell survival (due to PI3K
and mTOR inhibition). These results have two important clinical
implications. On one hand, considering the significant impairment
of DDRs resulting from NVP-BEZ235 treatment, combining the drug
with genotoxic chemotherapy could result in significant systemic toxic-
ity and limit therapeutic gain. On the other hand, radiotherapy, like
surgery, is a local treatment and its efficacy could be significantly en-
hanced by the use of potent radiosensitizers like NVP-BEZ235, which
confers radiosensitization even at radiation doses used in conventional
fractionation schemes (1.8-2 Gy per fraction). Thus, highly conformal
techniques, such as intensity-modulated radiotherapy, might be able to
exploit the radiosensitizing potential of the drug while minimizing nor-
mal tissue toxicity. Current inhibitors of DNA-PKcs or ATM have not
1/2 trials, and some preliminary clinical data already exist showing that
the drug is well tolerated . Thus, it can be more readily tested as a
radiosensitizer for GBMs and other solid tumors in the clinic. Further
preclinical evaluation is warranted regarding the effects of NVP-
BEZ235 in combination with chemotherapy or radiation in mouse
tumor models of GBMs and other cancers.
The authors thank Matthew Porteus for providing cells for the NHEJ
assay and Kum Kum Khanna for cells for the HR assay.The authors also
thank David Chen for facilitating laser microirradiation experiments and
Donglai Qi for preliminary work on the DNA-PKcs kinase assay.
 Liu P, Cheng H, Roberts TM, and Zhao JJ (2009). Targeting the phosphoinositide
3-kinase pathway in cancer. Nat Rev Drug Discov 8, 627–644.
 Garcia-Echeverria C and Sellers WR (2008). Drug discovery approaches target-
ing the PI3K/Akt pathway in cancer. Oncogene 27, 5511–5526.
 Maira SM, Stauffer F, Brueggen J, Furet P, Schnell C, Fritsch C, Brachmann S,
Chene P, De Pover A, Schoemaker K, et al. (2008). Identification and char-
acterization of NVP-BEZ235, a new orally available dual phosphatidylinositol
3-kinase/mammalian target of rapamycin inhibitor with potent in vivo anti-
tumor activity. Mol Cancer Ther 7, 1851–1863.
 Brachmann SM, Hofmann I, Schnell C, Fritsch C, Wee S, Lane H, Wang S,
Garcia-Echeverria C, and Maira SM (2009). Specific apoptosis induction by the
dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA
mutant breast cancer cells. Proc Natl Acad Sci USA 106, 22299–22304.
 Chiarini F, Grimaldi C, Ricci F, Tazzari PL, Evangelisti C, Ognibene A,
Battistelli M, Falcieri E, Melchionda F, Pession A, et al. (2010). Activity of
the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin
inhibitor NVP-BEZ235 against T-cell acute lymphoblastic leukemia. Cancer
Res 70, 8097–8107.
 Eichhorn PJ, Gili M, Scaltriti M, Serra V, Guzman M, Nijkamp W, Beijersbergen
RL, Valero V, Seoane J, Bernards R, et al. (2008). Phosphatidylinositol 3-
kinase hyperactivation results in lapatinib resistance that is reversed by the
mTOR/phosphatidylinositol 3-kinase inhibitor NVP-BEZ235. Cancer Res
 Engelman JA, Chen L, Tan X, Crosby K, Guimaraes AR, Upadhyay R, Maira M,
McNamara K, Perera SA, Song Y, et al. (2008). Effective use of PI3K and MEK
inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung
cancers. Nat Med 14, 1351–1356.
 Konstantinidou G, Bey EA, Rabellino A, Schuster K, Maira MS, Gazdar AF,
Amici A, Boothman DA, and Scaglioni PP (2009). Dual phosphoinositide
3-kinase/mammalian target of rapamycin blockade is an effective radiosensitizing
strategy for the treatment of non–small cell lung cancer harboring K-RAS muta-
tions. Cancer Res 69, 7644–7652.
