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

描述（由申请人提供）：本项目的总体目标是确定白血病起始（干）细胞自我更新和存活的分子机制，这些细胞可能是人费城染色体阳性（Ph+）白血病治愈性治疗的靶点。由BCR-ABL癌基因诱导的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），是最常见的骨髓增生性恶性肿瘤。异基因骨髓移植，唯一确定的治愈性治疗慢性粒细胞白血病，是不可行的，对大多数患者。BCR-ABL酪氨酸激酶抑制剂包括甲磺酸伊马替尼（Gleevec）对治疗慢性期CML患者非常有效，但对更晚期的CML急变和Ph+ B-ALL患者无效。此外，临床上对伊马替尼产生耐药性，伊马替尼不能完全根除白血病干细胞，这表明使用BCR-ABL激酶抑制剂如伊马替尼作为单一药物不能预防最终的疾病进展或治愈CML。需要新的治疗策略，特别是对于老年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的分子途径介导的，而不是刺激白血病细胞增殖，靶向白血病干细胞是必不可少的Ph+白血病的治愈性治疗。本项目使用高效、准确的Ph+白血病小鼠模型，通过以下三个具体目标来鉴定和表征白血病干细胞：1）确定BCR-ABL转导的骨髓细胞的造血谱系转移Ph+白血病，影响白血病干细胞功能，并对BCR-ABL激酶抑制剂敏感。白血病小鼠将用BCR-ABL激酶抑制剂伊马替尼或BMS-354825治疗，然后使用流式细胞术分析白血病干细胞，以鉴定和分选不同的细胞谱系，用于疾病的过继转移和检查干细胞的存活/自我更新。2)确定BCR-ABL激活2-连环蛋白的造血谱系，检测Src激酶是否参与这些谱系中2-连环蛋白的激活并对这些细胞的存活具有积极影响，并确定2-连环蛋白在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-连环蛋白条件性敲除小鼠将用于研究2-连环蛋白在BCR-ABL白血病发生中的作用。3)确定新型抗干细胞药物BMS-214662对表达BCR-ABL的HSC存活的抑制机制，并测试该化合物与BCR-ABL激酶抑制剂联合是否可为Ph+白血病提供治愈性治疗。白血病小鼠将用BMS-214662单独或与双重BCR-ABL/Src激酶抑制剂BMS-254825一起治疗，然后分析小鼠中白血病干细胞的存活/自我更新。这项研究将有助于了解白血病干细胞的生物学，并开发治疗Ph+白血病的新型抗干细胞策略。

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.