Transcription Elongation Defect Potentiates Anti-Tumor Immunity

Over the past decade,immunotherapy has rapidly transformed the treatment landscape of cancer.However,as clinical applications have progressed,the development of resistance to immunotherapy has become inevitable.Additionally,while immunotherapy can generate durable and even curative clinical responses,the majority of patients lack a response.The essence of the lack of response and resistance to immunotherapy lies in the immune evasion mechanisms evolved by tumor cells.Therefore,there is an urgent need to deepen our understanding of the immune evasion mechanisms of tumor cells in order to provide new insights for increasing the response rate of immunotherapy and overcoming therapeutic resistance.Regulation of gene expression is closely related to the initiation and development of various cancers.We sought to investigate whether genes involved in transcriptional regulation could mediate tumor immune evasion.Thus,we transduced Hepa1-6 liver cancer cells with an sgRNA library enriched for regulators of transcription,and orthotopically transplanted the resulting cells into the livers of mice for in vivo highthroughput genetic screening.By comparing the survival of Hepa1-6 cells with individual gene knockouts in vivo and in vitro,we discovered that SCAF8 could promote tumor immune evasion.Loss of Scaf8 did not affect the in vitro proliferation of cells.However,in wild-type mice,the growth of Scaf8-deficient tumors was significantly inhibited.The growth inhibition of Scaf8-deficient Hepa1-6 tumors in vivo could be partially relieved by clearance of macrophages.The growth inhibition of Scaf8-deficient MC38 tumors was completely relieved in immunodeficient mice.Through single-cell sequencing analysis of CD45+ immune cells in Scaf8-deficient MC38 tumors,we found that there was a significant increase in Ml macrophages and a significant decrease in MDSCs in Scaf8-deficient tumors.Scaf8-deficient tumors showed significantly higher expression of pro-inflammatory genes such as Cxcl9,Cxcl10,and Ccl5 and significantly lower expression of anti-inflammatory genes such as Argl,Cxcl2,and Vegfa in infiltrating immune cells compared to control tumors.Due to the limited number of lymphocytes in single-cell sequencing,we further used flow cytometry to analyze infiltrating lymphocytes in the tumor microenvironment.We found that Scaf8-deficient resulted in a significant increase in the number of CD8+ T cells and NK cells.These results indicate that Scaf8 deficiency reshapes the tumor immune microenvironment to be more anti-tumor.IFNγ and TNFa are two major cytokines in the tumor microenvironment.We found that Scaf8-deficient tumor cells were more sensitive to IFNγ+TNFα treatment.Tumor cells often evade immune surveillance by downregulating MHC Ⅰ levels.We found that Scaf8deficient tumor cells significantly upregulated MHC Ⅰ levels and were more sensitive to Tcell killing.We used RNA-seq to evaluate the transcriptome changes upon Scaf8 loss under IFNy+TNFα stimulation.We found that antigen presentation genes such as H2-K1,Tap1,and Psmb9,as well as pro-inflammatory genes such as Cxcl9、Ccl5 and Zbpl,were significantly upregulated.Gene length distribution analysis revealed that differential gene expression is gene length-dependent,with upregulated genes tending to be shorter and downregulated genes tending to be longer.We also observed gene length-dependent differential gene expression mediated by SCAF8 in the human cell line HeLa.To provide a mechanistic explanation for this phenomenon,we performed RNA PolⅡ CTD Ser2P ChIP-seq and TT-seq on Scaf8-deficient cells.We found that RNA Pol ⅡCTD Ser2P signals and TT-seq nascent RNA signals were elevated at most short genes,while they gradually decreased from 5’ to 3’ at long genes.Notably,we found that the nascent RNA levels of short genes such as H2-K1、Cxcl9 and Ccl5 were significantly increased.These results suggest that Scaf8 deficiency promotes the expression of short genes by affecting transcription elongation,thereby mediating anti-tumor immune responses.We also found that other transcription elongation factors like CDK13 could mediate gene length-dependent differential gene expression.Loss of Cdk13 and treatment of Cyclin K molecular glue compound significantly upregulated MHC Ⅰ levels and sensitize tumors cells to T-cell killing.The commonly used chemotherapeutic agent CPT is a DNA topoisomerase Ⅰ inhibitor that causes transcription elongation defects.We found that lowdose CPT treatment of cells under IFNy+TNFa stimulation also leads to gene lengthdependent differential gene expression.Based on RNA-seq analysis,low-dose CPT treatment induced similar upregulation of many immune-related short genes,and also resulted in upregulation of MHC Ⅰ levels and increased sensitivity to T-cell killing.These results indicate that the transcription elongation process can regulate differential gene expression based on gene length.In summary,this study identified SCAF8 as a factor that promotes immune evasion.Mechanistically,Scaf8 deficiency leads to impaired transcription elongation,thereby mediating gene length-dependent differential gene expression,with upregulation of short genes and downregulation of long genes.Scaf8 deficiency leads to upregulation of antigen presentation and various pro-inflammatory short genes,enhancing anti-tumor immune responses.The study also found that deficiencies in other transcription elongation factors,such as CDK13 and treatment with Cyclin K molecular glue compound and the chemotherapy drug CPT,can induce gene length-dependent differential gene expression,highlighting the potential of transcription elongation modulation in enhancing anti-tumor immunity.The findings of this study provide a new strategy for immunotherapy:targeting transcription elongation to enhance anti-tumor immunity.Our study also reveals that perturbation of transcription elongation is a novel mechanism for global transcriptional regulation and may play important roles in other physiological and pathological processes.