(AZD6244) in Combination with Vorinostat in KRAS-Mutant Colorectal Cancer Models

Published OnlineFirst December 15, 2011; DOI: 10.1158/1078-0432.CCR-11-1507

Clinical Cancer Cancer Therapy: Preclinical Research

Preclinical Activity of the Rational Combination of Selumetinib (AZD6244) in Combination with Vorinostat in KRAS-Mutant Colorectal Cancer Models

M. Pia Morelli1,3,4, John J. Tentler1, Gillian N. Kulikowski1, Aik-Choon Tan1, Erica L. Bradshaw-Pierce2, Todd M. Pitts1, Amy M. Brown1, Sujatha Nallapareddy1, John J. Arcaroli1, Natalie J. Serkova2, Manuel Hidalgo4, Fortunato Ciardiello3, and S. Gail Eckhardt1

Abstract Purpose: Despite the availability of several active combination regimens for advanced colorectal cancer (CRC), the 5-year survival rate remains poor at less than 10%, supporting the development of novel therapeutic approaches. In this study, we focused on the preclinical assessment of a rationally based combination against KRAS-mutated CRC by testing the combination of the MEK inhibitor, selumetinib, and vorinostat, a histone deacetylase (HDAC) inhibitor. Experimental Design: Transcriptional profiling and gene set enrichment analysis (baseline and posttreatment) of CRC cell lines provided the rationale for the combination. The activity of selumetinib and vorinostat against the KRAS-mutant SW620 and SW480 CRC cell lines was studied in vitro and in vivo. The effects of this combination on tumor phenotype were assessed using monolayer and 3-dimensional cultures, flow cytometry, apoptosis, and cell migration. In vivo, tumor growth inhibition, 18F-fluoro-deoxy-glucose positron emission tomography (FDG-PET), and proton nuclear magnetic resonance were carried out to evaluate the growth inhibitory and metabolic responses, respectively, in CRC xenografts. Results: In vitro, treatment with selumetinib and vorinostat resulted in a synergistic inhibition of proliferation and spheroid formation in both CRC cell lines. This inhibition was associated with an

increase in apoptosis, cell-cycle arrest in G1, and reduced cellular migration and VEGF-A secretion. In vivo, the combination resulted in additive tumor growth inhibition. The metabolic response to selumetinib and vorinostat consisted of signiﬁcant inhibition of membrane phospholipids; no signiﬁcant changes in glucose uptake or metabolism were observed in any of the treatment groups. Conclusion: These data indicate that the rationally based combination of the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, selumetinib, with the HDAC inhibitor vorinostat results in synergistic antiproliferative activity against KRAS-mutant CRC cell lines in vitro. In vivo, the combination showed additive effects that were associated with metabolic changes in phospholipid turnover, but not on FDG-PET, indicating that the former is a more sensitive endpoint of the combination effects. Clin Cancer Res; 18(4); 1051–62. 2011 AACR.

Introduction

Selumetinib (AZD6244; ARRY-142886) is a small mol- Authors' Afﬁliations: 1Division of Medical Oncology, 2Department of ecule, orally available, noncompetitive inhibitor of mito- Anesthesiology and Radiology, University of Colorado School of Medicine, gen-activated protein kinase (MAPK)/extracellular signal- Anschutz Medical Campus, Aurora, Colorado; 3Dipartimento Medico-Chir- urgico di Internistica Clinica e Sperimentale "F. Magrassi e A. Lanzara," regulated kinase 1/2 (MEK1/2; ref. 1). The RAS/RAF/MEK/ Seconda Universita degli Studi di Napoli, Napoli, Italy; and 4Gastro-Intes- ERK kinase cascade occupies a central role in mediating tinal Tumor Unit, Clinical Research Programme, Spanish National Cancer Research Center (CNIO), Madrid, Spain signal transduction from extracellular growth factors, cytokines, and proto-oncogenes. Alterations in the control of Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). RAS/MAPK pathway, resulting in its constitutive activation, have a well-established role in oncogenesis and tumor Corresponding Author: M. Pia Morelli, Gastro-Intestinal Tumor Unit, Clinical Research Programme, Spanish National Cancer Centre (CNIO), growth, mostly related to uncontrolled cell proliferation Melchor Fernandez Almagro 3, Madrid, Spain. Phone: 91-732-8000 (ext. and suppression of apoptosis (2). The hyperactivation of 2921); Fax: 34-91-224-6931; E-mail: [email protected] this cascade is mediated through various mechanisms, doi: 10.1158/1078-0432.CCR-11-1507 including tyrosine kinase receptor overexpression or muta- 2011 American Association for Cancer Research. tions, or to KRAS- or BRAF-activating mutations. KRAS

