Synergistic Inhibition of Erbb Signaling by Combined Treatment

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 inhibitor seliciclib could synergize with agents that target ErbB receptors and to elucidate the molecular 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 and Methods 1:5,000 dilution in PBST containing 3% (w/v) milk. Membranes were washed thrice in PBST before development using an enhanced Cell lines, cell culture, and reagents. MCF7, A549, H460, SkBr3, chemiluminescence kit (Amersham Corporation). Bands corresponding H1650, H292, and H358 cells were purchased from American Type to each protein were scanned, and the band intensities were measured 1 Culture Collection. Cell cultures were maintained in DMEM, except for using NIH image. H1650, H292, and H358 cells, which were grown in RPMI. Cells were Flow cytometry. H358, H292, or H1650 cells were seeded in 10 cm Â 5 cultured at 37jCin a humidified atmosphere of 5% CO in media plates at f5 10 per plate and left to settle overnight. Cells were 2 Â containing 10% (v/v) FCS, 100 units/mL penicillin, and 100 Ag/mL treated with 1 IC50 seliciclib, AG1478, or both drugs together. After streptomycin. All reagents were purchased from Sigma, unless stated 72 h treatment, cells were harvested by trypsinization, washed twice in otherwise. PBS, and then fixed overnight in 70% (v/v) ethanol at -20jC. Cells were Drug combination analysis. Experiments were done in 96-well washed twice in PBS containing 1% (w/v) bovine serum albumin and plates. SkBr3 and MCF7 cells were seeded at a density of 5,000 per then incubated with 50 Ag/mL propidium iodide and 50 Ag/mL RNase well in media containing 1% (v/v) FCS. H358 and H460 cells were A for 20 min at room temperature. Cells were analyzed for DNA seeded at 3,000 per well and A549 cells at 2,000 per well in media content by flow cytometry using the CellQuest program on a Becton containing 10% (v/v) FCS. Stock solutions of seliciclib (Cyclacel Ltd.) Dickinson LSR flow cytometer. and AG1478 (Tocris Bioscience) were prepared in DMSO, whereas In vivo studies. All in vivo experiments were done at Institute for Â trastuzumab (Genentech, Inc.) was dissolved in 0.9% (w/v) sterile saline Drug Development. Female (nu/nu) mice were injected s.c. with f1 7 solution. Serial dilutions (1.5-fold) for each compound were prepared, 10 H358 cells per mouse at a single site on their flanks. Tumors were f 3 with the concentration range spanning the IC50 value of each drug. allowed to grow to 110 mm before being pair-matched by tumor To assess potential synergistic interactions, the concomitant treat- size into treatment groups (nine mice per group). One group was ment regimen involved simultaneous treatment of cells with seliciclib treated with seliciclib (50 mg/kg) as a twice daily i.p. injection for 5 and either trastuzumab or AG1478 for 72 h, alongside suitable controls consecutive days followed by a 2 day break; the treatment was then of cells treated with the individual compounds alone for 72 h. In the repeated for a total of four cycles. Erlotinib (100 mg/kg) was given daily sequential treatment regimens, one drug was added to the cells 2 h after by oral gavage for 28 consecutive days. The group treated with the plating and left for 24 h. Media were then aspirated, replaced with fresh combination was dosed in the same manner as both single-agent media containing the second drug, and incubated for a further 72 h. groups. Mice were weighed at least twice a week to assess toxicity of the The two individual treatment controls for the sequential treatment regimen involved substituting one of the drug treatments with drug-free media. All treatments were done in triplicate wells. 1http://rsb.info.nih.gov/ij/

www.aacrjournals.org 4327 Clin Cancer Res 2008;14(13) July 1, 2008 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical

Fig. 1. Seliciclib plus trastuzumab down-regulates HER2 levels in SkBr3 cells. A, SkBr3 cells were treated for 72 h using increasing concentrations of seliciclib and trastuzumab, either alone or in a fixed ratio of 1,400:1, as described in Materials and Methods.The data were analyzed using the Calcusyn program, and a graph from a representative experiment is shown. CI values are defined in the legend forTable 1.SkBr3 cells were incubated with the indicated concentration of trastuzumab (B), seliciclib (C), or seliciclib + trastuzumab (D) for 24 h before harvesting. Protein lysates (30 Ag) were resolved on 3% to 8% acrylamideTris-acetate gels or 10% acrylamide Bis-Tris gels, transferred to nitrocellulose membranes, and probed with the antibodies shown. Results are representative of two independent experiments. treatments, and the tumors were measured with calipers at least twice a Endogenous peroxidase activity was blocked by treating the sections week to determine tumor growth. During the first week of treatment, with 0.1% (v/v) hydrogen peroxide in PBS for 20 min, followed there was some weight loss in the animals. However, this seemed to be by two 5 min washes in PBS. The sections were boiled for associated with the initial vehicle used for seliciclib (50 mmol/L HCl), 10 min in 10 mmol/L citrate buffer (pH 6.0) containing 0.05% (v/v) as the weight loss also occurred in the vehicle control group (max Tween 20. The slides were allowed to cool for 10 min and then washed weight loss, 11% between days 8 and 11). On day 10, the vehicle was twice in PBS. Immunohistochemistry staining of the sections was done changed to 10% cremophor EL, 10% ethanol, and 80% saline for the using the Vectastain avidin-biotin complex method kit (Vector control and the two groups receiving seliciclib; all three groups started Laboratories) following the manufacturer’s instructions. The primary gaining weight immediately. Tumor measurements were converted into antibody was raised against cyclin D1 (SP4, Abcam) and was used at a volumes using the formula: tumor volume (mm3) = width2 (mm) Â 1:50 dilution. On completion of the immunostaining process, sections length (mm) Â 0.52. The percentage of tumor growth inhibition was were washed for 5 min in water, counterstained for 30 s with Mayer’s determined with the formula: 1 - (mean change in treated tumor hematoxylin, and washed for 10 min in water. Sections were then volume / mean change in control tumor volume) Â 100. For each dehydrated through 70% (v/v) alcohol and 95% (v/v) alcohol, twice for group, statistical significance was determined by comparing to the 5 min in each solution and Histoclear twice for 10 min. Finally, sections control group using a one-way ANOVA followed by a Dunnett’s test. were mounted with a drop of DPX resin (BDH) and a coverslip. Slides Significance between different treatment groups was determined using a were viewed using a Nikon eclipse E800 light microscope, and images two-sided unpaired Student’s t test. were taken with a DS-Fi1 Nikon color digital camera. Axiovision viewer Immunohistochemical studies. Female (nu/nu) mice were injected was used to prepare images of the sections. s.c. with f1 Â 107 H358 cells per mouse at a single site on their flanks. 3 Tumors were allowed to grow to f110 mm before being pair-matched Results and Discussion by tumor size into treatment groups (two mice per group). Treatment was for 5 consecutive days; erlotinib (100 mg/kg) was given daily by The aim of this series of experiments was to determine oral gavage, whereas seliciclib (100 mg/kg) was given as a twice daily whether agents that target either CDKs or ErbB receptors can be i.p. injection. On day 5, the mice were sacrificed and the tumors combined synergistically. Recent experiments have shown that removed, fixed overnight in formalin, and then preserved in paraffin blocks. the anti-HER2 antibody trastuzumab synergizes with the CDK9 Slices (4 Am) were cut from the paraffin blocks; wax was removed inhibitor alvocidib (19–21). The data presented herein extend from the tissue sections by washing twice for 10 min in Histoclear these findings by exploring the synergy between seliciclib, an (National Diagnostics). The sections were then rehydrated in 95% (v/v) inhibitor of CDK2, CDK7, and CDK9, and either trastuzumab alcohol, 70% (v/v) alcohol, and PBS twice for 5 min in each solution. or the EGFR kinase inhibitors, AG1478 and erlotinib. The

