A Small-Molecule Inducer of PDX1 Expression Identified by High

Chemistry & Biology Article A Small-Molecule Inducer of PDX1 Expression Identified by High-Throughput Screening Yuan Yuan,1,2 Kate Hartland,3 Zarko Boskovic,1,2,4 Yikai Wang,1 Deepika Walpita,1 Philippe A. Lysy,5 Cheng Zhong,1 Damian W. Young,1 Young-kwon Kim,1 Nicola J. Tolliday,3 Etienne M. Sokal,5 Stuart L. Schreiber,1,2,4 and Bridget K. Wagner1,* 1Chemical Biology Program, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA 2Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA 3Chemical Biology Platform, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA 4Howard Hughes Medical Institute, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA 5Laboratory of Pediatric Hepatology and Cell Therapy, Catholic University of Leuven, Brussels 1200, Belgium *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2013.10.013 SUMMARY cells and through transdifferentiation from mature non-b cells such as liver, acinar, and ductal cells (Akinci et al., 2012; Ferber Pancreatic and duodenal homeobox 1 (PDX1), a et al., 2000; Kaneto et al., 2005; Karnieli et al., 2007; Kojima et al., member of the homeodomain-containing transcrip- 2002; Kubo et al., 2011; Motoyama et al., 2009; Wu et al., 2007; tion factor family, is a key transcription factor impor- Yamada et al., 2001; Yang et al., 2002). A notable recent tant for both pancreas development and mature b success in the transdifferentiation field was the generation of b cell function. The ectopic overexpression of Pdx1, functional insulin-producing cells from acinar cells in mice Neurog3, and MafA in mice reprograms acinar cells after viral transduction of the pancreas with Pdx1, Neurog3, and MafA (Zhou et al., 2008). These results were repeated in to insulin-producing cells. We developed a quantita- cell culture using the rat exocrine cell line AR42J (Akinci et al., tive PCR-based gene expression assay to screen 2012), suggesting that in vitro cell models can enable the study more than 60,000 compounds for expression of of cellular reprogramming. each of these genes in the human PANC-1 ductal From a therapeutic perspective, viral introduction of exoge- carcinoma cell line. We identified BRD7552, which nous genetic material raises concerns regarding genomic inte- upregulated PDX1 expression in both primary human gration and tumorigenicity (Barrilleaux and Knoepfler, 2011). islets and ductal cells, and induced epigenetic An attractive alternative is to use small molecules that achieve changes in the PDX1 promoter consistent with temporal high expression of transcription factors. Recent transcriptional activation. Prolonged compound efforts to this end have focused on the factors required for the treatment induced both insulin mRNA and protein generation of induced pluripotent stem (iPS) cells (Takahashi and also enhanced insulin expression induced by and Yamanaka, 2006). This approach identified kenpaullone as a potential replacement for Klf4 (Lyssiotis et al., 2009), the three-gene combination. These results provide a transforming growth factor b receptor inhibitor as a potential a proof of principle for identifying small molecules replacement for Sox2 and c-Myc (Ichida et al., 2009), and Src that induce expression of transcription factors to inhibitors as other replacements for Sox2 (Staerk et al., 2011). control cellular reprogramming. These results suggest that novel screening methods can identify compounds that aid in the reprogramming process. Herein we report the development of a gene expression-based INTRODUCTION assay to perform high-throughput screening of 60,752 compounds for endogenous expression of PDX1, NEUROG3,or Pancreatic and duodenal homeobox 1 (PDX1) is a key tran- MAFA. We identified BRD7552, a compound that increased scription factor involved in pancreas development and b cell PDX1 mRNA levels in a dose- and time-dependent manner in function (Babu et al., 2007; McKinnon and Docherty, 2001). human PANC-1 cells, primary human islets, and human duct- Homozygous disruption of Pdx1 in mice and PDX1 in human derived cells (HDDCs). BRD7552 modified histone H3 tail modi- patients causes pancreatic agenesis. Heterozygous mutation fications associated with transcriptional activation, suggesting is associated with both type 2 diabetes and maturity onset dia- that the compound may induce PDX1 expression through betes of the young, type 4 (Ahlgren et al., 1997; Oliver-Krasinski either direct or indirect epigenetic control. Further mechanism et al., 2009; Stoffers et al., 1997). In the adult pancreas, PDX1 is of action studies suggest a role for the transcription factor expressed in b cells and d cells and controls the expression FOXA2 in BRD7552-induced PDX1 transcriptional activation. of key b cell markers such as insulin, glucose