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

项目概要 缺乏治疗三阴性乳腺癌（TNBC）的具体靶点是一个重大挑战，因为 许多 TNBC 对细胞毒性化疗没有反应。免疫检查点封锁（ICB）已产生 在晚期和早期 TNBC 中都取得了有希望的结果，预计将大大改善整体 患有这种疾病的患者的预后。然而，由于 TNBC 本身不具有免疫原性，因此重要的是 确定最能从免疫治疗中受益的患者，并确定可以启动肿瘤的药物 微环境，增强治疗效果。众所周知，TNBC 表现出高水平的复制压力， 当DNA复制机器遇到阻碍复制过程的障碍时就会发生这种情况。在 正常细胞中，复制应激会激活复制应激反应（RSR）以维持基因组完整性。 有缺陷的 RSR 允许具有高复制压力的细胞存活和增殖。最近，我们发现了一个 代表 RSR (RSRD) 缺陷的基因特征。我们发现这个 RSRD 签名高度丰富 TNBC 细胞。此外，RSRD 高的 TNBC 细胞积累细胞质 DNA 并诱导 STING 依赖性 细胞因子的产生，这是 ICB 有效性所必需的。有趣的是，RSRD 签名分数 与同基因小鼠模型中 TNBC 对 ICB 的反应完美相关，并且可以准确预测 ICB 对 5 个低突变负荷肿瘤谱系的反应。所有这些有趣的发现都支持这些假设 RSRD 可能是低突变负担癌症（包括 TNBC）中 ICB 结果的关键决定因素， RSRD 增强药物可能会使 ICB 耐药的 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。我们将使用高度多重成像质谱流式细胞术面板来确定 RSRD 如何重塑 TNBC 的免疫微环境并诱导对 ICB 的易感性。此外，我们将操纵 RSR TNBC 细胞的状态，以评估 RSR 缺陷与免疫治疗反应之间的关系。 (2) 至 确定 TNBC 中 RSRD 高介导的 ICB 反应性的驱动因素。我们的初步研究表明 RSR 缺陷可能通过免疫刺激性胞质的积累来驱动免疫治疗反应 单链DNA（ssDNA）。因此，我们将寻求操纵 TNBC 模型中的胞质 ssDNA 水平 以确定胞浆 ssDNA 是否确实是 TNBC 中 ICB 反应性的驱动因素。此外， 为了理解为什么我们的 RSRD 基因特征可以预测 TNBC 对 ICB 的反应，我们将应用体内 CRISPR 筛选以确定我们的 RSRD 基因特征中包含的哪些转录变化导致了这种情况 回应。 (3) 开发新的联合疗法，将 RSRD-low 的 TNBC 转化为 RSRD-high 的 TNBC，以改善其 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 位最有前途的候选人并确定最佳候选人 可以有效地使 RSRD 低的 TNBC 对 ICB 敏感的化合物。

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.