Inflammatory gene expression following genotoxic cancer therapy is well documented, yet the events underlying its induction remain poorly understood. Inflammatory cytokines modify the tumor microenvironment by recruiting immune cells and are critical for both local and systemic (abscopal) tumor responses to radiotherapy1. An enigmatic feature of this phenomenon is its delayed onset (days), in contrast to the acute DNA damage responses that occur in minutes to hours. Such dichotomous kinetics implicate additional rate limiting steps that are essential for DNA-damage induced inflammation. Here, we show that cell cycle progression through mitosis following DNA double-strand breaks (DSBs) leads to the formation of micronuclei, which precede activation of inflammatory signaling and are a repository for the pattern recognition receptor cGAS. Inhibiting progression through mitosis or loss of pattern recognition by cGAS-STING impaired interferon signaling. Moreover, STING loss prevented the regression of abscopal tumors in the context of ionizing radiation and immune checkpoint blockade in vivo. These findings implicate temporal modulation of the cell cycle as an important consideration in the context of therapeutic strategies that combine genotoxic agents with immune checkpoint blockade.

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 No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint .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....

SUMMARY MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection and signaling; yet, how its endo- and exonuclease activities regulate DSB repair by non-homologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors we examined repair pathway choice at DSBs generated in G2 following radiation exposure. Whilst endo- or exonuclease inhibition impairs radiation-induced RPA chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, whilst exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exo and EXO1/BLM bidirectional resection towards and away from the DNA end, which commits to HR.

Migration through micron-size constrictions has been seen to rupture the nucleus, release nuclear-localized GFP, and cause localized accumulations of ectopic 53BP1 – a DNA repair protein. Here, constricted migration of two human cancer cell types and primary mesenchymal stem cells (MSC) increases DNA breaks throughout the nucleoplasm as assessed by endogenous damage markers and by electrophoretic ‘comet’ measurements. Migration also causes multiple DNA repair proteins to segregate away from DNA, with cytoplasmic mis-localization sustained for many hours as is relevant to delayed repair. Partial knockdown of repair factors that also regulate chromosome copy numbers is seen to increase DNA breaks in U2OS osteosarcoma cells without affecting migration and with nucleoplasmic patterns of damage similar to constricted migration. Such depletion also causes aberrant levels of DNA. Migration-induced nuclear damage is nonetheless reversible for wild-type and sub-cloned U2OS cells, except for lasting genomic differences between stable clones as revealed by DNA arrays and sequencing. Gains and losses of hundreds of megabases in many chromosomes are typical of the changes and heterogeneity in bone cancer. Phenotypic differences that arise from constricted migration of U2OS clones are further illustrated by a clone with a highly elongated and stable MSC-like shape that depends on microtubule assembly downstream of the transcription factor GATA4. Such changes are consistent with reversion to a more stem-like state upstream of cancerous osteoblastic cells. Migration-induced genomic instability can thus associate with heritable changes.