Single Cell Analysis of Sphingosine Kinase Activity in Human Leukemia Stem Cells

描述(申请人提供)：急性髓系白血病(AML)是成人白血病中最致命和最常见的形式。在美国，每年有超过10,000人被诊断为急性髓系白血病。虽然大多数患者在最初的化疗后癌症得到缓解，但大约80%的患者随后复发。只有不到30%的AML患者在复发后存活超过一年。AML的高复发率被归因于一小部分被称为白血病干细胞的细胞。LSCs对多种化疗药物高度耐药，在最初的化疗中存活下来。他们有能力积极地恢复白血病细胞数量。开发一种靶向和杀死LSCs的方法将使AML患者受益匪浅。然而，由于可以从患者身上分离出的数量较少，LSC生物学研究的进展一直受到限制。这项研究将开发一种技术，可以在单细胞水平上研究LSCs的鞘氨醇激酶(SphK)活性。SphK是一种致癌激酶，已知会增加白血病细胞对化疗药物的耐药性。这一提议的中心假设是SphK在LSCs中上调，这将使其成为药物治疗的有希望的靶点。SphK活性将通过在细胞中负载荧光报告程序来测量，该荧光报告程序可以被活性的SphK修饰。单细胞毛细管电泳法(CE)将用于在每个细胞中分离修饰和未修饰的报告。毛细管电泳法是一种极其灵敏的技术，使其成为单个细胞生物分析的理想方法。然而，单细胞CE的一个主要限制是吞吐量低(每天5-35个细胞)。该项目的目标是开发一个自动化的单细胞CE平台，该平台将显著提高吞吐量，使每天能够分析数百到数千个细胞。然后，这个自动化的CE仪器将被用于测量ML患者LSCs中的SphK活性。这项研究将是第一次研究单个患者体内人类LSCs的生物信号通路。确定LSCs中SphK活性的特征有望有助于开发专门针对LSCs的治疗方法，从而防止AML复发。 公共卫生相关性：来自急性髓系白血病(AML)患者的白血病干细胞(LSCs)将表征致癌激酶的活性。我们的目标是确定一种可以靶向降低LSC对化疗药物耐药性的激酶通路，从而防止AML复发。

DESCRIPTION (provided by applicant): Acute myelogenous leukemia (AML) is the deadliest and most common form of adult leukemia. Each year, over 10,000 people in the United States are diagnosed with AML. While most patients experience a remission of the cancer after initial chemotherapy treatments, approximately 80% subsequently relapse. Fewer than 30% of AML patients survive past one year after relapsing. The high rate of AML relapse has been attributed to a small fraction of cells termed leukemia stem cells. LSCs are highly resistant to multiple chemotherapeutic drugs, and survive initial chemotherapy treatments. They are capable of aggressively restoring the leukemia cell population. Developing a method to target and kill LSCs would benefit AML patients tremendously. However, progress in studying LSC biology has been limited because of the low numbers that can be isolated from patients. This research will develop technology that can investigate, on the single cell level; the sphingosine kinase (SphK) activity of LSCs. SphK is an oncogenic kinase that is known to increase resistance to chemotherapeutic drugs in leukemia cells. The central hypothesis of this proposal is that SphK is up regulated in LSCs, which would make it a promising target for drug therapy. SphK activity will be measured by loading cells with a fluorescent reporter, which can be modified by active SphK. Single-cell capillary electrophoresis (CE) will be used to separate the modified from the unmodified reporter in each cell. CE is an extremely sensitive technique, making it ideal for the biological analysis of individual cells. However, a major limitation of single-cell CE is low throughput (5-35 cells per day). The aim of this project is to develop an automated single-cell CE platform that will dramatically improve throughput, enabling hundreds to thousands of cells to be analyzed per day. This automated CE instrument will then be used to measure SphK activity in LSCs from ML patients. This study will be the first to investigate a biological signalng pathway in human LSCs within individual patients. Characterizing SphK activity in LSCs is expected to contribute to the development of therapies that specifically target LSCs and therefore prevent AML relapse. PUBLIC HEALTH RELEVANCE: The activity of an oncogenic kinase will be characterized in leukemia stem cells (LSCs) from patients with acute myeloid leukemia (AML). The goal is to identify a kinase pathway that can be targeted to decrease LSC resistance to chemotherapeutics, and therefore prevent AML relapse.