-
Small Molecule Therapeutics
Dual Targeting of Hypoxia and Homologous RecombinationRepair
Dysfunction in Triple-Negative Breast Cancer
Francis W. Hunter, Huai-Ling Hsu, Jiechuang Su, Susan M. Pullen,
William R. Wilson, and Jingli Wang
AbstractTriple-negative breast cancer (TNBC) is an aggressive
malignancy with poor clinical outcome and few
validated drug targets. Two prevalent features of TNBC, tumor
hypoxia and derangement of homologous
recombination (HR) repair, are potentially exploitable for
therapy. This study investigated whether
hypoxia-activated prodrugs (HAP) of DNA-damaging cytotoxins may
inhibit growth of TNBC by simul-
taneously addressing these two targets. We measured in vitro
activity of HAP of DNA breakers (tirapa-
zamine, SN30000) and alkylators (TH-302, PR-104, SN30548) in
TNBC cell lines and isogenic models, and
related this to measures of HR repair and expression of
prodrug-activating enzymes. Antitumor activity of
HAP was examined in isogenic BRCA2-knockout xenograft models and
compared with platinum chemo-
therapy. All five HAP selectively inhibited growth of TNBC cell
lines under hypoxia. Sensitivity to HAP
was not strongly associated with BRCA1 genotype. However, HAP
sensitivity was enhanced by suppres-
sion of HR (assessed by radiation-induced RAD51 focus formation)
when BRCA1 and PALB2were knocked
down in a common (MDA-MB-231) background. Furthermore, knockout
of BRCA2 markedly sensitized
DLD-1 cells to the clinical nitrogen mustard prodrugs TH-302 and
PR-104 and significantly augmented
sterilization of clonogens by these agents in xenografts, both
as monotherapy and in combination with
radiotherapy, but had less effect on activity of the
benzotriazine di-N-oxide SN30000. PR-104 monotherapy
was more effective than cisplatin at inhibiting growth of
BRCA2-knockout tumors at equitoxic doses. This
study demonstrates the potential for HAP of nitrogen mustards to
simultaneously exploit hypoxia and
HR defects in tumors, with translational implications for TNBC
and other HR-deficient malignancies. Mol
Cancer Ther; 13(11); 2501–14. �2014 AACR.
IntroductionBreast cancer is a disease characterized by
substantial
histologic and molecular heterogeneity, with wide dispa-rities
in prognosis and response to therapy. Triple-nega-tive breast
cancer (TNBC), defined by negative clinicalassays for expression of
estrogen receptor (ER), proges-terone receptor and amplification of
HER2, accounts for10% to 24%of invasive breast cancers (1)
andencompassesan aggressive albeit heterogeneous subtype
associatedwith young age at diagnosis (1), high histologic
grade(2), visceral and central nervous system (CNS) metastasis(3),
and worse prognosis than hormone receptor–positivetumors (4). Most
patients relapse within 3 years of pri-
mary diagnosis with aggressive, chemoresistant metasta-ses and
rapid progression to death (5, 6). TNBC is alsoclosely related to
the poor prognosis basal-like breastcancer (BLBC) subtype defined
by PAM50 gene expres-sion analysis. Although these classifications
are not syn-onymous, approximately 80% of TNBC are also BLBC
(7).Antagonists of ER and HER2 signaling, which have dras-tically
improved outcomes for ER-positive and HER2-positive breast cancers,
are not indicated for TNBC, andchemotherapy is the sole modality
for systemic manage-ment of advanced disease.
There has been significant recent interest in exploitingthe link
between TNBC and the BRCA1 pathway fortherapy. The vast majority of
mammary carcinomas inwomen carrying germline BRCA1 mutations are
triple-negative, and although BRCA1 is infrequently mutated
insporadic TNBC (8, 9), suppression by miRNA (10), epi-genetic
silencing (11), and other nongenetic causes of"BRCAness" phenotypes
may implicate dysfunction ofgenes epistatic with BRCA1 more widely
in TNBC.BRCA1 plays a critical function in resolution of
DNAdouble-strand breaks (DSB) by homologous recombina-tion (HR)
repair, particularly DSB associated with cross-links at DNA
replication forks (12). As a result, TNBCshow deficiency in HR and
may be more sensitive to
Auckland Cancer Society Research Centre, Faculty of Medical and
HealthSciences, University of Auckland, Auckland, New Zealand.
Note: Supplementary data for this article are available at
Molecular CancerTherapeutics Online
(http://mct.aacrjournals.org/).
Corresponding Author: William R. Wilson, Auckland Cancer
SocietyResearch Centre, Faculty of Medical and Health Sciences,
University ofAuckland, 85Park Road, Grafton, Private Bag 92019,
Auckland, 1142, NewZealand. Phone: 64-9-9236883; Fax: 64-9 3737571;
E-mail:[email protected]
doi: 10.1158/1535-7163.MCT-14-0476
�2014 American Association for Cancer Research.
MolecularCancer
Therapeutics
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-
therapies that generate cross-links or DSB (13),
includingplatinum drugs, alkylating agents, anthracyclines andPARP
inhibitors, although the efficacy of this approachis yet to be
validated in definitive clinical studies.
Hypoxia is an adverse pathologic feature of manytumors including
breast cancer (14).Although earlydefin-itive characterization of
tumor hypoxia preceded molec-ular classification of breast cancer
(15), recent compellingevidence across multiple technology
platforms links hyp-oxia specifically with TN/BL subtypes, where it
maynegatively influence treatment outcome (16–24), raisingthe
possibility that drugs targeted to tumor hypoxia maybe an effective
strategy for TNBC. Several classes ofhypoxia-activated prodrugs
(HAP) have been rationallydeveloped to exploit tumor hypoxia (14).
These includethe clinical stage benzotriazine di-N-oxide HAP
tirapa-zamine (25) and nitrogen mustard prodrugs TH-302 (26)and
PR-104 (27), in addition to advanced preclinical com-pounds such as
the tirapazamine analogue SN30000 (28)and a
nitro-chloromethylbenzindoline (nitroCBI) that is aprodrug of a
potent DNA minor groove alkylator (29).These agents are
enzymatically reduced in hypoxic tumortissue to DNA-damaging
metabolites that are selectivelytoxic to hypoxic cells.
By deploying in vitro isogenic models, we (30, 31) andothers
(26, 32) have demonstrated that several HAP arecapable of
exploiting HR defects analogous to those fre-quently observed in
TNBC (8, 10). A comparison of chem-ical classes in Rad51d-knockout
Chinese hamster cellssuggested that the DNA cross-linking HAP
(TH-302 andPR-104) may have greater selectivity for HR
dysfunctionthan benzotriazine-di-N-oxides or nitroCBI (30). HAPmay
therefore be uniquely positioned to simultaneouslyexploit hypoxia
and HR dysfunction in TNBC, anapproach that is further supported by
observations thathypoxia itself downregulatesHR repair in tumors
(33, 34).Here, we investigate the potential for HAP to inhibittumor
growth by dual targeting of hypoxia andHR repairdefects in
preclinical models.
