2407 - Validating Dual Oxygenase 2 as a Modulator of Radiosensitivity and Expression of the Immune Checkpoint Program Death-Ligand 1 (PD-L1) in Esophageal Cancer
Presenter(s)
A. J. Allen1, M. B. Martins2, S. Azarbarzin3, J. K. Molitoris1, and F. Carrier1,4; 1Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, MD, 2University of Maryland Baltimore, Baltimore, MD, 3Department of Radiation Oncology, University of Maryland School of Medicine, baltimore, MD, 4University of Maryland, Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, MD
Purpose/Objective(s): The mechanisms underlying association of increased radiosensitivity with immune activation are still ill defined. Our data indicate that Dual Oxygenase 2 (DUOX2), an NADPH enzyme mediating H2O2 production, increases radiosensitivity by down regulating the Hypoxia Inducible Factor 1a and consequently represses expression of the immune checkpoint PD-L1 in gastric and esophageal cancer cell lines. Our objective is to investigate whether DUOX2 expression in archived gastroesophageal tumors samples correlate with patients’ response to radio- and/or immunotherapy.
Materials/Methods:
DUOX2 was down regulated by shRNA or CRISPR-Cas9 in stomach and esophageal cancer cell lines. Protein expression was assessed by Western Blots. Cells were exposed to increasing radiation doses and levels of Ceramide and glutathione (GSH) were measured by ELISA and enzymatic assay respectively. Cells viability was measured by the WST-1 and clonogenic survival assays. RNASeq data were obtained from Public European Nucleotide Archive (ENA) at the EMBL’s European Bioinformatics Institute (EMBL-EBI). DUOX2 and PD-L1 levels were quantitatively assessed and expressed as Transcripts Per Million (TPM). Over 200 archived biopsies from gastroesophageal cancer patients treated with Tri-modalities at our institution are being retrospectively analyzed for DUOX2, HIF1-a and PD-L1 expression on Tumor microarrays (TMA).Results: DUOX2 expression significantly increased cancer cells radiosensitivity at every radiation dose tested (0-4 Gy). DUOX2 increases radio-sensitivity by repressing HIF-1a and increasing Ceramide levels. In addition, DUOX2 expression represses PD-L1. Moreover, treating DUOX2-expressing cells with the Reactive Oxygen Species scavenger N-Acetylcysteine led to PD-L1 upregulation. Similarly, down regulation of DUOX2 also resulted in PD-L1 upregulation as well as GSH upregulation and increased radio-resistance in cancer cell lines. Analysis of publicly available RNA-Seq data sets indicated an inverse relationship between DUOX2 and PD-L1 expressions in subsets of stomach and esophageal cancer patients’ samples. Of the 256 patients who underwent Tri-modality therapy at our institution, 202 underwent esophagectomy that were archived for residual tumors TMA processing. Expression of DUOX2, HIF-1a and PD-L1 are currently being analyzed and will be evaluated in the context of patients’ response to radiation and immuno- therapies.
Conclusion: Pre-clinical studies indicate that DUOX2 expression contributes to radiosensitivity and represses an immune checkpoint. These findings warrant further clinical investigation of DUOX2 expression as a potential biomarker for radiation therapy and/or immune checkpoint inhibitor response.