An in vivo large-scale chemical screening platform using Drosophila for anti-cancer drug discovery

Abstract

Anti-cancer drug development involves enormous expenditure and risk. For rapid and economical identification of novel, bioavailable anti-tumour chemicals, the use of appropriate in vivo tumour models suitable for large-scale screening is key. Using a Drosophila Ras-driven tumour model, we demonstrate that tumour overgrowth can be curtailed by feeding larvae with chemicals that have the in vivo pharmacokinetics essential for drug development and known efficacy against human tumour cells. We then develop an in vivo 96-well plate chemical screening platform to carry out large-scale chemical screening with the tumour model. In a proof-of-principle pilot screen of 2000 compounds, we identify the glutamine analogue, acivicin, a chemical with known activity against human tumour cells, as a potent and specific inhibitor of Drosophila tumour formation. RNAi-mediated knockdown of candidate acivicin target genes implicates an enzyme involved in pyrimidine biosynthesis, CTP synthase, as a possible crucial target of acivicin-mediated inhibition. Thus, the pilot screen has revealed that Drosophila tumours are glutamine-dependent, which is an emerging feature of many human cancers, and has validated the platform as a powerful and economical tool for in vivo chemical screening. The platform can also be adapted for use with other disease models, thus offering widespread applications in drug development.

Figures

Fig. 1.

PD0325901 inhibits tumour development in vivo. (A) Western blot of S2 cell lysates, prepared 30 minutes after treatment of cells with carrier alone (0.1% DMSO) or various MEK inhibitors (final concentration, 10 μM), and probed with antibodies to phosphorylated ERK (p-ERK), total ERK (t-ERK) and α-tubulin (α-Tub). (B) Quantification of phosphorylated ERK levels compared with total ERK levels from the western blot, normalised to DMSO alone. (C) Representative examples of 9-day-old larvae, bearing GFP-expressing (white) RasV12 scrib1 tumours, after 5 days of exposure to carrier alone (0.5% DMSO) or MEK inhibitor (final concentration, 50 μM). (D) Quantification of GFP-positive tumour burden per larva after MEK inhibitor exposure, as measured by the number of white pixels (from binarised images of GFP fluorescing larvae) within a defined area from the anterior end of each larva (one way ANOVA, *P<0.005; n=6; error bars indicate s.e.m.).

Fig. 2.

The Drosophila chemical screening platform and summarised screen results. (A) Schematic of the chemical screening protocol. The chemical library, stored as 96-well plates, was diluted with water and used to reconstitute powdered instant Drosophila media in 96-well deep-well plates (final compound concentration, 50 μM, DMSO <0.5%). Seven 4-day-old RasV12 scrib1 tumour-bearing larvae were then added to each well. The plate was sealed with fine mesh, held firmly in place by a perspex lid (containing a hole over every well, to allow air access to each well). After 5 days of incubation at 25°C, sucrose solution was added to each well, resulting in all larvae and pupae floating to the surface, before imaging. The number of larvae and pupae in each well were then scored from a white light image. A binarised GFP image was used to calculate the white pixel count per larva in each well. These data were stored in a database, from which positive hits were identified on the basis of a decrease in pixel count. (B) Example screening data showing white light, GFP and binary images captured for an entire plate. Note the location of compound 2Q1E3 in the third column, fifth row (circled). (C) Histogram summary of the complete screen data, showing the average pixel count per larva for each compound (n=2 replicates for each compound), and potential hits on the basis of a decreased pixel count.

Fig. 3.

Acivicin inhibits tumour development, and CTP synthase could be a key anti-tumorigenic target. (A) Nine-day-old larvae, bearing GFP-labelled RasV12 scrib1 tumours, after 5 days exposure to carrier alone (0.5% DMSO) or 50 μM acivicin. A representative eye-antennal imaginal disc pair, dissected from these larvae, is shown below. (B) Representative examples of w1118 flies two days after receiving transplantation into their abdominal cavity of GFP-labelled RasV12 scrib1 tumour tissue from 9-day-old larvae (day 0 of treatment regime), and 4 days later after treatment with either carrier alone (DMSO) or 50 μM acivicin (day 4). On the right is shown quantification of fold increase in tumour area in adult flies after 4 days of treatment (t-test, P=0.0007; n=10). (C) Quantification of GFP-positive tumour overgrowth in a RasV12 dlgRNAi tumour model (Willecke et al., 2011) upon knockdown of candidate glutamine-dependent amidotransferases (n=6 pupae). The overexpression of dap (Drosophila p21 homologue) was used as a positive control. Knockdown of CTPsyn and CG9674 (a glutamate synthase with no mammalian orthologue) exerted a significant reduction in tumour overgrowth. Representative images of the tumour-bearing pupae are shown on the right. (D,E) Quantification of pixel counts at day 9, upon knockdown of CTPsyn in GFP-labelled RasV12 scrib1 tumour-bearing larvae (D), and after 5 days exposure of RasV12 scrib1 tumour-bearing larvae to 50 μM acivicin or 500 μM 3-deazauridine (E); one way ANOVA, *P<0.0001; n=8 wells (D) and n=3 wells (E). (F) Above: Drosophila glutamine-dependent amidotransferases involved in nucleotide biosynthesis potentially targeted by acivicin, with CTPsyn highlighted as a potentially key biologically relevant target. Below: simplified pathway of glutamine utilisation for energy production in tumours that is inhibited by AOA. Error bars in B-E indicate s.e.m.