Clin Cancer Res 2008;14(13) July 1,2008 4328 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

receptor levels by seliciclib was most likely due to its inhibitory Table 1. Summary of combination studies effects on CDK7 and CDK9 (25, 26), resulting in decreased involving seliciclib and either trastuzumab or the phosphorylation of the carboxy-terminal domain of RNA EGFR inhibitor AG1478 polymerase II with a subsequent reduction in transcription of the receptor mRNA and loss of the HER2 protein through Cell line Schedule CI at ED50 degradation (27). The effect of seliciclib on transcription was SkBr3 Seliciclib + trastuzumab 0.66 confirmed by examining the extent of phosphorylation of the MCF7 Seliciclib + trastuzumab 0.96 H358 Seliciclib/AG1478 0.96 carboxy-terminal domain of RNA polymerase II (Fig. 1C). At AG1478/seliciclib 0.81 concentrations above the IC50 value, the level of phosphory- Seliciclib + AG1478 0.74 lation was dramatically reduced, as were the levels of the Mcl-1 A549 Seliciclib/AG1478 0.96 protein, an established marker for seliciclib-induced transcrip- AG1478/seliciclib 1.13 tional inhibition (Fig. 1C; ref. 11). When given together, Seliciclib + AG1478 1.36 H460 Seliciclib/AG1478 1.33 trastuzumab and seliciclib produced greater down-regulation of AG1478/seliciclib 1.21 the HER2 receptor than either single agent, suggesting that the Seliciclib + AG1478 1.34 drug combination enhanced the down-regulation of receptor levels (Fig. 1D). NOTE: Seliciclib was tested in combination with either trastuzu- Mitogenic stimuli transduced by the HER2 signaling mab or AG1478 in various cell lines, using the protocol described in pathway result in the expression of cyclin D1 and the Materials and Methods. Concomitant (+) and sequential (/) consequent activation of CDKs leading to cellular proliferation treatment schedules were tested, and the resulting CI values (3). Indeed it has been shown that cyclin D1 is required for were shown for ED50 (the point on the curve where the cell number is 50% of that in the untreated controls). Results are the average transformation by the HER2 receptor (28) and that cyclin of at least three independent experiments. The CI value definitions D1–deficient mice are resistant to HER2-mediated tumori- are as follows: 1.45 to 1.2 is moderately antagonistic, 1.2 to 1.1 is genesis (29, 30). At the concentrations tested in these slightly antagonistic, 1.1 to 0.9 is additive, 0.9 to 0.85 is slightly experiments, trastuzumab had no effect on cyclin D1 levels synergistic, 0.85 to 0.7 is moderately synergistic, and 0.7 to 0.3 is synergistic. (Fig. 1B), whereas seliciclib significantly decreased cyclin D1 levels (f60% reduction at 18 Amol/L) in a dose-dependent manner (Fig. 1C); like Mcl-1, cyclin D1 also has a very short results show synergistic suppression of tumor growth both half-life (14) and is subject to transcriptional control by either in vitro and in vivo. alvocidib or seliciclib (12, 14). The loss of cyclin D1 was In vitro analysis of the combination of seliciclib and slightly enhanced by combining seliciclib with trastuzumab trastuzumab. To evaluate the interaction between seliciclib (f75% reduction at 18 Amol/L; Fig. 1D), presumably as a and trastuzumab, combination experiments were done in two consequence of decreased HER2 signaling and seliciclib- breast cancer cell lines: SkBr3, which overexpresses HER2, and mediated inhibition of cyclin D1 transcription. These data MCF7, which expresses low levels of HER2 (19). Incubation indicate that in SkBr3 cells, seliciclib enhanced the efficacy of with trastuzumab (1-100 nmol/L) reduced the proliferation of trastuzumab by promoting the down-regulation of the HER2 SkBr3 cells by up to 40%, but had no significant effect on receptor and loss of the cell cycle regulator cyclin D1, which MCF7 cells (data not shown), in agreement with previous data has been shown to be critical for propagating the HER2 (19). Seliciclib inhibited the growth of both cell lines with IC50 mitogenic signal and transformation (28–30). values of 16.3 and 10.0 Amol/L in SkBr3 and MCF7 cells, In vitro analysis of the combination of seliciclib and respectively. Co-incubation of SkBr3 cells with seliciclib and AG1478. A second ErbB receptor family member, EGFR trastuzumab resulted in a synergistic inhibition of cell growth (ErbB1), has also been the focus of significant anticancer drug (Fig. 1A and Table 1), generating a Calcusyn CI of 0.66 at ED50 development. Erlotinib, an EGFR tyrosine kinase inhibitor, has (the point at which the combination inhibits cell growth by been approved for the treatment of advanced NSCLC, having 50%). In contrast, the inhibition of cell growth was only shown survival benefit in the treatment of NSCLC (15), additive in MCF7 cells (CI = 0.96). These findings are whereas cetuximab, a monoclonal antibody that targets the consistent with earlier observations, which showed that external domain of EGFR, has shown survival benefit in trastuzumab is synergistic with alvocidib in cells that over- combination with irinotecan for the treatment of colorectal express HER2 (19–21). cancer (31). To date, there have been no published studies To understand the mechanism behind the synergistic evaluating combinations between CDK inhibitors and EGFR seliciclib/trastuzumab combination, analysis of the effects on inhibitors. To extend the analysis of synergistic interactions the HER2 signaling pathway was done. SkBr3 cells were between seliciclib and inhibitors of the ErbB family, seliciclib incubated with seliciclib, trastuzumab, or the combination of was evaluated in combination with the EGFR tyrosine kinase both agents for 24 hours, cell lysates were prepared, and the inhibitor AG1478 (32, 33), an analogue of erlotinib. levels of HER2 and cyclin D1 proteins were determined. The combination between seliciclib and AG1478 was Trastuzumab treatment had no effect on HER2 levels at evaluated initially in three NSCLC cell lines: H358, A549, and concentrations of up to 28 nmol/L (Fig. 1B), which was H460. These cell lines express different levels of wild-type EGFR consistent with previous findings demonstrating that this drug with A549 cells expressing the highest levels and H358 cells does not down-regulate HER2 levels in SkBr3 cells (23). In expressing the lowest levels (Fig. 2A). The IC50 values for contrast, treatment with seliciclib decreased HER2 levels in a AG1478 were 4, 6.6, and 10.4 Amol/L in H358, A549, and dose-dependent manner (Fig. 1C) as has previously been H460 cells, respectively, demonstrating that there was no reported for alvocidib (20, 24). Down-regulation of the HER2 correlation between EGFR protein levels and sensitivity to

