Determining the role of nuclear envelope reformation proteins in regulating the cGAS/STING innate immune response in cancer

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PROJECT SUMMARY/ABSTRACT: In cancer cells, exposure of self DNA to the cytosol is driven by a variety of genomic instabilities such as micronuclei, chromatin bridges, and nuclear ruptures. This cytosolic DNA can be recognized by cytosolic DNA sensors such as cGAS (cyclic GMP-AMP synthase), which triggers a downstream innate immune response. Interestingly and confoundingly, the activation of the cGAS/STING innate immune pathway can protect or sensitize tumors to immunotherapeutic interventions depending on the specific context. Therefore, insight into the ways in which cGAS/STING signaling is regulated in cancer can inform targeted intervention. Sources of cytosolic DNA in cancer cells arise primarily from defects in mitosis that lead to the enclosure of chromosomes in micronuclei that are prone to rupture. These ruptured micronuclei recruit cGAS and nuclear envelope reformation (NER) factors—such as LEM2, CHMP7, and BAF—but it remains unknown how, or if, these NER factors impact cGAS/STING signaling but there is emerging evidence in published and in our preliminary data that there is potential crosstalk between cGAS/STING signaling and NER proteins. The goal of this proposal is to provide key insights into the regulation of the innate immune response to cytosolic DNA in cancer cells by nuclear envelope reformation factors. In order to achieve this goal, I will use transfected herring testes (HT) DNA and transfected DNA-coated beads as models for cytosolic DNA as this can be more readily controlled compared to the stochastic formation of micronuclei, only some of which are unstable and prone to rupture. With this model, I will use CRISPR/Cas9 gene-editing and the auxin-inducible-degron (AID) conditional degradation system to probe the roles of NER factors in cGAS/STING signaling in response to transfected HT DNA and DNA beads. This proposal will address fundamental aspects of cell biology and innate immune signaling that will shed light on immunotherapeutic targets for cancer.