High PRMT5 levels, maintained by KEAP1 inhibition, drive chemoresistance in ovarian cancer [CRISPR screen]

Protein arginine methyl transferases (PRMTs) are generally upregulated in cancers. However, the mechanisms leading to this upregulation and its biological consequences are poorly understood. Here, we identify PRMT5, the main symmetric arginine methyl transferase, as a critical driver of chemoresistance in high-grade serous ovarian cancer (HGSOC). Cancer transcriptome analysis shows that HGSOC tumors express the highest levels of PRMT5. Furthermore, PRMT5 levels and its enzymatic activity are further induced in a Platinum (Pt)-resistant state at the protein level. To reveal potential regulators of high PRMT5 protein levels, we optimized intracellular immunostaining conditions and performed an unbiased CRISPR screening to identify regulators of PRMT5 levels in OC cells. These results led to several critical regulators, including KEAP1, a top-scoring negative regulator of PRMT5 protein levels. Our mechanistic studies show that KEAP1 directly interacts with PRMT5, leading to its ubiquitin-dependent degradation under normal physiological conditions. However, Pt-treatment-mediated stress leads to KEAP1 inhibition, resulting in elevated levels of PRMT5, sufficient to confer cells resistant to Pt. At the genomic level, our chromatin immunoprecipitation and sequencing (ChIP) studies show that elevated PRMT5 directly interacts with the promoters of stress response genes and positively regulates their transcription. In line with increased PRMT5 levels in Pt-resistant cells, pharmacological inhibition of PRMT5 selectively renders these cells selectively sensitive to PRMT5 inhibition compared to chemo-naive or normal fallopian epithelial cells. As such, combined PRMT5 inhibition with Pt results in synergistic cellular cytotoxicity in vitro and tumor volume reduction in vivo in otherwise Pt-resistant patient-derived xenograft tumors. Overall, the findings from this study identify PRMT5 as a critical therapeutic target in Pt-resistant HGSOC cells and reveal the molecular mechanisms that lead to high PRMT5 levels in Pt-treated and chemo-resistant tumors. Overall design: To identify and characterize potential regulators of PRMT5, we conducted a CRISPR-Cas9-based genetic screen using chemoresistant COV362 cells.

蛋白质精氨酸甲基转移酶（PRMTs）在癌症中普遍上调。然而，导致该上调的分子机制及其生物学效应迄今仍未被充分阐明。本研究鉴定出主要的对称型精氨酸甲基转移酶PRMT5是高级别浆液性卵巢癌（HGSOC）化疗耐药的关键驱动因子。癌症转录组分析显示，HGSOC肿瘤的PRMT5表达水平最高。此外，在铂类（Pt）耐药状态下，PRMT5的蛋白水平及其酶活性会进一步升高。为了揭示PRMT5蛋白高表达的潜在调控因子，我们优化了细胞内免疫染色条件，并在卵巢癌细胞中开展了无偏倚CRISPR筛选，以鉴定PRMT5水平的调控因子。该筛选结果得到多个关键调控因子，其中得分最高的负调控因子包括KEAP1。我们的机制研究显示，KEAP1可直接与PRMT5相互作用，在正常生理条件下介导其泛素依赖性降解。然而，铂类药物处理介导的应激会抑制KEAP1的功能，进而导致PRMT5水平升高，足以使细胞获得铂类耐药性。在基因组层面，我们的染色质免疫共沉淀测序（ChIP）研究显示，高表达的PRMT5可直接结合应激反应基因的启动子，并正向调控其转录。鉴于铂类耐药细胞中PRMT5水平升高，与未接受化疗的细胞或正常输卵管上皮细胞相比，PRMT5的药理学抑制可选择性地使这类细胞对PRMT5抑制产生敏感性。因此，将PRMT5抑制与铂类药物联合使用，可在体外产生协同细胞毒性，并在铂类耐药的患者来源异种移植瘤模型中实现体内肿瘤体积的缩小。综上，本研究的发现确定PRMT5是铂类耐药HGSOC细胞的关键治疗靶点，并揭示了铂类处理及化疗耐药肿瘤中PRMT5高表达的分子机制。总体实验设计：为鉴定并表征PRMT5的潜在调控因子，我们利用铂类耐药的COV362细胞开展了基于CRISPR-Cas9的遗传筛选。