Materials and MethodsCompounds
SN30000, tirapazamine, TH-302, mechlorethamine(HN2), PR-104,
PR-104A, PR-104H, the nitroCBI SN30548,the corresponding aminoCBI
SN30550, FSL-61, pimoni-dazole, cisplatin, and olaparib were either
synthesized atthe Auckland Cancer Society Research Centre
(Auckland,New Zealand) or purchased from suppliers as indicatedin
Supplementary Table S1. Purity of batches synthesizedin-housewas
confirmedbyhigh-performance liquid chro-matography (HPLC).Drug
stock solutions (solvents listedin Supplementary Table S1) were
stored at �80�C.
Cell linesTNBC lines with known BRCA1 genotype (35) were
obtained fromAsterand (SUM1315MO2, SUM149PT, andSUM159PT), ATCC
(HCC1937 and MDA-MB-436), Cali-
per Life Sciences (MDA-MB-231-D3H2LN, a highly met-astatic
subclone of MDA-MB-231; henceforth, D3H2LN),Dr. A. Patterson
(University ofAuckland,Auckland,NewZealand; MDA-MB-468), and Dr. G.
Krissansen (Univer-sity of Auckland; BT549). The TNBC lines were
propa-gated in culture as described in Supplementary Table
S2.HEK293 cells were obtained from Open Biosystems andcultured in
RPMI with 10% FCS. DLD-1 cells with homo-zygous knockout of BRCA2
(line HD105-007, henceforth,DLD-1 BRCA2�/�) and isogenic
BRCA2wild-typeDLD-1cells were licensed from Horizon Discovery. The
DLD-1lines were maintained in McCoy’s 5A modified mediumwith 10%
FCS and 2 mmol/L L-glutamine. All cell lineswere cultured in
humidified CO2 incubators at 37
�C for�2months cumulative passage fromauthenticated frozenstocks
confirmed to be Mycoplasma negative by PCR-ELISA (Roche). Cell
lines that were not obtained from acommercial supplier were
authenticated by short tandemrepeat profiling (CellBank
Australia).
RNAi-mediated suppression of HR genesTRIPZ lentiviral plasmids
carrying doxycycline-induc-
ible shRNA were purchased from Open Biosystems. Sev-en
BRCA1-targeted shRNAs and four PALB2-targetedshRNAs were screened
by comparing induction of theturboRFP reporter gene, using a
fluorescence plate reader,and depletion of target mRNA, measured by
quantitativereal-time PCR, in transiently transfected HEK293
cellsinduced with 0.5 mg/mL doxycycline for 48 hours. PCRprimer
sequences for measuring BRCA1 and PALB2mRNA are given in
Supplementary Table S3. The shRNAsequences selected on the basis of
these screens,V2THS_254648 (henceforth, shBRCA1),
V3THS_369350(henceforth shPALB2), together with a nonsilencingTRIPZ
shRNA (TRIPZ control), were packaged into len-tiviruses and
transduced into D3H2LN cells using amultiplicity of infection of 1.
Stable transductants wereselected in puromycin and induced with 0.5
mg/mLdoxycycline for 48 hours before aseptically sorting
thebrightest 30% of turboRFP-expressing cells (12–16 � 103cells in
total) using a BD FACSAria II flow cytometer.Sorted pools were
maintained without doxycycline untilexperimentation. Fluorescence
and phase contrast micro-graphs were captured using a Nikon TE2000E
invertedmicroscope with attached Nikon Digital Sight (DS-5Mc)cooled
color camera.
Cytotoxicity assaysIn vitro antiproliferative activity of drugs
was mea-
sured using well-established (30) IC50 assays with
asulforhodamine B (SRB; Sigma-Aldrich) colorimetricendpoint. For
each cell line, the linearity of SRB stainingand associated optimal
seeding density (electronic par-ticle counter; Beckman Coulter;
Supplementary TableS4) was determined by titrating cell number. To
assaysensitivity to the PARP inhibitor olaparib, cells
werecontinuously exposed to drug for 5 days under aerobicconditions
before SRB staining. All other drug exposures
Hunter et al.
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Therapeutics2502
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were for 4 hours, with 4 to 5 days regrowth in drug-freemedium.
To assay drug sensitivity in D3H2LN cells withshRNA-mediated
knockdown of HR genes, log-phasecultures were induced with 2 mg/mL
doxycycline for72 hours before drug treatment. Doxycycline was
alsopresent in the regrowth medium after removal ofdrugs. Hypoxic
incubations were performed in a H2/Pdcatalyst–scrubbed anaerobic
chamber (Coy LaboratoryProducts)withmediumandconsumables
preequilibratedfor >3 days to remove residual oxygen. Hypoxic
cytotox-icity ratio (HCR) was defined as (IC50 oxic)/(IC50
hypoxic).Hypersensitivity factor (HF) for cell lines with
shRNAknockdown or genetic deletion of HR genes was definedas (IC50
HR-proficient line)/(IC50 HR-defective line), where
theHR-proficient line was D3N2LN-TRIPZ control or DLD-1 wild-type,
respectively. All ratios (HCR and HF) areintraexperiment
comparisons.
Liquid chromatography/tandem mass spectrometryanalysis of
SN30000 metabolismMetabolic depletion of SN30000, and production
of
the corresponding stable 1-oxide and nor-oxide
reducedmetabolites, in hypoxic and aerobic TNBC cells wasquantified
using a validated liquid chromatography/tan-dem triple-quad mass
spectrometry assay (LC/MS-MS)as described previously (36).
Western immunoblottingLysates were harvested from log-phase cell
cultures
using radioimmunoprecipitation assay buffer and totalprotein
concentration measured by bicinchoninic acid(BCA) assay.
Immunoblotting for expression of reduc-tases in cell lines used
well-validated mouse monoclonalprimary antibodies for POR (sc25263;
Santa Cruz Biotech-nology; ref. 37) andAKR1C3 (NP6.G6.A6;
Sigma-Aldrich;ref. 38) as described previously. For RAD51
immunoblot-ting, 30 mg samples of 2-mercaptoethanol- and
heat-dena-tured proteinwere resolved on 4% to
12%polyacrylamidegradient gels (Invitrogen), blocked, transferred
to poly-vinylidenedifluoride (PVDF) membrane, and probedwith an
anti-RAD51 primary antibody (rabbit polyclonalab63801; Abcam) that
we have previously validatedfor immunofluorescent detection of
radiation-inducedRAD51 foci in cell lines (30). Themembraneswere
probedwith anti-rabbit secondary antibody (Invitrogen).
Theseconditionsdetected abandat 37 kDa, corresponding to
theexpected molecular weight of RAD51, with weaker non-specific
bands at approximately 30, 50, 55, and 70 kDa insome cell lines
(Supplementary Fig. S1). For all blots,chemiluminescent images were
acquired using a LAS-4000 ImageQuant (GE Healthcare). Expression of
POR,RAD51, and AKR1C3 in cell lines was quantified bycomparing band
density normalized against ACTB(mouse monoclonal MAB1501R;
Chemicon) or TUBA(mouse monoclonal B-5-1-2; Sigma-Aldrich) as
loadingcontrols in ImageJ using unprocessed, unsaturatedimages.
