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。调节这种平衡的生化信号网络是复杂的，非直观的，也不是很清楚。更复杂的是，血细胞作为不同的谱系存在，每个谱系中有罕见但关键的子集。传统的流式细胞仪分析有限的细胞表面标志物面板已经创建了一个错误的概念，限制子集，突然过渡的谱系轨迹。这种有限的子集分类（和本文中的信号分析）阻碍了对调节增殖和分化之间的细胞平衡的生化网络的功能的充分理解。我们最近开创的单细胞质谱仪（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，有100多个可识别的子集。我们的确定性和随机计算模型探索了Ras信号传导的拓扑结构，预测了Ras激活的不同模式，作为Ras激活蛋白Rasgrp和Sos的功能。通过对这些假设的检验，我们发现淋巴细胞中可以出现类似的Rasgrp 1- Ras-ERK或双峰的Sos-Ras-ERK信号。我们的新小鼠模型现在表明，Rasgrp 1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，血细胞通过一个连续的发展在一个平衡的方式作为一个功能的拓扑结构和性质的Ras信号网络。 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，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酸流分析。我们还提出了几条证据，表明Ras激活剂Rasgrp 1塑造了Ras网络的特征，以平衡增殖和分化。Rasgrp 1的缺失导致免疫缺陷。我们目前的数据来自我们最近的2013年出版物的致癌Ras突变或Ras激活剂Rasgrp 1的过表达引起的T细胞白血病，以及一个点突变的Rasgrp 1Anaef等位基因的小鼠模型中的狼疮样自身免疫表型。 在这项提案中，我们将结合联合收割机计算假设生成，高分辨率的分析方法，高维度的CyTOF数据，高通量的生化分析，从小鼠模型与不同的Ras信号和人类白血病样本的原代血细胞，以了解拓扑结构的Ras信号网络在T淋巴细胞适当过渡通过正常的连续体在骨髓（目标1）和胸腺（目标2）。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循环，我们在这里专注于T细胞谱系，以确保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.