Even if Viagra is not needed, it is possible that the doctor will be able to determine the etiology of erectile dysfunction and prescribe appropriate treatmen https://vgraustralia.net it doesn't pay to forget about sexual activeness even at the first sings of malfunction.
Mct-14-0013 2203.2214
Published OnlineFirst July 15, 2014; DOI: 10.1158/1535-7163.MCT-14-0013
Cancer Biology and Signal Transduction
Quinacrine Overcomes Resistance to Erlotinib by InhibitingFACT, NF-kB, and Cell-Cycle Progression in Non–Small CellLung Cancer
Josephine Kam Tai Dermawan1, Katerina Gurova2, John Pink3, Afshin Dowlati4, Sarmishtha De1,Goutham Narla5, Neelesh Sharma4, and George R. Stark1
Erlotinib is a tyrosine kinase inhibitor approved for the treatment of patients with advanced non–small cell
lung cancer (NSCLC). In these patients, erlotinib prolongs survival but its benefit remains modest becausemany tumors express wild-type (wt) EGFR or develop a second-site EGFR mutation. To test drug combinationsthat could improve the efficacy of erlotinib, we combined erlotinib with quinacrine, which inhibits the FACT(facilitates chromatin transcription) complex that is required for NF-kB transcriptional activity. In A549(wtEGFR), H1975 (EGFR-L858R/T790M), and H1993 (MET amplification) NSCLC cells, this drug combinationwas highly synergistic, as quantified by Chou–Talalay combination indices, and slowed xenograft tumorgrowth. At a sub-IC50 but more clinically attainable concentration of erlotinib, quinacrine, alone or incombination with erlotinib, significantly inhibited colony formation and induced cell-cycle arrest andapoptosis. Quinacrine decreased the level of active FACT subunit SSRP1 and suppressed NF-kB–dependentluciferase activity. Knockdown of SSRP1 decreased cell growth and sensitized cells to erlotinib. Moreover,transcriptomic profiling showed that quinacrine or combination treatment significantly affected cell-cycle–related genes that contain binding sites for transcription factors that regulate SSRP1 target genes. As potentialbiomarkers of drug combination efficacy, we identified genes that were more strongly suppressed by thecombination than by single treatment, and whose increased expression predicted poorer survival in patientswith lung adenocarcinoma. This preclinical study shows that quinacrine overcomes erlotinib resistance byinhibiting FACT and cell-cycle progression, and supports a clinical trial testing erlotinib alone versus thiscombination in advanced NSCLC. Mol Cancer Ther; 13(9); 2203–14. 2014 AACR.
a better understanding of the pathogenesis of NSCLC,
Metastatic non–small cell lung cancer (NSCLC) is the
coupled with high-throughput genomic technologies
most common cause of cancer death in the United States.
applied to patient tumor samples, has led to a molecular
Cytotoxic chemotherapy has historically been the main-
classification of NSCLC (and a new generation of "preci-
stay of therapy but is associated with only modest
sion" therapies). This paradigm is best illustrated by the
improvements in patient survival. Over the past decade,
identification of activating mutations in EGFR as driversof lung cancer development and progression and thesubsequent demonstration of the clinical benefit of anti-
1Department of Molecular Genetics, Cleveland Clinic Lerner Research
EGFR therapies such as erlotinib (Tarceva), a reversible
Institute, Cleveland, Ohio. 2Department of Cell Stress Biology, Roswell
tyrosine kinase inhibitor (TKI) of EGFR (1). On the other
Park Cancer Institute, Buffalo, New York. 3Division of General Medical
hand, the clinical benefit of erlotinib is modest in patients
Sciences-Oncology, Case Comprehensive Cancer Center, Cleveland,Ohio. 4Case Comprehensive Cancer Center, University Hospitals Seidman
with wild-type (wt) EGFR, particularly in those with
Cancer Center, Case Western Reserve University, Cleveland, Ohio.
5
concurrent KRAS mutations (2, 3); in addition, even in
Department of Medicine, Institute for Transformative Molecular Medicine,
Case Western Reserve University, Cleveland, Ohio.
the initially sensitive EGFR mutation–positive patients,population resistance invariably develops through the
Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).
development of second-site EGFR mutations, for example,T790M (4), activation of alternative receptor tyrosine
Corresponding Authors: George R. Stark, Department of MolecularGenetics, Cleveland Clinic Lerner Research Institute/NE20, 9500 Euclid
kinases, for example, MET amplification (5), and other
Avenue, Cleveland, OH 44195.Phone: 216-444-6062; Fax: 216-444-0512;
mechanisms including transformation from non–small
E-mail:
[email protected]; and Neelesh Sharma, Division of Hematology/
cell to small cell histology (6).
Oncology, University Hospitals Case Medical Center, 11100 Euclid Ave-nue, Cleveland, OH 44106. Phone: 216-844-0363; Fax: 216-844-5234; E-
Quinacrine was widely used during World War II as an
antimalarial agent. Over the last four decades, it has been
used for the treatment of giardiasis, tapeworm infesta-
2014 American Association for Cancer Research.
tions, and connective tissue diseases, for example, lupus
Published OnlineFirst July 15, 2014; DOI: 10.1158/1535-7163.MCT-14-0013
Dermawan et al.
erythematosus and rheumatoid arthritis (7, 8). Recently, a
resuscitation from frozen stocks, and were maintained in
chemical screen identified 9-aminoacridines, including
DMEM (A549 and H1975) or RPMI-1640 (H1993) medium
quinacrine, as activators of p53 and inhibitors of NF-kB
supplemented with 5% FBS. All cells were kept at 37C in a
(9, 10). NF-kB regulates the expression of genes encoding
humidified atmosphere with 5% CO2. A549 has wtEGFR
proinflammatory and antiapoptotic proteins. In contrast
and mutant KRAS (G61H), H1975 has the activating
with the situation in normal cells, it is usually constitu-
EGFRL858R mutation as well as the second site EGFRT790M
tively active in tumor cells and plays a key role in
mutation, which decreases the affinity of the receptor for
promoting tumorigenesis, including resistance to many
erlotinib, and H1993 has wtEGFR and MET amplification.
cancer therapies (11–13). Indeed, a recent report showedthat inhibition of NF-kB sensitizes NSCLC cells to erloti-
Cell proliferation
nib-induced cell death (14). Thus, NF-kB is an attractive
Cells were seeded in 96-well plates at 1 to 2 103 per
target for cancer therapy (12, 15).
well, allowed to attach overnight, and treated with var-
Quinacrine is thought to act by intercalating into DNA
ious concentrations of erlotinib, quinacrine, or a combi-
through its planar acridine ring, whereas its diaminobutyl
nation of both in a 5:1 or 10:1 molar ratio. After 72 hours,
side chain extends into the DNA minor groove (8). Recent-
cell viability was determined by the MTT assay (17). The
ly, it was reported that quinacrine and its derivatives
combination index (CI) was assessed by using CalcuSyn
suppress NF-kB by causing chromatin trapping of the
software (Biosoft; refs. 18, 19).
