Abstract A69: An epigenetic-focused CRISPR/Cas9 screen to identify regulators of IFNγ-induced PD-L1 expression
Authors: Luster Troy A. , Kulkarni Meghana , Fitzpatrick Erica , Badalucco Lauren , English Jessie , Wong Kwok-Kin , Bittinger Mark 
Published: May 2016

PD-L1 (B7-H1, CD274) is a clinically validated immuno-oncology target, which is often over-expressed on the surface of tumor cells. PD-L1 binds to PD-1 expressed on T cells generating an immunosuppressive signaling response that limits T cell activation and facilitates immune evasion. The tumor microenvironment often recruits immune cells that produce a number of secreted factors, including IFNγ, a potent inducer of PD-L1 expression on tumor cells. Blocking IFNγ-induced PD-L1 expression with small molecules could be a potential alternative to antibody-based PD-L1/PD-1 blockade. Recent studies on human patient samples indicate that the level of PD-L1 expression on tumor cells is inversely related to the level of DNA methylation at the PD-L1 promoter (Gettinger et al, 2015), suggesting that PD-L1 expression is epigenetically silenced in tumor cells with low PD-L1 expression. Therefore, cytokines that induce PD-L1 expression on tumor cells, such as IFNγ, may regulate the activity of epigenetic silencing factors at the PD-L1 promoter, and identification of these epigenetic factors could provide novel therapeutic targets to block PD-L1 expression on tumor cells.

CRISPR/Cas9 gene editing has recently emerged as a powerful technology for phenotypic screening. To identify potential epigenetic regulators of IFNγ-induced PD-L1 on tumor cells, several murine tumor cell lines were treated with IFNγ and PD-L1 expression was monitored by flow cytometry. The ovarian cancer cell line ID8 demonstrated high PD-L1 expression following IFNγ stimulation, and stable Cas9 expressing clones were generated. A high expressing Cas9 clone was selected for follow-up transduction with a sgRNA library targeting &gt;350 known epigenetic factors, plus numerous positive and negative controls. Library transduced cells were evaluated in two separate screening streams: i) an enrichment screen to identify genes regulating IFNγ-induced PD-L1 expression, and ii) a depletion screen to identify genes essential for the growth of ID8 tumor cells. For the enrichment screen, sgRNA transduced cells were treated with IFNγ, and FACS was performed to collect cells with low PD-L1 expression i.e. cells refractory to IFNγ-induced PD-L1 expression. Genomic DNA was then isolated from the sorted cells and sgRNA sequences were quantified by next-generation sequencing (NGS). As an important validation of the FACS-based screening format, the most highly enriched sgRNAs in the low PD-L1 population were PD-L1 itself, and the canonical mediators of IFNγ signaling JAK1/2 and STAT1. For the depletion screen, sgRNA transduced cells were cultured for up to 14 days, with cell pellets collected on day 0, 3, 7 and 14 for NGS quantification of sgRNAs. All positive and negative controls scored as expected and several epigenetic factors were strongly depleted indicating an essential role for ID8 cell growth. In summary, CRISPR/Cas9 gene editing is a powerful screening technology for the identification of factors essential for cell growth, and when paired with FACS, is a useful methodology to identify factors regulating expression of immune checkpoint molecules.

Gettinger et al, Journal of Clinical Oncology, 2015 ASCO Annual Meeting, Vol 33, No 15 (May 20 supplement), 2015: 3015

Citation Format: Troy A. Luster, Meghana Kulkarni, Erica Fitzpatrick, Lauren Badalucco, Jessie English, Kwok-Kin Wong, Mark Bittinger. An epigenetic-focused CRISPR/Cas9 screen to identify regulators of IFNγ-induced PD-L1 expression. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A69.