Prostate cancer-associated SPOP mutations modulate innate immune response and immune checkpoint therapy

前列腺癌相关的 SPOP 突变调节先天免疫反应和免疫检查点治疗

• 批准号: 10314069
• 负责人: SAMIR M HANASH
• 金额: $ 18.56万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-08 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10314069
• 关键词: Antitumor Response; Binding; Binding Proteins; Biochemical; CD276 gene; Cells; Combined Modality Therapy; Consensus; DNA Damage; DNA Double Strand Break; Development; Double Strand Break Repair; Doxycycline; Family; Foundations; Future; Gene Expression; Gene Family; Genes; Genetic; Genetic Transcription; Genome Stability; Genomics; Human; Immune; In Vitro; Infiltration; Innate Immune Response; Lead; Link; Malignant neoplasm of prostate; Mediating; Methods; Missense Mutation; Molecular; Mus; Mutate; Mutation; Oncogenic; Output; PARP inhibition; Pathway interactions; Phosphorylation; Predictive Value; Prostate Cancer therapy; Protein Family; Proteins; Proteomics; Regulation; Role; Signal Induction; Signal Transduction; Signaling Protein; Stimulator of Interferon Genes; T-Lymphocyte; TBK1 gene; Testing; Therapeutic; Tissues; Tumor Suppressor Proteins; Tumor Tissue; Ubiquitination; anti-PD-L1; base; cell type; checkpoint therapy; cytotoxic; cytotoxicity; differential expression; experimental study; genetic approach; immune checkpoint; immune checkpoint blockade; in vivo; inducible gene expression; inhibitor; member; mutant; mutational status; novel; predictive marker; programmed cell death ligand 1; prostate cancer cell; prostate cancer cell line; prostate cancer model; prostate cancer progression; protein expression; research clinical testing; response; single-cell RNA sequencing; treatment effect; treatment group; treatment response; tumor; tumor-immune system interactions; ubiquitin ligase

PROJECT SUMMARY/ABSTRACT Speckle-type POZ protein (SPOP), a substrate adaptor of the cullin3 (Cul3)-RING ubiquitin ligase (CRL3), demonstrates tumor- and context-specific oncogenic or tumor suppressor functions in various human tumors. The potential role of SPOP in prostate cancer (PCa) gained recognition through the results of multiple genomic studies during recent years that identified heterozygous missense mutations in the SPOP substrate-binding cleft (MATH domain) in up to 15% of PCas. However, the functional significance of PCa-associated SPOP mutants in PCa progression and their impact on current PCa treatment options are largely unknown. Interestingly, SPOP has been shown to regulate genomic stability through modulation of DNA double-strand break repair in primary PCa, and specific SPOP mutations can potentiate the cytotoxic effects of PARP inhibition. We have identified SPOP-binding consensus (SBC) motifs in multiple protein components of the cGAS-STING pathway (cGAS, STING, TBK1), and members of B7 family immune checkpoint proteins (PD-L1, CD276, VISTA). We have also shown that expression of SPOP mutation (F102C) can stabilize STING, TBK1, and PD-L1 and increase STING phosphorylation (p-S366/S365) in human and mouse PCa cells. We hypothesize that the presence of PCa- associated SPOP mutations leads to activation of cGAS-STING signaling and induction of B7 family immune checkpoint proteins including PD-L1, by reducing SPOP substrate ubiquitination. We further hypothesize that the presence of PCa-associated SPOP mutations represents a therapeutic vulnerability that can be exploited by PARP inhibition combined with immune checkpoint blockade targeting of these B7 family proteins (such as anti– PD-L1) to maximize direct intrinsic DNA damage–induced cytotoxicity and cGAS-STING–mediated T-cell anti- tumor response. We will use biochemical methods, genetic approaches [including SPOP wild-type and SPOP mutants (F133V and F102C) doxycycline-inducible expression human and mouse PCa cell lines], and novel syngeneic mouse PCa models to test our hypothesis. Importantly, state-of-the-art proteomics and single cell RNA-seq analyses will be used to determine the molecular pathways and tumor microenvironment immune cell treatment responses altered by SPOP-F133V and SPOP-F102C mutations, to identify novel, relevant SPOP targets (markers) in our PCa models. Overall, the results of these studies will establish PCa-associated SPOP mutations as critical regulators of PARP inhibitor and anti–PD-L1 combination therapies, and define a subset of PCas that respond to this therapeutic approach. More broadly, these studies may lead to the development and clinical testing of specific SPOP mutations as predictive markers for DNA damage response–targeting and immune checkpoint blockade combination therapies for PCa.

项目概要/摘要 斑点型 POZ 蛋白 (SPOP)，cullin3 (Cul3)-RING 泛素连接酶 (CRL3) 的底物接头， 展示了各种人类肿瘤中的肿瘤和背景特异性致癌或肿瘤抑制功能。 SPOP 在前列腺癌 (PCa) 中的潜在作用通过多基因组结果得到认可 近年来的研究发现 SPOP 底物结合裂口存在杂合错义突变 （MATH 结构域）存在于高达 15% 的 PCa 中，然而，PCa 相关 SPOP 突变体的功能意义。 PCa 进展及其对当前 PCa 治疗方案的影响在很大程度上尚不清楚。 已被证明可以通过调节原代DNA双链断裂修复来调节基因组稳定性 我们已经发现 PCa 和特定的 SPOP 突变可以增强 PARP 抑制的细胞毒性作用。 cGAS-STING 通路的多个蛋白质成分中的 SPOP 结合共有 (SBC) 基序（cGAS、 STING、TBK1）以及 B7 家族免疫检查点蛋白的成员（PD-L1、CD276、VISTA）。 研究表明，SPOP 突变 (F102C) 的表达可以稳定 STING、TBK1 和 PD-L1，并增加 STING 我们捕获了人和小鼠 PCa 细胞中 PCa- 的磷酸化 (p-S366/S365)。 相关的 SPOP 突变导致 cGAS-STING 信号传导激活并诱导 B7 家族免疫 通过减少 SPOP 底物泛素化，我们进一步研究了包括 PD-L1 在内的检查点蛋白。 PCa 相关 SPOP 突变的存在代表了一种治疗脆弱性，可以利用 PARP 抑制结合针对这些 B7 家族蛋白（例如抗- PD-L1）以最大限度地提高直接内在 DNA 损伤诱导的细胞毒性和 cGAS-STING 介导的 T 细胞抗 我们将使用生化方法、遗传学方法[包括SPOP野生型和SPOP]。 突变体（F133V 和 F102C）强力霉素诱导表达的人和小鼠 PCa 细胞系]，以及新型 重要的是，我们使用最先进的蛋白质组学和单细胞模型来检验我们的假设。 RNA-seq分析将用于确定分子途径和肿瘤微环境免疫细胞 SPOP-F133V 和 SPOP-F102C 突变改变的治疗反应，以确定新的相关 SPOP 总的来说，这些研究的结果将建立 PCa 相关的 SPOP。 突变作为 PARP 抑制剂和抗 PD-L1 联合疗法的关键调节因子，并定义了一个子集 更广泛地说，这些研究可能会促进对这种治疗方法的反应。 特定 SPOP 突变作为 DNA 损伤反应预测标记的临床测试——靶向和 PCa 的免疫检查点阻断联合疗法。