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，以实现自我维持的血液系统，如免疫系统。调节这种平衡的生化信号网络是复杂的、非直觉的，也没有被很好地理解。增加复杂性的是，血细胞以不同的谱系存在，每个谱系中有罕见但关键的亚群。传统的FACS分析使用有限的细胞表面标记板，创造了限制子集的错误概念，并在谱系轨迹中发生突变。这种有限的子集分类(以及这里对信号的分析)阻碍了对调节细胞增殖和分化之间平衡的生化网络功能的充分理解。我们最近开创的单细胞质量细胞术(CyTOF)方法打破了这一僵局，揭示了骨髓中的造血是一个由100多个可识别亚群组成的连续体。众所周知，异常的生化网络可以形成人类疾病的基础，如癌症、自身免疫性疾病或免疫缺陷。我们探索RAS信号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。检验这些假说，我们发现在淋巴细胞中可以出现类似的RASGRP1-RAS-ERK或双峰SOS-RAS-ERK信号。我们的新小鼠模型现在表明，RASGRP1中的不同扰动会导致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，我们假设血细胞以平衡的方式通过连续体发育，这是RAS信号网络的拓扑和特征的函数。在初步结果中，我们讨论了我们的RAS信号的常微分方程式(ODE)和随机模拟编译(SSC)计算模型，我们的CyTOF数据收集和计算SPADE和ACCENSE分析方法的细节，以及我们对定义的淋巴细胞亚群的生化磷流动分析。我们还提出了几条证据表明，RAS激活剂RASGRP1塑造了RAS网络的特征，以平衡增殖和分化。RASGRP1的缺失会导致免疫缺陷。我们提供了2013年发表的关于T细胞白血病的数据，这些数据是由致癌的RAS突变或RAS激活剂RASGRP1的过度表达以及带有点突变的Rasgrp1Anaef等位基因的小鼠模型中的狼疮样自身免疫表型引起的。在这项提议中，我们将结合计算假设生成、高维CyTOF数据的高分辨率分析方法以及对具有不同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T淋巴细胞中RAS信号网络的拓扑结构，该网络通过骨髓(目标1)和胸腺(目标2)的正常连续体进行正确转换。我们还将描述网络特征的扰动如何导致免疫缺陷、自身免疫或T细胞白血病。利用这三个学科之间的重复循环，我们将重点放在T细胞谱系上，以确保一个富有成效的研究计划，同时也将产生与所有造血血统相关的新见解，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.