FACT (facilitates chromatin transcription) complex (10), aheterodimer of the structure-specific recognition protein
(SSRP1) and suppressor of Ty 16 (SPT16). The normal
Cells were seeded in 6-well plates at 500 per well,
function of FACT is to promote reorganization of nucleo-
allowed to attach overnight, and treated with erlotinib,
somes in front of RNA polymerase II during transcription
quinacrine, or the combination in triplicate. Drugs were
elongation. However, FACT is often expressed in aggres-
replaced every 72 hours. After 14 days, cells were fixed
sive, undifferentiated cancers, and neoplastic (but not
with 100% methanol and stained with 1% crystal violet.
normal) cell growth depends on FACT activity (16). Chro-
Colonies were quantified using the cell counter plugin of
matin trapping of FACT results in increased phosphory-
the NIH ImageJ software (v.1.48).
lation of p53 by the FACT-associated kinase CK2, andreduced NF-kB–dependent transcription because of the
Cell-cycle analysis
depletion of free active FACT (10).
Cells were treated with 1 mmol/L erlotinib, 3 or 5 mmol/L
To improve the clinical benefit of erlotinib in the treat-
quinacrine, or a combination of both for 96 or 120 hours,
ment of advanced NSCLC, we investigated whether com-
and then fixed with 100% cold ethanol at 20C for 1 hour,
bination with quinacrine potentiates the ability of erloti-
and stained with 3 mmol/L propidium iodide (PI; Invitro-
nib to mediate cell death, and the mechanism underlying
gen, #P3566) in the presence of RNase for 15 minutes at
the observed synergistic effect in NSCLC cells. As a result
room temperature. Cell-cycle distribution was assessed
of our findings, we are conducting a phase I/II clinical
by FACScan (BD Biosciences) analysis.
trial to test the combination of erlotinib and quinacrine inadvanced or metastatic (stage IIIB/IV) NSCLC patients
Analysis of apoptosis
who have failed at least one prior platinum-based che-
Staining was performed using Annexin V-APC
motherapy regimen (NCT01839955).
(eBioscience, #88–8007) in conjunction with PI accordingto the manufacturer's protocol, and was assessed by
Materials and Methods
FACScan. Apoptosis was validated by PARP cleavage
and analyzed by the Western method.
Erlotinib was obtained from Selleck Chemicals (# S1023)
and dissolved in DMSO. Quinacrine, from Sigma-Aldrich
NF-kB luciferase assay
(# Q3251), was dissolved in PBS as a 10 mmol/L stock
A549 or H1975 cells were infected with the kB-luciferase
solution. Dilutions to the required concentrations were
lentiviral construct pLA-NFkB-mCMV-luc-H4-puro (or
made in DMEM or RPMI-1640 medium. Mouse monoclo-
hygro) and stably selected with puromycin or hygromy-
nal SSRP1 antibody (# 609701) was from BioLegend.
cin. This NF-kB reporter lentiviral vector consists of a
Rabbit polyclonal PARP antibody (# 9542) was from Cell
firefly luciferase reporter gene under the control of a
Signaling Technology. Mouse monoclonal b-actin anti-
minimal (m)CMV promoter and six NF-kB–responsive
body (# A5316) was from Sigma. Goat polyclonal Lamin
elements from the immunoglobulin light chain gene
B (# sc-6216) and mouse monoclonal GAPDH antibody
(ref. 20; kind gift from Dr. Peter Chumakov, Russian
(# sc-32233) were from Santa Cruz Biotechnology.
Academy of Sciences, Moscow, Russia). The reporter cellswere then seeded in 96-well plates at 1 to 2 103 per well,
allowed to attach overnight, and then treated with drugs
The human non–small cell lung adenocarcinoma cell
and/or IL1. Cells were then harvested in reporter lysis
lines A549, H1975, and H1993 were obtained from ATCC
buffer (Promega) and assayed for luciferase activity using
and passaged for less than 6 months following receipt or
the luciferase assay system (Promega).
Mol Cancer Ther; 13(9) September 2014
Molecular Cancer Therapeutics
Published OnlineFirst July 15, 2014; DOI: 10.1158/1535-7163.MCT-14-0013
Combination of Quinacrine and Erlotinib in NSCLC
DNA-binding assay
normalization, and log2 transformation). Probes were
The ability of compounds to alter the mobility of plasmid
annotated using the HuGene 2.1 st hg19 probeset anno-
DNA was tested by incubating plasmid DNA in Tris-EDTA
tation files downloaded from the Affymetrix website. Low
buffer (pH 8.0) with 10 mmol/L quinacrine or chloroquine
intensity probes (probes whose log2 expression levels in
at room temperature for 20 minutes followed by electro-
the untreated sample were less than the median expres-
phoresis (1% agarose gel, 1.5 V/cm constant for 16 hours).
sion level across all probes) were filtered out. Hierarchical
Gels were stained with ethidium bromide (0.5 mg/mL) and
clustering (average linkage method with Euclidean dis-
visualized with short-wavelength UV light.
tance metrics) and principal component analysis wasperformed using Cluster 3.0 and visualized with the Java
shRNA-mediated knockdown
TreeView or JMP 10 software (SAS Institute). Differential
Lentiviral plasmids encoding shRNAs targeting GFP or
gene expression analysis among treatment groups was
SSRP1 (TRCN0000019270, "#2"; TRCN0000019272, "#4")
performed using Bayesian Analysis of Variance for Micro-
were from Sigma-Aldrich. Viruses were packaged in HEK
arrays (BAMarray) 3.0 (21), and the resulting gene lists
293T cells using the second-generation packaging con-
were further narrowed down using STEM v. 1.3.8 (Short
structs pCMV-dR8.74 and pMD2G (a kind gift from
Time-series Expression Miner; ref. 22) into genes whose
Dr. Mark Jackson, Case Western Reserve University, Cle-
expression showed more than 2-fold changes compared
veland, OH). Supernatant media containing virus were
with 0 hour and significant temporal profiles. DAVID v6.7
collected after 48 hours and supplemented with 1 mg/mL
(Database for Annotation, Visualization and Integrated
polybrene before being used to infect cells for 6 hours.
