Cyclin F-dependent degradation of E2F7 is critical for DNA repair and G2-phase progression.

E2F7 and E2F8 act as tumor suppressors via transcriptional repression of genes involved in S-phase entry and progression. Previously, we demonstrated that these atypical E2Fs are degraded by APC/CCdh1 during G1 phase of the cell cycle. However, the mechanism driving the downregulation of atypical E2Fs during G2 phase is unknown. Here, we show that E2F7 is targeted for degradation by the E3 ubiquitin ligase SCFcyclin F during G2. Cyclin F binds via its cyclin domain to a conserved C-terminal CY motif on E2F7. An E2F7 mutant unable to interact with SCFcyclin F remains stable during G2. Furthermore, SCFcyclin F can also interact and induce degradation of E2F8. However, this does not require the cyclin domain of SCFcyclin F nor the CY motifs in the C-terminus of E2F8, implying a different regulatory mechanism than for E2F7. Importantly, depletion of cyclin F causes an atypical-E2F-dependent delay of the G2/M transition, accompanied by reduced expression of E2F target genes involved in DNA repair. Live cell imaging of DNA damage revealed that cyclin F-dependent regulation of atypical E2Fs is critical for efficient DNA repair and cell cycle progression.

Feedback regulation between atypical E2Fs and APC/CCdh1 coordinates cell cycle progression.

E2F transcription factors control the oscillating expression pattern of multiple target genes during the cell cycle. Activator E2Fs, E2F1-3, induce an upswing of E2F targets, which is essential for the G1-to-S phase transition, whereas atypical E2Fs, E2F7 and E2F8, mediate a downswing of the same targets during late S, G2, and M phases. Expression of atypical E2Fs is induced by E2F1-3, but it is unknown how atypical E2Fs are inactivated in a timely manner. Here, we demonstrate that E2F7 and E2F8 are substrates of the anaphase-promoting complex/cyclosome (APC/C). Removal of CDH1, or mutating the CDH1-interacting KEN boxes, stabilized E2F7/8 from anaphase onwards and during G1. Expressing KEN mutant E2F7 during G1 impairs S phase entry and eventually results in cell death. Furthermore, we show that E2F8, but not E2F7, interacts also with APC/C(C) (dc20). Importantly, atypical E2Fs can activate APC/C(C) (dh1) by repressing its inhibitors cyclin A, cyclin E, and Emi1. In conclusion, we discovered a feedback loop between atypical E2Fs and APC/C(C) (dh1), which ensures balanced expression of cell cycle genes and normal cell cycle progression.

Mechanisms used by cancer cells to tolerate drug-induced replication stress.

Activation of oncogenes in cancer cells forces cell proliferation, leading to DNA replication stress (RS). As a consequence, cancer cells heavily rely on the intra S-phase checkpoint for survival. This fundamental principle formed the basis for the development of inhibitors against key players of the intra S-phase checkpoint, ATR and CHK1. These drugs are often combined with chemotherapeutic drugs that interfere with DNA replication to exacerbate RS and exhaust the intra S-phase checkpoint in cancer cells. However, drug resistance impedes efficient clinical use, suggesting that some cancer cells tolerate severe RS. In this review, we describe how an increased nucleotide pool, boosted stabilization and repair of stalled forks and firing of dormant origins fortify the RS response in cancer cells. Notably, the vast majority of the genes that confer RS tolerance are regulated by the E2F and NRF2 transcription factors. These transcriptional programs are frequently activated in cancer cells, allowing simultaneous activation of multiple tolerance avenues. We propose that the E2F and NRF2 transcriptional programs can be used as biomarker to select patients for treatment with RS-inducing drugs and as novel targets to kill RS-tolerant cancer cells. Together, this review aims to provide a framework to maximally exploit RS as an Achilles' heel of cancer cells.

Oncogenic RAS sensitizes cells to drug-induced replication stress via transcriptional silencing of P53.

