A targeted genetic modifier screen in Drosophila uncovers vulnerabilities in a genetically complex model of colon cancer

Abstract

Kinases are key regulators of cellular signal transduction pathways. Many diseases including cancer are associated with global alterations in protein phosphorylation networks, as a result, kinases are frequent targets of drug discovery efforts. However, target identification and assessment, a critical step in targeted drug discovery which involves identifying essential genetic mediators of disease phenotypes, can be challenging in complex, heterogeneous diseases like cancer where multiple concurrent genomic alterations are common. Drosophila is a particularly useful genetic model system to identify novel regulators of biological processes through unbiased genetic screens. Here, we report two classic genetic modifier screens focusing on the Drosophila kinome to identify kinase regulators in two different backgrounds: KRAS TP53 PTEN APC, a multigenic cancer model that targets four genes recurrently mutated in human colon tumors and KRAS alone, a simpler model that targets one of the most frequently altered pathways in cancer. These screens identified hits that are shared by both models as well as those unique to each one, emphasizing the importance of capturing the genetic complexity of human tumor genome landscapes in experimental models. Our follow-up analysis of two hits from the KRAS only screen suggest that classical genetic modifier screens in heterozygous mutant backgrounds that result in a modest, non-lethal reduction in candidate gene activity in the context of a whole animal —a key goal of systemic drug treatment— may be a particularly useful approach to identify most rate limiting genetic vulnerabilities in disease models as ideal candidate drug targets.

A polycistronic transgene design for combinatorial genetic perturbations from a single transcript in Drosophila

“…RNA and protein were extracted from whole experimental and control larvae at the late 3rd instar stage using previously established protocols [ 14 , 50 ] (6 larvae/biological replicate; 3 biological replicates/genotype). For evaluating the knockdown efficacy of exogenous proteins, animals ubiquitously expressing GFP ( Fig 1D ), β-gal ( Fig 2D ), and CD2 ( Fig 3F ) were used as controls.…”

“…RNA and protein were extracted from whole experimental and control larvae at the late 3rd instar stage using previously established protocols [14,50] For all others, w 1118 ; tub-gal4 tub-gal80 ts /+ animals were used as controls. Western blot analyses were performed as described previously [14,50] using the following primary and secondary antibodies: mouse anti-OctA(FLAG) antibody (1:1000, Santa Cruz Biotechnology, sc-166355), rat anti-HA High-Affinity Antibody (1:1000, ROAHAHA, MilliporeSigma), rabbit anti-GFP N-terminal antibody (1:4000, G1544, MilliporeSigma), mouse anti-p53 antibody (1:1000, Developmental Studies Hybridoma Bank, #dmp53 H3), and mouse anti-Syntaxin antibody (1:1000, Developmental Studies Hybridoma Bank, #8C3) as a loading control, goat anti-mouse and goat-anti-rat IgG HRP-linked secondary antibodies (1:5000, Cell Signaling Technology #7076 and #7077, respectively). qPCR analyses were performed as described previously [14] using the housekeeping gene rpl32 as an internal control.…”