Genome instability in leukemia stem cell

描述(由申请人提供)：bcr/abl融合酪氨酸激酶是t(9；22)染色体相互易位的结果，只有在具有自我更新能力的造血干细胞(HSCs)中表达，从而诱导白血病干细胞(LSC)时，才能产生白血病。LSC能够产生大量的白血病祖细胞(LPCs)，这些LPCs不能自我更新并最终分化为成熟细胞，导致慢性粒细胞白血病慢性期(CML-CP)。CML-CP是一种相对良性的干细胞来源的疾病，但由祖细胞驱动。在实验模型中，BCR/ABL激酶促进了ROS依赖的基因组不稳定性，这可能导致对ABL激酶抑制剂甲磺酸伊马替尼(IM)的耐药性，并导致CML-CP向侵袭性原始细胞危机(CML-BC)的恶性进展。对IM的耐药性通常是由编码BCR/ABL激酶氨基酸替换的突变引起的。从CML-CP到CML-BC的恶性进展与额外染色体异常的积累有关。耐药和恶性进展是bcr/abl阳性白血病的主要问题。在茎和早期富集祖细胞的池中检测到对IM具有抗性的bcr/abl突变体，这些突变可能表现出不同的致白血病能力，并增加了与新型酪氨酸激酶抑制剂耐药相关的额外突变积累的可能性。因此，这项应用的主要目的是确定ROS诱导的基因组不稳定性(1)起源于LSCs还是LPCS，以及(2)在表达IM耐药bcr/abl突变体的CML细胞中进一步增强。出于这个原因，我们将使用免疫荧光和生化方法检测和比较携带bcr/abl野生型或IM耐药突变的CML-CP和CML-BC患者的LSCs和LPCs中的ROS水平和氧化DNA损伤，以及来自健康捐赠者的HSCs和祖细胞。免疫缺陷NOG小鼠的LSCs和LPCs将检测和比较免疫缺陷NOG小鼠的LSCs和LPCs中的IM抗性BCR/ABL激酶点突变和染色体畸变率。NOG小鼠接种CML-CP和CML-BC患者细胞，携带BCR/ABL野生型和IM抗性突变体，保持在标准或降低氧化应激下。在表达bcr/abl野生型或IM耐药突变体的LSCs和/或LPCs中，鉴定ROS是导致DNA氧化损伤导致基因组不稳定的原因，将对了解疾病的一般情况、抗氧化剂与IM联合用作治疗药物以及在未来研究产生ROS的机制和/或损害DNA修复的保真度时针对特定的白血病细胞亚群具有重要影响。公共卫生相关性：bcr/abl致癌酪氨酸激酶将造血干细胞(HSCs)转化为白血病干细胞(LSCs)，并诱导慢性期慢性粒细胞白血病(CML-CP)。LSC能够产生大量的白血病前体细胞(LPC)，最终分化为成熟的成分。然而，BCR/ABL促进了额外的基因变化，导致对ABL激酶抑制剂甲磺酸伊马替尼(IM)的耐药性，以及CML-CP向侵袭性原始细胞危机(CML-BC)的恶性进展。这项应用的主要目标是确定这些问题是否起源于LSC或LPCS，以及IM耐药的CML细胞是否表现出更高的累积额外突变的敏感性。这些研究将在研究慢性粒细胞白血病和其他白血病的LSCs以及测试新的治疗策略方面开辟新的方向。

DESCRIPTION (provided by applicant): BCR/ABL fusion tyrosine kinase results from t(9;22) reciprocal chromosomal translocation and is leukemogenic only when expressed in a hematopoietic stem cells (HSCs) with self-renewal capacity thereby inducing leukemia stem cell (LSC). LSCs are capable to generate large numbers of leukemic progenitor cells (LPCs), which cannot self-renew and eventually differentiate to mature elements, causing chronic myelogenous leukemia in chronic phase (CML-CP). CML-CP is a relatively benign stem cell-derived but a progenitor-driven disease. In the experimental models BCR/ABL kinase promotes ROS-dependent genomic instability, which may lead to resistance to ABL kinase inhibitor imatinib mesylate (IM), and to malignant progression of CML- CP to aggressive blast crisis (CML-BC). Resistance to IM is often caused by mutations encoding amino acid substitutions in BCR/ABL kinase. Malignant progression from CML-CP to CML-BC is associated with accumulation of additional chromosomal aberrations. Resistance to the inhibitors and malignant progression are the major problems in BCR/ABL-positive leukemias. IM-resistant BCR/ABL mutants are detected in stem and early progenitor -enriched pools, which may display different leukemogenic capability and higher likelihood of accumulation of additional mutations associated with resistance to novel tyrosine kinase inhibitors. Therefore the main goals of this application are to determine if ROS-induced genomic instability (1) originate in LSCs or in LPCs, and (2) is further enhanced in CML cells expressing IM-resistant BCR/ABL mutants. For this reason ROS levels and oxidative DNA damage will be examined and compared in LSCs and LPCs from CML-CP and CML-BC patients carrying BCR/ABL wild-type or IM-resistant mutant, and in HSCs and progenitors from healthy donors, using immunofluorescent and biochemical approaches. IM-resistant BCR/ABL kinase point-mutants and chromosomal aberrations will be examined and compared in LSCs and LPCs harvested from immunodeficient NOG mice inoculated with CML-CP and CML-BC patient cells carrying BCR/ABL wild-type and IM-resistant mutants, which were kept under standard or reduced oxidative stress. Identification of ROS as causative element for oxidative DNA damage leading to genomic instability in LSCs and/or LPCs expressing BCR/ABL wild-type or IM-resistant mutant should have a significant impact on general understanding of the disease, potential use of anti-oxidants combined with IM as therapeutic agents, and targeting the particular leukemia cell sub-population in future investigations of the mechanisms generating ROS and/or compromising the fidelity of DNA repair. PUBLIC HEALTH RELEVANCE: BCR/ABL oncogenic tyrosine kinase transforms hematopoeitic stem cells (HSCs) into leukemia stem cells (LSCs) and induces chronic myeloid leukemia in chronic phase (CML-CP). LSCs are capable to generate large numbers of leukemic progenitors (LPCs), which eventually differentiate to mature elements. However, BCR/ABL promotes additional genetic changes causing resistance to ABL kinase inhibitor imatinib mesylate (IM), and malignant progression of CML-CP to the aggressive blast crisis (CML-BC). The main goal of this application is to determine if these problems originate in LSCs or in LPCs and if IM-resistant CML cells display higher susceptibility to accumulate additional mutations. These studies will break ground toward new directions in studying LSCs in CML and other leukemias, and in testing of novel therapeutic strategies.