www.aacrjournals.org 4329 Clin Cancer Res 2008;14(13) July 1, 2008 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical

AG1478. A similar pattern of sensitivity has been reported for pathway. This was confirmed by Western blot analysis; A549 erlotinib, leading to the description of these three cell lines as cells expressed relatively high levels of phosphorylated ERK, sensitive, intermediate, and resistant, respectively (34). It has whereas H460 cells contained high levels of phosphorylated been reported that A549 and H460 cells contain high levels of protein kinase B (Fig. 2A). Neither pathway seemed to be constitutively active ERK or protein kinase B, respectively (35), activated to such an extent in H358 cells (Fig. 2A) in line with which are two downstream effectors of the EGFR signaling its greater sensitivity to EGFR inhibitors. Seliciclib IC50 values

Fig. 2. Analysis of the combination of seliciclib and AG1478 in NSCLC cells. A, lysates (30 Ag) from untreated A549, H460, and H358 cells were resolved on 3% to 8% acrylamideTris-acetate gels or 10% acrylamide Bis-Tris gels, transferred to nitrocellulose membranes, and probed with the antibodies shown. Results are representative of two independent experiments. B, H358 cells were treated for 72 h using increasing concentrations of seliciclib and AG1478, either alone or in a fixed ratio of 2.125:1, as described in Materials and Methods.The resultant data were analyzed using the Calcusyn program, and graphs from a representative experiment for each treatment schedule are shown. CI values are defined in the legend forTable1. C, H358 cells were incubated in the presence (+) or absence (-)of either seliciclib orAG1478 for 72 h before harvesting. In H358 cells, the IC50 values for seliciclib and AG1478 were 8.5 and 4.0 Amol/L, respectively.Western blots were done as in A.

Clin Cancer Res 2008;14(13) July 1, 2008 4330 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

