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

描述(申请人提供)：染色体易位t(9；22)是编码bcr/abl融合酪氨酸激酶的癌基因出现的原因，该癌基因可导致慢性粒细胞白血病(CML)和一组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，发展为侵袭性疾病-原始细胞危象(CML-BC)。这种疾病的恶性转化与额外的遗传错误的积累有关。甲磺酸伊马替尼(IM)是一种BCR/ABL激酶的小分子抑制剂，它使CML-CP的治疗发生了革命性的变化。不幸的是，患者可能会对这种药物产生抗药性，这是由于编码BCR/ABL激酶域的氨基酸替换的点突变造成的。总之，慢性粒细胞白血病细胞表现出基因组不稳定，导致对IM的抵抗和疾病的恶性进展。这项建议的重点是确定导致这些现象的机制，并随后预防/抑制IM耐药和CML-BC的发展。BCR/ABL激活多种信号通路，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的生长因子非依赖性增殖和抗凋亡中发挥着重要作用。在这里，我们建议研究PI-3k及其下游效应因子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。利用遗传方法(显性负性突变体、siRNA、反义cDNA、基因敲除小鼠)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物种(ROS)，这反过来可能导致DNA氧化损伤，促进基因组不稳定。慢性粒细胞白血病的长期体外培养和小鼠模型将在这里应用。IM耐药性将通过克隆试验和测序BCR/ABL激酶结构域来检测，染色体异常将通过SNPs和Sky来检测。如果成功完成，这些实验将决定是否应该使用PI-3k途径抑制剂来提高IM的治疗效果，防止/延缓CML进展为致命的原始细胞危机。 公共卫生相关性：尽管酪氨酸激酶抑制剂(TKIs)彻底改变了慢性粒细胞白血病(CML)的治疗方法，但患者可能对该药物产生抗药性，并进展为致命的恶性白血病危象。这两种现象都与致癌的bcr/abl激酶引起的遗传不稳定性有关。我们将研究导致TKI耐药的BCR/ABL激酶突变和导致BLCs危象的染色体异常的机制。Bcr/abl依赖的磷脂酰肌醇(PI-3k)介导的活性氧(ROS)产生导致DNA氧化损伤、突变和染色体异常的分子机制将被确定。

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.