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Aneuploidy, while detrimental to untransformed cells, is notably prevalent in cancer. Aneuploidy is found as an early event during tumorigenesis which indicates that cancer cells have the ability to surmount the initial stress responses associated with aneuploidy, enabling rapid proliferation despite aberrant karyotypes. To generate more insight into key cellular processes and requirements underlying adaptation to aneuploidy, we generated a panel of aneuploid clones in p53-deficient RPE-1 cells and studied their behavior over time. As expected, de novo-generated aneuploid clones initially display reduced fitness, enhanced levels of chromosomal instability (CIN), and an upregulated inflammatory response. Intriguingly, after prolonged culturing, aneuploid clones exhibit increased proliferation rates while maintaining aberrant karyotypes, indicative of an adaptive response to the aneuploid state. Interestingly, all adapted clones display reduced CIN and reduced inflammatory signaling, suggesting that these are common aspects of adaptation to aneuploidy. Collectively, our data suggests that CIN and concomitant inflammation are key processes that require correction to allow for fast proliferation in vitro. Finally, we provide evidence that amplification of oncogenic KRAS can promote adaptation., (© 2024. The Author(s).)

Purpose: Tumor hypoxia imposes a main obstacle to the efficacy of anti-cancer therapy. Understanding the cellular dynamics of individual hypoxic cells before, during and post-treatment has been hampered by the technical inability to identify and trace these cells over time., Methods and Materials: Here, we present a novel lineage-tracing reporter for hypoxic cells based on the conditional expression of a HIF1a-CreER T2 -UnaG biosensor that can visualize hypoxic cells in a time-dependent manner and trace the fate of hypoxic cells over time. We combine this system with multiphoton microscopy, flow cytometry, and immunofluorescence to characterize the role of hypoxic cells in tumor relapse after irradiation in H1299 tumor spheroids and in vivo xenografts., Results: We validate the reporter in monolayer cultures and we show that tagged cells colocalize in spheroids and human tumor xenografts with the hypoxic marker pimonidazole. We found that irradiation of H1299-HIFcreUnaG spheroids leads to preferential outgrowth of cells from the hypoxic core. Similarly, in xenografts tumors, although initially UnaG-positive-cells coincide with pimonidazole-positive tumor areas and they are merely quiescent, upon Irradiation UnaG-positive cells enrich in regrowing tumors and are mainly proliferative., Conclusions: Collectively, our data provide clear evidence that the hypoxic cells drive tumor relapse after irradiation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break in different chromatin environments. We then measured Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 58 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g. PCI-24781), while others were only effective in euchromatin and H3K27me3-marked regions (e.g. apicidin). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a resource to improve Cas9 editing more selectively at the desired location., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress response programs that counteract the inherent toxicity of such aberrant signaling. Although inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of protein phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor-suppressive resistance. Significance: A therapy consisting of deliberate hyperactivation of oncogenic signaling combined with perturbation of the stress responses that result from this is very effective in animal models of colon cancer. Resistance to this therapy is associated with loss of oncogenic signaling and reduced oncogenic capacity, indicative of tumor-suppressive drug resistance., (©2024 The Authors; Published by the American Association for Cancer Research.)

DNA double-strand breaks are repaired by multiple pathways, including non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ). The balance of these pathways is dependent on the local chromatin context, but the underlying mechanisms are poorly understood. By combining knockout screening with a dual MMEJ:NHEJ reporter inserted in 19 different chromatin environments, we identified dozens of DNA repair proteins that modulate pathway balance dependent on the local chromatin state. Proteins that favor NHEJ mostly synergize with euchromatin, while proteins that favor MMEJ generally synergize with distinct types of heterochromatin. Examples of the former are BRCA2 and POLL, and of the latter the FANC complex and ATM. Moreover, in a diversity of human cancer types, loss of several of these proteins alters the distribution of pathway-specific mutations between heterochromatin and euchromatin. Together, these results uncover a complex network of proteins that regulate MMEJ:NHEJ balance in a chromatin context-dependent manner., (© 2024. The Author(s).)