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several cell lines (14–16). Because of their relative specificity Translational Relevance toward cancer cells, HDACi represent a new class of cancer treatment agents that are generally well tolerated. One such Colorectal cancer (CRC) is the third leading cause of compound, vorinostat [suberoylanilide hydroxamic acid cancer worldwide. Although current therapeutic regi- (SAHA)] has shown encouraging activity in early studies mens incorporate active chemotherapy and biologic against several cancers, including B-cell lymphoma (17), agents, patients eventually relapse without options for colon cancer (18, 19), NSCLC (20), and head and neck effective salvage therapy. Recent analysis of numerous cancer and is currently approved for the treatment of cuta- clinical trials evaluating response rates of advanced CRC neous T-cell lymphoma (21). Several phase II studies have to epidermal growth factor receptor inhibitors showed a been conducted for breast (22), colon, and lung (23, 24), correlation between activating mutations of the Kristen head and neck (25), and ovarian cancer patients (26); rat sarcoma viral oncogene homolog (KRAS) gene and however, no consistent antitumor activity of vorinostat as resistance to these agents, thus identifying a large subset single agent has been observed (11). The prevailing view is of patients in urgent need of alternative therapeutic that further investigations to evaluate the safety and activity approaches. In this study, we utilized gene set enrich- of vorinostat as a combination partner are needed to better ment analyses to support the rational combination evaluate the potential of this agent in cancer treatment (27). of the mitogen-activated protein kinase/extracellular Several reports in the literature have shown synergistic signal-regulated kinase 1/2 inhibitor, selumetinib interaction between MEK and HDAC inhibitors. Yu and (AZD6244), and the histone deacetylase inhibitor, vor- colleagues (28, 29) showed that combining HDAC with inostat (SAHA), against preclinical models of KRAS- MEK inhibitors resulted in increased apoptosis, through the mutant CRC. Our data show that this combination induction of oxidative damage and ROS generation (well- exhibits potent, synergistic antitumor effects both in vitro known response markers for HDAC), as well as enhanced and in vivo. These findings strongly support the clinical lethality in leukemia cells expressing the ABL/BCR mutation development of this rational combination in CRC and resistance to imatinib. patients with KRAS-mutated tumors. On the basis of these data, and our own transcriptional profiling and gene set enrichment analysis, we hypothesized that targeting the MEK pathway would inhibit signal transduction pathways involved in CRC tumor cell proliferation, mutations are common in many different tumors, particu- survival, and angiogenesis, and that this could be potenti- larly in pancreatic (90%) and colon cancers (50%; refs. 3, ated by combination with vorinostat (30). Furthermore, we 4). Although BRAF mutations are more common in mela- focused these preclinical studies on CRC tumors expressing noma (63%), papillary thyroid cancer (45%), and low- KRAS mutations, as they are associated with resistance to grade ovarian cancers (50%), the BRAF V600E mutation other signal transduction inhibitors and represent a subtype has also been found in colorectal cancers (CRC) that also of CRC that is in dire need of new therapeutic strategies. exhibit defective DNA mismatch repair (5, 6). The RAS/ Finally, we aimed to establish sensitive in vivo endpoints for MAPK cascade is one of the major downstream signaling MEK and HDAC inhibitors in a murine CRC xenograft networks linking the epidermal growth factor (EGF) recep- model. tor (EGFR), insulin-like growth factor-I (IGF-I) receptor (IGF-1R), and VEGF receptor-2 (VEGFR-2) pathways to Materials and Methods nuclear proteins. The safety profile and tolerability of selumetinib has been evaluated in a 2-part, multicenter, ascend- Drugs ing dose, phase I clinical study (7). This trial showed the Selumetinib (AZD6244) and vorinostat (SAHA) were tolerability of selumetinib, with the most common treat- generously provided by AstraZeneza Pharmaceuticals LP ment-related toxicities being rash, diarrhea, nausea, and and Merk Sharp and Dohme Corporation, respectively, and fatigue. Several phase II trials are ongoing to evaluate the National Cancer Institute, NIH. Selumetinib (AZD6244) activity of selumetinib, as single agent or in combination was prepared as a 10 mmol/L stock solution in dimethyl with chemotherapy in non-small-cell lung carcinoma sulfoxide (DMSO) for the in vitro experiments. For the in vivo (NSCLC), melanoma, and CRC (8–10). studies, selumetinib was prepared in a solution of 0.5% Histone deacetylation by histone deacetylases (HDAC) is methyl cellulose/0.1% Tween 80 in water. Vorinostat a posttranslational modification of lysine residues in nucle- (SAHA) was prepared as a 10 mmol/L stock solution in osomal histone proteins that affects chromatin structure DMSO for the in vitro studies and as a solution of 45% and, thereby, gene regulation (11). Recently, HDAC activity PEG400 in water for the in vivo studies. has been shown to be upregulated in cancer cells, and it has been theorized that this results in repression of tumor Culture of cell lines and assessment of cytotoxicity to suppressor gene products such as p53, making HDACs an selumetinib, vorinostat, or the combination attractive drug target (12, 13). In cell culture models, HDAC The following human colon cancer cell lines were inhibitors (HDACi) have been shown to decrease prolifer- obtained from American Type Culture Collection: HCT116 ation and induce apoptosis or autophagy-related death of (KRAS mut), HCT15 (KRAS mut), HCT8 (KRAS mut), HT29

