Cancer Therapy: Preclinical Synergistic Inhibition of ErbB Signaling by Combined Treatment with Seliciclib and ErbB-Targeting Agents IanN.Fleming,MoragHogben,SheelaghFrame,StevenJ.McClue,andSimonR.Green Abstract Purpose: The aims of this studywere to investigate whether the cyclin-dependent kinase inhi- bitor seliciclib could synergize with agents that target ErbB receptors and to elucidate the mole- cular mechanism of the observed synergy. Experimental Design: Synergy between seliciclib and ErbB receptor targeted agents was in- vestigated in various cell lines using the Calcusyn median effect model.The molecular mechanism of the observed synergy was studied in cultured cells, and the combination of seliciclib and the epidermal growth factor receptor (EGFR) inhibitor erlotinib was evaluated in an H358 xenograft model. Results: Seliciclib synergized with the anti-HER2 antibody trastuzumab in a breast cancer cell line, which overexpresses the HER2 receptor, and with the erlotinib analogue AG1478 in non ^ small cell lung cancer cell lines. In the H358 non ^ small cell lung cancer cell line, synergy involved decreased signaling from the EGFR, with AG1478 directlyinhibiting kinase activitywhile seliciclib decreased the levels of keycomponents of the receptor signaling pathway,resulting in enhanced loss of phosphorylated extracellular signal-regulated kinase and cyclin D1.The combination of seliciclib and erlotinib was evaluated further in an H358 xenograft and shown to be significantly more active than either agent alone. An enhanced loss of cyclin D1was also seen in vivo. Conclusions: This is the first report that investigates combining seliciclib with an EGFR inhibitor. The combination decreased signaling from the EGFR in vitro and in vivo and was effective in cell lines containing either wild-type or mutant EGFR, suggesting that it may expand the range of tumors that respond to erlotinib, and therefore, such combinations are worth exploring in the clinic. Protein kinases are key regulators of essential cellular ErbB receptor tyrosine kinase signaling has been documented processes, many of which are involved in the pathogenesis of in a number of human cancers, including overexpression of cancer. This important role in tumorigenesis, together with the HER2 (ErbB2) in up to 30% of breast cancer patients (4), fact that kinases contain a distinct active site, which is amenable whereas epidermal growth factor receptor (EGFR; ErbB1) is for targeting with small molecules, has made kinases an overexpressed in ovarian carcinomas (35-60%), head and neck attractive area for drug discovery (1). However, it was not until tumors (70-100%), and non–small cell lung cancer (NSCLC; the successful clinical development of imatinib, a small 50-90%; refs. 5, 6). Two main approaches have been pursued molecule inhibitor of BCR-ABL, that the first targeted protein for the development of drugs that target the ErbB family. kinase inhibitor became an established cancer treatment (2). Humanized monoclonal antibodies, such as trastuzumab and Members of the ErbB family, like many other growth factor cetuximab, bind to the HER2 and EGFRs, respectively, thereby receptors, dimerize upon ligand stimulation and undergo blocking receptor dimerization/activation and facilitating re- autophosphorylation through their cytoplasmic tyrosine kinase moval of these proteins from the cell surface (3). In contrast, domains. Once activated, the ErbB kinases transduce their erlotinib and gefitinib are small molecules that bind directly to signals via a number of cellular protein kinases, including the EGFR ATP-binding active site, blocking tyrosine kinase extracellular signal-regulated kinase (ERK) and protein kinase activity (7). For each of these agents, the subsequent loss of B, to ultimately result in up-regulation of cyclin D1 levels mitogenic signaling results in the cessation of cellular proli- leading to activation of cyclin-dependent kinases (CDK) and feration. the initiation of cellular proliferation (3). Overstimulation of The CDKs are a second kinase family that have attracted a considerable amount of interest from a drug discovery perspective; with the three most advanced compounds, Authors’ Affiliation: Cyclacel Ltd., Dundee, United Kingdom seliciclib (CYC202, R-roscovitine), alvocidib (flavopiridol), Received 10/15/07; revised 2/6/08; accepted 4/2/08. and SNS-032 (formerly BMS-387032), all currently in phase Requests for reprints: Ian N. Fleming, Cyclacel Ltd., 1 James Lindsay Place, II clinical development (8, 9). Through their key role of Dundee, DD15JJ, United Kingdom. Phone: 44-1382-206062; Fax: 44-1382- 206067; E-mail: [email protected]. phosphorylating proteins involved in the regulation of cell F 