NEDD8-targeting drug MLN4924 elicits DNA rereplication by stabilizing Cdt1 in S phase, triggering checkpoint activation, apoptosis, and senescence in cancer cells

Abstract

MLN4924 is a first-in-class experimental cancer drug that inhibits the NEDD8-activating enzyme, thereby inhibiting cullin-RING E3 ubiquitin ligases and stabilizing many cullin substrates. The mechanism by which MLN4924 inhibits cancer cell proliferation has not been defined, although it is accompanied by DNA rereplication and attendant DNA damage. Here we show that stabilization of the DNA replication factor Cdt1, a substrate of cullins 1 and 4, is critical for MLN4924 to trigger DNA rereplication and inhibit cell proliferation. Even only 1 hour of exposure to MLN4924, which was sufficient to elevate Cdt1 for 4-5 hours, was found to be sufficient to induce DNA rereplication and to activate apoptosis and senescence pathways. Cells in S phase were most susceptible, suggesting that MLN4924 will be most toxic on highly proliferating cancers. Although MLN4924-induced cell senescence seems to be dependent on induction of p53 and its downstream effector p21(Waf1), we found that p53(-/-) and p21(-/-) cells were even more susceptible than wild-type cells to MLN4924. Our results suggested that apoptosis, not senescence, might be more important for the antiproliferative effect of MLN4924. Furthermore, our findings show that transient exposure to this new investigational drug should be useful for controlling p53-negative cancer cells, which often pose significant clinical challenge.

©2010 AACR.

Figures

Figure 1. Cdt1 protein level is important for MLN4924 induced re-replication in HCT116 cells

(A) HCT116 cells were transfected twice with siGL2 or siCdt1 at 0- and 24-hour time point and incubated for a total of 48 hours before the addition of 0.3μM MLN4924 or DMSO. Cells were harvested for PI FACS after 20 hours of treatment. The percentage of cells containing >4N DNA was shown. (B) Total cell lysates from (A) were blotted with indicated protein antibodies. (*: non-specific band) (C) HCT116 WT or e83 cells were treated with DMSO or 0.3μM MLN4924 for 20 hours before harvested for FACS. The percentage of cells containing >4N DNA was shown. (D) Similar assay as described in (A) was performed with siMcm7. The percentage of cells containing >4N DNA was shown. (E) Similar assay as in (A) was performed with siCdc6.

Figure 2. MLN4924 induces re-replication through inhibition of CRL4Cdt2

(A) HCT116 cells were tranfected with GL2, Cdt2, Geminin siRNA and treated with MLN4924 as described in Fig. 1(A). Cell lysates were harvested and blotted with indicated antibodies. (B) DNA contents of the cells treated in (A) were determined using FACS and plotted in horizontal bar graph. Representative FACS data from siGL2 treated cells indicating different DNA contents measured.

Figure 3. Transient exposure to MLN4924 induces re-replication in HCT116 cells

(A) HCT116 cells were treated with DMSO or 1μM MLN4924 for indicated hours. Cells were washed with PBS twice, incubated in fresh medium and harvested 24 hours after initial addition of the chemicals. Percentage of cells containing >4N DNA contents was plotted. (B) HCT116 cells were treated with DMSO or 1μM MLN4924 for 4 hours. Cells were then washed and harvested at 0, 2, 4 and 20 hours after the wash-out as indicated. Cell lysates were blotted with Cdt1 or Actin antibodies.

Figure 4. S phase cells are more susceptible to MLN4924 induced re-replication

(A) Schematic of experimental procedures of (B) to (D). (B) FACS profiles of control samples harvested at indicated time points. (C) Total lysates from cells harvested 20 hours after the drug wash-out were blotted with indicated antibodies. (D) FACS profiles from above cells were shown. The percentage of cells with >4N DNA is plotted in the bar graphs below. (E) Schematic of experiment of (F). (F) FACS profiles were shown as indicated. (Dashed line: DMSO; solid line: MLN4924) Percentages of re-replicating cells after MLN4924 treatment are indicated.

Figure 5. Both checkpoint and apoptosis pathways are activated upon short exposure of cells to MLN4924

(A) HCT116 cells were treated with DMSO, 1μM or 3μM MLN4924 for 8 hours. Cells were harvested 24 or 72 hours after the drug wash-out. Cell lysates were blotted with indicated protein antibodies. (*: nonspecific band) (B) Cells from above were harvested for PI FACS. Percentages of cells containing <2N (left) or >4N DNA (right) are shown.

Figure 6. The senescence pathway is induced in HCT116 cells after transient exposure to MLN4924

(A) HCT116 cells were treated with 1μM MLN4924 continuously (top panel) or only for 8 hours (middle and lower panels). Movie images of the cells at indicated time points are shown. (B) HCT116 cells were treated as described in the text. Cell survival rate in the colony formation assay is shown. Error bar represents three independent experiments. (C) HCT116 cells were treated with 1μM MLN4924 for indicated hours before wash-out. SA β-Gal staining assay was then performed after 72 hours. Positive stained cells were counted and plotted as percentage of total cell numbers. Mean±standard deviation of three different experiments. (D) Representative SA-β-gal staining for indicated samples. (E) HCT116 cells were transfected twice with siGL2 or siCdt1 as described in Figure 1(A). 48 hours after initial transfection, cells were treated with 1μM MLN4924 for 8 hours. Cells were either harvested for FACS analysis after 24 hours, or subjected to SA-β-gal staining assay after 72 hours. (F) HCT116 cells were treated with 1μM MLN4924 8 hours. Cells were harvested for western blots of p53 and p21 at different time points after wash-out. (G) HCT116 WT, p53−/− or p21−/− cells were treated with 1μM MLN4924 for 8 hours. SA-β-gal staining assay was performed 72 hours after the wash-out. Percentage of positive stained cells is shown. Mean±standard deviation of 3 experiments. * indicates statistical significance (p

Figure 7. p53 mutant cells are susceptible to transient treatment with MLN4924

(A) HCT116 WT, p53−/− or p21−/− cells were treated with 0, 0.1, 0.3, 0.9, 1.8 or 2.7μM of MLN4924. Cell survival rates were measured as described in Figure 6(B). Mean and standard deviation from triplicates. * indicates statistical significance (p