Values plotted are mean and SEM of the intraex-periment
antigen/actin ratio for two independent experi-
ments. For clarity, blots in the main text have beencropped to
retain six bandwidths above and below anti-gens. Full, uncropped
replicate blots with molecular sizemarkers and quantitation are
provided in SupplementaryData. Borders of cropped blots are
indicated by blackmargins.
RAD51 immunofluorescenceFor analyzing induction of RAD51 foci in
response to
ionizing radiation (IR) in vitro, cells were cultured onsterile
poly-D-lysine–coated glass coverslips (BD Bios-ciences) and treated
with 8 or 10 Gy IR (Eldorado modelG 60Co radiotherapymachine) or
shamradiation. The cellswere fixed in 2% paraformaldehyde
(Sigma-Aldrich)10 hours after irradiation, rehydrated in ice-cold
PBS,permeabilized in 0.25% Triton X-100 (Sigma-Aldrich) inPBS for
20 minutes, and blocked using 5% goat serum(Invitrogen) in 0.1%
PSB–Tween 20 (Global Science) for 30minutes at 20�C. The specimens
were then probed withanti-RAD51 primary antibody (rabbit polyclonal
ab63801;Abcam) diluted at 1:1,000 in blocking buffer for 1 hour
at20�C. The coverslips were washed thoroughly in PBS andprobed with
either Cy3- (TNBC wild-type and DLD-1cells) or Alexa Fluor
488–conjugated (shRNA-expressingD3H2LN cells) anti-rabbit secondary
antibodies (bothfrom Invitrogen) diluted at 1:500 in blocking
buffer for30minutes in darkness at 20�C. Cells were
counterstainedwith 2.5 mg/mL 40,6-diamidino-2-phenylindole
(DAPI;Sigma-Aldrich) for 1minute andmounted on glassmicro-scope
slides using ProLongGold (Invitrogen). Slideswereair-dried before
storing at 4�C. Images of random fieldswere captured using a
LeicaDMRmicroscopewithNikonDigital Sight DS-U1 camera and 100�
objective lens withstandardized exposure conditions. Nuclei
presenting �2RAD51 foci were scored as positive by manual
counting.Typically, >150 nuclei were scored per slide in
eachindependent experiment. To assay induction of RAD51foci in
D3H2LN cells with shRNA-mediated knockdownof BRCA1 and PALB2, cells
were cultured on coverslips inthe presence of 2 mg/mL doxycycline
for 72 hours beforeirradiation.
POR enzyme activityPOR enzymatic activity in cellular S-9
fractions was
determinedby spectrophotometric assay as cyanide-resis-tant,
NADPH-dependent reduction of cytochrome c asreported elsewhere
(39). Total protein in S-9 fractions wasmeasured by BCA assay.
FSL-61 fluorogenic assaysEnzymatic activation of the fluorogenic
one-electron
reductase probe FSL-61 was measured as before (40).Briefly, 106
cells were seeded into non-tissue culture–treated 24-well plates in
0.5-mL preequilibrated PhenolRed–free MEMa with 5% FCS inside an
anaerobic cham-ber, and incubated for 30minutes. The cells were
exposedto 300 mmol/L FSL-61 for 3 hours and then stored indarkness
on ice for
-
LSRII flow cytometer with BD FACSDiva software (Bec-tonDickson).
The excitationwavelengthwas 355 nm,withemission at 425 to 475
nm.
Xenograft modelsAnimal studies were performed in accordance with
the
New Zealand Animal Welfare Act 1999 and ResearchApproval 001190
from the Animal Ethics Committee ofthe University of Auckland.
DLD-1 (1.5 � 106) or DLD-1BRCA2�/� (3 � 106) cells were inoculated,
in 0.1 mL of30% Matrigel/MEMa (BD Biosciences), into the subcutisof
anesthetized female, 18 to 21 gNIH-III nudemice (bredat the
University of Auckland). Tumor growth was mon-itored by
calipermeasurement using the formula: volume¼ 0.5 � length �
width2.
For ex vivo clonogenic assays, tumors were grown totreatment
size of 300 to 500 mm3 and stratified to cohortsthat were dosed
with SN30000, TH-302, or PR-104, byintraperitoneal (i.p.)
injection, at 155, 150, and 578mg/kg,respectively. These doses
corresponded to 75% of empir-ically determined MTD in this mouse
strain. In the drugand radiation combination cohorts, drugs were
adminis-tered 5 minutes after 10 Gy single-dose,
whole-bodyradiotherapy (Eldorado 78 60Co radiotherapy machine)or
sham irradiation. Tumors were excised 18 hours laterand
mechanically and enzymatically disaggregated tosingle cells, which
were then plated in dilution series intriplicate for evaluation of
clonogenic survival. Colonieswere scored 10 days thereafter by
crystal violet staining.Sterilization of tumor clonogens by
treatments is reportedas Log10 Cell Killing, defined
as�log10(Surviving Fraction)by reference to plating efficiency of
cells derived fromuntreated tumors. Cohort sizes were 3 for
drug-onlygroups and 4 for combination therapy groups.
For tumor growth delay, xenografts were grown to 250to 400mm3
and stratified to cohorts thatwere treatedwith10 Gy local-tumor
radiotherapy or single i.p. injection ofPR-104 or cisplatin at 578
and 5.1 mg/kg, respectively,which corresponded to 75% of MTD. Tumor
growthkinetics was evaluated by caliper measurement asdescribed
above. Survival analysis was performed usinglog-rank tests with the
endpoint defined as tumor volume>3-fold higher than volume on
the day of treatment.Cohort sizes were 5 for DLD-1 and 8 for
DLD-1BRCA2�/�.
Pimonidazole immunohistochemistryMice bearing subcutaneousDLD-1
orDLD-1BRCA2�/�
xenografts with mean volume of 350 mm3 were dosedwith
pimonidazole at 60 mg/kg or saline by i.p. injec-tion. The tumors
were excised 2 hours thereafter andfixed in 4% paraformaldehyde for
24 hours, washedthree times in PBS, and then cryoprotected using
20%(w/v) sucrose-PBS followed by 30% sucrose-PBS. Thetissue was
embedded in optimal cutting temperature(OCT) and frozen for
cryosectioning. Eight-micrometersections were stained with
anti-pimonidazole antibody(Hypoxyprobe 1-Mab1; HPI, Inc.),
counterstained with
DAPI, and imaged using a Leica DMR microscope withNikon Digital
Sight DS-U1 camera and 25� objectivelens with standardized exposure
conditions.
Statistical analysisUnless otherwise indicated in figure
legends, values are
mean and SEM of multiple independent experiments.Student t
tests, ANOVA,Mann–WhitneyU tests, log-ranktests, and Spearman
correlations were computed inSigmaPlot v12 (Systat Software). � P
< 0.05; ��, P < 0.01;���, P < 0.001.
