Transcriptional Profiling Identifies Cyclin D1 As a Critical Downstream Effector of Mutant Epidermal Growth Factor Receptor Signaling

Research Article

Transcriptional Profiling Identifies Cyclin D1 as a Critical Downstream Effector of Mutant Epidermal Growth Factor Receptor Signaling

Susumu Kobayashi,1 Takeshi Shimamura,3,4 Stefano Monti,5 Ulrich Steidl,1 Christopher J.Hetherington, 1 April M.Lowell, 3 Todd Golub,5,6,7 Matthew Meyerson,2,3,7 Daniel G.Tenen, 1 Geoffrey I.Shapiro, 3,4 and Bala´zs Halmos1,8

1Division of Hematology/Oncology, Beth Israel Deaconess Medical Center and 2Department of Pathology, Harvard Medical School; 3Department of Medical Oncology, Dana-Farber Cancer Institute; 4Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School; 5Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School; 6Howard Hughes Medical Institute, Boston, Massachusetts; 7The Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts;and 8University Hospitals of Cleveland and Case Western Reserve University, Cleveland, Ohio

Abstract of advanced non–small cell lung cancer (NSCLC). These com- Activating mutations in the epidermal growth factor receptor pounds show particular promise in certain subsets of patients, (EGFR) tyrosine kinase domain determine responsiveness to including women, nonsmokers, younger patients, patients with EGFR tyrosine kinase inhibitors in patients with advanced adenocarcinoma histology, and Asian populations (1, 2). Somatic non–small cell lung cancer (NSCLC). The modulation of EGFR mutations have been identified in such patients at a high transcriptional pathways by mutant EGFR signaling is not frequency and it seems that responsiveness to this class of agents strongly correlates with the presence of these EGFR mutants (3–7). fully understood. Previously, we and others identified a single base pair change leading to a threonine to methionine (T790M) Although it has been reported that these mutations mediate amino acid alteration in the ATP-binding pocket of the EGFR oncogenic effects by altering downstream signaling and antiapop- as a common mechanism of acquired resistance. The gefitinib- totic mechanisms (8), alterations in transcriptional pathways are resistant, T790M-mutant H1975 NSCLC cell line undergoes not fully understood. prominent growth arrest and apoptosis when treated with the Despite the initial success of EGFR inhibitors, resistance irreversible EGFR inhibitor, CL-387,785. We did a transcrip- emerges in the majority of patients over time. Recently, we and tional profiling study of mutant EGFR target genes that are others (9, 10) identified a single base pair change leading to a differentially expressed in the ‘‘resistant’’ gefitinib-treated and threonine to methionine (T790M) amino acid alteration in the the ‘‘sensitive’’ CL387,785-treated H1975 cells to identify the ATP-binding pocket of the EGFR, which leads to steric hindrance pivotal transcriptional changes in NSCLC with EGFR-activat- caused by the introduction of a bulkier methionine residue ing mutations. We identified a small subset of early gene interfering with drug binding and demonstrating high-level changes, including significant reduction of cyclin D1 as a result resistance against gefitinib and erlotinib. Although we showed that an alternative, irreversible anilinoquinazoline EGFR inhibitor, of EGFR inhibition by CL-387,785 but not by gefitinib. The reduction in cyclin D1 transcription was associated with CL-387,785, can overcome the resistance conferred by T790M subsequent suppression of E2F-responsive genes, consistent (9, 11), it is predictable that novel resistance mutations would with proliferation arrest. Furthermore, cyclin D1 expression emerge over time, limiting the efficacy of these irreversible was higher in EGFR-mutant lung cancer cells compared with inhibitors (12, 13). The H1975 NSCLC cell line carries a double cells with wild-type EGFR. EGFR-mutant cells were routinely L858R/T790M mutation and is highly resistant to gefitinib, whereas sensitive to the cyclin-dependent kinase inhibitor flavopiridol, prominent growth arrest and apoptosis result after treatment with confirming the functional relevance of the cyclin D axis. These CL-387,785 (10, 11). Based on these observations, we hypothesized studies suggest that cyclin D1 may contribute to the emergence that the study of genes that are differentially expressed in the of EGFR-driven tumorigenesis and can be an alternative target ‘‘resistant’’ gefitinib-treated and the ‘‘sensitive’’ CL-387,785-treated H1975 cells may allow the identification of pivotal downstream of therapy. (Cancer Res 2006;66(23): 11389-98) target genes in EGFR-driven cancers, especially in NSCLC with EGFR-activating mutations, and provide a novel strategy to Introduction overcome and/or prevent the emergence of the resistance. To this Development of anilinoquinazoline epidermal growth factor end, we did a transcriptional profiling study of mutant EGFR target (EGF) receptor (EGFR) inhibitors has greatly affected the treatment genes using H1975 cells and identified cyclin D1 as a critical downstream effector of mutant EGFR. These results suggest that attenuation of cyclin/cyclin-dependent kinase (CDK) pathways Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). may be an alternative therapeutic target in EGFR-mutant NSCLC. S. Kobayashi, T. Shimamura, and S. Monti contributed equally to this work and should be considered co–first authors. Requests for reprints: Daniel G. Tenen, Harvard Institutes of Medicine, Room 954, 77 Materials and Methods Avenue Louis Pasteur, Boston, MA 02115. Phone: 617-667-5561;Fax: 617-667-3299;E-mail: [email protected] or Geoffrey I. Shapiro, Department of Medical Oncology, Reagents. Stock solutions for gefitinib, erlotinib, CL-387,785, and Dana-Farber Cancer Institute, Dana 810A, 44 Binney Street, Boston, MA 02115. Phone: flavopiridol were prepared as previously described (11, 14). 617-632-4942;Fax: 617-632-1977;E-mail: [email protected]. I2006 American Association for Cancer Research. Cell culture. Calu-1, A549, H1975, and H460 were obtained from the doi:10.1158/0008-5472.CAN-06-2318 American Type Culture Collection (Rockville, MD) and were maintained in www.aacrjournals.org 11389 Cancer Res 2006; 66: (23). December 1, 2006

