PERSpECTIVES

is multidrug resistance protein 1,

Opinion
MDR1. The murine homologue was
found to confer resistance to doxorubicin,
Revisiting the role of ABC transporters colchicine and vincristine when transfected
into drug-​sensitive LR23 hamster cells13.
in multidrug-​resistant cancer These seminal findings launched the study
of ABC transporters, with a family of
48 human membrane transporters identified
Robert W. Robey, Kristen M. Pluchino, Matthew D. Hall   , Antonio T. Fojo, and shown to be involved in diverse
Susan E. Bates and Michael M. Gottesman physiological processes14.
The second member of the ABC
Abstract | Most patients who die of cancer have disseminated disease that has transporter family that was identified,
become resistant to multiple therapeutic modalities. Ample evidence suggests that multidrug resistance-​associated protein 1
the expression of ATP-​binding cassette (ABC) transporters, especially the multidrug (MRP1), encoded by ABCC1, was first
resistance protein 1 (MDR1, also known as P-​glycoprotein or P-​gp), which is reported in 1992 (ref. ). It was found in
15

cell line models to mediate resistance to

encoded by ABC subfamily B member 1 (ABCB1), can confer resistance to cytotoxic doxorubicin, etoposide and vincristine
and targeted chemotherapy. However, the development of MDR1 as a therapeutic among others16, but evidence of ubiquitous
target has been unsuccessful. At the time of its discovery , appropriate tools for the expression and lack of convincing evidence
characterization and clinical development of MDR1 as a therapeutic target were that it plays a role in clinical drug resistance
lacking. Thirty years after the initial cloning and characterization of MDR1 and the has meant it is unlikely to be a suitable
implication of two additional ABC transporters, the multidrug resistance-​ target for anticancer therapy. The third
member of the ABC transporter family
associated protein 1 (MRP1; encoded by ABCC1)), and ABCG2, in multidrug that was identified, ABCG2 (also known
resistance, interest in investigating these transporters as therapeutic targets has as breast cancer resistance protein, BCRP),
waned. However, with the emergence of new data and advanced techniques, we encoded by ABCG2, was reported by three
propose to re-​evaluate whether these transporters play a clinical role in multidrug different groups within the span of a few
resistance. With this Opinion article, we present recent evidence indicating that it months . These findings increased
17–19

interest in the study of ABC transporters

is time to revisit the investigation into the role of ABC transporters in efficient drug but added complexity to the definition
delivery in various cancer types and at the blood–brain barrier. of multidrug resistance. Although the
substrates and key roles for most of these
Despite heroic efforts to develop new in actinomycin D to select for resistance, transporters have been identified, the extent
anticancer drugs and biological therapies the selected cells were resistant not only to which these transporters play a role in
and to catalogue and study hundreds of to actinomycin D but also to vinblastine, clinical multidrug resistance has not been
potential mechanisms of resistance to these vincristine and daunomycin7. Another clarified yet. Despite the clinical failure of
treatment modalities1, most patients with study showed that the agent daunomycin MDR1 inhibitors, recent evidence suggests
metastatic cancer will die from multidrug-​ was actively transported out of multidrug-​ that expression of ABC transporters plays
resistant disease. Development of multidrug resistant mouse Ehrlich ascites cells, a role in clinical multidrug resistance in
resistance — the acquisition of resistance to suggesting the existence of a promiscuous some settings. In the following sections, we
multiple, structurally unrelated compounds membrane transporter that confers argue that a contemporary understanding
— is a frequent problem in the treatment multidrug resistance8. This transporter and reanalysis of target biology and
of cancer and should be distinguished from was later identified in multidrug-​resistant the identification and development of
resistance to anticancer drugs with precise Chinese hamster ovary cells and called efficient biomarkers using advanced
targets and immune therapies that are not ‘P-​glycoprotein’ because transporter technologies could identify settings in
examples of multidrug resistance. There expression was associated with altered drug which transporters involved in multidrug
are several extensive reviews detailing the permeability in resistant cells9. The gene resistance could be considered important
history and development of this field2–5. encoding P-​glycoprotein in Chinese hamster therapeutic targets.
Acquired multidrug resistance has been ovary cells was subsequently cloned10; the
intensively studied, and the basic science human homologue was reported soon after Structure and function
is well established. Over 50 years ago, a and the gene termed multidrug resistance The 48 human ABC transporter genes are
HeLa subline was described that exhibited (MDR) in the respective study11,12. The classified into seven subfamilies (termed
actinomycin D resistance following selection human gene is from here on referred to as ABC subfamily A through ABC subfamily
in 0.1 µg ml−1 of the drug6. When Chinese ATP-​binding cassette (ABC) subfamily B G)20,21. Structurally, ABC transporters are
hamster lung and fibroblast cells were grown member 1, ABCB1, and its protein product typified by a characteristic four-​domain

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© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Perspectives

architecture consisting of two cytoplasmic
nucleotide-​binding domains (NBDs)
that bind and hydrolyse ATP and two
transmembrane domains (TMDs) that
recognize and transport substrates (Fig. 1a).
Whereas the structure and function of NBDs
are similar throughout families, TMDs are
highly heterogeneous, allowing transporters
to recognize diverse substrates and use the
energy from ATP hydrolysis to translocate
molecules across membranes, irrespective
of the prevailing concentration gradient22.
Recent work suggests that while the energy
from ATP can help translocate engaged
substrates, basal ATP hydrolysis drives a
continuously changing conformation that
may facilitate MDR1 binding and transport
of a wide range of substrates23 (Fig. 1b).
High-​resolution structures of MRP1 (ref.24)
and ABCG2 (ref.25) determined using
cryo-​electron microscopy have helped to
clarify the drug-​binding domains but have
left the precise translocation mechanism
unresolved.
ABC transporters regulate cellular levels of
hormones, lipids, ions, xenobiotics and other
small molecules by transporting molecules
across cell membranes and serve a range of
physiological roles, including intracellular
regulation of organelles such as the
mitochondrion26, lysosome27, endoplasmic
reticulum28 and Golgi apparatus29. Loss of
function of a particular ABC transporter
via germline mutation is associated with a
number of heritable diseases, including cystic
fibrosis (associated with mutation
of cystic fibrosis transmembrane conductance
regulator (CFTR), encoded by CFTR (also
known as ABCC7)), pseudoxanthoma
elasticum (associated with mutation of
MRP6, encoded by ABCC6), Stargardt
macular degeneration (associated with
mutation of ABCA4), Tangier disease
(associated with mutation of ABCA1),
sitosterolaemia (associated with mutation of
ABCG5 or ABCG8) and harlequin ichthyosis
(associated with mutation of ABCA12)30.
The research and management of these
disorders present considerable biological and
clinical challenges, as the development of
small molecules or gene therapy is required
that enables normalization of mutant
transporters via strategies such as ribosomal
readthrough, stabilization of messenger
mRNA31, correction of folding defects32,
correction of trafficking defects33,34, allosteric
activation35, modulation of protein–protein
interactions36, control of post-​translational
regulation37, regulation of protein degradation
pathways37,38 or induction of compensatory
mechanisms39. Prospects are improved
somewhat given that restoration of activity
to as little as 5% of that of wild-​type basal
activity can be sufficient to partially
ameliorate disorder phenotypes40,41.
It should be noted that among the 48 ABC
transporters, some have very narrow substrate
specificity, whereas others have a broad
substrate specificity. It is the transporters
with broad substrate specificity that have the
potential to transport a range of anticancer
agents, and the expression level of these

