Replication stress response defects predict and enhance immune checkpoint therapy response in triple negative breast cancer

项目摘要 缺乏治疗三阴性乳腺癌(TNBC)的具体目标是一个重大挑战，因为 许多TNBCs对细胞毒性化疗没有反应。免疫检查点封锁(ICB)已屈服 在TNBC的晚期和早期阶段都取得了可喜的结果，预计将大幅改善整体 本病患者的预后。然而，由于TNBC本身不具有免疫原性，因此重要的是 确定哪些患者将从免疫治疗中获益最多，并确定可以启动肿瘤的药物 微环境，提高治疗效果。已知TNBC表现出高水平的复制应激， 当DNA复制机制遇到阻碍复制过程的障碍时，就会发生这种情况。在……里面 在正常细胞中，复制应激激活复制应激反应(RSR)以维持基因组的完整性。 有缺陷的RSR可以让高复制压力的细胞存活和增殖。最近，我们发现了一种 代表RSR(RSRD)缺陷的基因签名。我们发现这个RSRD签名在 TNBC细胞。此外，RSRD-High TNBC细胞积聚细胞质DNA，并诱导刺痛依赖 细胞因子的产生，这是ICB的有效性所必需的。有趣的是，RSRD签名得分 在同基因小鼠模型中，与TNBC对ICB的反应完全相关，并且它准确地预测 ICB在5个低突变负担肿瘤系中的反应。所有这些耐人寻味的发现都支持这些假说 RSRD可能是包括TNBC在内的低突变负担癌症ICB结局的关键决定因素， RSRD增强型药物可能使耐ICB的TNBC对免疫治疗敏感。这些假设将 通过3个具体目标进行测试。(1)确定RSR缺陷的免疫微环境是如何改变的 TNBC。我们将使用高度多元化的成像质量细胞仪面板来确定RSRD如何重塑 TNBC的免疫微环境，并诱导对ICB的敏感性。此外，我们将操纵RSR 以评估RSR缺陷与免疫治疗反应之间的关系。(2)至 确定RSRD的致病驱动因素--TNBC中高介导的ICB反应。我们的初步研究表明 RSR缺陷可能通过免疫刺激胞浆的积聚来驱动免疫治疗反应 单链DNA(SsDNA)。因此，我们将寻求在TNBC模型中操纵胞浆中的单链DNA水平 以确定胞浆单链DNA是否确实是TNBC中ICB反应的致病驱动因素。此外, 为了理解为什么我们的RSRD基因签名可以预测TNBC对ICB的反应，我们将应用体内CRISPR 筛选以确定我们的RSRD基因签名中包含的哪些转录变化导致了这种情况 回应。(3)开发新的联合治疗方法，将RSRD-low TNBC转化为RSRD-High，以改善其疗效 对ICB的回应。使用尖端系统和生物信息学方法，我们已经确定了许多潜在的 RSRD诱导剂。我们将评估6位最有希望的候选人，并确定最佳候选人 能有效增敏RSRD-LOW TNBC的化合物。

Project Summary The lack of specific targets for the treatment of triple-negative breast cancer (TNBC) is a major challenge, as many TNBCs do not respond to cytotoxic chemotherapies. Immune checkpoint blockade (ICB) has yielded promising results in both advanced and early-stage TNBC and is expected to substantially improve the overall prognosis of patients with this disease. However, since TNBC is not inherently immunogenic, it is important to identify patients who would benefit most from immunotherapy and to identify agents that can prime the tumor microenvironment to enhance the therapeutic effects. TNBC is known to exhibit high levels of replication stress, which occurs when the DNA replication machinery encounters obstacles that impede the replication process. In normal cells, replication stress activates the replication stress response (RSR) to maintain genome integrity. Defective RSR allows cells with high replication stress to survive and proliferate. Recently, we have identified a gene signature that represents defects in RSR (RSRD). We found this RSRD signature to be highly enriched in TNBC cells. Furthermore, RSRD-high TNBC cells accumulate cytoplasmic DNA and induce STING-dependent cytokine production, which is required for the effectiveness of ICB. Intriguingly, the RSRD signature score correlates perfectly with the response of TNBC to ICB in syngeneic mouse models, and it accurately predicts ICB response across 5 low–mutation-burden tumor lineages. All these intriguing findings support the hypotheses that RSRD may act as a key determinant of ICB outcomes in low–mutation-burden cancers, including TNBC, and that RSRD-enhancing drugs may sensitize ICB-resistant TNBC to immunotherapy. These hypotheses will be tested via 3 specific aims. (1) To determine how the immune microenvironment is modified in RSR-defective TNBC. We will use a highly multiplexed imaging mass cytometry panel to determine how RSRD remodels the immune microenvironment of TNBC and induces susceptibility to ICB. In addition, we will manipulate the RSR status in TNBC cells to assess the relationship between RSR defects and immunotherapy response. (2) To identify causative drivers of RSRD-high–mediated ICB responsiveness in TNBC. Our preliminary studies suggest that RSR defects may drive immunotherapy response through accumulation of immunostimulatory cytosolic single-stranded DNA (ssDNA). We will, therefore, seek to manipulate the cytosolic ssDNA level in TNBC models to determine whether cytosolic ssDNA is indeed a causative driver of ICB responsiveness in TNBC. In addition, to understand why our RSRD gene signature predicts response to ICB in TNBC, we will apply an in vivo CRISPR screen to determine what transcriptional changes contained within our RSRD gene signature cause this response. (3) To develop novel combination therapy to convert RSRD-low TNBC to RSRD-high to improve their response to ICB. Using cutting-edge systems and bioinformatics approaches, we have identified many potential RSRD-inducing agents. We will assess the 6 most promising candidates and identify the best candidate compound that can effectively sensitize RSRD-low TNBC to ICB.