Discovery) was used to analyze gene ontology processes
Knockdown efficiency was evaluated by the Western
for genes that were significantly affected by erlotinib-
method 48 hours after infection.
quinacrine combination treatment (23). Differentially reg-ulated genes were analyzed for overrepresented tran-
Protein extraction and Western analysis
scription factor binding sites (TFBS) compared with the
Soluble protein fractions were prepared by incubat-
background gene set using oPOSSUM 3.0. A z-score (rate
ing cell pellets with occasional vortexing in lysis buffer
of occurrence of a TFBS in target gene set vs. background
containing 50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl,
set) greater than mean þ SD and a fisher score (proportion
1.0% NP-40 with protease inhibitors and then centri-
of genes in target gene set containing a TFBS vs. that in
fuged at 20,000 g for 10 minutes, discarding the crude
background set) greater than 75% percentile were used as
nuclear pellet. Chromatin fractions were extracted
the cutoff to determine significant overrepresentation of
according to Gasparian and colleagues (10). Briefly,
TFBS (24). The Kaplan–Meier plotter (cancer survival
after removal of soluble cytoplasmic fraction, chroma-
analysis; www.kmplot.com) was used to assess the effect
tin-bound proteins from the insoluble nuclear pellets
of gene expression on lung cancer survival by download-
were extracted with using a high salt lysis buffer con-
ing the Kaplan–Meier curves, HRs, and log-rank P values
taining 2 mol/L NaCl followed by sonication (3 15
of gene expression and survival data with relevant Affy-
seconds, 30 seconds off). Cell extracts containing equal
metrix probe IDs (25).
quantities of proteins, determined by the Bradfordmethod, were separated by 10% SDS-PAGE and trans-
Real time reverse transcription-PCR analysis
ferred to polyvinylidene difluoride membranes (Milli-
Archive cDNA was prepared using the ABI High-
pore). Primary antibodies were detected with goat anti-
Capacity cDNA Archive Kit (Applied Biosystems, Inc.,
mouse or goat anti-rabbit conjugated to horseradish
ABI) using 1 mg total RNA for each sample as starting
peroxidase (Rockland), using enhanced chemilumines-
material in a 100 mL reverse transcription reaction in an
cence (PerkinElmer). Densitometry quantification of
ABI 9700 Sequence Detection System. Of note, 384-well
immunoblot analyses was performed using the NIH-
plates were set up to accommodate triplicate reactions
ImageJ software (v. 1.48).
for all assays. An endogenous control assay was used tocontrol for RNA loading and to produce the normalized
Total RNA extraction and microarray analysis
signal. TaqMan assays for genes of interest (selected
RNA was isolated from cells treated with 1 mmol/L
genes suppressed significantly by combination treatment
erlotinib, 3 or 5 mmol/L quinacrine or a combination of
from the microarray analysis) were purchased from ABI.
both for 6, 12, 24, or 48 hours using the RNeasy Mini Kit
Spectral data, gathered during the PCR run, were con-
(QIAGEN) according to the manufacturer's instructions
verted into numerical data using ABI SDS (sequence
(48-hour treatment samples were available only for erlo-
detection system) 2.3 proprietary software. All real-time
tinib or combination treatment). Microarray analysis was
reverse transcriptase PCR (RT-PCR) reactions were per-
performed using the Affymetrix Human Gene 2.1 ST
formed at the Gene Expression Array Core Facility of the
Array at the Gene Expression & Genotyping Core Facility
Case Comprehensive Cancer Center. Relative quantifica-
at Case Comprehensive Cancer Center. Raw CEL files
tion of gene expression changes was calculated by the 2-
were preprocessed using the Affymetrix Expression Con-
DDCt method, where DCt value ¼ [Ct (gene of interest)
sole Software 1.30 with Robust Multi-array Average
Ct (Endogenous Control)], and DDCt ¼ [DCt (treated) DCt
(RMA) normalization (background correction, quantile
(untreated at 0 hour)].
Mol Cancer Ther; 13(9) September 2014
Published OnlineFirst July 15, 2014; DOI: 10.1158/1535-7163.MCT-14-0013
Dermawan et al.
Tumorigenicity assay
acrine in each cell line, which are between 5 mmol/L and
NCr nu/nu athymic nude mice were obtained from
12 mmol/L and 1 to 2 mmol/L, respectively. On the basis of
Taconic. Studies were conducted under an approved
their IC50 ratios, we combined erlotinib with quinacrine
Institutional Animal Care and Use Committee protocol
at a 5 to 1 or 10 to 1 ratio and measured cell viability
by the Case Comprehensive Cancer Center Athymic Ani-
after treatment. We then quantified the degrees of syn-
mal and Xenograft Core. A549 cells were suspended at a
ergism using the median-drug effect analysis method
density of 2 106 cells in 100 mL DMEM medium contain-
developed by Chou and Talalay (18, 19). This method
ing 5% FBS. Cell suspensions were subcutaneously
quantifies the CIs of two drugs based on the growth
injected into the rear flanks bilaterally of 6-week-old male
inhibition curves of each drug alone or their combina-
mice (n ¼ 5, 10 tumors per group). Tumor volume (mm3)
tion (Fig. 1A). The combination of erlotinib and quin-
was calculated with the formula 0.525 W2 L, where W
acrine was synergistic in A549, H1975, and H1993 cells
and L were the smallest and largest diameters of the tumor
when combined at both 5:1 or 10:1 ratios [effective dose
in mm, measured every other day. Tumors were grown to
(ED)50: 0.61 (0.42–0.81); ED75: 0.53 (0.40–0.67); ED90:
at least 200 mm3 before start of treatment. Tumors that
0.63 (0.54–0.71); Fig. 1B].
failed to engraft (reach double digit diameter) were
To determine the in vivo effect of this drug combi-
excluded from the study. Thereafter, mice received daily
nation, we measured tumor growth in an A549 xeno-
oral gavage of vehicle control (0.5% w/v methyl cellu-
graft model treated with oral gavage of vehicle (0.5%
lose), erlotinib (30 mg/kg/d), quinacrine (100 mg/kg
methyl cellulose), erlotinib (30 mg/kg/d), and quinacrine
loading dose at day one followed by 50 mg/kg/d),
(50 mg/kg/d with a 2-fold initial loading dose), or a
or combination of erlotinib (30 mg/kg/d) plus quina-
combination of erlotinib (30 mg/kg/d) plus quinacrine
crine (100 mg/kg initial dose followed by 50 mg/kg/d).
(50 mg/kg/d with a 2-fold initial loading dose). The
Mice were sacrificed when tumors reached 17 mm in
combination significantly inhibited in vivo tumor growth
compared with vehicle control or single drug adminis-tration of quinacrine (Fig. 1C).