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Research the manufacturer-specified growth medium. The HCC827 cell line was presence or absence of inhibitors. To monitor expression of RNA obtained from Dr. Pasi A. Ja¨nne (Lowe Center for Thoracic Oncology and polymerase II and its phosphorylated (phospho-) forms (Ser2 and Ser5), Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, cells were plated at 1 Â 106 per 10-cm dish and treated with 300 nmol/L MA) and was maintained as described previously (15, 16). H3255 cells were flavopiridol for the indicated times. Forty micrograms of proteins were maintained as described previously (17). separated on 8% or 12.5% SDS-polyacrylamide gels. The anti-cyclin D1 Oligonucleotide array analysis. H1975 cells were grown to 60% antibody was from EMD Biosciences (San Diego, CA). Total EGFR antibody confluence in RPMI containing 10% fetal bovine serum. Pentuplicate plates and total RNA-Pol II (N20) were purchased from Santa Cruz Biotechnology were incubated by the addition of either gefitinib (1 Amol/L) or CL-387,785 (Santa Cruz, CA). Total extracellular signal-regulated kinase 1/2 (ERK1/2) (1 Amol/L), or DMSO (0.01%) as a control for 6 and 24 hours. Cells were antibody was purchased from BD Transduction Laboratories (Lexington, collected at the same time, and total cellular RNA was isolated using the KY). Phospho-EGFR (pTyr1068), phospho-AKT (pS473), phospho-ERK1/2 RNeasy kit (Qiagen). RNA specimens were then processed and hybridized to (pT202/pY204), and total AKT antibodies were purchased from Cell Affymetrix HG-U133A microarrays and scanned. The expression value for Signaling Technology (Danvers, MA). Anti-phospho-RNA Pol II (Ser2) and each gene was calculated using Affymetrix GeneChip software and the anti-phospho-RNA Pol II (Ser5) were from Covance Research Products robust multichip average method for signal extraction that is part of (Berkeley, CA). Antibodies were used according to the manufacturers’ BioConductor (18). recommended conditions. Preprocessing, filtering, and statistical analysis. The raw expression Growth inhibition assay. Growth inhibition was assessed by 3-(4,5- data consisted of the ‘‘signal’’ units as obtained by application of the robust dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tet- multichip average method to the ‘‘.CEL’’ files produced by the Affymetrix razolium, inner salt (MTS) assay using CellTiter 96 AQueous One solution scanner (18, 19). Agglomerative hierarchical clustering was preliminarily proliferation kit (Promega, Madison, WI) as previously described (11). done on the entire data set projected on the space of a reduced set of 2,307 Briefly, HCC827 stable cells were transferred to triplicate wells at 10,000 per probe sets/‘‘genes’’ [filter: 0.2VCVV10;P-callsz10%;avg(EXP) z50 in at least well in 96-well flat-bottomed plates. The next day, the cells were incubated 10% of the samples]. We used Pearson correlation, as the distance measure, with various concentrations of inhibitors for 72 hours. and a centroid-based agglomeration rule. The software package dChip was Cell cycle analysis. Cell cycle was assessed as previously described (17). used for this analysis (20). Differential analysis was done on a set of 5,570 Briefly, following fixation and treatment in 500 Ag/mL RNase A, cells were genes with median absolute deviation of the signal across chips above the resuspended in 69 Amol/L propidium iodide (1 mL) in 30 Amol/L sodium 75th percentile. Genes differentially expressed with respect to the binary citrate. Cells were analyzed for DNA content by flow cytometry using the phenotype of interest were identified by computing their variance- ModFit program (Verity Software House, Topsham, ME). thresholded t statistic (with the threshold set to the minimum variance Apoptosis analysis. Cells were plated at 1 Â 105 per well in a six-well observed within the replicate controls). Empirical, gene-specific P values plate and treated with DMSO or flavopiridol. Apoptosis was assessed using were computed by permutation test. When both time points (6 and 24 an Annexin-V–FLUOS staining kit (Roche) as previously described (9, 11). hours) were pooled in the analysis, restricted permutations aimed at controlling for the potential confounding effect of the time points were carried out (21, 22). Because several thousand genes were tested, the Results nominal P values were corrected for multiple hypotheses testing by the false CL-387,785 induces G1 arrest and apoptosis in gefitinib- discovery rate (FDR) procedure (23). The analysis was carried out using resistant H1975 cells. Previously, we have shown that H1975 cells GenePattern (24) and a set of ad hoc R scripts. harboring a double L858R/T790M mutation are highly resistant to Gene Ontology (GO) annotation was done by testing (based on the gefitinib, whereas the irreversible EGFR inhibitor, CL-387,785, hypergeometric distribution) for the overrepresentation of GO categories in overcomes the resistance. To confirm this observation, we did each of the marker lists of interest. The software package GeneMerge was fluorescence-activated cell sorting analysis using propidium iodide. used for this purpose (25). Real-time PCR assay. The mRNA levels of genes were measured by Apoptotic DNA fragmentation as well as cell cycle distribution was SYBR green real-time PCR. DNase-treated RNA was reverse transcribed and analyzed by propidium iodide staining of ethanol-permeabilized subsequently amplified using an ABI Prism 7700 Sequence Detector cells (17, 26). H1975 cells were treated for 6 and 24 hours with (Applied Biosystems) by the following variables: 50jC (2 minutes), 95jC gefitinib (1 Amol/L) or CL-387,785 (1 Amol/L). Control cells were (10 minutes) followed by 40 cycles of 95jC (15 seconds), and 60jC grown in the presence of 0.01% DMSO. After exposure to CL- (60 seconds). 387,785 for 24 hours, the cells showed a significantly higher G1 peak Primers for human cyclin D1 were as follows: forward primer, consistent with G1 arrest compared with DMSO- or gefitinib- ¶ ¶ ¶ 5 -ACCTGAGGAGCCCCAACAA 3 ;reverse primer, 5 -TCTGCTCCTG- treated cells. After 48 hours, CL-387,785–treated cells showed a ¶ ¶ GCAGGCC-3 . Human cyclin D3: forward primer, 5 -TGGATGCTGGAGG- dramatic increase in G content together with increased sub-G TATGTG-3¶;reverse primer, 5 ¶-CGTGGTCGGTGTAGATGC-3¶. Human G -G 1 1 0 1 population consistent with induction of apoptosis (Fig. 1). These switch gene 2 (G0S2): forward primer, 5¶-CGCCGTGCCACTAAGGTC-3¶; results suggest that CL-387,785 induces cell cycle arrest in the G1 reverse primer, 5¶-GCACACAGTCTCCATCAGGC-3¶.Humancyclin G2: forward primer, 5¶-ATCGTTTCAAGGCGCACAG-3¶;reverse primer, 5 ¶-CAAC- phase and subsequent apoptosis in H1975 cells. CCCCCTCAGGTATCG-3¶. Human dual-specificity phosphatase 6 (DUSP6): Transcriptional profiling of gefitinib versus CL-387,785- forward primer, 5¶-CAGTGGTGCTCTACGACGAG-3¶;reverse primer, treated H1975 cells. To identify the genes differentially expressed 5¶-GCAATGCAGGGAGAACTCGGC-3¶. Human glyceraldehyde-3-phosphate in gefitinib-treated and CL-387,785–treated H1975 cells, a tran- dehydrogenase (GAPDH) were used for internal control: forward primer, scriptional profiling study was done on total cellular RNA using 5¶-GAAGGTGAAGGTCGGAGTC-3¶;reverse primer, 5 ¶-GAAGATGGTGATGG- Affymetrix HG-U133A chips. The raw expression data were ¶ GATTTC-3 . preprocessed, rescaled, filtered, and analyzed as described in EGFR-mutant constructs and transfections. EGFR-mutant constructs Materials and Methods. Experiments were carried out in pentupli- were generated as described previously (9) and used to generate stable cates, with two time points for each experiment (6 and 24 hours). HCC827 cell lines using FuGene6 transfection (Roche, Basel, Switzerland) followed by selection in 1 mg/mL G418. Hierarchical clustering with a correlation-based, centroid-linkage Antibodies and Western blotting. Whole-cell lysates were prepared as algorithm was done for exploratory purposes. The visualization of previously described (17). To assess cyclin D1 expression and the the correspondingly sorted data set clearly shows the separation of phosphorylation level of the proteins, the cells were serum-starved for the CL-387,785–treated samples from the rest, as well as the much 24 hours and were then stimulated with 100 ng/mL EGF for 3 hours in the weaker distinction of the gefitinib-treated samples. Formal

