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Therapeutics, Targets, and Chemical Biology
MET-Independent Lung Cancer Cells Evading EGFR KinaseInhibitors
Are Therapeutically Susceptible to BH3 MimeticAgents
Weiwen Fan1, Zhe Tang2, Lihong Yin1, Bei Morrison1, Said
Hafez-Khayyata3, Pingfu Fu4,8,Honglian Huang6, Rakesh Bagai2, Shan
Jiang2, Adam Kresak8, Scott Howell9, Amit Vasanji7,Chris A.
Flask8,10, Balazs Halmos11, Henry Koon2,8, and Patrick C.
Ma1,5,8
AbstractTargeted therapies for cancer are inherently limited by
the inevitable recurrence of resistant disease after
initial responses. To define early molecular changes within
residual tumor cells that persist after treatment, weanalyzed
drug-sensitive lung adenocarcinoma cell lines exposed to reversible
or irreversible epidermal growthfactor receptor (EGFR) inhibitors,
alone or in combination with MET-kinase inhibitors, to characterize
theadaptive response that engenders drug resistance. Tumor cells
displaying early resistance exhibited dependenceon MET-independent
activation of BCL-2/BCL-XL survival signaling. Further, such cells
displayed a quiescence-like state associated with greatly retarded
cell proliferation and cytoskeletal functions that were readily
reversedafter withdrawal of targeted inhibitors. Findings were
validated in a xenograft model, showing BCL-2 inductionand
p-STAT3[Y705] activation within the residual tumor cells surviving
the initial antitumor response totargeted therapies. Disrupting the
mitochondrial BCL-2/BCL-XL antiapoptotic machinery in early
survivor cellsusing BCL-2 Homology Domain 3 (BH3) mimetic agents
such as ABT-737, or by dual RNAi-mediated knockdownof BCL-2/BCL-XL,
was sufficient to eradicate the early-resistant lung-tumor-cells
evading targeted inhibitors.Similarly, in a xenograft model the
preemptive cotreatment of lung tumor cells with an EGFR inhibitor
and aBH3 mimetic eradicated early TKI-resistant evaders and
ultimately achieved a more durable response withprolonged
remission. Our findings prompt prospective clinical investigations
using BH3-mimetics combinedwith targeted receptor kinase inhibitors
to optimize and improve clinical outcomes in lung-cancer
treatment.Cancer Res; 71(13); 4494–505. �2011 AACR.
Introduction
Lung cancer is the second most common cancer andcontinues to
have the highest cancer-mortality rates. Receptortyrosine kinase
(RTK) is a main class of druggable molecular
targets, such as epidermal growth factor receptor (EGFR; 1,
2),MET (3, 4), which can be therapeutically inhibited in
humancancer therapy. EGFR tyrosine kinase inhibitors (TKIs),
gefi-tinib and erlotinib, are approved targeted agents
againstnonsmall cell lung cancer (NSCLC), with enhanced
efficacytoward tumors that express somatic sensitizing kinasedomain
mutations (e.g., L858R, exon 19 deletions; 5–7). Oneof the most
formidable challenges of targeted therapy is theinvariable tumor
secondary resistance after initial response.MET genomic
amplification has been implicated in about 20%of acquired EGFR TKI
resistance (8, 9) whereas the EGFRgatekeeper T790M kinase mutation
(10–12) accounts forapproximately half of the resistant cases.
Further targetingstrategies to overcome EGFR TKI resistance include
the use ofirreversible TKIs (10, 13, 14), pan-EGFR/ERBB kinase
inhibi-tors (15), and MET inhibitors (8, 16). The MET receptor
hasbeen shown to be an important molecule in a variety
ofmalignancies (3, 17) and has recently been validated as
anattractive therapeutic target in cancer therapy, including
lungcancer (4, 18–23). Reversible small molecule inhibitors
totarget against MET have been developed for novel
anticancertherapeutic intervention (20, 21, 24–26). Studies from
ourgroup and others have recently showed the cross-talk signal-ing
network between EGFR andMET, and also the role of MET
Authors' Affiliations: 1Department of Translational Hematology
andOncology Research, Taussig Cancer Institute, Cleveland Clinic;
2Divisionof Hematology/Oncology, Department of Medicine,
3Department ofPathology, and 4Department of Biostatistics,
5Cleveland Clinic LernerCollege of Medicine, Case Western Reserve
University; 6Department ofImmunology, 7Animal Imaging Core, Lerner
Research Institute, ClevelandClinic; 8Case Comprehensive Cancer
Center; 9Center of Visual Research,10Case Center of Imaging
Research, University Hospitals Case MedicalCenter, Cleveland, Ohio;
11Division of Hematology/Oncology, Departmentof Medicine, Columbia
University, New York, New York
Note: W. Fan and Z. Tang have contributed equally to the
work.
Current address for Z. Tang: First Affiliated Hospital of
Zhengzhou Uni-versity, Zhengzhou, Henan, China 450001.
Note: Supplementary data for this article are available at
Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
Corresponding Author: Patrick C. Ma, Taussig Cancer Institute,
Cleve-land Clinic, 9500 Euclid Avenue, R40, Cleveland, OH 44195.
Phone: 216-445-5545; Fax: 216-636-2498; E-mail: [email protected]
doi: 10.1158/0008-5472.CAN-10-2668
�2011 American Association for Cancer Research.
CancerResearch
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inhibition in combination with EGFR inhibitor in lung cancerin
overcoming MET amplified resistance (8) or T790M–EGFRmediated
resistance (16) to EGFR–TKI.Further knowledge into additional
mechanisms of tumor-
cell resistance to targeted inhibitors should prove to be of
greatsignificance in the quest for novel effective treatment
strategiesto impact the long-term prognosis of lung cancer.
Majority ofthe reported studies investigating mechanisms of tumor
resis-tance centered on late time window after chronic exposure
toTKIs at escalating dosing concentrations when secondaryresistant
clones ultimately arose and propagated from theparental
drug-sensitive cell populations. Nonetheless, a deepunderstanding
of the entire spectrum of tumor cells mechan-istic strategies to
escape or evade targeted therapeutics inresistance, especially
during the early inhibitory phase, remainsto be better defined at
present (27, 28). Here, we investigatedthe "early" molecular events
in lung tumor cells under targetedEGFR alone or combined with
MET-kinase inhibitors treat-ment. Our results identified that a
resurgence of prosurvival-antiapoptotic signalingwas evident in the
surviving tumorwithearly evasion against the targeted kinase
inhibitors, thatinvolved a TKI-induced dependence of activated
STAT3, andits transcriptional target BCL-2/BCL-XL, with
therapeutictranslational values. Our results show that
proapoptoticBCL-2 Homology Domain 3 (BH3)-mimetic, such as ABT-737,
can be effective in eradicating these "early" TKI-resistantlung
tumor evader cells, thereby potentially enhancing thelong-term
efficacy of targeted EGFR lung cancer therapy.