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(KRAS wt), SW480 (KRAS mut), SW620 (KRAS mut), representing the same gene were collapsed based on max- SW1417 (KRAS wt), LoVo (KRAS mut), LS513 (KRAS mut), imum values. Next, the gene expression levels were con- LS180 (KRAS mut), LS174T (KRAS mut), LS1034 (KRAS verted to a rank-based matrix and standardized (mean ¼ 0, mut), Colo205 (KRAS wt), and the RKO (KRAS wt). Cells SD ¼ 1) for each microarray. Using this preprocessing were grown in RPMI medium supplemented with 10% FBS, method, the same cell lines from different data sets were 1% nonessential amino acids, 1% penicillin/streptomycin, clustered based on their gene expression profiles. Data and maintained at 37C in an incubator under an atmo- analyses were carried out on this rank-based matrix. sphere containing 5% CO2. The cells were routinely screened for the presence of Mycoplasma (MycoAlert; Cam- Gene set enrichment analysis brex Bio Science). Cytotoxic effects were determined using Gene set analysis was carried out using the GSEA software the sulforhodamine B (SRB) method as previously Version 2.0.1 obtained from the Broad Institute (http:// described (19). Briefly, cells in logarithmic growth phase www.broad.mit.edu/gsea). Gene set permutations were were transferred to 96-well flat bottom plates with lids. carried out 1,000 times for each analysis. We used the One hundred microliter of cell suspensions containing nominal P value and Normalized Enrichment Score (NES) viable HCT116 (1,500 cells per well), HCT15 (2,000 cells to sort the pathways enriched in each phenotype. We used per well), HCT8 (1,500 cells per well), GEO (2,000 cells the 313 pathways defined by BioCarta database as the gene per well), SW837 (1,500 per well), HT29 (3,000 cells per set in this study (www.biocarta.com). One hundred and well), SW480 (2,500 cells per well), SW620 (3,000 cells per eighty-eight gene sets passed the gene set size filter criteria well), SW1417 (4,000 cells per well), LoVo (5,000 cells (min ¼ 10, max ¼ 500). per well), LS513 (5,000 cells per well), GEO (2,000 cells per well), SW837 (1,500 per well), LS180 (3,000 cells per well), In vitro evaluation of the combination effects of LS174T (3,500 cells per well), LS1034 (6,000 cells per selumetinib and vorinostat well), Colo205 (8,000 cells per well), and RKO (1,500 cells Synergy was determined for the KRAS-mut cell lines per well) cells were plated into each well and incubated SW837, LS1034, HCT116, GEO, SW480, and SW620. These overnight before exposure to selumetinib or vorinostat. cell lines were selected because all of them contain KRAS Initially, all of the cell lines were exposed for 24, 48, and 72 mutations but exhibit divergent sensitivity to the single hours to increasing concentrations of selumetinib (0–5 agents. To evaluate synergy, cells were exposed for 72 hours mmol/L) and vorinostat (0–10 mmol/L). Post drug treat- to vorinostat at 0.375, 0.75, and 1.5 mmol/L and selume- ment media was removed and cells were fixed with cold tinib at 0.125, 0.250, 0.5, and 1 mmol/L (SW480), or 0.06, 10% trichloroacetic acid for 30 minutes at 4C. Cells were 0.125, 0.250, and 0.5 mmol/L (SW620), in all possible then washed with water and stained with 0.4% SRB (Fisher combinations. The results of the combined treatment were Scientific) for 30 minutes at room temperature, washed analyzed according to the isobolographic method of Chou again with 1% acetic acid, followed by stain solubilization and Talalay (32), using the Calcusyn software program with 10 mmol/L TRIS at room temperature on shaker for (Biosoft). The resulting combination index (CI) was used 15 minutes. The plates were then read on a plate reader as a quantitative measure of the degree of interaction (Biotek Synergy 2) set at an absorbance wavelength of 565 between the 2 drugs. A CI equal to 1 denotes additivity, CI nm, and cell proliferation curves were derived from the greater than 1 antagonism, and CI values less than 1 indicate raw absorbance (OD) data. The cells were classified as synergism. sensitive to the drug if their IC50 was lower than 1 mmol/L The effect of selumetinib and vorinostat was further or resistant if their IC50 was higher than 1 mmol/L as evaluated in the SW480 cell line which is KRAS mut, previously described (31). resistant to selumetinib as well as vorinostat (R/R), and in the SW620 cell line, which is KRAS mut but sensitive to both Transcriptional profiling of the CRC cell lines drugs (S/S) using a 3-dimensional (3D) culture system. The baseline gene expression of all of the human CRC cell Briefly, 100 mL of Matrigel (BD Biosciences) was plated in lines was assessed using the Affymetrix U133 Plus 2.0 8-well chamber slides (BD Biosciences) to form a base layer. microarrays. In addition, posttreatment profiling was car- Cells were then seeded at the density of 2,500 cells per well ried out on HCT8 (selumetinib-R/vorinostat-S), HT29 in 2% Matrigel-supplemented media to form the upper (selumetinib-S/vorinostat-R), SW480 (R/R), and SW620 layer. After an initial incubation to allow for tumor spheroid (S/S) cells, treated with vorinostat and selumetinib as single formation, vehicle, or drugs were added to media and agents and in combination. The cells were plated in T75 changed every 3 days for a total of 15 days. The SW480 flasks for 24 hours and then treated with vorinostat or (R/R) were exposed to vorinostat (0.750 mmol/L) or selu- selumetinib for 72 hours. All cell lines were treated at doses metinib (0.125 mmol/L) or the combination, whereas equivalent to the IC50 of the most sensitive cell line. After SW620 (S/S) cells were treated with selumetinib (0.06 treatment, RNA was extracted with a RNeasy Plus Mini Kit mmol/L), vorinostat (0.750 mmol/L), or the combination. (Qiagen Inc.) and analyzed by the Gene Array Core at the After 15 days of treatment, the plates were photographed at University of Colorado Cancer Center. Absolute intensity 20 magnification on an inverted microscope. Spheroids signals from the microarray gene expression profiles were with a diameter higher than 1 mm were counted. All of the in extracted using Affymetrix Power Tools, and probe sets vitro experiments were conducted in triplicate.

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Flow cytometric analysis of cell-cycle distribution Effects of selumetinib and vorinostat on ligand- For cell-cycle analysis, cells were seeded in 6-well induced cell migration plates (2.5 105 cells per well) for 24 hours. The cells The effects of selumetinib and vorinostat on SW620 and were then treated with vorinostat at 0.750 mmol/L, or SW480 cell migration were measured using the modified selumetinib at doses of 0.125 mmol/L for SW480 (R/R) Boyden chamber assay. Assays were carried out using and 0.06 mmol/L for SW620 (S/S), or with the respective uncoated 8.0 mm transwell inserts placed in 24-well plates combination for 24 hours. Culture media was removed (Becton Dickinson). Cells were resuspended in 250 mLof and the cells were washed in PBS, harvested, centrifuged standard growth media (RPMI supplemented with 10% for 5 minutes at 1,500 rpm, and resuspended in Krishan’s FBS, 1% nonessential amino acids, 1% penicillin/strepto- stain. After incubation at 4C for 24 hours, the samples mycin) and seeded at a density of 10 104 cells per well were analyzed by flow cytometry at the University of onto the inserts (upper chamber of the well), and the lower Colorado Cancer Center Flow Cytometry Core Facility. chambers of each well were filled with 600 mL of standard Experiments were conducted in triplicate. growth media. After 24 hours, the media in the upper chambers was replaced with media containing 0.1% FBS, in presence or absence of selumetinib or vorinostat or the Apoptosis assay combinations (selumetinib at the doses of 0.125 mmol/L SW480 and SW620 cells were seeded in 96-well, white- and vorinostat at the dose of 0.750 mmol/L). The media in walled plates and allowed to plate for 24 hours. SW480 the bottom of the 24-well plate was replaced with fresh (R/R) cells were treated with vorinostat (0.750 mmol/L) or media containing 10% FBS with or without the drugs selumetinib (0.125 mmol/L) or the combination for 24, (selumetinib 0.125 mmol/L or vorinostat, at the dose of 48, and 72 hours, after which apoptosis was determined 0.750 mmol/L). After 48 hours, the filters were fixed with 4% by the measurement of caspase 3/7 activity using a lumi- formaldehyde for 15 minutes, washed 3 times with D-PBS, nometric Caspase-Glo 3/7 assay (Promega) according to and migrating cells were stained with 40,6-diamidino-2- the manufacturer’s protocol and read using a 96-well phenylindole (DAPI) for 30 minutes, washed with D-PBS plate reader. Cellular apoptosis was expressed as the 3 15 minutes, and photographed at 20 magnification on fold-increase over untreated control cells. Similarly, an inverted microscope. Five fields per well were counted SW620 (S/S) cells were treated with selumetinib (0.06 manually and averaged. Selumetinib, vorinostat, and the mmol/L) or vorinostat (0.750 mmol/L) or the combina- combination effects on EGF- or IGF-I–induced migration tion for 24, 48, and 72 hours and apoptosis was measured was explored used SW480 (R/R) cells because the SW620 as described earlier. Experiments were conducted in cells were not sufficiently migratory. Briefly, 10 104 cells triplicate. per well were resuspended in 250 mL of standard growth media and plated in the upper chamber. The lower chamber Immunoblotting of downstream effector proteins of the wells was filled with 600 mL of standard growth SW480 and SW620 cells were seeded into 6-well plates media. After 24 hours, the media in the upper and lower (2.5 105 cells per well) for 24 hours before treatment chambers of the plate was replaced with 0.1% FBS media and exposed to each drug alone or in combination for an and incubated overnight in the presence or absence of additional 24 hours. After treatment, cells were recovered treatment. Cell migration was then stimulated by replacing in radioimmunoprecipitation assay buffer containing media in the lower chamber with media containing either protease inhibitors, EDTA, NaF, and sodium orthovana- 10% FBS or EGF (100 ng/mL), IGF (200 ng/mL) in absence date. The total protein in samples was determined using or presence of treatment (selumetinib at the dose of 0.125 the BioRad DC Protein Assay (BioRad). Fifty micrograms mmol/L and vorinostat at the dose of 0.750 mmol/L). Cell of total protein was loaded onto a 4% to 20% gradient gel, migration was evaluated after 48 hours from growth factor electrophoresed, and then transferred to nitrocellulose stimulation, as described earlier. After 48 hours, the filters using the I-Blot (Invitrogen). Membranes were blocked were fixed with 4% formaldehyde for 15 minutes and for 1 hour in blocking buffer (0.1% casein solution in 0.2 washed 3 times with D-PBS, and migrating cells were PBS), before overnight incubation at 4Cwithoneofthe stained with DAPI for 30 minutes, washed with D-PBS 3 following primary antibodies: phospho-ERK1/2, cleaved 15 minutes, and photographed at 20 magnification on PARP, acetylated histone H3, or b-actin (Cell Signaling an inverted microscope. Five fields per well were counted Technology). Following primary antibody incubation, manually and averaged. membranes were then washed 4 times (10 minutes each) in TBS-Tween (0.1%) and then incubated with the appro- Assessment of the selumetinib and vorinostat priate secondary anti-rabbit or anti-mouse IgG1 horse- combination in vivo radish peroxidase–linked antibody at 1:15,000 (Jackson Female athymic nude mice, 4 to 6 weeks old, were ImmunoResearch) for 1 hour at room temperature. After purchased from Harlan Laboratories (Harlan Laboratories 4 additional washes, blots were developed using the online). Animals were housed in polycarbonate cages and Odyssey Infrared Imaging System (LI-COR Biosciences). maintained on a 12-hour light/dark cycle in the University Immunoblot experiments were done in triplicate for each of Colorado Center for Comparative Medicine, a facility antibody. accredited by the American Association for Accreditation of

