Genome instability in leukemia stem cell

描述（由申请人提供）：BCR/ABL融合酪氨酸激酶由t（9;22）相互染色体易位产生，并且仅当在具有自我更新能力的造血干细胞（HSC）中表达时具有致白血病性，从而诱导白血病干细胞（LSC）。LSC能够产生大量的白血病祖细胞（LPC），其不能自我更新并最终分化为成熟成分，导致慢性期慢性髓细胞性白血病（CML-CP）。CML-CP是一种相对良性的干细胞衍生疾病，但却是一种祖细胞驱动的疾病。在实验模型中，BCR/ABL激酶促进ROS依赖性基因组不稳定性，这可能导致对ABL激酶抑制剂甲磺酸伊马替尼（IM）的抗性，以及导致CML-CP恶性进展为侵袭性原始细胞危象（CML-BC）。对IM的抗性通常由编码BCR/ABL激酶中的氨基酸取代的突变引起。从CML-CP到CML-BC的恶性进展与其他染色体畸变的积累相关。对抑制剂的耐药性和恶性进展是BCR/ABL阳性白血病的主要问题。在干细胞和早期祖细胞富集池中检测到IM耐药BCR/ABL突变体，这些突变体可能表现出不同的致白血病能力，并且与新型酪氨酸激酶抑制剂耐药相关的其他突变累积的可能性更高。因此，本申请的主要目的是确定ROS诱导的基因组不稳定性（1）是否起源于LSC或LPC，以及（2）是否在表达IM抗性BCR/ABL突变体的CML细胞中进一步增强。因此，将使用免疫荧光和生物化学方法，在来自携带BCR/ABL野生型或IM抗性突变体的CML-CP和CML-BC患者的LSC和LPC中，以及在来自健康供体的HSC和祖细胞中，检查和比较ROS水平和氧化DNA损伤。将在从接种携带BCR/ABL野生型和IM耐药突变体的CML-CP和CML-BC患者细胞的免疫缺陷NOG小鼠中收获的LSC和LPC中检查和比较IM耐药BCR/ABL激酶点突变体和染色体畸变，这些细胞保持在标准或降低的氧化应激下。将ROS鉴定为导致表达BCR/ABL野生型或IM抗性突变体的LSC和/或LPC中的基因组不稳定性的氧化DNA损伤的致病因素，这对疾病的一般理解、抗氧化剂与IM组合作为治疗剂的潜在用途、并在未来研究产生ROS和/或抗氧化剂的机制中靶向特定的白血病细胞亚群。或损害DNA修复的保真度。公共卫生相关性：BCR/ABL致癌酪氨酸激酶可将造血干细胞（HSC）转化为白血病干细胞（LSC），并诱导慢性期慢性髓性白血病（CML-CP）。LSC能够产生大量的白血病祖细胞（LPC），其最终分化为成熟元件。然而，BCR/ABL促进额外的遗传变化，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恶性进展为侵袭性急变（CML-BC）。本申请的主要目的是确定这些问题是否起源于LSC或LPC，以及IM耐药CML细胞是否表现出更高的累积额外突变的易感性。这些研究将为研究CML和其他白血病中的LSC以及测试新的治疗策略开辟新的方向。

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.