Mechanisms of Multidrug-Resistance in Cancer - NIH Clinical Center

Learning Some New Tricks
From a Multidrug Transporter
Michael M. Gottesman, M.D.
Chief, Laboratory of Cell Biology
Center for Cancer Research, NCI
National Institutes of Health
January 15, 2009

Estimated New Cancer Cases & Deaths, 2008
Sites New Cases Deaths* %
All Sites 1,437,180 565,650 39%
Prostate 186,320 28,660 15%
Breast 184,450 40,930 22%
Digestive System 202,720 79,090 39%
Pancreas, Liver & Gall Bladder 68,570 56,040 82%
Lung & Bronchus 215,020 161,840 75%
Bladder 68,810 14,100 20%
Kidney & Renal Pelvis 54,390 13,010 24%
Ovary 21,650 15,520 72%
Cervical Cancer 11,070 3,870 35%
Lymphoma & Leukemia 118,610 42,220 36%
Brain & Nervous System 21,810 13,070 60%
*Virtually all deaths are due to chemotherapy resistance CA Cancer J Clin, 2008

Drug Resistance in Cancer
• May affect multiple drugs used simultaneously:
known as multidrug resistance (MDR)
• Affects all classes of drugs, including newly
designed targeted drugs
• Just as oncogene targets have been catalogued,
we need to enumerate all mechanisms of drug
resistance in cancer to solve this problem and
circumvent resistance

Mechanisms of resistance to
anti-cancer drugs
Decreased
uptake
Reduced apoptosis
Altered cell cycle checkpoints
Increased metabolism of drugs
Increased or altered targets
Increased repair of damage
Compartmentalization
Increased
efflux

Ultimate Goals
1. Molecular analysis of human cancers
to predict response to therapy
2. Use this information to develop novel
drugs to treat cancer and new imaging
modalities for cancer
3. To learn more about cellular
pharmacology and pharmacokinetics
of drugs, including drug uptake,
distribution, and excretion

ABC transporters: Domain organization
ABCB1
TM Domain
ATP binding
TM Domain
ATP binding
ABCC1
ABC
G2

Hypothetical Model of Human P-glycoprotein
100
OUT
MEMBRANE
200
IN
300
ATP SITE
ATP SITE
A
B
700
1000 1200
A B
400
C
600
P
800
P
P
P
900
1100
C
1
500
POINT MUTATIONS ( ),
PHOTOAFFINITY LABELED
REGIONS ( ), AND
PHOSPHORYLATION SITES ( P )
1280

Substrates and Reversing Agents of Pgp
N
H
H 3 COOC
N
H
OH
N
CH 2 CH 3
H 3 CO
H 3 CO
H 3 CO
OCH 3
NHCOCH 3
L-Pro
D-Val
L-Thr
Sar
O
L-M eva l
O
C
N
L-Pro
D-Val
C
O
Sar
L-T
hr
NH 2
L-M eva l
O
H 3 CO
N
HO
CH 3
Vinblastine
CH 2 CH 3
COOCH 3
COOCH 3
O
Colchicine
O
O
O
CH 3 CH 3
Actinomycin D
O
OH
H
OCH 3 O OH
H
OH
H
H 3 C
H
NH 2
O
H
O
H
CHOCH 3
OH
Daunorubicin
O
C
H 3 CO
H 3 CO
NH
O O
H
C C
H
C
CN
O
H 3 C
H 3 C
C C C C N
H 2 H 2 H 2
CH(CH 3 ) 2
C
HO
Taxol
O
O
O
CH 3
CH 3
CH 3
O
OH
CH 2 O
C CH 3
CH 3
C
H 2
C
H 2
OCH 3
Verapamil
O
OCH 3
HO
H 3 CO
O
HO
H 3 C
H 3 C
CH 3
O O
OH
N
H 3 C
O
O
H 3 CO
O
H
O OCH 3 C
3
CH 3
CH 3
Rapamycin

P-glycoprotein removes hydrophobic substrates
directly from the plasma membrane
+
OUT
+
MEMBRANE
ATP
ATP
+
IN

Homology model of human P-
glycoprotein based on the
structure of bacterial ABC
transporter Sav1866 of S. aureus
N-TMD
Plasma membrane
C-TMD
Cell
N-NBD
C-NBD

