Ionizing radiation (IR) can cause to significant tumor cell death, however resistance to radiotherapy can limit its efficacy. IR induces DNA double-strand breaks (DSBs) that can lead to toxic chromosomal rearrangements;such that limiting these rearrangements may be critical for radioresistance. Such IR-induced chromosomal rearrangements likely form via end joining (EJ) that uses incorrect DSB ends during repair. Thus, our long-term goal is to define the factors that limit rearrangements during EJ, and thereby develop therapeutic targets for tumor radiosensitization. To advance this goal, we developed a unique assay to quantify the use of correct versus incorrect ends during EJ of multiple DSBs. Using a chromosomal reporter with two tandem DSBs, and our technical innovation of generating site-specific non-cohesive DSBs, we can measure EJ that uses proximal ends that flank a single DSB versus EJ that uses distal ends of two DSBs. Distal end use during EJ is incorrect, since it causes a deletion rearrangement. Using this system, we found that the DNA damage response factors RAD50 and DNA-PKcs are important to limit incorrect end use during EJ. Since these factors also promote radioresistance, we propose to test our central hypothesis that the role of RAD50 and DNA-PKcs in limiting incorrect end use during EJ is critical for cellular radioresistance. A corollary of this hypothesis is that the funcion of these factors in limiting incorrect end use during EJ is a target for radiosensitization.
Aim 1 : To determine the importance to radioresistance of RAD50 function in limiting incorrect end use during EJ, as compared to its other roles in DNA repair. For this, we will examine a series of RAD50 mutants for the ability to complement a set of DNA repair functions, and promote radioresistance, in RAD50-deficient human cells.
Aim 2 : To determine the importance to radioresistance of DNA-PKcs function in limiting incorrect end use during EJ, as compared to its other roles in DNA repair. Using a similar approach as Aim 1, we will examine a series of DNA-PKcs mutants for the ability to complement DNA repair functions, and promote radioresistance, in DNA-PKcs-deficient mammalian cells.
Aim 3. To determine how increasing the distance between two tandem DSBs affects incorrect end use during EJ, and the relative requirement of RAD50 and DNA-PKcs for limiting such EJ-mediated deletion rearrangements. These proposed studies are significant, because they will provide novel insight into how RAD50 and DNA-PKcs promote radioresistance, which is critical for their development as therapeutic targets for tumor radiosensitization. Our study is innovative because it will establish a new paradigm for understanding the role of RAD50 and DNA-PKcs in promoting radioresistance, and because we propose in Aim 3 to develop a new reporter system to examine how the distance between DSBs affects end use during EJ. Our proposed study is also innovative because it will provide insight into the importance of incorrect end use during EJ for radiation toxicity, which will lead to new therapeutic strategies for tumor radiosensitization.

Public Health Relevance

The proposed research is relevant to public health, because determining how DNA repair proteins limit radiation toxicity will lead to new therapeutic strategies for tumor radiosensitization. Thus, the proposed study supports the NIH mission for improving patient outcomes from cancer treatment.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA120954-08
Application #
8676686
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Pelroy, Richard
Project Start
2006-09-01
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
8
Fiscal Year
2014
Total Cost
$210,451
Indirect Cost
$85,182
Name
City of Hope/Beckman Research Institute
Department
Type
DUNS #
027176833
City
Duarte
State
CA
Country
United States
Zip Code
91010
Muñoz, Meilen C; Yanez, Diana A; Stark, Jeremy M (2014) An RNF168 fragment defective for focal accumulation at DNA damage is proficient for inhibition of homologous recombination in BRCA1 deficient cells. Nucleic Acids Res 42:7720-33
Gu, Long; Smith, Shanna; Li, Caroline et al. (2014) A PCNA-derived cell permeable peptide selectively inhibits neuroblastoma cell growth. PLoS One 9:e94773
Liu, Liang; Zhou, Weiying; Cheng, Chun-Ting et al. (2014) TGF? induces "BRCAness" and sensitivity to PARP inhibition in breast cancer by regulating DNA-repair genes. Mol Cancer Res 12:1597-609
Cannon, Brian; Kuhnlein, Jeffrey; Yang, Soo-Hyun et al. (2013) Visualization of local DNA unwinding by Mre11/Rad50/Nbs1 using single-molecule FRET. Proc Natl Acad Sci U S A 110:18868-73
Wang, Z; Yuan, H; Roth, M et al. (2013) SIRT1 deacetylase promotes acquisition of genetic mutations for drug resistance in CML cells. Oncogene 32:589-98
Reczek, Colleen R; Szabolcs, Matthias; Stark, Jeremy M et al. (2013) The interaction between CtIP and BRCA1 is not essential for resection-mediated DNA repair or tumor suppression. J Cell Biol 201:693-707
Zhao, Zhen; Oh, Soohwan; Li, Dapeng et al. (2012) A dual role for UVRAG in maintaining chromosomal stability independent of autophagy. Dev Cell 22:1001-16
Gunn, Amanda; Bennardo, Nicole; Cheng, Anita et al. (2011) Correct end use during end joining of multiple chromosomal double strand breaks is influenced by repair protein RAD50, DNA-dependent protein kinase DNA-PKcs, and transcription context. J Biol Chem 286:42470-82
Laulier, Corentin; Cheng, Anita; Stark, Jeremy M (2011) The relative efficiency of homology-directed repair has distinct effects on proper anaphase chromosome separation. Nucleic Acids Res 39:5935-44
Dumitrache, Lavinia C; Hu, Lingchuan; Son, Mi Young et al. (2011) Trex2 enables spontaneous sister chromatid exchanges without facilitating DNA double-strand break repair. Genetics 188:787-97

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