ResultsHypoxia-selective cytotoxicity of HAP in TNBC
celllines
To compare the potential of HAP representing mul-tiple chemical
classes to inhibit growth of TNBC cells,we examined in vitro
sensitivity of eight TNBC cell linesof known BRCA1 genotype
(Supplementary Table S2) tofive HAP (benzotriazine di-N-oxides
tirapazamine andSN30000; alkylator prodrugs TH-302, PR-104A,
andnitroCBI SN30548) under hypoxia (Fig. 1A). TH-302was the most
potent hypoxic cytotoxin (mean IC50 for8 cell lines 0.071 mmol/L),
followed by SN30548 (0.40mmol/L), tirapazamine (3.0 mmol/L),
PR-104A (3.2mmol/L), and SN30000 (3.9 mmol/L). Cytotoxicity
wasstrongly suppressed by oxygen in all cases (Fig. 1B) withHCR
(Fig. 1B and Supplementary Fig. S2) greatest forTH-302 (range,
150–880) and least for PR-104A (range,7.9–73). There was no obvious
relationship betweenBRCA1 mutational status and cytotoxic potency
or hyp-oxia selectivity. Sensitivity to the active metabolites
ofPR-104A (i.e., PR-104H) and SN30548 (i.e., SN30550)again showed
no clear relationship with BRCA1 geno-type, as was also the case
for HN2, which we used as amodel for the aliphatic mustard active
metabolite fromTH-302, bromo-isophosphoramide mustard (41).
Cis-platin showed a similar cell line dependence to HN2(r ¼ 0.83; P
¼ 0.01). The BRCA1-mutant MDA-MB-436was the most sensitive line to
the cross-linking agents(Fig. 1C), and was also exquisitely
sensitive to thePARP1/2 inhibitor olaparib (Fig. 1D). However,
therewas no statistically significant relationship betweenBRCA1
genotype (wild-type vs. mutant) and eitheraerobic or hypoxic
potency of any of the agents tested(Supplementary Table S5).
One-electron reductase activity in TNBC cell linesGiven that HAP
activation in hypoxic cells requires
one-electron reduction (14), variation in reductase activ-ity
could contribute to cell line differences in HAPsensitivity.
Comparison of SN30000 reduction toits one-oxide and nor-oxide
metabolites showed >97%inhibition by oxygen in all cell lines,
with a difference ofonly approximately 2-fold in rates of anoxic
metabolicreduction across the panel (Fig. 2A). The
well-charac-terized one-electron reductase POR was expressed in
Hunter et al.
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all cell lines, with significant variation (range, 0.3–1.2as the
ratio of POR/ACTB; Fig. 2B; Supplementary S3and S4); protein
expression correlated significantly withPOR enzymatic activity in
the same cells (r ¼ 0.89; P ¼2 � 10�7; Fig. 2C). Activation of the
one-electron reduc-tase flow cytometry probe FSL-61 in hypoxic
cells(Fig. 2D) showed larger variation between lines, anddid not
correlate with POR activity (r ¼ 0.0; P ¼ 1.0),
which is consistent with its reported activation by mul-tiple
one-electron reductases (40). With the exceptionof SN30000, for
which hypoxic activation correlatedwith POR expression and
sensitivity correlated withPOR enzymatic activity (Supplementary
Fig. S5), noneof these measures of one-electron reductase
activitycorrelated with sensitivity of TNBC cells to other HAPin
univariate analyses (Supplementary Table S6).
TPZ
SN30
000
PR-1
04A
TH-3
02
nitro
CBI
Hyp
oxic
IC50
(µm
ol/L
)
10–3
10–2
10–1
100
101
102
103
104
BT549D3H2LNMDA-MB-468SUM159PT
TPZ
SN30
000
PR-1
04A
TH-3
02
nitro
CBI
Aer
obic
IC50
(µm
ol/L
)
10–310–210–1100101102103104105
HCC1937MDA-MB-436SUM1315MO2SUM149PT
PR-1
04H
HN2
Amino
CBI
Cisp
latin
Aer
obic
IC50
(µm
ol/L
)
10–3
10–2
10–1
100
101
102
BRCA1 wt BRCA1 mutant
68–408-fold65–308-fold12–180-fold149–884-fold8–73-fold
Olaparib10–1
100
101
102
103
A
B
DC
Figure 1. Hypoxia-selectivecytotoxicity of HAP in TNBC celllines
in vitro. Antiproliferativeactivity of the prodrugstirapazamine
(TPZ), SN30000, PR-104A, TH-302, and nitroCBI(SN30548) in a panel
of eight TNBCcell lines, four carrying BRCA1mutations and four with
wild-typeBRCA1, exposed for 4 hours underhypoxic (A) or aerobic
(B)conditions. The values shown aremean þ SEM of three to
sixindependent determinations ofhalf-maximal
inhibitoryconcentration (IC50). The range ofHCR observed for each
compoundis indicated numerically above thebars in B. C,
antiproliferativeactivity of PR-104H (an activemetabolite of
PR-104A),mechlorethamine (HN2; ananalogue of the active
metaboliteof TH-302), aminoCBI (SN30550;the active metabolite of
nitroCBI),and cisplatin in TNBC cell linesexposed to compounds
underaerobic conditions for 4 hours. Thevalues plotted are mean þ
SEM ofthree to six independent IC50determinations. D,
antiproliferativeactivity of the clinical PARPinhibitor olaparib in
TNBC cellsexposed continuously to drug for120 hours. The values are
mean þSEM of three to ten independentdeterminations.
Targeting Hypoxia and HR Repair Defects in TNBC
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HR repair and its relationship to HAP sensitivity inTNBC
cells
Although BRCA1 genotype did not show an obviousrelationship with
HAP sensitivity above, the mutationsinvestigated may have
significant phenotypic differencesand HR status may also be
influenced by other mutationsand epigenetic changes in these cells.
