Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biology

Abstract

The AF9 protein is one of the most common chromosomal translocation partners of the MLL gene in MLL leukemia. Wild-type AF9 is a member of the pTEFb transcription elongation complex, and interacts with gene regulatory proteins such as AF4/AF5q31, DOT1L, Pc3/CBX8 and BCoR. These interactions are retained in the oncogenic MLL-AF9 fusion protein, and may be required for leukemic transformation.

Using bone marrow progenitor cells isolated from conditional Af9 knockout mice, we examined in vitro differentiation of hematopoietic progenitor cells to the erythroid, myeloid and megakaryocytic lineages in the presence or absence of Af9. Based on previously published studies, we hypothesized that loss of Af9 would result in a loss of erythroid colony formation. Our results indicate no significant difference in erythroid, myeloid or megakaryocytic colony formation ability in cells expressing or null for Af9. Expression of transcription factor genes associated with erythroid or myeloid lineage commitment were also unaltered in cells null for Af9 compared to control. There was a statistically significant decrease in cell surface expression of early erythroid cell surface marker Ter119 in Af9-null cells compared to control mice, indicating a potential role for Af9 in early erythroid lineage commitment.

To examine the role of AF9-mediated protein interactions in leukemic transformation, we engineered point mutants in the C-terminus of AF9 in the context of MLL-AF9 fusion protein predicted to disrupt specific protein-protein interactions.

Hematopoietic progenitor cells were isolated from the bone marrow of wild type mice and retrovirally transduced with constructs encoding wild type or mutant MLL-AF9 fusion proteins, or empty vector control. In vitro colony assay experiments indicate that mutants MLL-AF9(D544R) and MLL-AF9(D546R) dramatically reduced colony formation ability and expression of MLL target genes Hoxa9 and Meis1 compared to MLL-AF9(WT). Coimmunoprecipitation studies show that the D546R mutation in the context of amino acids 376-568 of AF9 significantly reduces binding to Dot1l while maintaining interaction with Af4. These results indicate that disruption of AF9 interaction with AF4 and DOT1L decreases colony formation and target gene expression in in vitro leukemia assays. These interactions are attractive targets for designing small molecule inhibitors for the treatment of MLL leukemia.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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