Â Fig. 3. Seliciclib plus AG1478 induces increased apoptosis. A, duplicate flasks of cells were treated with 2.25 IC50 seliciclib, AG1478, or both compounds for 72 h before harvesting. The DNA content of the cells was analyzed by flow cytometry after propidium iodide staining. Sub-G 1cells contain less DNA than normal diploid cells and are considered to represent apoptotic cells. In H358 cells, the IC50 values for seliciclib and AG1478 were 8.5 and 4.0 Amol/L, respectively; in H292 cells, the IC50 values for seliciclib and AG1478 were 15.0 and 5.0 Amol/L, respectively; in H1650 cells, the IC50 values for seliciclib and AG1478 were 10.4 and 13.0 Amol/L, respectively. Results are Â representative of two independent experiments. B, lysates (30 Ag) from cells treated for 72 h at 2.25 IC50 value were resolved on 3% to 8% acrylamideTris-acetate gels or 10% acrylamide Bis-Tris gels, transferred to nitrocellulose membranes, and probed with the antibodies shown. Results are representative of two independent experiments. were determined for H358, A549, and H460 cells and To elucidate the molecular mechanism involved in the corresponded to 8.5, 10, and 14 Amol/L, respectively. The synergistic effects of seliciclib and AG1478, H358 cells effect of the combination on cell growth was examined by were treated with either agent alone or in combination. addition of the compounds, either concomitantly or sequen- These experiments were done with concomitant treatment, as tially. In H358 cells, moderate synergy was seen in the this seemed to be the best regimen. Cell lysates were prepared, combination treatments, whereas the combination had, at and proteins were analyzed by Western blotting (Fig. 2C). best, an additive effect on cell growth in either A549 or H460 Initially the effects of the compounds on the levels of the EGFR cells (summarized in Table 1). Thus, the combination of and its phosphorylation status were studied. Treatment with seliciclib and AG1478 generated the best synergistic interaction either AG1478 or seliciclib decreased modestly the level of the in H358 cells, the EGFR inhibitor-sensitive cell line, and was EGFR in a dose-dependent manner; at 2.25 times the IC50 value, less effective in cells with constitutively active components both single-agent treatments resulted in a f30% reduction in downstream of the EGFR. A general sequence dependence for EGFR levels. When AG1478 and seliciclib were applied combinations involving either CDK inhibitors or EGFR together, the EGFR protein levels were decreased more inhibitors has been reported previously, for example, alvocidib substantially (f60% reduction in signal) than with either of and paclitaxel (36, 37) or erlotinib and pemetrexed (38, 39). In the two individual drug treatments (Fig. 2C). In addition, each case, the sequence dependence has been hypothesized to AG1478 treatment resulted in a dose-dependent decrease in be due to one treatment blocking the cells in a phase of the cell EGFR phosphorylation, whereas seliciclib treatment had a cycle where the second treatment is less effective. A degree of limited effect on phosphorylated EGFR levels. The reduction in sequence dependence was observed with the combination the levels of EGFR phosphorylation was enhanced by the between seliciclib and AG1478. When the different combina- concomitant treatment (Fig. 2C), suggesting that the combination schedules were examined in H358 cells, concomitant tion lowered both the quantity and activation status of the treatment and pretreatment with AG1478 followed by seliciclib EGFR. caused moderately synergistic inhibition of cell growth (average To further explore the effects of the combination of seliciclib ED50 CI values of 0.74 and 0.81, respectively), whereas and AG1478 on the EGFR signaling pathway, the phosphory- treatment with seliciclib followed by AG1478 produced only lation status of ERK, and cyclin D1 protein levels were an additive drug interaction (ED50 CI = 0.96; summarized in examined. Neither seliciclib nor AG1478 had any significant Table 1 and a representative experiment shown in Fig. 2B). The effect on ERK phosphorylation at the concentrations tested. fact that concomitant treatment resulted in the best synergy was However, the combination was very effective at reducing ERK also confirmed when the data was analyzed using a nonlinear phosphorylation to barely detectable levels, suggesting an regression fit of the data to the equation of Greco (data not enhanced inhibition of EGFR signaling (Fig. 2C). As mentioned shown; ref. 40). above, cyclin D1 is a key regulator of the cell cycle and lies

www.aacrjournals.org 4331 Clin Cancer Res 2008;14(13) July 1, 2008 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical downstream of the majority of ErbB receptor signaling path- as a marker of apoptosis, Western blot analysis showed that at ways. Indeed, a reduction in cyclin D1 levels may be required to the concentrations tested in H358 cells, AG1478 caused little achieve a response to EGFR inhibitors both in vitro and in apoptosis (Fig. 2C). In contrast, seliciclib induced a dose- patients (41, 42). Seliciclib concentrations above cellular IC50 dependent increase in cleaved PARP, which was not enhanced in values produced a modest decrease in cyclin D1 levels in H358 the presence of AG1478 (Fig. 2C). Taken together, these data Â Â cells (reductions of 5% at 1.5 IC50 and 45% at 2.25 IC50), suggest that in H358 cells, the combination of seliciclib and whereas at the concentrations tested AG1478 had very little AG1478 caused a synergistic down-regulation of the EGFR effect on the levels of this protein. When applied together, the signaling pathway, resulting in a cessation of cell proliferation two drugs dramatically reduced cyclin D1 expression in a dose- rather than increased apoptotic cell death. dependent manner equivalent to f90% and f98% reduction To explore the induction of apoptosis in more detail and to at the two concentrations (Fig. 2C). The loss of cyclin D1, one address whether the combination was also synergistic in cell of the key drivers of the cell cycle, will have contributed to the lines expressing mutant EGFR, these studies were expanded to effect of the combination on cellular proliferation. include two additional NSCLC cell lines. H1650 cells express The combination of seliciclib and AG1478 can induce apoptosis mutant EGFR; they are sensitive to erlotinib and undergo in cell lines that express either wild-type or mutant EGFR. Seli- apoptosis after treatment (47). H292 cells express wild-type ciclib treatment is known to cause apoptotic cell death (11, 43), EGFR and are very sensitive to erlotinib both in vitro and in vivo but significant induction of apoptosis is not observed in many (34), although erlotinib fails to induce apoptosis in this cell cell lines treated with AG1478, erlotinib, or trastuzumab (21, line (47). Initially, cytotoxicity experiments were done to show 44–46). Using poly(ADP)-ribose polymerase (PARP) cleavage that the combination was synergistic in these cell lines. In

Fig. 4. The effect of the combination of seliciclib and erlotinib on the growth of an H358 xenograft. A, mice (nu/nu) were injected s.c. with f1 Â107 H358 cells per mouse at a single site on their flanks.Treatment was initiated when tumors were f110 m m 3 and was continued for 28 d. Points, mean tumor volume for each treatment group (nine mice per group); bars, SE; Represent treatment with vehicle, seliciclib (50 mg/kg) as a twice dailyi.p. injection for 5 consecutive daysfollowed bya 2-d b reak and then repeated for a total of four cycles, erlotinib (100 mg/kg) daily by oral gavage for 28 consecutive days, or the combination of both agents.B, mice (nu/nu) were injected s.c. with f1 Â107 H358 cells per mouse at a single site on their flanks. Treatment was initiated when tumors were f110 m m 3 and continued for 5 consecutive days. Dosing regimens were erlotinib (100 mg/kg) dailybyoral gavage and seliciclib (100 mg/kg) as a twice dailyi.p. injection. At the end of the treatment period, tumors were re moved, fixed, and stored in paraffin. Cut sections were then immunostained with an antibodyraised against cyclin D1. Panels show representative images from mice in each trea tment group. C, bars represent individual mice, showing the percentage of positivelystained cells averaged from four fields of view. Cells were scored as positive if the level of staining was greater than that observed in sections stained with secondaryantibodyalone.