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Figure 1. CL-387,785 induces G1 arrest and subsequent apoptosis through down-regulation of cyclin D1.H1975 cell lines were treated with 0.01%DMSO as a control; 1 Amol/L gefitinib or 1 Amol/L CL-387,785 for 24 and 48 hours; and harvested for flow cytometry.Note that only CL-387,785 induced G 1 cell cycle arrest with appearance of a sub-G1 peak consistent with the induction of apoptosis. differential analysis based on a variance-thresholded t statistic and 6 hours to identify the earliest wave of transcriptional changes permutation test confirms the hierarchical clustering results. This most likely highly enriched in direct targets of mutant EGFR. analysis clearly distinguished gefitinib and CL-387,785–treated Because no significant difference was seen between the DMSO and samples (Fig. 2A). The genes altered by CL-387,785 treatment are gefitinib-treated samples, it was prudent to pool these samples to shown as Table 1 (6 hours) and Supplementary Tables S1 and S2 provide more power for our analysis. Then, we used very stringent (24 hours). The marker signatures of the differently treated samples criteria to select genes that are both statistically different in the was further examined by assessing the marker overlap between sets two groups and made biological sense based on substantial fold with the use of a strict permutation based FDR value of V0.01 changes. We used a variance-thresholded t statistic to rank genes. (Supplementary Table S3). This analysis showed that 1,641 marker From the ranked list of genes, we excluded genes with a FDR > 0.01 genes were down-regulated and 1,948 genes were up-regulated (representing no association). We further narrowed this list down when all CL-387,785–treated samples were compared with the rest by limiting our analysis to genes differing at least 2-fold. The top of the samples, whereas only three genes were down-regulated and genes thus obtained are shown in Table 1. This analysis identified one gene was up-regulated when gefitinib-treated samples were only five known up-regulated genes by CL-387,785 in this set, cyclin compared with the rest of the samples. These results illustrate the G2, DRE1, programmed cell death 4, aquaporin 3, and pellino striking changes as a result of CL-387,785 treatment and also show homologue 1. We confirmed the up-regulation of cyclin G2 by that gefitinib has essentially no ‘‘off-target’’ effect on these cells and quantitative reverse transcription-PCR analysis on the RNA used shows great selectivity of the drug at the concentration used for for the array analysis (Fig. 2B). Of note is that cyclin G2 is involved EGFR. Furthermore, the same comparison at 6 hours of treatment in negative cell cycle regulation (27, 28). identified 615 down-regulated and 688 up-regulated genes The analysis of the highly down-regulated genes identified 18 distinguishing CL-387,785–treated samples from the rest, whereas genes falling into three major clusters. First, many of the down- at 24 hours, the corresponding analysis identified 1,923 down- regulated genes code for growth factors, growth factor receptors, regulated genes (477 of these overlapped between 6 and 24 hours, or proangiogenic molecules, such as transforming growth factor 77%) and 2,607 up-regulated genes (582 overlapping, 85%). This a (TGFa), interleukin 11, Cyr61 (also called CCN1), thrombo- latter comparison is based on relatively small sample sizes in each plastin, EphA2, and vascular endothelial growth factor. Many of group and therefore should be considered more representative these factors are regulated by EGFR signaling corroborating the than definitive. validity of our findings and also suggesting the presence of a Next, we were interested in identifying those genes manifesting a positive feedback loop in EGFR signaling (29). A second group of differential expression between DMSO/gefitinib and CL-387,785 at genes comprises genes encoding DUSPs, such as DUSP4, DUSP5, www.aacrjournals.org 11391 Cancer Res 2006; 66: (23). December 1, 2006