 Liu TJ, Koul D, LaFortune T, Tiao N, Shen RJ, Maira SM, Garcia-Echevrria C,
and Yung WK (2009). NVP-BEZ235, a novel dual phosphatidylinositol 3-kinase/
mammalian target of rapamycin inhibitor, elicits multifaceted antitumor activities
in human gliomas. Mol Cancer Ther 8, 2204–2210.
 Schnell CR, Stauffer F, Allegrini PR, O’Reilly T, McSheehy PM, Dartois C,
Stumm M, Cozens R, Littlewood-Evans A, Garcia-Echeverria C, et al. (2008).
Effects of the dual phosphatidylinositol 3-kinase/mammalian target of rapamycin
inhibitor NVP-BEZ235 on the tumor vasculature: implications for clinical
imaging. Cancer Res 68, 6598–6607.
 Serra V,Markman B,ScaltritiM,EichhornPJ,ValeroV,GuzmanM,BoteroML,
Llonch E, Atzori F, Di Cosimo S, et al. (2008). NVP-BEZ235, a dual PI3K/
mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells
with activating PI3K mutations. Cancer Res 68, 8022–8030.
 Abraham RT (2004). PI 3-kinase related kinases: “big” players in stress-induced
signaling pathways. DNA Repair (Amst) 3, 883–887.
 Mukherjee B, Choy H, Nirodi C, and Burma S (2010). Targeting nonhomol-
ogous end-joining through epidermal growth factor receptor inhibition: rationale
and strategies for radiosensitization. Semin Radiat Oncol 20, 250–257.
 Begg AC, Stewart FA, and Vens C (2011). Strategies to improve radiotherapy
with targeted drugs. Nat Rev Cancer 11, 239–253.
 Golding SE, Morgan RN, Adams BR, Hawkins AJ, Povirk LF, and Valerie K
(2009). Pro-survival AKT and ERK signaling from EGFR and mutant EGFRvIII
enhances DNA double-strand break repairin human gliomacells.Cancer BiolTher
 Jiang Z, Pore N, Cerniglia GJ, Mick R, Georgescu MM, Bernhard EJ, Hahn SM,
Gupta AK, and Maity A (2007). Phosphatase and tensin homologue deficiency in
glioblastoma confers resistance to radiation and temozolomide that is reversed by
the protease inhibitor nelfinavir. Cancer Res 67, 4467–4473.
 Kao GD, Jiang Z, Fernandes AM, Gupta AK, and Maity A (2007). Inhibition
of phosphatidylinositol-3-OH kinase/Akt signaling impairs DNA repair in glio-
blastoma cells following ionizing radiation. J Biol Chem 282, 21206–21212.
 McEllin B, Camacho CV, Mukherjee B, Hahm B, Tomimatsu N, Bachoo RM,
and Burma S (2010). PTEN loss compromises homologous recombination
repair in astrocytes: implications for glioblastoma therapy with temozolomide
or poly(ADP-ribose) polymerase inhibitors. Cancer Res 70, 5457–5464.
 Mukherjee B, McEllin B, Camacho CV, Tomimatsu N, Sirasanagandala S,
Nannepaga S, Hatanpaa KJ, Mickey B, Madden C, Maher E, et al. (2009).
EGFRvIII and DNA double-strand break repair: a molecular mechanism for
radioresistance in glioblastoma. Cancer Res 69, 4252–4259.
 Veuger SJ, Curtin NJ, Richardson CJ, Smith GC, and Durkacz BW (2003).
Radiosensitization and DNA repair inhibition by the combined use of novel
inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1.
Cancer Res 63, 6008–6015.
 Hickson I, Zhao Y, Richardson CJ, Green SJ, Martin NM, Orr AI, Reaper PM,
Jackson SP, Curtin NJ, and Smith GC (2004). Identification and characteriza-
tion of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase
ATM. Cancer Res 64, 9152–9159.
 Ding J, Miao ZH, Meng LH, and Geng MY (2006). Emerging cancer therapeu-
tic opportunities target DNA-repair systems. Trends Pharmacol Sci 27, 338–344.
