Altered Glycolytic Pathway in in vitro Models of leukemia developed in hypoxia

描述(申请人提供)：大多数癌症患者糖酵解水平升高，即使在轻度有氧条件下也是如此。这种偏向有氧糖酵解的现象，被称为沃堡效应，是癌症的一个标志，它调节着包括药物敏感性在内的各种细胞属性。许多研究表明，调节糖酵解的主要途径是PI3K/Akt/mTor途径。我们假设，在生理性低氧条件下建立的癌细胞系可以更准确地模拟体内的白血病，而针对PI3K/Akt/mTOR通路的药物在生理性氧气中的体外研究将更接近体内的结果。我们的目标是1)证明在不同氧气条件下建立的急性淋巴细胞白血病(ALL)中，糖酵解途径基因受到氧气条件的影响，因此，在低氧条件下建立的细胞系可以看到更多的葡萄糖消耗和更多的乳酸产生；以及2)利用在低氧和直接异种移植中建立的细胞系，我们将评估针对糖酵解途径的药物。通过比较新的儿童急性淋巴细胞白血病细胞株在大气(20%O2)和生理骨髓(5%O2)氧浓度下的差异，我们将证明大气氧压抑制糖酵解相关基因的表达、葡萄糖消耗、乳酸产生、细胞增殖，并改变DUG敏感性。生理性和非生理性(即“标准”培养条件)氧浓度对糖酵解的影响不能通过将细胞培养从一种氧气条件切换到另一种条件而轻易逆转，这表明需要在生理氧气条件下建立细胞系，以便为研究癌细胞代谢提供准确的体外模型。我们的初步观察表明，在白血病中保存Warburg效应需要从细胞最初建立起就在生理氧浓度下培养细胞。通过建立和评估两种不同氧气条件下的大量白血病细胞系，以及作为免疫低下小鼠的直接异种移植的比较，我们将提供对体外肿瘤模型中代谢网络培养条件的重要性的理解。此外，我们将通过评估新的免疫抑制较少的PI3K抑制剂与目前治疗ALL的方法(如地塞米松和另一种代谢抑制剂L-天冬酰胺酶)来评估糖酵解途径是否能为ALL的治疗提供可行的靶点。该项目还将通过COG信息库(www.COGcell.org)向其他研究人员提供一套独特的、经过充分表征和验证的实验室模型，用于研究癌症生物学和临床前治疗。 公共卫生相关性：淋巴系恶性肿瘤是美国儿童和青少年最常见的癌症，其中最常见的是急性淋巴细胞性白血病(ALL)，其发病率在过去25年中逐渐上升。在儿童初级治疗方面的重大改进都导致了总体治愈率约为80%。然而，在20%复发的患者中，大多数人死亡。我们建议的最终目标是确定白血病细胞是如何产生能量来生长和增殖的，并找到可以攻击白血病细胞能量产生机制的药物。

DESCRIPTION (provided by applicant): The majority of cancers employ an elevated level of glycolysis, even under mildly aerobic conditions. This bias towards aerobic glycolysis, known as the Warburg effect, is a hallmark of cancer that regulates various cellular attributes including drug sensitivity. Many studies show that the major pathway regulating glycolysis is PI3K/Akt/mTOR pathway. We hypothesize that cancer cell lines established in physiological hypoxia can more accurately mimic leukemia in vivo, and that in vitro studies in physiological O2 of agents targeting the PI3K/Akt/mTOR Pathway in ALL will more closely resemble in vivo results. Our goals are 1) to show that the glycolytic pathway genes are affected by oxygen conditions in acute lymphoblastic leukemia (ALL) established in different oxygen conditions and therefore, the more glucose consumption and more lactate production are seen in cell lines established in hypoxia; and 2) using the cell lines established in hypoxia and direct xenografts we will evaluate drugs targeting the glycolytic pathway. By comparing new childhood acute lymphoblastic leukemia cell lines in atmospheric (20% O2) and physiologic bone marrow (5% O2) oxygen concentrations, we will demonstrate that atmospheric oxygen tension inhibits glycolysis-related gene expression, glucose consumption, lactate production, cell proliferation, and alters dug sensitivities. The effects of physiological and non-physiological (i.e. "standard" culture conditions) oxygen concentrations on glycolysis are not readily reversible by switching cell cultures from one oxygen condition to another, indicating the need to establish cell lines in physiological oxygen conditions in order to provide accurate in vitro models for studying cancer cell metabolism. Our preliminary observations suggest that preservation of the Warburg effect in leukemia requires culturing cells at physiologic oxygen concentrations from the time of their initial establishment. By establishing and evaluating a large panel of leukemia cell lines in two different oxygen conditions, and also for comparison as direct xenografts in immunocompromised mice, we will provide an understanding on the importance of culture conditions on metabolic networks in cancer in vitro models. In addition, we will evaluate whether glycolytic pathway could provide feasible targets for ALL treatment by evaluating new PI3K inhibitor with less immunosuppression in combination with current therapy of ALL (e.g. dexamethasone and L-asparaginase, another metabolism inhibitor). This project will also make available for other investigators via the COG repository (www.COGcell.org) a unique set of well characterized and validated laboratory models for studying cancer biology and preclinical therapeutics. PUBLIC HEALTH RELEVANCE: Lymphoid malignancies are the most common cancer in children and adolescents in the United States, and the most prevalent of these is acute lymphoblastic leukemia (ALL) with the incidence being gradually increasing over the last 25 years. Significant improvements in primary therapy for childhood ALL have led to an overall cure rate of approximately 80 %. However, of the 20% of patients who relapse, the majority die. The ultimate goals of our proposal are to identify how leukemia cells make energy to grow and proliferate, and to find drugs that can attack the energy generating mechanisms of leukemia cells.