Dissecting the roles of Runx3 and Mll1 in dysfunctional T cell responses to tumors

项目摘要/摘要： CD8+T细胞分化为细胞毒性T淋巴细胞(CTL)，直接与恶性细胞接触，导致其 通过细胞毒性颗粒的胞吐作用进行裂解。然而，这些对肿瘤的反应背后的监管是病态的- 确定的和通常不成功的对急性病毒感染的反应进行比较。此外，研究还表明， 研究表明，虽然抑制信号阻断可以恢复一些T细胞功能，但它不会导致全球表观遗传 对功能失调的细胞重新编程。然而，澄清这些因素和机制的基础研究 控制CD8T细胞对肿瘤和病毒感染的染色质结构和转录可能是 确定如何实现这一目标。 为了解决这些问题，我感兴趣的是定义转录因子Runx3和染色质调节因子M111是如何 单独和共同作用影响全基因组转录表达、染色质可及性和抑制 三阴性乳腺癌中CTL受体的表达我提供证据证明Runx3和Runx3 MLL1的表达水平可以操纵PD-1的表达，并且该MLL1的表达与Runx3相连。我 假设通过研究这两个基因是如何独立和协同工作的，我可以定义 一个基因调控和染色质可及性网络，编程PD-1的表达，更广泛地说， 更多地了解CTL在非淋巴组织中的遗传结构。为了做到这一点，我提出了两个 具体目的：阐明Runx3如何在抗肿瘤反应中编程CD8+T细胞分化 了解MLL1在确定肿瘤微环境中CTL功能中的作用。 这两个目标都将利用带有淋巴细胞标记的小鼠三阴性乳腺癌模型E0771 脉络膜脑膜炎病毒抗原GP33，以允许在拟议的实验中进行抗原特异性肿瘤靶向。在……里面 目标1，我将确定Runx3发挥作用的关键遗传和表观遗传环境以及它们之间的关系 至PD-1功能。先前的研究表明，Runx3是产生非淋巴组织和肿瘤所必需的 并对PD-1等抑制基因的表达进行编程。通过研究这些特点，我可以 确定顶端转录因子通过什么途径发挥作用，以及它如何影响PD-1的表达。在……里面 目的2，我将研究M111在类似途径中所起的作用。MLL1是通过体外RNAi鉴定的 筛选调节共抑制基因的染色质调节剂。从这个屏幕上看，M111和基因 上调MLL1/2复合体，与抑制PD-1的表达密切相关。通过查看Mll1在 TME中的CTL以及它在可访问性和甲基化方面促进了哪些变化，我可以理解如何 染色质的这些变化决定了肿瘤中CTL的命运。结合了解Runx3和 基因之间的关系，这将为基本遗传结构提供详细的证据 潜在的CTL在肿瘤内的积累和维持。

Project Summary/Abstract: CD8+ T cells differentiate into cytotoxic T lymphocytes (CTLs), to directly engage malignant cells and cause their lysis via exocytosis of cytotoxic granules. However, the regulation underlying these responses to tumors is ill- defined and generally unsuccessful compared responses to acute viral infections. In addition, studies have shown that while inhibitory signal blockade can restore some T cell function, it does not lead to global epigenetic reprogramming of dysfunctional cells. Nevertheless, basic studies to clarify the factors and mechanisms that control chromatin structure and transcription in CD8 T cells reacting to tumors and viral infection are likely to identify how this can be achieved. To address these issues, I am interested in defining how transcription factor Runx3 and chromatin regulator Mll1 work separately and together to effect genome wide transcript expression, chromatin accessibility, and inhibitory receptor expression in CTLs infiltrating triple negative breast cancers. I provide evidence that both Runx3 and Mll1 expression levels can manipulate PD-1 expression and that Mll1 expression is linked to Runx3. I hypothesize that by studying how these two genes work independently and in a coordinated fashion I can define a network of gene regulation and chromatin accessibility that programs PD-1 expression, and more broadly, understand more about the genetic architecture of CTLs infiltrating non-lymphoid tissues. To do, I propose two specific aims: Elucidate how Runx3 programs CD8+ T cell differentiation during anti-tumor responses & Understand the role of Mll1 in determining CTL function in tumor microenvironments. Both aims will utilize a model of murine triple negative breast cancer, E0771, tagged with lymphocytic choriomeningitis virus antigen GP33 to allow for antigen specific tumor targeting in proposed experiments. In Aim 1, I will identify the key genetic and epigenetic landscapes through which Runx3 acts and how this relates to PD-1 function. Previous research has shown that Runx3 is necessary to generate non-lymphoid and tumor residency and also programs the expression of inhibitory genes such as PD-1. By studying these features, I can identify what pathways this apical transcription factor works through and how this effects PD-1 expression. In Aim 2, I will be investigating the role Mll1 plays in similar pathways. Mll1 was identified through an in vitro RNAi screen for chromatin regulators that modulated co-inhibitory genes. From this screen Mll1, and genes that make up the MLL1/2 complex, are heavily implicated in inhibiting PD-1 expression. By looking at how Mll1 functions in CTLs within the TME and what changes it promotes both in accessibility and methylation, I can understand how these changes in chromatin directs CTL fates in tumors. Combined with understanding Runx3 and the relationship between the genes, this will provide detailed evidence into the fundamental genetic architecture underlying CTLs accumulation and maintenance within tumors.