Abstract 1649: In vivo CRISPR-Cas9 library screen for immune checkpoint regulators

Therapeutic antibodies blocking immune checkpoints such as programmed cell death protein 1 (PD1) have proven clinically beneficial in multiple cancer types. However, anti-PD1 drugs approved by FDA are neither effective in treating all cancer types nor in all patients in a responsive cancer type. Therefore, understanding the mechanism underlying the efficacy of immune checkpoint blockade is critical for our rational design of synergistic treatment combinations. Here we describe how we employ the latest CRISPR-Cas9 genome-wide knockout screening strategies to identify targets in cancer cells or in T lymphocytes that control the sensitivity or resistance to anti-PD1 treatment. We constructed a sgRNA library targeting the functional domains of 116 mouse methyltransferases and demethylases, which was infected into the mouse Cloudman S91 melanoma cell line. Syngeneic mouse xenograft models derived from these infected cells were treated with anti-mPD1 monoclonal antibody, and the xenograft tumors were harvest at different time points after antibody treatment. Deep sequencing of the integrated sgRNA sequences in the tumors has identified potential druggable targets to sensitize or antagonize the tumors resistance to anti-mPD1 immunotherapy. Alternatively, we introduced the same library to naive T cells purified from the OT-1 transgenic mice. The T cell library was then injected to B16-OVA melanoma syngeneic mouse models, followed by anti-mPD1 treatment. Deep sequencing of the integrated sgRNA sequences in the tumor-infiltrating T cells would allow us to uncover novel signaling molecules in the immune system that can potentially be manipulated to enhance the anti-PD1 therapy. In summary, we provide in vivo knockout screen strategies that can be applied to identify and validate combination therapies with immune checkpoint antibodies. Citation Format: Sascha Karassek, Min Long, Xi Li, Zhengang Peng, Qunsheng Ji, Yong Cang. In vivo CRISPR-Cas9 library screen for immune checkpoint regulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1649. doi:10.1158/1538-7445.AM2017-1649