Iman Khan, Michelle Melanson, Myungsun Shin, Isabella Stelter, Raie Bekele, Jean-Bernard Lazaro, Joe Mancias, Nadine Elowe, Kent Mouw

Abstract PR002: Functional screens identify a role for CHK1 in early nucleotide excision repair

  • Cancer Research
  • Oncology
Save to CiteDrive Show PDF

Abstract Nucleotide excision repair (NER) is a highly conserved pathway that repairs DNA intrastrand adducts caused by genotoxins such as UV radiation and platinum chemotherapies. The NER pathway is activated when DDB2 binds at damage sites and recruits XPC and additional NER factors. However, the molecular regulatory events that govern the earliest steps in NER are poorly understood. In order to uncover novel mediators of DNA damage-induced NER activation, we developed an immunofluorescence-based DDB2 microscopy assay and adapted it for high-throughput genetic and drug screens. These functional screens converged on an unexpected role for CHK1 activity in NER regulation. CHK1 depletion or pharmacologic inhibition resulted in aberrant DDB2 retention at UV-induced DNA damage sites, therapy preventing recruitment of XPC and other downstream NER proteins. This effect was measurable within minutes of DNA damage and was independent of the cell cycle. ATR inhibition, but not ATM inhibition, also led to retention of DDB2 at UV damage sites and faulty XPC recruitment, consistent with an ATR-dependent role for CHK1 in early NER. Subsequent mechanistic analyses revealed that CHK1 inhibition delayed ubiquitination of DDB2 by Cullin 4 (CUL4), thereby preventing DDB2 release from DNA. CUL4A is an E3 ubiquitin ligase that is part of the Cullin 4-RING E3 ubiquitin ligase (CRL) 4 complex which also includes a RING domain protein, the adaptor protein DDB1, and a substrate recognition protein. In NER, DDB2 serves as the substrate recognition protein for the CRL4 complex. Activation of CRL4 ubiquitin ligase activity requires post-translational modification of CUL4 via neddylation and concomitant release of the COPS9 signalosome from the CRL4-DDB2 complex. CHK1 inhibition was sufficient to prevent CUL4 neddylation and COPS9 release from the CRL4-DDB2 complex. We therefore searched for potential CHK1 phosphorylation sites with the CRL4-DDB2 complex and identified two putative CHK1 phosphorylation sites (R-x-x-S/T) in DDB2 at S24 and S26. Mutating these serines to alanines led to a dramatic decrease in UV-induced DDB2 ubiquitination and an increase in retention of DDB2 at damage sites, similar to the effects of CHK1 inhibition, suggesting that CHK1-mediated phosphorylation of DDB2 is required for DDB2 ubiquitination and subsequent steps of NER. We are now testing whether CHK1 is able to directly phosphorylate DDB2 at S24 and S26. Together, our data identify a novel role CHK1 in NER. ATR-dependent CHK1 activity is required for CUL4-mediated ubiquitination and subsequent degradation of DDB2 at NER damage sites. When CHK1 is lost or inhibited, DDB2 is retained at damage sites and prevents timely recruitment of downstream NER factors, leading to increased cellular sensitivity to NER damaging agents. These data have important implications for clinical development of ATR and CHK1 inhibitors in both the monotherapy and combination therapy settings. Citation Format: Iman Khan, Michelle Melanson, Myungsun Shin, Isabella Stelter, Raie Bekele, Jean-Bernard Lazaro, Joe Mancias, Nadine Elowe, Kent Mouw. Functional screens identify a role for CHK1 in early nucleotide excision repair [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR002.

Need a simple solution for managing your BibTeX entries? Explore CiteDrive!

  • Web-based, modern reference management
  • Collaborate and share with fellow researchers
  • Integration with Overleaf
  • Comprehensive BibTeX/BibLaTeX support
  • Save articles and websites directly from your browser
  • Search for new articles from a database of tens of millions of references
Try out CiteDrive

More from our Archive