We therefore eval-uated HR function by quantifying
radiation-inducedRAD51 focus formation, which showed marked
differ-
ences between cell lines (Fig. 3A). MDA-MB-436 showedthe lowest
HR activity with no detectable induction ofRAD51 foci, consistent
with its marked sensitivity toolaparib (Fig. 1D). Overall, lines
with BRCA1 mutationsshowed a reduced proportion of nuclei with
RAD51 foci(mean 18% vs. 56% of irradiated cells; Fig. 3B) but
thisdifference was not statistically significant in our smallpanel
(P ¼ 0.06, Mann–Whitney U test). We also testedRAD51 protein
expression (Fig. 3C and Supplementary
SUM
159P
T
D3H2
LN
MDA
-MB-
468
SUM
1315
MO2
SUM
149P
T
HCC1
937
MDA
-MB-
436C
ytoc
hrom
e c
redu
ctio
n (n
mol
.min
−1 .m
g−1
)
0
5
10
15
20
25
30
D3H2
LN
SUM
159P
T
MDA
-MB-
468
SUM
149P
T
HCC1
937
MDA
-MB-
436
SUM
1315
MO2
FS
L-61
red
uctio
n(g
eom
etric
mea
n of
fluo
rese
nce
area
)
0
100
200
300
400
500
600
700
SUM
159P
T
D3H2
LN
MDA
-MB-
468
BT54
9
SUM
149P
T
MDA
-MB-
436
HCC1
937
SUM
1315
MO2S
N30
000
met
abol
ism
(am
ol.c
ell−
1 .h−1
. µM
−1)
0
50
100
150
200
250
BRCA1 wt
BRCA1 mutant
BRCA1 wt, hypoxic
BRCA1 mutant, hypoxic
Aerobic
BRCA1 wt
BRCA1 mutant
BA
DC
MDA
-MB-
468
D3H2
LN
BT54
9
SUM
159P
T
SUM
1315
MO2
SUM
149P
T
MDA
-MB-
436
HCC1
937
SEM
POR76 kDa
42 kDa
0.4
0.03 0.02 0.11 0.3 0.04 0.31 0.07 0.00
0.4 1.0 0.3 0.4 1.2 0.3 0.3
ACTIN
POR:ACTIN
Figure 2. One-electron reductase activity in TNBC cells. A,
metabolic activation of SN30000 by TNBC cells under hypoxic and
aerobic conditions. The rate ofsummed production of stable 1-oxide
and nor-oxide metabolites was normalized for cell density and
actual (i.e., measured) SN30000 concentration. Valuesare mean þ SEM
from three independent experiments, each measuring three separate
cultures. B, evaluation of POR protein expression in TNBC cells
byWestern blot analysis. POR:ACTIN band densitometry ratios,
normalized against MDA-MB-468 cells, are shown numerically below
the image, where bluecoloring corresponds to BRCA1 wild-type lines
and red to BRCA1-mutant lines, and are the mean and SEM
determination of two experiments. C,POR enzymatic activity in TNBC
cell lines measured as cyanide-resistant, NADPH-dependent reduction
of cytochrome c by spectrophotometric assay.Values are mean þ SEM
of determinations from two biologic replicates, each with three
technical replicates. D, reductive activation of the fluorogenic
agentFSL-61 measured by flow cytometry in TNBC cells. Values are
mean þ SEM of geometric mean of fluorescence in three independent
experiments.
Hunter et al.
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-
Figs. S1 and S6) given that increased RAD51 can
partiallycompensate for HR dysfunction (42). Although
RAD51expression trended higher in BRCA1-mutant lines,
thisdifference was not significant (Supplementary Table S5).At
least one surrogate marker of HR—RAD51 foci, ola-parib or cisplatin
sensitivity—was strongly correlated toTH-302, PR-104A, HN2, and
PR-104H sensitivity underhypoxia and to tirapazamine and SN30000
sensitivityunder aerobic conditions (Supplementary Table S6).
Col-lectively, these data suggested that HR repair competencemay
influence sensitivity of TNBC cell lines to someclasses of HAP,
although other determinants are likely tocontribute across a panel
of genetically diverse cell lines.
RNAi-mediated suppression of HR repair sensitizesTNBC cells to
HAP in vitroTo further investigate HR repair as a determinant
of
sensitivity toHAP,we turned to isogenicmodels inwhichthis
variable could be isolated.Wegenerateddoxycycline-inducible
lentiviral shRNA vectors to suppress the HRgenesBRCA1 andPALB2
inHR-competentD3H2LNcells.Hairpins that efficiently suppressed
BRCA1 or PALB2upon induction with doxycycline were identified
byscreening transiently transfectedHEK293 cells for expres-sion of
the turboRFP reporter gene, using a fluorescenceplate reader
(Supplementary Fig. S7), and depletion oftarget mRNA, measured by
quantitative real-time PCR(Supplementary Fig. S8). The most
effective shRNA
against each target, in addition to a nonsilencing TRIPZshRNA,
were stably transduced into D3H2LN cells, andpoolswith high
expression of the bicistronic cassette wereisolated by
fluorescence-activated cell sorting of thebrightest 30% of
turboRFP-expressing cells (Supplemen-tary Fig. S9). Exposure to
doxycycline for 72 hours gaveoptimal turboRFP induction without
cytotoxicity at 2 mgdoxycyline/mL (Supplementary Fig. S10). These
condi-tions efficiently elicited expression of the linked
turboRFPreporter gene (Fig. 4A) and resulted in partial
suppressionof BRCA1 (47% of noninduced) and PALB2 transcripts(42%
of noninduced) with no effect of the control vector(Fig. 4B).
Suppression of BRCA1 and PALB2 resulted inreduction ofHRactivity
asdemonstratedby the radiation-induced RAD51 focus assay, although
this did not reachstatistical significance for PALB2 (Fig. 4C).
This loss of HRwas associated with a 2- to 5-fold increase in
sensitivity toHN2, chlorambucil, cisplatin, and PR-104Hunder
aerobicconditions and 2- to 3-fold increased sensitivity to
TH-302,PR-104A, SN30000, and cisplatin under hypoxic condi-tions
(Fig. 4D).
Genetic deletion of BRCA2 markedly augmentscytotoxicity and
antitumor activity of the nitrogenmustard prodrugs TH-302 and
PR-104
As demonstrated above, shRNA knockdown only par-tially
suppressed BRCA1 and PALB2 expression and HRrepair activity in
D3H2LN cells, resulting in modest
RA
D51
-pos
itive
nuc
lei (
%)
0
20
40
60
80
100MDA-MB-436
SUM1315MO2
SUM149PT
MDA-MB-468
HCC1937
BT-549
SUM159PT
D3H2LN
10 GyMock IR
BA
BRCA1 status
RA
D51
-pos
itive
nuc
lei (
%)
0
20
40
60
80
100
mutwt
P = 0.06
C
MDA
-MB-
468
D3H2
LN
BT54
9
SUM
159P
T
SUM
1315
MO2
SUM
149P
T
MDA
-MB-
436
HCC1
937
37 kDaRAD51
ACTIN 42 kDa
RAD51:ACTIN
SEM
1.0
0.00 0.11 0.42 0.66 0.61 1.12 3.22 1.50
1.8 2.2 1.6 1.8 4.0 6.5 5.6
Figure 3. HR repair activity in TNBCcells. A, induction of
nuclearRAD51 foci in TNBC cells 10 hoursafter treatment with either
10 Gy IRor mock radiation. The values aremean þ SEM percentage
nucleipresenting �2 RAD51 foci in twoindependent experiments.
B,comparison of induction of RAD51foci in irradiated TNBC cell
lineswith either wild-type or mutantBRCA1. The position of
groupmeans is indicated by blue lines.Statistical significance of
thiscomparison was assessed usingthe Mann–Whitney U test.
C,evaluation of RAD51 expression inTNBC cell lines by Western
blotanalysis. RAD51:ACTIN banddensitometry ratios,
normalizedagainst MDA-MB-468 cells, areshown numerically below
theimage and are the mean and SEMdetermination of two
experiments.In all panels, blue coloringrepresents BRCA1 wild-type
celllines and red corresponds toBRCA1-mutant lines.