Role of P-glycoprotein in cancer
• Approximately 50% of human cancers express P-glycoprotein at
levels sufficient to confer MDR
• Cancers which acquire expression of P-gp following treatment of the
patient include leukemias, myeloma, lymphomas, breast, ovarian
cancer; preliminary results with P-gp inhibitors suggest improved
response to chemotherapy in some of these patients
• Cancers which express P-gp at time of diagnosis include colon,
kidney, pancreas, liver; these do not respond to P-gp inhibitors alone
and have other mechanisms of resistance
• Being able to image P-gp in cancer (and ultimately other transporters
that contribute to resistance) could help guide therapy

Physiologic Role of P-glycoprotein

ABC transporters determine oral bioavailability, excretion, penetration
and protect the organism against airborne xenobiotics
BRAIN
B1, C1, C2, G2
BBB
BLOOD
MILK
G2
CSF
B1
C1
choroid plexus
C1, C3-5
C2, B1, B4, B11, G2
C1
GI TRACT
oral
aerosol
LUNG
LIVER
C2, B1,
G2
B1, C1,
A3,G21
PLACENTA
FETUS
B1, G2
KIDNEY
B1, C2, G2
BTB
URINE
C1
C1
TESTIS

Polymorphisms in the human
MDR1 1 gene
1. More than 50 SNPs have been reported in the MDR1 gene.
14 of them are silent polymorphisms.
2. 5 common coding (non-synonymous) polymorphisms have no
demonstrable effect on drug transport function.
3. The synonymous SNP in exon 26 (C3435T) was the first associated
with altered MDR1 function and is often part of a haplotype including
another synonymous (C1236T) and a nonsynonymous SNP (G2677T).

The C1236T, G2677T, C3435T haplotype has
been linked to several different phenotypes
•Altered digoxin and fexofenadine
pharmacokinetics
•Altered toxicity in transplant patients from
cyclosporine A, tacrilimus
•Altered incidence of Crohn’s disease,
colon cancer, and Parkinson’s disease

MDR1 1 wild-type, SNPs, and
haplotypes show similar P-gp P
total
and surface expression
pTM1
MDR1
C1236T
G2677T
C3435T
C1236T-G2677T
C1236T-C3435T
G2677T-C3435T
C1236T-G2677T-C3435T
pTM1
X3
WT
P-gp
10 0 10 1 10 2 10 3 10 4
Fluorescence Intensity

MDR1 1 wild-type and the haplotype
(1236-2677
2677-3435) do not exhibit similar
Bodipy-verapamil
accumulation
WT
X3
pTM1
10 0 10 1 10 2 10 3 10 4
Fluorescence Intensity

MDR1 1 wild-type and the haplotype exhibit
different patterns using rhodamine 123 efflux with
cyclosporin A reversing agent
MDR1 WT
pTM1 control
1236-2677-3435
10 0 10 1 10 2 10 3 10 4
Fluorescence Intensity
5 mM CsA

MDR1 1 wild-type and haplotype show the same P-gp P
cell
surface expression using MRK16 and 17F9, but not UIC2
- a conformational sensitive antibody
pTM1
WT
1236-2677-3435

MDR1 1 wild-type and haplotype show different
trypsinization patterns confirming altered
conformation

Polymorphic forms of P-gp with alleles that
don’t change amino acid sequence change the
conformation of P-gp
1 . In transient transfection experiments, the amount of P-gp
mRNA, protein, and protein localization on the cell
surface is unchanged.
2. The conformation of polymorphic P-gp is altered as
shown by tryptic peptide analysis and conformationspecific
MoAbs.
3. Translational toeprint experiments show a major delay in
translation at the site of the “silent” polymorphism.

mRNA
no ribosome
(Control)
Primer extension inhibition (toeprint) assay – to
detect presence of ribosome stalling sites
32 P labeled primer
for reverse transcription
Control
Wild-type
+ribosome
Mutant
+ribosome
G A U C
5’
CAP
Wild type mRNA
+ ribosome
CHX
ribosome
complex
CHX
ribosome
complex
CAP
Mutant mRNA
+ ribosome
CHX
ribosome
complex
CHX
ribosome
complex
CHX
ribosome
complex
CAP
Reverse transcription products
were resolved by
polyacrylamide gel
electrophoresis
3’

Toeprint analysis of MDR1 mRNA
around the 3435 SNP
A
C
In vitro
translated
MDR1
Toeprint
signal
B
Wild-type SNP 3435T SNP 3435A
Nucleotide
AUC
AUU
AUA
Codon usage
47% 35% 18%