a
ABCG2 MDR1 MRP1

Extracellular
domain

Transmembrane
domain

Intracellular
domain

b Drug
efflux

Transmembrane
domain Substrate ATP ADP ATP ADP
binds to hydrolysis release hydrolysis release
NBD binding site

ATP binds Conformational Conformational ATP and substrate

ATP ADP Substrate to NBD change reset bind and process
repeats

Fig. 1 | Structure and mechanism of three aBC transporters. a | High-​
resolution 3D structures of ATP-​binding cassette (ABC) subfamily G member
2 (ABCG2)25 (PDB ID 5NJ3), multidrug resistance protein 1 (MDR1)23 (PDB ID
5KPI) and multidrug resistance-​associated protein 1 (MRP1)24 (PDB ID 5UJ9).
Although the structure for MRP1 is that of Bos taurus, the protein identity is
91%, and the structure is likely similar to that of human MRP1. Structures
were generated using PyMol138 with data from RCSB PDB (see Related links).
b | Schematic representation of the proposed pumping action of MDR1. The
substrate binds to the binding pocket, and ATP binds to the two binding
sites in the nucleotide-​binding domains (NBDs). This is followed by the
hydrolysis of ATP, which generates a conformational change, allowing
the substrate to be released from the protein. The second molecule of ATP
is hydrolysed, allowing for a conformational reset where substrate and
ATP can bind again so the process can repeat.

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transporters in tumour cells may determine
the ability to confer drug resistance42. It has
been difficult to determine which transporters
deserve the most scrutiny to define their role
in multidrug resistance, as the use of cell lines
with high ABC transporter expression levels
in some cases led to an overestimation of their
role in cancer. The reader is referred to several
reviews compiling substrate lists of a range of
transporters43–45. One detailed compilation
notes that 19 of the 48 ABC transporters
have been shown to efflux anticancer agents
in some context43. We focus on the subset
of ABC transporters that were first reported
as multidrug efflux pumps (MDR1, MRP1
and ABCG2), what we have learned about
their basic science, how clinical application
has faltered and what might constitute a path
forward, focusing on the most recent work in
this field.
MDR1, MRP1 and ABCG2 have
excretory and/or protective physiological
capacities by transporting substrates
across biological membranes. At the
blood–brain barrier (BBB), the blood–
testis barrier and the blood–placental
barrier, expression of ABC transporters
in the capillary endothelial cells serves to
prevent entry of exogenous molecules46,47.
A consequence of these protective roles
is that these transporters can affect
pharmacokinetic parameters of drug
absorption, distribution, metabolism,
excretion and toxicity48. Inhibition of
ABC transporters often leads to toxicities
or pharmacokinetic changes owing to
drug–drug interactions48, and the US Food
and Drug Administration (FDA) offers
guidance on how investigational drugs
should be characterized with regard to their
ability to interact with ABC transporters49.
The multidrug resistance hypothesis
Nearly 40 years of findings from cell
culture and animal models indicate that
the efflux activity of ABC transporters
mediates multidrug resistance. The potential
importance of ABC transporters in multidrug
resistance is illustrated by the numerous
anticancer agents that have been identified
as substrates, including anthracyclines,
taxanes, vinca alkaloids, camptothecins,
epipodophyllotoxins and tyrosine kinase
inhibitors1,50. Although there is considerable
overlap among the substrate profiles of
the various ABC transporters, there are
some differences. MRP1 has been shown
to transport various neutral and anionic
hydrophobic compounds and products of
phase II drug metabolism, including many
glutathione and glucuronide conjugates51.
ABCG2 transports the anticancer drugs
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© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
methotrexate, mitoxantrone, topotecan,
irinotecan and flavopiridol52.
In addition to efforts to define ABC
transporter structure and function, efforts
have been made to define the clinical roles
of ABC transporters in multidrug resistance,
primarily for MDR1, the first transporter
to be discovered. These studies generally
used RNA-​based or immunohistochemical
methods of detection and examined
association with outcomes53. Numerous
studies reported the presence of MDR1
mRNA and protein in clinical samples — in
leukaemias and in kidney, colon, breast
and lung cancers — and, typically, MDR1
expression portendeds a poor response to
chemotherapy53–55. These results led
to the development of clinical trials to test
the multidrug resistance hypothesis that
inhibitors of MDR1 could improve response
to chemotherapy and outcome via increased
drug accumulation mediated by inhibition
of drug transport56,57.
Targeting MDR1
After the discovery of MDR1, a number
of first-​generation inhibitors, including
verapamil, quinidine, amiodarone and
cyclosporine A, were identified and
added to chemotherapy regimens in
clinical trials56. However, these agents
were not very potent or were toxic in
their own right, and their ability to inhibit
MDR1 was not verified in patients5. The
second-​generation agents valspodar and
dexverapamil were more potent MDR1
inhibitors56. Surrogate assays were used
to confirm that serum concentrations of
inhibitors such as valspodar were adequate
to inhibit rhodamine 123 transport in
MDR1-positive circulating CD56+ cells after
inhibitor administration58,59. However, at
that point in time, neither the expression
nor the function of MDR1 in tumours had
been verified, nor whether the inhibitors
actually blocked drug efflux in tumour cells.
Additionally, pharmacokinetic interactions
with the inhibitors, such as the inhibition
of cytochrome P450 by valspodar, required
chemotherapy dose reductions, leading
to potential underdosing of patients60. A
third generation of inhibitors, including
dofequidar, zosuquidar, tariquidar, elacridar
and biricodar, were developed specifically
as MDR1 inhibitors. These were more
potent and displayed fewer pharmacokinetic
interactions than inhibitors of previous
generations but caused some toxicity in
combination with chemotherapy, potentially
owing to inhibition of MDR1 expressed
in normal tissue57. Importantly, some
showed cross reactivity with MRP1 and/or
Perspectives
ABCG2 (refs61–64). Elacridar and tariquidar
were found to inhibit both MDR1 and
ABCG2 (refs61,64) while cyclosporine A and
biricodar were found to inhibit MDR1,
MRP1 and ABCG2 (refs62,63). Interestingly,
the combination of cyclosporine A with
chemotherapy led to increased response
in acute myeloid leukaemia (AML) in one
study65; however, these results could not be
duplicated in studies with other inhibitors66.
Unfortunately, despite a few early successes,
the majority of clinical trials with MDR1
inhibitors, even third-​generation inhibitors,
did not confirm clinical benefit57.
The lack of success of these inhibitors
in clinical trials resulted in a nearly
complete shutdown of study in the field
after these trials were completed, and
the clinical validation and development
of specific inhibitors for other ABC
transporters potentially involved in
multidrug resistance was not pursued.
Further clinical trials using MDR1
inhibitors were vehemently discouraged67.
However, some experimental work on
mechanistic and functional aspects of ABC
transporters continued, including efforts
to define substrates and inhibitors of ABC
transporters, the role of ABC transporters
in the BBB and the role of
ABC transporters in pharmacology. As
noted above, the FDA now offers guidance
on measuring the interaction of novel
therapies with ABC transporters during
clinical development49 because ABC
transporters can be critical determinants
of drug pharmacokinetics, including
oral availability, drug–drug interactions
and drug toxicity. Of note, recent reports
of unexpected toxicity in response to
chemotherapy combined with receptor
tyrosine kinase (RTK) inhibitors may be
explained by interactions of RTK inhibitors
with ABC transporters68–70. Indeed,
several RTK inhibitors, such as lapatinib,
apatinib and nilotinib, have been shown
to inhibit ABC transporters71–73, whereas
some, such as ceritinib, crenolanib and
imatinib, were found to be substrates
of ABC transporters74–76. In some cases,
these interactions might be beneficial. For
example, when patients were treated with
the chemotherapeutic agent topotecan
in combination with the RTK inhibitor
pazopanib, total topotecan exposure in
these patients was 1.7-fold higher than in
patients treated with topotecan alone. This
was primarily because pazopanib inhibits
ABCG2, thus increasing the absorption of
orally administered topotecan77.
Unfortunately, these studies did not
confirm the role of transporter-​mediated
Perspectives