Clin Cancer Res 2008;14(13) July 1, 2008 4332 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

H1650 cells, the combination showed moderate synergy with 46 Amol/L (data not shown), a concentration above that an optimum CI of 0.71 at ED90, whereas for H292 cells, the needed to see synergy in vitro but significantly below the combination showed synergy with an ED50 CI of 0.37, maximum tolerated dose. Based on published reports, erlotinib indicating improved synergism over that in both H358 and (100 mg/kg) was dosed by oral gavage once per day for 28 H1650 cell lines. To evaluate the effect of the combination on consecutive days (34); for combination treatment, mice apoptosis, the three cell lines were treated with the compounds received both compounds on the same schedules as for each at 2.25 times their appropriate IC50 value and analyzed by flow single agent. Control mice received seliciclib vehicle on a twice cytometry. In all three cell lines, seliciclib treatment caused an daily schedule. On day 49, 3 weeks after all treatments had increase in the percentage of cells with a sub-G1 DNA content ceased, control mice (nine of nine mice alive) had tumors with 3 (apoptotic cells) and in the number of cells in the G2-M phase a mean volume of 700 mm , which represented a >6-fold of the cell cycle (Fig. 3A), which is in agreement with previous increase in tumor size (Fig. 4A). Mice treated with single-agent reports (48). After AG1478 treatment, the predominant effect seliciclib or erlotinib (seven of nine and eight of nine mice on the cell cycle was to cause an increase in the number of cells alive, respectively) had mean tumor volumes of 717 and 3 in G1; only in the mutant EGFR expressing H1650 cells was 444 mm , respectively, which indicated that neither agent had there a large increase in the number of apoptotic cells, significant activity on their own at these doses. The relatively consistent with previous findings (47). The observation that low level of erlotinib activity (44% tumor growth inhibition) in these three cell lines significant apoptosis was only induced correlated with the modest activity reported previously for this by AG1478 in the cell line expressing mutant EGFR was also in tumor model (34). In stark contrast, by day 49, mice treated line with the clinical finding that patients with EGFR mutations with the combination of seliciclib and erlotinib (eight of nine were more likely to show a response to EGFR inhibitors than mice alive) had a mean tumor volume of 153 mm3, patients that express wild-type EGFR (49, 50). In H358 cells, representing 93% tumor growth inhibition. The ratio of the flow cytometry results agreed well with the cleaved PARP median treated tumor volume to median control tumor volume data (Fig. 2C) in that the combination of the two agents did not for the combination was 22% on day 49, demonstrating that result in a greater induction of apoptosis than seliciclib the combination of the two compounds led to significant treatment alone (Fig. 3A). In H1650 cells, the combination of tumor growth delay. When the treated groups were compared drugs led to an even greater increase in the apoptotic with the vehicle control using the one-way ANOVA test population compared with the single-agent treatments and followed by Dunnett’s multiple comparison test, the only represented at least an additive induction of apoptosis group that was statistically different was the combination group (Fig. 3A). Finally, in H292 cells, the combination treatment (P = 0.001). If the individual groups were compared against resulted in a substantial increase in the number of cells with a each other using an unpaired Student’s t test, then the sub-G1 DNA content above that seen with seliciclib treatment combination was also significantly different from the single- alone (Fig. 3A). This equated to a synergistic induction of cell agent seliciclib and erlotinib treatments (P < 0.002). death, which was particularly significant, given that AG1478 To explore the proposed mechanism of action in vivo,a alone failed to induce apoptosis in this cell line. Moreover, the second H358 xenograft was done; two mice per treatment enhanced synergistic induction of apoptosis in the H292 cell group were dosed for 5 days with erlotinib orally daily line also correlated with the Calcusyn data, which suggested (100 mg/kg), seliciclib i.p. twice a day (100 mg/kg), or the that the synergy was greatest in this cell line. combination of the two agents. After 5 days, the mice were As a second method of monitoring the induction of sacrificed and the tumors were removed for immunohisto- apoptosis, protein lysates extracted from cells treated with chemical staining with an antibody raised against cyclin D1. compounds were analyzed by Western blot for the appearance Representative fields of view are shown in Fig. 4B and graphed of cleaved PARP (Fig. 3B). There was excellent correlation in Fig. 4C. It was clear that the combination treatment resulted between the two methods of measuring apoptosis. In H1650 in a dramatic reduction in the amount of cyclin D1 staining cells, both single-agent treatments resulted in the appearance of compared with either single-agent treatment, in terms of both cleaved PARP whereas the combination treatment generated an the number of cells stained and the intensity of staining in the enhanced signal. In H292 cells, seliciclib alone caused an cells. An unpaired Student’s t test suggested that the number of induction of PARP cleavage whereas AG1478 had no effect. The cells stained in the combination samples was significantly combination treatment induced an increase in the amount of lower than the vehicle control or either of the single-agent PARP cleavage clearly greater than the seliciclib treatment treatments (P < 0.0001). Thus, the mechanistic in vivo results alone, suggesting, once again, that the combination treatment mirror the results seen in vitro; targeting one of the key drivers resulted in a synergistic induction of cell death in H292 cells. of cell proliferation, cyclin D1, by blocking growth factor Although these data suggest that the extent of apoptosis signaling and preventing its transcription has synergistic obtained with the combination treatment can vary depending antitumor activity both in vitro and in vivo. on the cell line, importantly, the combination of the two drugs With the exception of the recent data on alvocidib in B-CLL was beneficial regardless of the EGFR status of the cell line. (18), to date CDK inhibitors have had relatively modest single- In vivo evaluation of the combination of seliciclib and agent activity in the clinic. This is also true of several ErbB erlotinib. The combination of seliciclib and erlotinib was inhibitors, which have been clinically approved despite their evaluated in vivo in an H358 NSCLC xenograft model. Mice relatively low response rate. For example, the EGFR inhibitor bearing H358 tumors of f110 mm3 were given seliciclib erlotinib had a response rate of only 9% in the pivotal single- (50 mg/kg) i.p. twice per day for 5 consecutive days, and then agent NSCLC trial (15). Likewise, trastuzumab, which was after a 2 day treatment break, this schedule was repeated for a given only to patients that overexpress the HER2 receptor, total of 28 days. At this dose level, seliciclib can reach a Cmax of achieved just an 11% response rate in the original phase II

