Cell Cycle Regulation and Leukemogenesis by CBFb-SMMHC

描述（由申请人提供）：核心结合因子的AML1或cbfβ亚基在30%的急性髓性白血病（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。CBF的抑制阻断了分化，减缓了G1到S细胞周期的进程。刺激G1的突变可能阻止CBF癌蛋白对细胞周期的抑制，并增强其阻碍分化的能力。目的1：确定细胞周期中AML1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。从逆转录病毒载体在正常或AML1（+/-）祖细胞中表达siRNA对细胞周期动力学的影响将被评估。当32D c13细胞进入S时，内源性AML1水平急剧增加，外源性AML1也观察到这一点，这意味着蛋白质稳定性受到调节。cdks和其他激酶、蛋白：蛋白相互作用和泛素化在这一过程中的作用将被确定。目的2：确定cbfβ - smmhc和p15INK4b缺失是否共同诱导AML， p15缺失是否特异性影响髓系祖细胞增殖，以及p15缺失是否阻止AML1活性降低对细胞周期的抑制。cbfβ - smmhc与p16p19缺失协同诱导小鼠淋巴性白血病。在80%的aml中，p15启动子因甲基化而失活，而p16p19异常很少见。C57BL/6 p15（-/-）小鼠的骨髓将被cbfβ - smmhc转导并移植。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。AML1 siRNA和cbfβ - smmhc对p15（-/-）祖细胞周期动力学的影响将被评估。目的3：确定转化是否需要cbfβ - smmhc组装能力域，确定ACD功能的关键残基，并确定其在辅抑制因子结合中的作用。cbfβ - smmhc c端附近的28个残基片段ACD的缺失阻止了多聚化，抑制了AML1反激活，抑制了细胞增殖。我们建议在Aim 2中开发的AML模型中评估这种缺失，以确定ACD中阻止多聚化的点突变，评估它们对AML1转激活和增殖的影响，并确定它们是否与mSin3a或HDAC8结合，就像cbf - smmhc一样。

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.