reduced drug accumulation in clinical
drug resistance. However, gene expression,
gene mutation and single-​nucleotide
polymorphism (SNP) studies have
provided new evidence supporting the
potential benefit of a re-​examination
of ABC transporters in clinical drug
resistance. Additionally, the use of
genetically engineered mouse models
(GEMMs) has provided new impetus for
examining the role of ABC transporters
in acquired resistance to certain drugs,
such as doxorubicin, topotecan and
some poly(ADP-​ribose) polymerase
(PARP) inhibitors. Imaging studies and
mouse models examining the role of
ABC transporters in the gastrointestinal
tract and at the BBB have consistently
demonstrated that the activities of MDR1
and ABCG2 are significant impediments
to anticancer agent oral absorption and
brain exposure78. A summary of the new
evidence supporting the role of ABC
transporters in drug resistance is given in
the next section.
ABC transporters in resistance
MDR1 overexpression is associated with
resistance in some tumour subsets. Many
of the early clinical trials to test the MDR1
hypothesis were designed for patients with
AML56. Although largely unsuccessful, we
now recognize their design was optimistic
and success unlikely given that the focus
was only on MDR1, and patients were not
selected based on MDR1 expression or
potential MDR1 involvement in the patient’s
resistance to therapy55. For example, gene
expression profiling in 170 AML samples
showed that only 13% of the samples
were refractory and positive for ABCB1
or ABCG2 expression79. Whole-​genome
sequencing of 92 patients with high-​grade
serous ovarian carcinoma with primary
and matched resistant disease showed
that amplification of cyclin E1 (CCNE1)
and reversion of BRCA1 and/or BRCA2
mutations were potential mechanisms
of resistance. Interestingly, the authors
also found recurrent promoter fusions
associated with overexpression of ABCB1
(ref.80). These promoter fusions, occurring
in about 8% of patients with resistant
disease, lead to ABCB1 overexpression.
The underlying gene rearrangements place
a more active promoter at the 5′ end of
the ABCB1 transcript81,82 (Fig. 2). These
gene rearrangements are monoallelic, with
the remaining alleles being transcribed
normally81,82. This mechanism has first
been reported in cell culture models and
in clinical samples from patients with
refractory acute lymphoblastic leukaemia81,82.
Of note, paclitaxel and docetaxel, the main
therapies used in ovarian cancer, which were
administered to patients in this study before
recurrence, are substrates of MDR1 (ref.80).
In our view, these observations suggest
that the ABCB1 gene rearrangements were
selected for in the drug-​resistant phenotype;
however, the low occurrence rate of these
gene rearrangements argues that they
might occur only in a particular molecular
background. As previously reported using
a TaqMan low-​density array, only a small
percentage of ovarian cancers show high

+1 –194 +1 ATG –194 +1 ATG

Constitutively ABCB1 ABCB1

1 1 2 3 1 2 3
active promoter promoter promoter

Gene X ABCB1 gene (7q21, allele 1) ABCB1 gene (7q21, allele 2)

(any chromosome)
Monoallelic gene
rearrangement

+1 –194 +1 ATG –194 +1 ATG

Constitutively ABCB1 ABCB1

1 1 2 3 1 2 3
active promoter promoter promoter

Gene X ABCB1 gene (7q21, allele 1) ABCB1 gene (7q21, allele 2)

(any chromosome)
Increased Normal
transcription transcription

Fusion ABCB1 mRNA Normal ABCB1 mRNA

+1 –194 +1 AUG +1 AUG

ABCB1 1 2 3
1 1 2 3
promoter
Conserved AUG start codon
ensures normal protein
translation

Fig. 2 | Upregulation of ABCB1 via promoter capture. Resistance medi-
ated by ATP-​binding cassette subfamily B member 1 (ABCB1) occurs not
by acquired mutations but by increased expression. The figure depicts an
acquired mechanism of ABCB1 overexpression. Although highly expressed
in some cells, such as those arising in the adrenal cortex, expression in the
majority of cells is low or absent. In the latter, expression can be augmented
by having expression controlled and/or driven by a constitutively active
promoter that ‘captures’ ABCB1 following a rearrangement that leads to the
fusion of the active promoter proximal to the transcription start site. The
capture is monoallelic and leads to a hybrid and/or fusion mRNA comprising
sequences from the capturing gene (light green box) and promoter proximal
to the ABCB1 promoter but with the conserved ATG ensuring a normal start
site for ABCB1 translation and a normal protein (ATG transcribed to AUG in
mRNA). Figure adapted with permission from ref.139, John Wiley & Sons.