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Laboratory Animal Care. Animals were housed 3 to 5 per time between the injection and scan time and is cage and food and water were provided ad libitum. All presented as the percentage of the baseline scan of respec- studies were conducted in accordance with the NIH Guide- tive tumor. lines for the Care and Use of Laboratory Animals. SW620 By the end of the study (day 22), CRC xenografts were (S/S) and SW480 (R/R) cell lines were harvested in expo- collected, snap frozen in liquid nitrogen, and extracted for nential growth phase and resuspended in a 1:1 mixture of an expanded metabolic profile by proton nuclear magnetic media:Matrigel. One million SW620 and 5 million of resonance (1H-NMR). The exact extraction and NMR acqui- SW480 per injection were injected subcutaneously in both sition protocols were extensively reported previously by our flanks of each mouse. When tumors reached an average of group (33, 34). All quantitative data sets (presented as mmol 150 to 300 mm3 of volume, mice were randomized into 4 of metabolite per gram tissue) were included in custom- groups (n ¼ 8 tumor per group). Animals received either build metabolic fluxes analyzers and data interfaces and vehicle, 25 mg/kg selumetinib, 75 mg/kg vorinostat, or a presented as metabolic heatmaps (35). combination of 25 mg/kg selumetinib and 75 mg/kg vorinostat. Selumetinib was administered by oral gavage twice Statistical analysis a day, 7 days per week. Vorinostat was administered by The statistical analyses of the in vitro and in vivo data were intraperitoneal injection daily, 5 days per week. For com- carried out using Prism version 4.02 program (GraphPad bination treatment, animals received selumetinib followed Software, Inc.). A 1-way ANOVA test was carried out to immediately by vorinostat injection. Mice were monitored evaluate statistically significant changes between the study daily for signs of toxicity and were weighed twice weekly. groups. P < 0.05 was considered statistically significant. Bar Tumor size was evaluated twice per week by caliper mea- represents the mean of 3 independent experiments carried surements using the following formula: tumor volume ¼ out in triplicate. [length width2]/0.52. Tumor volume and body weight data were collected using the Study Director Software pack- Results age (Studylog Systems). Animals were treated for 28 days and then euthanized by isoflurane anesthesia. These studies Gene array and pathway analyses were carried out under an approved University of Colorado To identify genes and pathways that are correlated with Animal Care (IACUC) protocol. single-agent vorinostat sensitivity, we compared the baseline gene expression across the CRC cell line panel using Tumor metabolic response of SW480 xenografts by GSEA. HDAC pathway was among the top enriched path- 18FDG-PET and 1H-NMR ways in the sensitive lines as compared with the resistant 18 F-fluoro-2-deoxy-D-glucose positron emission tomog- lines. We next sought to identify which genes and pathways raphy (FDG-PET) was carried out on SW620 (S/S) xeno- were enriched in the resistant cell lines after vorinostat grafts to measure the effect of selumetinib, vorinostat, or treatment. Comparing the baseline and posttreatment the combination on tumor metabolic activity. When expression profiles of the 2 resistant cell lines (HT29 and tumors reached an average of 150 to 200 mm3,mice SW480) revealed that MAPK signaling pathway is among were randomized into 4 groups (n ¼ 8 tumors per group). the top enriched pathways after vorinostat treatment Treatment groups were the same as the first in vivo study (Fig. 1A). The core genes of the MAPK signaling pathway and animals were treated for a total of 21 days as are illustrated in Fig. 1B and C. This suggested that treating described in the previous section. FDG-PET was carried these vorinostat-resistant cell lines with selumetinib may out the day before the start of treatment (baseline; BL) synergize the combination drug effects. Indeed, when we and on days 4 (D4) and 21 (D21) of treatment. Animals compared the core genes in the MAPK signaling pathway of were fasted for 4 to 8 hours before FDG injection and the 2 vorinostat-resistant cell lines after selumetinib and the glucose blood levels were monitored. Approximately 250 combination, the gene expression patterns were altered mCi of FDG, obtained through the University of Colorado (Fig. 1B). Hospital (PetNet Solutions), was administered by tail vein injection (intravenously) to conscious animals. Ani- Effects of the selumetinib and vorinostat combination mals were maintained in cages on a heated water pad for 1 on proliferation of KRAS-mutant CRC cell lines in vitro hour to allow for FDG uptake in tumors. Under isoflurane To seek for a new active treatment for KRAS-mut CRC anesthesia (2.5%), animals were placed on a warm pad patient, we studied the combination of selumetinib and (m2m Imaging) and a 10-minute emission scan was vorinostat on GEO, HCT116, SW837, SW620, and LS1034. acquired with a Siemens Inveon micro-PET scanner (Sie- In an initial study, conducted on a panel of CRC cell lines to mens Medical). Image analysis was conducted with Asi- evaluate the activity of selumetinib and vorinostat as single ProVM (Concorde Microsystems) software. Regions of agent, we observed that the SW837 and the SW480 were interest were drawn with the trace command around resistant to both selumetinib and vorinostat (R/R), the GEO the tumors on axial slices, and the total activity of all were resistant to selumetinib and sensitive/intermediate for tumor slices was summed. Total activity was divided by vorinostat (R/I-R), the LS1034 were sensitive to selumetinib the time-corrected dose delivered (time-corrected dose and resistant to vorinostat (S/R), and the SW620 were ¼ dose injected exp(0.006317 t), where t is the sensitive to both drugs (S/S) as shown in Supplementary

