Chromosomal DMA double strand breaks (DSBs) are induced by a variety of factors and occur constantly in every cell. The proper repair of DSBs is essential for maintaining genomic stability and preventing cellular transformation. We recently demonstrated that the Ataxia Telangiectasia Mutated (ATM) protein, which is the master regulator of the cellular DSB response, is required during chromosomal DSB repair to maintain broken DNA ends in repair complexes. An immediate substrate of ATM is H2AX, a core histone variant that is phosphorylated in chromatin around DSBs. We have demonstrated that expression of both allelic copies of H2ax is essential for error-free DSB repair, maintanence of genomic stability, and suppression of cancer. Notably, deletion or loss of heterozygosity (LOH) of the H2AX gene appears commonly in a wide variety of human tumors, suggesting similar dosage-dependent H2AX functions in man. In this application, we propose to elucidate mechanisms by which H2AX functions downstream of ATM and related kinases to promote error-free DSB repair, maintain genomic stability, and suppress malignant transformation. The specific hypothesis to be investigated is that one critical function of H2AX is to hold broken DNA strands together and ensure that DNA ends are properly repaired.
In Specific Aim 1, we will test our hypothesis that H2AX functions during V(D)J recombination to hold together broken DNA strands upon G1 to S transition and, thereby, prevent antigen receptor locus translocations. We will employ novel cell lines and primary thymocytes to induce DSB intermediates at specific genomic locations in G1 phase cells and monitor their repair during continued cell cycle progression.
In Specific Aim 2, we will test our hypothesis that ATM independent H2AX functions downstream of other kinases are essential for normal DSB repair, prevention of genomic instability, and suppression of cancer. We will utilize cell lines and thymocyes deficient for H2AX and ATM to elucidate the roles of ATM independent H2AX functions.
In Specific Aim 3, we will test our hypothesis that H2AX functions at replication-associated DSBs induced during normal and oncogene-driven proliferation to prevent p53 deletions that drive malignant transformation. We will directly evaluate the ability of H2AX to suppress tumors with p53 deletions. Relevance: Our studies will lead to a greater understanding of the molecular mechanisms through which genomic integrity is maintained and how defects in these can lead to malignant transformation. This knowledge and the animal models generated will contribute to the development of more effective diagnostic and/or therapeutic tools for human cancers. Consequently, our proposed studies will have broad implications for human disease.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA125195-03
Application #
7624244
Study Section
Cancer Genetics Study Section (CG)
Program Officer
Pelroy, Richard
Project Start
2007-07-01
Project End
2012-05-31
Budget Start
2009-06-01
Budget End
2010-05-31
Support Year
3
Fiscal Year
2009
Total Cost
$312,550
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Balestrini, Alessia; Nicolas, Laura; Yang-Lott, Katherine et al. (2016) Defining ATM-Independent Functions of the Mre11 Complex with a Novel Mouse Model. Mol Cancer Res 14:185-95
Ehrlich, Lori A; Yang-Iott, Katherine; DeMicco, Amy et al. (2015) Somatic inactivation of ATM in hematopoietic cells predisposes mice to cyclin D3 dependent T cell acute lymphoblastic leukemia. Cell Cycle 14:388-98
DeMicco, Amy; Naradikian, Martin S; Sindhava, Vishal J et al. (2015) B Cell-Intrinsic Expression of the HuR RNA-Binding Protein Is Required for the T Cell-Dependent Immune Response In Vivo. J Immunol 195:3449-62
Rupp, Levi J; Brady, Brenna L; Carpenter, Andrea C et al. (2014) The microRNA biogenesis machinery modulates lineage commitment during ?? T cell development. J Immunol 193:4032-42
Horowitz, Julie E; Bassing, Craig H (2014) Noncore RAG1 regions promote V? rearrangements and ?? T cell development by overcoming inherent inefficiency of V? recombination signal sequences. J Immunol 192:1609-19
Steinel, Natalie C; Fisher, Megan R; Yang-Iott, Katherine S et al. (2014) The ataxia telangiectasia mutated and cyclin D3 proteins cooperate to help enforce TCR? and IgH allelic exclusion. J Immunol 193:2881-90
DeMicco, Amy; Yang-Iott, Katherine; Bassing, Craig H (2013) Somatic inactivation of Tp53 in hematopoietic stem cells or thymocytes predisposes mice to thymic lymphomas with clonal translocations. Cell Cycle 12:3307-16
Brady, Brenna L; Rupp, Levi J; Bassing, Craig H (2013) Requirement for dicer in survival of proliferating thymocytes experiencing DNA double-strand breaks. J Immunol 190:3256-66

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