Fig 2: Effects of pevonedistat treatment on cell cycle progression, p21 and CDT1 accumulation. (a) Cell cycle analysis by double staining with (propidium iodide) PI and pH3 showing an accumulation of cells in the S or G2 phase of the cell cycle predominantly in sensitive cells (Mero-82, MSTO211H and ONE58) treated with 0.3-µM pevonedistat for 24 h compared to control (treated only with dimethylsulfoxide (DMSO)). We noted a drastically increased accumulation of cells containing >4N DNA after the treatment, predominantly in the sensitive cell lines, without increased accumulation of cells undergoing mitosis (pH3+, circles). (b) Cells were treated with an increasing concentration of pevonedistat for 24 h and subjected to Western blot analysis for the indicated proteins. Number above each line of the blots represents protein expressions (ratio densitometry of the bands normalized with β-actin) relative to no pevonedistat treatment. Arrows indicate neddylated CUL4A or CUL4B. (c) A panel of MPM cell lines treated with DMSO or 0.6-µM pevonedistat for 24 h shows an accumulation of CDT1 and p21 in both sensitive (underlined) and resistant cell lines. Number above each line of the blots represents protein expressions (ratio densitometry of the bands normalized with β-actin) relative to no pevonedistat treatment.

Fig 3: In vitro response of MPM cell lines to pevonedistat. (a) IC50 showing the pevonedistat concentration (µM) required for the inhibition of 50% cell survival after 3 days of drug exposure (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay). The graph depicts the mean IC50 from at least two independent experiments from each cell line. (b) Gene expressions of CUL4A and CUL4B in MPM cell lines normalized to the expression of the nonmalignant mesothelial cell line SDM104. (c,d) Western blot analysis showing protein expressions of CUL4A and CUL4B in MPM cell lines and nonmalignant mesothelial cell lines (Met5A and SDM104). The upper band (see arrows) of CUL4A and CUL4B represent the neddylated form of both proteins. Treatment with 0.6-µM pevonedistat for 24 h sufficiently inhibited the neddylation of CUL4A and CUL4B in most of the cell lines. The number above each line of the blots represents the protein expression (ratio densitometry of the bands normalized with β-actin) relative to untreated SDM104. Underlined are the cell lines sensitive to the neural precursor cell expressed, developmentally downregulated (NEDD) inhibition by pevonedistat.

Fig 4: Effect of pevonedistat treatment on the expression of CCL2. (a) Comparison of the baseline expression levels of CCL2 in four different MPM cell lines in vitro. Lower Ct is equivalent to a higher expression. (b) qPCR analysis using mouse and human-specific CCL2 primers (using human-specific β-actin and mouse-specific β-2-microglobulin as a reference gene) showing a significant reduction of human CCL2 in the ACC-Meso-1 tumor treated with pevonedistat. (c) Change in the expression of human CCL2 in vitro after pevonedistat treatment in MPM cell lines. (d) Scheme illustrating the rationale and findings of our current study. CUL4A and CUL4B are shown to be regulated by Merlin dysfunction; here, we sought to investigate the importance of CUL4A and CUL4B expressions in MPM progression and tested the efficacy of pevonedistat, a small molecule inhibiting cullins via protein neddylation. We observed that a high CUL4B expression was associated with the worst clinical outcome. The treatment of MPM cells with pevonedistat resulted in an accumulation of CDT1 and p21, known targets of CUL4 and CUL1, leading to cell cycle arrest. Reduced nuclear factor- κB (NF-κB) pathway resulting from CUL1 inhibition caused a reduced CCL2 expression in tumor cells and reduced macrophage infiltration in tumors. Via an unknown mechanism, a reduced macrophage infiltration may result in more tumor cell death. It is important to note that CUL4A and CUL4B are shown to regulate several ubiquitination targets; thus, these targets may be important for MPM progression, and this remains to be investigated. In addition to cullins, pevonedistat also can affect some other proteins that are also regulated by neddylation. # not significant; * p < 0.05, Ct, threshold cycle and ND, not done.