Materials and Methods
Cell culture and immunoblottingLung cancer cell lines were
obtained directly from American
Type Culture Collection (ATCC) and grown under standardcell
culture conditions. Cell lines characterization and authenti-cation
were carried out by the ATCCMolecular AuthenticationCenter, using
cytochrome c oxidase subunit I (COI) for inter-species
identification and short tandem repeat (STR) anlaysis(DNA
fingerprinting) for intraspecies identification.
Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis (SDS—PAGE)and
Western blotting were carried out as previously described(16, 29).
The primary antibodies used are as follows:
phospho-MET[Y1234/1235]: Cell Signaling Technology (CST), MET(C-12,
Santa Cruz Biotechnology), phospho-EGFR[Y1068]; CST,EGFR (Santa
Cruz), phosphotyrosine (p-Tyr), phospho-AKT[S473], AKT,
phospho-MAPK(ERK1/2)[T202/Y204]—all
fromCST,MAPK(ERK1/2):Biosource,phospho-STAT3[Y705];CST, STAT3,
BCL-2, BCL-XL (all fromZymed), cleaved-Caspase-3[Asp175],
cleaved-PARP[Asp214], phospho-STAT5[Y694]—allfrom CST, survivin
(Zymed) and actin (Santa Cruz).
Chemicals and inhibitorsEGFR inhibitor (reversible) erlotinib
was prepared as pre-
viously described (29, 30). MET inhibitors SU11274,PHA665752 and
EGFR inhibitor (irreversible) CL-387,785 wereobtained from
EMD–Calbiochem. BCL-2 family inhibitorsABT-737, obatoclax mesylate
(GX15–070), and HA14–1 wereobtained from Selleck.
Cellular cytotoxicity, viability, and survival assaysCellular
cytotoxicity and viability assays were carried out
using CellTiter 96 AQueous. One Solution Cell Proliferation(MTS)
assay (Promega), according to the manufacturer'sinstruction at 72
hours after treatment with indicated inhi-bitors in 10% fetal
bovine serum (FBS) media. For the studiesof cells under 9 days of
pretreatment under targeted (EGFR/MET) inhibitors, the indicated
inhibitors in the culture mediawere replenished at least every 2–3
days (which was verified tobe indistinguishable from daily TKI
replacement) prior to cellharvesting at the end of the inhibitory
culture for subsequentcellular assays.
For cell survival assay using crystal violet staining
method,H1975 cells or HCC827 cells were treated as described
inSupplementary Materials and Methods with indicated TKIsfor 6
days, followed by indicated BH3-mimetic inhibition withor without
concurrent TKI for 3 extra days.
Time-lapsed video microscopy: Image analysis ofcytoskeletal
functions
HCC827 cells were plated on cell-culture dishes in a
tem-perature-controlled chamber at 37�C in an atmosphere of 5%CO2
for time-lapsed video microscopy (TLVM) analysis ofcytoskeletal
functions and determination of cellular mitoticactivities as
previously described (16) and also in Supplemen-tary Materials.
In vivo xenograft model and bioluminescence imagingof human lung
cancer
Lung cancer xenograft. Firefly-luciferase(luc)-expressingHCC827
and H1975 lung cancer cells, and their correspondingmurine
xenograft models were established as previouslydescribed (see also
Supplementary Materials and Methods)according to
institution-approved protocols and guidelines(16).
Immunohistochemical (IHC) analysis of the tumor xeno-graft was
carried out in the Tissue Procurement and HistologyCore Facility,
Case Comprehensive Cancer Center, using anti-human BCL-2 (Abcam),
antihuman p-STAT3[Y705] (rabbitmonoclonal antibody, D3A7, CST)
primary antibodies. Fordetails see also Supplementary Materials and
Methods.
Tumor microarrayHuman lung cancer tumor microarray was purchased
from
Zymed-Invitrogen (MaxArray Human Lung Cancer TissueMicroarray
Slides, Cat. No. 75–4083). IHC staining usingantihuman BCL-2
antibody was carried out as describedabove, and graded using 4-tier
scoring system (0, 1þ, 2þ,and 3þ) by a dedicated thoracic
pathologist (S. H.-K.). For thelung cancer tumor microarray (TMA)
analysis, the TMAused in the analysis consisted of the followings:
SquamousCell (n ¼ 25), Adenocarcinoma (n ¼ 21), Large Cell (n ¼
3),SCLC (n ¼ 5), Carcinoid (n ¼ 2), Mesothelioma (n ¼ 2).
BCL-2/BCL-XL DNA transfection and RNA interferencestudies
Human BCL-2 plasmid vector was a generous gift from Dr.Clark
Distelhorst (Case Western Reserve University). Trans-fection of the
BCL-2 expression vector into HCC827 cells was
BH3-Mimetic Susceptible Early TKI-Resistant Tumor Cells
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carried out using Fugene 6 according to the
manufacturer'sinstructions (Roche). RNA interference (RNAi)
knockdownstudies were conducted using the Thermo
Scientific/Dharma-con RNAi Technologies, including siGENOME
siRNA-NT (non-targeting; Cat.#D-001210–02), siRNAs against human
BCL-2(Cat.#L-003307–00), and BCL-XL (Cat.#L-003458–00). ForHCC827
cells (Fig. 6A–B), cells were plated at full confluenceon 48-well
plates, then cultured for 9 days in serum-containingmedia (a)
without inhibitor, or with treatment of (b) Erlotinibalone for 9
days, or (c–f) Erlotinib together with the followingsin
combination: (c) ABT-737 (2 mmol/L) concurrently at Day 0,(d)
siRNA-nontargeting (siNT), (e) siRNA-BCL-2, and (f)
dualsiRNA-BCL-2/BCL-XL RNAi knockdown. Cells were then fixedin
methanol and stained with 0.1% crystal violet as above atthe end of
day 9 to visualize the early TKI-resistant tumorsurvivor cells
emerged under various conditions. Experimentswere carried out in
triplicate.
Statistical analysisIn the BCL-2 transfection study and
erlotinib cellular cyto-
toxicity assay in the HCC827 cells (Fig. 5D), the results
undereach transfection condition were first summarized by the
areaunder the curve (AUC). The differences of AUC
betweentransfection conditions were then examined by Z-test.
