Transcript Slide 1

Resistance in the Clinical Setting
Dr. Wilson H. Miller, Jr
Potential Conflict of Interest
Resistance in the Clinical Setting
Wilson H. Miller Jr., M.D., Ph.D.
Segal Cancer Center
SMBD Jewish General Hospital
McGill University, Montreal, Quebec Canada
Mechanisms of Cellular Drug Resistance
Intrinsic Resistance Mechanisms
Host factors
•Decreased intracellular drug accumulation (poor absorption, rapid
metabolism, or excretion).
•Inefficient delivery of a drug to its target (tumor cells).
Specific genetic and epigenetic drivers
•Malignant cell growth is associated with tumor-specific activation of
oncogenic pathways and inactivation of tumor suppressor genes.
•Specific drug targets may or may not be relevant to growth of a given
tumor.
The wrong target cell?
•Stem cell resistance
Mechanisms of Cellular Drug Resistance
Acquired Resistance Mechanisms
Decreased accumulation of drugs within cells
• Increased drug efflux.
• Reduced drug uptake.
Changes in drug-target interactions
• Mutations in targeted oncogenes.
• Changes in target gene expression.
Changes in signaling pathways that drive growth
• Replacement of one TK pathway with another.
• Interchangeable pro-angiogenic factors and pathways.
• Multiple interdependent cell survival pathways.
• Loss of checkpoints.
Three Main Mechanisms of Cellular Drug Resistance
(1) Decrease in intracellular
drug concentrations
(2) Changes in drug-target
interactions
(3) Changes in signal
transduction pathways
Mutation
Cell cycle arrest and repair
Decrease in Intracellular Drug Concentrations
Decrease of drug influx
• Alterations of cell membrane structures.
• Most chemotherapeutic drugs enter cells by passive diffusion.
Increase of drug efflux
• Overexpression of transmembrane proteins (ABC superfamily of
transporters).
LRP/MVP
Is the major
component of the
Vault protein
Involved in cellular
traffic
Examples of Chemotherapeutic Drugs with
Increased Delivery to Tumors
SarCNU Rationale
• SarCNU is a novel chloroethylnitrosourea which
demonstrates selective cytotoxicity against primary
human gliomas in-vitro.
• Selective uptake via the extraneuronal catecholamine
uptake carrier allows increased concentration in
tumor cells.
• Preclinical toxicity studies confirm that SarCNU is
less toxic than BCNU, the standard treatment of
gliomas.
Examples of Chemotherapeutic Drugs with
Increased Delivery to Tumors
SarCNU
• Phase I and pharmacokinetics study in advanced
solid tumor malignancy.
• 43 patients enrolled.
• Myelosuppression and some pulmonary toxicity
observed in patients.
Examples of Chemotherapeutic Drugs with
Increased Delivery to Tumors
Darinaparsin: Organic Arsenic
• First in a new class of molecules.
• Potentially safer and more active for cancer treatment
than approved inorganic arsenic.
H3C
O
S
O
HO
As
O
H
N
N
H
NH2
CH3
O
OH
Darinaparsin (DAR) is more potent than As2O3 at inducing
apoptosis in a variety of leukemia and lymphoma cell lines.
NB4 (APL)
IM9 (NHL)
AsR2 (As-resistant APL)
CCRF-CEM (NHL)
Diaz et al, 2009 Feb;23(2):431
DAR induces more cellular oxidative stress than As2O3.
3
***
2
1
**
Protein Carbonyls
(nmol/mg protein)
NB4
(APL)
NB4
(APL)
NB4
cells
AR2
cells
AsR2
(APL)
AsR2
(APL)
***
2
1
*
AR
D
1
M
2O
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As
1
M
D
0.
5
M
As
2O
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5
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As
2O
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2O
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tro
NB4 (APL)
AR
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C
Protein Carbonyls
(nmol/mg protein)
3
AsR2 (APL)
Diaz et al, 2009 Feb;23(2):431
Increased ABCC1 exporter expression causes resistance
to As2O3 but not DAR in the arsenic-resistant cell line AsR2.
NB4 cells 10.0
5.0
ABCC1/GAPDH
Relative quantity
Arsenic (ppb)
7.5
2.5
0.0
10
10
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6
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AsR2 cells
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0
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AR
M
1.
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As
M
1.
0
D
2O
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TL
0
C
Arsenic (ppb)
12
NB4
AsR2
ATO-resistant NB4-AR2 cells, are sensitized to ATO, but
not DAR, by co-treatment with an ABCC1 inhibitor.
A.
B.
Total intracellular As in AsR2 treated for 24hrs.
17.5
7
15.0
6
12.5
5
10.0
7.5
**
As (ppb)
cell number (x10 4 cell/ml)
Viable cell number in AsR2 treated for 24hrs.
