AG

AA

bst

ract

sTu1892

Chronomodulated Administration of Capecitabine Reduces Side Effects ofTherapy in Patients With Colorectal CancerGisela Paul, Stefanie Rauch, Reiner Wiest, Johannes Grossmann, Frank Kullmann, UlrikeStrauch

Aim: The aim of the present study was to evaluate whether chronomodulated administrationof capecitabine would reduce toxicity of the drug in patients with metastatic colorectalcancer. Patients & Methods: 27 patients with advanced colorectal cancer were randomizedto receive capecitabine (2500mg/KOF daily) either in standard administration with 50% ofthe dose in themorning and 50% in the evening separated by 12 hours or as chronomodulateddose-regime with 25% morning-dose and 75% late evening-dose. Treatment was continueduntil thirteen treatment-cycles were finished or progress of cancer or limitation of therapydue to side effects occurred. Results: Overall, response rates to chemotherapy were similarin both treatment groups (p= 0,296). However, within the group of patients with chronomo-dulated application of capecitabine, reduction of drug-doses due to side effects was lessfrequently necessary (14.8 vs. 19.3%, p=0,026) and more patients were able to finish allthirteen chemotherapy-cycles (41.7 vs. 7.1%, p=0,007). While there was no statisticallysignificant difference in the frequency of hand-foot-syndrome in both treatment-arms (51.7vs. 41.5%, p=0,119) other side effects as nightly nausea, tiredness and sleeplessness weresignificantly reduced in patients receiving chronomodulated therapy (p=0,035, p=0,001 and0,009, respectively). Additionally, there was a trend to lower frequency of nausea, diarrheaand stomatitis within this group compared to standard treatment (9.0 vs. 19.5%, 19.1 vs.25.6%, and 4.5 vs. 8.5%) Conclusion: The chronomodulated capecitabine schedule achievedsimilar response rate and better tolerability regarding various side effects compared withstandard application of the drug in patients with colorectal cancer. This schedule couldenable patients to sustain on capecitabine therapy for a longer time-period.

Tu1893

Tumor Suppressor XAF1 and TRAIL Cooperate to Inhibit Migration andInvasion of Colon Cancer CellsYanfei Ding, Xiaojiao Cheng, Liming Zhu, MInmin Qiao, Jing Ye, Shihu R. Jiang,Shuiping Tu

Background: X-chromosome-linked IAP (XIAP) has been shown to be associated with tumormetastasis and TRAIL receptors function primarily as the metastasis suppressor. XIAP associ-ated factor 1 (XAF1), an antagonist of XIAP, can down-regulate expression of XIAP and up-regulate expression of TRAIL receptors DR4 and DR5. In this study, we investigated theeffect and mechanism of XAF1 on tumor cell migration and invasion in human colon cancer.Materials and methods: The adeno-XAF1 virus was packaged with Ad5/F35 vector. Coloncancer SW620 cells were pre-infected with Ad5/F35-XAF1 adenovirus or control virus for4 hours and then incubated with recombinant human (rh) TRAIL peptide at the dose of 5ng/mL for 48 hours. The capacity of tumor migration and invasion in vitro was determinedby a transwell cell migration and invasion assay respectively. Expression of metastasis-relatedgenes and epithelial-to-mesenchymal related genes were determined by RT-PCR, WesternBlot and immunofluorescence staining. Results: Immunofluorescence staining showed thatthe Ad5/F35-XAF1 treatment upregulated the expression of E-cadherin and decreased theactivation of vimentin, indicating that XAF1 inhibited EMT of cancer cells. RT-PCR andWestern Blot further showed that Ad5/F35-XAF1 decreased the expression of MTA1,KIAA1199, TGF-β1 and vimentin. The combined treatment of Ad5/F35-XAF1 with TRAILmarkedly inhibited the expression of MTA1, KIAA1199, TGF- β1 and vimentin comparedto the treatment with Ad5/F35-XAF1 or TRAIL alone. The combined treatment of Ad5/F35-XAF1 and rhTRAIL significantly inhibited the migration and invasion of colon cancer SW620cells. Furthermore, FACS analysis results showed that adeno-XAF1 up-regulated expressionof DR4 and DR5. Conclusion: Our results, for the first time, show that XAF1 inhibits tumormigration and invasion through the inhibition of metastasis-related genes and EMT and theupregulation of DR4 and DR5 expression. The combined treatment of Ad5/F35-XAF1 withTRAIL synergistically inhibits migration and invasion of colon cancer cells, and may be apotential strategy for the prevention of cancer metastasis. (This study is supported by NSFC81172159 to SPTU).

Tu1894

Ultrahigh-Throughput Screening Strategies for Identification of Small-MoleculeCompounds That Target the Colon Cancer Oncogene KrüPpel-Like Factor 5Agnieszka Bialkowska, Melissa L. Crisp, Thomas D. Bannister, Yuanjun He, SarwatChowdhury, Stephan Schürer, Peter Chase, Timothy Spicer, Franck Madoux, PeterHodder, Daniel Zaharevitz, Vincent W. Yang

