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  1. 1. Small-molecule inducers of insulin expression in pancreatic -cells Dina Fomina-Yadlina,b,c,1 , Stefan Kubiceka,b,1 , Deepika Walpitab , Vlado Dancikb,d , Jacob Hecksher-Srensenb,2 , Joshua A. Bittkerb , Tanaz Sharifniab,e , Alykhan Shamjib , Paul A. Clemonsb , Bridget K. Wagnerb , and Stuart L. Schreibera,b,f,3 a Howard Hughes Medical Institute, b Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA 02142; c Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138; d Mathematical Institute, Slovak Academy of Sciences, Koice, 040 01, Slovakia; e Department Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115; and f Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138 Contributed by Stuart L. Schreiber, July 15, 2010 (sent for review June 22, 2010) High-content screening for small-molecule inducers of insulin expression identied the compound BRD7389, which caused -cells to adopt several morphological and gene expression features of a -cell state. Assay-performance prole analysis suggests kinase inhibition as a mechanism of action, and we show that biochemical and cellular inhibition of the RSK kinase family by BRD7389 is likely related to its ability induce a -cell-like state. BRD7389 also in- creases the endocrine cell content and function of donor human pancreatic islets in culture. BRD7389 | pancreatic islets | Rsk kinase | transdifferentiation | beta cells Type 1 diabetes is an autoimmune disease characterized by the loss of insulin-producing -cells in pancreatic islets of Lang- erhans. Islet transplantation into the liver can effectively cure the disease (1), but is not an ideal treatment due to limited donor material and immunological complications. An alternative ap- proach, not yet feasible, is to create new -cells (2), either by stepwise differentiation of undifferentiated stem or stem-like cells (3), or by transdifferentiation (4), the heritable change of cell identity to an insulin-producing (-like) cell. The latter approach could result in a replacement source for the decient cell type directly from patient material (either in vivo or ex vivo). Increasing -cell mass by small-molecule drug-induced transdifferentiation is a speculative but exciting approach to treating diabetesone that is signicantly different from currently available small-molecule drugs that increase insulin secretion in existing -cells and are therefore ineffective in the later stages of type 1 diabetes, in which most -cell mass has been lost. Cell-type specication in the pancreas is regulated by a set of master regulatory transcription factors that control the transition from one progenitor cell state to the next, ultimately yielding mature endocrine cell types in islets (5). Recently, it has been shown that misexpression of these master regulatory transcription factors causes direct transdifferentiation between cell types. For example, ectopic overexpression of a single transcription factor (Arx) is sufcient for in vivo conversion of -cells to -cells in the adult mouse pancreas (6). Similarly, viral delivery of three tran- scription factors (Pdx1, Ngn3, MafA) to an adult mouse pancreas causes the transdifferentiation of acinar cells to -cells (7). Finally, in vivo conversion of -cells to -cells has recently been achieved in mature mouse -cells by ectopic overexpression of Pax4 (8). Results Because a single gene is sufcient to induce transdifferentiation of -cells to -cells, we sought to determine whether a small molecule could have the same effect. Possible readouts for induction of a - cell state include insulin production and insulin secretion. We chose to target the production of insulin protein because we imagined that this would be more feasible to achieve in the course of a 3-d small-molecule treatment than insulin secretion. To that end, we developed a high-content, cell-based assay to detect in- sulin protein expression in the mouse -cell line TC1. Normal mouse -cells are insulin negative, but have the ability to adopt a -cell phenotype after extreme -cell loss (9). Similarly, the -cell line we used spontaneously reexpressed small but detectable levels of insulin, despite being a subclone selected for low insulin protein (10). During assay development and optimization, we could show, by spiking in -cells and by antibody competition, that our assay was sensitive enough to reliably detect insulin levels in as few as 3% of cells, and at 15-fold lower levels than in -cells (Fig. S1). We screened 30,710 compounds for induction of insulin pro- duction using this assay and identied a molecule, BRD7389 (Fig. 1A), that after 3-d treatment induced insulin expression in mouse -cells. BRD7389 induced a dose-dependent up-regulation of Ins2 mRNA, peaking at 0.85 M; 5-d treatment with BRD7389 resulted in greater induction of insulin gene expression, about 50- fold at 0.85 M (Fig. 1B), which could not be further