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）染色体互易易位产生，只有在具有自我更新能力的造血干细胞（hsc）中表达时才会导致白血病干细胞（LSC）。LSCs能够产生大量的白血病祖细胞（leukemia progenitor cells, LPCs），这些细胞不能自我更新并最终分化为成熟元件，导致慢性粒细胞白血病慢性期（chronic myelgenous leukemia in chronic phase, CML-CP）。CML-CP是一种相对良性的干细胞衍生但由祖细胞驱动的疾病。在实验模型中，BCR/ABL激酶促进ros依赖的基因组不稳定性，这可能导致对ABL激酶抑制剂甲甲酸伊马替尼（imatinib mesylate， IM）的耐药性，并导致CML- CP恶性进展为侵袭性母细胞危像（CML- bc）。对IM的抗性通常是由编码BCR/ABL激酶氨基酸取代的突变引起的。从CML-CP到CML-BC的恶性进展与额外染色体畸变的积累有关。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的细胞池中检测到抗im的BCR/ABL突变体，它们可能表现出不同的白血病发生能力，并且更有可能积累与新型酪氨酸激酶抑制剂耐药相关的额外突变。因此，本应用的主要目的是确定ros诱导的基因组不稳定性(1)是否起源于LSCs或LPCs，以及(2)是否在表达抗im的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化方法检测和比较携带BCR/ABL野生型或im抗性突变体的CML-CP和CML-BC患者的LSCs和LPCs，以及来自健康供体的hsc和祖细胞中的ROS水平和氧化DNA损伤。将免疫缺陷NOG小鼠的LSCs和LPCs接种携带BCR/ABL野生型和im抗性突变体的CML-CP和CML-BC患者细胞，并将其保存在标准或减少氧化应激下，检测并比较抗im的BCR/ABL激酶点突变体和染色体畸变。在表达BCR/ABL野生型或IM抗性突变体的LSCs和/或LPCs中，鉴定ROS是导致氧化性DNA损伤的致病因素，这将对疾病的总体认识、抗氧化剂与IM联合作为治疗剂的潜在使用，以及在未来研究产生ROS和/或损害DNA修复保真度的机制时针对特定的白血病细胞亚群产生重大影响。公共卫生相关性：BCR/ABL致癌酪氨酸激酶将造血干细胞（hsc）转化为白血病干细胞（LSCs）并诱导慢性粒细胞白血病慢性期（CML-CP）。LSCs能够产生大量的白血病祖细胞（LPCs），最终分化为成熟细胞。然而，BCR/ABL促进额外的遗传变化，导致对ABL激酶抑制剂甲甲酸伊马替尼（IM）的耐药性，以及CML-CP恶性进展到侵袭性母细胞危象（CML-BC）。该应用程序的主要目的是确定这些问题是源于LSCs还是LPCs，以及抗im CML细胞是否对积累额外突变表现出更高的易感性。这些研究将为研究LSCs在CML和其他白血病中的作用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.