Synonymous SNPs affect P-glycoprotein conformation and function
Wild-type Pgp
tRNAs
mRNA
G
G
G
G
G
G G
A
A
A A
A
A A
GGCXXXXXXXXGCTXXXXXXXXATCXXX
1236 2677 3435
I
I
The Pgp Haplotype
(rare tRNAs) or altered
mRNA structure
I
I
I
I
I
Wild-type: normal
kinetics of
translation
ATP
ATP
Drug-substrate
sites show different
conformations
tRNAs
G
G
I
A
G A
A
I
I
Haplotype: altered
kinetics of
translation
ATP
ATP
mRNA
GG XXXX CTXX XXXXXXAT X
TXX XXT TXX
SNP
(synonymous)
SNP
(non-synonymous)
SNP
(synonymous)
Interaction with antibody
and modulators is affected

P-gp expression on cell surface (MRK16)
of stably transfected LLC-PK1 cells
1 0 Antibody: MRK16
2 0 Antibody: Goat antimouse
FITC

P-gp expression on cell surface (UIC2) of
transfected LLC-PK1 cells
Conformation-sensitive
Monoclonal antibody

Vinca-alkaloids
Survival (%)
150
125
100
75
50
Vincristine
V
WT
3H
3HA
Survival (%)
150
125
100
75
50
Vinblastine
V
WT
3H
3HA
25
25
0
-12 -11 -10 -9 -8 -7 -6 -5
Concentration (M)
0
-11 -10 -9 -8 -7 -6 -5 -4
Concentration (M)

Taxol
Paclitaxel
Docetaxel
Survival (%)
125
100
75
50
V
WT
3H
3HA
Survival (%)
150
100
50
V
WT
3H
3HA
25
0
-11 -10 -9 -8 -7 -6 -5 -4 -3
Concentration (M)
0
-11 -10 -9 -8 -7 -6 -5 -4
Concentration (M)

Anthracyclines
Doxorubicin
Daunorubicin
Survival (%)
125
100
75
50
25
V
WT
3H
3HA
Survival (%)
125
100
75
50
25
V
WT
3H
3HA
0
-10 -9 -8 -7 -6 -5 -4 -3
Concentration (M)
0
-10 -9 -8 -7 -6 -5 -4 -3
Concentration (M)
Doxorubicin Daunorubicin

Implications
• Explains conservation of third position for many
codons
• Might explain some non-Mendelian inheritance
• Might explain linkage of phenotypes to other
synonymous polymorphisms
• For P-gp, the haplotype could have selective
advantage and/or affect drug distribution
• For cancers, could affect pattern of MDR and ability
to respond to specific inhibitors

ABC Transporters
Confer Resistance to
Anti-Cancer Drugs
Confers resistance
Selected
Doesn’t transport

Can we discover new drugs that interact with
ABC transporter genes?
• Use Real Time (RT)-PCR to measure ABC
mRNA levels for 48 ABC transporters and 23
solute carrier proteins
• Exploit NCI-60 cell line database, with known
resistance to 100,000 different drugs, to
correlate patterns of drug-resistance and
expression of these transporters

.
Expression of ABC-transporters in the NCI60 panel

Search for MDR1-potentiated
compounds in DTP’s s database
17K
Sensitivity (dlogGI50)
1
0.5
0
-0.5
-1
NSC 363997
-5 0 5 10 15
ABCB1 expression (dCP)
NSC 73306
NSC 363997
140
cell survival (%)
120
100
80
60
40
20
0
0.1 1 10 100 1000 10000
-1
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
-0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
concentration (uM)
Substrates
MDR1-potentiated compounds?

The cytotoxicity of NSC 73306 is
increased in KB-V1 cells
Sensitivity (dlogGI50)
1.5
1
0.5
0
-0.5
r = 0.54
-1
-1.5
-5 0 5 10 15
ABCB1 expression (dCP)
Cell survival (%)
120
100
80
60
40
20
0
1 10 100 1000
Concentration (µM)
KB-3-1
(MDR1-)
KB-V1
(MDR1+)
KB-3-1
+PSC 833
KB-V1
+PSC 833
NSC73306

Potential Clinical Utility of Discovery of
Compounds that Specifically Kill MDR1-
Expressing Cells
Can be used in combination with standard
chemotherapy to eliminate MDR1-expressing cell
populations
Preclinical development of thiosemicarbazones and
search for additional compounds with similar
properties is underway