Statistical analysis
All statistical analyses (except microarray data) were
The combination of quinacrine with erlotinib induced
conducted using GraphPad Prism 5. Results are repre-
apoptosis and cell-cycle arrest
sented by mean SD. Statistical significance was assumed
We treated A549 or H1975 cells with 1 mmol/L
for a two-tailed P value less than 0.05 using ANOVA with
erlotinib and either 5 or 3 mmol/L quinacrine in most
the Bonferroni or Dunnett posthoc test, compared with
of the subsequent experiments. The sub-IC50 concen-
untreated controls or nontargeted shRNA.
tration of erlotinib was chosen because at the standarddosage of erlotinib (150 mg/d) used in the clinical
setting, the maximum concentration of erlotinib (Cmax)
Microarray data in the form of raw CEL and RMA
achievable in humans is much lower than the IC50
normalized matrix files were deposited on the NCBI Gene
of erlotinib in these resistant cell lines (26, 27). On the
Expression Omnibus database under the accession num-
other hand, the Cmax of quinacrine reaches 3 to 5 mmol/L
ber GSE57422.
in patients (unpublished data), and quinacrine isknown to accumulate at high concentrations in tissues
(especially in liver and lung) with a volume of distri-
The combination of erlotinib and quinacrine is
bution of approximately 50,000 L (7), and thus these
synergistic in several NSCLC lung adenocarcinoma
concentrations were chosen because they could be
cell lines and inhibits in vivo NSCLC tumor cell
achieved in vivo.
Because the combination shows synergy when erlotinib
Constitutive NF-kB activation is known to mediate
and quinacrine are used at their IC50 ratios (erlotinib:
survival and drug resistance in cancer, and its inhibition
quinacrine ¼ 5:1 or 10:1), at a concentration of 1 mmol/L
has been reported to increase sensitivity to cancer thera-
erlotinib and a concentration of 3 to 5 mmol/L quinacrine
pies including EGFR-TKIs (12, 14). To test whether inhi-
in our erlotinib-resistant cell lines, the two drugs were no
bition of NF-kB is synergistic with erlotinib, a major
longer synergistic and quinacrine showed potent single-
EGFR-TKI used in NSCLC treatment, we tested the effects
agent activity in this combination. Quinacrine alone or
of the combination of erlotinib and quinacrine, an NF-kB
addition of quinacrine to erlotinib induced similar levels
inhibitor, on cell viability in three NSCLC cell lines: A549
of cell death, as demonstrated by increased Annexin V-PI
(wtEGFR, mutant KRAS), H1975 (EGFRL858R/T790M), and
staining in A549 and H1975 cells after 48 hours of treat-
H1993 (MET amplification). Each of these cell lines harbor
ment (Fig. 2A), and when we increased erlotinib to
genetic aberrations that represent three major mechan-
5 mmol/L (which is still below its IC50), combination
isms driving resistance to anti-EGFR therapy in advanced
treatment induced higher levels of cell death than quin-
acrine alone (Fig. 2A). We further confirmed this result by
We first determined the individual IC50 values (half
observing a time-dependent increase in PARP cleavage
maximal inhibitory concentrations) for erlotinib and quin-
induced by quinacrine (Fig. 2B). Quinacrine alone or
Mol Cancer Ther; 13(9) September 2014
Molecular Cancer Therapeutics
Published OnlineFirst July 15, 2014; DOI: 10.1158/1535-7163.MCT-14-0013
Combination of Quinacrine and Erlotinib in NSCLC
Figure 1. The combination of erlotinib and quinacrine is synergistic in several NSCLC lung adenocarcinoma cell lines and inhibits in vivo xenograft tumorgrowth. A, A549 (wtEGFR), H1975 (EGFR-L858R/T790M), and H1993 (MET amplification) cells were treated with erlotinib, quinacrine, or the combination ofboth agents at a 5:1 or 10:1 molar ratio. After 72 hours, cell viability was determined by the MTT assay (effect% ¼ 100% cell viability%). The experimentswere repeated three times. B, the CI was assessed using CalcuSyn software to determine drug interaction (additivity, synergism). A CI < 1.0 isconsidered to be synergistic. All values represent mean SD. C, growth curves of A549 lung adenocarcinoma xenograft in NCr nu/nu athymic mice. Followingan initial growth period of 35 days, group tumor volume reached at least 200 mm3 before treatment (P ¼ 0.5273 between groups). Tumor diameterswere measured every other day. Treatment continued for at least 20 days before the mice were sacrificed; , significance of P < 0.001 compared with vehiclecontrol or quinacrine only treatment (ANOVA followed by Dunnett multiple comparison test).
addition of quinacrine to erlotinib treatment also signif-
phosphorylation in cells treated with either erlotinib or
icantly inhibited in vitro cell proliferation, as measured by
quinacrine, finding that only erlotinib inhibited AKT or
colony formation (Fig. 2C).
ERK activation (data not shown). This result suggests that
Next, we measured the effect of quinacrine plus erlo-
the effect of quinacrine on cell survival is mediated
tinib on cell-cycle progression, using flow cytometry. Both
through a pathway other than the PI3K/AKT or MAPK
quinacrine alone and combination treatment induced
similar levels of marked G1–S and G2–M cell-cycle arrest
A recent report suggested that chloroquine can over-
in A549 and H1975 cells. This effect was dominated
come erlotinib resistance in NSCLC cells overexpressing
by the action of quinacrine when the low concentration
wtEGFR by inhibiting autophagy (29). Both chloroquine
of 1 mmol/L erlotinib was used (Fig. 3A–D).
and quinacrine are known to inhibit autophagy (30), butwhether both of these antimalarial drugs inhibit NF-kB
Quinacrine, but not chloroquine, suppresses NF-kB–
remains uncertain. To address this issue, we determined
driven luciferase activity
whether quinacrine or chloroquine inhibits NF-kB activity
Next, we analyzed how quinacrine overcomes erlotinib
in A549 or H1975, utilizing an NF-kB-luciferase reporter
resistance in NSCLC cells. Because the PI3K/AKT and
assay. Luciferase expression driven by either constitutive-
MAPK pathways are known to be important for cell
ly active NF-kB (Fig. 4A) or IL1 treatment (Fig. 4B) was
survival and are determinants of EGFR-TKI sensitivity
significantly suppressed by quinacrine but not by equal
in EGFR-driven cancers (28), we analyzed AKT and ERK
concentrations of chloroquine.
Mol Cancer Ther; 13(9) September 2014
Published OnlineFirst July 15, 2014; DOI: 10.1158/1535-7163.MCT-14-0013
Dermawan et al.
Figure 2. The combination of quinacrine with erlotinib induced in vitro NSCLC cell apoptosis and inhibited cell growth. A, A549 and H1975 cells were untreatedor treated with 1 mmol/L (left) or 5 mmol/L (right) erlotinib, 5 mmol/L quinacrine, or both for 48 hours. Apoptosis was assessed by Annexin V-PI double stainingfollowed by flow-cytometric analysis. B, H1975 cells were treated with 5 mmol/L quinacrine for the indicated time-points. Western analysis was used todetect PARP cleavage as an indicator of apoptosis. C, A549 cells were seeded in 6-well plates and treated with erlotinib, quinacrine, or a combination of both intriplicate at the indicated concentrations. Drugs were replaced every 72 hours. After 14 days, colonies were stained and quantified. Statistical analysis of thedifferences in colony formation between the treated cells and untreated controls were conducted using one-way ANOVA followed by Bonferroni multiplecomparison test (, P < 0.05; , P < 0.01; , P < 0.001). All values represent mean SD.