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A Name BioCarta pathway Size NES NOM P H_PGC1APATHWAY Regulation of PGC-1a 11 –1.64 0.0218 H_MALPATHWAY Role of MAL in Rho-mediated activation of SRF 19 –1.61 0.0219 H_RAC1PATHWAY Rac 1 cell motility signaling pathway 20 –1.41 0.0625 H_MAPKPATHWAY MAPKinase signaling pathway 81 –1.30 0.0625 Figure 1. Pathway analysis of CRC H_FMLPPATHWAY fMLP induced chemokine gene expression in HMC-1 cells 25 –1.42 0.0776 cell lines at baseline and after treatment with vorinostat, B C selumetinib, or the combination. ––++ –– ++ SAHA FASL, A, top 5 pathways enriched in the Growth factor, UV, Stress, osmotic –– –– ++++ AZD6244 shock, γ-radiation, inflammatory, trophic factors, cytokines, UV, vorinostat-resistant cell lines anisomycin etc etc (HT29 and SW480) after vorinostat

HT29 SW480 HT29 SW480 HT29 SW480 HT29 SW480 HRAS RAC1 TRADD treatment. B, heatmap of the core RAPGEF2 MAP4K1 SHC MAP4K2 PAK2 TRAF2 TRADD PAK1 genes in the HDAC and MAP

TRAF2 MAP4K kinase signaling pathway at HRAS BRAF baseline, after treatment with PAK1 MAP3K6 BRAF MAP3K8 MAP3K4 MAP3K5 vorinostat, selumetinib, and the MAP4K1 MAP4K MAP4K2 MAP3K combination. Pathway diagram MAP3K4 derived from BioCarta where the MAP2K1/2 MAP2K5 MAP2K7 MAP2K4 MAP3K6 MAP3K5 MAP3K MAP3K6 core genes are circled. Red and MAP3K8 MAP2K green in the heatmap represent MAP2K5 MAP2K MAPK14 MAP2K6 MAPK1/2 MAPK7 MAPK8/9/10 gene overexpression and MAPK14 MAPK underexpression, respectively. MAPK CEBPA C, pathway diagram derived from CREB1 MAPKAPK5 ELK1 RPS6KA1 RPS6KA3 RPS6KA2 BioCarta where the core genes are Targets of Kinases FOS MKNK1 MKNK2 RPS6KA5 ELK1 CEBPA colored in red. MAPKAPK5 MAPK

MAPK CREB1 RPS6KB2 RPS6KB1 RPS6KB3 FOS

MKNK1 of Targets RPS6KA1 Factors Transcription RPS6KA3 RPS6KB2 Relative gene expression

Low High

Fig. S1. Then, cells were treated concurrently for 72 hours at Effects of selumetinib and vorinostat on cell-cycle clinically relevant doses, according to phase I trial results distribution, apoptosis, and downstream signaling (7, 36, 37). Our results, as calculated by the method of pathways Chou and Talalay (32), showed that selumetinib and To further elucidate the mechanism(s) responsible for the vorinostat treatment resulted in clear synergy for the all observed synergistic effects on CRC cell proliferation, we cell lines analyzed (Supplementary Fig. S2), with a stron- investigated the impact of selumetinib in combination with gest effect observed for the SW620 (S/S) cell line (Fig. 2A vorinostat on cell-cycle distribution, apoptosis, and intra- and B) with CI values between 0.354 and 0.736, and for cellular signaling pathways. By flow cytometry, cell-cycle the SW480 (R/R) cell line (Fig. 2C and D) with CI values analysis following treatment of SW620 S/S cells with single- of 0.07 to 0.777. On the basis of these results, we chose to agent selumetinib or vorinostat resulted in a significant use for the subsequent experiments, the SW620 and the increase in the percentage of cells in G1, which was further SW480, treated at the following doses of selumetinib and increased in the combination and associated with a reduc- vorinostat for subsequent experiments: SW620 (S/S): tion of cells in S phase (Fig. 4A). By contrast, in the SW480, 0.06 mmol/L selumetinib and 0.750 mmol/L vorinostat; R/R cells, G1 arrest was induced by treatment with selume- SW480 (R/R): 0.125 mmol/L selumetinib and 0.750 tinib which was maintained in the combination (Fig. 4B). mmol/L vorinostat. Next, we evaluated the ability of selumetinib, vorinostat, or To assess the efficacy of selumetinib, vorinostat, and the the combination to induce apoptosis in SW620 and SW480 combination in a more physiologic model, we next carried cells after 24 hours of exposure to these agents using a out a 3D culture analysis with tumor spheroid formation in caspase 3/7 activity assay. Although apoptosis was modestly Matrigel as the endpoint. As depicted in Fig. 3A–D, the increased by the single agents, the combination showed a SW620 S/S and SW480 R/R cells generated spheroids when significant induction of apoptosis in both cell lines, as grown in Matrigel, which were inhibited by both single depicted in Fig. 4C and D. agents and, more strikingly, by the combination. Interest- Analysis of downstream signaling pathways by immuno- ingly, the SW480 R/R cells displayed a sensitive phenotype blotting showed that in both cell lines selumetinib reduced to selumetinib and vorinostat in 3D culture, but a resistant ERK phosphorylation and vorinostat induced histone H3 phenotype in monolayer growth, suggesting that these acetylation, as expected (Supplementary Fig. S3). Interest- agents require more intact tumor architecture to exert their ingly, H3 acetylation was markedly increased in the com- full effects. bination treatment (Supplementary Fig. S3). Increased

