Investigation Of The Function And Mechanism Of Cancer Common Mutations In Cancer Immunotherapy Response

Cancer is the second leading cause of death worldwide.Following traditional therapies such as surgery,radiotherapy,chemotherapy and targeted therapies,the success of immunotherapies,particularly the immune checkpoint blockade(ICB),initiated a new era of cancer treatment.However,most patients fail to respond to immunotherapies,and many of those initial responders develop resistance or relapse eventually.It is therefore urgent to understand the genetic heterogeneity of tumors that expose their vulnerability to immune-mediated clearance.As a fundamental hallmark of cancer,genetic mutations enable cancer cells to sustain abnormal proliferation and survive extrinsic suppression including evasion from immunotherapies.To explore the association between cancer common mutations and caner immunotherapy response,we designed and constructed a mouse guide RNA(gRNA)library targeting ?300 cancer common mutation genes,introduced the library to a mouse colorectal cancer cell line,and performed in vivo CRISPR screen in immune competent mouse syngeneic models.By evaluating the representation of sgRNAs targeting these genes in tumors with or without receiving an antibody blocking programmed cell death protein 1(anti-PD-1),we identified multiple genes governing cancer response to ICB,including 5 genes(Stk11,H2-K1,B2 m,Pten and Smarca4)that have been reported to cause immunotherapy resistance when mutated,and 10 more genes(Kmt2d,Arid2,Lztr1,Pbrm1,Erbb3,Fat1,Ptpn11,Keap1,Smad4 and Nras)whose mutations have been implicated in sensitizing tumors to immunotherapy.More importantly,we found that the abundance of gRNAs targeting Kdm6a was significantly reduced in anti-PD-1-treated tumors.KDM6A is a histone 3lysine 27(H3K27)demethylase,and complexes with KMT2D,a histone 3 lysine 4(H3K4)methyltransferase,to regulate expression of many genes controlling development and differentiation.We demonstrated that deletion of Kmt2d or Kdm6a in cancer cells significantly increased T cell infiltration in tumor and enhanced tumor antigen presentation,leading to better response to anti-PD-1 treatment.Mechanistically,both KMT2D and KDM6A maintain cellular H3K4me1 levels and DNA repair integrity,and their deficiency renders cancer cell vulnerable to killing by cytotoxic T cells or type ? and ? interferons and tumor necrosis factor.Analysis of TCGA datasets shows that patients with KMT2D or KDM6A mutations benefit more from ICB therapies than those without such mutations.Taken together,our data suggest that KMT2D and KDM6A mutations can serve as prognostic biomarkers for cancer ICB therapies.Finally,we explored the role of KMT2D mutations in tumor progression.We found that KMT2D mutations cannot promote tumor growth,but its inactivation conferred a competitive survival advantage on tumor cells,and the underlying mechanisms are still being investigated.In summary,our findings indicate that KMT2D and KDM6A mutations are potential biomarkers for ICB therapy,and more researches are needed for future clinical application.