Figure 2. Transcriptional profiling identifies cyclin D1 as one of the most down-regulated genes. A, hierarchical cluster analysis based on the correlation-based, centroid-linkage algorithm. GEF, gefitinib; CL, CL-387,785. B, quantitative real-time PCR analysis.H1975 were treated and RNA was extracted as in ( A).For standardization, we first measured the relative expression level of each gene against GAPDH. Bars, SD. C, time course of expression of cyclin D1 protein.H1975 were treated as in Fig.1 and whole lysates were subjected to Western blot analysis.CL-387,785 treatment led to down-regulation of cyclin D1, whereas gefitinib did not. and DUSP6. This finding suggests the presence of a strong five categories: nucleus, cell cycle, mitosis, DNA replication, and À negative feedback loop toward the mitogen-activated protein cell division (Bonferroni-corrected P < 1.0e 11;Supplementary (MAP) kinase (MAPK) pathway induced by oncogenic EGFR Table S4). This finding strongly suggests that the marked gene signaling (30). The third group of genes includes activator changes observed at 24 hours are a reflection of dramatic cell protein-1 (AP-1) components, such as c-Jun and FOS-like cycle and proliferation arrest. Next, we examined whether we antigen-1. Because the AP-1 complex is one of the major might be able to identify a specific cyclin D1–mediated signature mediators of proliferative signals through MAPK and signal within this group. The D-type cyclins, D1, D2, and D3, associate transducers and activators of transcription (STAT) signaling, with CDK4 and CDK6 and play a critical role early in the G1 phase this also seems very consistent with an EGFR signaling effect of the cell cycle. These complexes phosphorylate the retinoblas- (31, 32). Last, one of the most highly suppressed genes is cyclin toma protein and inactivate its ability to act as a transcriptional D1. This finding is of particular importance because cyclin D1 is repressor in a complex with E2F. The release of E2F leads to known to play a major role in cell cycle progression and has transcriptional induction of genes required for progression from previously also been described as a target of EGFR and ErbB2 G1 to S phase, most notably cyclin E. Therefore, cyclin D1 down- signaling (33–36). Three of these genes, cyclin D1, G0S2, and regulation should lead to shutdown of E2F-mediated transcrip- DUSP6 were selected and confirmed by quantitative real-time tional activity. A previous study identified E2F-regulated gene PCR (Fig. 2B). changes using transcriptional profiling of E2F-inducible model Next, we did the same analysis for the 24-hour gene set using systems. A comparison of the E2F-regulated genes identified the same selection criteria. Here, our analysis led to the through the study of Ishida et al. (37) to the gene set down- identification of a much larger number of regulated genes regulated in the CL-387,785–treated specimens very remarkably (Supplementary Tables S1 and S2). Given the large number of showed a perfect match between the two sets for the genes that genes, we next did GO annotation analysis of the marker genes were present on both the mouse arrays used in that study and the identifying CL-387,785–treated samples to arrive at a compre- HG-U133A human arrays used in our current study (Table 2). hensive view of the gene changes observed. This analysis showed These results further suggest that CL-387,785 induces G1 cell cycle that the list of down-regulated markers in the CL-387,785– arrest (Fig. 1) mediated by repression of cyclin D1 and E2F treated samples is significantly enriched with members of these followed by apoptosis.

