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.

项目总结/摘要 斑点型POZ蛋白（SPOP），cullin 3（Cul 3）-RING泛素连接酶（CRL 3）的底物衔接子， 在各种人类肿瘤中显示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研究的结果得到了认可。 近年来的研究发现SPOP底物结合裂隙中存在杂合错义突变 (MATH在15%的PCAs中。然而，PCa相关SPOP突变体的功能意义 PCa进展及其对当前PCa治疗选择的影响在很大程度上是未知的。有趣的是， 已显示通过调节原发性肿瘤中DNA双链断裂修复来调节基因组稳定性， PCa和特异性SPOP突变可增强PARP抑制的细胞毒性作用。我们已经确定 cGAS-STING途径的多个蛋白质组分中的SPOP结合共有（SBC）基序（cGAS， STING、TBK 1）和B7家族免疫检查点蛋白的成员（PD-L1、CD 276、VISTA）。我们还 显示SPOP突变（F102 C）表达可以稳定STING、TBK 1和PD-L1并增加STING 在人和小鼠PCa细胞中的磷酸化（p-S366/S365）。我们假设前列腺癌的存在- 相关的SPOP突变导致cGAS-STING信号传导的激活和B7家族免疫的诱导。 检查点蛋白，包括PD-L1，通过减少SPOP底物泛素化。我们进一步假设， PCa相关SPOP突变的存在代表了治疗的脆弱性， PARP抑制与靶向这些B7家族蛋白（如抗- PD-L1）以最大化直接内在DNA损伤诱导的细胞毒性和cGAS-STING介导的T细胞抗PD-L1 肿瘤反应我们将使用生物化学方法、遗传学方法[包括SPOP野生型和SPOP 突变体（F133 V和F102 C）多西环素诱导表达的人和小鼠PCa细胞系]，和新的 同源小鼠PCa模型来检验我们的假设。重要的是，最先进的蛋白质组学和单细胞 RNA-seq分析将用于确定免疫细胞的分子途径和肿瘤微环境。 SPOP-F133 V和SPOP-F102 C突变改变的治疗反应，以确定新的相关SPOP 我们的PCa模型中的目标（标记）。总体而言，这些研究的结果将确定PCA相关的SPOP 突变作为PARP抑制剂和抗PD-L1联合治疗的关键调节因子，并定义了 对这种治疗方法有反应的前列腺癌。更广泛地说，这些研究可能会导致发展和 特异性SPOP突变作为DNA损伤反应靶向的预测标志物的临床试验， 免疫检查点阻断联合治疗前列腺癌。