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levels of ABCB1 expression after paclitaxel or
docetaxel treatment83. These studies suggest
that patient selection in any clinical trial
targeting MDR1 should include validation of
MDR1 expression in the patients’ tumours.
In a recent observation, investigators
identified unexpectedly high levels of
ABCB1 in samples obtained from a patient
whose anaplastic lymphoma kinase (ALK)-
rearranged lung cancer had developed
resistance to ceritinib, a drug used to treat
lung cancers with mutations in the ALK
gene, without a new ALK mutation. The
investigators established cell lines from
different metastatic sites in this patient
and found that both the post-​ceritinib-
treated tumour and cell lines derived from
it exhibited high levels of MDR1 (ref.74).
The authors also found that MDR1 was
highly expressed in 3 of 11 ALK-​rearranged
refractory lung cancers in which secondary
ALK mutations were absent74, indicating a
correlation between MDR1 expression and
ceritinib resistance. Importantly, owing to a
lack of analytical tools, lung cancer studies
conducted with MDR1 inhibitors in the
1990s2 could not have envisioned the subset
stratification that would be needed to find
the small subset of patients in whom MDR1
is both highly expressed and likely to confer
drug resistance.
Expression of multiple ABC transporters. It
is now clear that multiple ABC transporters
may be expressed in a single tumour type.
A study that examined expression of all
48 human ABC transporters in 281 AML
samples found in a multivariate analysis that
expression of ABCB1, ABCG1 and ABCG2
was linked to overall survival, and the overall
survival decreased with increasing number
of transporter genes co-​expressed84. Similar
results were observed in childhood AML,
where ABCA3, ABCB1, ABCC3 and ABCG2
mRNA expression levels were measured by
real-​time PCR, and the increasing number
of co-​expressed ABC transporters correlated
with shorter relapse-​free survival85. Finally,
in a set of 11 paired samples taken from
patients with AML at diagnosis and relapse,
a twofold overexpression of at least one
ABC transporter capable of transporting
anthracyclines or vinca alkaloids was found
in ten of the paired samples86. Although
the links between ABC transporter co-​
expression and clinical outcome are
correlative and have not been shown to be
causal, together these results suggest that
inhibition of multiple transporters might be
required to achieve clinical benefit. This may
be especially true for primitive leukaemic
CD34+CD38- cells, the putative stem cell
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population. This cell population has been
shown to express high levels of ABCG2 as
well as potentially MDR1 and MRP1 in some
patient samples87. Expression of ABCG2 and
MDR1 in this cell population has been found
to correlate with response to chemotherapy
both clinically and when measured ex vivo
in leukaemic blast samples88.
It remains to be determined whether
all or a subset of these ABC transporters
may be involved in the efflux of anticancer
drugs in the clinical setting. Support for the
idea that the expression of multiple ABC
transporters correlates with a resistance
phenotype has also been found in solid
tumours. A study of all ABC transporters
in pancreatic cancer found significant
upregulation of ABCB4, ABCB11, ABCC1,
ABCC3, ABCC5, ABCC10 and ABCG2
at the mRNA level in macrodissected
tumours relative to normal tissue, although
the study did not attempt to correlate
transporter expression with chemotherapy
response89. However, the fact that tumour
samples were not microdissected may be a
confounding factor. ABCB1 mRNA, which
is physiologically expressed at high levels
in cells of the pancreatic duct and acinar
cells90, was not upregulated in tumour tissue
compared with normal tissue. Upregulation
of ABC transporter expression beyond the
high levels found in normal tissue may not
be necessary to confer drug resistance, as in
hepatocellular carcinoma, where expression
levels of multiple ABC transporters have
been reported to be similar to the already
high levels of expression in normal liver
cells91,92.
With the advent of genomic analysis, we
have an unbiased approach to measuring
ABC transporter mRNA expression. It is
now possible to measure expression of all
transporters simultaneously rather than
focusing on certain transporters that we
hypothesize might be involved in resistance.
Analysis of RNA sequencing (RNA-​seq)
data from The Cancer Genome Atlas
(TCGA) database found that expression
of both ABCB1 and ABCG2 in a variety
of tumour types ranges over 1,000-fold
(Fig. 3), a far greater range of expression
than was reported in studies using older
technologies. Although a frequent criticism
of early MDR1 studies was that in vitro
levels of ABCB1 are much higher than those
found in patient tumours93, this broad range
suggests very high levels in some tumours.
However, it has been reported that in the
case of some tumours, such as breast cancer,
ABCB1 expression is found primarily in
tumour-​associated macrophages and not in
the tumour samples themselves94, and high
Perspectives
stromal expression of ABC transporters
could skew expression values in RNA-​seq
data from tumour samples. Discordance
between RNA and immunohistochemical
data is thought to be responsible for this
problem in some cases95, but doubts in
regards to antibody specificity have also
been expressed96. Despite this caveat, it
is interesting that tumour types most
refractory to chemotherapy are among
those with the highest levels of ABCB1 or
ABCG2 expression. Tumour tissues from
hepatocellular carcinomas and kidney
cancers show the highest levels of expression
of both ABCB1 and ABCG2 compared
with other cancer types (Fig. 3). While RTK
inhibitors are approved for the treatment
of both of these types of cancer97, these
cancer types show frequent resistance to
the mainstay chemotherapeutics such as
vincristine and doxorubicin, which are
MDR1 substrates53. Of note, the ability
to detect RNA transcript levels more
accurately via newer technologies does
not really overcome the deficiencies in
our understanding of the role of ABC
transporters in multidrug resistance. These
deficiencies include the lack of a direct
demonstration of how ABC transporter
activity affects drug accumulation in cancer
cells and the clinical significance of that
activity.
The studies outlined above indicate that
only a fraction of cancers express ABC
transporters at levels that could potentially
be linked to drug resistance. MDR1 is
expressed in only 13% of AML, 8% of
ovarian cancers and 30% of ALK-​positive
non-​small-cell lung carcinoma samples,
suggesting that MDR1 should have been
targeted as cancer mutations are today,
where patients are carefully screened and
selected for expression of the target in
tumour cells. In addition, expression of
more than one ABC transporter is common.
Clinical studies of MDR1 inhibitors in the
past did not routinely include molecular
characterization of tumour tissues56 and
thus were likely confounded by the inclusion
of patients who had low tumour levels of
MDR1, and in whom MDR1 inhibition
would have had no predicted clinical benefit.
Single-​nucleotide polymorphism studies
to determine the role of ABC transporters.
One way to determine whether MDR1 plays
a role in anticancer drug resistance is to
evaluate the impact of polymorphic variants
on chemotherapy response or cancer
outcome. Polymorphic variants of ABC
transporter genes that impair substrate efflux
could be associated with a higher cancer
Perspectives