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Cyclin D1 is down-regulated by CL-387,785, but not by constructs because they were found in a patient resistant to gefitinib or erlotinib, in H1975 cells. To confirm the findings of tyrosine kinase inhibitors (TKI;ref. 9). The EGFR constructs were the oligonucleotide array analysis, the repression of cyclin D1 was engineered with a hemagglutinin tag at the COOH-terminal tail so confirmed by real-time quantitative PCR analysis (Fig. 2B). When that ectopic and endogenous EGFR could be distinguished (Fig. H1975 cells were treated by CL-387,785, expression of the cyclin 3A). As shown in Fig. 3B, MTS assays showed that the introduction D1 gene on the RNA level was repressed to 31.4 F 4.8% (mean F of T790M led to resistance to erlotinib. In this system, T790M SD) of gefitinib-treated or DMSO-treated control cells (Fig. 2B). In receptors likely lead to resistance either by forming heterodimers contrast, expression of the cyclin D3 gene was repressed only with endogenous EGFR and/or through the formation of modestly to 66.9 F 4.9% (Fig. 1C), suggesting that down- homodimers. In contrast, proliferation was still compromised in regulation of the cyclin D1 is more correlated with drug response to CL-387,785, possibly due to cell cycle arrest. To sensitivity. In addition, Western blotting analysis showed that examine whether this growth inhibition was correlated with down- cyclin D1 was down-regulated as early as 2 hours into CL-387,785 regulation of cyclin D1 as well as the phosphorylation of EGFR and treatment, whereas it stayed unchanged in response to gefitinib its main downstream signaling effectors AKT and ERK1/2, we did (Fig. 2C). These results suggest that cyclin D1 is down-regulated Western blot analysis. In HCC827/Del cells, erlotinib inhibited by suppression of EGFR signaling at both mRNA and protein cyclin D1 expression as well as phosphorylation of EGFR and levels. its examined downstream signaling effectors (Fig. 3C, left). In Down-regulation of cyclin D1 is correlated with sensitivity HCC827/Del-TM cells, erlotinib led to only modest inhibition of to EGFR tyrosine kinase inhibitors. Next, to ask whether down- cyclin D1 expression and EGFR, AKT, and ERK1/2 phosphoryla- regulation of cyclin D1 is directly correlated with sensitivity to tion, whereas CL-387,785 potently inhibited cyclin D1 expression EGFR inhibitors, we established isogenic cell lines that express and EGFR, AKT, and ERK1/2 phosphorylation in HCC827/Del-TM EGFR-Del747-752 (HCC827/Del) or EGFR-Del747-752-T790M cells (Fig. 3C, middle and right). Identical results were obtained (HCC827/Del-TM) in a lung adenocarcinoma cell line, HCC827. when these cells were treated with gefitinib (data not shown). The HCC827 is heterozygous for the E746_A750 mutation in exon 19 of down-regulation of cyclin D1 as well as phosphorylation of EGFR the EGFR gene and is very sensitive to gefitinib and erlotinib and its downstream targets correlated with the results of the treatment (15, 16). We used Del747-752 and Del747-752-T790M growth inhibition assays done (Fig. 3B), suggesting that the efficacy

Table 1. Genes altered by CL-387,785 at 6 hours

Rank Score FDR Fold change Description Gene ID

Up-regulated genes 1 21.15 0.0041 2.78 DRE1 protein 221985_at 2 18.03 0.0041 2.57 Programmed cell death 4 202731_at 3 15.05 0.0041 2.01 KIAA0582 protein 212675_s_at

4 11.82 0.0041 3.12 Cyclin G2 202769_at 5 10.01 0.0041 2.21 Pellino (Drosophila) homologue 1 218319_at 6 9.65 0.0041 2.01 DKFZP586A0522 protein 207761_s_at 7 9.54 0.0041 2.22 Aquaporin 3 39248_at Down-regulated genes 1 32.45 0.0041 2.24 Immediate early response 3 201631_s_at 2 29.7 0.0041 2.13 Pleckstrin homology-like domain, family A, member 2 209803_s_at 3 26.36 0.0041 2.53 High-mobility group protein isoform I-C 208025_s_at 4 25.15 0.0041 2.21 Dual-specificity phosphatase 5 209457_at 5 25.05 0.0041 2.5 Transforming growth factor, a 205016_at 6 24.94 0.0041 4.19 Dual specificity phosphatase 6 208892_s_at 7 24.89 0.0041 2.3 V-jun sarcoma virus 17 oncogene homologue (avian) 201464_x_at 8 23.06 0.0041 3.64 Dual-specificity phosphatase 4 204014_at

9 20.8 0.0041 2.36 Putative lymphocyte G0G1 switch gene 213524_s_at 10 20.32 0.0041 2.14 EphA2 203499_at 11 20 0.0041 2.26 CYR61 210764_s_at 12 19.78 0.0041 3.05 FOS-like antigen-1 204420_at 13 16.77 0.0041 2.38 Cyclin D1 208712_at 14 15.36 0.0041 2.08 Vascular endothelial growth factor 210512_s_at 15 14.82 0.0041 2.5 Interleukin 11 206924_at 16 14.77 0.0041 2.1 CX3C chemokine precursor 823_at 17 14.16 0.0041 2.94 Coagulation factor III 204363_at 18 11.63 0.0041 2.58 Novel MAFF-like protein 36711_at

NOTE: List of the top up-regulated and down-regulated genes based on an analysis comparing CL-387,785 versus pooled, DMSO/gefitinib–treated samples at 6 hours of treatment. Ranking was based on t score. Fold-change was determined by the fold difference between the means of grouped specimens, and the genes demonstrating a >2-fold difference are shown.

