Researchers at The Wistar Institute say they have discovered a new pathway that allows the detection of DNA in the cytoplasm and triggers inflammation and cellular senescence. This pathway may be modulated during senescence-inducing chemotherapy to affect cancer cell response to checkpoint inhibitors, say the scientists who published their study (“Topoisomerase 1 cleavage complex enables pattern recognition and inflammation during senescence”) online in Nature Communications.

“Cyclic cGMP-AMP synthase (cGAS) is a pattern recognition cytosolic DNA sensor that is essential for cellular senescence. cGAS promotes inflammatory senescence-associated secretory phenotype (SASP) through recognizing cytoplasmic chromatin during senescence. cGAS-mediated inflammation is essential for the antitumor effects of immune checkpoint blockade. However, the mechanism by which cGAS recognizes cytoplasmic chromatin is unknown. Here we show that topoisomerase 1-DNA covalent cleavage complex (TOP1cc) is both necessary and sufficient for cGAS-mediated cytoplasmic chromatin recognition and SASP during senescence,” write the investigators.

Rugang Zhang, PhD
Lead researcher Rugang Zhang, PhD, and scientists in The Wistar Institute’s Zhang lab.

“TOP1cc localizes to cytoplasmic chromatin and TOP1 interacts with cGAS to enhance the binding of cGAS to DNA. Retention of TOP1cc to cytoplasmic chromatin depends on its stabilization by the chromatin architecture protein HMGB2. Functionally, the HMGB2-TOP1cc-cGAS axis determines the response of orthotopically transplanted ex vivo therapy-induced senescent cells to immune checkpoint blockade in vivo. Together, these findings establish a HMGB2-TOP1cc-cGAS axis that enables cytoplasmic chromatin recognition and response to immune checkpoint blockade.”

Cellular senescence is a natural tumor suppression mechanism that stably halts proliferation of damaged or premalignant cells. Senescent cells also represent a trigger of inflammation and immune reaction as they produce an array of inflammatory molecules collectively known as senescence-associated secretory phenotype (SASP).

“Uncovering an important step that mediates the senescence response and enables the SASP, we identified a novel molecular pathway involved in immunotherapy response,” said lead researcher Rugang Zhang, PhD, deputy director of The Wistar Institute Cancer Center, professor and co-program leader of the gene expression and regulation program. “We suggest that this pathway might be targeted to modulate senescence-inducing effects of cancer therapeutics and affect response of senescent cancer cells to immunotherapy.”

Cells that have been exposed to various stressors and have suffered substantial DNA damage, for example during chemotherapy, transport pieces of DNA from the nucleus to the cytoplasm as a way to signal that something is wrong. cGAS senses cytosolic DNA and activates senescence and immunity by triggering a cascade of cellular events that culminate with production of the SASP. How cGAS senses DNA was unknown.

To investigate this, the Zhang lab focused on the proteins attached to cytoplasmic DNA in senescent cells and identified topoisomerase 1 (TOP1) as the missing link between cGAS and DNA. TOP1 is an enzyme that unwinds the DNA helix to facilitate its replication and transcription to RNA. It has the ability to attach to DNA forming TOP1cc. According to the new study, TOP1 also interacts with cytosolic DNA and cGAS, connecting the two and facilitating the DNA-sensing activity of cGAS.

Importantly, researchers also found that HMGB2, a protein that regulates chromatin structure and orchestrates the SASP at the gene expression level, enhances the interaction of TOP1 with DNA by stabilizing the DNA-bound form TOP1cc and is required for senescence and SASP.

The research team went on to establish that the HMGB2-TOP1cc-cGAS pathway is essential for the antitumor effect of immune checkpoint blockade therapy in a mouse model, as knockdown of HMGB2 abated response to anti-PD-L1 treatment. Treating tumors with a TOP1 inhibitor that stabilizes the TOP1cc-DNA binding and mimics HMGB2 restored treatment response and increased survival.

“TOP1 inhibitors are clinically used for cancer therapy,” said Bo Zhao, PhD, first author of the study and a postdoctoral researcher in the Zhang Lab. “We suggest they may have additional applications to sensitize tumors to immunotherapy, especially targeting cancer cells that become senescent in response to therapies such as chemotherapy or radiotherapy.”

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