Abstract A180: Development of a high-throughput screening (HTS) assay to detect splicing inhibitors.
Authors: Arslan Ahmet Dirim , He Xiaolong , Wang Minxiu , Rumschlag-Booms Emily , Rong Lijun , Beck William T. 
Published: June 2012

Background and Hypothesis: Alternative splicing (AS) of RNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract binding protein (PTB) is overexpressed in ovarian tumors (He et al, Clin Cancer Res, 10:4652–60, 2004), compared to matched normal controls, and knockdown (KD) of PTB expression by shRNA impairs ovarian tumor cell growth, colony formation and invasiveness (He et al, Oncogene 26:4961–8, 2007). PTB is a widely expressed RNA binding protein whose main molecular function is regulating alternative splicing. Because KD of PTB with shRNA impairs ovarian cancer cell growth and other malignant properties, we wished to identify small molecule inhibitors of PTB that will have the same effect as the shRNA. We hypothesized that PTB is a drugable therapeutic target and that its activity in live cells can be monitored by measuring the splicing of a PTB target gene. Accordingly, we have developed a cell-based fluorescent reporter assay for monitoring RNA splicing by using a GFP minigene. This assay allows HTS of small molecule libraries to identify splicing modulator agents that inhibit PTB.

Materials and Methods: In this pilot study, we screened 89 NCI-approved oncology drugs (http://.goo.gl/Lf1Jl) in engineered A2780 epithelial ovarian tumor sublines that carried the splicing reporter minigene construct and/or an inducible shRNA against PTB. Three different sublines were used for this assay: (i) a positive control that carries the splicing reporter construct and an inducible shRNA against PTB; (ii) a negative control that carries the splicing reporter construct only; and (iii) a blank control that consist of parental cells only. For the assay, all cells were seeded in 96-well plates (black, clear bottom) at 1.5×106 cells/mL. After 24 h, compounds were added using a PerkinElmer Janus workstation (http://goo.gl/zbK71), and plates were incubated with compounds (final concentration, 1μM) for 48 h at 37C, 5% CO2. Plates were washed inside the sterile workstation with 1× PBS solution before measuring activity. Fluorescence measurements were carried out on a PerkinElmer EnVision plate reader (http://goo.gl/ZxDN8), using filters at 485-nm for excitation and 520-nm for emission. The activation percent was assessed by quantifying fluorescence intensity changes in the compound-treated cells in relation to the positive and negative control cells.

Results: We found that our cell-based splicing reporter assay can be use to detect PTB activity in live cells. Moreover, because compounds in this library are intrinsically cytotoxic, 18% (16 of 89) showed toxicity in the A2780 cells. Of importance, this assay was able to detect cytotoxic compounds. These compounds were separated for further investigation to test them at lower concentrations, and to determine whether any of them affect splicing in the reporter assay.

Conclusions: This study describes the preliminary assessment of a novel HTS approach to identify small molecule inhibitors of the splicing factor PTB. This cell-based HTS allows large numbers of agents to be tested against engineered cell lines to identify small molecule inhibitors of PTB with the goal of translating positive hits to the clinic.

Supported in part by grants RO1 CA40570 and RO1 CA138762 (to WTB), by OCRF (to XH), and by UIC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A180.