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 HRAS(G12V) 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 HRAS(G12V) 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-beta pathway components which consequently dampens P53 target gene expression. Accordingly, live cell imaging showed that HRAS(G12V) 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

Inferring single-cell protein levels and cell cycle behavior in heterogeneous cell populations

Individual cells in a genetically identical population can show highly variable behavior. Single-cell measurements allow us to study this variability, but the available measurement techniques have limitations: live-cell microscopy is typically restricted to one or a few molecular markers, while techniques that simultaneously measure large numbers of molecular markers are destructive and cannot be used to follow cells over time. To help overcome these limitations, we present here scMeMo (single cell Mechanistic Modeler): a mechanistic modeling framework that can leverage diverse sets of measurements in order to infer unobserved variables in heterogeneous single cells. We used this framework to construct a model describing cell cycle progression in human cells, and show that it can predict the levels of several proteins in individual cells, based on live-cell microscopy measurements of only one marker and information learned from other experiments. The framework incorporates an uncertainty calibration step that makes the posterior distributions robust against partial model misspecification. Our modeling framework can be used to integrate information from separate experiments with diverse readouts, and to infer single cell variables that may be difficult to measure directly

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

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)

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)

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

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

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

Graphical Abstract Highlights d Individual cycling cancer cells display enhanced E2F target gene expression d E2F7/8 deletion or E2F3 overexpression overrides cell-cycle exit after DNA damage d Elevated levels of the E2F target Emi1 prevent DNA-damage-induced cell-cycle exit d The cell-cycle exit after DNA damage is transient and leads to endoreplicatio