SETD2 loss sensitizes kidney cancer cells to DNA hypomethylating agents

Large-scale sequencing efforts in Clear cell renal cell carcinoma (ccRCC) have found a high prevalence of mutations in chromatin-related genes.  Prominent within this group is SETD2, which is mutated in 15% of ccRCC and is associated with aggressive disease. SETD2 is a methyltransferase responsible for trimethylating lysine 36 on histone H3 (H3K36me3). Although it is not completely understood how SETD2 loss contributes to ccRCC tumorigenesis, it is thought that it reprograms the epigenetic landscape of the cell. Here we explore the impact that SETD2/H3K36me3 loss has on the DNA methylome in ccRCC cells. DNA methylation was measured using the EPIC DNA methylation assay in 786-O ccRCC cells and non-cancerous transformed proximal tubule kidney cells (HKC) with and without SETD2. Sensitivity to DNA hypomethylating agents was assessed by dose-response assay using 5-aza-2'-deoxycytidine. Apoptosis was measured via Annexin-V/PI staining by flow cytometry. Mitochondrial fitness was evaluated by electron microscopy and flow cytometry. Moreover, activity of 5-aza-2'-deoxycytidine, a DNA hypomethylating agent, in was assessed in SETD2 WT/KO xenografts in NOD-Scid mice. SETD2 loss resulted in DNA hypermethylation in HKC cells and to a greater extent in 786-O. Dose-response assays showed that SETD2-null ccRCC cells are sensitive to 5-aza-2'-deoxycytidine. Furthermore, Annexin-V/PI staining revealed more apoptotic and necrotic cells in SETD2-null cells following 5-aza-2'-deoxycytidine treatment, which was rescued using a Caspase inhibitor. In addition, 5-aza-2'-deoxycytidine induced profound changes in mitochondria in SETD2-null cells, including loss of membrane potential and size reduction. Indeed, in vivo experiments verified increased SETD2-null xenografts’ sensitivity to 5-aza-2'-deoxycytidine. We show that SETD2 loss in ccRCC cells causes DNA hypermethylation, creating a synthetic lethal dependency with DNA hypomethylating agents. RNAseq profiles in HKC and 786-0 kidney cells treated with DMSO or Decitabine (100nM or 300nM)\

针对透明细胞肾细胞癌（Clear cell renal cell carcinoma, ccRCC）的大规模测序研究发现，染色质相关基因存在高频率突变。该类突变基因中以SETD2尤为突出，其在15%的ccRCC病例中发生突变，且与疾病的侵袭性密切相关。SETD2是一种负责对组蛋白H3的赖氨酸36进行三甲基化（H3K36me3）的甲基转移酶。尽管目前尚未完全阐明SETD2缺失如何促进ccRCC的肿瘤发生，但普遍认为其会重编程细胞的表观遗传景观。

本研究探究了SETD2/H3K36me3缺失对ccRCC细胞DNA甲基化组的影响。研究采用EPIC DNA甲基化检测技术，对是否敲除SETD2的786-O ccRCC细胞与非癌转化肾近端小管细胞（HKC）进行了DNA甲基化水平检测。通过5-氮杂-2'-脱氧胞苷（5-aza-2'-deoxycytidine）开展剂量反应实验，评估细胞对DNA去甲基化剂的敏感性。采用Annexin-V/PI染色联合流式细胞术检测细胞凋亡情况，通过电子显微镜与流式细胞术评估线粒体功能状态。此外，本研究还在NOD-Scid小鼠的SETD2野生型/敲除异种移植瘤模型中，评估了5-氮杂-2'-脱氧胞苷的抗肿瘤活性。

实验结果显示：SETD2缺失可导致HKC细胞发生DNA高甲基化，且该效应在786-O细胞中更为显著；剂量反应实验证实，SETD2敲除的ccRCC细胞对5-氮杂-2'-脱氧胞苷具有敏感性。Annexin-V/PI染色结果表明，经5-氮杂-2'-脱氧胞苷处理后，SETD2敲除细胞中的凋亡与坏死细胞比例显著升高，且该效应可被半胱天冬酶抑制剂逆转。此外，5-氮杂-2'-脱氧胞苷可诱导SETD2敲除细胞的线粒体发生显著改变，包括膜电位丧失与体积缩小。体内实验进一步证实，SETD2敲除的异种移植瘤对5-氮杂-2'-脱氧胞苷的敏感性显著提升。

本研究证实，ccRCC细胞中的SETD2缺失会引发DNA高甲基化，从而使其产生与DNA去甲基化剂相关的合成致死依赖性。本研究还对经二甲基亚砜（DMSO）或地西他滨（Decitabine，100nM或300nM）处理的HKC与786-O肾细胞进行了RNA测序（RNAseq）分析。