A novel chemical screening strategy in zebrafish identifies common pathways in embryogenesis and rhabdomyosarcoma development

Abstract

SUMMARYThe zebrafish is a powerful genetic model that has only recently been used to dissect developmental pathways involved in oncogenesis. We hypothesized that operative pathways during embryogenesis would also be used for oncogenesis. In an effort to define RAS target genes during embryogenesis, gene expression was evaluated in Tg(hsp70-HRAS G12V ) zebrafish embryos subjected to heat shock. dusp6 was activated by RAS, and this was used as the basis for a chemical genetic screen to identify small molecules that interfere with RAS signaling during embryogenesis. A KRAS G12D-induced zebrafish embryonal rhabdomyosarcoma was then used to assess the therapeutic effects of the small molecules. Two of these inhibitors, PD98059 and TPCK, had anti-tumor activity as single agents in both zebrafish embryonal rhabdomyosarcoma and a human cell line of rhabdomyosarcoma that harbored activated mutations in NRAS. PD98059 inhibited MEK1 whereas TPCK suppressed S6K1 activity; however, the combined treatment completely suppressed eIF4B phosphorylation and decreased translation initiation. Our work demonstrates that the activated pathways in RAS induction during embryogenesis are also important in oncogenesis and that inhibition of these pathways suppresses tumor growth.

An FDA-Approved Drug Screen for Compounds Influencing Craniofacial Skeletal Development and Craniosynostosis

“…Our confirmed drug hit rate was 2.2% of the tested compounds. This is consistent with previously reported perturbation screens [Peterson et al, 2004;Stern et al, 2005;North et al, 2007;Kitambi et al, 2009;Ishizaki et al, 2010;Paik et al, 2010;Rihel et al, 2010;Ni et al, 2011;Peal et al, 2011;Rovira et al, 2011;Namdaran et al, 2012;Ridges et al, 2012;Gut et al, 2013;Le et al, 2013;Kong et al, 2014;Mork and Crump, 2015] suggesting that this screen design had good sensitivity. As discussed in the introduction, while published perturbation screens have an average 29-fold increased hit rate as compared to modifier screens, their success in potential clinical translation is likely to depend on selection of a specific cell type assessed in the screen as well as further testing in mammalian disease models.…”

“…Approximately 30 unbiased in vivo drug screens using zebrafish have been reported to date to our knowledge, and all but 4 of these were based on the perturbation design [Peterson et al, 2004;Stern et al, 2005;North et al, 2007;Kitambi et al, 2009;Ishizaki et al, 2010;Paik et al, 2010;Rihel et al, 2010;Ni et al, 2011;Peal et al, 2011;Rovira et al, 2011;Namdaran et al, 2012;Ridges et al, 2012;Gut et al, 2013;Le et al, 2013;Kong et al, 2014;Mork and Crump, 2015]. This suggests a reporting bias such that most of the many modifier screens that we know to have been conducted in the field have not yielded any hits.…”

“…This suggests a reporting bias such that most of the many modifier screens that we know to have been conducted in the field have not yielded any hits. Of 4 published modifier screens, an average confirmed drug hit rate of 0.086% (range 0.17-0.06%) was reported [Peterson et al, 2004;Stern et al, 2005;Paik et al, 2010;Peal et al, 2011], whereas for perturbation screens that reported their results in full, an average confirmed hit rate of 2.50% (range 8.2-0.08%) was achieved [North et al, 2007;Kitambi et al, 2009;Ishizaki et al, 2010;Rihel et al, 2010;Ni et al, 2011;Rovira et al, 2011;Namdaran et al, 2012;Ridges et al, 2012;Gut et al, 2013;Le et al, 2013;Kong et al, 2014;Mork and Crump, 2015]. Thus a typical screen of 1,000 small molecules, which is achievable in most labs using nonautomated zebrafish screens, is expected to produce 25 hits in a perturbation screen but none in a modifier screen.…”

Glycogen synthase kinase 3 inhibitors induce the canonical WNT/β-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma

“…Importantly, the zebrafish ERMS model also shows morphologic features resembling the human counterpart, expresses clinical diagnostic markers, and shares common molecular pathways with human and mouse disease as assessed by expression profiling, array CGH studies, and cross-species functional analysis (14,16). Building on these findings and the use of the zebrafish ERMS model for in vivo drug discovery (17), novel therapies that modulate TPC function and/or induce differentiation will likely provide new treatment options for relapsed or metastatic ERMS.…”

“…The confirmed hits identified in the human differentiation screen (12 compounds) were combined with the top hits from a zebrafish embryonic screen (83 compounds) that identified compounds with anti-RAS activity (17). This panel of 95 drugs was assessed for their effect on growth in transplanted zebrafish RAS-induced ERMS (schematic in Fig.…”