Cancer cells often experience high basal levels of DNA replication stress (RS), for example due to hyperactivation of oncoproteins like MYC or RAS. Therefore, cancer cells are considered to be sensitive to drugs that exacerbate the level of RS or block the intra S-phase checkpoint. Consequently, RS-inducing drugs including ATR and CHK1 inhibitors are used or evaluated as anti-cancer therapies. However, drug resistance and lack of biomarkers predicting therapeutic efficacy limit efficient use. This raises the question what determines sensitivity of individual cancer cells to RS. Here, we report that oncogenic RAS does not only enhance the sensitivity to ATR/CHK1 inhibitors by directly causing RS. Instead, we observed that HRASG12V dampens the activation of the P53-dependent transcriptional response to drug-induced RS, which in turn confers sensitivity to RS. We demonstrate that inducible expression of HRASG12V sensitized cells to ATR and CHK1 inhibitors. Using RNA-sequencing of FACS-sorted cells we discovered that P53 signaling is the sole transcriptional response to RS. However, oncogenic RAS attenuates the transcription of P53 and TGF-ß pathway components which consequently dampens P53 target gene expression. Accordingly, live cell imaging showed that HRASG12V exacerbates RS in S/G2-phase, which could be rescued by stabilization of P53. Thus, our results demonstrate that transcriptional control of P53 target genes is the prime determinant in the response to ATR/CHK1 inhibitors and show that hyperactivation of the MAPK pathway impedes this response. Our findings suggest that the level of oncogenic MAPK signaling could predict sensitivity to intra-S-phase checkpoint inhibition in cancers with intact P53.

Genome-Protective Topoisomerase 2a-Dependent G2 Arrest Requires p53 in hTERT-Positive Cancer Cells.

Topoisomerase 2a (Topo2a)-dependent G2 arrest engenders faithful segregation of sister chromatids, yet in certain tumor cell lines where this arrest is dysfunctional, a PKCε-dependent failsafe pathway can be triggered. Here we elaborate on recent advances in understanding the underlying mechanisms associated with this G2 arrest by determining that p53-p21 signaling is essential for efficient arrest in cell lines, in patient-derived cells, and in colorectal cancer organoids. Regulation of this p53 axis required the SMC5/6 complex, which is distinct from the p53 pathways observed in the DNA damage response. Topo2a inhibition specifically during S phase did not trigger G2 arrest despite affecting completion of DNA replication. Moreover, in cancer cells reliant upon the alternative lengthening of telomeres (ALT) mechanism, a distinct form of Topo2a-dependent, p53-independent G2 arrest was found to be mediated by BLM and Chk1. Importantly, the previously described PKCε-dependent mitotic failsafe was engaged in hTERT-positive cells when Topo2a-dependent G2 arrest was dysfunctional and where p53 was absent, but not in cells dependent on the ALT mechanism. In PKCε knockout mice, p53 deletion elicited tumors were less aggressive than in PKCε-replete animals and exhibited a distinct pattern of chromosomal rearrangements. This evidence suggests the potential of exploiting synthetic lethality in arrest-defective hTERT-positive tumors through PKCε-directed therapeutic intervention. SIGNIFICANCE: The identification of a requirement for p53 in stringent Topo2a-dependent G2 arrest and engagement of PKCε failsafe pathways in arrest-defective hTERT-positive cells provides a therapeutic opportunity to induce selective synthetic lethality.

Collection of cells for single-cell RNA sequencing using high-resolution fluorescence microscopy.

FACS sorting followed by single-cell RNA-sequencing (SORT-Seq) is a popular procedure to select cells of interest for single-cell transcriptomics. However, FACS is not suitable for measurement of subcellular distribution of fluorescence or for small samples (<1,000 cells). The VYCAP puncher system overcomes these limitations. Here, we describe a workflow to capture, image, and collect fluorescent human retina pigment epithelium cells for SORT-Seq using this system. The workflow can be used for any cell type with a diameter of ∼5-50 µm. For complete details on the use and execution of this protocol, please refer to Segeren et al. (2020).

Excessive E2F Transcription in Single Cancer Cells Precludes Transient Cell-Cycle Exit after DNA Damage.

E2F transcription factors control the expression of cell-cycle genes. Cancers often demonstrate enhanced E2F target gene expression, which can be explained by increased percentages of replicating cells. However, we demonstrate in human cancer biopsy specimens that individual neoplastic cells display abnormally high levels of E2F-dependent transcription. To mimic this situation, we delete the atypical E2F repressors (E2F7/8) or overexpress the E2F3 activator in untransformed cells. Cells with elevated E2F activity during S/G2 phase fail to exit the cell cycle after DNA damage and undergo mitosis. In contrast, wild-type cells complete S phase and then exit the cell cycle by activating the APC/CCdh1 via repression of the E2F target Emi1. Many arrested wild-type cells eventually inactivate APC/CCdh1 to execute a second round of DNA replication and mitosis, thereby becoming tetraploid. Cells with elevated E2F transcription fail to exit the cell cycle after DNA damage, which potentially causes genomic instability, promotes malignant progression, and reduces drug sensitivity.