Small-molecule exploitation of ZBP1-driven nuclear necroptosis for cancer immunotherapy

项目总结/摘要。 免疫检查点阻断（ICB）和其他免疫疗法已经彻底改变了癌症治疗，但 大多数癌症对基于ICB的单一疗法的无反应性仍然是一个重要的问题。一个主要原因 因为这些所谓的“冷”肿瘤的无反应性在于它们缺乏免疫原性肿瘤 因此，尽管表达ICB靶标，T细胞仍然可以在微环境（TME）中存活并因此逃脱T细胞杀伤。如何选择性 增强TME的免疫原性一直是一个未解决的挑战。在这里，我们提出了一种新的小分子 这是一种激活坏死性凋亡并在TME中触发强大先天免疫反应的方法。这条新的大道 源自我们对甲型流感病毒（IAV）的研究。我们的早期研究结果表明，IAV激活坏死性凋亡， 被感染的细胞坏死性凋亡杀死受感染的细胞并且具有高度免疫原性。当病毒RNA激活 宿主蛋白ZBP 1。最近，与癌症免疫治疗高度相关的是，我们发现ZBP 1激活了 细胞核坏死性凋亡这种“核坏死性凋亡”比常规的免疫原性更强。 （细胞质引发的）坏死性凋亡，因为它使细胞核破裂并释放出高度炎性的核 DAMPs进入细胞外空间。我们还发现激活ZBP 1的病毒RNA是Z-RNA。虽然 这些独特的ZBP 1激活剂应该是ICB的极好佐剂，Z-RNA不稳定并且难以在缺乏ZBP 1的情况下产生。 病毒感染。然而，Z-DNA在结构上几乎与Z-RNA相同，以相同的亲和力结合ZBP 1， 可以通过将DNA扭曲成Z-构象而在真核细胞中稳定产生。这表明， 一种可以在细胞中产生Z-DNA的化合物会激活ZBP 1并触发按需核坏死性凋亡 而不需要病毒感染。这样的化合物将填补对坏死性凋亡激动剂的长期未满足的需求， 用于癌症免疫治疗。我们现在已经发现了一种小分子，curaxin，它可以诱导Z-DNA ZBP 1在活细胞中形成，并直接激活ZBP 1以触发TME细胞中的“按需”核坏死性凋亡。 这些和其他发现使我们能够提出假设，curaxin改变染色质结构，并诱导 Z-DNA的形成;这种Z-DNA将ZBP 1募集到细胞核并触发细胞核坏死性凋亡;以及 Curaxin诱导的核坏死性凋亡将大大改善ICB治疗结果。在这份提案中，我们将要求 curaxin如何触发Z-DNA形成（Aim 1），Z-DNA如何激活ZBP 1和核坏死性凋亡（Aim 2），以及 Curaxin诱导的核坏死性凋亡是否与ICB在临床相关性 黑色素瘤小鼠模型（Aim 3）。这些目标的成功完成将勾勒出一个全新的小- 基于分子的策略，以激活高度炎性形式的坏死性凋亡并增强基于ICB的 免疫疗法，具有重要的临床意义。

PROJECT SUMMARY/ABSTRACT. Immune checkpoint blockade (ICB) and other immunotherapies have revolutionized cancer treatment, but the non-responsiveness of most cancers to ICB-based monotherapy remains a significant problem. A major reason for the non-responsiveness of these so-called ‘cold’ tumors is that they lack an immunogenic tumor microenvironment (TME) and thus escape T-cell killing despite expressing ICB targets. How to selectively intensify the immunogenicity of the TME has been an unmet challenge. Here we propose a new small-molecule approach that activates necroptosis and triggers robust innate immune responses in the TME. This new avenue derives from our work on influenza A virus (IAV). Our early findings showed that IAV activates necroptosis in infected cells. Necroptosis kills infected cells and is highly immunogenic. It is initiated when viral RNAs activating the host protein ZBP1. Recently, and highly relevant for cancer immunotherapy, we found that ZBP1 activates necroptosis from the nucleus. Such ‘nuclear necroptosis’ is significantly more immunogenic than conventional (cytoplasm-initiated) necroptosis because it ruptures the nucleus and releases hyper-inflammatory nuclear DAMPs into the extracellular space. We also found that the viral RNAs that activate ZBP1 are Z-RNAs. Although these unique ZBP1 activators should be superb adjuvants for ICB, Z-RNA is unstable and hard to produce absent virus infection. Z-DNA, however, is structurally almost identical to Z-RNA, binds ZBP1 with the same affinity, and can be stably produced in eukaryotic cells by distorting DNA into the Z-conformation. This suggested that a compound that can generate Z-DNA in cells would activate ZBP1 and trigger on-demand nuclear necroptosis without need for virus infection. Such a compound would fill the long-unmet need for a necroptosis agonist for use in cancer immunotherapy. We have now identified a small molecule, curaxin, which induces Z-DNA formation in live cells and directly activates ZBP1 to trigger ‘on-demand’ nuclear necroptosis in cells of the TME. These and other findings allow us to propose the hypotheses that curaxin alters chromatin structure and induces the formation of Z-DNA; that such Z-DNA recruits ZBP1 to the nucleus and triggers nuclear necroptosis; and that curaxin-induced nuclear necroptosis will greatly improve ICB treatment outcomes. In this proposal, we will ask how curaxin triggers Z-DNA formation (Aim 1), how Z-DNA activates ZBP1 and nuclear necroptosis (Aim 2), and whether induction of nuclear necroptosis by curaxin has combinatorial benefit with ICB in clinically-relevant mouse models of melanoma (Aim 3). The successful completion of these Aims will outline an entirely new small- molecule based strategy to activate a highly inflammatory form of necroptosis and potentiate ICB-based immunotherapies, with important clinical ramifications.