Fig. 5: Modulation of cancer-driven immunity by neddylation inhibitors.

a MDA-MB-231 cells were treated with pevonedistat for 24 h and the NEDD8 expression was assessed in Western Blotting. Representative image of 3 independent repeats was shown. b CellTrace Violet (CTV)-pulsed primary human lymphocytes were incubated with pevonedistat in the presence of αCD3/28 activation beads ±rhIL2 (100 ng/ml). The resulting cell proliferation was quantified by flow cytometry. Representative experiment from 3 independent donors was shown. c Primary human lymphocytes were co-cultured with MDA-MB-231 cells in the presence of pevonedistat ± nivolumab or durvalumab (10 μg/ml). Soluble granzyme B and IFNγ were quantified by ELISA on day 5. Six independent blood donors were included and data was shown at mean ± SD, unpaired two-tailed T-test. d Effect of pevonedistat on parental or pevonedistat resistant MDA-MB-231 cells was tested in a live-cell imaging system. Representative experiment of 3 independent repeats was shown. e Primary human lymphocytes were co-cultured with parental or pevonedistat-resistant MDA-MB-231 cells ± nivolumab or durvalumab (10 μg/ml). Levels of soluble granzyme B and IFNγ were measured by ELISA on day 5. Five independent donors were included and data were shown with mean ± SD, unpaired two-tailed T-test. f NEDD8-activating enzyme 1 (NAE1) protein expression in control MDA-MB-231 cells or knock-out (KO) clones was measured using Western Blotting. Representative image of 2 independent repeats was shown. g The NAE1 KO MDA-MB-231 clone was treated with pevonedistat and cell proliferation was measured using live-cell imaging. Representative graph of 2 independent repeats was shown. h MDA-MB-231 cells were treated with 1000, 100 nM pevonedistat or 0.1% DMSO for 24 h and the total ubiquitin was tested using Western Blotting. Representative image of 2 independent repeats was shown. Control MDA-MB-231 cells were treated with 1000, 400, 100 or 10 nM of TAK-243 (i) or 1000, 100, 10 nM of pevonedistat (j) or 0.1% DMSO for 24 h, and NAE1 protein expression was measured using Western Blotting. Representative image of 2 independent repeats was shown. Source data are provided as a source data file.