Statis-tical data analysis of the in vivo study using
HCC827-lucxenograft murine model (Fig. 6E) was carried out using
themixed model to examine the difference of
read-out[bioluminescence imaging (BLI) region of interest
(ROI)]among the four study groups (I–IV), by the in vivo
xenograftgrowth rate–changing rate over time [i.e., the change of
read-out (BLI ROI) divided by time (day)]. To ensure the
normalityassumption for the mixed model used is satisfied, the
read-outs were transformed by natural log function, i.e.,
loge(read-out), prior to fitting the data using Mixed Model.
Tumorrecurrence was defined as 20% increase of tumor BLI-fluxfrom
the nadir and the difference of recurrence rates betweenGroup II
(ABT-737 alone), Group III (Erlotinib alone), andGroup IV (ABT-737
plus Erlotinib) was examined by Fisher'sexact test. All tests were
two-sided and P-values � 0.05 wereconsidered statistically
significant.
Results
Tumor resistance emerged "early" from
EGFR-reversible-TKI-sensitive lung adenocarcinoma
evadingerlotinib:MET-independent BCL-2/BCL-XL signaling
The lung adenocarcinoma cell lines HCC827 and PC-9 areboth
highly sensitive to reversible-EGFR inhibitors
(erlotinib/gefitinib), owing to the oncogenic sensitizing EGFR exon
19deletion (E746_A750 del). Here, we focused to study the
"early"molecular alterations in tumor cells under TKI treatment,
inan attempt to uncover potential therapeutic "Achilles’ heel"for
the tumor cells that may survive the TKI within the
earlytime-window.
We first adopted the HCC827 cell line in the in vitro
"early"TKI-resistance studies, with the cells cultured under
ongoingerlotinib (1 mmol/L) inhibitory treatment up to 9 days.We
chose the concentration of erlotinib to be used at
approximately IC70–75 in the 72-hrs cell viability assay. Byday
9 of inhibition, there were cell subpopulations ("early"survivors,
HCC827_ERL-D9.R) that evaded and survived theTKI treatment. These
"early" survivor cells exhibited a dra-matic shift of
TKI-sensitivity phenotype toward higherresistance (�100-fold),
compared with the TKI-naïve par-ental cells (Fig. 1A). After an
initial inhibited state, there wasalso reactivated BCL–2/BCL–XL,
within the background of atyrosine-phosphoproteomic reactivated
cellular state of aunique profile different from the parental cells
(Fig. 1B–C).Importantly, the tumor cells that survived up to days
6–9 ofthe EGFR–TKI treatment evidently signaled independentlyof
EGFR and MET (Fig. 1C). Following an initial inhibitoryperiod, we
observed a rather early p-STAT3[Y705] reactiva-tion despite ongoing
erlotinib treatment. These restoredprosurvival-antiapoptotic
markers correlated well withtime-dependent downregulated
cleaved-caspase 3 andcleaved-PARP, indicative of a suppressed
caspase-dependentintrinsic apoptosis mechanism among these
TKI-evadercells. These results were further verified in PC-9 cell
line,with the erlotinib-surviving PC-9 cells (PC-9_ERL-D9.R)
atDay-9 inhibition exhibiting MET-independent
upregulatedp-STAT3[Y705]/BCL-2/BCL-XL signaling, and "early"
TKI-resistance (Supplementary Fig. S1).
EGFR-irreversible-TKI-sensitive lung adenocarcinomaH1975 cells
also showed "early" tumor resistant evasionfrom CL-387,785
We also tested the H1975 cell line against the
irreversible-EGFR-TKI CL-387,785 as an alternate model. Similarly,
the"early" TKI-surviving H1975 cells (H1975_CL-D9.R) that
wereharvested after 9 days of CL-387,785 (1mmol/L) exposure
werefound remarkably more resistant (Fig. 2A), accompanied witha
prosurvival-antiapoptotic signaling upregulation as early asbeyond
day 3 of inhibition (Fig. 2B). Consistent with thefindings in both
HCC827 and PC-9 cells, the H1975_CL-D9.R cells also signaled
independent of MET. Intriguingly, theH1975_CL-D9.R cells were found
to exhibit reactivated p-EGFR[Y1068] beyond day 3 and onward
despite ongoingCL-387,785, in the presence of complete p-MET
inhibition.Thus, the downstream prosurvival-antiapoptotic signaling
inthe survivor cells may still be at least partially driven by
EGFRsignaling. We verified that the reactivated EGFR
phosphoryla-tion, and its downstream signals emerged in the
H1975_CL-D9.R cells, could be inhibited by a higher concentration
(5mmol/L) of the same inhibitor (Fig. 2C). Nonetheless, p-STAT3and
BCL-2 were not effectively inhibited by the
retreatment,highlighting their potential importance in promoting
thecellular resistant survival, independent of both EGFR andMET
signaling.
Early EGFR–TKI resistance exhibited "adaptive"phenotypes that
were highly "reversible"
Using TLVM analysis, we found that the HCC827_ERL-D9.R exhibited
a cellular "quiescence-like" state, with dramati-cally inhibited
proliferative and cytoskeletal functions whilein evasion against
erlotinib. Promptly after erlotinib-with-drawal, we found that the
early resistant cells could readily
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be reverted to a highly activated state of cellular
motility(Fig. 3A) and mitotic proliferation (Fig. 3B; see also
Supple-mentary Movies). We further tested to see if the Day-9
"early"resistant cells could maintain their resistant phenotype
aftera brief period of TKI withdrawal. Interestingly, after only7
days of withdrawal of the corresponding TKIs, bothHCC827_ERL-D9.R
and H1975_CL-D9.R cells quicklyreverted back to a highly
TKI-sensitive phenotype, indis-tinguishable from parental-cell
populations, respectively(Fig. 3C and E). Importantly, upon a
washout period ofTKI-withdrawal, these early resistant escape
survivor tumorcells reexhibited TKI-induced p-EGFR inhibition as in
theTKI-naïve parental cells (Fig. 3D).
