Genetic screening reveals a link between Wnt signaling and antitubulin drugs

Abstract

The antitubulin drugs, paclitaxel (PX) and colchicine (COL), inhibit cell growth and are therapeutically valuable. PX stabilizes microtubules, while COL promotes their depolymerization. But, the drug concentrations that alter tubulin polymerization are hundreds of times higher than their clinically useful levels. To map genetic targets for drug action at single-gene resolution, we used a human radiation hybrid panel. We identified loci that affected cell survival in the presence of five compounds of medical relevance. For PX and COL, the zinc and ring finger 3 (ZNRF3) gene dominated the genetic landscape at therapeutic concentrations. ZNRF3 encodes an R-spondin regulated receptor that inhibits Wingless/Int (Wnt) signaling. Overexpression of the ZNRF3 gene shielded cells from antitubulin drug action, while small interfering RNA knockdowns resulted in sensitization. Further a potent pharmacological inhibitor of Wnt signaling, Wnt-C59, protected cells from PX and COL. Our results suggest that the antitubulin drugs perturb microtubule dynamics, thereby influencing Wnt signaling.

Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells

“…The number of loci increased significantly with longer growth periods, suggesting that the various exposure times recruit different genes ( Figures 4B, S20, S21B and S30, Tables S10, S11, S14, S15 and S17–S19, Supporting Information ). This observation is consistent with our previous findings using a panel of human RH clones (Khan et al 2016), as well as findings in yeast (Wang and Kruglyak 2014). There were 38 unique paclitaxel loci in total.…”

“…We previously identified ZNRF3, a regulator of Wnt signaling, as a target for paclitaxel action using individual clones from the human G3 RH panel (Khan et al 2016). This locus was not replicated using the RH pools.…”

“…Human embryonic kidney 293 (HEK293) cells are female (Lin et al 2014) and were obtained from the American Type Culture Collection (Manassas, VA) around 2006. We validated these cells in a previous study using simple tandem repeat genotyping (Khan et al 2016).…”

“…As a new strategy to dissecting genotype/phenotype relationships in mammalian cells, we have been retooling radiation hybrid (RH) technology (Ahn et al 2009; Khan et al 2016; Lin et al 2010; Park et al 2008; Ranola et al 2010; Wang, Ahn, et al 2011). This venerable approach to genetic mapping employs lethal doses of radiation to break the genome of a human cell line into small fragments, which are then transferred to living hamster cells by cell fusion (Cox et al 1990; Goss and Harris 1975; Walter et al 1994).…”

“…Although RH panels were widely used in the genome projects, their potential for dissecting biological traits was not fully realized. Recently, we measured the growth of clones from a human RH panel in the presence of the antibulin drugs paclitaxel (Taxol®) and colchicine and identfied a single gene, ZNRF3, as important to the action of both agents (Khan et al 2016). However, statistical power was limited by the modest size of the RH panel (79 clones).…”