Reader TTS

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. Access through your institution Buy or

subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your institution Access Nature and 54 other Nature Portfolio journals Get

Nature+, our best-value online-access subscription $29.99 / 30 days cancel any time Learn more Subscribe to this journal Receive 12 print issues and online access $259.00 per year only

$21.58 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout

ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS GENETIC AND PHARMACOLOGICAL

INTERROGATION OF CANCER VULNERABILITY USING A MULTIPLEXED CELL LINE SCREENING PLATFORM Article Open access 02 July 2021 METABOLIC DRUG SURVEY HIGHLIGHTS CANCER CELL DEPENDENCIES AND

VULNERABILITIES Article Open access 14 December 2021 SYNTHETIC LETHAL STRATEGIES FOR THE DEVELOPMENT OF CANCER THERAPEUTICS Article 03 December 2024 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. REFERENCES * Horton, P. A., Koehn, F. E., Longley, R. E. & McConnell, O. J. Lasonolide A,

a new cytotoxic macrolide from the marine sponge _Forcepia_ sp. _J. Am. Chem. Soc._ 116, 6015–6016 (1994). Article  CAS  Google Scholar  * Wright, A. E. et al. Lasonolides C–G, five new

lasonolide compounds from the sponge _Forcepia_ sp. _J. Nat. Prod._ 67, 1351–1355 (2004). Article  CAS  Google Scholar  * Isbrucker, R. A., Guzman, E. A., Pitts, T. P. & Wright, A. E.

Early effects of lasonolide A on pancreatic cancer cells. _J. Pharmacol. Exp. Ther._ 331, 733–739 (2009). Article  CAS  Google Scholar  * Zhang, Y. W., Ghosh, A. K. & Pommier, Y.

Lasonolide A, a potent and reversible inducer of chromosome condensation. _Cell Cycle_ 11, 4424–4435 (2012). Article  CAS  Google Scholar  * Josse, R. et al. Activation of RAF1 (c-RAF) by

the marine alkaloid lasonolide A induces rapid premature chromosome condensation. _Mar. Drugs_ 13, 3625–3639 (2015). Article  CAS  Google Scholar  * Trost, B. M. et al. Total synthesis of

(−)-lasonolide A. _J. Am. Chem. Soc._ 138, 11690–11701 (2016). Article  CAS  Google Scholar  * Carette, J. E. et al. Haploid genetic screens in human cells identify host factors used by

pathogens. _Science_ 326, 1231–1235 (2009). Article  CAS  Google Scholar  * Carette, J. E. et al. Global gene disruption in human cells to assign genes to phenotypes by deep sequencing.

_Nat. Biotechnol._ 29, 542–546 (2011). Article  CAS  Google Scholar  * Dubey, R. et al. Chromatin-remodeling complex SWI/SNF controls multidrug resistance by transcriptionally regulating the

drug efflux pump ABCB1. _Cancer Res._ 76, 5810–5821 (2016). Article  CAS  Google Scholar  * Carette, J. E. et al. Ebola virus entry requires the cholesterol transporter Niemann–Pick C1.

_Nature_ 477, 340–343 (2011). Article  CAS  Google Scholar  * Simon, G. M. & Cravatt, B. F. Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study. _J.

Biol. Chem._ 285, 11051–11055 (2010). Article  CAS  Google Scholar  * Bachovchin, D. A. & Cravatt, B. F. The pharmacological landscape and therapeutic potential of serine hydrolases.

_Nat. Rev. Drug Discov._ 11, 52–68 (2012). Article  CAS  Google Scholar  * Hebenstreit, D. et al. RNA sequencing reveals two major classes of gene expression levels in metazoan cells. _Mol.

Syst. Biol._ 7, 497–497 (2011). Article  Google Scholar  * Goo, Y. H., Son, S. H., Kreienberg, P. B. & Paul, A. Novel lipid droplet-associated serine hydrolase regulates macrophage

cholesterol mobilization. _Arterioscler. Thromb. Vasc. Biol._ 34, 386–396 (2014). Article  CAS  Google Scholar  * Thiel, K. et al. The evolutionarily conserved protein CG9186 is associated

with lipid droplets, required for their positioning and for fat storage. _J. Cell Sci._ 126, 2198–2212 (2013). Article  CAS  Google Scholar  * Marchler-Bauer, A. et al. CDD: NCBI’s conserved

domain database. _Nucleic Acids Res._ 43, D222–D226 (2015). Article  CAS  Google Scholar  * Currall, B. B. et al. Loss of LDAH associated with prostate cancer and hearing loss. _Hum. Mol.

Genet._ 27, 4194–4203 (2018). Article  CAS  Google Scholar  * Kory, N. et al. Mice lacking lipid droplet-associated hydrolase, a gene linked to human prostate cancer, have normal cholesterol

ester metabolism. _J. Lipid Res._ 58, 226–235 (2017). Article  CAS  Google Scholar  * Olzmann, J. A. & Carvalho, P. Dynamics and functions of lipid droplets. _Nat. Rev. Mol. Cell Biol._

20, 137–155 (2019). Article  CAS  Google Scholar  * Brasaemle, D. L. & Wolins, N. E. Isolation of lipid droplets from cells by density gradient centrifugation. _Curr. Protoc. Cell

Biol._ 72, 3.15.1–3.15.13 (2016). Article  Google Scholar  * Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. _Nat.