a
Not sequenced
14 No mutation
Missense
ABCB1 expression RNA-seq V2 (log)

12 Nonsense
10 In frame
Splice
8

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10
Fig. 3 | expression of ABCB1 and ABCG2 in patient tumour
9
samples. Data from the cBioPortal website (see Related links)
ABCG2 (exp)

8 showing expression of ATP-​binding cassette (ABC) subfamily B

7 member 1 (ABCB1) (part a) or ABCG2 (part b) in cancers of various
6 origin, and data from the TCGA database (see Related links) show-
ing expression of both ABCB1 and ABCG2 in breast, thyroid, pan-
5
creas, liver and kidney tumours as well as glioblastomas (GBMs)
4 Breast (part c). The data in part c are viewed with tcgaMiner, an in-​
3 Thyroid house-developed, R Shiny-​based program. AC, adenocarcinoma;
Pancreas
2 Liver ACC, adenoid cystic carcinoma; AML , acute myeloid leukaemia;
1 Kidney ccRCC, clear cell renal cell carcinoma; chRCC, chromophobe RCC;
Glioblastoma CS, carcinosarcoma; DLBCL , diffuse large B cell lymphoma; exp,
0
0 2 4 6 8 10 12 14
RNA-​seq expression; PCPG, pheochromocytoma and paragangli-
ABCB1 (exp) oma; pRCC, papillary RCC; RNA-​seq, RNA sequencing; SCC,
squamous cell carcinoma; V2, version 2.

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© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

incidence owing to decreased xenobiotic
efflux and impaired normal tissue protection
because of decreased ABC transporter
efficacy, but they could also be associated
with a better outcome once a cancer is
diagnosed because of reduced chemotherapy
drug efflux. However, multiple variables
confound such associations: not all cancers
are linked to xenobiotic exposure; not all
cancer chemotherapeutics are substrates
for ABC transporters; current methods
provide incomplete information on the
transport activity of polymorphic variants
of ABC transporters; and clinical outcome
may be impacted by coexisting polymorphic
variants in multiple ABC transporter
genes. As a result of this complexity, the
literature on clinical outcomes associated
with polymorphic variants of ABCB1 is
contradictory98–101.
The largest and most reliable SNP
association study to date was based on RNA-​
seq data derived from the TCGA database,
examining RNA sequence and expression
data from 4,616 ovarian cancer patients
who had received any form of adjuvant
chemotherapy102. In particular, the correlation
of three common coding SNPs tagging either
C1236T (rs1128503), G2677T/A (rs2032582)
or C3435T (rs1045642) of ABCB1 in patients
with prior chemotherapy was determined102,
and only a marginal association of C1236T
with improved overall survival was found.
The study also reported that ABCB1 mRNA
overexpression in 143 serous ovarian tumours
was associated with a worse prognosis in
suboptimally debulked patients102.
Transporter expression in mouse models
of acquired resistance. Expression of ABC
transporters emerged as the principal
mechanism of resistance in an elegant
series of studies involving a GEMM of
hereditary breast cancer that arises in the
mammary epithelium of mice deficient
for Brca1 and Trp53 (ref.103). Treatment
of animals from this model with the
maximum tolerable dose of docetaxel
or doxorubicin, both MDR1 substrates,
led to an initial differential response in
mice, but, eventually, all tumours became
resistant to treatment. Gene expression
analysis was subsequently performed on
13 doxorubicin-​resistant tumours, and
upregulation of mRNA expression levels
of Abcb1a and/or Abcb1b (the murine
orthologues of ABCB1) were found in 11
of the 13 tumour samples compared with
the matched, sensitive tumours103. These
doxorubicin-​resistant tumours were also
resistant to docetaxel. In a subsequent study,
transport of 99mTc-​MIBI, a contrast agent
Nature Reviews | Cancer
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
used in cardiac imaging that is a substrate
of MDR1 (ref.104), was also demonstrated in
mice harbouring the doxorubicin-​resistant
tumours generated from the mice described
above103 that overexpressed Abcb1a and
Abcb1b but not in control tumours105.
Similar results were obtained when the mice
deficient in Brca1 and Trp53 were treated
with the PARP inhibitor olaparib: all tumours
responded initially to a 28-consecutive-​
day treatment, but, eventually, all tumours
acquired resistance. When compared with
their olaparib-​responsive counterparts,
recurring tumours overexpressed Abcb1a
and/or Abcb1b106. The addition of tariquidar
to olaparib treatment resensitized tumours
expressing Abcb1a and/or Abcb1b to olaparib,
indicating a role of MDR1 in olaparib
resistance in these tumours, whereas the
addition of tariquidar alone had no effect on
growth106. In doxorubicin-​resistant tumours
arising in this model, moderate increases
in MDR1 expression — as little as twofold
compared with treatment-​naive tumours —
was found in 11 of 13 tumours compared
with untreated tumours. In some of these
resistant tumours, resistance to doxorubicin
could be overcome by the addition of
tariquidar to doxorubicin treatment, whereas
tariquidar itself had no effect107.
In a separate study using the same mouse
model, when tumour-​bearing mice were
treated with the topoisomerase I inhibitor
topotecan, heterogeneous responses were
observed, but, eventually, all tumours
developed resistance to topotecan108. Of the
20 topotecan-​resistant tumours that were
examined, 9 expressed at least twofold higher
levels of Abcg2 compared with matched
untreated control tumours. When Abcg2-null
alleles were introduced into the mice deficient
in Brca1 and Trp53, the resulting tumours
were transplanted into syngeneic wild-​type
animals, which were subsequently treated
with topotecan. Mice bearing tumours
deficient in Brca1, Trp53 and Abcg2 had an
increased overall survival compared with
mice bearing tumours deficient in Brca1 and
Trp53 and expressing Abcg2, suggesting that
Abcg2 contributed to topotecan resistance
in this model103. Of note, expression of
Abcc1 and Abcc4, which have also been
implicated in topotecan resistance, were not
found to be increased in resistant tumours
compared with matched untreated control
tumours108. In order to test the efficacy of
the ABCG2 inhibitor Ko143 on topotecan-​
resistant Brca1 and Trp53-deficient tumours
that overexpressed Abcg2, tumours were
transplanted into Abcg2-deficient mice so as
to overcome the effects of Abcg2 on topotecan
clearance109. Compared with tumours treated
Perspectives
with topotecan alone, the addition of Ko143
was not able to completely overcome tumour
resistance to topotecan, as overall survival
only moderately increased from 52 to 71.5
days. This could be due to the short plasma
half-​life of Ko143. To further explore the
role of Abcg2 in drug resistance, the efficacy
of EZN-2208 (a pegylated form of SN-38)
was compared with irinotecan. The active
metabolite of EZN-2208, SN-38, is an ABCG2
substrate108. Pegylation of SN-38 allows for
sustained release of SN-38 and may overcome
ABCG2-mediated resistance108. Overall
survival of mice treated with EZN-2208 was
doubled compared with mice treated with
irinotecan alone108, suggesting that thwarting
the activity of ABCG2 increases sensitivity to
substrate drugs.
Similar observations have been reported
in other GEMMs. In a mouse model of
mammary tumours deficient in Brca2 and
Trp53, Abcb1a and/or Abcb1b expression
was higher in three of four chemo-​naive
tumours with a sarcomatoid phenotype,
referred to as carcinosarcoma, compared
with the carcinomas that predominantly
arise from this model; all carcinosarcomas
expressed higher levels of Abcg2 compared
with the carcinomas110. Additionally, when
four treatment-​naive carcinosarcomas were
treated with the maximum tolerated dose
of topotecan, docetaxel, doxorubicin or
olaparib, none of the tumours responded110.
When mice bearing the carcinosarcoma
with the highest level of Abcb1a and Abcb1b
were treated with olaparib, docetaxel or
doxorubicin in the presence of tariquidar,
significantly higher growth delay was
observed compared with mice treated
with chemotherapy or tariquidar alone110.
After genetically sequencing a panel of
ten carcinomas and four carcinosarcomas
derived from this same model, unsupervised
clustering was performed using an
epithelial-​to-mesenchymal transition
(EMT) genetic signature110. Interestingly, the
carcinosarcomas, which express higher levels
of Abcb1a, Abcb1b and Abcg2, were found to
have higher expression of mesenchymal genes
and lower expression of epithelial genes110.
An examination of ABCB1 expression in
human triple-​negative breast cancer samples
mined from a previous study111 demonstrated
that tumours with an EMT phenotype (low
expression of the EMT marker claudin)
had high basal levels of ABCB1 expression
compared with basal-​like tumours112.
Despite these elegant studies in mice that
implicate MDR1 as a potential resistance
mechanism in breast cancer, the role of
MDR1 in mediating resistance has not been
as clearly implicated in human breast cancer.
Perspectives

