CRISPR library screens identify frequent cancer mutations regulating PD-1 blockade response

AACR Annual meeting 2021

Wenrong Zhou1,* , Min Long2,3* , Tianyu Song3,* , Qiangqiang Fan1 , Zhengang Peng1 , Yong Cang3 , Qunsheng Ji1 1 Oncology and Immunology Unit, Research Service Division, WuXi AppTec (Shanghai) Co., Ltd., China; 2 Life Sciences Institute, Zhejiang University, Hangzhou, China; School of Life Science and Technology, ShanghaiTech University, Shanghai, china

Abstract

Background

Immune checkpoint blockade therapy has proven clinically beneficial in multiple cancer types. However, immune checkpoint blockade drugs approved by FDA are neither effective in treating all cancer types nor in all patients in a responsive cancer type. Cancer mutations play essential roles in tumor development by promote cell proliferation or immune evasion. But, cancer cells with these mutation burden might also expose their vulnerability to immune-based therapies such as checkpoint blockade.Therefore, understanding the correlation between these mutations and the efficacy of immune checkpoint blockade is critical for our rational design of synergistic treatment combinations.

Results

Here we performed CRISPR knockout library screens in syngeneic mouse xenograft models on cancer driver mutation genes, and identified a set of genes (Kmt2d, Arid2, Pbrm1, Rhob, Eloc, Kdm6a, and Ptpn11 ) which have high frequency mutation in multiple human cancer types and their deficiency made tumor cells better respond to anti-PD-1 therapy. Furthermore, we conducted a sub library screen by co culture edited tumor cells with CD8+ T cells, it revealed that some of these immunotherapy regulators sensitize tumor cells to cytotoxic T cells when deletion, such as Ptpn11, Eloc and Kmt2d . Kmt2d was reported as a major modulator of immune checkpoint blockade and we verified the function of KMT2D in sensitivity to T cells by co culture assay. Consistently, KMT2D mutation in cancer patients predicted better clinical benefits from immunotherapy according to TCGA database.

Conclusion

In summary, our screen on cancer driver mutation genes provided a way to distinguish the mutations accumulated in cancers that can synergize the efficacy of immune checkpoint blockade therapy.Our approach utilizing small focused libraries instead of genome wide ones results in a high coverage/ sgRNA and a robust correlation between tumor samples.

Cancer diver genes in vivo screening identified genes related to immune evasion.

Loss-of-function screening recovered targets related to the response of immunotherapy.

CD8+ T cells co culture screen revealed the regulators of cytotoxic T cell sensitivity

Kmt2d deficiency sensitized tumor cells to T cells killing and synergized the efficacy of immune checkpoint blockade therapy.

Summary & Conclusion

We provided a way to distinguish cancer driver mutations that can synergize the efficacy of immune checkpoint blockade therapy.
We put forwarded a possibility to take advantage of the hallmark of cancer, that mutations driving caner development may make tumor be vulnerable to immunotherapy.
By utilizing those focused sgRNA libraries we can achieve a high coverage per sgRNA and produce good correlation results so as to increased reliability of candidates.
Conducting another sub-library screen by co culture with immune cells, such as CD8+ T cells or NK cells and macrophages, is great helpful to figure out the mechanisms and application.
Collaboration with industry and academia is welcome

For more information and possible collaboration please contact: Dr. Zhengang Peng