In vivo activated STAT3/BCL-2 prosurvival-antiapoptotic signal
axis in "early" TKI-resistant lungtumor survivor cells
We extended our studies using in vivo xenograft model toexamine
tumor cells that survived initial treatment withtargeted
RTK-inhibitors. HCC827 xenograft was inhibited witherlotinib for 3
days, during which remarkable tumor responsewas evident as
expected. Consistent with our in vitro data, theSTAT3 downstream
transcriptional target BCL-2 expressionwas found induced in the
TKI-evading survivor cells (Fig. 4A).Interestingly, these early
TKI-resistant cells were localizedalong the peripheral rind of the
tumor xenograft (Fig. 4A–B). P-STAT3[Y705] was predominantly
membranous and less
A
B
C
W.B.
W.B.
EGFR (Total)P-EGFR
EGFR-TKl:
Erlotinib
P-MET
P-STAT3
BCL-2
BCL-XL
P-AKT
P-MAPK
c-Caspase3
c-PARP
P-STAT5
Survivin
Cyclin D1
Actin
HCC827
HCC827
Hours
WB: anti-p-Tyr
kDa
250
150
100
75
50
37
25
Erlotinib (1 µmol/L)
6 D
ays
9 D
ays
72482484210
Hours
6 D
ays
9 D
ays
72482484210
EGFR-TKl:
Erlotinib (1 µmol/L)
Hours
6 D
ays
9 D
ays
72482484210
HCC827HCC827_ERL-D9.R
HCC827
Erlotinib Conc. (µmol/L)
Per
cent
cel
l via
bilit
y (M
TS
Ass
ay)
(%)
100
90
80
70
60
50
40
30
20
10
00 0.001 0.01 0.1 1 10
110
120
MET (Total)
STAT3 (Total)
AKT (Total)
MAPK (Total)
Figure 1. Targeted inhibition of EGFR-TKI-sensitive HCC827 lung
adenocarcinoma cells resulted in emergence of "early" tumor
TKI-resistant evadercells with MET-independent
prosurvival-antiapoptotic signaling. A, significant shift of cell
viability toward insensitivity in HCC827 cells that survived 9 days
oferlotinib (1mmol/L) treatment. HCC827_ERL-D9.R cells displayed
�100-fold increase in IC50 corresponding with a dramatically higher
resistant phenotypeagainst the EGFR-TKI. B, activated
tyrosine-phosphoproteome of early resistant HCC827 cells
(HCC827_ERL-D9.R) evading erlotinib up to 9 days.C, activation of
p-STAT3[Y705]/BCL-2/BCL-XL signaling in HCC827_ERL-D9.R cells.
BH3-Mimetic Susceptible Early TKI-Resistant Tumor Cells
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so cytoplasmic in the untreated HCC827 cells. Conversely,
theactivated p-STAT3 signal was predominantly nuclear in
theresidual HCC827 tumor early survivor cells circumscribingalong
the xenograft periphery (Fig. 4B).
Past studies suggested that non-T790M-EGFR mediatedacquired
gefitinib/erlotinib resistance in sensitive-lung can-cer cells may
include genomic MET amplification (8, 9) orhepatocyte growth factor
(HGF) overexpression (31). Cur-rently, there are various clinical
trials investigating thestrategy of combining EGFR- and
MET-inhibitors to enhancetherapeutic efficacy and overcome acquired
EGFR-TKI resis-tance in NSCLC. We have previously characterized the
METinhibitors SU11274 (20) and PHA665752 (21) in lung cancernovel
therapy, which were utilized in the present study.Similar to the
EGFR-TKI monotherapy model above, therewas an upregulated
BCL-2/BCL-XL prosurvival-antiapopto-tic signaling in H1975
TKI-evader cells after 9 days ofdual CL-387,785/PHA665752
inhibition, alongwith restoredp-STAT3 activation (Fig. 4C), and
associating with a moreTKI-resistant phenotype in the evader cells
(P ¼ 0.0118;Supplementary Fig. S2).
We recently reported the efficacy of combined SU11274-erlotinib
(MET-EGFR/ERBB TKI) in vivo H1975 xenograftmodel in overcoming
T790M-EGFR drug-resistance, withresultant near-complete
BLI-radiographic complete response(16). Here, we further evaluated
the microscopic residualTKI-evading H1975-luc tumor cells and found
they residedprimarily along the tumor periphery juxtaposing the
murinehost-microenvironment. These TKI-evading survivor tumorcells
also exhibited upregulated BCL-2 expression (Fig. 4D).
BCL-2 signal pathway expression and its potentialtherapeutic
utility in NSCLC inhibition by BH3-mimetic
BCL-2 was found to be expressed at varying levels in NSCLCcell
lines and lung tumor tissues, albeit at a significantlylower level
range than in SCLC (Fig. 5A–B). Interestingly,unlike BCL-2, the
expression levels of BCL-XL appeared tobe more comparable among
NSCLC and SCLC cell lines(Fig. 5A). TMA analysis showed that BCL-2
expression inNSCLC was primarily nuclear, whereas that in SCLC
wasstrongly positive both nuclear and cytoplasmic. Stronger
P-EGFR
P-EGFR
P-MET
P-STAT3
P-STAT3
BCL-2
BCL-2
BCL-XL
BCL-XL
P-AKT
P-AKT
P-MAPK
P-MAPK
c-PARP
Survivin
Actin
Actin
H1975
H1975
–
–
+ +
(+)–
–
–
+ +
(+)–
H1975
100
75
50
25
0
Per
cent
cel
l via
bilit
y (M
TS
Ass
ay)
(%)
CL-387,785 (µmol/L)
CL-387,785 (D1–9)
CL-387,785 (D9)
0 0.001 0.01 0.1 51 10
EGFR (Total)
EGFR-TKl:
CL-387,785 (1 µmol/L)
EGFR-TKl:
CL-387,785
MET (Total)
STAT3 (Total)
AKT (Total)
MAPK (Total)
W.B.
W.B.
W.B.
A
C
B
W.B.
0 1 4 24 3 90 1 4 24 3 9
DaysHrHr Days
EGFR (Total)
EGFR/ERBB-TKl:
1 µmol/L (D1–9)
5 µmol/L (at D9, x8h)
STAT3 (Total)
AKT (Total)
MAPK (Total)
H1975
H1975_CL-9D.R
Figure 2. Targeted inhibition of irreversible EGFR-TKI-sensitive
H1975lung adenocarcinoma cells also results in "early’ emergence of
tumorresistant escape survivor cells with activated
prosurvival-antiapoptoticsignaling. A, significant resistant shift
of cell viability in H1975 cellsthat survived 9 days of CL-387,785
treatment. B, activation ofp-STAT3[Y705]/BCL-2/BCL-XL signaling in
H1975 cells that survived9 days of CL-387,785 treatment. C,
reactivated p-EGFR anddownstream signaling in day
9-resistant-survivors H1975 cells againstCL-387,785 (1 mmol/L)
could be inhibited by higher dose ofCL-387,785 (5 mmol/L) in
retreatment, but not p-STAT3 orBCL-2.
