Survival Mechanisms of Cancer-initiating (Stem) Cells in Ph+ Leukemia

描述(申请人提供)：这个项目的总体目标是确定白血病起始(干细胞)细胞自我更新和存活的分子机制，这些细胞可能是人类费城染色体阳性(Ph)白血病治疗的靶标。由bcr-abl癌基因引起的Ph白血病，包括慢性粒细胞白血病(CML)和B细胞急性淋巴细胞白血病(B-ALL)，是最常见的骨髓增殖性恶性肿瘤之一。异基因骨髓移植是CML唯一公认的根治方法，对大多数患者来说是不可行的。BCR-ABL酪氨酸激酶抑制剂包括甲磺酸伊马替尼(Gleevec)对慢性期CML患者非常有效，但对更晚期的CML急变期和PHB-ALL患者无效。此外，临床上会出现对伊马替尼的耐药性，而且伊马替尼并不能完全根除白血病干细胞，这表明将bcr-abl激酶抑制剂(如伊马替尼)作为单一药物使用不会阻止最终的疾病进展或治愈慢性粒细胞白血病。需要新的治疗策略，特别是对老年CML患者。我们的总体假设是，这些白血病干细胞的自我更新和存活是由不同于刺激白血病细胞增殖的分子途径介导的，靶向白血病干细胞是治疗Ph白血病的关键。本项目使用高效、准确的Ph白血病小鼠模型来鉴定和表征白血病干细胞，主要集中在三个特定的目标：1)确定哪些BCR-ABL转导的骨髓细胞系转移Ph白血病，影响白血病干细胞功能，以及对BCR-ABL激酶抑制剂敏感。白血病小鼠将接受bcr-abl激酶抑制剂伊马替尼或bms-354825治疗，然后使用多色流式细胞仪分析白血病干细胞，以鉴定和分类用于过继转移疾病的不同细胞系，并检查干细胞的存活/自我更新。2)确定bcr-abl激活2-catenin的造血谱系，检测在这些谱系中Src激酶是否参与2-catenin的激活，并对这些细胞的存活起积极作用，并确定2-catenin在bcr-abl白血病发生中的作用。SRC激酶缺陷小鼠和Src激酶抑制剂，以及2-连环蛋白条件性基因敲除小鼠将被用于研究2-连环蛋白在bcr-abl白血病发生中的作用。3)探讨新型抗干细胞药物BMS-214662对表达bcr-abl的造血干细胞存活的抑制作用机制，并检测该化合物与bcr-abl激酶抑制剂联合治疗Ph白血病的疗效。白血病小鼠将接受bms-214662单独或与bcr-abl/src双抑制剂bms-254825联合治疗，然后分析白血病干细胞在小鼠体内的存活/自我更新。这项研究将有助于了解白血病干细胞的生物学，并开发针对Ph白血病的新的抗干细胞策略。

DESCRIPTION (provided by applicant): The overall goal of this project is to identify molecular mechanisms for self-renewal and survival of leukemic initiating (stem) cells that may be targets for curative therapy of human Philadelphia chromosome-positive (Ph+) leukemias. Ph+ leukemias induced by the BCR-ABL oncogene, including chronic myeloid leukemia (CML) and the B-cell acute lymphoblastic leukemia (B-ALL), are among the most common myeloproliferative malignancies. Allogeneic bone marrow transplantation, the only established curative therapy for CML, is not feasible for most patients. The BCR-ABL tyrosine kinase inhibitors including imatinib mesylate (Gleevec) are highly effective in treating chronic phase CML patients, but not more advanced CML blast crisis and Ph+ B-ALL patients. Moreover, clinical resistance to imatinib develops and imatinib does not completely eradicate leukemic stem cells, suggesting that use of a BCR-ABL kinase inhibitor such as imatinib as a single agent will not prevent eventual disease progression or cure CML. New therapeutic strategies are needed, especially for older CML patients. Our overall hypothesis is that self-renewal and survival of these leukemic stem cells are mediated by different molecular pathways than those that stimulate leukemic cell proliferation, and targeting leukemic stem cells is essential to curative therapy of Ph+ leukemias. This project uses the efficient, accurate mouse models for Ph+ leukemias to identify and characterize leukemic stem cells by focusing on three specific aims: 1) To determine which hematopoietic lineages of BCR-ABL-transduced bone marrow cells transfer Ph+ leukemias, affect leukemic stem cell function, and are sensitive to BCR-ABL kinase inhibitors. Leukemic mice will be treated with a BCR-ABL kinase inhibitor imatinib or BMS-354825, followed by the analysis of leukemic stem cells using multicolor FACS to identify and sort different cell lineages for adoptive transfer of the diseases and for examination of survival/self-renewal of the stem cells. 2) To determine the hematopoietic lineages in which 2-catenin is activated by BCR-ABL, to test whether Src kinases are involved in 2-catenin activation in these lineages and have a positive effect on survival of these cells, and to establish the role of 2-catenin in BCR-ABL leukemogenesis. Src kinase-deficient mice and Src kinase inhibitors, and 2-catenin conditional knockout mouse will be used to address the role of 2-catenin in BCR-ABL leukemogenesis. 3) To determine the inhibitory mechanism of a novel anti-stem cell agent BMS-214662 on survival of BCR-ABL-expressing HSCs and to test whether this compound in combination with a BCR-ABL kinase inhibitor provides a curative therapy for Ph+ leukemias. Leukemic mice will be treated with BMS-214662 alone or together with a dual BCR- ABL/Src kinase inhibitor BMS-254825, followed by the analysis of survival/self-renewal of leukemic stem cells in mice. This study will help to understand the biology of leukemia stem cells and develop novel anti-stem cell strategies for Ph+ leukemias.