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AB SW620 (S/S) Dose–effect curve

1.1 1.00

0.9 0.80

0.7 0.60 CI 0.5 Effect 0.40 Figure 2. Selumetinib in 0.5 μmol/L combination with vorinostat shows 0.3 0.20 0.25 μmol/L synergistic inhibition against CRC 0.125 μmol/L Selumetinib cell lines. SW620 S/S (A, B) and 0.1 0 0.106 μmol/L SW480 R/R (C, D) were plated in 96- 0 0.5 1.0 1.5 0.375 μ Dose well plates for 24 hours and treated mol/L 0.75 μmol/L 1.5 Combo Selumetinib Vorinostat for 72 hours with varying doses of μmol/L Vorinostat selumetinib and vorinostat. Proliferation was assessed by the CD SRB assay. Raw proliferation data SW480 (R/R) were expressed as percent of viable Dose–effect curve cells and CI values were analyzed 1.1 0.80 according to the Chou and Talalay method for drug interactions using 0.9 the Calcusyn software package. 0.60 0.7 CI 0.5 0.40 Effect μ 0.3 1 mol/L 0.5 μmol/L 0.20 μ Selumetinib 0.1 0.25 mol/L 0.125 μmol/L 0 0 0.5 1.0 1.5 0.375 μmol/L 0.75 Dose μmol/L 1.5 μmol/L Combo Selumetinib Vorinostat Vorinostat

PARP cleavage was observed in both cell lines after treat- these drugs in a murine xenograft model. Tumor growth ment with vorinostat or selumetinib, however, levels of curves for these studies are depicted in Supplementary Fig. cleaved PARP were markedly increased in SW620 S/S line S5 for the SW480 R/R xenograft and in Fig. 5A for the SW620 after the combination treatment and, to a lesser extent, in S/S xenograft. In the SW620 S/S model, vorinostat alone the resistant cell line, SW480 (R/R), consistent with an showed little antitumor growth effects, whereas selumetinib increase in apoptosis as observed using the caspase 3/7 and the combination were similar, showing a trend toward activity assay (Supplementary Fig. S3; Fig. 4C and D). significant tumor growth inhibition compared with control (, P < 0.05). Treatment of the SW480 R/R xenograft resulted Effects of selumetinib, vorinostat, and the combination in more modest growth inhibitory effects, which also on ligand-induced migration of SW480 cells appeared to be dominated by the single-agent effects of As cancer cell migration is mediated by growth factor selumetinib. pathways such as the RAS/RAF/MEK/ERK pathway (38–44), we evaluated the effects of selumetinib and vorinostat alone Assessment of SW620 S/S xenograft tumor metabolic and in combination on cell migration using a modified response by 1H-NMR and 18FDG-PET Boyden chamber assay. As depicted in Supplementary Fig. An expanded metabolic profile, calculated from 1H-NMR S4, the combination of selumetinib and vorinostat signif- extracts of treated and untreated tumors, showed that no icantly reduced both FBS- and IGF-stimulated migration changes in lactate, the end product of glycolysis, were compared with EGF, which exhibited little induction of observed in the treatment groups (Fig. 5B). In fact, no migration, regardless of treatment. significant metabolic changes were detected for the HDAC inhibitor, vorinostat. The most striking changes observed Antitumor activity of selumetinib in combination with with MEK inhibition by selumetinib were related to highly vorinostat in SW620 S/S and SW480 R/R xenograft decreased levels of membrane phospholipids (such as models phosphatidylcholine, its precursors phosphocholine, and To further evaluate the efficacy of selumetinib in combi- phosphatidylinositol). Accumulation of nucleosides and nation with vorinostat as a potential treatment option for adenosines were also seen in the selumetinib-treated group. CRC, we tested the tumor growth inhibitory potential of The combination group mostly reflected the metabolic

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molecules related to cancer cell biology to achieve a more A B specific and effective treatment, and at the same time, avoid SW620 (S/S) some of the systemic toxicities associated with chemotherapy (45, 46). To optimize this therapeutic approach, patient 60 selection for clinical trials has become of critical importance 50 (47–50). For example, recent studies have shown a corre- 40 Selumetinib lation between CRC tumors with activating KRAS mutations * Control 30 and resistance to EGFR-targeted therapies. Therefore, there 20 * * * is an emerging need for alternative therapeutic approaches 10 ** ** for this patient population (5, 50, 51). In this study, we # Colonies per field 0 utilized in vitro and in vivo models of CRC to evaluate the 2 1 3 456 7 8 efficacy of selumetinib in combination with vorinostat as a 7 d 15 d Vorinostat Combo potential therapeutic strategy for the treatment of CRC patients expressing mutant KRAS. Our data show that this CD combination exerts potent antitumor effects on in vitro SW480 (R/R) models of CRC, including synergistic inhibition of cell proliferation, G1 cell-cycle arrest, induction of apoptosis, 19 18 17 and spheroid formation in 3D culture. Interestingly, this 16 15 14 combination inhibited CRC cell migration, which suggests 13 12 11 Control Selumetinib that this treatment not only inhibits tumor growth but may 10 * 9 In vivo 8 play a role in the metastatic process as well. studies 7 * * 6 5 * using KRAS-mutant CRC xenograft models in nude mice 4 3 confirmed the antitumor effects of the combination of 2 ** ** # Colonies per field 1 0 selumetinib and vorinostat, although synergy was not 132 5678 4 observed, likely because of the single-agent effects of 7 d 15 d Vorinostat Combo selumetinib. In our initial studies, we evaluated selumetinib and vorinostat as single agents against a panel of CRC cell lines Figure 3. Effects of selumetinib and vorinostat on CRC spheroid formation that were selected based on differential KRAS, BRAF, and in 3D culture. CRC cell lines were plated in 8-well chamber slides on p53 Matrigel pads at a density of 2,500 cell per well resuspended in 2% mutational status. Several reports have suggested a Matrigel-supplemented RPMI plus 10% FBS. SW620 (S/S) and SW480 relationship between KRAS and BRAF mutation status and R/R cells were treated with indicated doses of selumetinib, vorinostat, or response to MEK inhibitors (1). Other investigators show the combination for 15 days. SW620 (A, B) and SW480 (C, D) spheroids that the blockage of the MAPK pathway with a MEK inhib- were photographed and counted after 7 and 15 days of treatment. Spheroids with a diameter greater than 1 mm were counted. Data itor induced cell death only in cells that were KRAS mutant represent the mean of 3 independent experiments. , P < 0.05 but p53 wild type (52). Although we failed to show any selumetinib, vorinostat, or combination treatment versus control; significant correlations between cell line sensitivity to selu- , P < 0.05 combination versus control, and single agents. Bars represent metinib or vorinostat and KRAS, BRAF, or p53 status (data the mean of 3 independent experiments carried out in triplicate. not shown), a recent study by Dry and colleagues showed that a gene signature of functional MEK activation was consistently elevated in cell lines sensitive to selumetinib, effects of single-agent selumetinib, with broader decreases whereas BRAF/KRAS mutation or ERK activation varied in membrane phospholipids. To assess the effects of selu- across cell lines, indicating that expression profiling may metinib in combination with vorinostat on glucose uptake, be a better predictor of drug response than individual 18FDG-PET was carried out on mice bearing SW620 tumors biomarkers or gene mutations for this class of agents at baseline, before starting treatment, after 4 days of treat- (53). Consistent with this report, we observed constitutive ment to evaluate early drug effects and after 21 days of overexpression of HDAC and MAPK pathways in the cell treatment with either vehicle, selumetinib, vorinostat, or lines sensitive to vorinostat or selumetinib, respectively. the combination. No significant decreases in glucose uptake Interestingly, our data showed that cell lines resistant to were observed in either study group when compared with vorinostat exhibited a constitutive overexpression of the untreated controls after 3 days of treatment (data not MAPK pathway and that the MAPK pathway was upregu- shown), whereas a trend of decrease in glucose uptake was lated in response to treatment with vorinostat, providing a observed on day 21 in the selumetinib and combo group functional rationale for exploring the use of vorinostat in compared with vorinostat (Fig. 5C and D; , P < 0.05). combination with selumetinib in our preclinical models of CRC. Discussion To further evaluate the synergistic effects of this combination, we chose to focus on several KRAS-mutant CRC Recent efforts in cancer therapeutics have focused on the cell lines. Our results showed that the combination of development of targeted therapies directed toward specific selumetinib and vorinostat resulted in a synergistic