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BCL-2 nuclear expression was observed in squamous cellcomparing
with adenocarcinoma subtype. Our results aboveprovide a rationale
to target BCL-2 family signaling throughproapoptotic BH3-mimetic,
such as ABT-737 (32–34), in orderto optimize targeted therapies.
ABT-737 has been well-char-acterized recently and shown to
antagonize BCL-2/BCL-XL,thereby inducing a proapoptotic effect
through the mitochon-drial intrinsic apoptosis pathway. NSCLC cell
lines wererelatively insensitive to ABT-737 (IC50 > 5 mmol/L),
whereasthe SCLC H345 cell line tested was as expected highly
sensitive(IC50 < 0.2 mmol/L; Fig. 5C). HCC827 cells with forced
over-expression of transfected BCL-2 was sufficient to induce a
significantly higher erlotinib-resistance, with
�100-foldincrease in IC50 (Fig. 5D).
ABT-737 inhibition in concert with targeted kinaseinhibitors,
both in vitro and in vivo, eradicated "early"TKI-resistant tumor
evaders and further inhibitedsubsequent tumor recurrence
We hypothesized that preemptive inhibition and eradica-tion of
"early" resistant tumor cells in RTK targeted therapymay impact on
the long-term outcome of targeted therapy. Weadopted RNAi knockdown
of BCL-2/BCL-XL using siRNAmethods here to test in parallel with
ABT-737 (Fig. 6A–B).
A D
B C E
“Quiescence”
H1975
– + +
– – +
n/a
CL-387,785(1 µmol/L x 9 Days)
CL-387,785 Re-Treatment(1 µmol/L, 8 h)
Withdrawal CL-387,785 x 7 Days
“Proliferative” with “Activated cytoskeletal functions”
HCC827-TLVM
HCC827
Parental cell
TKI x 9D -> Washout x 7D
TKI x 9D -> Washout x 7DParental cell
P = N.S. P = N.S.
H1975
HCC827
50
40
30
Mito
tic in
dex
(%)
20
10
0 Per
cent
cel
l via
bilit
y (M
TS
Ass
ay)
Per
cent
cel
l via
bilit
y (M
TS
Ass
ay)
0
0 0.001 0.01 0.1
Erlotinib (µmol/L)
1 100
100
75
50
25
0
CL-387,785 (µmol/L)10310.30.1
25
50
75
100
Pare
ntal
Erlot
inib
x9D
Off-T
KI x3
D
Off-T
KI x1
1D
OFFErlotinib
(i)
(ii)
(iii)
W.B.
P-EGFR
P-MET
P-MAPK
Cleaved-PARP
Actin
Erlotinib(1 µmol/L)x9 Days
Erlotinibwithdrawalx11 Days
Figure 3. "Adaptive" emergence of "early" TKI-resistant tumor
survivor cells in HCC827 under erlotinib, and H1975 under
CL-387,785 inhibitorypressure. HCC827 cells (A–C): A, TLVM:
HCC827_ERL-D9.R cells exhibited a "quiescence-like" state with
highly inhibited cell proliferation as wellas cellular cytoskeletal
functions that were both readily reversible upon TKI-washout for 11
days. B, cell mitotic index (M.I.) of HCC827 cells under
varioustreatment conditions. Error bar, S.E.M. (n¼ 4). *, P <
0.0001; **, P¼ 0.002; †, P¼ 0.03; z, P¼ 0.008. C, HCC827_ERL-D9.R
cells were resensitized to erlotinibinhibition after 7 days of
TKI-washout culture conditions, indistinguishable from the parental
HCC827 cells sensitivity (P > 0.05, N.S.). H1975 cells (D–E):D,
H1975_CL-D9.R cells were resensitized to TKI in cellular signaling
inhibition promptly upon withdrawal of CL-387,785. E, H1975_CL-D9.R
cells wereresensitized to CL-387,785 inhibition upon 7 days of
TKI-washout, indistinguishable from the parental H1975 cells
sensitivity (P > 0.05, N.S.).
BH3-Mimetic Susceptible Early TKI-Resistant Tumor Cells
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-
Dramatic reduction in the early TKI-resistant tumor
survivorcells was achievable by dual BCL-2/BCL-XL RNAi knockdownin
conjunction with erlotinib, but not by mere knockdown ofBCL-2
alone. ABT-737, when used concurrently with erlotinibto inhibit
HCC827 cells, also dramatically reduced emergenceof early
TKI-resistant tumor survivor cells against erlotinib(Fig. 6B).
We further carried out in vitro ABT-737 inhibition studieson the
NSCLC H1975 TKI-evading tumor cells that wereprimed to upregulate
BCL-2/BCL-XL prosurvival signalingby (i) EGFR/ERBB inhibitor
(CL-387,785; Fig. 6C), and (ii) dualEGFR–MET inhibitors (erlotinib
plus SU11274; Fig. 6D). ABT-737, at a concentration relatively
insensitive against H1975parental cells, completely eradicated the
early CL-387,785-resistant H1975 evader cells (Fig. 6C, top),
either alone or incombination with CL-387,785. Importantly, we
showed thatthe "early" TKI-resistant tumor cells were primed to be
moresusceptible to ABT-737 inhibition, exhibiting a muchenhanced
proapoptotic marker cleaved-PARP induction bythe BH3-mimetic (Fig.
6C, bottom). Moreover, the dual-TKI-resistant H1975_ERL/SU-D9.R
tumor cells could also be tar-
geted by ABT-737 to further induce apoptosis (Fig. 6D).
OtherBH3-mimetic BCL-2 family inhibitors tested in our
study,obatoclax (35) and HA14–1 (36), also showed efficacy(Fig.
6D). Similar to H1975 cells, early TKI-evading resistantHCC827
cells also displayed therapeutic susceptibility to BH3-mimetic in
vitro (Fig. 6E).