 Tomimatsu N, Mukherjee B, and Burma S (2009). Distinct roles of ATR and
DNA-PKcs in triggering DNA damage responses in ATM-deficient cells.
EMBO Rep 10(6), 629–635.
 Peterson SR, Kurimasa A, Oshimura M, Dynan WS, Bradbury EM, and Chen
DJ (1995). Loss of the catalytic subunit of the DNA-dependent protein kinase
in DNA double-strand-break-repair mutant mammalian cells. Proc Natl Acad
Sci USA 92, 3171–3174.
NVP-BEZ235 Inhibits DNA RepairMukherjee et al.Neoplasia Vol. 14, No. 1, 2012
 D’Anna JA, Valdez JG, Habbersett RC, and Crissman HA (1997). Association
of G1/S-phase and late S-phase checkpoints with regulation of cyclin-dependent
kinases in Chinese hamster ovary cells. Radiat Res 148, 260–271.
 Bolderson E, Tomimatsu N, Richard DJ, Boucher D, Kumar R, Pandita TK,
Burma S, and Khanna KK (2010). Phosphorylation of Exo1 modulates homol-
ogous recombination repair of DNA double-strand breaks. Nucleic Acids Res
 Sharma GG, So S, Gupta A, Kumar R, Cayrou C, Avvakumov N, Bhadra U,
Pandita RK, Porteus MH, Chen DJ, et al. (2010). MOF and histone H4 acety-
lation at lysine 16 are critical for DNA damage response and double-strand
break repair. Mol Cell Biol 30, 3582–3595.
DJ (1999). Requirement for the kinase activity of human DNA-dependent protein
 Mukherjee B, Kessinger C, Kobayashi J, Chen BP, Chen DJ, Chatterjee A, and
Burma S (2006). DNA-PK phosphorylates histone H2AX during apoptotic
DNA fragmentation in mammalian cells. DNA Repair (Amst) 5, 575–590.
 Burma S and Chen DJ (2004). Role of DNA-PK in the cellular response to
DNA double-strand breaks. DNA Repair (Amst) 3, 909–918.
 Morrison C, Sonoda E, Takao N, Shinohara A, Yamamoto K, and Takeda S
(2000). The controlling role of ATM in homologous recombinational repair of
DNA damage. EMBO J 19, 463–471.
 Goodarzi AA, Jeggo P, and Lobrich M (2010). The influence of heterochromatin
on DNA double strand break repair: getting the strong, silent type to relax. DNA
Repair (Amst) 9, 1273–1282.
 Lavin MF (2008). Ataxia-telangiectasia: from a rare disorder to a paradigm for
cell signalling and cancer. Nat Rev Mol Cell Biol 9, 759–769.
 Beucher A, Birraux J, Tchouandong L, Barton O, Shibata A, Conrad S,
Goodarzi AA, Krempler A, Jeggo PA, and Lobrich M (2009). ATM and Artemis
promote homologous recombination of radiation-induced DNA double-strand
breaks in G2. EMBO J 28, 3413–3427.
 Mukherjee B, Camacho CV, Tomimatsu N, Miller J, and Burma S (2008).
Modulation of the DNA-damage response to HZE particles by shielding.
DNA Repair (Amst) 7, 1717–1730.
 Chen BP, Uematsu N, Kobayashi J, Lerenthal Y, Krempler A, Yajima H,
Lobrich M, Shiloh Y, and Chen DJ (2007). Ataxia telangiectasia mutated
(ATM) is essential for DNA-PKcs phosphorylations at the Thr-2609 cluster
upon DNA double strand break. J Biol Chem 282, 6582–6587.
 Kong D, Yaguchi S, and Yamori T (2009). Effect of ZSTK474, a novel phos-
phatidylinositol3-kinase inhibitor,onDNA-dependent protein kinase.BiolPharm
Bull 32, 297–300.
 Azad A, Jackson S, Cullinane C, Natoli A, Neilsen PM, Callen DF, Maira SM,
Hackl W, McArthur GA, and Solomon B (2011). Inhibition of DNA-dependent
protein kinase induces accelerated senescence in irradiated human cancer cells.