Targeting Hypoxia and HR Repair Defects in TNBC
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-
Rel
ativ
e P
ALB
2 m
RN
A
0.0
0.2
0.4
0.6
0.8
1.0
TRIP
Z co
ntro
l
shBR
CA1
shPA
LB2
Hyp
erse
nsiti
vity
fact
or
0
1
2
3
4
5
6HN2CHLCisPtPR-104H
TRIP
Z co
ntro
l
shBR
CA1
shPA
LB2
Hyp
erse
nsiti
vity
fact
or
0
1
2
3
4
5TH-302 PR-104A SN30000 CisPt
** *
***
**
*
*
*
***
**
*
* *****
*
8 GyUntreated
RA
D51
-pos
itive
nuc
lei (
%)
0
20
40
60
80
100Noninduced+ doxycycline
8 GyUntreated0
20
40
60
80
100
8 GyUntreated0
20
40
60
80
100
TRIP
Z co
ntro
l
shBR
CA1
TRIP
Z co
ntro
l
shPA
LB2
Rel
ativ
e B
RC
A1
mR
NA
0.0
0.2
0.4
0.6
0.8
1.0 Noninduced+ doxycycline
** NS
****
shPALB2shBRCA1Nonsilencing
Doxycycline (2 µg/mL)Noninduced
C
D Aerobic
B
A
Hypoxic
RFP RFPPC PC
Figure 4. RNAi-mediated suppression of HR repair sensitizes TNBC
cells to HAP in vitro. A, phase-contrast (PC) and fluorescence
micrographs illustratinginduction of shRNA expression, with
concomitant induction of turboRFP reporter expression, in D3H2LN
cells stably transduced with shRNA to BRCA1exposed to doxycycline
for 72 hours. Analogous images were obtained for shPALB2 and TRIPZ
control lines but have been excluded for simplicity.
B,doxycycline-induced, shRNA-mediated suppression of target mRNA in
stably transduced D3H2LN cells. Changes in abundance of BRCA1 and
PALB2transcriptsweremeasured by quantitative real-timePCR in
reference toACTB using the relative quantificationmethod, and are
plotted asmeanþSEMof foldchanges relative towild-typeD3H2LNcells
assayed in parallel. Statistical significance of changes in
transcript abundancewas evaluated byone-wayANOVA.C, quantitation of
RAD51 foci in doxycycline-induced and noninduced TRIPZ control,
shBRCA1, and shPALB2 D3H2LN cells 10 hours after treatment with8 Gy
IR. The values plotted are mean þ SEM of two independent cultures.
Statistical significance was assessed using two-way ANOVA. D,
increasedsensitivity of D3H2LN cells to mechlorethamine (HN2),
chlorambucil (CHL), cisplatin (CisPt), and PR-104H under aerobic
conditions following doxycycline-induced knockdownofBRCA1 andPALB2
(left); increased sensitivity of D3H2LN cells to TH-302, PR-104A,
SN30000, and cisplatin under hypoxic
conditionsfollowingdoxycycline-induced knockdownofBRCA1 andPALB2
(right). D,HFwasdefinedas the intraexperiment quotient (IC50
Noninduced/IC50 Induced) and themeanþ SEM from four to seven
independent experiments is plotted. Statistical significance of
effects of BRCA1 and PALB2 knockdown on drug sensitivitywas
established by comparing HF distributions for each compound in
shBRCA1/shPALB2 to TRIPZ control cells using Student two-tailed t
tests. �, P < 0.05;��, P < 0.01; ���, P < 0.001.
Hunter et al.
Mol Cancer Ther; 13(11) November 2014 Molecular Cancer
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-
hypersensitivity to cross-linking agents. As a furtherisogenic
model of HR deficiency, we investigated aDLD-1 colorectal
adenocarcinoma cell line with homozy-gous deletion of exon 11 of
BRCA2 (43). These cellsdemonstrated complete loss of
radiation-induced RAD51foci compared with parental DLD-1 cells
(Fig. 5A), with ahighly significant difference between the two
lines(Fig. 5B). Both lines were in the upper range of one-electron
reductase activity as compared with the TNBCpanel for reductive
activation of FSL-61 (SupplementaryFig. S11), and showed very weak
expression of the aldo-keto reductase AKR1C3 (Supplementary Figs.
S12 andS13) that has been shown to mediate
oxygen-insensitivetwo-electron activation of PR-104A (38). BRCA2�/�
cellswere 18- to 28-fold more sensitive to HN2,
PR-104H,chlorambucil, and cisplatin than their isogenic
counter-part under aerobic conditions (Fig. 5C), with 10- to
13-foldincreased sensitivity to TH-302, PR-104A, and cisplatinunder
hypoxic conditions (Fig. 5C) without compromis-ing hypoxia
selectivity of the HAP (Fig. 5D). BRCA2�/�
cells were only modestly (2-fold) more sensitive toSN30000 under
normoxia and were not significantlymore sensitive under hypoxic
conditions (two-wayANOVA, P ¼ 0.01 and 0.9, respectively). The
absoluteIC50 values for the DLD-1 line and its BRCA2-null
deriv-ativewere in the range for theTNBCcell lines
investigatedabove, and for other cell lines studied in our
laboratory(Supplementary Figs. S14 and S15), suggesting thatthe
model recapitulates variability in HAP sensitivityobserved in
wild-type cancer cell lines.To examine effects of HR derangement on
antitumor
activity of HAP, we grew DLD-1 BRCA2�/� and wild-type xenografts
subcutaneously in female NIH-III nudemice and used IR as a tool to
distinguish the radiation-resistant hypoxic tumor fraction. IHC
analysis demon-strated that both DLD-1 and DLD-1 BRCA2�/�
tumorscontainpimonidazole-bindinghypoxic cell fractions
char-acteristic ofmany xenograft models (Fig. 6A). To
compareradiosensitivity of the two xenograft models, and toaddress
this potentially confounding variable, we com-pared tumor growth
following administration of a single10 Gy dose of localized
external beam radiotherapy orsham irradiation (Supplementary Fig.
