Melissa Crisp

Identification of Small-Molecule Inhibitors of the Colorectal Cancer Oncogene Krüppel-Like Factor 5 Expression by Ultrahigh-Throughput Screening

The transcription factor Krüppel-like factor 5 (KLF5) is primarily expressed in the proliferative zone of the mammalian intestinal epithelium, where it regulates cell proliferation. Studies showed that inhibition of KLF5 expression reduces proliferation rates in human colorectal cancer cells and intestinal tumor formation in mice. To identify chemical probes that decrease levels of KLF5, we used cell-based ultrahigh-throughput screening (uHTS) to test compounds in the public domain of NIH, the Molecular Libraries Probe Production Centers Network library. The primary screen involved luciferase assays in the DLD-1/pGL4.18hKLF5p cell line, which stably expressed a luciferase reporter driven by the human KLF5 promoter. A cytotoxicity counterscreen was done in the rat intestinal epithelial cell line, IEC-6. We identified 97 KLF5-selective compounds with EC50 < 10 μmol/L for KLF5 inhibition and EC50 > 10 μmol/L for IEC-6 cytotoxicity. The two most potent compounds, CIDs (PubChem Compound IDs) 439501 and 5951923, were further characterized on the basis of computational, Western blot, and cell viability analyses. Both of these compounds, and two newly synthesized structural analogs of CID 5951923, significantly reduced endogenous KLF5 protein levels and decreased viability of several colorectal cancer cell lines without any apparent impact on IEC-6 cells. Finally, when tested in the NCI-60 panel of human cancer cell lines, compound CID 5951923 was selectively active against colon cancer cells. Our results show the feasibility of uHTS in identifying novel compounds that inhibit colorectal cancer cell proliferation by targeting KLF5. Mol Cancer Ther; 10(11); 2043–51. ©2011 AACR.

Tu1894 Ultrahigh-Throughput Screening Strategies for Identification of Small-Molecule Compounds That Target the Colon Cancer Oncogene KrüPpel-Like Factor 5

Aim: The aim of the present study was to evaluate whether chronomodulated administration of capecitabine would reduce toxicity of the drug in patients with metastatic colorectal cancer. Patients & Methods: 27 patients with advanced colorectal cancer were randomized to receive capecitabine (2500mg/KOF daily) either in standard administration with 50% of the dose in themorning and 50% in the evening separated by 12 hours or as chronomodulated dose-regime with 25% morning-dose and 75% late evening-dose. Treatment was continued until thirteen treatment-cycles were finished or progress of cancer or limitation of therapy due to side effects occurred. Results: Overall, response rates to chemotherapy were similar in both treatment groups (p= 0,296). However, within the group of patients with chronomodulated application of capecitabine, reduction of drug-doses due to side effects was less frequently necessary (14.8 vs. 19.3%, p=0,026) and more patients were able to finish all thirteen chemotherapy-cycles (41.7 vs. 7.1%, p=0,007). While there was no statistically significant difference in the frequency of hand-foot-syndrome in both treatment-arms (51.7 vs. 41.5%, p=0,119) other side effects as nightly nausea, tiredness and sleeplessness were significantly reduced in patients receiving chronomodulated therapy (p=0,035, p=0,001 and 0,009, respectively). Additionally, there was a trend to lower frequency of nausea, diarrhea and 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 achieved similar response rate and better tolerability regarding various side effects compared with standard application of the drug in patients with colorectal cancer. This schedule could enable patients to sustain on capecitabine therapy for a longer time-period.