Finally, we tested if the addition of the BH3-mimetic ABT-737 in
vivo would prolong the duration of response inerlotinib-treated
drug-sensitive HCC827-luc xenograft(Fig. 6E). The in vivo ABT-737
treatment dosage in our studywas chosen based on reported
literature on the therapeuticrange in the tested sensitive cancer
models (37). The tumorgrowth rate in recurrence of the
ABT-737þErlotinib-treatedgroup (IV) was significantly lower than
that of the Erlotinib-alone group–III [P ¼ 0.0009; Fig. 6E, Table
1; also Supple-mentary Fig. S3]. ABT-737-alone (Group II) did not
accountfor the tumor regression and abrogation of tumor
recurrenceas seen in the ABT-737þErlotinib group (Group IV; P
¼0.0004). Finally, the HCC827 tumor recurrence rates atday 18 and
day 32 for Group III (Erlotinib-alone) animalswere 50% (P ¼ 0.014)
and 62.5% (P ¼ 0.004), respectively,
A
HC
C82
7H
CC
827
B
C
D
BCL-2Control
H1975
BCL-2
BCL-2
Erlotinib
HCC827 Xenograft: BCL-2
HCC827 Xenograft: P-STAT3[Y705]
MET/EGFR-TKIs:
PHA665752 (1 µmol/L)CL-387,785 (1 µmol/L)
P-EGFR
EGFR
P-MET
MET
P-STAT3
STAT3
BCL-2
BCL-XL
Actin
W.B.
ControlControl
SU11274 + Erlotinib
Erlotinib
BCL-2P-STAT3
H19
75 BCL-2
BCL-2
10x
10x
Hr
0 1 4 24 72 9 D
ays
5x
5x
5x
20x
10x
10x
P-STAT3
P-STAT3
10x
10x20x 5x
Figure 4. In vivo xenograft lung tumor survivor cells evading
against targeted inhibitors, with activated p-STAT3/BCL-2
antiapoptotic signaling. In vivoHCC827 xenograft model treated with
erlotinib (n ¼ 6) revealed "early" TKI-resistant tumor survivor
cells (A) with induced BCL-2 expression especiallyin the nuclear
subcellular localization (red arrows), and (B) with nuclear
translocation (red arrows) of p-STAT3[Y705] from the untreated
cytoplasmic localization(yellow arrows). C, combined MET–EGFR TKIs
treatment of H1975 cells in vitro also led to an induction of
MET-independent activated p-STAT3[Y705],and BCL-2/BCL-XL signaling
in TKI-evading survivor cells against 9 days of combined-TKIs
(PHA665752 [1 mmol/L] and CL-387,785 [1 mmol/L]). D, inducedBCL-2
expression in H1975-xenograft residual dual
SU11274/erlotinib-TKI-evader survivor cells.
Fan et al.
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-
both significantly higher than Group IV (ABT-737þErlotinib;0%;
Fig. 6E; Table 1).
Discussion
In recent years, molecularly-targeted cancer therapy hasrenewed
our hope for cancer cure. Nonetheless, the challengesof clinical
tumor resistance, both intrinsic and acquired,remain formidable and
substantially limit long-term efficacy.Classic secondary mutational
resistance [e.g., T790M-EGFRagainst gefitinib/erlotinib in lung
cancer (11)], and receptorkinase class-switching [e.g., from
EGFR-addiction to MET/HGF (8, 9, 16), IGF1-R (27, 38), or AXL (39)
signaling] have beenidentified in earlier studies that emphasized
on "acquired"drug-resistance at "late-stages" of chronic drug
treatment. Ourstudy here focuses on the molecular changes of
drug-sensitivetumor cell population within the "early" time-window
oftargeted TKI treatment. We identified and further character-ized
the "early" adaptive functional TKI-resistant lung adeno-carcinoma
cells that survived and evaded EGFR/MET-TKI,as early as within 9
days of therapy. During our manu-script preparation, Settleman and
colleagues reported theidentification of "drug-tolerant state" in
cancer cell subpopu-lations that was maintained through engagement
of IGF-1Rsignaling and an epigenetic alteration of chromatin state
that
requires the histone demethylase RBP2/KDM5A/Jarid1A (27).Our
report here lends further support to the emerging evi-dence of the
existence of tumor cell subpopulations withadaptive
resistant-escape under therapeutic inhibitory stress.These early
adaptive resistant survivors likely serve as thefounder population
as minimal residual disease in solidcancers under therapeutic
pressure, which ultimately leadsto frankly recurrent resistant
disease on therapy in the future.
Despite the new insights into nonmutational early resis-tance
(28), detailed underlying regulatory mechanism(s) thatdirectly
mediate the emergence of such early resurgent resis-tant cells
against the inhibitors remain to be fully defined. Ourstudy here
provided the first evidence that the "early" emer-gence of
resistant tumor survivors evading EGFR/ERBB-METTKIs is independent
of MET receptor signaling activation,contrasting previous reports
ofMET genomic amplification asacquired resistance mechanism in
HCC827 cells that escape"chronic" dose-escalating gefitinib
inhibition at "late stages"after many months of treatment (8, 9).
We present findingshere that the BCL-2-family signaling in the
mitochondrial(intrinsic) programmed-cell death pathway may indeed
repre-sent the central mechanism as tumor cells’
newly-dependentaddiction, in promoting "early tumor evasion" to
survivetargeted therapeutics. Here, we also provide additional in
vivotherapeutic study evidence to validate the efficacy of
targeting
ANSCLC
Exposure:
(–)
“Light”
“Heavy”
BCL-2
BCL-2
TMA: IHC
TMA analysis
W.B.
Small cell Adenocarcinoma Squamous cell
Human lung cancer
Lung cancer TMA: BCL-2
BCL-2 Expression
HCC827-BCL-2
ABT-737 (µmol/L)
00.
078
0.15
60.
313
0.62
51.
25 2.5 5
HCC827
(†, ‡)
(*)
BCL-2
ParentalMock
SCLC
NSCLC
0 1+ 2+ 3+
3+
2+
1+
0
BCL-XL
HC
C82
7
PC
-9
A54
9
H59
5
H14
37
H19
75
H19
93
H69
H28
H20
52
MS
TO-2
11H
H34
5
H69
H28
H20
52
MS
TO-2
11H
H34
5
H18
38
H14
37
H19
75
H19
93
H18
38
H44
1
HC
C82
7
PC
-9
A54
9
H44
1
SCLC MesotheliomaB C
D1009080
7060
5040
3020
100
Per
cent
TM
A tu
mor
sam
ple
(%)
Per
cent
cel
l via
bilit
y (M
TS
)
Squa
mou
s cell
Larg
e ce
ll
Aden
ocar
cinom
a
100
50
00 0.001 0.01
Erlotinib (µmol/L)0.1 1 10
Moc
kPa
rent
al
BCL-
2 Tra
nsfec
ted
BCL-2
Actin
0
25
50
75
100
H441
H1975
H345
HCC827
Cel
l via
bilit
y (%
Con
trol
)
Figure 5. BCL-2/BCL-XL signaling expression and inhibition in
lung cancer. A, BCL-2/BCL-XL expression in lung cancer. Top, BCL-2
and BCL-XL expressionin thoracic malignancy cell lines. Bottom,
BCL-2 TMA-IHC staining in SCLC, adenocarcinoma, and squamous cell
carcinoma of the lung. B, pattern of lungcancer TMA BCL-2
expression. C, cell viability assay by ABT-737 in HCC827, H441 and
H1975 (NSCLC), and H345 (SCLC) cells. D, forced BCL-2overexpression
in HCC827 cells desensitized the cells to erlotinib.