www.aacrjournals.org 4333 Clin Cancer Res 2008;14(13) July 1, 2008 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical single agent trial in this preselected population (16). Evaluation new oncogenic pathway, such as c-MET, still depends on the of the potential for combining either of these classes of agents is production of cyclin D1 as one of the main drivers of cell therefore of great value to try and increase the clinical options proliferation, the enhanced reduction in the levels of cyclin D1 for patients. by either the erlotinib/bexarotene or potentially the erlotinib/ This report discusses the potential synergistic effects of using seliciclib combination could result in extended response times two agents to cooperatively repress cyclin D1 expression and by limiting the ability to switch receptor dependency. thereby limit cellular proliferation. A proof-of-principle clinical In summary, preclinically, both alvocidib and seliciclib have trial has been done recently based on a similar hypothesis using shown in vitro synergy with a wide range of agents, including erlotinib and the rexinoid bexarotene; the latter agent contrib- both standard chemotherapies and targeted agents; as with utes by increasing the proteosomal degradation of cyclin D1 other novel targeted therapies, combination therapy may turn (51). The combination of erlotinib and bexarotene in out to be the optimum way to exploit these agents in the clinic. aerodigestive tract cancers was well tolerated, and in a 24- In this study, we present the first report of synergy between patient population, four achieved partial responses and nine CDK inhibitors and EGFR inhibitors. The combination of had stable disease (51). In addition, reductions in cyclin D1 seliciclib and AG1478 was synergistic in vitro in cells that protein levels were detected in buccal scrapes taken posttreat- expressed either mutant EGFR or wild-type EGFR and resulted ment. Significantly, the patients that achieved clinical benefit in dramatic synergy in vivo in a NSCLC xenograft model. At the were atypical erlotinib responders; they were predominately molecular level, synergy seemed to involve augmented down- male, current or former smokers, and infrequently had regulation of ErbB receptor levels and inhibition of down- bronchioalveolar carcinoma histology. This provides proof-of- stream signaling, resulting in an enhanced loss of cyclin D1, concept clinical support for the idea that combinations of one of the downstream components of ErbB signaling path- agents that target cyclin D1 can expand the clinical activity of ways, a key regulator of cellular proliferation, and a protein that EGFR inhibitors beyond the typically responsive population. has been highlighted already as a potential biomarker of A second finding of this study was the median overall clinical response to erlotinib (42). These data suggest that the survival time, which was 14.1 months for the combination combination of these agents could potentially improve the compared with 6.7 months for single-agent erlotinib (51). This efficacy of ErbB inhibitors and is worthy of further investigation is of particular significance because frequently patients that in the clinic. respond to EGFR inhibitors have relatively transient responses and eventually relapse with disease progression (52). Resistance DisclosureofPotentialConflictsofInterest to the agent is often due to the appearance of ‘‘gatekeeper’’ mutations (e.g., T790M) at the active site of the kinase that I. Fleming, S. McClue, S. Frame, S. Green, and M. Hogben are employed by block inhibitor binding (53). Recently, a second mechanism of Cyclacel Limited. resistance that involves the amplification of an alternative proto-oncogene, such as the c-MET receptor, which interacts Acknowledgments directly with the ErbB3 receptor activating the phosphatidylinositol 3-kinase pathway and stimulating cellular proliferation, We thank our Cyclacel colleagues for insightful discussions and critical reading of has been identified (54). However, because the emergence of a this manuscript and Dr. Robert Jackson for help with the combination analysis.