S16). Radiotherapysignificantly delayed growth of both DLD-1 and
DLD-1BRCA2�/� xenografts (log-rank tests,P¼ 0.006 and
0.003,respectively). The median time to endpoint ratio (IR/sham)
was 2.8 for both models, indicating equivalentsensitivity to
radiotherapy. Next, we measured steriliza-tion of clonogens in
DLD-1 and DLD-1 BRCA2�/� xeno-grafts by ex vivo culturing of single
cells recovered fromtumors 18 hours after treatment with a single
i.p. dose ofSN30000, TH-302, or PR-104 (thewater-soluble
phosphatepre-prodrug of PR-104A) at equivalent toxicity (75%
ofMTD), either as monotherapy or 5 minutes after admin-istering 10
Gy whole-body radiotherapy (Fig. 6B).SN30000, TH-302, and PR-104
were all inactive as singleagents in HR-competent DLD-1 tumors
(two-wayANOVA, P > 0.5). Consistent with our in vitro
cytotoxicity
data, BRCA2 deletion did not significantly affect antitu-mor
activity of SN30000 as a single agent (P ¼ 0.9);however, TH-302 and
PR-104 had marked monotherapyactivity inHR-deficient tumors (P <
0.001 for both agents),with surviving fractions of 6� 10�3 and 1�
10�3, respec-tively. PR-104 was modestly active in DLD-1
wild-typetumors in combination with radiotherapy (one additionallog
of cell killing; P > 0.001), whereas TH-302 (P¼ 0.2) andSN30000
(P¼ 0.7)were inactive in this context. Deletion ofBRCA2
dramatically increased sterilization of radiother-apy-resistant
tumor cells bybothTH-302 andPR-104,withcell killing beyond the
dynamic range of the assay (sur-viving fraction
-
HN2
PR-1
04H
Chlor
ambu
cil
Cisp
latin
Hyp
erse
nsiti
vity
fact
or
100
101
102
8 GyMock
RA
D51
-pos
itive
nuc
lei (
%)
0
20
40
60
80
100
DLD-1
DLD-1 BRCA2–/–
***
TH-3
02
PR-1
04A
SN30
000
Cisp
latin
Hyp
oxic
cyt
otox
icity
rat
io
100
101
102
103DLD-1
DLD-1 BRCA2–/–
TH-3
02
PR-1
04A
SN30
000
Cisp
latin
Hyp
erse
nsiti
vity
fact
or100
101
102
Aerobic Hypoxic
A
DAPI
DAPI
DAPI
DAPI
RAD51
RAD51
RAD51
RAD51
Merge
Merge
Merge
Merge
8 GyMockB
RC
A2−
/−W
T
CB
D
Figure 5. Genetic deletion ofBRCA2 sensitizes tumor cells toHAP
in vitro. A, fluorescencemicrographs of DLD-1 andDLD-1BRCA2�/�
cells fixed and stainedfor induction of nuclear RAD51 foci 8 hours
after exposure to either 8 Gy IR or mock radiation. B, proportion
of irradiated and unirradiated DLD-1 and DLD-1BRCA2�/� nuclei
presenting with �2 RAD51 foci. Values are mean þ SEM of two
independent experiments. Significance was assessed using
two-wayANOVA. ���, P < 0.001. C, enhanced sensitivity of DLD-1
BRCA2�/� cells to cytotoxins under aerobic conditions (left) and to
HAP under aerobic and hypoxicconditions (right). HFwas definedas
the intraexperiment quotient (IC50 DLD-1/IC50 DLD-1 BRCA2�/�) and
themeanþSEM from three to six independent assays isshown. D, HCR of
TH-302, PR-104A, SN30000, and cisplatin in DLD-1 and DLD-1 BRCA2�/�
cells in vitro. HCR was defined as the intraexperimentquotient
(IC50 aerobic/IC50 hypoxic) and the mean þ SEM from three
independent assays is shown.
Hunter et al.
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-
No d
rug
SN30
000
TH-3
02
PR-1
04
Log
10 c
ell k
ill
0
1
2
3
4
5
DLD-1
DLD-1 BRCA2–/–
IR o
nly
IR +
SN3
0000
IR +
TH-
302
IR +
PR-
104
Log
10 c
ell k
ill0
2
4
6
8
3/4 4/4
NS
*****
***
***
B
Pimonidazole
DLD
-1D
LD-1
BR
CA
2–/–
Control
DAPI
DAPI
DAPI
DAPI
Pimo
Pimo
Pimo
Pimo
A
DLD-1 BRCA2–/–
Time (d)806040200
Sur
viva
l
0.0
0.2
0.4
0.6
0.8
1.0
Control
Cisplatin
PR-104
DLD-1
Time (d)403020100
Sur
viva
l
0.0
0.2
0.4
0.6
0.8
1.0
Control
Cisplatin
PR-104
DLD-1 BRCA2–/–
Days after treatment
3020100
Tum
or v
olum
e (m
m3 )
0
200
400
600
800
1,000
1,200
1,400
1,600
ControlCisplatinPR-104
DLD-1
Days after treatment
86420–2–4–6
Tum
or v
olum
e (m
m3 )
0
200
400
600
800
1,000
ControlCisplatinPR-104
C
D
Figure 6. Genetic deletion ofBRCA2 markedly augmentsantitumor
activity of the nitrogenmustard prodrugs TH-302 and PR-104. A,
fluorescence micrographsof thin sections from DLD-1 andDLD-1
BRCA2�/� tumorsadministered pimonidazole by i.p.injection at 60
mg/kg andimmunostained 2 hours thereafterfor hypoxia.
Representativeimages are shown. B, sterilizationof clonogens in
DLD-1 and DLD-1BRCA2�/� tumors administeredSN30000 (155 mg/kg),
TH-302(150 mg/kg), or PR-104 (578mg/kg) by single i.p. injection
eitheras monotherapy (left) or 5 minutesfollowing 10 Gy IR (right).
Thesedrug doses corresponded to 75%of murine MTD
determinedempirically in the current study. Thesurviving fraction
(SF) for eachtreatment was determined byindexing plating efficiency
againstunirradiated tumors treatedwith nodrug. Log10 Cell Kill was
defined as�log10(SF), and the mean þ SEMfor three to four
(monotherapy) orfour to five (combination therapy)tumors is shown.
Statisticalsignificance was evaluated usingtwo-way ANOVA. In
thecombination setting, cell killing inthree of four tumors treated
with IRþ TH-302 and four of four tumorstreated with IR þ PR-104
wasbeyond the assay limit (SF < 10�5).��, P < 0.01; ���, P
< 0.001. Tumorgrowth delay (C) and Kaplan–Meiersurvival analysis
(D) ofmice bearing DLD-1 or DLD-1BRCA2�/� tumors andadministered
cisplatin (5.1 mg/kg)or PR-104 (578 mg/kg)monotherapy, which was
75% ofMTD in this mouse strain, by singlei.p. injection. Cohort
sizes were 5(DLD-1) and 8 (DLD-1 BRCA2�/�),and mean þ SEM of tumor
volumeis shown. Growth delay curveswere truncated when the
firstanimal in each cohort wassacrificed for humane reasons.
Thestudy endpoint for survival analysiswas defined as tumor volume
�3-fold tumor volume on the day oftreatment. Statistical
significanceof differences in survival wasevaluated using log-rank
tests.
Targeting Hypoxia and HR Repair Defects in TNBC
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-
chronic hypoxia downregulates expression of key com-ponents of
the HR machinery, offsetting chemo- andradioresistance (33, 34).
Our present finding that DNAcross-linking HAP are able to exploit
HR dysfunctionsimilarly to the widely used clinical cross-linkers
cis-platin and chlorambucil in human tumor cell cultures(Figs. 4D
and 5C), and are more active than nitroCBI orbenzotriazine
di-N-oxides in this context, is consistentwith earlier studies in
Chinese hamster ovary (CHO)models (30). The correlation between
sensitivity to eachof the DNA cross-linking agents across cell
lines sug-gests that cellular sensitivity is dominated by
DNA-damage responses that are generic across these diverseagents.
SN30000 and tirapazamine also show similarcell line dependence
under aerobic conditions, consis-tent with the idea that
replication fork arrest is a com-mon lesion across both the
benzotriazine di-N-oxides(32) and cross-linking agents (27). We
note that our datado not prove that compromised cross-link repair
issolely responsible for the observed hypersensitivity
ofHR-defective cells; higher endogenous levels of DNAlesions and a
correspondingly lower threshold to exog-enous agents might also
contribute.
