Synthetic Lethal Targeting of SETD2 in Renal Cell Carcinoma

摘要 肾透明细胞癌(CcRCC)约占肾癌的75%，是肾癌的第8大病因。 美国的癌症死亡人数。在癌症基因组图谱(TCGA)项目完成后，临床 几乎在每个实体肿瘤中都发现了可操作的突变。然而，一个主要的例外是RCC， 目前的护理标准、检查点抑制剂和抗血管内皮生长因子治疗没有考虑到这一点 约50%的肾癌存在染色质调节基因突变。一线治疗后，有效率为20%， 目前还没有FDA批准的针对染色质调节器的疗法，这突显了确定如何 功能丧失的基因类型可以作为治疗的靶点。表观基因组在癌症中被严重破坏 包括中国铁路总公司。除了几乎无处不在的vhl的丧失外，ccrcc的突变格局占主导地位。 通过表观遗传调节因子，包括SETD2、BAP1和PBRM1的功能丧失突变。SETD2损失有 现在被认为与不良预后和转移密切相关。H3K36me3基因失控的分子表型 在SETD2-突变体ccRCC中，从合成致死性的角度来看，这是一个理想的场景，因为它 引发必须补偿的全球表观遗传变化，创造独特的脆弱性。瞄准 显示已知癌症突变的遗传上位性驱动合成致命表型的因素是 经过验证的治疗方法。我们进行了无偏见的CRISPR/CAS9筛查，以确定表现出 具有SETD2功能丧失的综合致命性。表观遗传因子NSD1，H3K36me1/2作用的编写者 通过H3K36途径被鉴定。基于这些发现，我们假设抑制 H3K36轴以其表观遗传编写者的形式(SETD2丢失上下文中的NSD1)驱动合成致死 伴随不相容表达缺陷的不同增强子重塑所介导的表型 细胞存活率。NSD1的鉴定，这是一个更大的家族的三个相关蛋白(NSD1/2/3)的一部分 反过来，H3K36信号轴将导致针对SETD2的个体化治疗的新方法 功能丧失，经典定义为“无法下药”。我们将通过三个目标来解决这一假设。在目标1中 我们对H3K36编写者NSD1、NSD2和H3K36的合成致死表型进行了功能表征 NSD3在等基因SETD2 ccRCC细胞系中的表达。在目标2中，我们展示了药物抑制剂的用途。 H3K36信号在SETD2突变细胞合成致死性中的作用及其生物学意义 支撑点。最后，在目标3中，我们验证了遗传和药物靶向的有效性和特异性。 利用小鼠模型研究H3K36信号轴诱导SETD2突变的体内合成致死性。我们的 研究将揭示表观遗传调控因子和H3K36轴如何具体推动癌症和 解除管制时的转移。预计这将对人类健康产生积极的影响， 有证据表明，治疗SETD2突变的ccRCC的新方法将使非靶标副作用最小化，并增强 CcRCC患者的生存，特别是那些有更多侵袭性/转移性疾病的患者。

ABSTRACT Clear cell renal cell carcinoma (ccRCC) accounts for ~75% of kidney cancers and is the 8th leading cause of cancer death in the United States. After completion of The Cancer Genome Atlas (TCGA) Project, clinically actionable mutations were identified in virtually every solid tumor. One major exception, however, is RCC, where the current standard of care, checkpoint inhibitor and anti-VEGF therapy, does not take into account that ~50% of RCCs have mutations in chromatin regulators. After first-line therapy, response rates are 20% and there are no FDA-approved therapies that target chromatin regulators, highlighting the need to identify how loss-of-function genotypes can be therapeutically targeted. The epigenome is profoundly disrupted in cancers including ccRCC. Aside from the near ubiquitous loss of VHL, the mutational landscape of ccRCC is dominated by loss-of-function mutations in epigenetic regulators, including SETD2, BAP1, and PBRM1. SETD2 loss has now been firmly linked to poor outcome and metastasis. The molecular phenotype of H3K36me3 deregulation in SETD2-mutant ccRCC makes this an ideal scenario to study from the angle of synthetic lethality because it induces global epigenetic changes that must be compensated for, creating unique vulnerabilities. Targeting factors that exhibit genetic epistasis with known cancer mutations to drive a synthetic lethal phenotype is a proven therapeutic approach. We performed an unbiased CRISPR/Cas9 screen to identify factors that exhibit synthetic lethality with SETD2 loss-of-function. The epigenetic factor NSD1, a writer of H3K36me1/2 acting through the H3K36 pathway, was identified. Based on these findings, we hypothesize that suppression of the H3K36 axis in the form of its epigenetic writers (NSD1 in a SETD2 loss context) drives a synthetic lethal phenotype mediated by distinct enhancer remodeling accompanied by expression defects incompatible with cell viability. Identification of NSD1, which is part of a larger family of three related proteins (NSD1/2/3) within the H3K36 signaling axis in turn, will lead to novel approaches for individualized therapeutics to target SETD2 loss-of-function, classically defined as ‘undruggable’. We will address this hypothesis with three aims. In aim 1 we functionally characterize the synthetic lethal phenotypes associated with H3K36 writers NSD1, NSD2, and NSD3 in isogenic SETD2 ccRCC cell lines. In aim 2 we demonstrate the utility of pharmacologic inhibitors of H3K36 signaling in driving synthetic lethality in SETD2-mutant cells and elucidate their biological underpinnings. Finally, in aim 3 we validate the efficacy and specificity of genetic and pharmacologic targeting of the H3K36 signaling axis to induce SETD2-mutant synthetic lethality in vivo using mouse models. Our studies will shed new light on how epigenetic regulators and the H3K36 axis specifically, drive cancer and metastasis when deregulated. This is expected to positively affect human health by generating preclinical evidence for new ways to treat SETD2-mutant ccRCC that will minimize off-target side effects, and enhance survival for patients with ccRCC, particularly those with more aggressive/metastatic disease.