To enable a collective effort that generates a new level of UNderstanding CANcer (UNCAN.eu) [Cancer Discov (2022) 12 (11): OF1], the European Union supports the creation of a sustainable platform that connects cancer research across Member States. A workshop hosted in Heidelberg gathered European cancer experts to identify ongoing initiatives that may contribute to building this platform and discuss the governance and long-term evolution of a European Federated Cancer Data Hub., (©2023 The Authors; Published by the American Association for Cancer Research.)

Acquired drug resistance is a major problem in the treatment of cancer. hTERT-immortalized, untransformed RPE-1 cells can acquire resistance to Taxol by derepressing the ABCB1 gene, encoding for the multidrug transporter P-gP. Here, we investigate how the ABCB1 gene is derepressed. ABCB1 activation is associated with reduced H3K9 trimethylation, increased H3K27 acetylation, and ABCB1 displacement from the nuclear lamina. While altering DNA methylation and H3K27 methylation had no major impact on ABCB1 expression, nor did it promote resistance, disrupting the nuclear lamina component Lamin B Receptor did promote the acquisition of a Taxol-resistant phenotype in a subset of cells. CRISPRa-mediated gene activation supported the notion that lamina dissociation influences ABCB1 derepression. We propose a model in which nuclear lamina dissociation of a repressed gene allows for its activation, implying that deregulation of the 3D genome topology could play an important role in tumor evolution and the acquisition of drug resistance., Competing Interests: Declaration of interests E.d.W. is a co-founder of Cergentis B.V., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Faithful and timely repair of DNA double-strand breaks (DSBs) is fundamental for the maintenance of genomic integrity. Here, we demonstrate that the meiotic recombination co-factor MND1 facilitates the repair of DSBs in somatic cells. We show that MND1 localizes to DSBs, where it stimulates DNA repair through homologous recombination (HR). Importantly, MND1 is not involved in the response to replication-associated DSBs, implying that it is dispensable for HR-mediated repair of one-ended DSBs. Instead, we find that MND1 specifically plays a role in the response to two-ended DSBs that are induced by irradiation (IR) or various chemotherapeutic drugs. Surprisingly, we find that MND1 is specifically active in G2 phase, whereas it only marginally affects repair during S phase. MND1 localization to DSBs is dependent on resection of the DNA ends and seemingly occurs through direct binding of MND1 to RAD51-coated ssDNA. Importantly, the lack of MND1-driven HR repair directly potentiates the toxicity of IR-induced damage, which could open new possibilities for therapeutic intervention, specifically in HR-proficient tumors., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

"UNCAN.eu" refers to a collective European effort seeking to enable a leap forward in our understanding of cancer. This initiative, which includes the creation of a European cancer research data hub, will pave the way to new advances in cancer care. Starting on September 1, 2022, a 15-month coordination and support action will generate a blueprint for UNCAN.eu. Here, we summarize the cancer research issues that the blueprint will propose to tackle at the European level., (©2022 American Association for Cancer Research.)

Technologies to study DNA double-strand break (DSB) repair have traditionally mostly relied on fluorescence read-outs, either by microscopy or flow cytometry. The advent of high throughput sequencing (HTS) has created fundamentally new opportunities to study the mechanisms underlying DSB repair. Here, we review the suite of HTS-based assays that are used to study three different aspects of DNA repair: detection of broken ends, protein recruitment and pathway usage. We highlight new opportunities that HTS technology offers towards a better understanding of the DSB repair process., Competing Interests: Conflict of interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cells respond to double-strand breaks (DSBs) by activating DNA damage response pathways, including cell cycle arrest. We have previously shown that a single double-strand break generated via CRISPR/Cas9 is sufficient to delay cell cycle progression and compromise cell viability. However, we also found that the cellular response to DSBs can vary, independent of the number of lesions. This implies that not all DSBs are equally toxic, and raises the question if the location of a single double-strand break could influence its toxicity. To systematically investigate if DSB-location is a determinant of toxicity we performed a CRISPR/Cas9 screen targeting 6237 single sites in the human genome. Next, we developed a data-driven framework to design CRISPR/Cas9 sgRNA (crRNA) pools targeting specific chromatin features. The chromatin context was defined using ChromHMM states, Lamin-B1 DAM-iD, DNAseI hypersensitivity, and RNA-sequencing data. We computationally designed 6 distinct crRNA pools, each containing 10 crRNAs targeting the same chromatin state. We show that the toxicity of a DSB is highly similar across the different ChromHMM states. Rather, we find that the major determinants of toxicity of a sgRNA are cutting efficiency and off-target effects. Thus, chromatin features have little to no effect on the toxicity of a single CRISPR/Cas9-induced DSB., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition., Competing Interests: R.B is the founder of the company Oncosence (https://www.oncosence.com), which aims to develop senescence-inducing and senolytic compounds to treat cancer. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