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Table 2. E2F target genes down-regulated by CL-387,785 at 24 hours

Score Fold change Description Gene ID

Replication enzyme* 38.78 3.18 POLA2: polymerase (DNA directed), a2 204441_s_at 16.24 2.41 POLA, polymerase (DNA directed), a 204835_at 30.6 5.06 PCNA: proliferating cell nuclear antigen 201202_at 23.41 2.37 TK1: thymidine kinase 1, soluble 202338_at 36.17 4.26 TYMS: thymidylate synthetase 202589_at 35.69 6.72 RRM2: ribonucleotide reductase M2 polypeptide 201890_at 16.33 2.21 TOP2A: topoisomerase (DNA) II a 170 kDa 201292_at 23 2.24 TOP2A: topoisomerase (DNA) II a 170 kDa 201291_s_at 28.56 4.07 FEN1: flap structure-specific endonuclease 1 204767_s_at 28.1 4.02 FEN1: flap structure-specific endonuclease 1 204768_s_at 20.09 2.47 PRIM1: primase, polypeptide 1, 49 kDa 205053_at 16.27 2.22 DUT: dUTP pyrophosphatase 208955_at 24.75 3.04 RFC5: replication factor C (activator 1) 5, 36.5 kDa 203209_at 23.01 3.99 RFC3: replication factor C (activator 1) 3, 38 kDa 204127_at 29.46 3.21 RFC4: replication factor C (activator 1) 4, 37 kDa 204023_at 24.75 3.04 RFC5: replication factor C (activator 1) 5, 36.5 kDa 203209_at 34.87 2.91 RFC5: replication factor C (activator 1) 5, 36.5 kDa 203210_s_at 18.74 2.67 RFC2: replication factor C (activator 1) 2, 40 kDa 203696_s_at 28.46 2.67 RFC2: replication factor C (activator 1) 2, 40 kDa 1053_at Origin factors 34.23 8.62 CDC6: CDC6 cell division cycle 6 homologue 203967_at 27.12 6.81 CDC6: CDC6 cell division cycle 6 homologue 203968_s_at 19.12 3.32 ORC1L: origin recognition complex, subunit 1-like 205085_at 32.05 3.14 MCM7 210983_s_at 43.96 2.7 MCM7 208795_s_at 36.74 4.33 MCM3 201555_at c Activation 22.49 5.28 CCNE2: cyclin E2 205034_at 22.49 5.28 CCNE2: cyclin E2 205034_at 37.96 2.64 CDK2: cyclin-dependent kinase 2 204252_at 17.29 2.56 CDK2: cyclin-dependent kinase 2 211804_s_at Repair and other 18.73 2.35 RAD51: RAD51 homologue 205024_s_at Transcription factors 31.6 3.08 HMGA2: high-mobility group AT-hook 2 208025_s_at 20.61 2.56 HMGB2: high-mobility group box 2 208808_s_at 16.35 2.47 EZH2: enhancer of zeste homologue 2 203358_s_at b Mitotic activators

49.24 3.72 CDC2: cell division cycle 2, G1-S and G2-M 203214_x_at 49.05 3.67 CDC2: cell division cycle 2, G1-S and G2-M 210559_s_at 31.86 3.63 CDC2: cell division cycle 2, G1-S and G2-M 203213_at 22.18 2.34 CDC20: CDC20 cell division cycle 20 homologue 202870_s_at 30.65 3.2 BUB1 215509_s_at 23.72 2.85 BUB1 209642_at 18.88 2.31 CCNB1: cyclin B1 214710_s_at 33.5 4.06 CCNA2: cyclin A2 203418_at 19.22 3.75 CCNA2: Cyclin A2 213226_at

NOTE: List of the down-regulated E2F target genes based on an analysis comparing CL-387,785 versus pooled DMSO/gefitinib–treated samples at 24 hoursof treatment. Fold change was determined by the fold difference between the means of grouped specimens. Of note, 23 of the previously identified 27 E2F target genes were down-regulated. The remaining four genes are not represented on the HG-U133A arrays, i.e., 23 of 23 (100%) of the identifiable genes matched. *DNA ligase was not detected. cDbf4 was not detected. bStathmin and importin a2 were not detected.

of TKIs is at least in part associated with decreased cyclin D1 H1975 cells suggests that the cyclin D1-CDK4/6 axis is critical in expression. EGFR-mutant NSCLC. Consistent with this hypothesis, cell lines Cyclin D1 is up-regulated in EGFR-mutant NSCLC cell lines. harboring EGFR mutations (HCC827 and H1975) had higher levels The down-regulation of cyclin D1 in response to EGFR inhibition in of cyclin D1 expression when compared with the cell lines

