Screens In Haploid Cells Research Articles

Lipid droplets can promote drug accumulation and activation.

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.

Abstract 2059: Identification of novel paclitaxel sensitivity genes via lentiviral CRISPR/Cas9 screening in haploid human cells

Abstract Some cancers lack clearly druggable driver mutations and might also have too few neo-antigen generating mutations to provoke a tumor-specific immune response, even in the presence of antibodies targeting immune checkpoint mechanisms. Synthetic lethal/non-oncogene addiction represents an alternative approach to the therapy of such cancers, as exemplified by the recent approval of olaparib. RNA interference has identified several putative synthetic lethal targets, but few, if any, of these have have withstood vigorous validation. The recent application of RNA-guided gene editing to mammalian cells offers new prospects for target identification. The complete ablation of gene expression, which this technology permits allows the identification of targets that have no phenotype when partially depleted by RNA interference. Horizon has recently established a capability for CRISPR-Cas9 KO screening. Screens are performed by transducing a pool of cells with a complex lentiviral library. Next generation sequencing is then used to identify which sgRNAs are accumulating or being depleted from the population. As there are multiple sgRNAs per gene, we can rank genes for their likelihood of having a phenotype under the conditions used in the screen. Our proof-of-principle small molecule resistance and sensitivity screens using both whole-genome libraries and custom subset libraries have successfully identified previously published hits and also yielded novel hits. Horizon has also developed a fully haploid cell line (eHAP), which has the potential to provide additional clarity in genetic screening. To evaluate the power of CRISPR-Cas9 KO screening in haploid cells, we have used our screening platform to identify genes required for survival in low doses of paclitaxel. Whole-genome screening using the GeCKOv2 library identified three kinesins amongst the most prominent of the hits whose depletion sensitised cells to this microtubule stabilising agent. Intriguingly, two of these kinesins, KIF2A and KIF2C are thought to destabilise mitotic microtubules, providing an attractive explanation for the synthetic lethality. We will also present results from a second genome-wide CRISPR KO screen in haploid cells to identify genes required for survival under glucose starvation. Prominent hits included components of the electron transport chain and the tuberous sclerosis complex. Citation Format: Jessica R. Hunt, Steffen Lawo, David Walter, Isabelle Nett, Joanne Yarker, Benedict C S Cross, Jonathan D. Moore. Identification of novel paclitaxel sensitivity genes via lentiviral CRISPR/Cas9 screening in haploid human cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2059.

Abstract 2743: Finding determinants of PARP inhibitor sensitivity using genome-wide and focused CRISPR screens

Abstract We are using genomic approaches to study the mechanism of action of targeted cancer drugs, particularly poly(ADP-ribose) polymerase (PARP) inhibitors. One of these drugs, olaparib was recently approved for use in ovarian cancers with germline BRCA1/2 mutations and many other clinical trials with this and other PARP inhibitors are underway. Experimental study of potential resistance mechanisms can inform the ongoing clinical development of these drugs. We previously carried out a loss of function mutagenesis screen in haploid cells, which showed that PARP1 was required for cellular toxicity of olaparib and other PARP inhibitors. This supported the hypothesis that PARP inhibitor toxicity occurs via a poisoning mechanism where inhibited PARP is tightly bound to DNA, forming a toxic lesion. Recently we carried out a similar screen using genome-wide lentiviral CRISPR-Cas9 mutagenesis in diploid cells. Many PARP inhibitor resistant clones had loss of function Parp1 mutations as expected. However, we also isolated a point mutation affecting a single amino acid in the Parp1 DNA binding domain. This mutant encodes a stable Parp1 protein that cannot bind DNA and does not become trapped in the presence of inhibitors. Thus the CRISPR screen implicated PARP1 DNA binding directly in determination of PARP inhibitor toxicity and was extremely informative about the mechanism of action compared to conventional loss-of-function mutagenesis. We extended this approach by synthesising a high-density focused sgRNA library targeting only PARP1. We developed a reporter cell line that allows us to selectively isolate in-frame mutations that preserve PARP1 protein expression. By deep sequencing mutagenised and appropriately selected cells we identified a series of subtle mutations in PARP1 that result in PARP inhibitor resistance, giving us a detailed insight into structure-function relationships in PARP1. The approach is applicable to other drug targets, providing a framework for understanding drug action and predicting clinical resistance. Citation Format: Stephen Pettitt, Dragomir B. Krastev, Feifei Song, Alan Ashworth, Christopher J. Lord. Finding determinants of PARP inhibitor sensitivity using genome-wide and focused CRISPR screens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2743.

Attachment ofChlamydia trachomatisL2 to host cells requires sulfation

Chlamydia trachomatis is a pathogen responsible for a prevalent sexually transmitted disease. It is also the most common cause of infectious blindness in the developing world. We performed a loss-of-function genetic screen in human haploid cells to identify host factors important in C. trachomatis L2 infection. We identified and confirmed B3GAT3, B4GALT7, and SLC35B2, which encode glucuronosyltransferase I, galactosyltransferase I, and the 3'-phosphoadenosine 5'-phosphosulfate transporter 1, respectively, as important in facilitating Chlamydia infection. Knockout of any of these three genes inhibits Chlamydia attachment. In complementation studies, we found that the introduction of functional copies of these three genes into the null clones restored full susceptibility to Chlamydia infection. The degree of attachment of Chlamydia strongly correlates with the level of sulfation of the host cell, not simply with the amount of heparan sulfate. Thus, other, as-yet unidentified sulfated macromolecules must contribute to infection. These results demonstrate the utility of screens in haploid cells to study interactions of human cells with bacteria. Furthermore, the human null clones generated can be used to investigate the role of heparan sulfate and sulfation in other settings not limited to infectious disease.