Abstract
Genetic screens in cultured human cells represent a powerful unbiased strategy to identify cellular pathways that determine drug efficacy, providing critical information for clinical development. We used insertional mutagenesis-based screens in haploid cells to identify genes required for the sensitivity to lasonolide A (LasA), a macrolide derived from a marine sponge that kills certain types of cancer cells at low nanomolar concentrations. Our screens converged on a single gene, LDAH, encoding a member of the metabolite serine hydrolase family that is localized on the surface of lipid droplets. Mechanistic studies revealed that LasA accumulates in lipid droplets, where it is cleaved into a toxic metabolite by LDAH. We suggest that selective partitioning of hydrophobic drugs into the oil phase of lipid droplets can influence their activation and eventual toxicity to cells.
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Data availability
The complete lists of the hits from the genetic screens are given in Supplementary Data 1. RNA-seq data from Hap1 cells is freely available at NCBI GEO, under accession no. GSE75515. The GI50 data for LasA and the RNA-seq data for cancer cell lines is publicly available (accession numbers given in the appropriate Methods section). Software for analysis of screen results has been described previously9,10 and is freely available on github: https://github.com/RohatgiLab/BAIMS-Pipeline.
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Acknowledgements
We thank D. Herschlag for bringing the LasA project to our attention, C. Pataki and R. Kopito for comments and advice on lipid droplet fractionation experiments and A. Lebensohn for advice on the project. The work was funded by DP2 GM105448 (R.R.), R35 GM118082 (R.R.), DP2 AI104557 (J.E.C.), American Heart Association Transformational Research Projects no. 18TPA34230103 (A.P.) and no. 18TPA34230086 (Y.-H.G.), and Dominic Ferraioli Foundation (A.P.). R.R. is a Josephine Q. Berry Faculty Scholar in Cancer Research at Stanford, J.E.C. is a David and Lucile Packard Foundation fellow and R.D. was supported by fellowships from the Stanford Dean’s Fund and Alex’s Lemonade Stand Foundation.
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Contributions
R.R. and R.D. designed the project. B.M.T. and C.E.S. designed and synthesized LasA, LasF, Ces-73, Ces-24a and Ces-24b. R.D. and J.E.C. executed the haploid genetic screens. R.D. and H.Q.N. performed the mass spectrometry experiments. R.D., A.P. and Y.-H.G. designed and constructed the LDAH variants. R.D. performed all other experiments and analyses presented in the paper. R.R. and R.D. wrote the paper and all the authors edited and commented on the paper.
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Supplementary Information
Supplementary Figs. 1–12.
Supplementary Video 1
Movie of live Hap1 cells expressing LDAH-GFP.
Supplementary Data 1
Compiled data from the haploid screens (see Fig. 1c and Supplementary Figs. 1 and 2a).
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Dubey, R., Stivala, C.E., Nguyen, H.Q. et al. Lipid droplets can promote drug accumulation and activation. Nat Chem Biol 16, 206–213 (2020). https://doi.org/10.1038/s41589-019-0447-7
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DOI: https://doi.org/10.1038/s41589-019-0447-7
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