Background: Krüppel-like factor 5 (KLF5) is a zinc finger transcription factor highly expressedin the rapidly dividing crypt epithelial cells of the intestine and colon. Results from our labhave shown that KLF5 is necessary for the tumor initiating activity of β-catenin duringintestinal adenoma formation in both ApcMin/+ and ApcMin/+/KRASV12G mice (McConnell etal. [2009] Cancer Res. 69:4125, Nandan et al. [2010] Molecular Cancer 9:63, respectively).These studies indicate that KLF5 has a crucial role in promoting intestinal tumorigenesis.Aim: To identify compounds with therapeutic potential for the prevention or treatment ofcolon cancer. Methods: We devised an ultrahigh-throughput screening (uHTS) strategy toidentify compounds with potent KLF5-specific inhibitory activity in the NIH MolecularLibraries Small Molecule Repository, eliminating false positives using a cytotoxicitycounterscreen with the nontransformed IEC-6 intestinal epithelial cells. Based on hits identi-fied during the primary screen we initiated structure-activity relationship analyses and anin-house synthesis campaign. The final lead compound was characterized using standard invitro drug metabolism and pharmacokinetics (DMPK) methods. Results: Using uHTSapproach, we identified a lead compound 5951923 (compound ID or CID) that specificallyreduces levels of KLF5 in colon cancer cells. This compound was characterized on the basisof computational, Western blot, and cell viability analyses. In-house chemistry efforts weresuccessful in augmenting the potency and selectivity of CID 5951923 and resulted in the

S-874AGA Abstracts

synthesis of a compound fully meeting the probe criteria (see below) for KLF5 inhibition:ML264. This compound does not inhibit kinases associated with the KLF5 pathway, asdetermined using a panel of 47 selected kinases. Furthermore, in a screen for off-targetactivity, ML264 showed no significant inhibition against 67 protein targets of therapeuticand/or toxicological interest. Western blot analysis shows that ML264 significantly reducesKLF5 expression in colon cancer cells. When tested in an NCI60 panel of cancer cell lines,ML264 inhibits proliferation of mostly colon cancer cell lines, which contain higher levelsof KLF5 compared to other cancer cell lines in the panel. ML264 is highly stable in thepresence of mouse, rat, and human hepatic microsomes, has favorable properties associatedwith drug-likeness, and does not inhibit many cytochrome P450 (CYP) enzymes. Preliminaryin vivo DMPK studies indicate a 3 hour half-life for ML264 in mice with no cytotoxic effectsunder tested conditions. Conclusion: Due to its favorable properties in concert with highcellular potency and selectivity, ML264 is potentially an excellent candidate for colon cancerpreclinical trials.

Tu1895

Antibody Directed Phototherapy (ADP) With HuHMFG1:PS1 Is a Novel andPotentially Curative Therapeutic Modality for MUC1 Expressing OesophagealAdenocarcinomasMohammed A. Butt, Hayley Pye, Ioanna Stamati, Gokhan Yahioglu, Rehan J. Haidry,Eleanor S. Bloom, Dahmane Oukrif, Marco Novelli, Mahendra Deonarain, Laurence Lovat

Introduction: The incidence of oesophageal adenocarcinoma (OA) is associated with anappalling 5 year survival rate under 15% as most patients are either unfit for standardtherapy, or relapse with no remaining curative option. Antibody Drug Conjugates (ADC)are a rapidly expanding therapeutic modality, with notable successes in the CD-30 targetingAdcetris and HER2 targeting transtuzumab-DM1. Antibody Directed Phototherapy (ADP)is an extension of this technology, combining the selectivity of ADC's with the loco-regionalphotoactivation of the drug. This study summarises the efficacy of a first in class ADP agent,targeting a cancer associated form of the epithelial glycoprotein MUC1. Method: We havepreviously reported how an aberrant MUC1 isoform is expressed in 36% of OA's using theantibody HuHMFG1. HuHMFG1 was first conjugated with the photosensitiser PS1. PS1 isa water soluble photosensitiser absorbing in the near infrared. Binding of the HuHMFG1:PS1ADC to oesophageal (OE-19), ovarian (SKOV-3), breast (MCF-7) and colon (HT-29) cancercell lines were demonstrated with FACS. The efficacy of HMFG1:PS1 ADC's on these lineswere compared with equivalent concentrations of PS1 alone in the presence or absence oflaser "light" activation at a dose of 0.33J/cm2. Power was deliberately set low to highlightthe efficacy of ADP. Cell viability was then measured with standard MTS assay (PromegaCell Titre-96) and the plates read on an ELISA plate reader at 490nm. Results: In thepresence of HMFG1:PS1, FACS confirmed binding to OE19, SKOV-3 and MCF-7 cells butnot HT-29 cells. Cell viability counts in all plates were initially corrected for the untreatedcell survival controls in the plate itself and then plotted on a log scale to produce dosedependent response curves. This confirmed superior cytotoxic efficacy of HuHMFG1:PS1over PS1 in SKOV-3 (F=104.93, p=0.00051) and OE-19 (F=11.13, p=0.0125) cell linesusing linear regression analysis and an F test to compare treatments. HuHMFG1:PS1 didnot kill HT29 effectively over control (p=0.84) or differ significantly from PS1 in its efficacy(F=0.155, p=0.71) confirming cytotoxicity to be selective for HuHMFG1 binding cells.Conclusion: This proof-of-concept study demonstrates the potential of ADP for OA usingHuHMFG1:PS1. We further demonstrated that HuHMFG1:PS1 can effectively kill otherMUC1 overexpressing tumours. Absence of effect in the negative control colon cancer cellline confirmed selectivity of HuHMFG1:PS1 to HuHMFG1 expressing cells. ADP could offera curative option for the majority of patients who are unfit for standard therapy, andthose who have relapsed. The deep tissue penetration of near infrared light to activateHuHMFG1:PS1, extends the indication for this drug to include those OA's with locoregionallymph node spread.