Cell Cycle Regulation and Leukemogenesis by CBFb-SMMHC

描述（由申请人提供）：核心结合因子的AML 1或CBF β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CBF β-SMMHC，将CBF β连接至平滑肌肌球蛋白重链。抑制CBF阻断分化并减慢G1至S细胞周期进程。刺激G1期的突变可能阻止CBF癌蛋白对细胞周期的抑制，并增强其阻止分化的能力。目标1：确定细胞周期中AML 1表达变化的调控途径，并确定AML 1是否调控正常祖细胞的细胞周期。将评估在正常或AML 1（+/-）祖细胞中表达来自逆转录病毒载体的siRNA对细胞周期动力学的影响。内源性AML 1水平急剧增加32 D c13细胞进入S，这也观察到与外源性AML 1，涉及调节蛋白质的稳定性。cdks和其他激酶的作用，蛋白质：蛋白质相互作用，在这一过程中的泛素化将被确定。目标二：确定CBFbeta-SMMHC和p15 INK 4 b缺失是否协同诱导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AML 1活性降低导致的细胞周期抑制。CBFbeta-SMMHC与p16 p19缺失协同诱导小鼠淋巴样白血病在80%的AML中，p15启动子因甲基化而失活，而p16 p19异常很少见。来自C57 BL/6 p15（-/-）小鼠的骨髓将用CBFbeta-SMMHC转导并移植。将比较p15（+/+）、（+/-）和（-/-）小鼠骨髓、淋巴和红系祖细胞的细胞周期特征。将评估AML 1 siRNA和CBFbeta-SMMHC对p15（-/-）祖细胞周期动力学的影响。目标3：确定CBFbeta-SMMHC组装感受域是否是转化所必需的，以鉴定ACD功能的关键残基，并确定其在辅阻遏物结合中的作用。CBFbeta-SMMHC C端附近28个残基片段ACD的缺失可防止多聚化、抑制AML 1反式激活和抑制细胞增殖。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，评估其对AML 1反式激活和增殖的影响，并确定它们是否与mSin 3a或HDAC 8结合，就像CBFbeta-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.