Mitotic progression following DNA damage enables pattern recognition within micronuclei

Abstract

Radiotherapy and many chemotherapeutics rely on DNA double strand break (DSB) formation to drive the killing of tumor cells over several cell division cycles 2,3 . Concomitant with this protracted cell death schedule, inflammatory cytokine production increases over days following the insult. As a host of cytokines and inflammatory signals are produced following ionizing radiation (IR) 4,5 , we used STAT1 phosphorylation at Y701 as a surrogate for inflammatory pathway activation ( Fig. 1a and 1b). MCF10A mammary epithelial cells showed STAT1activation between 3 and 6 days post-IR with a dose-dependent threshold of at least 5Gy. TotalAll rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/156414 doi: bioRxiv preprint first posted online Jun. 27, 2017; STAT1 protein and the mRNA levels of multiple inflammatory genes were also detected in a time dependent manner (Fig. 1c. and Extended data 1a). Using inducible nucleases ( Fig. 1d and Extended data 1b and 1c), we observed a delayed accumulation of active STAT1 and inflammatory gene expression, confirming these signals are driven by DSBs.We reasoned that if residual DSBs were driving inflammatory signals then failure of nonhomologous end-joining (NHEJ) DSB repair should amplify the response. Paradoxically, we observed that inhibition of DNA-PKcs (DNA-PKi) or CRISPR-Cas9 knockout of multiple NHEJ components diminished STAT1 activation in MCF10A and prostate epithelial cells (Fig 1e and Extended data 1c and 1d). STAT1 activation through exogenous IFNβ1 was unaffected by DNAPKi (Extended data 1e), ruling out a direct role in STAT1 phosphorylation. Inhibition of ATM kinase on the other hand had little influence over the level of STAT1 activation (Extended data 1f).DSB-induced STAT1 activation correlated with the appearance of aberrantly shaped nuclei and micronuclei (Fig 1f and 2a). The DSB marker γH2AX was increased in micronuclei however 53BP1 was absent, consistent with their reported defects in DSB signaling (Fig 1f) 6,7 . Nuclei of NHEJ knockout and DNA-PKi treated cells were morphologically normal despite being replete with DSBs (Fig 1f and 2a). As micronuclei are byproducts of mitotic progression, these data suggest that STAT1 activation occurred following mitosis 8 . Flow cytometry showed progression of parental cells from G2 into G1 between 24 and 48h post-IR, whereas NHEJ knockouts were static over this time (Extended data 2a). This corresponds to a wave of parental cells moving into mitosis as evidenced by phospho-histone H3 staining that is absent in the NHEJ knockout cellsAll rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/156414 doi: bioRxiv preprint first posted online Jun....

Regulation of programmed death‐ligand 1 expression in response to DNA damage in cancer cells: Implications for precision medicine

“…10 (Figure 2). 25,26 The notion that the cGAS/STING pathway is involved in the immune response within the tumor environment is supported by the finding that a specific Chk1 inhibitor used in clinical trials upregulates cGAS/STING signaling subsequent to the formation of micronuclei, possibly due to defective G 2 /M checkpoint arrest. 27 Micronuclei-induced cGAS/STING signaling under Chk1 inhibition stimulates the immune response, including PD-L1 expression.…”

“…In addition, from the clinical point of view, cGAS/STING-dependent immune activation is considered to be involved in promoting the abscopal effect (a systemic antitumor response distant from the X-ray-irradiated tumors). 25 The ATR/Chk1-dependent upregulation of PD-L1 expression occurs during G 2 /M checkpoint arrest, that is, at an early time after DNA damage. However, we propose that the cGAS/STING-dependent immune response is induced at the mid-phase of the immune response after DNA damage because micronuclei are generated following the release of cells from G 2 /M checkpoint arrest.…”

Trial watch: Immunogenic cell death induction by anticancer chemotherapeutics

“…3 At the level of cancer cells, ICD depends upon the timely emission of a constellation of immunomodulatory damage-associated molecular patterns (DAMPs). 40,83 In the case of chemotherapy-induced ICD, these include (but may not be limited to): (1) surface-exposed endoplasmic reticulum (ER) chaperones including calreticulin (CALR) [84][85][86] ; (2) extracellular ATP; [87][88][89][90][91] (3) extracellular high mobility group box 1 (HMGB1) 13,92 ; (4) extracellular annexin A1 (ANXA1) 55 ; (5) secreted type I interferon; [93][94][95][96] and (6) extracellular nucleic acids. 97 That said, ICD triggered by stimuli other than chemotherapy (e.g., radiation therapy, photodynamic therapy) is not necessarily associated with the same DAMPs.…”

Critical questions in ovarian cancer research and treatment: Report of an American Association for Cancer Research Special Conference

“…56 DNA damage can induce inflammatory cytokine signaling that might augment effects of immunotherapy. 57 Inflammatory cytokines modify the tumor microenvironment by recruiting immune cells that are critical for both local and systemic responses to immunotherapy and radiotherapy in preclinical murine cancer models. 58 Cell cycle progression through mitosis following ionizing radiation or PARPi is essential to activate type 1 interferon responses.…”