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Updated: Nov 19, 2025
Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo
Published on: September 18, 2014
Targeting DNA damage response pathways to activate the STING innate immune signaling pathway in human cancer cells
Joanne Wayne1, Teresa Brooks1, Alexandra Landras1
1Vernalis (R&D) Ltd, Cambridge, UK.
Abstract:
Activating stimulator of interferon genes to turn immunologically refractive cold tumor hot is an exciting therapeutic approach to increase the clinical responsiveness of some human cancers to immune checkpoint inhibitors. DNA damaging drugs and PARP inhibitors are two types of agents that have demonstrated this potential. Inhibitors of Chk1 or Wee1 induce DNA damage in cancer cells in predominantly the S-phase population. Increased cytoplasmic single-stranded and double-stranded DNA (dsDNA) from this DNA damage resulted in increased tank-binding kinase 1 (TBK1) phosphorylation in a range of cancer cell lines. However, despite robust increases in pTBK1, no downstream consequences of TBK1 phosphorylation were observed (namely no increase in pIRF3/7, interferon regulatory factor (IRF)-dependent gene expression or a type I IFN response). In combination with cytotoxic chemotherapy such as gemcitabine or camptothecin (CPT), Chk1 inhibition increased cytoplasmic dsDNA compared with the cytotoxic alone but attenuated the cytotoxic chemotherapy-induced increase in IRF1 protein and STAT1 phosphorylation through inhibition of nuclear RelB translocation. Despite increased cytoplasmic DNA and TBK1 activation, inhibition of Chk1, ataxia telangiectasia and Rad3-related protein, or Wee1 failed to activate a type I IFN response. We discuss the potential underlying mechanisms for this lack of IRF-dependent gene response and how this might influence the clinical strategies of combining Chk1 or Wee1 inhibitors with immune checkpoint inhibitors.
Insights
Inhibitors of Chk1 or Wee1 induce DNA damage, increasing cytoplasmic DNA and TBK1 activation in cancer cells. However, these agents failed to trigger a type I interferon response, impacting combination strategies with immune checkpoint inhibitors.
Area of Science:
- Oncology
- Immunology
- Molecular Biology
Background:
- Immune checkpoint inhibitors (ICIs) show promise in treating cancers, but many tumors are refractory.
- Activating interferon (IFN) genes can enhance tumor immunogenicity, potentially increasing ICI efficacy.
- DNA-damaging agents, including PARP inhibitors and Chk1/Wee1 inhibitors, are explored for their ability to 'heat' cold tumors.
Purpose of the Study:
- To investigate the effects of Chk1 and Wee1 inhibitors on DNA damage, TBK1 activation, and type I IFN responses in cancer cells.
- To evaluate the impact of combining Chk1 inhibition with cytotoxic chemotherapy on DNA damage and IFN signaling.
- To explore mechanisms underlying the failure to elicit an IFN response and its implications for combination therapies.
Main Methods:
- Treatment of cancer cell lines with Chk1 inhibitors, Wee1 inhibitors, and cytotoxic chemotherapy (gemcitabine, CPT).
- Assessment of cytoplasmic single-stranded and double-stranded DNA (dsDNA) levels.
- Measurement of tank-binding kinase 1 (TBK1) phosphorylation, interferon regulatory factor (IRF) activation, and type I IFN response markers.
Main Results:
- Chk1/Wee1 inhibition increased cytoplasmic DNA and TBK1 phosphorylation in cancer cells.
- Despite increased pTBK1, downstream IFN regulatory factor (IRF)-dependent gene expression and type I IFN responses were not observed.
- Combination of Chk1 inhibition with cytotoxic chemotherapy increased cytoplasmic dsDNA but attenuated chemotherapy-induced IRF1 and STAT1 activation.
Conclusions:
- Inhibition of Chk1 or Wee1 alone or in combination with cytotoxic chemotherapy fails to activate a type I IFN response, despite inducing DNA damage and TBK1 activation.
- Potential mechanisms for the lack of IRF-dependent gene response need further investigation.
- These findings may necessitate adjustments in clinical strategies for combining Chk1/Wee1 inhibitors with ICIs.
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