Deciphering the interplay between SETD2 mediated H3K36me3 and RNA N6-methyladenosine in clear cell renal cell carcinoma (ccRCC) [MeRIP-seq]

RNA N6-methyladenosine (m6A) plays diverse roles in RNA metabolism and its deregulation contributes to tumor initiation and progression. Clear cell renal cell carcinoma (ccRCC) is characterized by near ubiquitous loss of VHL followed by mutations in epigenetic regulators PBRM1, SETD2, and BAP1. Mutations in SETD2, a histone H3 lysine 36 trimethylase (H3K36me3), are associated with reduced survival, greater metastatic propensity, and metabolic reprogramming. While m6A and H3K36me3 deregulation are separately implicated in renal tumorigenesis, H3K36me3 may participate directly in m6A targeting, but the m6A-H3K36me3 interplay has not been investigated in the context of ccRCC. Using RCC-relevant SETD2 isogenic knockout and rescue cell line models, we demonstrate a dynamic redistribution of m6A in the SETD2 depleted transcriptome, with a subset of transcripts involved in metabolic reprogramming demonstrating SETD2 dependent m6A and expression level changes. Using a panel of six histone modifications we show that m6A redistributes to regions enriched in gained active enhancers upon SETD2 inactivation. Finally, we demonstrate a reversal of transcriptomic programs involved in SETD2 loss mediated metabolic reprogramming, and reduced cell viability through pharmacological inhibition or genetic ablation of m6A writer METTL3 specific to SETD2 deficient cells. Thus, targeting m6A may represent a novel therapeutic vulnerability in SETD2 mutant ccRCC. Overall design: SETD2WT and SETD2KO ccRCC (786-O) and surrogate normal (RPTEC) cell lines were profiled for m6A methylome to determine the impact of SETD2 inactivation on m6A methylation patterns

RNA N6-甲基腺苷（RNA N6-methyladenosine，m6A）在RNA代谢中发挥多种重要功能，其表达失调可促进肿瘤发生与进展。透明细胞肾细胞癌（Clear cell renal cell carcinoma，ccRCC）的典型特征为几乎普遍存在VHL基因缺失，随后伴随表观遗传调控因子PBRM1、SETD2及BAP1的突变。SETD2作为组蛋白H3赖氨酸36三甲基化酶（histone H3 lysine 36 trimethylase，H3K36me3），其突变与患者生存期缩短、转移潜能增强及代谢重编程显著相关。尽管m6A与H3K36me3的失调已被分别证实参与肾肿瘤发生过程，但H3K36me3可能直接参与m6A的靶向调控，然而二者的互作关系尚未在ccRCC的研究背景中得到阐释。
本研究依托RCC相关的SETD2同基因敲除与回复细胞系模型，证实SETD2缺失的转录组中m6A修饰发生动态重分布，其中参与代谢重编程的部分转录本呈现SETD2依赖的m6A修饰及表达水平变化。通过针对六种组蛋白修饰的检测分析，我们发现SETD2失活后，m6A重分布至新增的活性增强子富集区域。最后，本研究证实SETD2缺失介导的代谢重编程相关转录组程序可被逆转，且通过对SETD2缺陷细胞特异性的m6A写入酶METTL3进行药物抑制或基因敲除，可显著降低细胞活力。因此，靶向m6A或可成为SETD2突变型ccRCC的新型治疗脆弱靶点。
总体实验设计：本研究对SETD2野生型（SETD2WT）、SETD2敲除型（SETD2KO）ccRCC细胞系（786-O）及替代正常细胞系（RPTEC）开展m6A甲基化组测序，以明确SETD2失活对m6A甲基化模式的影响。