Download O-13928 DD Combining Targeted Agents Poster Handouts 2nd

Combining targeted agents against cell signaling components involved in cancer:
Beta-lactamase’s robustness and versatility as a functional cell-based reporter
Combining targeted agents against cell signaling components involved in cancer: Beta-lactamase’s robustness and versatility as a functional cell-based reporter
Michael O’Grady¹, Bonnie Hanson¹, George Hanson¹, and Debasish Raha²
1: Invitrogen Corporation • 501 Charmany Drive • Madison, Wisconsin 53719 • USA
2: Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, California 92008 • USA
introduction
The important role protein kinases play in cellular pathways has led to their becoming the
EGF
EGF
EGF
EGF
second most important group of drug targets
hibitors of pathway components for elucidation of the kinase involved and possible therapeutic effects. Resistance
to such inhibitors has already been seen (2), guiding the
EGF Receptor
Stealth™ RNAi
Cell Membrane
EGF Receptor
(1). Currently, efforts have centered on small molecule in-
Extracellular
GRB2 SOS-1
GTP
RAS
GDP P
Shc
GDP
RAS
GTP
GAP
GRB2
P
prediction of their use in combination therapies with other
SOS-1
targeted agents such as RNAi (3,4). In addition, interrogating
RAF1
MEKK1
MEK1
JNKK1
ERK
JNK1
cellular signaling pathways with RNAi to identify additional
U0126
targets that act synergistically with a specific kinase inhibitor is a technical approach that may aid in directing kinase
c-FOS
ELK-1
drug discovery. The use of a functional cell based reporter
system to analyze the effects of RNAi on the dose response
AP-1
of inhibitors towards components of the epidermal growth
c-FOS c-JUN
factor receptor (EGFR) pathway shows the potential of this
ELK-1
activated
c-JUN
DNA
AP-1 Response Element
Nucleus
Cytoplasm
“cocktail” method.
EGFR pathway shown with an EGF agonist stimulated signal cascade leading to
AP-1 activation. The RNAi (EGFR) and small molecule inhibitor (U0126) targeted
agents are shown at the point in the cascade which they effect.
Figure 2—EGF dose response
3
Blue/Green Ratio
EC80 = 1 ng/ml
2
1
0
0.001
0.01
0.1
1
10
100
1000
[EGF] (ng/ml)
1 ng/ml EGF
0 ng/ml EGF
A stable ME180 cell line was established using the CellSensor™ technology for analysis of the
EGFR signaling pathway.
A dose response curve was performed using an EGF agonist in complete media with serum.
Subsequent inhibition studies were performed at EC₈₀ of 1 ng/ml.
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Combining targeted agents against cell signaling components involved in cancer:
Beta-lactamase’s robustness and versatility as a functional cell-based reporter
Figure 3—kinase inhibitor panel
100
90
80
60
50
40
30
20
10
0
DMSO
Rott
KN62
SB202190
PD98059
UO126
AG1478
AG490
PD153035
CL387785
-10
AG183
% Inhibition
70
Inhibition @ 500 nM
AG183
U0126
PD153035
DMSO
A panel of small molecule kinase inhibitors was analyzed for the ability to inhibit the EGFR pathway. Inhibitors targeting EGFR (CL387785, PD153035, AG1478) or MEK1 (U0126) kinase exhibit
potency in the functional cell-based assay.
3
Figure 4—EGFR RNAi analysis
150
16 hr
Normalized Response
40 hr
60 hr
100
50
0
EGFR
Med GC
beta-lactamase
RNAi
40 hr
60 hr
EGFR
Med GC control
beta-lactamase
RNAi targeting the EGFR was used for further validation of pathway components and effects on functional AP-1 response. Timecourse studies indicate that knockdown of EGFR
expression leads to a loss of AP-1 activation by EGF.
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Combining targeted agents against cell signaling components involved in cancer:
Beta-lactamase’s robustness and versatility as a functional cell-based reporter
Figure 5—combining RNAi against EGFR and a small molecule inhibitor towards MEK1
4
U0126 alone
EGFR RNAi + U0126
Blue/Green Ratio
3
Med GC control
2
EGFR RNAi alone
1
beta-lactamase RNAi alone
0
0.001
0.01
0.1
1
10
100
[U0126] (µM)
2 µM
0.2 µM
U0126 Alone
IC50 = 543 nM
U0126 + EGFR RNAi
IC50 = 192 nM
Blue Cells: beta-lactamase expressing; Green Cells: non-expressing
RNAi and a small molecule kinase inhibitor shown to have inhibitory properties in the AP-1 CellSensor™ assay were used in tandem. RNAi targeting the EGFR was kept constant while U0126
targeting MEK1 kinase was used in a dose response manner to examine potency effects of the
combined treatment. Controls for RNAi transfection (Med GC), EGFR knockdown (EGFR RNAi),
and beta-lactamase knockdown were used to set standards for transfection, single treatment,
and maximal effects respectively. The shift in IC₅₀ indicates the “cocktail” effect seen with the
dual targeted treatment.
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Combining targeted agents against cell signaling components involved in cancer:
Beta-lactamase’s robustness and versatility as a functional cell-based reporter
Results and conclusions
• Stable cell lines incorporating the beta-lactamase reporter gene were established for analysis of the EGFR pathway. The
Activator Protein-1 (AP-1) response element (5) was used for the functional study of EGF-stimulated signal transduction
events in this pathway of interest.
• Kinase inhibitors as well as RNAi were used for validation of the pathway, and to analyze possible combinatorial effects
when used in tandem.
• Small molecule kinase inhibitors tested against components of the EGFR pathway show potency at the receptor level (e.g.
AG1478, PD153035) as well as cytosolic level (U0126) when assayed using an AP-1 functional cell-based assay. RNAi targeting
EGFR showed a >70% reduction in AP-1 activation using the same functional beta-lactamase assay, indicating potential
therapeutic use of RNAi.
• To determine whether there were additive effects, which could be relevant in therapeutic efficacy and prevention of drug
resistance (3,4), we performed experiments using small molecule inhibitors in combination with RNAi. U0126, a known
MEK1 inhibitor (6), exhibits an IC₅₀ of 543 nM when used in isolation on the AP-1 cell line. Upon combination with RNAi
against EGFR, the IC₅₀ for the MEK1 inhibitor shifts to 192 nM indicating possible benefits for combining kinase inhibitors
with other targeted agents such as Iressa, Herceptin, or RNAi for EGFR.
References
1. Cohen, P. (2002) Nature Reviews: Drug Discovery 1: 309-15.
2. Arteaga, C., and Baselga, J. (2004) Cancer Cell 5: 525-31.
3. Melnikova, I., Golden, J. (2004) Nature Reviews: Drug Discovery 3: 993-4.
4. Daub, H., Specht, K., and Ullrich, A. (2004) Nature Reviews: Drug Discovery 3: 1001-10.
5. Turner, R. and Tjian, R. (1989) Science 243: 1689-94.
6. Duncia, J.V., et al. (1998) Bioorgan. Med. Chem. Lett. 8: 2839.
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