Quinacrine mediates cell killing and overcomes
as shown by the ability of quinacrine, but not chloroquine,
resistance to erlotinib by targeting FACT
to reduce mobility of plasmid DNA (Supplementary
Gasparian and colleagues showed that a series of anti-
Fig. S1C). To test whether the anticancer activity of quin-
cancer compounds including quinacrine suppresses NF-
acrine in NSCLC is due to inhibition of FACT, we knocked
kB activation by causing chromatin trapping of the FACT
SSRP1 down in A549 or H1975 cells. Loss of SSRP1
complex (10). This finding is supported by our observa-
significantly decreased cell survival (Fig. 4E and F) and
tion that treatment of A549 or H1975 cells with quinacrine,
increased sensitivity to erlotinib (Fig. 4G and H) in both
but not chloroquine, rapidly depletes SSRP1, a FACT
subunit, from the soluble cytoplasmic fraction (Fig. 4Cand D), and led to SSRP1 accumulation in the insoluble
The quinacrine and erlotinib combination inhibits
chromatin fraction (Fig. 4D), which has also been shown
the expression of SSRP1-regulated genes and cell-
by Gasparian and colleagues to be an indicator of chro-
cycle genes that predict worse survival in patients
matin trapping of FACT (10). Because the overall level of
with lung adenocarcinoma
SSRP1 from whole-cell lysates remained unchanged (Sup-
To further elucidate the mechanisms of the effect of
plementary Fig. S1A), the decrease of SSRP1 from the
combined quinacrine and erlotinib treatment in NSCLC,
cytoplasmic fraction was not due to protein degradation.
we performed global transcriptomic profiling of A549 and
Quinacrine and chloroquine are structurally related com-
H1975 cells treated with 1 mmol/L erlotinib alone, 5 or
pounds known to interact with DNA, but with different
3 mmol/L quinacrine alone, or combinations of 1 mmol/L
affinities, due to the stronger drug-DNA ring–ring stack-
erlotinib and 5 or 3 mmol/L quinacrine for 0, 6, 12,
ing interaction with quinacrine, which has a 3-ring acri-
or 24 hours, using an Affymetrix microarray platform.
dine moiety, compared with chloroquine, which has a 2-
(48-hour treatment samples were available for erlotinib
ring quinolone moiety (Supplementary Fig. S1B; ref. 31),
alone or combination treatment). Principal component
Mol Cancer Ther; 13(9) September 2014
Molecular Cancer Therapeutics
Published OnlineFirst July 15, 2014; DOI: 10.1158/1535-7163.MCT-14-0013
Combination of Quinacrine and Erlotinib in NSCLC
Figure 3. The combination of quinacrine and erlotinib inhibits G1–S and G2–M cell-cycle progression. A549 (A and C) and H1975 (B and D) cells were untreatedor treated with erlotinib only, quinacrine only, or erlotinib plus quinacrine for 72 or 96 hours at the indicated concentrations. Cell-cycle analysis was thenperformed using PI-staining followed by flow-cytometric analysis. G1–S and G2–M cell-cycle arrest was determined by quantifying relative G0–G1, S,and G2–M phase percentages. Statistical analysis of the differences in relative cell-cycle phase percentages between the treated cells and untreated controlswere conducted using two-way ANOVA followed by Dunnett multiple comparison test (, P < 0.01; , P < 0.001). The experiment was repeated three times.
All values represent mean SD.
analysis revealed that, relative to untreated cells at 0 hour,
ing our functional analysis showing that quinacrine plus
gene expression profiles diverged most significantly with
erlotinib induced significant cell-cycle arrest and inhib-
increased treatment time (24 and 48 hours) and with
ited tumor growth.
quinacrine or combination treatment (Supplementary
Next, we analyzed the enrichment of TFBSs among
Fig. S2). We next determined genes that were differen-
those genes whose expression was significantly affected
tially expressed between treatment groups. Differential
by quinacrine or by erlotinib plus quinacrine. Comparison
gene expression analysis was used to identify genes that
of our data with the ChIP-SEQ results for SSRP1-enriched
were significantly induced or suppressed by combination
genes reported by Garcia and colleagues (16) showed that
treatment compared with either single drug treatment
many of the genes affected by quinacrine or combination
and whose expression levels showed a >2-fold change
treatment were regulated by the same transcription fac-
relative to 0 hour and significant temporal profiles (Fig. 5A
tors that were also involved in regulating expression of
and B). Gene ontology analysis showed that genes signif-
SSRP1-enriched genes. These transcription factors belong
icantly affected by the combination were most highly
to the EGR (EGR1), ETS (ELK1, ELK4, GABPA, SPI1),
enriched for those encoding proteins involved in cell-
MYC (MYC, MYCN), and SP/KLF (SP1, KLF4) families
cycle progression or DNA metabolism (Table 1), confirm-
(Supplementary Table S1). This result supports our
Mol Cancer Ther; 13(9) September 2014
Published OnlineFirst July 15, 2014; DOI: 10.1158/1535-7163.MCT-14-0013
Dermawan et al.
Figure 4. Quinacrine, but not chloroquine, suppresses NF-kB–driven luciferase activity, mediates cell killing, and overcomes resistance to erlotinib by targetingFACT. A, luciferase units relative to untreated control (RLU) were quantified in A549 or H1975 cells stably expressing NF-kB luciferase reporter after 4 hours oftreatment with increasing concentrations of quinacrine or chloroquine in triplicate. Statistical analysis of the differences in RLU between cells treated withdifferent drug concentrations and untreated controls were conducted using two-way ANOVA followed by Dunnett multiple comparison test (, P < 0.001).
B, RLU was quantified in A549 or H1975 stable NF-kB luciferase reporter cells pretreated with 10 mmol/L quinacrine or 10 mmol/L chloroquine for 1hour, andthen stimulated with 10 ng/mL IL1b for 6 hours in quadruplicate. Statistical analysis of the differences in RLU between IL1-treated or -untreated cellswere conducted using two-way ANOVA followed by Bonferroni posttest (, P < 0.001). All values represent mean SD. C, A549 or H1975 cells weretreated with quinacrine or chloroquine for 1 hour at the indicated concentrations. Soluble protein fractions were then extracted by mild cell lysis and theSSRP1 subunit of the FACT complex was analyzed by the Western method. D, A549 or H1975 cells were treated with 20 mmol/L quinacrine for 3 hours. Thelevels of SSRP1 in the cytoplasmic and chromatin fractions were analyzed by the Western method and quantified by densitometry. b-actin or lamin B served asloading controls. E, H1975 cells were transduced with shRNA lentiviruses against GFP or SSRP1. Cells were plated in quadruplicate and cell viabilitywas measured by MTT assay 5 days after infection. F, A549 cells were infected with shRNA against GFP or SSRP1. Cells were plated in 6-well plates intriplicate and cell colonies were quantified after 2 weeks by crystal violet staining. H1975 (G) or A549 (H) cells were transiently infected with shSSRP1 andplated in 96-well plates and treated with DMSO or increasing concentrations of erlotinib over 72 hours in quadruplicate. Cell viability was measured by MTTassay. Statistical analyses of the differences in cell viability or colony formation between SSRP1 or GFP knockdown cells were conducted using one-way ortwo-way ANOVA followed by Dunnett multiple comparison test (, P < 0.01; , P < 0.001). All values represent mean SD.
