Balanced signaling cues to guide cell transitions in the blood lineage continuum

描述（由申请人提供）：血细胞需要以平衡的方式自我更新，增殖和分化，以使自我维持的血液系统（如免疫系统）成为可能。调节这种平衡的生化信号网络是复杂的，非直觉的，并且不被很好地理解。更复杂的是，血细胞以不同的谱系存在，每个谱系中都有罕见但关键的亚群。传统的FACS分析与有限的细胞表面标记面板产生了限制子集的错误概念，在谱系轨迹中突然转变。这种有限的子集分类（以及这里的信号分析）阻碍了对调节细胞增殖和分化平衡的生化网络功能的充分理解。我们最近首创的单细胞大量细胞术（CyTOF）方法打破了这一僵局，并揭示了骨髓中的造血是一个连续体，具有超过100个可识别的亚群。众所周知，异常的生化网络可以形成癌症、自身免疫性疾病或免疫缺陷等人类疾病的基础。我们探索Ras信号传导拓扑的确定性和随机计算模型预测了Ras激活蛋白Rasgrp和Sos的不同激活模式。为了验证这些假设，我们发现类似的Rasgrp1- Ras-ERK或双峰Sos-Ras-ERK信号可以在淋巴细胞中发生。我们的新小鼠模型现在表明，Rasgrp1的不同扰动导致Ras信号的重塑，并导致癌症、自身免疫性疾病或免疫缺陷。在这里，我们假设血细胞以一种平衡的方式通过连续体发育，作为Ras信号网络的拓扑结构和特征的功能。在初步结果中，我们讨论了Ras信号的常微分方程（ODE）和随机模拟编译（SSC）计算模型，详细介绍了我们的CyTOF数据收集和计算SPADE和ACCENSE分析方法，以及我们对淋巴细胞亚群的生化磷流分析。我们还提出了几条证据，表明Ras激活子Rasgrp1塑造了Ras网络的特征，以平衡增殖和分化。缺失Rasgrp1导致免疫缺陷。我们提供了2013年发表的关于由致癌Ras突变或Ras激活子Rasgrp1过表达引起的T细胞白血病以及在具有点突变Rasgrp1Anaef等位基因的小鼠模型中狼疮样自身免疫表型的数据。在这个提议中，我们将结合计算假设生成，高维细胞tof数据的高分辨率分析方法，以及来自具有不同Ras信号的小鼠模型和人类白血病样本的原代血细胞的高通量生化分析，以了解在骨髓（Aim 1）和胸腺（Aim 2）正常连续体中正常过渡的T淋巴细胞中的Ras信号网络的拓扑结构。我们还将描述网络特性的扰动如何导致免疫缺陷、自身免疫或T细胞白血病。使用三个学科之间的重复循环，我们在这里专注于T细胞谱系，以确保一个富有成效的研究计划，但也将产生与所有造血血液谱系相关的新见解，以刺激未来的研究。

DESCRIPTION (provided by applicant): Blood cells need to self-renew, proliferate, and differentiate in a balanced fashion to enable self-sustaining blood systems such as the immune system. The biochemical signaling network that regulates this balance is complex, non-intuitive, and not well understood. Adding to the complexity, blood cells exist as diverse lineages with rare, but critical, subsets within each lineage. Traditional FACS analyses with limited cell surface marker panels have created the false notion of restricted subsets, with abrupt transitions in a lineage trajectory. Such limited subset classification (and analyses of signals herein) has obstructed a full understanding of the function of biochemical networks that regulate the cellular balance between proliferation and differentiation. Our recently pioneered single-cell mass cytometry (CyTOF) method broke this impasse and has revealed that hematopoiesis in the bone marrow is a continuum with over a hundred identifiable subsets. It is known that aberrant biochemical networks can form the basis for human diseases like cancer, autoimmune diseases, or immunodeficiency Our deterministic and stochastic computational models that explored the topology of Ras signaling predicted distinct patterns of Ras activation as a function of the Ras activator proteins Rasgrp and Sos. Testing these hypotheses, we found that analog Rasgrp1- Ras-ERK or bimodal Sos-Ras-ERK signals can occur in lymphocytes. Our new mouse models now indicate that different perturbation in Rasgrp1 lead to reshaping of the Ras signals and result in cancer, autoimmune diseases, or immunodeficiency. Here we hypothesize that blood cells develop through a continuum in a balanced manner as a function of the topology and character of the Ras signaling network. In Preliminary Results, we discuss our ordinary differential equation (ODE) and Stochastic simulation compile (SSC) computational models of Ras signaling, details of our CyTOF data collection and computational SPADE and ACCENSE analysis methods, as well as our biochemical phospho-flow analyses on defined subsets of lymphocytes. We also present several lines of evidence that the Ras activator Rasgrp1 shapes the character of the Ras network to balance proliferation and differentiation. Loss of Rasgrp1 leads to immunodeficiency. We present data from our recent 2013 publications on T cell leukemia caused by oncogenic Ras mutations or overexpression of the Ras activator Rasgrp1 as well as a lupus-like autoimmune phenotype in a mouse model with a point-mutated Rasgrp1Anaef allele. In this proposal we will combine computational hypothesis generation, high-resolution analytic approaches of high-dimensional CyTOF data, and high-throughput biochemical analyses of primary blood cells from mouse models with distinct Ras signals and human leukemia samples to understand the topology of the Ras signaling network in T lymphocytes properly transitioning through the normal continuum in the bone marrow (Aim 1) and thymus (Aim 2). We will also characterize how perturbations of the network's character can lead to immunodeficiency, autoimmunity, or T cell leukemia. Using reiterative loops between the three disciplines, we focus on the T cell lineage here to ensure a productive research plan but will also generate new insights relevant for all hematopoietic blood lineages to spur future investigations.