ESTRO 2024 - Abstract Book

S5177

Radiobiology - Immuno-radiobiology

ESTRO 2024

In addition to these cell intrinsic effects, it is possible that the DNA damage associated with loss of redox homeostasis could stimulate inflammatory signaling. Accumulation of unresolved DNA damage can generate the release of cytosolic DNA which constitutes a potent trigger for the innate immune system, governing the production of secreted factors including pro-inflammatory chemokines and cytokines that drive immune cell infiltration, polarization and activation. We herein hypothesize that DNA damage induced upon loss of redox hoemostasis by targeting PRDX4 can generate cytosolic DNA and activate pro-inflammatory signaling to reshape the pancreatic tumor microenvironment and improve sensitivity to radio-immune therapy.

Material/Methods:

We genetically engineered human PANC-1 and mouse PDAC cells to express doxycycline (DOX)-inducible shRNA targeting PRDX4. Mouse cells were established from tumors that spontaneously developed in KPC (KrasG12D/+; Trp53R172H/+; Pdx1-Cre) and KC (KrasG12D, Pdx1-Cre) mice. Cytosolic DNA was detected and quantified using immunofluorescence from an antibody against double-strand (ds)DNA. Expression of a subset of genes encoding pro-inflammatory factors (CCL5, CXCL10, CCL20, ISG54, IFNB1) was assessed by quantitative PCR (qPCR). Immune factor secretion was detected by specific and multiplex ELISA assays. Isolation of specific immune cell populations (macrophages, dendritic cells, T cells) and co-cultures with tumor cells in vitro are ongoing to assess how PRDX4 loss +/- radiation may reshape the tumor microenvironement. Finally, the survival of mice knocked-out for PRDX4 in a KC and KPC context is currently under study.

Results:

We show that DNA damage induced by PRDX4 loss in human PANC-1 and mouse pancreatic cancer cells leads to substantial accumulation of cytosolic DNA, both in micronuclei and present as free double-stranded DNA in the cytosol. Micronuclei that emerge after cells go through mitosis with unresolved DNA damage have been showed to activate the pattern-recognition receptor cyclic GMP-AMP synthase (cGAS) and the downstream Stimulator of interferon genes (STING) regulator. Consistently, we show colocalization of micronuclei and cGAS upon PRDX4 depletion as well as activation of STING-target genes, such as Interferon Stimulated Genes (ISGs) ISG54 or OAS1. PRDX4 loss also results in the transcriptional upregulation and protein secretion of key cytokines involved in antitumor responses, like CXCL10, CCL20 and CCL5. Interestingly, this is dependent on STING, as treatment with an inhibitor or siRNA-mediated depletion substantially blunts this upregulation. Finally, PRDX4 loss combined with radiation significantly increased the expression of pro-inflammatory genes and impaired cell growth and survival compared to single treatment alone.

Conclusion:

Disruption of redox homeostasis generates substantial amounts of cytosolic DNA and results in an upregulation of specific pro-inflammatory genes. Of note, these effects are significantly strengthened in combination with radiation, compared to either treatment alone. Multiplexed ELISA assays reveal an increased secretion of key chemokines, including CXCL10 and CCL20, both involved in the recruitment of T cells under these conditions. Taken together, our data suggest that exploiting vulnerable redox homeostasis may cooperate with radiation to result in immune modulation, potentially for the benefit of immune therapy response in PDAC.

Keywords: Redox homeostasis, cytosolic DNA, inflammation

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