In prostate cancer, androgen receptor (AR)-targeting agents are very effective in various disease stages. However, therapy resistance inevitably occurs, and little is known about how tumor cells adapt to bypass AR suppression. Here, we performed integrative multiomics analyses on tissues isolated before and after 3 months of AR-targeting enzalutamide monotherapy from patients with high-risk prostate cancer enrolled in a neoadjuvant clinical trial. Transcriptomic analyses demonstrated that AR inhibition drove tumors toward a neuroendocrine-like disease state. Additionally, epigenomic profiling revealed massive enzalutamide-induced reprogramming of pioneer factor FOXA1 from inactive chromatin sites toward active cis-regulatory elements that dictate prosurvival signals. Notably, treatment-induced FOXA1 sites were enriched for the circadian clock component ARNTL. Posttreatment ARNTL levels were associated with patients' clinical outcomes, and ARNTL knockout strongly decreased prostate cancer cell growth. Our data highlight a remarkable cistromic plasticity of FOXA1 following AR-targeted therapy and revealed an acquired dependency on the circadian regulator ARNTL, a novel candidate therapeutic target., Significance: Understanding how prostate cancers adapt to AR-targeted interventions is critical for identifying novel drug targets to improve the clinical management of treatment-resistant disease. Our study revealed an enzalutamide-induced epigenomic plasticity toward prosurvival signaling and uncovered the circadian regulator ARNTL as an acquired vulnerability after AR inhibition, presenting a novel lead for therapeutic development. See related commentary by Zhang et al., p. 2017. This article is highlighted in the In This Issue feature, p. 2007., (©2022 American Association for Cancer Research.)

Advances in genome sequencing have revealed a type of extrachromosomal DNA, historically named double minutes (also referred to as ecDNA), to be common in a wide range of cancer types, but not in healthy tissues. These cancer-associated circular DNA molecules contain one or a few genes that are amplified when double minutes accumulate. Double minutes harbor oncogenes or drug resistance genes that contribute to tumor aggressiveness through copy number amplification in combination with favorable epigenetic properties. Unequal distribution of double minutes over daughter cells contributes to intratumoral heterogeneity, thereby increasing tumor adaptability. In this review, we discuss various models delineating the mechanism of generation of double minutes. Furthermore, we highlight how double minutes are maintained, how they evolve, and discuss possible mechanisms driving their elimination., (© 2022. The Author(s).)

Aneuploidy and chromosomal instability are both commonly found in cancer. Chromosomal instability leads to karyotype heterogeneity in tumors and is associated with therapy resistance, metastasis and poor prognosis. It has been hypothesized that aneuploidy per se is sufficient to drive CIN, however due to limited models and heterogenous results, it has remained controversial which aspects of aneuploidy can drive CIN. In this study we systematically tested the impact of different types of aneuploidies on the induction of CIN. We generated a plethora of isogenic aneuploid clones harboring whole chromosome or segmental aneuploidies in human p53-deficient RPE-1 cells. We observed increased segregation errors in cells harboring trisomies that strongly correlated to the number of gained genes. Strikingly, we found that clones harboring only monosomies do not induce a CIN phenotype. Finally, we found that an initial chromosome breakage event and subsequent fusion can instigate breakage-fusion-bridge cycles. By investigating the impact of monosomies, trisomies and segmental aneuploidies on chromosomal instability we further deciphered the complex relationship between aneuploidy and CIN., Competing Interests: No, there is no conflict of interest. My manuscript contains the following statement: "The authors declare that they have no conflict of interest."

The discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its development as a genome editing tool has revolutionized the field of molecular biology. In the DNA damage field, CRISPR has brought an alternative to induce endogenous double-strand breaks (DSBs) at desired genomic locations and study the DNA damage response and its consequences. Many systems for sgRNA delivery have been reported in order to efficiently generate this DSB, including lentiviral vectors. However, some of the consequences of these systems are not yet well understood. Here, we report that lentiviral-based sgRNA vectors can integrate into the endogenous genomic target location, leading to undesired activation of the target gene. By generating a DSB in the regulatory region of the ABCB1 gene using a lentiviral sgRNA vector, we can induce the formation of Taxol-resistant colonies. We show that these colonies upregulate ABCB1 via integration of the EEF1A1 and the U6 promoters from the sgRNA vector. We believe that this is an unreported CRISPR/Cas9 on-target effect that researchers need to be aware of when using lentiviral vectors for genome editing., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Upon exposure to genotoxic stress, cells activate DNA damage response (DDR) that coordinates DNA repair with a temporal arrest in the cell cycle progression. DDR is triggered by activation of ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related protein kinases that phosphorylate multiple targets including tumor suppressor protein tumor suppressor p53 (p53). In addition, DNA damage can activate parallel stress response pathways [such as mitogen-activated protein kinase p38 alpha (p38)/MAPK-activated protein kinase 2 (MK2) kinases] contributing to establishing the cell cycle arrest. Wild-type p53-induced phosphatase 1 (WIP1) controls timely inactivation of DDR and is needed for recovery from the G2 checkpoint by counteracting the function of p53. Here, we developed a simple in vitro assay for testing WIP1 substrates in nuclear extracts. Whereas we did not detect any activity of WIP1 toward p38/MK2, we confirmed p53 as a substrate of WIP1. Inhibition or inactivation of WIP1 in U2OS cells increased phosphorylation of p53 at S15 and potentiated its acetylation at K382. Further, we identified Deleted in breast cancer gene 1 (DBC1) as a new substrate of WIP1 but surprisingly, depletion of DBC1 did not interfere with the ability of WIP1 to regulate p53 acetylation. Instead, we have found that WIP1 activity suppresses p53-K382 acetylation by inhibiting the interaction between p53 and the acetyltransferase p300. Newly established phosphatase assay allows an easy comparison of WIP1 ability to dephosphorylate various proteins and thus contributes to identification of its physiological substrates., (© 2021 Federation of European Biochemical Societies.)

Basal-like breast cancers (BLBC) are characterized by defects in homologous recombination (HR), deficient mitotic checkpoint, and high-proliferation activity. Here, we discover CIP2A as a candidate driver of BLBC. CIP2A was essential for DNA damage-induced initiation of mouse BLBC-like mammary tumors and for survival of HR-defective BLBC cells. CIP2A was dispensable for normal mammary gland development and for unperturbed mitosis, but selectively essential for mitotic progression of DNA damaged cells. A direct interaction between CIP2A and a DNA repair scaffold protein TopBP1 was identified, and CIP2A inhibition resulted in enhanced DNA damage-induced TopBP1 and RAD51 recruitment to chromatin in mammary epithelial cells. In addition to its role in tumor initiation, and survival of BRCA-deficient cells, CIP2A also drove proliferative MYC and E2F1 signaling in basal-like triple-negative breast cancer (BL-TNBC) cells. Clinically, high CIP2A expression was associated with poor patient prognosis in BL-TNBCs but not in other breast cancer subtypes. Small-molecule reactivators of PP2A (SMAP) inhibited CIP2A transcription, phenocopied the CIP2A-deficient DNA damage response (DDR), and inhibited growth of patient-derived BLBC xenograft. In summary, these results demonstrate that CIP2A directly interacts with TopBP1 and coordinates DNA damage-induced mitotic checkpoint and proliferation, thereby driving BLBC initiation and progression. SMAPs could serve as a surrogate therapeutic strategy to inhibit the oncogenic activity of CIP2A in BLBCs. SIGNIFICANCE: These results identify CIP2A as a nongenetic driver and therapeutic target in basal-like breast cancer that regulates DNA damage-induced G 2 -M checkpoint and proliferative signaling., (©2021 The Authors; Published by the American Association for Cancer Research.)

