Hypoxia-inducible factor 2α (HIF2α) antagonists are effective against clear cell renal cell carcinomas (ccRCCs) that highly express HIF2α. To identify potential drug targets in HIF2α^low/− ccRCC, we constructed an epigenetic-focused single-guide RNA library and performed an in vivo CRISPR-Cas9 knockout screen in BALB/c nude mice transplanted with 786-O (HIF2α^high) or Caki-2 (HIF2α^low/−) cells. We found that the m6A demethylase fat mass and obesity-associated (FTO) gene was indispensable to the growth of HIF2α^low/− but not HIF2α^high ccRCC. Activation of FTO in HIF2α^low/− ccRCC was caused by an increased intracellular α-ketoglutarate–to-succinate ratio and stabilized bromodomain-containing protein 9 (BRD9) messenger RNA via m6A demethylation. RNA sequencing and chromatin immunoprecipitation sequencing profiling further revealed that SRY-box transcription factor 17 (SOX17) recruited BRD9 to de novo super enhancers associated with genes that feature prominently in ccRCC pathogenesis, including CCND1, VEGFR2, CDC20, SRC, and MAPK6. BRD9 knockdown or the BRD9-selective antagonist I-BRD9 suppressed the growth of HIF2α^low/− but not HIF2α^high ccRCC cells in vitro. In BALB/c nude mice bearing HIF2α^low/− ccRCC cell line–derived xenografts and patient-derived tumor xenografts, I-BRD9 administration effectively inhibited tumor growth and prolonged the survival of tumor-bearing mice with greater efficacy than sunitinib. Together, these findings indicate that BRD9 is a druggable target for treating HIF2α^low/− ccRCC.

中文翻译：

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m6A 去甲基化酶 FTO 的异常激活使 HIF2αlow/− 透明细胞肾细胞癌对 BRD9 抑制剂敏感

缺氧诱导因子 2α (HIF2α) 拮抗剂可有效对抗高表达 HIF2α 的透明细胞肾细胞癌 (ccRCC)。为了识别 HIF2α ^low/− ccRCC 中的潜在药物靶点，我们构建了一个以表观遗传学为重点的单向导 RNA 文库，并在移植了 786-O (HIF2α high ) 或 Caki 的 BALB/c 裸鼠中进行了体内 CRISPR-Cas9 敲^除筛选-2（HIF2α^低/−）个细胞。我们发现 m6A 去甲基化酶脂肪量和肥胖相关 ( FTO ) 基因对于 HIF2α^低/− ccRCC 的生长是不可或缺的，但对于 HIF2α^高ccRCC 的生长却不是必不可少的。HIF2α 中 FTO 的激活^low/−ccRCC 是由细胞内 α-酮戊二酸与琥珀酸比例增加以及通过 m6A 去甲基化稳定含溴结构域蛋白 9 ( BRD9 ) 信使 RNA 引起的。RNA测序和染色质免疫沉淀测序分析进一步揭示，SRY-box转录因子17 (SOX17)将BRD9招募到与ccRCC发病机制中显着特征的基因相关的从头超级增强子，包括CCND1、VEGFR2、CDC20、SRC和MAPK6。BRD9敲除或 BRD9 选择性拮抗剂 I-BRD9 抑制 HIF2α ^low/−的生长，但不抑制 HIF2α ^{high 的生长}ccRCC 细胞体外。^{在携带 HIF2α low/−} ccRCC 细胞系来源的异种移植物和患者来源的肿瘤异种移植物的BALB/c 裸鼠中，I-BRD9 给药可有效抑制肿瘤生长并延长携带肿瘤小鼠的生存期，其疗效优于舒尼替尼。总之，这些发现表明 BRD9 是治疗 HIF2α ^low/− ccRCC 的药物靶标。