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A B SW620 SW480 100 100 G1 G1 ** S * * S 75 * * 75 G2–M G2–M

50 50 Figure 4. Selumetinib and vorinostat induce G1 cell-cycle arrest 25 25 and apoptosis in CRC cell lines. SW620 S/S (A, C) and SW480 R/R (B, D) CRC cell lines were treated with 0 0 selected doses of selumetinib and vorinostat for 24 hours. For cell-cycle ND ND analysis, cells were stained with Combo Combo Vorinostat Vorinostat Krishanaposs stain followed by ﬂow Selumetinib Selumetinib cytometric analysis. Apoptosis was 24 h 24 h analyzed by ﬂuorometric measurement of caspase-3 and caspase-7 activity. , P < 0.05 C D SW620 SW480 selumetinib, vorinostat, or 5 5 combination treatment versus control; , P < 0.05 combination 4 4 ** versus control and single agents. ** Bars represent the mean of 3 3 3 independent experiments carried out in triplicate. 2 2 Caspase 3/7 Caspase 3/7

1 1

0 0

ND ND Combo Combo Vorinostat Vorinostat Selumetinib Selumetinib 24 h 24 h

antiproliferative effects in all cell lines studied. Mecha- resulted in a G1 arrest, suggesting that the combination nistically, our results suggest that this antiprolifterative effects were mediated by selumetinib. effect of combination of selumetinib and vorinostat is Because cell proliferation is only one phenotypic end- mediated by an increase in apoptotic cell death, as mea- point of malignant behavior, we used other in vitro models sured by caspase 3/7 activity, cleaved PARP, and, perhaps, such as a modified Boyden chamber and 3D culture and increased acetylated histone H3 levels. This is in accor- found that the combination of these agents resulted in dance with previous reports showing that MEK inhibition significant decreases in cell motility and spheroid forma- was synergistic with HDAC inhibitor through the induction, respectively. As MEK is a downstream effector of the tion of cell death via apoptosis (29). We also evaluated EGFR, VEGFR, and IGF-1R signaling pathways, we hypoth- the effects of the combination on cell-cycle distribution esized that MEK activation could play a role in ligand– and apoptotic events. It has been previously reported that induced cell migration. For example, previous data have vorinostat increases the expression of cyclin-independent shown that treatment of NSCLC with HDAC inhibitors kinases such as p21CIP/WAF1 in bladder cancer, p27KIP1 in induces reexpression of E-cadherin mRNA, induction of gliomas, and induces G2/M cell-cycle arrest in CRC cell the epithelial phenotype, and decreased migratory activity lines, whereas selumetinib induced G1 cell-cycle arrest (55). By contrast, our findings indicated that stimulation of and induction of apoptosis in a CRC xenograft model EGFR pathway did not affect the migration of SW480 cells, (1, 19, 54). In our study, we observed a G1 cell-cycle arrest although both FBS and IGF-1 induced cell migration that in the SW620 S/S cell line after each single-agent treat- was inhibited by both single agents and substantially by the ment that was significantly increased after exposure to the combination. The IGF1R and other components of the IGF combination. In the SW480 R/R cell line, vorinostat system have been shown to be overexpressed in colon showed no effects on cell-cycle distribution, whereas cancer and are associated with advanced stage of disease, single-agent selumetinib and combination treatment metastasis, and reduced survival (56). These results suggest

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Figure 5. Antiproliferative and metabolic responses of the SW620 A B S/S cell line xenograft in vivo.A,

metabolism SW620: Day 21 Vehicle AZD6244 Vorino- Combo curves represent tumor growth

