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

项目摘要/摘要 斑点型POZ蛋白(SPOP)是culin3(CUL3)-环泛素连接酶(CRL3)的底物接头， 在各种人类肿瘤中显示肿瘤和上下文特定的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个基因组的结果得到了认可 近年来发现SPOP底物结合裂隙杂合性错义突变的研究 (数学领域)高达15%的PCAS。然而，与PCA相关的SPOP突变体的功能意义 PCa的进展及其对当前PCa治疗方案的影响在很大程度上是未知的。有趣的是，SPOP 已被证明通过调节初级DNA双链断裂修复来调节基因组的稳定性 PARP的特异性突变可增强PARP抑制的细胞毒作用。我们已经确定了 CGAS-Sting途径(cGAS， STING、TBK1)和B7家族免疫检查点蛋白(PD-L1、CD276、Vista)。我们还有 结果表明，SPOP突变(F102C)的表达可以稳定STING、TBK1和PD-L1，并增加STING 人和小鼠前列腺癌细胞中的磷酸化(p-S366/S365)。我们假设PCA的存在- 相关SPOP突变导致cGAS-STING信号激活并诱导B7家族免疫 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突变的存在代表了一种治疗漏洞，可以被 PARP抑制结合免疫检查点阻断靶向这些B7家族蛋白(如抗 PD-L1)最大限度地提高直接内源性DNA损伤诱导的细胞毒作用和cGAS-STING介导的T细胞抗 肿瘤反应。我们将使用生化方法，遗传方法[包括SPOP野生型和SPOP 突变体(F133V和F102C)多西环素可诱导表达的人和小鼠前列腺癌细胞系]和新的 同基因小鼠PCA模型来验证我们的假设。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，以识别新的、相关的SPOP 我们的主成分分析模型中的目标(标记)。总体而言，这些研究的结果将建立与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PCAS。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损伤反应靶向和DNA损伤预测标志的临床试验 免疫检查点阻断联合疗法治疗前列腺癌。

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.