Mol Cancer Ther; 13(9) September 2014
Molecular Cancer Therapeutics
Published OnlineFirst July 15, 2014; DOI: 10.1158/1535-7163.MCT-14-0013
Combination of Quinacrine and Erlotinib in NSCLC
Figure 5. Quinacrine and erlotinib combination treatment inhibits SSRP1-regulated genes and cell-cycle genes whose increased expression predicts poorersurvival in patients with lung adenocarcinoma. A549 (top) or H1975 (bottom) cells were treated with or without erlotinib, quinacrine, or combination for 6, 12, or24 hours. Differential gene expression analysis was used to identify genes that were significantly induced or suppressed by combination treatment comparedwith single drug treatment and showed a >2-fold change relative to 0 hour and significant temporal profiles, as represented by Venn diagrams (A) andhierarchical clustering (B). C, as potential biomarkers of drug combination efficacy in the ongoing clinical trial, cell-cycle–related genes that showed significanttemporal suppression by combination treatment compared with either single treatment alone in A549 cells were identified and represented in a heatmap. D,examples of the Kaplan–Meier survival curves of patients with lung adenocarcinoma from Supplementary Table S2 with high versus low KIFC1,
FOSL1, BIRC5, or HIST1H2BM expression.
observation that quinacrine targets and inhibits the FACT
in patients with NSCLC (HR ranges from 1.19 to 1.98),
complex (16). Interestingly, the levels of the FACT subunit
and this correlation was even more significant when
SSRP1 and SPT16 mRNAs were not affected by drug
only patients with lung adenocarcinoma were analyzed
treatment in our microarray study (data not shown),
(HR ranges from 1.58 to 2.92; Fig. 5D and Supplemen-
which corroborates previous reports showing that the
tary Table S2). This result is relevant to an ongoing
action of quinacrine on FACT is at a functional level, by
phase I/II clinical trial (NCT01839955) to test the com-
trapping the FACT complex onto chromatin.
bination of erlotinib and quinacrine in metastatic (stage
To identify potential biomarkers for erlotinib-quina-
IIIB-IV) NSCLC patients who failed first-line chemo-
crine synergy, we identified genes that were suppressed
therapy, the vast majority of whom have wtEGFR non–
more significantly by combination treatment than by
small cell lung adenocarcinoma tumors. Therefore, our
either drug alone in A549 lung adenocarcinoma cells
preclinical studies for this clinical trial not only identify
(Fig. 5C). The more potent suppression of this gene set
a potential set of treatment response pharmacodynamic
by combination treatment was verified by TaqMan-
biomarkers, but also suggest important biologic
based qRT-PCR analysis (Supplementary Fig. S3).
mechanisms regulating the potent single-agent activity
Because our functional analysis showed that the com-
of quinacrine activity in erlotinib-insensitive NSCLC
bination of erlotinib and quinacrine induced significant
cell-cycle arrest, we preferentially selected genes thatour gene ontology analysis showed to be involved in
cell-cycle progression. Importantly, increased expres-
Erlotinib is effective in patients with NSCLC with
sion of these genes was associated with poorer survival
known drug-sensitizing EGFR mutations (3), but its
Mol Cancer Ther; 13(9) September 2014
Published OnlineFirst July 15, 2014; DOI: 10.1158/1535-7163.MCT-14-0013
Dermawan et al.
Table 1. Gene ontology processes for genes that are significantly affected by erlotinib-quinacrinecombination treatment
Cell-cycle process
M phase of mitotic cell cycle
Mitotic cell cycle
Organelle fission
DNA metabolic process
Response to DNA damage stimulus
Cellular response to stress
Chromosome segregation
Negative regulation of macromolecule biosynthetic process
Notch signaling pathway
Negative regulation of macromolecule metabolic process
Negative regulation of transcription
Regulation of RNA metabolic process
Pyrimidine deoxyribonucleotide metabolic process
Negative regulation of gene expression
Regulation of DNA metabolic process
clinical efficacy in patients with wtEGFR or acquired
and treatment of malaria and other parasitic diseases, are
resistance to TKIs due to secondary mutations remains
well tolerated (7, 8). A future experiment looking at
modest (3, 6). We show here that the addition of quina-
whether overexpression of FACT itself could overcome
crine to erlotinib in several patient-derived erlotinib-resis-
resistance to erlotinib or other EGFR-TKI in NSCLC
tant NSCLC cell lines overcomes resistance to erlotinib. A
would be valuable.
major advantage of our strategy is the pairing of a highly
We are currently conducting a phase I/II clinical trial
specific small-molecule kinase inhibitor, erlotinib, to a
(NCT01839955) to test erlotinib alone versus erlotinib plus
broadly acting DNA intercalator, quinacrine, thereby
quinacrine in patients with locally advanced or metastatic
decreasing the chance of emergence of resistance against
(stage IIIB/IV) NSCLC with either: (i) wtEGFR, with
targeted therapies.
disease progression after previous platinum-based che-
Quinacrine has been shown to reduce the availability of
motherapy, for which erlotinib was approved as a second-
the FACT complex by causing its trapping on chromatin
line monotherapy; or (ii) documented EGFRL858R/T790M
(10), and FACT was shown by the same group to promote
mutation or EML4–ALK fusion gene, with subsequent
tumor survival and growth (16). Because the ability of
progression on first-line erlotinib or crizotinib and che-
quinacrine to inhibit FACT and subsequently modulate
motherapy. Using the Chou–Talalay method, we deter-
NF-kB–dependent transcriptional activity is not depen-
mined the two drugs to be synergistic at their respective
dent on direct binding to these targets but is mediated by
IC50 ratios in erlotinib-resistant cell lines, when erlotinib
binding to DNA (10), it may be less likely for drug
was used at a much higher concentration (5:1 or 10:1) than
resistance to arise. Even though quinacrine intercalates
quinacrine. However, based on preexisting pharmacoki-
into DNA, it is not genotoxic (10), and its side effects and
netics data, in these erlotinib-resistant patients, erlotinib is
general toxicity, which have been well documented over
likely to reach only a sub-IC50 serum concentration (26,
several decades due to its extensive use in the prevention
27), whereas quinacrine is known to accumulate in tissues
Mol Cancer Ther; 13(9) September 2014
Molecular Cancer Therapeutics
Published OnlineFirst July 15, 2014; DOI: 10.1158/1535-7163.MCT-14-0013
Combination of Quinacrine and Erlotinib in NSCLC
(7). When we selected drug doses more representative of
independent pathways is exponentially smaller than the
those achievable clinically, we observed potent single-
probability of resistance to a single agent. Our novel
agent activity of quinacrine in the erlotinib-resistant
combination offers a promising therapy in advanced
NSCLC cell lines. At such concentrations, quinacrine
NSCLC, for which there are currently few effective treat-
dominates the cell killing activity of the combination, as
ment options after the tumors have progressed during
indicated by our colony formation, cell cycle, and apo-
first-line anticancer treatments.
