全基因组CRISPR筛选确定PHF8是KRAS或BRAF突变型结直肠癌的有效治疗靶点。
Genome-wide CRISPR screening identifies PHF8 as an effective therapeutic target for KRAS- or BRAF-mutant colorectal cancers.
作者信息
Liu Zhao, Li Yiqi, Wang Simeng, Wang Yubo, Sui Mengjun, Liu Jiaxin, Chen Pu, Wang Jianling, Zhang Yuchen, Dang Chengxue, Hou Peng
机构信息
Department of Endocrinology and International Joint Research Center for Tumor Precision Medicine of Shaanxi Province, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710061, P.R. China.
Department of Surgical Oncology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710061, P.R. China.
出版信息
J Exp Clin Cancer Res. 2025 Feb 25;44(1):70. doi: 10.1186/s13046-025-03338-2.
BACKGROUND
Mutations in KRAS and BRAF genes are prevalent in colorectal cancer (CRC), which strikingly promote tumorigenesis and lead to poor response to a variety of treatments including immunotherapy by activating the MAPK/ERK pathway. Thus, there is an urgent need to discover effective therapeutic targets and strategies.
METHODS
CRISPR-Cas9 lentiviral knockout library was used to screen the suppressors of anti-PD1 immunotherapy. Bioinformatic analysis was used to analyze the correlation between PHF8 expression and immune indicators in CRC. In vitro and in vivo experiments were utilized to determine the effects of PHF8 on the immune indexes and malignant phenotypes of CRC cells. qRT-PCR, western blotting, immunohistochemical (IHC) staining, and chromatin immunoprecipitation (ChIP)-qPCR assays were used to determine the regulatory effects of PHF8 on PD-L1, KRAS, BRAF, and c-Myc and the regulatory effect c-Myc/miR-22-3p signaling axis on PHF8 expression in CRC cells.
RESULTS
This study identified histone lysine demethylase PHF8 as a negative regulator for the efficacy of anti-PD1 therapy and found that it was highly expressed in CRCs and strongly associated with poor patient survival. Functional studies showed that PHF8 played an oncogenic role in KRAS- or BRAF-mutant CRC cells, but not in wild-type ones. Mechanistically, PHF8 up-regulated the expression of PD-L1, KRAS, BRAF, and c-Myc by increasing the levels of transcriptional activation marks H3K4me3 and H3K27ac and decreasing the levels of transcriptional repression mark H3K9me2 within their promoter regions, promoting immune escape and tumor progression. Besides, our data also demonstrated that PHF8 was up-regulated by the c-Myc/miR-22-3p signaling axis to form a positive feedback loop. Targeting PHF8 substantially improved the efficacy of anti-PD1 therapy and inhibited the malignant phenotypes of KRAS- or BRAF-mutant CRC cells.
CONCLUSION
Our data demonstrate that PHF8 may be an effective therapeutic target for KRAS- or BRAF-mutant CRCs.
背景
KRAS和BRAF基因的突变在结直肠癌（CRC）中普遍存在，这些突变显著促进肿瘤发生，并导致包括免疫治疗在内的多种治疗反应不佳，其通过激活MAPK/ERK途径实现。因此，迫切需要发现有效的治疗靶点和策略。
方法
使用CRISPR-Cas9慢病毒敲除文库筛选抗PD1免疫治疗的抑制因子。利用生物信息学分析来分析CRC中PHF8表达与免疫指标之间的相关性。采用体外和体内实验来确定PHF8对CRC细胞免疫指标和恶性表型的影响。使用qRT-PCR、蛋白质免疫印迹法、免疫组织化学（IHC）染色和染色质免疫沉淀（ChIP）-qPCR实验来确定PHF8对PD-L1、KRAS、BRAF和c-Myc的调控作用，以及c-Myc/miR-22-3p信号轴对CRC细胞中PHF8表达的调控作用。
结果
本研究确定组蛋白赖氨酸去甲基化酶PHF8是抗PD1治疗疗效的负调节因子，并发现其在CRC中高表达，且与患者的不良生存密切相关。功能研究表明，PHF8在KRAS或BRAF突变的CRC细胞中发挥致癌作用，但在野生型细胞中则不然。从机制上讲，PHF8通过增加其启动子区域的转录激活标记H3K4me3和H3K27ac水平，并降低转录抑制标记H3K9me2水平，上调PD-L1、KRAS、BRAF和c-Myc的表达，促进免疫逃逸和肿瘤进展。此外，我们的数据还表明，PHF8被c-Myc/miR-22-3p信号轴上调，从而形成正反馈回路。靶向PHF8可显著提高抗PD1治疗的疗效，并抑制KRAS或BRAF突变的CRC细胞的恶性表型。
结论
我们的数据表明，PHF8可能是KRAS或BRAF突变的CRC的有效治疗靶点。
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