The inability to translate MDR1 data derived
from mouse models to humans could be
because basal levels of MDR1 are higher
in rodents than in humans, which then
translates to MDR1 expression in response
to anticancer drug treatment in rodents113.
Another explanation is that relatively small
increases in MDR1 expression, which could
be clinically important, might not have
been detected owing to methodological
mouse models in which brain concentration
of these therapeutics is measured in wild-​
type versus ABC transporter-​deficient
mice78 (Fig. 4).
The deletion of either Abcb1a and
Abcb1b or Abcg2 alone or in combination
Plasma
typically has only minimal effects on
systemic blood levels of their substrates,
though steady-​state brain levels of substrates
are markedly higher when both transporters
are deleted rather than either alone78. As
one example, mice lacking Abcb1a and
Brain

Abcb1a/b–/–

Abcb1a/b–/–
limitations such as the technique used

Abcg2–/–

Abcg2–/–
to determine MDR1 expression113. To

TKO

TKO
overcome the limitations of animal models

WT

WT
Target and/or function Drug
in preclinical target characterization, human
Antiparasitic Ivermectin Fold change
breast epithelial organoids could be used to compared
Vinblastine
verify data derived from animal models114. DNA damage with WT
Doxorubicin ≥50
BMS-275 and BMS-183
Decreased oral bioavailability owing Taxanes
to ABC transporter activity. Although Cabazitaxel
not directly involved in drug resistance, Cobimetinib
MEK
ABC transporter expression in the Trametinib 25
gastrointestinal tract is known to affect the mTOR Everolimus
oral bioavailability of some chemotherapy Flavopiridol
CDKs
drugs that are ABC transporter substrates. Palbociclib
This has been shown for taxanes and JAK1 and JAK2 Momelotinib 1
topotecan, which are not given orally Dasatinib
owing to interactions with MDR1 and BCR–ABL Imatinib
ABCG2, respectively. The dual MDR1 Ponatinib
and ABCG2 inhibitor elacridar has been Axitinib
combined in exploratory trials with oral Multikinase
Sorafenib
taxol115 or topotecan116 to increase oral Sunitinib
bioavailability in patients; however, the Regorafenib
clinical efficacy of these combinations α1 blocker Prazosin
has not been reported. The expression of EPZ005687
ABC transporters in the gastrointestinal EZH2 EPZ-6438
tract might have the potential to cause GSK126
drug resistance. For example, when Caco-2 MDR1 Elacridar
intestinal cells were chronically exposed to Rucaparib
PARP
imatinib, MDR1 and ABCG2 expression Veliparib
was induced117. If this were to occur in Crizotinib
ALK
the gastrointestinal tract, it would limit Ceritinib
oral availability of imatinib, resulting in VEGF Cediranib
lower serum concentrations and resistance Lapatinib
to the drug, although this has yet to be HER2 and EGFR
Erlotinib
demonstrated in vivo or in the clinic. SN-38
Camptothecin
derivative Gimatecan
ABC transporters at the blood–brain Topotecan
barrier limit drug uptake. One of the Pictilisib
stunning discoveries made during the AKT and PI3K
Omipalisib
evolution of this field was the critical Vemurafenib
importance of MDR1 at the BBB, which BRAF Encorafenib
was first shown in mouse models in which Dabrafenib
deletion of Abcb1a and Abcb1b resulted in Aurora kinase Barasertib
central nervous system (CNS) toxicity from
ivermectin, an antiparasitic commonly Fig. 4 | effect of transporter deletion on plasma or brain levels of drugs. Fold increase in plasma
used on animals118. After the discovery of and brain drug levels in mice deficient for ATP-​binding cassette (ABC) subfamily B member 1a (Abcb1a)
and Abcb1b (Abcb1a/b-/-), ABC subfamily G member 2 (Abcg2) or all three (triple knockout (TKO)) trans-
ABCG2, mice lacking Abcb1a, Abcb1b and
porters is compared with wild-​type mice, which are assigned a value of 1. White blocks denote mice
Abcg2 were generated78. A systematic series not studied. ALK , anaplastic lymphoma kinase; CDK, cyclin-​dependent kinase; EGFR , epidermal
of investigations demonstrated the often growth factor receptor ; EZH2, enhancer of zeste homologue 2; HER2, human epidermal growth factor
overlapping and synergistic role of these receptor 2; MDR1, multidrug-​resistance protein 1; PARP, poly(ADP-​ribose) polymerase; VEGF, vascular
two transporters in restricting the entrance endothelial growth factor ; WT, wild type. Figure adapted with permission from ref.78, Springer, and
of anticancer therapeutics to the brain in compiled from refs118–121,140–168.