References 1. DanceyJ, Sausville EA. Issues and progress with pro- targets for cancer treatment. J Clin Oncol 2006;24: human breast carcinoma cells induced byflavopiridol. tein kinase inhibitors for cancer treatment. Nat Rev 1770 ^ 83. Cancer Res 1999;59:4634 ^ 41. Drug Discov 2003;2:296 ^ 313. 9. Senderowicz AM. Small-molecule cyclin-depen- 15. Shepherd FA, Rodrigues Pereira J, CiuleanuT, et al. 2. Kantarjian H, Sawyers C, Hochhaus A, et al. Hemato- dent kinase modulators. Oncogene 2003;22: Erlotinib in previouslytreated non-small-cell lung logic and cytogenetic responses to imatinib mesylate 6609^20. cancer. N Engl J Med 2005;353:123^ 32. in chronic myelogenous leukemia. N Engl J Med 2002; 10. Shapiro GI, HarperJW. Anticancer drug targets: cell 16. Baselga J,TripathyD, Mendelsohn J, et al. Phase II 346:645^52. cycle and checkpoint control. J Clin Invest 1999;104: studyof weeklyintravenous recombinant humanized 3. Harari D,YardenY. Molecular mechanisms underlying 1645 ^ 53. anti-p185HER2 monoclonal antibodyin patients with ErbB2/HER2 action in breast cancer. Oncogene 11. MacCallum DE, Melville J, Frame S, et al. Seliciclib HER2/neu-overexpressing metastatic breast cancer. 2000;19:6102^ 14. (CYC202, R-Roscovitine) induces cell death in mul- J Clin Oncol 1996;14:737 ^ 44. 4. SlamonDJ,ClarkGM,WongSG,LevinWJ,UllrichA, tiple myeloma cells by inhibition of RNA polymerase 17. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of McGuire WL. Human breast cancer: correlation of re- II-dependent transcription and down-regulation of chemotherapyplus a monoclonal antibodyagainst lapse and survival with amplification of the HER-2/ Mcl-1. Cancer Res 2005;65:5399^407. HER2 for metastatic breast cancer that overexpresses neu oncogene. Science 1987;235:177 ^ 82. 12. Whittaker SR, Walton MI, Garrett MD, Workman P. HER2. N Engl J Med 2001;344:783 ^ 92. 5. Tortora G, Ciardiello F. Anti-epidermal growth factor The cyclin-dependent kinase inhibitor CYC202 18. Byrd JC, LinTS, Dalton JT, et al. Flavopiridol admin- receptor drugs in cancer therapy. Expert Opin Investig (R-roscovitine) inhibits retinoblastoma protein phos- istered using a pharmacologicallyderived schedule is Drugs 2002;11:755 ^68. phorylation, causes loss of cyclin D1, and activates associated with marked clinical efficacyin refractory, 6. Salomon DS, Brandt R, Ciardiello F, Normanno N. the mitogen-activated protein kinase pathway. geneticallyhigh-risk chronic lymphocytic leukemia. Epidermal growth factor-related peptides and their Cancer Res 2004;64:262 ^ 72. Blood 2007;109:399 ^ 404. receptors in human malignancies. Crit Rev Oncol 13. Schubert KM, Duronio V. Distinct roles for extracel- 19. Wu K, Wang C, D’Amico M, et al. Flavopiridol and Hematol 1995;19:183 ^ 232. lular-signal-regulated protein kinase (ERK) mitogen- trastuzumab synergistically inhibit proliferation of 7. Silvestri GA, Rivera MP. Targeted therapyfor the activated protein kinases and phosphatidylinositol breast cancer cells: association with selective coope- treatment of advanced non-small cell lung cancer: a 3-kinase in the regulation of Mcl-1synthesis. Biochem rative inhibition of cyclin D1-dependent kinase and review of the epidermal growth factor receptor anta- J 2001;356:473 ^ 80. Akt signaling pathways. Mol Cancer Ther 2002;1: gonists. Chest 2005;128:3975 ^ 84. 14. Carlson B, LahusenT, Singh S, et al. Down-regula- 695 ^706. 8. Shapiro GI. Cyclin-dependent kinase pathways as tion of cyclin D1by transcriptional repression in MCF-7 20. Nahta R, Iglehart JD, Kempkes B, Schmidt EV.

Clin Cancer Res 2008;14(13) July 1, 2008 4334 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

