Targeting DNA damage response pathways to activate the STING innate immune signaling pathway in human cancer cells

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.

Checkpoint Kinase 1 (Chk1) inhibition fails to activate the Stimulator of Interferon Genes (STING) innate immune signalling in a human coculture cancer system

“…We have previously demonstrated the failure of Chk1 inhibitors to induce a Type I IFN response in solid tumour cell lines growing in culture [23]. Here we further expand these studies to leukaemia derived cell lines, and subsequently in a coculture system with pre-treated human solid cancer cell lines.…”

“…Twenty-four hours treatment with V158411, however, significantly increased the fraction of HT29 and HCC1937 but not U2OS nuclei staining anti-dsDNA antibody positive (Fig. 3b, adapted from [23]). This suggests that V158411 treatment of HT29 and HCC1937 cells resulted in reduced nuclear membrane integrity.…”

“…3-times the γH2AX EC 50 (3x γH2AX EC 50 ) of V158411 for 24 h increased cytoplasmic dsDNA in HT29 cells 4-5-fold compared to DMSO treated control cells (Fig. 3a, adapted from [23]). In U2OS cells, the increase in cytoplasmic dsDNA by V158411 was less robust being 2-4fold dependent on the mouse monoclonal antibody used.…”

“…The innate immune response has been demonstrated to be activated by cytoplasmic dsDNA resulting in a robust type I interferon response [34]. We have previously demonstrated that Chk1 inhibition in solid cancer cell lines such as HCC1937 or HT29 failed to activate the cGAS/ STING pathway downstream of TBK1 [23]. However, damage-associated molecular patterns (DAMPs) (endogenous danger molecules release for damaged or dying cells) can activate components of the innate immune system such as natural killer cells, dendritic cells, or monocytes/macrophages [35,36].…”

“…Chk1 inhibitors, including V158411, increase DNA damage and replication stress, as measured by increased pan-nuclear γH2AX, in HT29, U2OS or HCC1937 cells with EC 50 values (γH2AX EC 50 ) of 0.80, 0.64 and 0.26 μM respectively [23,33] (Supplementary Table 1). Increased DNA damage can result in increased cytoplasmic DNA.…”