Balance of uptake and efflux determines drug
accumulation in cancer cells

Solute Carriers are plausible uptake transporters
for anti-cancer drugs
OUT
IN
NH 2
1 2 3 4 5 6 7 8 9 10 11 12
SLCO: organic
anion transporting
polypeptide (OATP)
family
COOH
OUT
IN
W S L R N K R E EDTGALLSSGQKDYV NTS
H SN N
S
L
T VQLQNGEIWELSRCS
R P PGNVSQVVFH
C V H H
P
T
V
M F
G
A
V
L S
H I
Y
S G
I C
Q N
F
I A
F
C
RVLY
Q
G
R F
HF
G
V
H
D Y I
G
NH 2
M G S RHFE
G
Y
E
Y
T
G
S
K
K
E F P C VDG
N T W KST
Y I YDQ A
V
T
Q
W
N
L
V
D
C
R
K
T F M AAR
W
M A L
L Q I
L P
M F
F G
A F A V D YY F V
F
L V I A A M A L L V R T W W
L
S
G
V F T
F L F G
V
S A
G Y
L T G Y L Y
Y G
S S M V L L V A
M Q I T L S
F W A T S
G
V F
Y V
V G T L V T
V V L E
M F F A
V P L F I
F I H L H S C C
A S V
W V L
S
DR
R R
G
M K T W
L G S R
P
P
E
T
P
F
W
L L S E G R Y
V N L
S L N S
S Y S F
L G F
F T G
W L I W
L T V
F L N L L V L V G
I E
P T A F Y
C I
V
A
P
V I
L V V M
A C G
C S A
Y S L F
V L A
I L G V V T A M V G K F A I G A A F G L I Y L Y T A
P F S I L A I F R L A
P I Q V L S M V C G F
G S G
M T A S L L
S L A V L G V T
I V R L
S SIW
G G N EY
P Q K H Y S VDL
T K
DKV
R T E L Y P T L PETL
TKRT G R
A E E
Q
K
IVD
I M A K WN R A S S C K L W SI L
P
T L L
K
G
N Y
S
N T
K
S
S R
L
E
S L
D L
Q
G
L
P
V S N S
P T E V QK H N L S Y LF
NSGL
P
K L
P
S A
E T
L
N TWEEA
E G G S D E
R KTE E
P
S
COOH
T
E
I A
L K A
P
SLC22: organic
cation and anion
transporter (OCT
And OAT) families

Summary of SLCO and SLC22 Transporters
Most of the SLCO and SLC22 family members we tested are
expressed at some level in cancer cell lines.
By correlating the expression profiles with the growth
inhibitory profiles, expression of 3 of the SLCO and SLC22
family members were found to correlate with sensitivity to
specific drugs.
Expression of SLC22A4 in KB cells confers sensitivity to
mitoxantrone, doxorubicin, carboplatin and cisplatin.

Microfluidic technology using TaqMan Low Density Array
(TLDA)-based detection to study the mechanisms of
multidrug resistance in clinical samples
Goal: To correlate expression of drug-resistance genes with response to
chemotherapy in clinical samples
Method: a customized TLDA with probes for 380 drug-resistance genes enabling
high-throughput quantitative real-time PCR based on TaqMan chemistry
Port
1 to 8 samples
Channel
Each well contains a pair
of primers and a probe
12 to 380 targets
1, 2 or 4 replicates

The Anti-Cancer Drug Resistance Transcriptome

No ovarian cell line has a drug-resistance gene
expression profile similar to any clinical sample

Strategies for dealing with MDR1-
mediated multidrug resistance
Development of
specific inhibitors of P-
gp
Poor performance in
clinical trials for a
number of reasons:
-Poor trial design, e.g.,
cancers don’t
express MDR1
Can we use MDR1
Expression as an
Achilles
heel to kill MDR
cells?
-Side effects due to
inhibition of endogenous
functions
Drug structural variation
Dose escalation
Imaging of P-gp in vivo in cancers can
enable all of these strategies

Acknowledgements
• Gergely Szakacs
• Jean-Philippe Annereau
• Joe Ludwig
• Jean-Pierre Gillet
• Mitsunori Okabe
• Matthew Hall
• Chava Kimchi-Sarfaty
• Jung-Mi Oh
• Andy Fung
• Suresh Ambudkar
– Zuben Sauna
– InWha Kim
– Anna Calcagno
• Ira Pastan
• John Weinstein
• Carol Cardarelli
• Takaaki Abe
• Joe Covey
• Henry Fales