4
3
5.0
2
2.5
1
0
0
Examples of Chemotherapeutic Drugs with
Increased Delivery to Tumors
Hybrid Molecules –
Targeting the Oncogene with Two Therapeutic Agents
Me Me
Me
Me O
Retinoic Acid
O
OH
Me
Butyric Acid
OH
Me
Me
Me
Me
Me
O
O
O
RN1
O
Me
Me
Figure 2. Chemical structure of RN1 and it’s precursors.
RN1 induces growth arrest in NB4 and R4 cell lines.
R4
140
80
120
70
cell number (x 10,000)
cell number (x10,000)
NB4
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days
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days
NB4 and R4 cells were treated with media, 10-5 M RA, butyrate, RA plus
butyrate, or RN1. In NB4 cells, RA, RA plus butyrate, and RN1
significantly inhibited growth (P<0.001). In R4 cells, RN1 significantly
inhibited growth (P<0.02).
Imatinib Treatment in CML
Chronic Myeloid Leukemia (CML)
• Characterized by the
Philadelphia chromosome
t(9;22).
• Results in fusion of BCR and
ABL genes.
• Imatinib mesylate is the
frontline therapy.
• Imatinib is a selective inhibitor
of Bcr-Abl, PDGF-R, Kit.
Imatinib Treatment in CML Models Multiple
Resistance Mechanisms
 Imatinib has revolutionized treatment for CML but resistance is a problem
in a small percentage of patients.
Primary resistance
• Insufficient inhibition of BCR-ABL
• Low plasma levels of imatinib.
• Activity of drug pumps.
• Stem cells
Secondary resistance
• Imatinib-resistant BCR-ABL kinase-domain mutations.
• Overproduction of BCR-ABL (genomic amplification).
• BCR-ABL-independent mechanisms (not well understood).
• ? Activation of other kinases.
• ? Other molecular events.
BCR-ABL Mutations
Associated with Imatinib Resistance
V299L
L298V
E292V
P-loop
Activation loop
Most mutated clones, except for T315I, may be eradicated with appropriate
choice and combination among the second generation Abl TKIs (Dasatinib,
Nilotinib, Bosutinib).
CML Stem Cells – Resistance to TKI’s
Persistence of minimal residual disease
Possible mechanisms of stem cell resistance
• High levels of ABC drug transporters.
• Increased capacity for DNA repair.
• Accumulation of mutations.
• Quiescence.
Therapeutic Approaches for Stem Cell Resistance
• Targeting the ABC transporters.
• Targeting the different surface markers.
• Targeting the pathways in stem cell renewal.
• Targeting the quiescence.
Resistance in Signal Transduction Pathways –
HER2 (ERBB2)
HER2 (ERBB2) Driven Breast Cancer
• Overexpression of the Her2 (ErbB2) protein found in 18-20% of breast tumors.
• Correlates with more aggressive tumors.
Current targeted therapies
Trastuzumab (Herceptin) – monoclonal Ab specifically targets Her2.
• Treatment for early stage HER2+ breast cancer.
• Resistance in vast majority of patients occur within 1 year.
• HER2 mutations not commonly found.
Lapatinib -TKI inhibitor
• Inhibits Her2 and EGFR.
Current Therapies to Overcome Trastuzumab Resistance
Lapatinib -TKI inhibitor
• This combined inhibition can
overcome Herceptin resistance
in some cases.
LBH589 – Deacetylase inhibitor
• Induces degradation of Her2,
ER and pAKT.
• Phase Ib/IIa LBH589 in combo
with Trastuzumab for HER2+
metastatic breast cancer.
• Enhances Her2 inhibition in combo
with Trastuzumab or Lapatinib
Resistance in Signal Transduction Pathways:
The importance of KRAS, BRAF and EGFR mutations in
EGFR signaling in Colon Cancer
Ligand binding to EGFR
promotes heterodimerization,
activation and downstream
pathways;
• Ras-Raf
• MAPK
• PI3K-Akt
The importance of KRAS status in
Metastatic Colorectal Cancer
•Ab against EGFR (Cetuximab and
Panitumumab) inhibit downstream
pathways.
•Mutated KRAS or BRAF leads to
constitutive activated pathway.
•Mutated KRAS (~30% pts)
•Mutated BRAF (~10% pts)
•Cetuximab and Panitumumab
Only effective in KRAS and BRAF
wild type tumors.