transporter 2, BRD7552 can partially replace PDX1 in the genetic induction of MafA, glucokinase, and islet amyloid polypeptide (Babu et al., insulin expression in PANC-1 cells. These results lay a founda- 2007; McKinnon and Docherty, 2001). Furthermore, ectopic tion for the development of novel small molecules as useful overexpression of PDX1 is essential for b cell neogenesis, both tools with which to manipulate the endogenous expression of through direct differentiation from pluripotent or progenitor master regulatory transcription factors. Chemistry & Biology 20, 1513–1522, December 19, 2013 ª2013 Elsevier Ltd All rights reserved 1513 Chemistry & Biology Small Molecule-Induced PDX1 Expression RESULTS molecules that induce the endogenous expression of PDX1. We therefore developed a gene expression-based assay using Transcription Factor Target Validation in PANC-1 Cells real-time qPCR (Arany et al., 2008) to identify potential small- In order to develop an in vitro model for high-throughput chem- molecule inducers. Using transfection of all three genes as a ical screening, we assessed the suitability of human PANC-1 positive control, we observed that assay Z0-factor values, a ductal adenocarcinoma cells. Although not an acinar cell line, measure of statistical effect size and assay quality (Zhang PANC-1 cells are amenable to high-throughput screening and et al., 1999), were greater than 0.5 for each of the three genes provide a good framework for b cell neogenesis. After cotrans- (Figure 2A), indicating that the assays were suitable for high- fection of full-length PDX1, NEUROG3, and MAFA and antibiotic throughput screening. selection over 2 weeks, we observed an increase in C-peptide We then screened 60,752 compounds to identify inducers of immunofluorescence in a majority of cells (Figure 1A). Each transcription factor expression in PANC-1 cells after 3-day transcription factor was highly expressed in these cells (Figures treatment (Figures S2A and S2B). The screening collection 1B–1D). Interestingly, at the end of 2 weeks, the localization of included compounds from libraries prepared by diversity- each transcription factor was both nuclear and cytoplasmic, oriented synthesis (DOS) (Schreiber, 2009), as well as kinase- suggesting that protein export from the expected nuclear biased, chromatin-biased, commercial compound, bioactive, location may occur during this time. Cytokeratin-19 (CK19), a and natural product libraries. Although no hits were identified ductal marker highly expressed in PANC-1 cells, was signifi- in the NEUROG3 or MAFA screens, we identified BRD7552, cantly downregulated in these cells, consistent with reports of a glucose-derived DOS compound, as a hit in the PDX1 its negative regulation by PDX1 (Deramaudt et al., 2006)(Fig- screen (Figures 2B and S2C). BRD7552 increased PDX1 ure 1E). Quantitative PCR (qPCR) revealed that each of the mRNA levels two- to fourfold, with a maximal effect at 5 mM three transcription factors was highly upregulated 3 days after (Figure 2C). The expression of MAFA was only marginally transfection but decreased to lower levels after 2 weeks in increased by BRD7552, but no NEUROG3 induction was de- culture (Figure 1F). Insulin mRNA levels were induced greater tected (Figures S2D and S2E). than 1,000-fold in transfected cells, albeit from an undetectable Five- and nine-day treatment of cells with BRD7552 further basal state. On the other hand, hormones expressed in other increased PDX1 expression (Figure 2C), but effects were pancreatic endocrine cell types, such as glucagon, somato- observed as early as six hours after compound treatment statin, and pancreatic polypeptide, were induced less than and gradually increased over time (Figure 2D). After 3-day 10-fold (Figure 1F). Examination of the threshold cycle (Ct) compound washout, the expression level of PDX1 returned values of control PANC-1 cells, as well as the DCt values relative to original levels (Figure S3A), suggesting that the effects to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), of BRD7552 are reversible. Immunofluorescence revealed reveals that, with the exception of PDX1 (Ct 28.0), these hor- PDX1 to be cytoplasmic (Figure S3B), perhaps reflecting an mones and transcription factors are more or less not expressed immature or stressed state (Guo et al., 2013; Kawamori in PANC-1 cells. However, the 1,000-fold increase in PDX1, et al., 2003; Macfarlane et al., 1999). We then assessed the NEUROG3, and MAFA, as well as the 2-week 1,000-fold upregu- effects of BRD7552 on endocrine cells. We observed an lation of INS, would put these Ct values nearer to those of increase in PDX1 expression in mouse aTC cells (Figure S3C), GAPDH. These results suggest that an initial

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