E2F1 proteolysis via SCF‐cyclin F underlies synthetic lethality between cyclin F loss and Chk1 inhibition

Abstract Cyclins are central engines of cell cycle progression in conjunction with cyclin‐dependent kinases (CDKs). Among the different cyclins controlling cell cycle progression, cyclin F does not partner with a CDK, but instead forms via its F‐box domain an SCF (Skp1‐Cul1‐F‐box)‐type E3 ubiquitin ligase module. Although various substrates of cyclin F have been identified, the vulnerabilities of cells lacking cyclin F are not known. Thus, we assessed viability of cells lacking cyclin F upon challenging them with more than 180 different kinase inhibitors. The screen revealed a striking synthetic lethality between Chk1 inhibition and cyclin F loss. Chk1 inhibition in cells lacking cyclin F leads to DNA replication catastrophe. Replication catastrophe depends on accumulation of the transcription factor E2F1 in cyclin F‐depleted cells. We find that SCF‐cyclin F controls E2F1 ubiquitylation and degradation during the G2/M phase of the cell cycle and upon challenging cells with Chk1 inhibitors. Thus, Cyclin F restricts E2F1 activity during the cell cycle and upon checkpoint inhibition to prevent DNA replication stress. Our findings pave the way for patient selection in the clinical use of checkpoint inhibitors.

A U-2-OS cells transfected with the indicated siRNA and treated with Chk1i (LY2603618) for 20 h were harvested and lysed using SDS. Indicated proteins were resolved by SDS-PAGE and detected by Western blot (WB). B Percentage (%) of c-H2AX-positive cells in early S cells after Chk1i treatment (LY2603618). Early S phase cells were considered having 2n DAPI content and EdU + . Data from at least three independent experiments were plotted with mean % AE SD. C Percentage (%) of c-H2AX-positive cells in mid-late S phase after Chk1i (LY2603618) treatment. Mid-late S phase cells were considered having DAPI 4n staining and EdU + . Data from at least 3 independent experiments were plotted with mean % AE SD. D Representative plots of HeLa and CCNF K/O cells treated with 1 lM Chk1i for the indicated hours (h). Half an hour before harvesting, cells were incorporated with EdU (10 lM as final concentration). EdU by click reaction, c-H2Ax and RPA by immunostaining, and DAPI staining were detected by FACS analysis. G H Figure EV3. E2F1 accumulation mediates DNA damage in CCNF K/O cells treated with Chk1 and ATR inhibitors (related to Fig 4).
A HeLa and CCNF K/O cells were transfected with the indicated siRNA for 48 h were harvested and lysed using SDS. Indicated proteins were resolved by SDS-PAGE and visualised by WB. The image verifies knockdown efficiency of experiments presented in Fig 4A and B. TFIIH was used as a loading control. B WB of indicated proteins as in (A). Ponceau S staining is a loading control. C WB of indicated proteins as in (A). Ponceau S staining is a loading control. D WB of indicated proteins as in (A). H3 is a loading control. E Percentage (%) of c-H2AX-positive cells in HeLa and CCNF K/O after indicated siRNA transfection and 1 lM Chk1i for 20 h. Percent of c-H2AX-positive cells were plotted as mean % AE SD (**P < 0.005). Data were calculated from triplicate experiments using two-tailed paired t-test. F Representative FACS plots of c-H2AX versus DAPI signal of HeLa cells transfected with empty vector (EV) or HA-E2F1 untreated or treated with 1 lM Chk1i for 20 h. G Cells transfected with non-targeting siRNA (siNC) or indicated siRNAs were left untreated (DMSO) or treated with ATRi (VE-821, 1 lM) for 24 h. Cells were harvested and lysed using a Triton X-100-based lysis buffer. Indicated proteins were resolved by SDS-PAGE and visualised by WB. H Quantification of comet tails in alkaline gel. At least 100 cells, across two slides, were analysed in each condition in three biological replicates. Data are shown as medians, with 25/75% percentile range (box) and 10-90% percentile range (whiskers). P-values were calculated using the Mann-Whitney test (two-tailed). Olive tail moment = (Tail.mean À Head.mean)*Tail%DNA/100 ***P < 0.0005. I Cells transfected with indicated siRNAs are subjected to ATR (VE-821, 1 lM) inhibition for 24 h. Percentage survival of each cell line was assessed via resazurin viability assay and plotted as mean % AE SD (**P < 0.005). Data were calculated from triplicate experiments using two-tailed paired t-test. For simplification, survival rates were normalised to non-targeting control siNC treated with ATRi.
▸ Figure EV4. E2F1 is a ubiquitylation substrate of cyclin F at the G2/M transition and after checkpoint inhibition (related to Fig 5).
A A gene set enrichment analysis (GSEA) with enrichment of PID_E2F_PATHWAY, ISHIDA_E2F_TARGETS and HALLMARK_E2F_TARGETS gene signature in HeLa versus CCNF K/O. B HeLa and CCNF K/O cells were synchronised by double thymidine block and collected at the indicated hours (h) after release. Indicated proteins were resolved by SDS-PAGE and detected by WB. GAPDH was used as a loading control. C HEK293T cells were cotransfected with MYC-tagged ubiquitin, and HA-E2F1 in the presence of FLAG-cyclin F WT, FLAG-cyclin F (DF) and FLAG-cyclin F M309A as indicated (+). Indicated proteins were resolved by SDS-PAGE and detected by WB. H3 was used as a loading control. Correspond to the input of Fig 5E. D HeLa and CCNF K/O cells were treated with cycloheximide (CHX-50 lg/ml) for the indicated hours (h), harvested and lysed using SDS. Indicated proteins were resolved by SDS-PAGE and detected by WB. TFIIH was used as a loading control. E HeLa and CCNF K/O cells treated with Chk1i for 8 h and MG-132 or MLN4924 for 2 h at the indicated concentrations were harvested and lysed using SDS. Indicated proteins were resolved by SDS-PAGE and detected by WB. H2B was used as a loading control.
(Ub)n α-Myc   H3 Figure EV5. A mutant of E2F1 lacking a CY motif is not degraded by cyclin F at the G2/M transition (related to Fig 6).
A HEK293T cells were cotransfected with MYCtagged ubiquitin and HA-E2F1 or HA-E2F1 DRxL lacking the CY motif as indicated (+). Indicated proteins were resolved by SDS-PAGE and detected by WB. H3 was used as a loading control. Correspond to the input of Fig 6B. B HeLa cells stably expressing HA-E2F1 WT and HA-E2F1 DRxL under the control of a retroviral promoter were synchronised by double thymidine block and harvested at the indicated hours (h) after release. Indicated proteins were resolved by SDS-PAGE and detected by WB. GAPDH was used as a loading control.