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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 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. 2 w w w. i nv i t r o g e n . co m 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. 4 w w w. i nv i t r o g e n . co m 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. 5 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. These products may be covered by one or more Limited Use Label Licenses (See the Invitrogen catalog or www.invitrogen.com). By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. For research use only. Not intended for any animal or human therapeutic or diagnostic use. ©2005 Invitrogen Corporation. All rights reserved. Reproduction forbidden without permission. Printed in the U.S.A. 0-13928-r1 US 0305 0M w w w. i nv i t r o g e n . co m