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.