Treatment of Bcr/Abl caused leukemias with FTIs

描述(申请人提供)：尽管急性淋巴细胞性白血病(ALL)的治疗取得了实质性进展，但长期存活率已达到平台期，儿童约为80%，成人约为55%。我们为bcr和abl基因融合引起的ALL的一个亚型建立了转基因小鼠模型，并可以在与成纤维细胞饲养层共培养的情况下处理白血病细胞，以模拟药物治疗和出现耐药的体内环境。Bcr/abl白血病细胞如何与相应的正常非恶性白血病细胞精确地区别是一个持续深入的研究问题。蛋白质糖基化发生的规模很大，但由于很难研究和识别这些通常非常复杂的侧链，因此在很大程度上被忽视了。然而，所有细胞都被紧密的碳水化合物网络覆盖，这些碳水化合物附着在蛋白质、脂类和其他碳水化合物上--所谓的糖原--糖基化的存在、缺失或类型可能对生物功能产生巨大影响。我们的初步数据显示，许多“糖基因”的表达在耐药的bcr/abl淋巴母细胞和非耐药的bcr/abl淋巴母细胞之间以及在非白血病和白血病前B细胞之间存在显著差异。我们推测，bcr/abl淋巴母细胞和耐药的bcr/abl淋巴母细胞分别不同于非白血病正常细胞和药物敏感的相应细胞，其细胞表面的糖基不同，导致黏附相互作用异常。为了测试这一点，我们建议分析我们自己的微阵列数据中的“糖基因”，并验证Clec4d、Galectin-3、St3gal1、St8sia4和HEXA等基因的差异表达，以及从这一分析得出的其他基因。我们还将研究9-O-乙酰化和唾液酸化Lex在bcr/abl转基因小鼠白血病发生过程中的表达，以及在体外与酪氨酸激酶抑制剂尼洛替尼共培养的细胞耐药性演变过程中的表达。最后，我们将通过对野生型、白血病前期和完全白血病BCR/ABL转基因小鼠的AA41+、CD19+细胞以及与基质共培养的白血病细胞在体外诱导耐药之前、期间和之后使用糖链和凝集素阵列，比较正常PRO-B细胞和白血病对应细胞表面表达的糖和凝集素。结果将与糖基因表达相关。这些实验将提供关于BCR/ABL ALL细胞上特异表达的碳水化合物和碳水化合物结合蛋白的类型以及这些在药物治疗过程中是否发生变化的重要信息。除了提供新的诊断工具外，这些实验还可能在一类生物分子中产生非常新的药物治疗靶点，这些生物分子在Ph阳性ALL领域根本没有研究过。 公共卫生相关性：尽管超过80%的成人急性淋巴细胞性白血病患者可以实现完全缓解，但大多数患者会复发。骨髓是正常血细胞发育的地方，也是残留白血病细胞隐藏的地方，但我们对周围细胞如何促进和控制正常血细胞发育的了解还不完全。我们在此建议研究特定血细胞--B细胞系--如何在骨髓中正常发育，以更好地了解白血病细胞如何在耐药治疗中得到这些细胞的帮助

DESCRIPTION (provided by applicant): Despite substantial advances in the treatment of acute lymphoblastic leukemia (ALL), the rate of long-term survival has reached a plateau, at approximately 80 percent for children and 55 percent for adults. We developed a transgenic mouse model for a subtype of ALL caused by a fusion between the BCR and ABL genes and can treat the leukemic cells in co-culture with a fibroblast feeder layer, to model the in vivo circumstances of drug treatment and emerging drug resistance. How BCR/ABL leukemic cells precisely differ from their corresponding normal non-malignant counterparts is a matter of continued intense investigation. Protein glycosylation takes place on a massive scale but has been largely neglected because of the difficulty in studying and identifying these often exceedingly complex side chains. However, all cells are covered by a dense network of carbohydrates that are attached to proteins, lipids and other carbohydrates- the so-called glycocalx- and the presence, absence or type of glycosylation can have a tremendous impact on biological function. Our preliminary data show that expression of many "glyco-genes" differs significantly between drug-resistant BCR/ABL lymphoblasts and non-resistant counterparts, and between non-leukemic and leukemic pro-B cells. We hypothesize, that BCR/ABL lymphoblasts and drug-resistant BCR/ABL lymphoblasts differ from their non-leukemic normal and drug-sensitive counterparts, respectively, in the glycocalyx that covers their cell surface, resulting in abnormalities in adhesive interactions. To test this, we propose to analyze our own microarray data for "glyco-genes" and to validate the differential expression of Clec4d, Galectin-3, St3gal1, St8sia4 and HexA and other genes forthcoming from this analysis. We will also investigate the expression of 9-O-acetylation and of sialyl-Lex during the development of leukemia in BCR/ABL transgenic mice and during the evolution of drug resistance in vitro co-culture of cells treated with the tyrosine kinase inhibitor nilotinib. Finally, we will compare the sugars and lectins expressed on the surface of normal pro-B cells with their leukemic counterparts, by using glycome and lectin arrays on flow-sorted AA41+, CD19+ cells from wild type, preleukemic and fully leukemic BCR/ABL transgenic mice and on leukemic cells co-cultured with stroma before, during and after the induction of drug resistance in vitro. Results will be correlated with glycogene expression. These experiments will provide important information on the types of carbohydrates and carbohydrate-binding proteins that are specifically expressed on BCR/ABL ALL cells and if these change during drug treatment. In addition to providing new tools for diagnosis, these experiments may yield very novel targets for drug treatment within a class of biomolecules that have not been studied at all in the field of Ph-positive ALL. Public Health Relevance: Although over 80% of adults who develop acute lymphoblastic leukemia can achieve a complete remission, the majority of such patients relapse. The bone marrow is the place where the development of normal blood cells takes place and also the location where the residual leukemia cells hide, but our understanding of how the surrounding cells promote and control the development of normal blood cell development is incomplete. We here propose to investigate how the normal development of specific blood cells -the B-lineage- occurs in the bone marrow to better understand how leukemia cells are helped by these cells in withstanding drug treatment