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Figure 3. Cyclin D1 down-regulation is associated with sensitivity to EGFR TKIs in HCC827 stable cell lines. A, establishment of HCC827 stable cell lines.HCC827 cells were transfected with the pcDNA3.1 empty vector (Emp), EGFR-Del747-752 (Del), or EGFR-Del747-752-T790M (Del-TM).Western blotting shows strong expression of hemagglutinin (HA)–tagged EGFR in stably transfected HCC827 cells. B, dose-dependent growth inhibition of HCC827 stable cell lines treated with erlotinib or CL-387,785 detected by MTS assay. C, dose response of erlotinib and CL-387,785 on expression of cyclin D1 and phosphorylation of EGFR, AKT, and ERK1/2 in HCC827/Del or HCC827/Del-TM cells.The cells were starved for 24 hours and treated with erlotinib or CL-387,785 at indicated concentration s for 2.5 hours. Then, the cells were incubated for 3 hours in the presence of 100 ng/mL EGF. expressing wild-type EGFR (A549 and H460;Fig. 4 A). Interestingly, Discussion serum starvation led to down-regulation of cyclin D1 only in cells The reversible anilinoquinazoline EGFR TKIs erlotinib and with mutant EGFR (Fig. 4A). In addition, EGF-induced cyclin D1 gefitinib have shown dramatic responses in certain subsets of expression was inhibited by CL-387,785, which was correlated with patients with NSCLC (38, 39). Recurrent, oncogenic mutations of inhibition of EGFR downstream molecules, including AKT and ERK the EGFR gene were recently identified in such patients at high (Fig. 4B). frequency and it seems that responsiveness to this class of agents EGFR-mutant lung cancer cells are routinely sensitive to the strongly correlates with the presence of such somatic EGFR CDK inhibitor flavopiridol. The data in Figs. 3C and 4C suggest mutations (3–5, 40, 41). These EGFR mutants have increased and that cyclin D1 is a downstream target of mutant EGFR signaling. prolonged tyrosine kinase activity in response to the ligand Therefore, attenuation of cyclin D1 function and/or expression (EGF), are exquisitely sensitive to EGFR TKIs (3, 4), and are may lead to proliferation arrest and apoptosis. To test this oncogenic in transgenic mouse models (42, 43). The immediate hypothesis, we treated EGFR-mutant cell lines with flavopiridol, a effects of EGFR activation occur through the activation of pan-CDK inhibitor that inhibits both cell cycle and transcriptional downstream signaling pathways, such as the activation of STAT/ CDKs (37). Flavopiridol treatment resulted in dephosphorylation of AKT/MAPK pathways (44). EGFR-mediated signaling is ultimately 2 5 the COOH-terminal domain of RNA Pol II at Ser and Ser in all transduced to the nucleus via events such as STAT translocation NSCLC cells tested, suggesting uniform drug uptake (Fig. 5A). The and activation of downstream transcriptional effectors. The treatment of EGFR-mutant NSCLC cell lines with flavopiridol actual transcriptional signature of EGFR and, in particular, resulted in substantial apoptosis (Fig. 5B and C), whereas the same oncogenic, mutant-EGFR signaling remains poorly defined. Better treatment induced only minimal apoptosis in A549 and Calu-1 understanding of such transcriptional changes might identify cells, which express wild-type EGFR. These data suggest that the novel critical effectors of EGFR activation/blockade and provide cyclin D1-CDK4/6 axis plays a pivotal role in EGFR-mutant NSCLC new targets for therapeutic interventions. and also suggests that cyclin D1 may serve as an alternative target Clinical resistance to such reversible inhibitors often develops of therapy. through the emergence of the T790M secondary EGFR mutation www.aacrjournals.org 11395 Cancer Res 2006; 66: (23). December 1, 2006

(9, 10). However, irreversible anilinoquinazoline EGFR inhibitors, indicate that cyclin D1 is down-regulated by suppression of such as CL-387,785 and HKI-272, can overcome gefitinib/erlotinib EGFR signaling, resulting in G1 arrest at the G1 phase and resistance caused by the T790M mutation both in vitro and in subsequent apoptosis. Our results support that reduction of mouse models (9, 11, 45, 46). Because novel resistance mechanisms cyclin D1 expression may be a sensitive marker of TKI response. against these irreversible EGFR inhibitors will likely occur in the Of note, however, there was only mild growth inhibition of long run (12, 13), the identification of new targets remains a very HCC827/Del-TM cells treated with <1 Amol/L CL387,785, despite high priority. the reduction in phospho-EGFR, cyclin D1, phospho-AKT, and Our goal was to identify such novel targets by examining the phospho-ERK (Fig. 3B and C). These results suggest the presence transcriptional signature of gefitinib versus CL-387,785–treated of other prosurvival signals that contribute to resistance to EGFR L858R/T790M double-mutant, gefitinib-resistant H1975 cells. We inhibition. Further work will be required to identify these showed that only a very select number of genes were altered prosurvival factors. In addition, as shown in Fig. 4, it seems early, i.e., at 6 hours following EGFR blockade by CL-387,785. that cyclin D1 is overexpressed in lung cancer cells harboring These genes included a number of known EGFR targets mutant EGFR compared with cells with wild-type EGFR. It has validating our results, such as vascular endothelial growth factor, been reported that mutant EGFR preferentially activates the AP-1 family members, and cyclin D1. This analysis also showed AKT and STAT pathways compared with wild-type EGFR the presence of both negative as well as positive feedback loops (8, 16). Indeed, STAT3 promotes uncontrolled growth and in EGFR signaling, such as the EGFR blockade–induced down- survival through dysregulation of gene expression, including regulation of EGF agonists such as TGFa and the down- cyclin D1 (47). In addition, the Ras/MAP/ERK kinase/ERK– regulation of a number of DUSPs that play a role in dampening dependent pathway is also implicated in the expression of the MAPK activation. The late, i.e., 24-hour, signature included a cyclin D1 gene (48, 49). Further work is required to clarify the much larger number of genes corresponding to a prominent mechanism by which cyclin D1 is up-regulated in mutant EGFR proliferation arrest signature. This signature included suppression cells. of E2F target genes. Taken together, the data imply that cyclin Progression of the early to mid G1 phase is largely regulated by D1 down-regulation results in E2F inhibition with subsequent D-type cyclins, which associate with CDK4/6. We have shown that proliferation arrest and ultimate apoptosis. We confirmed cyclin EGFR-mutant NSCLC cell lines express high levels of cyclin D1. D1 down-regulation at both the RNA and protein level and also Therefore, inhibition of the cyclin D1-CDK4/6 pathway may have showed that down-regulation of cyclin D1 was strongly therapeutic benefit in this subset of NSCLCs. Flavopiridol directly correlated with sensitivity to EGFR inhibitors in isogenic inhibits CDK4 and CDK6, and also reduces the transcription of HCC827 stable cell lines as well as H1975 cells. Our results cyclin D1 via CDK9 and CDK7 inhibition (50). Although cyclin D1