ptosis assay, and our drug synergy quantification predictsthat a much higher erlotinib concentration would be
Disclosure of Potential Conflicts of Interest
needed to achieve synergy with quinacrine in these
N. Sharma received a commercial research grant from Incuron Inc. No
assays. This result suggests that the inclusion of quina-
potential conflicts of interest were disclosed by the other authors.
crine alone as one of the arms in a future clinical trialwould be valuable. Our findings also indicate that obser-
Authors' ContributionsConception and design: J.K.T. Dermawan, K. Gurova, A. Dowlati, S. De,
vation of drug synergy in in vitro studies might fail to
G. Narla, N. Sharma, G. Stark
translate to clinical trials when the same ratios are not
Development of methodology: J.K.T. Dermawan, K. Gurova, A. Dowlati,
attainable based on in vivo drug pharmacokinetics.
S. De, G. Narla, G. StarkAcquisition of data (provided animals, acquired and managed patients,
We also identified, as potential pharmacodynamic mar-
provided facilities, etc.): J.K.T. Dermawan, A. Dowlati, N. Sharma
kers for this clinical trial, a set of genes whose expression
Analysis and interpretation of data (e.g., statistical analysis, biostatis-
levels were significantly suppressed by combination ther-
tics, computational analysis): J.K.T. Dermawan, J.J. Pink, A. Dowlati,G. Narla, N. Sharma, G. Stark
apy and were shown to correlate with worse patient
Writing, review, and/or revision of the manuscript: J.K.T. Dermawan,
survival in existing gene expression databases. Because
A. Dowlati, S. De, G. Narla, N. Sharma, G. StarkAdministrative, technical, or material support (i.e., reporting or orga-
these are posttreatment rather than pretreatment biomar-
nizing data, constructing databases): J.J. Pink, N. Sharma, G. Stark
kers, the goal is to use these gene signatures to validate
Study supervision: N. Sharma, G. Stark
that the biologic efficacy of the combination over singledrug treatment during the early phase of treatment by
gene expression profiling of pre- and posttreatment biop-
The authors thank Drs. Martina Veigl and Patrick Leahy at the Gene
Expression Array Core Facility for their valuable input in the design and
sies from the subjects in our clinical trial, and then apply
analysis of the microarray study, Vai Pathak for conducting the ABI
this knowledge to select for patients who would truly
Expression Real-time RT-PCR experiments, Ian Lent at the Translational
benefit from the combination and thus would remain in
Research & Pharmacology Core Facility for performing the CalcuSynanalysis, and Cathy Shemo and Bunny Cotleur at the CCF Flow Cytometry
the trial through the entire course of treatment. For future
Core for excellent technical support for flow-cytometric data acquisition
studies, to identify pretreatment predictive pharmacody-
and analysis.
namics biomarkers, we would need to identify cell linesthat are resistant to the combination and compare their
gene signatures with those of cell lines that are sensitive to
This work was supported by the National Cancer Institute Grant P01
CA062220 (to G.R. Stark), Clinical & Translational Science Collaborative of
the combination, such as those used in the present study.
Cleveland Core Utilization pilot grant P30 CA043703-23 at Case Western
Recent discoveries of the high degree of intratumoral
Reserve University (to N. Sharma), and a Harrington Discovery Institute
and intermetastatic genetic heterogeneity among tumor
grant (to G. Narla).
The costs of publication of this article were defrayed in part by the
cells in cancer genomics projects suggest that the devel-
payment of page charges. This article must therefore be hereby marked
opment of resistance is inevitable in any targeted therapy
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.
for cancer (32). The use of combinatorial therapy is animportant means to circumvent this problem because the
Received January 10, 2014; revised May 29, 2014; accepted June 23, 2014;
probability of cancer cells becoming resistant to two
published OnlineFirst July 15, 2014.
Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA,
lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl
Brannigan BW, et al. Activating mutations in the epidermal growth
Acad Sci U S A 2007;104:20932–7.
factor receptor underlying responsiveness of non-small cell lung
Sequist LV, Waltman BA, Dias-Santagata D, Digumarthy S, Turke AB,
cancer to gefitinib. N Engl J Med 2004;350:2129–39.
Fidias P, et al. Genotypic and histological evolution of lung cancers
Garassino MC, Martelli O, Broggini M, Farina G, Veronese S, Rulli E,
acquiring resistance to EGFR inhibitors. Sci Transl Med 2011;3:
et al. Erlotinib versus docetaxel as second-line treatment of patients
with advanced non-small cell lung cancer and wild-type EGFR tumors
Ehsanian R, Van Waes C, Feller SM. Beyond DNA binding – a review of
(TAILOR): a randomized controlled trial. Lancet Oncol 2013;14:981–8.
the potential mechanisms mediating quinacrine's therapeutic activi-
Shepherd FA, Rodrigues PJ, Ciuleanu T, Tan EH, Hirsh V, Thongpra-
ties in parasitic infections, inflammation, and cancers. Cell Commun
sert S, et al. Erlotinib in previously treated non-small cell lung cancer. N
Signal 2011;9:13.
Engl J Med 2005;353:123–32.
Gurova K. New hopes from old drugs: revisiting DNA-binding small
Kobayashi S, Boggon TJ, Dayaram T, Janne PA, Kocher O, Meyerson
molecules as anticancer agents. Future Oncol 2009;5:1685–704.
M, et al. EGFR mutation and resistance of non-small cell lung cancer to
Gurova KV, Hill JE, Guo C, Prokvolit A, Burdelya LG, Samoylova E, et al.
gefitinib. N Engl J Med 2005;352:786–92.
Small molecules that reactivate p53 in renal cell carcinoma reveal a NF-
Bean J, Brennan C, Shih JY, Riely G, Viale A, Wang L, et al. MET
kB-dependent mechanism of p53 suppression in tumors. Proc Natl
amplification occurs with or without T790M mutations in EGFR mutant
Acad Sci U S A 2005;102:17448–53.
Mol Cancer Ther; 13(9) September 2014
Published OnlineFirst July 15, 2014; DOI: 10.1158/1535-7163.MCT-14-0013
Dermawan et al.
