Core binding factors (CBFs) are heterodimeric transcription factors consisting of DNA-binding RUNX (AML1) subunits and a non-DNA-binding CBFB subunit. All four genes that encode RUNX subunits (RUNX1, RUNX2, RUNX3) and the gene encoding the CBFB subunit are essential for normal development, and are mutated in human disease. RUNX1 and CBFB are required for hematopoiesis, and are mutated in 25%-30% of human leukemias. RUNX1 and CBFB are proto-oncogenes commonly activated in human leukemias. The reversion and translocations identified in these genes are associated with ~30% of de novo acute myeloid leukemias (AML) in humans. The t(8;21) associated with 12-15% of AML cases generates a novel fusion protein, AML1-ETO, containing the N-terminal 177 amino acids of RUNX1 including the Runt domain and virtually all (577 amino acids) of ETO. Heterozygous knock-in of AML1-ETO was lethal indicating that it is a dominant negative. An inducible mouse model of AML1-ETO shows that it highly predisposes mice to the development of leukemia. We are proposing to test the role of 2 functional domains of AML1-ETO.
Aim 1 : Structural characterization of functional domains of AML1-ETO (HHR and MYND domains) to test the hypothesis that each domain is essential for the dominant negative phenotype of AML1-ETO.
Aim 1 proposes to solve the structures of these two domains (HHR and MYND) by themselves or as functional complexes using NMR spectroscopy or x-ray crystallography.
Aim 2 : Biochemical characterization of functional domains of AML1-ETO (HHR and MYND domains) to test the hypothesis that each domain is essential for the dominant negative phenotype of AML1-ETO.
Aim 2 proposes to measure the binding constants of these individual domains and longer forms with interacting proteins and with DNA using isothermal titration calorimetry and surface plasmon resonance methods, including identification of hot spots for interaction by Ala point mutagenesis.
Aim 3 : In vivo characterization of functional domains of AML1-ETO (HHR and MYND domains) to test the hypothesis that each domain is essential for the dominant negative phenotype of AML1-ETO. Based on the identification of highly specific point mutations that disrupt particular interactions of AML1-ETO, we can surgically test the role of each of these functions in vivo.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA108056-04
Application #
7192478
Study Section
Special Emphasis Panel (ZRG1-ELB (02))
Program Officer
Knowlton, John R
Project Start
2004-03-04
Project End
2009-02-28
Budget Start
2007-03-01
Budget End
2008-02-28
Support Year
4
Fiscal Year
2007
Total Cost
$338,141
Indirect Cost
Name
University of Virginia
Department
Physiology
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
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DeKelver, Russell C; Yan, Ming; Ahn, Eun-Young et al. (2013) Attenuation of AML1-ETO cellular dysregulation correlates with increased leukemogenic potential. Blood 121:3714-7
Sinenko, Sergey A; Hung, Tony; Moroz, Tatiana et al. (2010) Genetic manipulation of AML1-ETO-induced expansion of hematopoietic precursors in a Drosophila model. Blood 116:4612-20
Corpora, Takeshi; Roudaia, Liya; Oo, Zaw Min et al. (2010) Structure of the AML1-ETO NHR3-PKA(RII?) complex and its contribution to AML1-ETO activity. J Mol Biol 402:560-77
Park, Sangho; Speck, Nancy A; Bushweller, John H (2009) The role of CBFbeta in AML1-ETO's activity. Blood 114:2849-50
Park, Sangho; Chen, Wei; Cierpicki, Tomasz et al. (2009) Structure of the AML1-ETO eTAFH domain-HEB peptide complex and its contribution to AML1-ETO activity. Blood 113:3558-67
Roudaia, Liya; Cheney, Matthew D; Manuylova, Ekaterina et al. (2009) CBFbeta is critical for AML1-ETO and TEL-AML1 activity. Blood 113:3070-9
Liu, Yizhou; Chen, Wei; Gaudet, Justin et al. (2007) Structural basis for recognition of SMRT/N-CoR by the MYND domain and its contribution to AML1/ETO's activity. Cancer Cell 11:483-97
Liu, Yizhou; Cheney, Matthew D; Gaudet, Justin J et al. (2006) The tetramer structure of the Nervy homology two domain, NHR2, is critical for AML1/ETO's activity. Cancer Cell 9:249-60
Li, Zhe; Lukasik, Steven M; Liu, Yizhou et al. (2006) A mutation in the S-switch region of the Runt domain alters the dynamics of an allosteric network responsible for CBFbeta regulation. J Mol Biol 364:1073-83