Cell Cycle Regulation and Leukemogenesis by CBFb-SMMHC

描述(申请人提供)：核心结合因子AML1或CBFβ亚基在30%的急性髓系白血病(AML)病例中发生突变或易位，LNV(16)编码CBFβ-SMMHC，将CBFβ连接到平滑肌肌球蛋白重链。抑制CBF可阻断细胞分化，延缓G1期对S细胞的周期进程。刺激G1期的突变可能会阻止CBF癌蛋白对细胞周期的抑制，并增强它们阻碍分化的能力。目的1：确定AML1在细胞周期中表达变化的调控途径，并确定AML1是否调节正常祖细胞的细胞周期。在正常或AML1(+/-)祖细胞中表达逆转录病毒载体的siRNA对细胞周期动力学的影响将被评估。随着32D c13细胞进入S，内源性AML1水平急剧升高，外源性AML1也观察到这一点，这意味着调控的蛋白质稳定性。CDKs和其他激酶、蛋白质：蛋白质相互作用和泛素化在这一过程中的作用将被确定。目的：探讨CBFbeta-SMMHC和p15INK4b缺失是否协同诱发AML，p15缺失是否特异性影响髓系祖细胞的增殖，以及p15缺失是否阻止AML1活性降低对细胞周期的抑制。CBFbeta-SMMHC协同p16p19基因缺失诱导小鼠淋巴细胞性白血病。在80%的AML中，p15启动子被甲基化失活，而p16p19异常很少见。将C57BL/6 p15(-/-)小鼠的骨髓用CBFbeta-SMMHC转导并移植。将比较p15(+/+)、(+/-)和(-/-)小鼠的髓系、淋巴系和红系祖细胞的细胞周期特征。将评估AML1 siRNA和CBFbeta-SMMHC对p15(-/-)前体细胞周期动力学的影响。目的3：确定CBFbeta-SMMHC组装功能域是否是转化所必需的，确定对ACD功能至关重要的残基，并确定它们在辅阻遏子结合中的作用。CBFbeta-SMMHC C末端附近的28个残基片段ACD的缺失阻止了多聚化，抑制了AML1的反式激活，并抑制了细胞增殖。我们建议在AIM 2中建立的AML模型中评估这一缺失，以确定ACD中阻止多聚化的点突变，评估它们对AML1反式激活和增殖的影响，并确定它们是否与CBFbeta-SMMHC一样与mSin3a或HDAC8结合。

DESCRIPTION (provided by applicant): The AML1 or CBFbeta subunits of Core Binding Factor are mutated or translocated in 30% of acute myeloid leukemia (AML) cases, lnv(16) encodes CBFbeta-SMMHC, linking CBFbeta to Smooth Muscle Myosin Heavy Chain. Inhibition of CBF blocks differentiation and slows G1 to S cell cycle progression. Mutations, which stimulate G1 may prevent cell cycle inhibition by CBF oncoproteins and potentiate their ability to impede differentiation. Aim 1: To identify the regulatory pathway responsible for variation in AML1 expression during the cell cycle and to determine whether AML1 regulates the cell cycle in normal progenitors. The effect of expressing an siRNA from a retroviral vector in normal or AML1(+/-) progenitors on cell cycle kinetics will be assessed. Endogenous AML1 levels increase sharply as 32D c13 cells enters S, and this is also observed with exogenous AML1, implicating regulated protein stability. The role of cdks and other kinases, protein:protein interaction, and ubiquitination in this process will be determined. Aim 2: To determine whether CBFbeta-SMMHC and loss of p15INK4b cooperate to induce AML, whether loss of p15 specifically affects myeloid progenitor proliferation, and whether lack of p15 prevents cell cycle inhibition from reduced AML1 activity. CBFbeta-SMMHC cooperates with loss of p16p19 to induce lymphoid leukemias in mice. The p15 promoter is inactivated by methylation in 80% of AMLs, whereas p16p19 abnormalities are rare. Marrow from C57BL/6 p15 (-/-) mice will be transduced with CBFbeta-SMMHC and transplanted. The cell cycle characteristics of myeloid, lymphoid, and erythroid progenitors from p15 (+/+), (+/-), and (-/-) mice will be compared. The effect of AML1 siRNA and of CBFbeta-SMMHC on p15 (-/-) progenitor cell cycle kinetics will be assessed. Aim 3: To determine whether the CBFbeta-SMMHC Assembly Competence Domain is required for transformation, to identify residues critical for ACD function, and to determine their role in corepressor binding. Deletion of a 28 residue segment, the ACD, near the C-terminus of CBFbeta-SMMHC prevents multimerization, inhibition of AML1 transactivation, and inhibition of cell proliferation. We propose to evaluate this deletion in the AML model developed in Aim 2, to identify point mutations in the ACD which prevent multimerization, to assess their effect on AML1 transactivation and on proliferation, and to determine whether they bind mSin3a or HDAC8, as does CBFbeta-SMMHC.