Abstract 3758: Quantitative high-throughput screening as a tool to identify novel therapies in bladder cancer: lessons from flavopiridol

Abstract

Abstract Introduction and Objectives: Bladder cancer (CaB) is the 4th most common cancer among men and 12th most common among women in United States. It is one of the most expensive malignancies to treat from diagnosis to death. No new pharmacological agents have been approved for the treatment of bladder cancer in the last two decades. Therefore, there is an urgent need for development of new treatment therapies. Quantitative high throughput screening (qHTS) of representative cancer cell lines with oncology drugs may identify new treatments and pathways. We utilized this technique to identify new targets and therapies in two primary bladder lines (T24 and UMUC3) and their metastatic derivatives (T24T, SLT3 and FL3 of T24 and LUL-2 for UMUC3). Methods: We screened 7 bladder cancer cell lines (T24T, SLT3, FL3, LUL-2, RT4, T24, and UMUC3) against 1,912 oncology drugs using a 48 hour cell proliferation assay with an ATP−based readout (CellTiterGlo) to determine activity and potency of compounds in a dose response manner. One of the candidate drugs inhibitory in all cell lines tested is flavopiridol, a pan-CDK inhibitor. We further characterized the mechanism of action of flavopiridol using various cell based assays such as cell proliferation, cell cycle analysis, apoptosis assays, and modified colony forming assays. Finally, mouse xenograft studies were carried out to elucidate the in vivo effects of flavopiridol. Results: The initial screen identified 95 compounds active in 7 cell lines. The top 50 compounds were further analyzed for molecular size of >200 g/mol and TPSA<90. These parameters are consistent with lipophilicity and may help identify compounds that can be used as intravesical agents. The selection parameters identified mitomycin C and 8 novel compounds. Cell proliferation assays of these 8 novel compounds in cell lines not part of the initial screen revealed that flavopiridol most consistently achieved IC50s of 100-150 nM, consistent with the qHTS data. Flavopiridol induces G2/M arrest; however, it induces little apoptosis as per the Annexin V FITC-PI assay. Growth in Low Attachment (GILA) assay showed inhibition of colony formation in the presence of flavopiridol. Xenograft studies using the UMUC-3 cell line implanted subcutaneously demonstrated cytostatic inhibition of tumor growth in the presence of systemically delivered flavopiridol. However, intravesical administration has not yet been evaluated. Conclusions: qHTS can identify novel compounds. Flavopiridol is an effective inhibitor both in vitro and in vivo. Although it merely caused cytostatic inhibition in a xenograft model when delivered systemically, its physical properties are most suited for intravesical use which may lead to it being more effective as higher doses can be administered into the bladder with minimal/no systemic toxicities. Studies are now underway to evaluate the use of flavopiridol as an intravesical agent. Citation Format: Reema Railkar, Achuth Nair, Keidren Lewi, Spencer Krane, Rajarshi Guha, Marc Ferrer, Craig Thomas, Piyush K. Agarwal. Quantitative high-throughput screening as a tool to identify novel therapies in bladder cancer: lessons from flavopiridol. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3758.