M5L3 DNA damage and the immune system

  • PRRs in innate immunity - best known PRRs = TLRs

    • Eg. recognition of bacterial cell wall components by TLR4 (interacts with LPS which activates inflammatory genes)

  • For each PRR, the more general a pathogen component that is recognised, the wider the range of protection

    • Genetic material is the most general component of pathogens

    • Pathogen DNA is detected by cGAS which triggers innate immune response

  • cGAS interacts with pathogen DNA (undergoes allosteric conformational change) and generates cGAMP (second messenger)

  • cGAMP interacts with STING (a dimer which acts as a signalling platform, stabilised by cGAMP)

    • Dimers get arranged in an array which help in signalling

  • STING is an ER membrane bound protein which gets shuttled to the Golgi upon binding to cGAMP

  • STING activates IKK and more importantly TBK1

    • Upon cGAMP engagement thgere is a rotation in the molecule relative to the TMD which releases the CTD of STINGto engage/activate TBK1

    • TBK1 in close proximity due to the array of STING dimers allows them to cross-phosphorylate and activate each other

  • Active TBK1 activates/phosphorylates IRF3, causing it to dimerise (generates type I interferons eg IFNb) whereas IKK phosphorylates NFkB (generates inflammatory cytokine production)

  • IGN drives expression of interferon stimulated genes (ISGs)

    • ISGs have diverse functions - limiting specific pathogen functions, activating adapative immunity, recruiting specific immune cells …

    • PD-L1 induction by IFN prevents overstimulation of immune response

  • cGAMP can be transmitted to neighbouring cells via gap junctions, interferons and cytokines are also diffusible

  • cGAS and STING expression are mildly stimulated by interferon, and expression of other DNA and RNA sensors are also stimulated by interferon (positive feedback)

  • Turning off signalling

    • ABCC1 (channel protein) removes cGAMP from intracellular space

    • Other cGAMP importers (SLC19A1, SLC46A2, LRCC8A) can allow STING to enter back into the same cell or other cells

    • Secreted ENPP1 converts cGAMP to AMP + GMP to prevent it from being imported back

    • STING activation also induces its own degradation via autophagy

    • TREX1 and other nucleases can also degrade DNA which removes the starting substrate for the cGAS pathway

  • Cancer cells rewire their cGAS-STING signalling

  • Why is cGAS not constantly activated by self-DNA?

    • cGAS is in the cytoplasm - however would it be able access chromosomal DNA?

    • Cytoplasmic cGAS enriches on chromosomes during mitosis

    • When nuclear envelope breaks down during mitosis then all of it gets ‘soaked up’ by chromosomes so it can be localised to the nucleus after the nuclear membrane reforms

    • Chromatin is a potential marker for self-DNA, nucleosomes may protect from cGAS binding

    • cGAS bind nucleosomes with higher affinity than naked DNA which inhibits it

  • Aicardi-Gutieres and Lupus syndromes - autoinflammatory diseases caused by genetic mutations eg cytoplasmic exonuclease TREX1 and DNA repair enzyme RNase H2

    • Lethality of TREX1 loss can be rescured by loss of cGAS

  • DNA damage, genome instability and other stressors can activate cGAS

    • Causing inflammation, apoptosis, senescence…

    • Under some conditions it may also drive survival and invasiveness

    • Depending on the context it may be pro- or anti-cancer

  • Hypothesis - cGAS may be activated by micronuclei (tiny, nucleus-like structures containing a chromosome fragment or whole chromosome that failed to incorporate into the main daughter nucleus during cell division, forming a small, membrane-bound body)

    • Micronuclei are a common consequence of chormosomal instability, DNA damage, chromosome mis-segregation, nuclear envelope assembly defects

      • attractive candidate for cGAS activation upon DNA damage

    • Micronuclei have fragile nuclear envelopes

    • Micronuclei accumulate cGAS

    • However, micronuclei contain chromatin which should inactivate cGAS

  • Alternative sources of cGAS activation - mitochondrial DNA

    • Radiotherapy damages not only nuclear DNA but also mitochondria which may activate cGAS

    • However not all data supports this

  • Genome instability is generally a poor stimulant of cGAS signalling, but their is evidence that at some point in tumour evolution there is a selection pressure to reduce cGAS signalling (reason unclear)