Most cancer patients receive radio- and/or chemotherapy that causes DNA damage, which blocks DNA replication. Normal and tumor cells respond to DNA damage and associated replication stress by activating DNA repair, cell cycle arrest (checkpoint) systems, and when damage is severe, programmed death pathways, collectively termed the DNA damage response (DDR). DDR proteins play crucial roles in tumor suppression and genome stabilization (cancer etiology) as well as tumor response to radio- and chemotherapy (cancer treatment). DDR pathways determine cell fates in response to DNA damage, including cell survival, genome stability, and cell death/permanent growth arrest via apoptosis, autophagy, necrosis, senescence, or mitotic catastrophe. Cells are particularly vulnerable to DNA damage during S phase because most DNA lesions stall replication forks, causing replication stress. This proposal focuses on several proteins with roles in DNA repair, checkpoint activation, and recovery from replication stress. Metnase and DNA-PK were both initially characterized for their roles in DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ). Recent studies demonstrate that Metnase and DNA-PK (along with replication protein A (RPA), ATM/ATR, Chk1, and others) also function in checkpoint activation and replication stress recovery. The replication checkpoint prevents new origin firing and stabilizes stalled replication forks to prevent fork collapse, allowing time for repair and for restart. Persistent replication stress can lead to fork collapse, producing one-ended DSBs marked by phosphorylated H2AX (?-H2AX). RPA accumulates on single-stranded DNA at stalled forks and the RPA32 subunit is phosphorylated at multiple sites by phosphoinositide 3-kinase-related protein kinases (PIKKs) DNA-PK, ATM and ATR, leading to Chk1 activation and replication arrest. Metnase also regulates Chk1 activation and replication arrest. Our central hypothesis is that Metnase, DNA-PK, and RPA operate within the DDR to influence cell fate after genotoxic stress, including cell survival, genome stability, and death pathway activation. We will determine roles of Metnase (Aim 1) and PIKK phosphorylation of RPA (Aim 2) in replication stress responses including replication arrest, fork restart, genome stability, cell survival and cell death by apoptosis.
In Aim 3 we will define epistatic relationships between Metnase and PIKK/RPA pathways, and test novel combinations of replication stress agents plus DDR inhibitors to enhance killing of breast, lung, pancreatic, colon, head and neck, and leukemic tumor cells. A better understanding of how DDR factors regulate cell fate decisions will drive the development of novel cancer therapies to improve local tumor control, and reduce the risk of therapy-induced tumor progression and secondary tumor induction.
Most cancer patients receive radiotherapy and/or chemotherapy that damages DNA and blocks DNA replication. Normal and tumor cells respond to these threats by activating DNA repair pathways and arresting cell cycle progression, collectively called the DNA damage response network. This project is focused on understanding how DNA damage response proteins determine cell fate after chemotherapy. These studies will help us design more effective therapeutic strategies that increase local tumor control, and minimize therapy- induced tumor progression and secondary tumor induction.
|Ashley, Amanda K; Shrivastav, Meena; Nie, Jingyi et al. (2014) DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation, fork restart, homologous recombination and mitotic catastrophe. DNA Repair (Amst) 21:131-9|
|Kim, Hyun-Suk; Chen, Qiujia; Kim, Sung-Kyung et al. (2014) The DDN catalytic motif is required for Metnase functions in non-homologous end joining (NHEJ) repair and replication restart. J Biol Chem 289:10930-8|
|Wray, Justin; Williamson, Elizabeth A; Singh, Sudha B et al. (2013) PARP1 is required for chromosomal translocations. Blood 121:4359-65|
|Allen, Christopher; Borak, Thomas B; Tsujii, Hirohiko et al. (2011) Heavy charged particle radiobiology: using enhanced biological effectiveness and improved beam focusing to advance cancer therapy. Mutat Res 711:150-7|
|Fnu, Sheema; Williamson, Elizabeth A; De Haro, Leyma P et al. (2011) Methylation of histone H3 lysine 36 enhances DNA repair by nonhomologous end-joining. Proc Natl Acad Sci U S A 108:540-5|
|Ponder, Jessica; Yoo, Byong Hoon; Abraham, Adedoyin D et al. (2011) Neoamphimedine circumvents metnase-enhanced DNA topoisomerase II? activity through ATP-competitive inhibition. Mar Drugs 9:2397-408|
|Allen, Chris; Ashley, Amanda K; Hromas, Robert et al. (2011) More forks on the road to replication stress recovery. J Mol Cell Biol 3:4-12|
|De Haro, Leyma P; Wray, Justin; Williamson, Elizabeth A et al. (2010) Metnase promotes restart and repair of stalled and collapsed replication forks. Nucleic Acids Res 38:5681-91|