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Abcb1b had 2.3-fold higher steady-​state
brain levels of vemurafenib, the mutant
BRAF inhibitor approved for the treatment
of melanoma, than wild-​type mice, while
Abcg2-deficient mice had no change in
vemurafenib brain levels compared with
wild-​type mice. However, steady-​state
brain levels of vemurafenib in mice lacking
both Abcb1a and Abcb1b and Abcg2 were
approximately 43-fold higher than in wild-​
type mice119. This suggests a remarkable
compensatory function for the two
transporters. Similarly, a recent report on
the ALK inhibitor ceritinib demonstrated
that steady-​state brain levels of ceritinib
are increased approximately 37-fold in the
absence of Abcb1a and Abcb1b, and 87-fold
in the absence of Abcb1a, Abcb1b and
Abcg2, compared with wild-​type mice120.
Knockout of Abcg2 alone did not increase
accumulation of ceritinib in the brain121.
Presumably for these drugs, the expression
of Abcb1a and Abcb1b is able to compensate
for the absence of Abcg2. Ceritinib and
vemurafenib are thus likely restricted
from the brain by MDR1 and ABCG2 and
therefore cannot control early metastatic
disease in the brain. This is particularly
troubling for vemurafenib, as high serum
concentrations are already necessary for
systemic treatment of melanoma122.
Imaging demonstrates utility of transport
inhibition at the blood–brain barrier.
Other in vivo models have used inhibitors
of ABC transporters to demonstrate their
role at the BBB using positron emission
tomography (PET) imaging. In mouse
models, the entrance of [11C]erlotinib into
the brain was restricted by expression
of Abcb1a and Abcb1b as well as Abcg2.
Deletion of Abcb1 and Abcb1b as well as
Abcg2 in mice led to higher brain levels of
[11C]erlotinib than in mice with deletion of
Abcb1a and Abcb1b, in mice with deletion
of Abcg2 or in wild-​type mice123. In non-​
human primates, coadministration of
[11C]erlotinib with elacridar resulted in
a 3.5-fold increase in brain penetration
compared with controls that received
only [11C]erlotinib124. Similarly, in healthy
human subjects, MDR1 inhibition by
high doses of tariquidar led to significant
increases in brain levels of the MDR1-
specific substrate (R)-[11C]verapamil
compared with untreated subjects. By
contrast, high doses of tariquidar did
not lead to increased brain levels of [11C]
tariquidar, a substrate of both MDR1 and
ABCG2, owing to the ability of ABCG2
to compensate for inhibition of MDR1
function125. Similar observations were
Nature Reviews | Cancer
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Perspectives
Substrate Apical BBB Brain signal intensity
MDR1 ABCG2 MDR1 and
inhibition inhibition ABCG2
inhibition
Cl
N
MDR1 –
OH O NH
[11C]dLop
O N
O
O N
MDR1
O
NH – –
ABCG2
[11C]Erlotinib
S N
O N S OH ABCG2 –
OH
D-luciferin
Fig. 5 | The utility and function of positron emission tomography radiotracers and other probes
for imaging aBC transporter function, using the central nervous system as a model. Combination
with inhibitors of known function or administration to knockout mice provides insight into the function
of the respective target molecules. Multidrug-​resistance protein 1 (MDR1): 11C-​labelled des-​methyl
loperamide ([11C]dLop) is a specific substrate of MDR1 that cannot pass through the blood–brain bar-
rier (BBB) when MDR1 is active; that is, no radio signal in the brain can be observed. Upon inhibition or
knockout of MDR1, high signal intensity in the brain is observed, whereas inhibition of ATP-​binding
cassette (ABC) subfamily G member 2 (ABCG2) has no effect. In the instance of a dual substrate of both
MDR1 and ABCG2 such as [11C]erlotinib, specifically blocking either MDR1 or ABCG2 results in a min-
imal increase in brain signal, and only dual inhibition or knockout produces an effect. An alternative
imaging strategy is presented by using the specific ABCG2 substrate D-​luciferin, with transgenic mice
expressing firefly luciferase in astrocytes. Brain bioluminescence signal was low , and specific inhibition
of ABCG2 but not MDR1 produced an elevated signal. Third-​generation inhibitors, such as tariquidar
and elacridar, are considered to primarily inhibit MDR1, while Ko143 (which has not been used in
humans) acts primarily on ABCG2. No gold-​standard probe for dual inhibition of ABCG2 and MDR1
exists. These imaging tools can act as the basis for studies of multidrug resistance in tumours and
efficacy and dose optimization of new inhibitors.
made in mouse models126. However, sevenfold increase in brain levels of [11C]
marked increases in brain levels of [11C] dLop compared with untreated controls129.
tariquidar were observed when individuals Clinical studies of [11C]dLop showed
carrying the C421A ABCG2 polymorphic that it had low brain retention130, and
variant were treated with high doses coadministration of tariquidar with [11C]
of tariquidar compared with treated dLop in healthy human subjects resulted
individuals with wild-​type ABCG2 (ref.125). in a twofold to fourfold increase of [11C]
This result is not surprising because the dLop brain levels compared with subjects
C421A polymorphism adversely affects the receiving [11C]dLop alone131.
activity of ABCG2 (refs127,128). Finally, D-​luciferin, the substrate
[N-​methyl-11C]N-​desmethyl-loperamide for firefly luciferase, was shown to be
(d-​loperamide; also known as dLop) was transported by ABCG2 (ref.132) and was
investigated as a potential PET imaging subsequently shown to be selectively
agent for the CNS. Like loperamide, its transported by ABCG2 rather than MDR1
metabolite, dLop, is also transported or MRP1 (ref.133). Indeed, when firefly
exclusively by MDR1 (ref.129). Mice lacking luciferase was expressed in a mouse model
Abcb1a and Abcb1b had a 3.5-fold higher in glia behind the BBB under the control
brain uptake of [11C]dLop compared with of the glial fibrillary acidic protein (GFAP)