2008 American Association for Cancer Research. cycle checkpoints, CDKs control the orderly progression of the doi:10.1158/1078-0432.CCR-07-4633 cell division cycle (8). In cancer cells, activation of CDKs, Clin Cancer Res 2008;14(13) July 1, 2008 4326 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. Synergy between Seliciclib and ErbB-Targeting Agents mainly by overexpression of their cognate partners, the cyclins, After drug treatment, the number of viable cells in each well was or loss of the endogenous inhibitors, such as p16, leads estimated by incubating in media containing 10% alamar blue (Roche) to inappropriate proliferation of cells containing genetic and measuring the absorbance at 488 to 595 nm. Drug interactions alterations, cells which would normally be arrested and either were analyzed using the Calcusyn software package (BioSoft), which is based on the median effect model of Chou and Talalay (22). A repaired or induced to undergo apoptosis at specific cell cycle combination index (CI) of 1 indicated an additive drug interaction, checkpoints (10). In addition to controlling the cell cycle, some whereas a CI of >1 was antagonistic and a score lower than 1 was CDKs, such as CDK7 and CDK9, regulate transcription by synergistic. phosphorylating the carboxy-terminal domain of RNA poly- Western blot analysis. Cells were seeded at f8 Â 105 per plate on merase II. Seliciclib, alvocidib, and SNS-032 all inhibit CDK7 10 cm plates and left to settle overnight. Compounds were added to the and/or CDK9, thereby leading to inhibition of transcription plates, and cells were incubated for the indicated times. Media were and down-regulation of proteins, such as Mcl-1 and cyclin D1 removed from each of the wells and centrifuged at 1,000 Â g for 5 min (11, 12), which have short half-lives (13, 14). to pellet any cells that had become detached from the plate surface. Inhibitors of both kinase families have shown some clinical Cells that remained attached to the plate were washed once with ice- activity, erlotinib in NSCLC (15), trastuzumab in breast cancer cold buffer A [50 mmol/L HEPES (pH 7.0), containing 20 mmol/L NaCl, 1 mmol/L DTT, and protease inhibitor cocktail (Merck)] and (16, 17), and alvocidib in B-cell chronic lymphocytic leukemia then scraped into 0.35 mL buffer B (buffer A containing 10 mmol/L (18). However, most agents will be used in the clinic ultimately sodium PPi, 10 mmol/L sodium fluoride, and 1 mmol/L sodium in combination with other anticancer drugs. It was recently orthovanadate). The resuspended cells were then pooled with the shown that the combination of trastuzumab and alvocidib was appropriate media cell pellet and lysed by sonication (2 Â 3 bursts synergistically cytotoxic in HER2-overexpressing breast cancer using Sanyo Soniprep 150 at 5 A). The protein concentration of each cell lines (19–21). To initially ascertain whether seliciclib could lysate was determined using the bicinchoninic acid assay (Perbio also be combined synergistically with trastuzumab, the Science). Lysates (20-30 Ag protein/well) were resolved on 3% to 8% combination was evaluated in the SkBr3 breast cancer cell line acrylamide Tris-acetate gels or 10% acrylamide Bis-Tris gels (Invitro- that overexpresses the HER2 receptor. These studies were then gen), and the proteins were transferred to nitrocellulose membranes extended to determine whether seliciclib could act synergisti- (Schleicher & Schuell) using the Invitrogen wet transfer system. Membranes were blocked for 1 h at room temperature in PBS cally in combination with agents, such as AG1478 and containing 0.02% (v/v) Tween 20 (PBST) and 5% (w/v) fat-free dried erlotinib, which target the EGFR, a second member of the ErbB milk. Primary antibody incubations were carried out overnight at 2 to family. The results from these studies showed that seliciclib 8jCin PBST containing 3% (w/v) dried milk, using the following synergized with trastuzumab in SkBr3 cells and with AG1478 in primary antibodies: HER2 and EGFR (Calbiochem), phosphorylated the NSCLC cell lines H358, H292, and H1650, which express Tyr1068 EGFR (New England Biolabs), cyclin D1 (Lab Vision), ERK2, wild-type or mutant EGFR. In each case, synergy involved and phosphorylated ERK1/ERK2 (Abcam), phosphorylated Ser473 inhibition of the ErbB signaling pathway, which could be protein kinase B (Upstate), Asp-214 cleaved poly(ADP)-ribose poly- shown both in vitro and in vivo in an NSCLC xenograft model. merase (BD PharMingen), Mcl-1 (Santa Cruz), CTD RNA polymerase II phosphorylated Ser2 (Covance), and h-actin. Membranes were washed thrice in PBST and then incubated for 1 h with the appropriate horseradish peroxidase–conjugated secondary antibody (Perbio) at Materials