Rev. Mol. Cell Biol._ 18, 285–298 (2017). Article  CAS  Google Scholar  * Greenwood, D. J. et al. Subcellular antibiotic visualization reveals a dynamic drug reservoir in infected

macrophages. _Science_ 364, 1279–1282 (2019). Article  CAS  Google Scholar  * Zheng, N., Tsai, H. N., Zhang, X. & Rosania, G. R. The subcellular distribution of small molecules: from

pharmacokinetics to synthetic biology. _Mol. Pharm._ 8, 1619–1628 (2011). Article  CAS  Google Scholar  * den Brok, M. H., Raaijmakers, T. K., Collado-Camps, E. & Adema, G. J. Lipid

droplets as immune modulators in myeloid cells. _Trends Immunol._ 39, 380–392 (2018). Article  Google Scholar  * Fowler, S., Shio, H. & Haley, N. J. Characterization of lipid-laden

aortic cells from cholesterol-fed rabbits. IV. Investigation of macrophage-like properties of aortic cell populations. _Lab. Investig._ 41, 372–378 (1979). CAS  PubMed  Google Scholar  *

Foley, P. Lipids in Alzheimer’s disease: a century-old story. _Biochim. Biophys. Acta_ 1801, 750–753 (2010). Article  CAS  Google Scholar  * Delikatny, E. J., Chawla, S., Leung, D.-J. &

Poptani, H. MR-visible lipids and the tumor microenvironment. _NMR Biomedicine_ 24, 592–611 (2011). CAS  Google Scholar  * Petan, T., Jarc, E. & Jusović, M. Lipid droplets in cancer:

guardians of fat in a stressful world. _Molecules_ 23, 1941 (2018). Article  Google Scholar  * Sundelin, J. P. et al. Increased expression of the very low-density lipoprotein receptor

mediates lipid accumulation in clear-cell renal cell carcinoma. _PLoS ONE_ 7, e48694 (2012). Article  CAS  Google Scholar  * Hager, M. H., Solomon, K. R. & Freeman, M. R. The role of

cholesterol in prostate cancer. _Curr. Opin. Clin. Nutr. Metab. Care_ 9, 379–385 (2006). Article  CAS  Google Scholar  * Aboumrad, M. H., Horn, R. C. Jr. & Fine, G. Lipid-secreting

mammary carcinoma. Report of a case associated with Paget’s disease of the nipple. _Cancer_ 16, 521–525 (1963). Article  CAS  Google Scholar  * Ramos, C. V. & Taylor, H. B. Lipid-rich

carcinoma of the breast. A clinicopathologic analysis of 13 examples. _Cancer_ 33, 812–819 (1974). Article  CAS  Google Scholar  * Rautio, J., Meanwell, N. A., Di, L. & Hageman, M. J.

The expanding role of prodrugs in contemporary drug design and development. _Nat. Rev. Drug Discov._ 17, 559–587 (2018). Article  CAS  Google Scholar  * Ran, F. A. et al. Genome engineering

using the CRISPR-Cas9 system. _Nat. Protoc._ 8, 2281–2308 (2013). Article  CAS  Google Scholar  * Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for

CRISPR screening. _Nat. Methods_ 11, 783–784 (2014). Article  CAS  Google Scholar  * Campeau, E. et al. A versatile viral system for expression and depletion of proteins in mammalian cells.

_PLoS ONE_ 4, e6529 (2009). Article  Google Scholar  * Trost, B. M. et al. A concise synthesis of (−)-lasonolide A. _J. Am. Chem. Soc._ 136, 88–91 (2014). Article  CAS  Google Scholar 

Download references 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. AUTHOR INFORMATION

AUTHORS AND AFFILIATIONS * Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA Ramin Dubey & Rajat Rohatgi * Department of Chemistry, Stanford

University, Stanford, CA, USA Craig E. Stivala & Barry M. Trost * Genentech, South San Francisco, CA, USA Craig E. Stivala & Huy Quoc Nguyen * Department of Molecular and Cellular

Physiology, Albany Medical College, Albany, NY, USA Young-Hwa Goo & Antoni Paul * Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA Jan

E. Carette * Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA Rajat Rohatgi Authors * Ramin Dubey View author publications You can also search for this

author inPubMed Google Scholar * Craig E. Stivala View author publications You can also search for this author inPubMed Google Scholar * Huy Quoc Nguyen View author publications You can also

search for this author inPubMed Google Scholar * Young-Hwa Goo View author publications You can also search for this author inPubMed Google Scholar * Antoni Paul View author publications

You can also search for this author inPubMed Google Scholar * Jan E. Carette View author publications You can also search for this author inPubMed Google Scholar * Barry M. Trost View author

publications You can also search for this author inPubMed Google Scholar * Rajat Rohatgi View author publications You can also search for this author inPubMed Google Scholar 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. CORRESPONDING AUTHOR Correspondence to Rajat Rohatgi. ETHICS DECLARATIONS COMPETING INTERESTS The authors

declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–12. REPORTING SUMMARY 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). RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download

citation * Received: 13 February 2019 * Accepted: 02 December 2019 * Published: 13 January 2020 * Issue Date: February 2020 * DOI: https://doi.org/10.1038/s41589-019-0447-7 SHARE THIS

ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard

Provided by the Springer Nature SharedIt content-sharing initiative