increased by longer treatments up to 21 d. This compound appears to be spe- cic to -cells, because a pancreatic ductal cell line (PANC-1) showed no induction, and a mouse -cell line (TC3) no further increase of insulin expression. In addition to insulin expression, BRD7389 signicantly up-regulated expression of Pdx1 (Fig. 1C), a master regulatory transcription factor that species pancreatic progenitors and directly activates the insulin promoter (11). We also observed a dose-dependent increase in the expression of other -cell markers, including Pax4, Iapp, and Npy, after a 5-d treatment with BRD7389 (Fig. S2). Treatment with BRD7389 caused a stable change in cell shape from a broblast-like morphology, characteristic of -cells, to a clustered state resembling -cells in culture (Fig. 1 DF, Left). Finally, we detected low levels of insulin protein in compound- treated -cells by immunouorescence (Fig. 1 DF, Right). Rela- tive to background uorescence in DMSO-treated -cells, insulin staining is induced 1.5-fold following 5-d treatment with BRD7389, compared with 4-fold higher levels in -cells. Both insulin mRNA and protein levels are signicantly increased from a basal -cell state in compound-treated cells, but do no reach levels detected in mature -cells. Therefore, although these cells have not achieved a -cell state, they have adopted several features of -cells. To identify the mechanism of action of BRD7389, we used screening data in ChemBank (12) to compare assay performance Author contributions: D.F.-Y., S.K., J.H.-S., B.K.W., and S.L.S. designed research; D.F.-Y., S.K., and T.S. performed research; D.W. and V.D. contributed new reagents/analytic tools; D.F.-Y., S.K., J.A.B., and P.A.C. analyzed data; and D.F.-Y., S.K., A.S., B.K.W., and S.L.S wrote the paper. The authors declare no conict of interest. Freely available online through the PNAS open access option. 1 D.F.-Y. and S.K. contributed equally to this work. 2 Present address: Hagedorn Research Institute, DK-2820 Gentofte, Denmark. 3 To whom correspondence should be addressed. E-mail: stuart_schreiber@harvard.edu. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1010018107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1010018107 PNAS | August 24, 2010 | vol. 107 | no. 34 | 1509915104 CELLBIOLOGY
  2. 2. of BRD7389 with 9,995 other small molecules in a total of 32 assays involving both BRD7389 and other compounds. This computa- tional method looks for similarity of biological assay-performance proles among a diverse set of compounds, including many known bioactives. We uncovered multiple connections of BRD7389 to known kinase inhibitors. Accordingly, we proled this compound at 10 M against a panel of 219 kinases, selected to represent a diverse subset of the human kinome (13). We observed signi- cant inhibition of a number of kinases, including FLT3, DRAK2, and the RSK family (Fig. 2A and Table S1). To validate these proling results, we obtained doseresponse curves and deter- mined half-maximal inhibitory concentration (IC50) values for BRD7389 and the most potently inhibited kinases (Fig. S3). The compound was most active against the entire RSK family of ki- nases, with IC50 values of 1.5 M, 2.4 M, and 1.2 M for RSK1, RSK2, and RSK3, respectively (Fig. 2B). Therefore, we focused on investigating the role of RSK kinases in -cells. In addition to measuring the biochemical in vitro inhibition of RSKs, we also determined the functional consequences of BRD7389 on Rsk activity in mouse -cells. All Rsk kinases consist of two functional domains, which are activated through a series of consecutive phosphorylation events (14). Kinase activity was measured using pan- and phospho-specic antibodies to detect total and active Rsk protein in TC1 cells. Western blot analy- sis revealed a 50% decrease in kinase activity, as measured by autophosphorylation of both N-terminal and C-terminal domains, at concentrations above 3.4 M (Fig. 2 C and E). Phosphorylation of ribosomal protein S6 at serines 235 and 236, direct targets of the Rsk kinases (15), was reduced by a similar amount after compound treatment (Fig. 2 D and F). These ndings conrm that BRD7389 has activity as an Rsk family kinase inhibitor in vitro and in cell culture. We then sought to determine whether knockdown of Rsk family members would have an effect on insulin production in -cells. We observed 2- to 4-fold increases in insulin expres- sion upon RNAi of individual Rsk proteins, especially Rsk2 and Rsk3, but the effect is not as strong as compound treatment with BRD7389 (Fig. 2G). The knockdown efciency was at least 50% for all constructs (Fig. 2H), and better knockdown did not cor- relate with stronger induction of insulin expression. Similar to compound treatment, which causes maximum induction of insulin expression at concentrations around the biochemical IC50 for Rsks, only partial knockdown of the enzymes seems optimal for insulin in