Anton Willer:
RNAi-mediated Therapy & Preliminary Data on The Role of TGIF in Hematopoiesis

Date: 15-01-2010    Supervisor: Olaf Nielsen

The Ph.d thesis comprise two projects dealing with different aspects of research into the hematopoietic system: (1) Probing the therapeutic potential of retroviral delivered shRNA hairpins targeting the MLL-AF9 fusion. (2) Identification of factors disrupting the function of the tumor suppressor CCAAT Enhancer Binding Protein alpha (C/EBPα).

The generation of fusion proteins by aberrant genetic events such as chromosomal translocations is a major pathway leading to hematological malignancies including leukemias and lymphomas. A frequently found oncogenic fusion protein is the fusion between the Mixed lineage leukaemia gene (MLL) and AF9. Oncogenic fusion proteins provide an obvious target for RNAi-mediated intervention, i.e. the fusion point. Ideally, shRNAs (small hairpin RNAs) should be able to target the fusion point of the messenger RNA encoding the fusion protein in a highly specific manner without interfering with the remaining normal alleles of the two fusion partners. To test the potential of this kind of therapeutic, MLL-AF9 immortalised cells were transduced with a retroviral vector expressing a hairpin targeting the fusion point. This resulted in repression of proliferation and induction of differentiation in vitro, and prolonged the survival of mice transplanted with transduced MLL-AF9 cells. In principle this demonstrate the feasibility of RNAi-mediated knock-down of the MLL-AF9 fusion protein using shRNAs delivered by retroviral vectors.

The CCAAT Enhancer Binding Protein alpha (C/EBPα), is a key transcription factor in the hematopoietic system where it is believed to act as master switch between uncommitted, proliferating cells, and differentiated growth arrested cell. Several studies have reported the presence of genomic CEBPA mutations in AML patients. Additionally several oncogenic determinants of AML exists that affect the level or function of C/EBPα protein, including PMLRARα, AML-ETO, BCR-ABL. Collectively this suggests that C/EBPα act as a tumor suppressor in the hematopoietic system. To identify factors disrupting the function of C/EBPα, we screened a cDNA library in NIH3T3 cells which were growth repressed by C/EBPα. From the screen we isolated the TGF-β induced factor (TGIF), a member of the three-amino acid loop extension (TALE) superclass of atypical homeodomains, and best described as an inhibitor of TGF-β signaling. Ectopic expression of TGIF suppressed C/EBPα-induced growth arrest in NIH3T3, validating the output of the screen. Surprisingly, forced expression of TGIF in hematopoietic cells resulted in repression of proliferation. In vitro we observed that TGIF expression resulted in reduced colonyforming capacity of hematopoietic stem and progenitor cells. In vivo TGIF-transduced bone marrow cells showed impaired reconstitution capacity. Forced expression of TGIF in MLL-AF9 immortalized cells induced growth arrest and monocytic differentiation, collectively suggesting that TGIF negatively regulate hematopoietic cell proliferation and may be involved in myeloid differentiation.