Mol Cancer Res 9(12), 1696–1707.
 Toledo LI, Murga M, Zur R, Soria R, Rodriguez A, Martinez S, Oyarzabal J,
Pastor J, Bischoff JR, and Fernandez-Capetillo O (2011). A cell-based screen
identifies ATR inhibitors with synthetic lethal properties for cancer-associated
mutations. Nat Struct Mol Biol 18(6), 721–727.
 Peyton JD, Rodon Ahnert J, Burris H, Britten C, Chen LC, Tabernero J, Duval
V, Rouyrre N, Silva AP, Quadt C, et al. (2011). A dose-escalation study with
the novel formulation of the oral pan-class I PI3K inhibitor BEZ235, solid dis-
persion system (SDS) sachet, in patients with advanced solid tumors. J Clin
Oncol 29, ASCO Annual Meeting Proceedings, Abstract 3066.
Neoplasia Vol. 14, No. 1, 2012 NVP-BEZ235 Inhibits DNA RepairMukherjee et al.
Table W1. Dilutions of Primary and Secondary Antibodies for Western Blot Analysis and IF.
Antibody Source WesternIF
HRP-conjugated secondary antibodies
Alexa488/568-conjugated secondary antibodies
Gift from Dr B. Chen
Table W2. Plating Efficiency of Human GBM Cell Lines after Treatment with Varying Concen-
trations of NVP-BEZ235.
Glioma LineConcentration of NVP-BEZ235 (nM)
0 1050 100500
Figure W1. Radiation survival of GBM cell lines after treatment with varying concentrations of NVP-BEZ235.
Figure W2. GBM cell lines were irradiated with 1 Gy of gamma rays after a 1-hour period of treatment with NVP-BEZ235, KU55933, or
NU7026 alone or with a combination of KU55933 and NU7026, as indicated. To quantify residual (unrepaired) DSBs, cells were immuno-
stained for 53BP1 foci at 24 hours after IR. Residual 53BP1 foci in irradiated cells were scored (average of 50 nuclei), and after subtracting
background (number of foci in mock-irradiated nuclei), average foci per nucleus were plotted against the indicated treatment conditions.
Figure W3. (A) AT5 cells were pretreated with NVP-BEZ235 and irradiated with 10 Gy of gamma rays. Autophosphorylation of DNA-PKcs
was assayed after 30 minutes by Western blot analysis with a α–phospho-DNA-PKcs (Ser2056) antibody. (B) M059J cells were pre-
treated with NVP-BEZ235 and irradiated with 10 Gy of gamma rays. Autophosphorylation of ATM was assayed after 30 minutes by
Western blot analysis with a α–phospho-ATM (Ser1981) antibody.
Figure W4. Inhibition of ATM kinase activity in irradiated human GBM lines after treatment with NVP-BEZ235 was analyzed by Western
blot analysis with α–phospho-ATM (Ser1981) antibody. Cells were irradiated (10 Gy) after 1 hour of drug treatment and harvested at
30 minutes after IR.
Figure W5. Inhibition of DNA-PKcs activity in irradiated human GBM lines after treatment with NVP-BEZ235 was analyzed by Western Download full-text
blot analysis with α–phospho-DNA-PKcs (Ser2056) antibody. Cells were irradiated (10 Gy) after 1 hour of drug treatment and harvested at
30 minutes after IR.
Figure W6. (A) Inhibition of PI3K/Akt signaling in mock-irradiated or
irradiated human U87-EGFRvIII cells after treatment with 100 nM
NVP-BEZ235 was analyzed by Western blot analysis with α–phospho-
Akt (Ser473) antibody. Cells were irradiated (10 Gy) after 1 hour of
drug treatment and harvested at 30 minutes after IR. (B) U87-EGFRvIII
cells were irradiated with 1 Gy of gamma rays after a 1-hour period
at 0.5 and 24 hours after IR. 53BP1 foci were scored at 0.5 and
24 hours after IR (average of 50 nuclei), and after subtracting back-
ground (number of foci in mock-irradiated nuclei), average foci per
nucleus were plotted against the indicated times.