Ras Pathway Mutations in Human Leukemia

描述(申请人提供)：超过80%的青少年粒单核细胞白血病(JMML)患者存在NF1、RAS或PTPN11基因突变，这些基因编码与RAS信号有关的蛋白质。这一信息以及观察到NF1或PTPN11胚系突变的儿童易患JMML，强烈暗示过度活跃的RAS在JMML的发病机制中发挥核心作用。在这项面向患者的K-22应用中，我建议研究JMML患者队列中的这些突变基因。首先，我将使用突变的RAS和PTPN11作为JMML患者疾病活动性的分子标志物。为此，我开发了一种新的最小残留病检测方法，它基于一种名为TaqMAMA的突变等位基因的荧光放大原理。其次，我将研究JMML患者，他们通过分析在克隆形成试验中生长的单个亚克隆，意外地发现PTPN11和NF1都存在多种突变。我将确定每个细胞中是否存在多个突变，或者是否一些细胞存在NF1杂合性缺失(LOH)，而另一些细胞则存在PTPN11突变。最后，在JMML标本中有相当大的细胞异质性，这使得在可能对白血病生长至关重要的细胞亚群中检测异常信号变得复杂。因此，目标3的假设是，当JMML患者暴露于GM-CSF等细胞因子时，患者骨髓中的细胞亚群具有明显的信号转导网络扰动。我将研究诊断时从JMML患者身上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，这种技术可以研究活细胞亚群中的信号转导。使用这项检测，我将能够识别同时用表面标记和细胞内磷酸蛋白染色的离散细胞群体，从而评估RAS效应器级联的激活状态。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单个患者使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。 这项研究对公众的重要性在于，我们将开发新的测试来跟踪癌症患者的治疗，并了解更多关于癌细胞沟通的方式--这可能导致副作用更少的改进药物以及患者的生存时间延长。

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.