Rate-limiting effects of cyclin D1in transformation by 32. Osherov N, Levitzki A. Epidermal-growth-factor- represses cyclin D1in aerodigestive tract cancers. Clin ErbB2 predicts synergy between herceptin and fla- dependent activation of the src-familykinases. Eur J Cancer Res 2004;10:7547 ^54. vopiridol. Cancer Res 2002;62:2267 ^ 71. Biochem 1994;225:1047 ^ 53. 43. McClue S, Blake D, Clarke R, et al. In vitro and 21. Nahta R, Trent S, Yang C, Schmidt EV. Epidermal 33. Levitzki A, Gazit A. Tyrosine kinase inhibition: an in vivo antitumor properties of the cyclin dependent growth factor receptor expression is a candidate tar- approach to drug development. Science 1995;267: kinase inhibitor CYC202 (R-roscovitine). Int J Cancer get of the synergistic combination of trastuzumab 17 8 2 ^ 8 . 2002;102:463 ^ 8. and flavopiridol in breast cancer. Cancer Res 2003; 34. Thomson S, Buck E, Petti F, et al. Epithelial to 44. Chinnaiyan P, Huang S,Vallabhaneni G, et al. Mech- 63:3626^31. mesenchymal transition is a determinant of sensitivity anisms of enhanced radiation response following epi- 22. ChouTC,TalalayP. Quantitative analysis of dose-ef- of non-small-cell lung carcinoma cell lines and xeno- dermal growth factor receptor signaling inhibition by fect relationships: the combined effects of multiple grafts to epidermal growth factor receptor inhibition. erlotinib (Tarceva). Cancer Res 2005;65:3328^ 35. drugs or enzyme inhibitors. Adv Enzyme Regul 1984; Cancer Res 2005;65:9455^62. 45. Huang S, Armstrong EA, Benavente S, Chinnaiyan 22:27 ^55. 35. Janmaat ML, Kruyt FAE, Rodriguez JA, Giaccone P, Harari PM. Dual-agent molecular targeting of the 23. Longva KE, Pedersen NM, Haslekas C, Stang E, G. Response to epidermal growth factor receptor epidermal growth factor receptor (EGFR): combining Madshus IH. Herceptin-induced inhibition of ErbB2 inhibitors in non-small cell lung cancer cells: limited anti-EGFR antibodywith tyrosine kinase inhibitor. signaling involves reduced phosphorylation of Akt antiproliferative effects and absence of apoptosis as- Cancer Res 2004;64:5355 ^ 62. but not endocytic down-regulation of ErbB2. Int J sociated with persistent activityof extracellular sig- 46. ZhouY, Brattain MG. Synergy of epidermal growth Cancer 2005;116:359 ^ 67. nal-regulated kinase or Akt kinase pathways. Clin factor receptor kinase inhibitor AG1478 and ErbB2 ki- 24. Li Y, Bhuiyan M, Alhasan S, Senderowicz AM, Cancer Res 2003;9:2316 ^ 26. nase inhibitor AG879 in human colon carcinoma cells Sarkar FH. Induction of apoptosis and inhibition of 36. Motwani M, DeloheryTM, Schwartz GK. Sequential is associated with induction of apoptosis. Cancer Res c-erbB-2 in breast cancer cells byflavopiridol. Clin dependent enhancement of caspase activation and 2005;65:5848^56. Cancer Res 2000;6:223 ^ 9. apoptosis byflavopiridol on paclitaxel-treated human 47. Yauch RL, JanuarioT, Eberhard DA, et al. Epithelial ver- gastric and breast cancer cells. Clin Cancer Res 1999; 25. Ljungman M, Paulsen MT. The cyclin-dependent ki- sus mesenchymal phenotype determines in vitro sensi- nase inhibitor roscovitine inhibits RNA synthesis and 5:1876 ^ 83. tivityand predicts clinical activityof erlotinib in lung triggers nuclear accumulation of p53 that is unmodified 37. Bible KC, Kaufmann SH. Cytotoxic synergy cancer patients. Clin Cancer Res 2005;11:8686^98. at Ser15and Lys382. Mol Pharmacol 2001;60:785 ^9. between flavopiridol (NSC 649890, L86-8275) and 48. Mihara M, Shintani S, Kiyota A, MatsumuraT,Wong various antineoplastic agents: the importance of 26. Dubois MF, Nguyen VT, Bellier S, Bensaude O. DTW. Cyclin-dependent kinase inhibitor (roscovitine) sequence of administration. Cancer Res 1997;57: Inhibitors of transcription such as 5,6-dichloro-1- suppresses growth and induces apoptosis byregulat- h 3375 ^ 80. -D-ribofuranosylbenzimidazole and ioquinoline ing Bcl-x in head and neck squamous cell carcinoma sulphonamide derivatives (H-8 and H-7) promote de- 38. Li T, Ling Y-H, Goldman ID, Perez-Soler R. Sched- cells. Int J Oncol 2002;21:95 ^ 101. phosphorylation of the carboxyl-terminal domain of ule-dependent cytotoxic synergism of pemetrexed 49. Lynch TJ, Bell DW, Sordella R, et al. Activating RNA polymerase II largest subunit. J Biol Chem 1994; and erlotinib in human non-small cell lung cancer cells 269:13331 ^6. 10.1158/1078-0432.CCR-06-2923. Clin Cancer Res mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer 27. Scott GK, Marden C, Xu F, Kirk L, Benz CC. 2007;13:3413^ 22. to gefitinib. N Engl J Med 2004;350:2129 ^ 39. Transcriptional repression of ErbB2 byhistone deace- 39. Davies AM, Ho C, Lara PN, Jr., Mack P, Gumerlock tylase inhibitors detected by a genomically integrated PH, Gandara DR. Pharmacodynamic separation of 50. Pao W, Miller V, Zakowski M, et al. EGF receptor ErbB2 promoter-reporting cell screen. Mol Cancer epidermal growth factor receptor tyrosine kinase gene mutations are common in lung cancers from Ther 2002;1:385 ^ 92. inhibitors and chemotherapyin non-small-cell lung ‘‘never smokers’’and are associated with sensitivityof tumors to gefitinib and erlotinib. Proc Natl Acad Sci U 28. Lee RJ, Albanese C, Fu M, et al. Cyclin D1is re- cancer. Clin Lung Cancer 2006;7:385 ^ 8. S A 2004;101:13306 ^ 11. quired for transformation byactivated Neu and is in- 40. Greco WR, Park HS, Rustum YM. Application of a duced through an E2F-dependent signaling pathway. new approach for the quantitation of drug synergism 51. Dragnev KH, PettyWJ, Shah S, et al. Bexarotene MolCellBiol2000;20:672^83. to the combination of cis-diamminedichloroplatinum and erlotinib for aerodigestive tract cancer. J Clin Oncol 2005;23:8757 ^ 64. 29. YuQ,GaengY,SicinskiP.Specificprotection and 1-{h}-D-arabinofuranosylcytosine. Cancer Res against breast cancers bycyclin D1ablation. Nature 199 0;50 :5318 ^ 27. 52. Sharma SV, Bell DW, Settleman J, Haber DA. Epi- 2001;411:1017^ 21. 41. Kalish LH, Kwong RA, Cole IE, Gallagher RM, dermal growth factor receptor mutations in lung can- 30. Landis MW, Pawlyk BS, Li T, Sicinski P, Hinds PW. Sutherland RL, Musgrove EA. Deregulated cyclin D1 cer. Nat Rev Cancer 2007;7:169 ^ 81. Cyclin D1-dependent kinase activity in murine deve- expression is associated with decreased efficacyof 53. Kobayashi S, Boggon TJ, Dayaram T, et al. EGFR lopment and mammarytumorigenesis. Cancer Cell the selective epidermal growth factor receptor tyro- mutation and resistance of non-small-cell lung cancer 2006;9:13^ 22. sine kinase inhibitor gefitinib in head and neck squa- to gefitinib. N Engl J Med 2005;352:786 ^ 92. 31. Cunningham D, HumbletY, Siena S, et al. Cetuximab mous cell carcinoma cell lines. Clin Cancer Res 2004; 54. Engelman JA, Zejnullahu K, MitsudomiT, et al. MET monotherapyand cetuximab plus irinotecan in irinote- 10 :7764 ^ 74. amplification leads to gefitinib resistance in lung can- can-refractorymetastatic colorectal cancer. N Engl J 42. PettyWJ,DragnevKH,MemoliVA,etal.Epidermal cer byactivating ERBB3 signaling. Science Med 2004;351:337^ 45. growth factor receptor tyrosine kinase inhibition 2007;316:1039 ^ 43.

www.aacrjournals.org 4335 Clin Cancer Res 2008;14(13) July 1, 2008 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. Synergistic Inhibition of ErbB Signaling by Combined Treatment with Seliciclib and ErbB-Targeting Agents

Ian N. Fleming, Morag Hogben, Sheelagh Frame, et al.

Clin Cancer Res 2008;14:4326-4335.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/14/13/4326

Cited articles This article cites 52 articles, 30 of which you can access for free at: http://clincancerres.aacrjournals.org/content/14/13/4326.full#ref-list-1

Citing articles This article has been cited by 5 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/14/13/4326.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/14/13/4326. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.