Glucose stat rate, expressed as "percent of day 18 FDG Uptake (PET) 969.2 ± 399.8 Total lactate 10.7 ± 1.5 one," when treated with either Aromatic amino acids 10.0 ± 2.3 Tyrosine 0.5 ± 0.2 vehicle, selumetinib (25 mg/kg), Metabolism Amino Acid Glutamine 0.7 ± 0.3 vorinostat (75 mg/kg), or the 1,750 Glutamate 2.0 ± 0.3 Vehicle Succinate 0.6 ± 0.2 combination. B, metabolic Lysine + Arginine 0.5 ± 0.1 1,500 Alanine 1.1 ± 0.3 heatmaps based on quantitative Selumetinib 1 Valine, Leucine, Isoleucine 2.6 ± 0.4 FDG-PET and H-NMR 1,250 Vorinostat Osmo, ROS Acetate 0.4 ± 0.1 spectroscopic data sets in SW620 Total glutathione 1.0 ± 0.2 Combo Taurine 6.1 ± 1.1 xenografts on day 22. The data 1,000 Myo-Inositol 1.1 ± 0.5 metabolism Polyols 16.6 ± 4.4 represents mean SEM of 8 Energy 750 Adenosines 1.5 ± 0.3 biopsies per group. The Nucleotides 1.6 ± 0.4 Total creatine 1.6 ± 0.2 metabolites, their ratios, and

500 Membrane P-lipids Phosphocholine (PCho) 4.6 ± 0.6 ﬂ Percent of day 1 metabolic uxes were grouped on Metabolism Glycerophosphocholine (GPC) 0.8 ± 0.2 250 (PCho: GPC) 5.8 ± 1.7 the basis of their biochemical ± Total phospholipids 6.6 0.9 relevance. For the control group, all 0 Phosphatidylcholine 4.7 ± 0.6 Phosphatidylinositol 1.0 ± 0.2 intracellular metabolite levels are 0510 15 20 25 30 Phosphatidylethanolamine 1.0 ± 0.2 Phosphatidylserine 0.2 ± 0.1 presented as mmol per cell wet Total lipids 74.5 ± 8.6 weights; metabolite ratios and Day Metabolism Total fatty acids (FA) 39.9 ± 4.4

Lipid Polyunsaturated FA (PUFA) 21.2 ± 2.5 glucose uptake are unitless. Monounsaturated FA (MUFA) 14.8 ± 3.9 Triacylglycerides 5.8 ± 0.8 Metabolic pathways, which were Cholesterol 5.3 ± 0.8 undisturbed by treatment, are presented as yellow blocks. A decrease in a metabolic endpoint is C D indicated by red, whereas an Vehicle Selumetinib Vorinostat Combo Day 4 increase is depicted in green. 900 Statistical significance for 800 metabolite changes are based on 700 600 multivariate analysis of metabolic 500 fluxes with , P < 0.02. The 400 interactive metabolic profile array 300 200 database was custom based (35). Baseline 100 C, representative PET images and 0 their quantification (D, presented Veh SVC as the percent of normalized Day 21 glucose uptake to the baseline) 3,500 acquired on days 4 and 21 3,000 posttreatment. Decreases of 2,500 18FDG uptake in tumors can be 2,000 measured as early as 4 days

Day 21 1,500 posttreatment, with no signiﬁcant 1,000 differences between either 500 treatment group compared with 0 control but with a trend of Veh SVC selumetinib compared with vorinostat measured at day 21.

that the combination of vorinostat and selumetinib may act untreated controls or vorinostat, but not to single-agent downstream of this pathway to block the phenotypic effects selumetinib. As would be expected from the 3D results, of IGF-induced CRC migration. In 3D culture, cancer cells the SW480 xenograft was sensitive to selumetinib in vivo, are able to form tumor spheroids that are thought to more which we hypothesize could be related to MEK-depen- closely recapitulate the cancer cell–extracellular matrix dent effects on the tumor microenvironment and tumor interactions that play a fundamental role in tumor growth cell–matrix interactions. Although the single-agent activ- and metastasis (57). The inhibitory effects of the combina- ity of selumetinib at 25 mg/kg obfuscated the detection of tion were confirmed in the 3D model in both the sensitive synergy in vivo, our assessment of metabolic endpoints led (SW620) and resistant (SW480) cell lines. Of interest was to some interesting findings. Surprisingly, no significant the finding that the SW480 cells appeared to be sensitive to inhibition of glucose uptake (FDG-PET) or metabolism selumetinib in the 3D model, supporting the limitation of (lactate from 1H-NMR) was observed for either selume- 2D assays in assessing more global impacts on cellular tinib or vorinostat, alone or in combination, compared phenotype in vitro. with untreated controls. Although HDAC inhibition did In our in vivo studies of both SW620 (S/S) and SW480 not appear to induce any metabolic changes, the MEK (R/R) cell line xenografts, combination treatment showed inhibitor selumetinib had a profound inhibitory effect on significant tumor growth inhibition compared with membrane phospholipid turnover (decrease in total

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phospholipids as well as phosphatidyl-choline and phos- testing of selumetinib in combination with vorinostat phatidyl-inositol). This, together with an accumulation of should be explored as a potential therapy for KRAS-mutant adenosine and nucleotides, is reflective of the antiproli- CRC. ferative effects observed in this study for selumetinib and the combination. These data show that effects on glucose Disclosure of Potential Conflicts of Interest metabolism are not observed with all targeted agents and that preclinical metabolic studies should be conducted to No potential conflicts of interest were disclosed. more precisely determine the most appropriate readout for functional imaging. For example, these results suggest Acknowledgments that in human clinical trials, incorporation of 18F-choline The authors thank Heather Selby and Kelly McPhillips for their excellent PET, rather than FDG-PET, might be the most relevant technical support. imaging endpoint for treatment response to MEK inhibitors. Grant Support In summary, we utilized gene array and gene set enrichment analyses to develop a rational basis for testing the This work was supported by research grants CA106349 (S.G. Eckhardt), combination of selumetinib and vorinostat as a novel CA079446 (S.G. Eckhardt), CA46934 (NCI Cancer Center Support Grant), and an Astra Zeneca Research Agreement (M.P. Morelli, S.G. Eckhardt). therapeutic option for KRAS-mutant CRC. Moreover, the The costs of publication of this article were defrayed in part by the preclinical findings described in this article, utilizing both in payment of page charges. This article must therefore be hereby marked vitro and in vivo models of CRC, show that dual inhibition of advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. MAPK signaling and HDAC activity results in inhibition of several biologic processes associated with tumor growth Received June 13, 2011; revised November 16, 2011; accepted December and progression. Together, these data suggest that clinical 9, 2011; published OnlineFirst December 15, 2011.

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1062 Clin Cancer Res; 18(4) February 15, 2012 Clinical Cancer Research

Preclinical Activity of the Rational Combination of Selumetinib (AZD6244) in Combination with Vorinostat in KRAS-Mutant Colorectal Cancer Models

M. Pia Morelli, John J. Tentler, Gillian N. Kulikowski, et al.

Clin Cancer Res 2012;18:1051-1062. Published OnlineFirst December 15, 2011.

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