P14: ART1, a mono-ADP-ribosyl transferase, is increased by chemotherapy and radiotherapy and is a novel target in colorectal carcinoma.

Mono-ADP-ribosyltransferase-1 (ART1) utilizes free NAD+ to mono-ADP-ribosylate (MARylate) arginine-rich target proteins. We previously observed ART1 blockade decreases tumor growth in murine lung and melanoma cancer models due to effects on CD8 T cell infiltration and activity.  Additionally, ART1 has been implicated in tumor progression of colorectal cancer cells.  Here we investigate ART1 expression and enzymatic activity in colorectal cancer cells following cytotoxic treatment and blockade by anti-ART1 antibody.


Methods: ART1 and MAR expression were evaluated by immunofluorescence in the murine CT26 colorectal cancer cell line. Cells were treated with Cisplatin (Cis) for 24 hours (15µM) and with 8Gy of radiotherapy (R) for 3 consecutive days with or without blockade of ART1 with a novel humanized monoclonal antibody (22C12), which binds to ART1 and inhibits MARylation. ART1 expression and MARylation(as measured by MABE1076 antibody) were compared using mean fluorescence index (MFI) per condition. (Using t-test or appropriate non-parametric test depending upon distribution). We also evaluated the antiproliferative effect of ART1 pathway blockade on CT26 cells (WST-1 assay). 


Results:

CT26 cells express ART1 and we observed MARylation in untreated cells. Following treatment with Cis, we observed a significant increase in surface ART1 (sART1) (2.04-fold ,p=0.0016) and surface MARylation (sMAR), (2.48-fold,p < 0.0001). The cisplatin-mediated increase in sMAR was inhibited by 22C12(0.50-fold, p< 0.0001) (Fig1A). Upon radiation treatment, sART1 expression was significantly increased compared to non-radiated CT26 cells (2.45-fold, p< 0.0001).  sMAR was also significantly increased with radiation.   22C12 antibody inhibited theses radiation induced changes (1.99-fold R vs C, p< 0.0001 and 0.68-fold Rad+22C12 vs C, p< 0.001, respectively)(Fig1B). Treatment with 22C12 decreased proliferation of CT26 cells compared to untreated controls (77.24% of control at 72 hours, p= < 0.001).


Conclusions: ART1 expression increases in the CT26 colorectal cancer cell line following treatment with chemotherapy or radiation suggesting MARylation of cell surface proteins activates cell signaling pathways and cell survival.  ART1 blockade may be a therapeutic adjunct to overcome treatment resistance in colorectal cancer and potential an immune response to tumor. Further investigation of the potential synergistic effects of anti-ART1 therapy with commonly used therapeutics are warranted.