10. Gasparian AV, Burkhart CA, Purmal AA, Brodsky L, Pal M, Saranadasa
22. Ernst J, Bar-Joseph Z. STEM: a tool for analysis of short time series
M, et al. Curaxins: anticancer compounds that simultaneously sup-
gene expression data. BMC Bioinformatics 2006;7:191.
press NF-kB and activate p53 by targeting FACT. Sci Transl Med
23. Sherman BT, Huang DW, Lempicki RA. Systemic and integrative
analysis of large gene lists using DAVID bioinformatics resources. Nat
11. Lu T, Sathe SS, Swiatkowski SM, Hampole CV, Stark GR. Secretion of
cytokines and growth factors as a general cause of constitutive NFkB
24. Ho Sui SJ, Mortimer JR, Arenillas DJ, Brumm J, Walsh CJ, Kennedy
activation in cancer. Oncogene 2004;23;2138–45.
BP, et al. oPOSSUM: identification of over-represented transcription
12. Nakanishi C, Toi M. Nuclear factor-kappaB inhibitors as sensitizers to
factor binding sites in co-expressed genes. Nucleic Acid Res
anticancer drugs. Nat Rev Cancer 2005;5:297–309.
13. Tanaka K, Babic I, Nathanson D, Akhavan D, Guo D, Gini B, et al.
rffy B, Surowiak P, Budczies J, Lanczky A. Online survival analysis
Oncogenic EGFR signaling activates an mTORC2-NF-kB pathway that
software to assess the prognostic value of biomarkers using tran-
promotes chemotherapy resistance. Cancer Discov 2011;1:524–38.
scriptomic data in non-small-cell lung cancer. PLoS ONE 2013;8:
14. Bivona TG, Hieronymus H, Parker J, Chang K, Taron M, Rosell R, et al.
FAS and NF-kB signaling modulate dependence of lung cancers on
26. Hidalgo M, Siu LL, Neumunaitis J, Rizzo J, Hammond LA, Takimoto C,
mutant EGFR. Nature 2011;471:523–6.
et al. Phase I and pharmacologic study of OSI-774, an epidermal
15. Brown M, Cohen J, Arun P, Chen Z, Van Waes C. NF-kB in carcinoma
growth factor receptor tyrosine kinase inhibitor, in patients with
therapy and prevention. Exp Opin Ther Targets 2008;12:1109–22.
advanced solid malignancies. J Clin Oncol 2001;19:3267–79.
16. Garcia H, Miecznikowski JC, Safina A, Commane M, Ruusulehto A,
27. Lu JF, Eppler SM, Wolf J, Hamilton M, Rakhit A, Bruno R, et al. Clinical
Kilpinen S, et al. Facilitates Chromatin Transcription Complex is an
pharmacokinetics of erlotinib in patients with solid tumors and expo-
"accelerator" of tumor transformation and potential marker and target
sure-safety relationship in patients with non-small cell lung cancer.
of aggressive cancers. Cell Rep 2013;4:159–73.
Clin Pharmacol Ther 2006;80:136–45.
17. van Meerloo J, Kaspers GJ, Cloos J. Cell sensitivity assays: the MTT
28. Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor
assay. Methods Mol Biol 2011;731:237–45.
receptor mutations in lung cancer. Nat Rev Cancer 2007;7:169–81.
18. Chou TC. Drug combination studies and their synergy quantification
29. Zou Y, Ling YH, Sironi J, Schwartz EL, Perez-Soler R, Piperdi B. The
using the Chou-Talalay method. Cancer Res 2010;70:440–6.
autophagy inhibitor chloroquine overcomes the innate resistance of
19. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships:
wild-type EGFR non-small-cell-lung cancer cells to erlotinib. J Thorac
the combined effects of multiple drugs or enzyme inhibitors. Adv
Enzyme Regul 1984;22:27–55.
30. Kimmelman AC. The dynamic nature of autophagy in cancer. Genes
20. Gasparian AV, Guryanova OA, Chebotaev DV, Shishkin AA, Yemelya-
nov AY, Budunova IV. Targeting transcription factor NF-kB: compar-
31. Bolte J, Demuynck C, Lhomme MF, Lhomme J, Barbet J, Roques BP.
ative analysis of proteasome and IKK inhibitors. Cell Cycle 2009;8:
Synthetic models related to DNA intercalating molecules: comparison
between quinacrine and chloroquine in their ring-ring interaction with
21. Ishwaran H, Rao JS, Kogalur UB. BAMarray: Java software for Bayes-
adenine and thymine. J Am Chem Soc 1982;104:760–65.
ian analysis of variance for microarray data. BMC Bioinformatics
32. Garraway LA, Lander ES. Lessons from the cancer genome. Cell
Mol Cancer Ther; 13(9) September 2014
Molecular Cancer Therapeutics
Published OnlineFirst July 15, 2014; DOI: 10.1158/1535-7163.MCT-14-0013
Quinacrine Overcomes Resistance to Erlotinib by Inhibiting FACT,
NF-
Small Cell Lung Cancer
B, and Cell-Cycle Progression in Non
Josephine Kam Tai Dermawan, Katerina Gurova, John Pink, et al.
2014;13:2203-2214. Published OnlineFirst July 15, 2014.
Mol Cancer Ther
Access the most recent version of this article at:
Access the most recent supplemental material at:
This article cites 32 articles, 8 of which you can access for free at:
This article has been cited by 3 HighWire-hosted articles. Access the articles at:
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at
To request permission to re-use all or part of this article, contact the AACR Publications Department at
Source: http://incuron.ru/doc/st/13.pdf
and Developing Academic and Behavioral Interventions for Students with Bipolar KIM KILLU AND R. MARK A. CRUNDWELL Despite significant advances in practices for effectively designing anddelivering instruction for students with disabilities, educators continueto face challenges addressing the needs of students with emotionaland behavioral disorders. Little information is available for educatorson accommodations and modifications that would serve the needs ofthese students and address the unique challenges they present in theclassroom. The educational, social, and behavioral needs of studentswith bipolar disorder are discussed along with suggestions for provid-ing effective accommodations and modifications in the classroom.
Liberty Global, Inc. Programa de conducta empresarial y cumplimiento del reglamento Este documento proporciona información sobre nuestro Código de conducta empresarial. Todos los directores, gerentes y empleados son responsables, individual y colectivamente, de la gestión de nuestras actividades comerciales conforme al Código y a la legislación pertinente. Por ello, le recomendamos que lea este documento con atención. Al aceptar un cargo o continuar ejerciendo sus funciones como director, gerente o empleado, se compromete a cumplir lo establecido en el presente Código. El desconocimiento del Código no exime a ningún empleado de la responsabilidad de atenerse a él, así como a la legislación pertinente y a cualquier otra normativa aplicable a sus actividades laborales.