Ras Pathway Mutations in Human Leukemia

描述（由申请人提供）：超过80%的青少年髓细胞白血病（JMML）患者在NF1、RAS或PTPN11基因中发生突变，这些基因编码参与RAS信号传导的蛋白质。这一信息以及NF1或PTPN11种系突变的儿童易患JMML的观察结果强烈暗示，过度活跃的RAS在JMML的发病机制中起着核心作用。在这个以患者为导向的K-22应用中，我建议在具有良好特征的JMML患者队列中研究这些突变基因。首先，我将使用突变体RAS和PTPN11作为JMML患者疾病活动性的分子标记。为此，我开发了一种基于荧光扩增突变等位基因TaqMAMA原理的新型最小残留疾病检测方法。其次，我将通过分析在集落形成试验中生长的单个亚克隆，研究那些在PTPN11和NF1中发现了令人惊讶的多种突变的JMML患者。我将确定每个细胞中是否存在多个突变，或者是否一些细胞携带NF1杂合性缺失（LOH），而其他细胞携带PTPN11突变。最后，在JMML标本中存在相当大的细胞异质性，这使得检测可能对白血病生长至关重要的细胞亚群中的异常信号变得复杂。因此，目的3的假设是，当暴露于细胞因子（如GM-CSF）时，JMML患者骨髓中的细胞亚群明显干扰信号转导网络。我将研究在诊断时从JMML患者获得的现有骨髓样本，使用一种新的多参数流式细胞术技术，该技术允许研究活细胞亚群中的信号转导。使用该分析，我将能够识别同时被表面标记物和细胞内磷酸化蛋白染色的离散细胞群，从而对Ras效应级联的激活状态进行评估。这项技术的长期影响可能是显著的，因为它可能允许研究人员对个体患者施用特定的信号分子抑制剂，如Akt、MEK或mTOR，并对相关原代细胞群体中的生化目标进行实时监测。

DESCRIPTION (provided by applicant): More than 80% of patients with juvenile myelomonocytic leukemia (JMML) have a mutation in the NF1, RAS, or PTPN11 genes, which encode proteins that are involved in RAS signaling. This information and the observation that children with germline mutations in either NF1 or PTPN11 are predisposed to JMML, strongly implicates hyperactive RAS as playing a central role in the pathogenesis of JMML. In this patient oriented K-22 application, I propose studying these mutated genes in well-characterized cohorts of patients with JMML. First, I will use mutant RAS and PTPN11 as molecular markers of disease activity in patients with JMML. Toward this end, I have developed a novel minimal residual disease assay based on the principles of fluorescence-based amplification of a mutant allele called TaqMAMA. Second, I will study JMML patients who have identified to surprisingly harbor multiple mutations in both PTPN11 and NF1 by analyzing individual subclones that are grown in colony-forming assays. I will determine if multiple mutations are present in each cell or if some cells harbor loss of heterozygosity (LOH) of NF1 while others harbor PTPN11 mutations. Finally, there is considerable cellular heterogeneity in JMML specimens, which complicates detecting aberrant signaling in subpopulations of cells that may be essential for leukemic growth. The hypothesis for aim 3, therefore, is that subsets of cells in the bone marrow of patients with JMML have distinctly perturbed signal transduction networks when exposed to cytokines, such as GM-CSF. I will study existing bone marrow samples obtained from JMML patients at diagnosis, using a novel technique of multi-parameter flow cytometry that permits investigations of signal transduction in subsets of living cells. Using this assay, I will be able to identify discrete populations of cells simultaneously stained with surface markers as well as intracellular phosphoproteins, thereby yielding an assessment of the activation status of Ras effector cascades. The long-term impact of this technology is potentially remarkable, as it might allow investigators to administer a specific inhibitor of a signaling molecule such as Akt, MEK, or mTOR to an individual patient, and perform real-time monitoring of the biochemical target in relevant populations of primary cells. The importance of this research to the general public is that we will develop new tests to follow patients with cancer on therapy, as well as to learn more about the ways that cancer cells communicate -- this may lead to improved medicines with fewer side effects as well as increased survival for patients.