Figure 4. Cyclin D1 is overexpressed in the cells harboring EGFR mutations. A, cyclin D1 expression in NSCLC cells.The cells were grown in the presence of 10% serum for 24 hours. B, dose response effect of erlotinib and CL-387,785 on EGF-induced cyclin D1 expression and phosphorylation of EGFR, AKT, and ERK1/2 in H1975 cells.Serum-starved cells were stimulated with 100 ng/mL EGF for 3 hours in the presence or absence of inhibitors.

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Figure 5. Flavopiridol induces apoptosis only in mutant EGFR cells. A, time course of dephosphorylation of RNA Pol II and its target proteins by flavopiridol.RasGAP was used for loading control. B, Annexin V apoptosis assay.Numbers in representative flow cytometry histograms are the percentages of cells in the appropriate quadrant. Left lower quadrant, viable cells; right lower quadrant, early apoptotic cells; right upper quadrant, late apoptotic cells. C, quantification of apoptosis.NSCLC cells were grown in the increasing doses of flavopiridol for 48 hours. Bars, SE (n = 3). was depleted in response to flavopiridol in all of the cell lines Nonetheless, the transcriptional profiling done here, as well as the studied, the reduction was more evident in H3255 and H1975 observed response to pharmacologic inhibition of cyclin D1– cells, which have higher baseline levels (data not shown). Over the dependent kinase activity, suggests that suppression of this 48-hour time period examined, flavopiridol induced substantial pathway will compromise the viability of EGFR-mutant NSCLC apoptosis in cell lines harboring EGFR mutation. A549 and Calu-1 cells. cells are representative of a larger panel of EGFR wild-type The identification of critical effectors of mutant EGFR, such as NSCLC cell lines that initially undergo cell cycle arrest in cyclin D1, should have immediate clinical implications. Impor- response to flavopiridol, followed by limited apoptosis at later tantly, the erlotinib/gefitinib–resistant H1975 cells remained time points (i.e., 72 hours), most evident after exposure to sensitive to flavopiridol. Active inhibitors of the cyclin D1-CDK4/ concentrations >500 nmol/L (14). Interestingly, these cell lines 6 axis (52), alone or in combination with EGFR inhibitors, should both carry K-ras mutations. Such mutations have been noted to be tested as a strategy to overcome or prevent resistance to EGFR be mutually exclusive with EGFR mutations and lead to primary TKI inhibitors in patients with EGFR-mutant NSCLCs. EGFR TKI resistance (51). It is possible that reduction of cyclin D1 in these cell lines arrests proliferation without affecting viability. Additional work will be required to determine whether K-ras mutant lung cancer cells are less dependent on cyclin D1 Acknowledgments for survival. Received 6/26/2006;revised 9/14/2006;accepted 10/5/2006. Of note, early apoptosis in response to flavopiridol is not Grant support: Specialized Programs of Research Excellence in Lung Cancer NIH grant PA20-CA090578-01A1 (D.G. Tenen);the Uehara Memorial Foundation restricted to EGFR-mutant NSCLC cells (14). Therefore, analysis of (S. Kobayashi);a Career Development Award as part of the Dana-Farber/Harvard additional cell lines will be necessary to confirm overall greater Cancer Center Specialized Programs of Research Excellence in Lung Cancer, NIH grant P20 CA90578 (T. Shimamura);NIH grant 1R01 CA116010 (M. Meyerson);and a Flight sensitivity of EGFR-mutant NSCLC cells to CDK inhibition. Attendant Medical Research Institute Young Clinical Scientist Award (B. Halmos). Flavopiridol has been shown to deplete a large variety of short The costs of publication of this article were defrayed in part by the payment of page half-life mRNAs, including those encoding antiapoptotic proteins, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. so that cell death may occur by mechanisms other than cyclin D1 We thank all members of the Tenen and Shapiro Laboratories for their helpful depletion, both in EGFR mutant– and wild-type–expressing cells. comments and technical advice.

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Susumu Kobayashi, Takeshi Shimamura, Stefano Monti, et al.

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