Response to Cetuximab According to the Presence or
Absence of KRAS Mutation in the Overall 114 Patients
KRAS mutation
Tumor Response
Wild type KRAS
No. of Patients
%
No. of Patients
%
P
CR
0
0
2
2.6
<
.001
PR
0
0
32
41.0
SD
14
38.9
26
33.3
PD
22
61.1
18
23.1
Total
36
100
78
100
Lievre, A. et al. J Clin Oncol; 26:374-379 2008
(A) Progression-free survival (B) overall survival according
to the presence or absence of KRAS mutation
PFS
32 vs. 9 weeks
P = 0.0000001
Lievre, A. et al. J Clin Oncol; 26:374-379 2008
OS
14.3 vs. 10.1 months
P = 0.0017
Copyright © American Society of Clinical Oncology
Signal Transduction Pathways:
The importance of KRAS, BRAF and EGFR mutations in EGFR
signaling in lung adenocarcinoma
Oncogene mutations in the EGFR
pathway in lung adenocarcinoma
• About 50% of lung adenocarcinoma harbor somatic
mutations of six genes that encode proteins in the EGFR
signaling pathway:
–
–
–
–
–
–
KRAS mutations
EGFR mutations
Her-2 mutations
Her-4 mutations
BRAF mutations
Phosphatidylinositol 3-kinase (PI3K) mutations
KRAS mutations in lung adenocarcinoma
• KRAS mutation in 30% of
lung adenocarcinoma.
• Association with smoking.
Poor prognostic factor in
resected tumors.
• Lack of sensitivity of KRAS
mutated tumors to geftinib
or erlotinib (EGFR
inhibitors).
Activating Mutations in the EGFR Correlate with
EGFR-TKI Sensitivity
EGFR mutations in lung adenocarcinoma
associated with sensitivity but additional mutations can
mediate resistance
Sharma, Nat Rev Cancer, 2007
Resistance in Angiogenic Targeted
Therapy
Current Angiogenic Inhibitors in Clinical Use
and Clinical Trials
•
•
•
•
•
Bevacizumab (Avastin)
Sunitinib (Sutent)
Sorafenib (Nexavar)
Cederanib (Recentin - AZD- 2171)
VEGF-Trap
Many others in development
Modes of Resistance to Anti-Angiogenic Therapy
Upregulation of pro-angiogenic signaling pathways
• FGF, ephrin and angiopoietin families.
Recruitment of BM derived cells
• Endothelial and pericyte progenitors are incorporated as
components of new vessels to build new blood vessels
• Pro-angiogenic monocytes fuel the tumors with cytokines,
growth factors and proteases.
Increased pericyte coverage protects tumor blood vessels
• Helps tumor endothelium to survive and function.
Overcoming Resistance to Anti-Angiogenic Therapy
• The combination of antiangiogenesis agents with cytotoxic
chemotherapy has increased the activity of chemotherapy in
breast, colon, lung cancer and in melanoma.
• Data on toxicity of targeted agents in older individuals are
limited: the risk of thrombosis with avastin and of serious
cutaneous reactions with cetuximab appears to increase with age.
Conclusions
Overcoming Resistance
• Targeted therapy has been very successful in situations
where a single or few targets are responsible to maintain
the disease (CML, HER2 positive breast cancer).
• Inhibiting a single target in a complex signaling pathway
is unlikely to provide sufficient therapeutic activity for the
treatment of most genetically unstable human cancers.
-Multiple activating signals and cross talk.
-Signals transmitted via multiple pathways.
• The combination of 2 or more targeting agents seems to
be more effective and safer, at least in the case of
inhibition of the signal transduction cascade.
Conclusions: More work needed
• Need to continue to characterize mechanisms of action,
mechanisms of resistance, signaling pathways.
• Continued research to improve our understanding of the
heterogeneity and complexity of the tumor microenvironment.
• Continue to identify mutations in targeted oncogenes and
targets in the downstream pathways.
• The use of technological advances in genomics, proteomics
and biomarker development to better predict tumor types and
patient subsets that may be particularly responsive to
treatment.
The Importance of Pharmacodynamic Markers
Anti-estrogens
ER, PR
Trastuzumab
Her2 FISH, IHC
EGFR inhibitors
?FISH, ?IHC, mutation status
Anti-VEGF agents
??
PI3K-Akt-mTOR
??
IGF-I receptor antagonists
??
Src inhibitors
??
Cdk/Cyclin D1 inhibitors
??
HDAC/DNMT inhibitors
??
Gene expression
Preclinical
Enzyme activity
Samples
Clinical
Tumor cell markers
Metabolomics
Experiments
Analysis
•Data processing
•Data integration
•Pathway linkage
•Analysis
•Data coherence
Informatics
B
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A
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Discovery
Translational research should be
part of the solution
The complexity of resistance in patients demonstrates the need for
• Developing new models of
– Multi-disciplinary and multi-institutional collaborations
– Academic and industrial partnerships
• Designing biomarker-driven clinical trials to
– Collect clinical samples
– Identify biomarkers predicting resistance
– Study mechanism of resistance identified in patients (vs. in cell
lines)
– Develop new or improved molecules
The Quebec – Clinical Research Organization in Cancer was designed
to answer these challenges.