Cell Cycle Regulation and Leukemogenesis by CBFb-SMMHC

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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.