Analysis of functional properties of the Acute Promylocytic Leukemia (APL) oncogene PML-RARalpha using a novel ex-vivo assay

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This proposal aims at analyzing functional properties of the leukemia-inducing PML-RARα fusion protein. Our previous data suggest, that both the RARα part, and also the PML part provide unique features, which prime them to become leukemia-inducing through their fusion in APL. We will use an in-vitro immortalization assay, which I have established, based on the observation, that expression of a fusion protein of murine PML and human RARα (mPR) by retroviral transduction can immortalize primary murine bone marrow cells, providing a rapid in vitro-assay (mPR assay). We want to use this assay to investigate the contribution of the different structural domains of mPML-RARα for this phenomenon, by performing domain swapping and mutagenesis experiments. Aim 1: determination of relevant features on the RARα side: Analysis of DNA binding and interaction with the cofactors RXR and SMRT. Aim 2: determination of relevant features on the PML side: Analysis of RING finger, B Boxes and coiled coil structural domains. Aim 3: Experiments correlating the immortalisation results with leukemogenesis, using classical transduction/ transplantation experiments in the mouse. If successful, our method will provide a fast way to determine effects of specific changes in the oncogene, saving time and significantly reducing necessary animal experiments.