wild-​type mice129. In non-​human primates, promoter, coadministration of D-​luciferin
inhibition of MDR1 with DCPQ yielded a and the ABCG2 inhibitor Ko143 resulted
Perspectives
in increased bioluminescent signal in
mouse brains as compared with mice
administered D-​luciferin alone133 (Fig. 5).
These studies pave the way for the use of
radiotracers in patients, which can help to
confirm the activity of inhibitors, monitor
the brain penetration of substrate drugs and
serve as possible biomarkers for assessing
the multidrug resistance status of patient
tumours.
Studies of the BBB have addressed the
ability to target ABC transporters in patients
to increase drug distribution into sanctuaries
such as the brain131. Some investigators have
attempted to demonstrate the ability to
target ABC transporters in human cancers
using radiolabelled substrates, particularly
[94Tc]sestamibi by either planar or PET
imaging in patients with MDR1-expressing
cancers before and after administration
of an MDR1 inhibitor. Except for a small
number of patients134, these studies have
never shown the type of differential
observed in laboratory models135,136, but the
caveats mentioned above concerning patient
selection and inhibitor choice apply to these
studies as well.
Although data from imaging studies are
limited, they represent the one method that
could be further developed to demonstrate
both the impact of drug efflux and the ability
to alter it and act as a biomarker for clinical
trials. Such studies have been needed for
a very long time, even in the absence of a
strategy to inhibit MDR1.
Conclusions
Investigations into the role of ABC
transporters in multidrug resistance have
been encumbered by the weight of a
succession of negative results from clinical
trials. But evolving technology and data lead
to the question of whether investigation
into the role of ABC transporters in clinical
drug resistance should be reopened. We
argue that further investigation is indeed
warranted in light of the recent data.
There are various examples of the effects
of MDR1 in restricting drug efficacy in
patients with select cancers74, of MDR1
or ABCG2 expression being associated
with poor clinical outcomes79 and of gene
rearrangements resulting in high expression
levels of MDR1 in patients whose tumours
exhibit drug resistance80.
Adequate and consistent drug delivery
has rarely been documented in clinical
oncology, and results of the few studies to
determine drug delivery efficacy suggest
highly variable drug penetration137.
Whether ABC transporters play a role
in variable drug delivery is at present
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
unknown. Clinical trials examining the
efficacy of inhibitors of ABC transporters
were conducted without selection of
patients whose tumours had high levels
of ABC transporter expression. With the
current tools developed for advancing
personalized medicine, the vision is
to identify patients whose cancer cells
overexpress an ABC transporter that
has been shown to reduce drug efficacy
in order to improve drug selection
and clinical outcome. Even if we are
unable to improve response to therapy
by using an ABC transporter inhibitor,
being able to predict clinical response to
certain drugs could be seen as a valuable
accomplishment.
To achieve clinical application of ABC
transporters as biomarkers and as targets
in combination therapy, ABC transporter
expression must first be reliably detected
in the tissue and/or cell type of interest,
and the inhibitors used in combination
with other drugs must then be safe for use
in patients. Therefore, clinically validated
methods to detect protein expression
of the respective ABC transporters
(or a highly correlated biomarker) and
clinically validated imaging assays to
detect ABC transporter function in
tumours are needed. As argued in this
Opinion article, a revival of research
interest into the ABC transporter field,
focusing on the most important questions
of the role of drug efflux in clinical
response and using advanced techniques,
could potentially lead to clinical use of
ATP transporters as biomarkers and
therapeutic targets.
Robert W. Robey1, Kristen M. Pluchino1,
Matthew D. Hall   2, Antonio T. Fojo3,4,
Susan E. Bates3,4 and Michael M. Gottesman1*
1
Laboratory of Cell Biology, National Cancer Institute,
National Institutes of Health, Bethesda, MD, USA.
National Center for Advancing Translational Sciences,
2
National Institutes of Health, Rockville, MD, USA.
3
Division of Hematology/Oncology, Department of
Medicine, Columbia University/New York Presbyterian
Hospital, Manhattan, NY, USA.
4
James J. Peters VA Medical Center, Bronx, NY, USA.
*e-​mail: mgottesman@nih.gov
https://doi.org/10.1038/s41568-018-0005-8
Published online xx xx xxxx
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Acknowledgements
The authors appreciate the help of S. Lusvarghi with figures
and the editorial assistance of G. Leiman. This research was
supported by the Intramural Research Program of the
National Institutes of Health, US National Cancer Institute.
The content of this publication does not necessarily reflect
the views of policies of the Department of Health and Human
Services, nor does the mention of trade names, commercial
products or organizations imply endorsement by the US
government.
Perspectives
Author contributions
R.W.R. researched the data for the article, provided substan-
tial contributions to discussions of its content, wrote the arti-
cle and undertook review and/or editing of the manuscript
before submission. K.M.P. researched data for the article.
M.D.H. and A.T.F. provided substantial contributions to dis-
cussions of the content and wrote the article. S.E.B. and
M.M.G. provided substantial contributions to discussions of
the content, wrote the article and reviewed and/or edited the
manuscript before submission.
Competing interests
The authors declare no competing interests.
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