Cross-linking agents, such as cisplatin, are
increasinglyadministered as part of first-line therapy for TNBC
andother HR-deficient tumors (44); however, toxicity pre-cludes
dosing cisplatin above 100 mg/m2 on a conven-tional 3-weekly
schedule.We showhere, for the first time,that dysfunction of HR
repair analogous to that observedin BRCA-related breast and ovarian
cancer drasticallyenhances antitumor activity of DNA cross-linking
HAPin xenografts (Fig. 6B). This observation raises the
possi-bility that HAP may provide an alternative to
platinumchemotherapy, with potential to address a clinically
chal-lenging subpopulation of hypoxic cells and to
amelioratetoxicity by limiting exposure of well-oxygenated
normaltissue to the active agent. Accordingly, we demonstratedthat
PR-104 is more effective than cisplatin at inhibitinggrowth of
BRCA2-null xenografts when administered atequivalent levels of
toxicity to mice (Fig. 6C and D). Thisresult must be qualified by
the observation that micetolerate PR-104 doses that provide higher
plasma phar-macokinetics than achieved in solid tumor
oncologypatients (46). Interspecies scaling of TH-302
toxicokineticsappears to be more favorable (47). Thus, our finding
thatTH-302 has similar selective activity to PR-104 in BRCA2-null
xenografts (Fig. 6B) suggests that it may be a bettercandidate for
exploiting HR dysfunction in humancancers that, such as DLD-1, do
not highly express thePR-104A–activating reductase AKR1C3.
We reasoned that the striking single-agent activity ofPR-104 and
TH-302 in BRCA2-null tumors despite acti-vation being restricted to
the minority hypoxic fraction(Fig. 6B) must reflect significant
bystander cell killingcaused by diffusion of active metabolites
into better-oxy-genated zones. This interpretation aligns with
spatiallyresolved pharmacokinetic/pharmacodynamic
modelingundertaken in our laboratory, which estimated such
bystander effects to contribute 30% and 50% of PR-104monotherapy
activity in SiHa and HCT116 tumors,respectively (48).
Interestingly, an efficient bystandereffect places central
importance onHR status in normoxiccells, suggesting that
dysfunction of HR through muta-tions in genes, such as BRCA1 and
BRCA2, rather thansuppression of HR by hypoxia, to be the more
relevanttherapeutic target. However, macroregional heterogene-ity
will place some cells beyond the reach of bystandereffects,
implying that HAP may be expected to offeradvantages over cisplatin
only in settings where hypoxialimits therapeutic outcome, an issue
that is not yet wellunderstood in breast cancer.
The finding that SN30000 has limited capacity to exploitHR
dysfunction in tumors, both as a single-agent and incombination
with radiation (Fig. 6B), agrees with cellculture data in the
present and previous studies (30, 32)and suggests that the
benzotriazine di-N-oxide class is lesssuited than cross-linkers to
exploiting this target. Thelatter may reflect the lesser dependence
on HR for reso-lution of lesions induced by SN30000 under
hypoxicconditions, and cell entrapment of the cytotoxic-free
rad-icalmetabolites of SN30000precluding efficient
bystandereffects. Interestingly, SN30000 showedno activity
inwild-typeDLD-1 tumors in combinationwith radiation
despitesignificant antitumor activity in HT29, SiHa, H1299,
andHCT116 xenografts in previous studies (28, 36, 49). Thelikely
explanation for this difference is thatDLD-1 cells areintrinsically
resistant to SN30000 in culture (18th mostsensitive of 21 cell
lines tested; Supplementary Fig. S14).
This study has translational implications beyondTNBC. Indeed
high-grade serous ovarian carcinoma mayprovide earlier
opportunities to clinically evaluate theactivity of HAP in
HR-deficient tumors. Platinum-taxanechemotherapy is well
established as standard-of-care inthe latter indication and many
patients already undergoroutine BRCAmutation testing to determine
eligibility forolaparib maintenance therapy in current phase III
trials(NCT01874353 and NCT01844986). We also note withinterest that
a subset of pancreatic adenocarcinomas har-bor mutations in BRCA2
(50), an indication in which TH-302 is currently undergoing phase
III evaluation (trialNCT01746979). Our study provides a strong
rationale forexplicitly evaluating a nitrogen mustard HAP in
humancancers with HR dysfunction.
Disclosure of Potential Conflicts of InterestW.R Wilson has
ownership interest (including patents) in and is a
consultant/advisory boardmember for Proacta, Inc. No potential
conflictsof interest were disclosed by the other authors.
Authors' ContributionsConception and design: F.W. Hunter, W.R.
Wilson, J. WangDevelopment of methodology: F.W. Hunter, H.-L. Hsu,
J. WangAcquisition of data (provided animals, acquired and managed
patients,provided facilities, etc.): F.W. Hunter, H.-L. Hsu, J. Su,
S.M. Pullen,J. WangAnalysis and interpretation of data (e.g.,
statistical analysis, biostatis-tics, computational analysis): F.W.
Hunter, H.-L. Hsu, J. WangWriting, review, and/or revision of the
manuscript: F.W. Hunter, J. Su,W.R. Wilson, J. Wang
Hunter et al.
Mol Cancer Ther; 13(11) November 2014 Molecular Cancer
Therapeutics2512
on June 24, 2021. © 2014 American Association for Cancer
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Administrative, technical, or material support (i.e., reporting
or orga-nizing data, constructing databases): F.W.Hunter, H.-L.
Hsu, S.M. PullenStudy supervision: W.R. Wilson, J. Wang
AcknowledgmentsThe authors thank Dr. Michael Hay for synthesis
of SN30000, tirapa-
zamine, TH-302, and FSL-61, Dr. Moana Tercel for synthesis of
SN30548and SN30550, and Mr. Stephen Edgar for assistance with
FACS.
Grant SupportF.W. Hunter was supported by Postgraduate
Scholarship, Genesis
Oncology Trust (grant 3627392); and Health Research Doctoral
Schol-arship, University of Auckland; H.-L. Hsu was supported by
Project
Grant, Health Research Council of New Zealand (grant 10/459); J.
Su byDoctoral Scholarship from University of Auckland; S.M. Pullen
byCancer Society, Auckland; W.R. Wilson by Project Grant,
HealthResearch Council of New Zealand (grant 10/459); and J. Wang
byProject Grant, Health Research Council of New Zealand (grant
10/459). This research was also supported by a grant 11-1103 from
theHealth Research Council of New Zealand.
The costs of publication of this article were defrayed in part
by thepayment of page charges. This article must therefore be
hereby markedadvertisement in accordance with 18 U.S.C. Section
1734 solely to indicatethis fact.
Received June 4, 2014; revised August 12, 2014; accepted August
25,2014; published OnlineFirst September 5, 2014.
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Dysfunction in Triple-Negative Breast CancerDual Targeting of
Hypoxia and Homologous Recombination Repair
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