BCL-2-transfected versus mock-transfected cells: †, P < 0.0001,
or versus parental cells:z, P < 0.0001. Mock-transfected versus
parental cells: *, P ¼ 0.99.
BH3-Mimetic Susceptible Early TKI-Resistant Tumor Cells
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-
HCC827A
B
E
C DR
NA
i
Moc
ksi
-NT
si-B
CL-
2si
-BC
L-2/
BCL-
XL
Moc
ksi
-NT
si-B
CL-
2si
-BC
L-2/
BCL-
XL
BCL-2
BCL-XL
Actin
BCL-2
6 Days
Treatment
H1975 cells
H1975
HCC827-luc in vivo bioluminescence imaging
W.B.
– – + +
– – + +– + – +
W.B.
DAY:Groups
I
I II
II
III
III A
B
A
B
IV
IV
I
II
III
A
B
A
B
Time
IV
0 4 18 32
– + +
– + ––
+
––– +
+
++
+
+
– – – +– +–
H1975
H1975 cells
U
U
U ER
L/S
U
Oba
tocl
ax
HA
14-1
HA
14-1
+ E
RL/
SU
Oba
tocl
ax +
ER
L/S
U
AB
T-73
7 +
ER
L/S
U
AB
T-73
7
UCL-387,785 x 6D
CL-387,785 (1 µmol/L)x 9 Days
SU11274 + Erlotinibx 9 Days
SU11274 + Erlotinib
Cleaved-PARPCleaved-PARP
ActinActin
CL-387,785 (1 µmol/L)
ABT-737 (2 µmol/L) ABT-737 (2 µmol/L)Oblatoclax (2 µmol/L)
Treatment at Days 9 (x8h): Treatment at Days 9 (x8h):
Erlotinib + SU11274 x 6D
CL-3
87,7
85
ABT-7
37 +
CL-
387,
785
ABT-7
37
Erlot
inib
Oblat
oclax
HA14
-1
HA14
-1 +
Erlo
tinib
Oblat
oclax
+ E
rlotin
ib
ABT-7
37 +
Erlo
tinib
ABT-7
37
Day 7–9:
Day 1–6: Day 1–6:
Treatment
Day 7–9:
Post-Transfection RNAi
(a) (b) (c)
(d) (e) (f)
2 Days
U ERL ERL+ABT-737
ERL+si-BCL-2/BCL-XL
HCC827 Cells
Treatment
(–)
(–) Erlotinib (1 µmol/L)
Day 7−9:
Day 1−6:
ERL+si-BCL-2ERL+si-NT
In vivo treatment groups:
BCL-XL
Actin
ImageMin=-2.8587e+06Max=8.2892e+06
p/sec/cm2/sr
Color barMin=-2000
Max=2.9905e+06
0
0.5
1.0
1.5
x106
2.0
2.5
bkg subflat-fieldedcosmic
Erlotinib
ABT-737
HCC827-luc xenograft
4000000
3000000
BLI
Flu
x (R
OI)
1600000
1200000
800000
400000
00 1 2 3 4
Weeks
EGFR-TKI ± BH3-Mimetics
5 6 7 8
* P = 0.0009† P < 0.0001†† P = 0.0004
* P = 0.0009† P < 0.0001
Figure 6. BH3-mimetic therapeutic inhibition of the BCL-2/BCL-XL
programmed cell death pathway "Achilles’ heel" to eradicate "early"
TKI-resistantlung tumor survivor cells. A, siRNA-mediated knockdown
of BCL-2 and BCL-XL in HCC827 cells. WCLs at day 2 and day 6
post-siRNA transfection were thenextracted for Western blotting to
verify efficient gene knockdown of the target protein(s)
expression. B, BCL-2/BCL-XL RNAi knockdown or BH3-mimeticABT-737 (2
mmol/L) in conjunction with erlotinib (1 mmol/L) remarkably
suppressed the emergence of "early" EGFR–TKI resistant tumor-evader
cells inHCC827. Representative photomicrographs from the triplicate
experiments are shown here. Mag: 50�. C, proapoptotic BH3-mimetic
ABT-737 eradicated theH1975 early tumor prosurvival resistance
against CL-387,785. H1975 cells that were pretreated with 6 days of
CL-387,785 (1 mmol/L) were replatedat full confluence, followed by
further treatments as indicated for 3 additional days in
triplicate, either with CL- 387,785 (1 mmol/L), ABT-787 (2
mmol/L),or ABT-737þCL-387,785, followed by crystal violet cell
staining (top). U, untreated control. Bottom, induction of
proapoptotic marker cleaved-PARPby BH3-mimetic in the
CL-387,785-resistant early tumor survivor H1975 cells. D, ABT-737,
Obatoclax, and HA14-1 eradicated the H1975 early tumorprosurvival
resistance against dual-TKIs inhibition by erlotinib/SU11274
(ERL/SU). The experiment was carried out with H1975 cells similar
to (C)above, except that cells were pretreated with dual EGFR–MET
inhibitors here, i.e., erlotinib (1 mmol/L)/SU11274 (1 mmol/L).
BH3-mimetic used in treatmentdays 7–9 were all 2 mmol/L in
concentration. Top, crystal violet cell survival staining assay.
Bottom, BH3-mimetic treatment of the dual ERL/SU-resistanttumor
cells induced a proapoptotic response. E, in vivo EGFR inhibition
with erlotinib in conjunction with BH3-mimetic ABT-737 led to
significantlymore durable tumor response and prolonged remission in
HCC827-luc lung adenocarcinoma xenograft. Left, schematic outline
of treatment conditions of thein vivo HCC827-luc xenografts. Middle
top, in vitro HCC827_ERL-D9.R early resistant TKI-evader cells
emerged after 6 days of erlotinib (1 mmol/L) treatment,and were
eradicated by cotargeting BH3-mimetic inhibition using ABT-737 (2
mmol/L), Obatoclax (2 mmol/L), or HA14-1 (2 mmol/L)þ/-ongoing
erlotinibfrom days 7 to 9. Middle bottom, in vivo HCC827- luc tumor
xenograft growth, under treatment conditions as in Groups I–IV, was
monitored by BLI asdescribed in the section Materials and Methods.
