"BCR/ABL-PI-3k-ROS pathway induce genomic instability ...."

描述（由申请人提供）：染色体易位t（9;22）导致编码BCR/ABL融合酪氨酸激酶的癌基因出现，该基因可诱导慢性髓性白血病（CML）和急性淋巴细胞白血病（ALL）。CML通常以相对良性的慢性期（CML- cp）开始，然后发展为侵袭性疾病-原细胞危象（CML- bc）。该疾病的恶性转化与额外遗传错误的积累有关。甲磺酸伊马替尼（Imatinib mesylate， IM）是一种BCR/ABL激酶的小分子抑制剂，彻底改变了CML-CP的治疗方法。不幸的是，患者可能由于编码BCR/ABL激酶结构域的氨基酸取代的点突变而对药物产生耐药性。总之，CML细胞表现出基因组不稳定性，导致对IM的抵抗和疾病的恶性进展。这一建议的重点是确定负责这些现象的机制，并随后预防/抑制IM耐药和CML-BC的发展。BCR/ABL激酶刺激多种信号通路，诱导和维持造血细胞的转化。我们和其他人发现磷脂酰肌醇-3激酶（PI-3k）在CML中不依赖生长因子的增殖和保护细胞凋亡中起重要作用。本研究拟研究PI-3k及其下游效应物Akt和Rac在CML干细胞和祖细胞群体基因组不稳定性中的作用。利用遗传学方法（显性阴性突变体、siRNA、反义cDNA、敲除小鼠）和小分子抑制剂（例如perifosine、NSC23766），我们将确定PI-3k及其下游效应物如何产生活性氧（ROS），而活性氧反过来可能导致DNA氧化损伤并促进基因组不稳定。本研究将采用CML的长期体外培养和小鼠模型。通过克隆分析和对BCR/ABL激酶结构域的测序来检测IM耐药性，通过snp和SKY检测染色体畸变。如果成功完成，这些实验将确定是否应该使用PI-3k途径抑制剂来提高IM的治疗效果，防止/延迟CML向致命的blast危象发展。

DESCRIPTION (provided by applicant): Chromosomal translocation t(9;22) is responsible for appearance of an oncogene encoding BCR/ABL fusion tyrosine kinase, which induce chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemia (ALL). CML usually starts as a relatively benign chronic phase (CML-CP), which progresses to an aggressive disease - blast crisis (CML-BC). Malignant transformation of the disease is associated with accumulation of additional genetic errors. Imatinib mesylate (IM), a small molecule inhibitor of BCR/ABL kinase, revolutionized the treatment of CML-CP. Unfortunately patients may develop resistance to the drug caused by point mutations encoding amino acid substitutions in the BCR/ABL kinase domain. In conclusion, CML cells display genomic instability leading to resistance to IM and malignant progression of the disease. This proposal is focused on determination the mechanisms responsible for these phenomena and subsequently on prevention/inhibition of the development of IM resistance and CML-BC. BCR/ABL kinase stimulates numerous signaling pathways to induce and maintain transformation of hematopoietic cells. We, and others found that phosphatidylinositol-3 kinase (PI-3k) play an essential role in growth factor independent proliferation and protection from apoptosis in CML. Here we propose to study the role of PI-3k and its downstream effectors Akt and Rac in genomic instability in CML stem and progenitor cell populations. Using genetic approach (dominant-negative mutants, siRNA, antisense cDNA, knockout mice) and small molecule inhibitors (for example perifosine, NSC23766) we will determine how PI-3k and its downstream effectors generate the reactive oxygen species (ROS), which in turn may cause oxidative DNA damage and facilitate genomic instability. Long-term in vitro culture and mouse models of CML will be applied here. IM resistance will be detected in clonogenic assays and by sequencing BCR/ABL kinase domain, and chromosomal aberrations will be detected by SNPs and SKY. If successfully accomplished, these experiments will determine if PI-3k pathway inhibitors should be used to improve therapeutic effect of IM and prevent/delay CML progression toward lethal blast crisis. PUBLIC HEALTH RELEVANCE: Although tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myelogenous leukemia (CML), patients can develop resistance to the drug and progress toward fatal malignant blast crisis of the disease. Both phenomena are associated with genetic instability caused by oncogenic BCR/ABL kinase. We will investigate the mechanisms responsible for mutations in BCR/ABL kinase causing TKI-resistance and for chromosomal aberrations leading to blast crisis. Molecular mechanisms of BCR/ABL-dependent phosphatidylinositol (PI-3k)-mediated production of reactive oxygen species (ROS) causing oxidative DNA damage, mutations, and chromosomal aberrations will be determined.