Error bar, � SEM. Erlotinib-alone (III) versus ABT-737þErlotinib
(IV): *, P ¼ 0.0009. Erlotinib-alone (III) versusDiluent Control
(I), P < 0.0001 (†); ABT-737þErlotinib (IV) versus Diluent
Control (I), P < 0.0001 (†). Group II (ABT-737-alone) versus
Group IV(ABT-737þErlotinib), P ¼ 0.0004 (††). Right, BH3-mimetic
ABT-737 in vivo treatment in conjunction with EGFR-TKI (Group IV)
significantly abolishedlung tumor recurrence.
Fan et al.
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-
the BCL-2 family antiapoptotic machinery in the residualtumor
survivors under TKI(s) therapeutic inhibition(Fig. 6E).
Collectively, our results further raise the promiseof the
feasibility in "drugging" the drug-resistant residualtumor cells
(40), particularly within an early
therapeuticwindow-of-opportunity. Hence, targeting the
mitochondrialantiapoptotic machinery as the secondary "Achilles’
heel"newly-emerged in the early-resistant tumor cells appears tobe
an attractive therapeutic strategy. On the other hand,concurrent
EGFR-TKI and ABT-737 treatment also signifi-cantly suppressed the
emergence of early TKI-resistantHCC827 cells, evident as early as
within 6 to 9 days (Fig. 6)with the efficacy lasting up to 4 to 6
weeks (data not shown).We believe that the novel therapeutic
strategy in targeting theadaptive drug-evader tumor survivor cells
emergent withinthe "early treatment time-window" is attractive, as
theseevader cells are most likely more "homogeneous"
molecularlythan those found eventually as overtly resistant disease
after"chronic" TKI inhibition for months. "Late" TKI-resistanttumor
cells likely already had undergone divergent resistantmolecular
evolution in progression, hence more heteroge-neous, during the
long time lapsed under chronic TKI stress.Our data suggest that the
early tumor survival against TKI is
an "adaptive"mechanism, rather than a selection of
preexistingresistant cell clones. It remains unclear at present as
to whatdefinitively regulates and determines the cell fates early
undertargeted inhibition, and which cells among the parental
drug-sensitive cell populationwould emerge as resistant survivors
inthe beginning of the tumor evolution under therapeutic
stres-sors. Nonetheless, the contribution of intrinsic
molecularheterogeneity and nongenetic variation within individual
cellsamong the parental cell population may still play at least
apartial role in the ultimate cell-fate determination.
Interest-ingly, BCL-2 has recently been implicated as inhibitor of
DNArepairmechanism (41–44), whichmay potentially enhance
andfacilitate themolecular evolution of tumor progression beyondthe
"early" nonmutational resistance.Persistent STAT3 activation has
been detected in a variety
of hematopoietic malignancies and solid tumors (45–47).
Weobserved phosphorylated-STAT3(p-STAT3) in the residualtumor
survivor cells both in vitro and in vivo under targetedkinase
inhibitors. Our results suggest that "early" reactivationof STAT3
at tyrosine-705 (important in STAT3 dimerizationand subsequent
nuclear translocation) may be an important
central transcriptional programming event prior to the ulti-mate
resurgence of resistant tumor survivors. Recent attemptsto develop
therapeutic inhibitors to target STAT3 have provento be rather
difficult. Nonetheless, a number of BH3-mimeticthat target the key
STAT3 downstream transcriptional targets,such as BCL-2/BCL-XL, have
shown promise in preclinical andclinical studies, including
ABT-737, and the newer pan-BCL-2family inhibitors ABT-263, and
obatoclax (37, 48, 49). Thelatter pan-BCL-2 family inhibitors may
potentially be moreadvantageous over ABT-737 in their effective
inhibition ofMCL-1, shown to induce ABT-737 resistance (50).
To our knowledge, our study represents the first in vivoevidence
that therapeutic targeting early resurgent resistanttumor survivor
cells evading cancer targeted inhibitors isfeasible through
inhibiting the mitochondrial antiapoptoticBCL-2/BCL-XL signaling in
NSCLC, impacting on the thera-peutic outcome. Our results here
provide support to furtherdevelop BH3-mimetic beyond basally BCL-2
overexpressingtumors such as SCLC and lymphomas, and extend to
NSCLCas a therapeutic strategy to unleash the full potential and
tooptimize long-term clinical outcome of oncogenic
kinaseinhibitors. We propose that the combinational approach
usingBH3-mimetic and RTK inhibitors should be investigatedfurther
in the context of NSCLC human clinical trial studies.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Drs. Zhenghe Wang, Stan Gerson, and George Stark for
helpfuldiscussion and suggestions. We also thank Mr. Joseph Molter
for technicalassistance.
Grant Support
This work is supported by National Institutes of Health/National
CancerInstitute–K08 Award (5K08-CA102545–05, 3K08-CA102545–05S1;
P.C. Ma), Cle-veland Clinic Taussig Cancer Institute, Sol Siegal
Lung Cancer Research GrantProgram, Case Comprehensive Cancer Center
(Gene Expression and Genotyp-ing Core, Confocal Microscopy Core,
Xenograft and Athymic Animal Core,Tissue Procurement and Histology
Core, and Animal Imaging Core Facilities;P30-CA43703–12), and
Northern-East Ohio Small Animal Imaging ResourcesProgram
(R24-CA110943).
Received July 21, 2010; revised May 3, 2011; accepted May 3,
2011;published OnlineFirst May 9, 2011.
Table 1. Inhibition of HCC827-luc tumor in vivo xenograft
recurrence rates by BH3-mimetic ABT-737treatment in conjunction
with EGFR-inhibitor (Fig. 6E)
Treatment Groups Xenograft Recurrence Rates(%)
Day 18 Day 32
III A 50% (2/4) 50%a (4/8) 75% (3/4) 62.5%b (5/8)B 50% (2/4) 50%
(2/4)
IV A 0% (0/6) 0% (0/12) 0% (0/6) 0% (0/12)B 0% (0/6) 0%
(0/6)
aP ¼ 0.014.bP ¼ 0.004.
BH3-Mimetic Susceptible Early TKI-Resistant Tumor Cells
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-
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