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments., Competing Interests: Declaration of interests B.v.S. is a member of the Advisory Board of Molecular Cell., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Double strand breaks (DSBs) are highly toxic to a cell, a property that is exploited in radiation therapy. A critical component for the damage induction is cellular oxygen, making hypoxic tumor areas refractory to the efficacy of radiation treatment. During a fractionated radiation regimen, these hypoxic areas can be re-oxygenated. Nonetheless, hypoxia still constitutes a negative prognostic factor for the patient's outcome. We hypothesized that this might be attributed to specific hypoxia-induced cellular traits that are maintained upon reoxygenation. Here, we show that reoxygenation of hypoxic non-transformed RPE-1 cells fully restored induction of DSBs but the cells remain radioresistant as a consequence of hypoxia-induced quiescence. With the use of the cell cycle indicators (FUCCI), cell cycle-specific radiation sensitivity, the cell cycle phase duration with live cell imaging, and single cell tracing were assessed. We observed that RPE-1 cells experience a longer G1 phase under hypoxia and retain a large fraction of cells that are non-cycling. Expression of HPV oncoprotein E7 prevents hypoxia-induced quiescence and abolishes the radioprotective effect. In line with this, HPV-negative cancer cell lines retain radioresistance, while HPV-positive cancer cell lines are radiosensitized upon reoxygenation. Quiescence induction in hypoxia and its HPV-driven prevention was observed in 3D multicellular spheroids. Collectively, we identify a new hypoxia-dependent radioprotective phenotype due to hypoxia-induced quiescence that accounts for a global decrease in radiosensitivity that can be retained upon reoxygenation and is absent in cells expressing oncoprotein E7.

Most Cyclin-dependent kinases (Cdks) are redundant for normal cell division. Here we tested whether these redundancies are maintained during cell cycle recovery after a DNA damage-induced arrest in G1. Using non-transformed RPE-1 cells, we find that while Cdk4 and Cdk6 act redundantly during normal S-phase entry, they both become essential for S-phase entry after DNA damage in G1. We show that this is due to a greater overall dependency for Cdk4/6 activity, rather than to independent functions of either kinase. In addition, we show that inactivation of pocket proteins is sufficient to overcome the inhibitory effects of complete Cdk4/6 inhibition in otherwise unperturbed cells, but that this cannot revert the effects of Cdk4/6 inhibition in DNA damaged cultures. Indeed, we could confirm that, in addition to inactivation of pocket proteins, Cdh1-dependent anaphase-promoting complex/cyclosome (APC/C Cdh1 ) activity needs to be inhibited to promote S-phase entry in damaged cultures. Collectively, our data indicate that DNA damage in G1 creates a unique situation where high levels of Cdk4/6 activity are required to inactivate pocket proteins and APC/C Cdh1 to promote the transition from G1 to S phase.

Background: The G1 checkpoint is a critical regulator of genomic stability in untransformed cells, preventing cell cycle progression after DNA damage. DNA double-strand breaks (DSBs) recruit and activate ATM, a kinase which in turn activates the CHK2 kinase to establish G1 arrest. While the onset of G1 arrest is well understood, the specific role that ATM and CHK2 play in regulating G1 checkpoint maintenance remains poorly characterized., Results: Here we examine the impact of ATM and CHK2 activities on G1 checkpoint maintenance in untransformed cells after DNA damage caused by DSBs. We show that ATM becomes dispensable for G1 checkpoint maintenance as early as 1 h after DSB induction. In contrast, CHK2 kinase activity is necessary to maintain the G1 arrest, independently of ATM, ATR, and DNA-PKcs, implying that the G1 arrest is maintained in a lesion-independent manner. Sustained CHK2 activity is achieved through auto-activation and its acute inhibition enables cells to abrogate the G1-checkpoint and enter into S-phase. Accordingly, we show that CHK2 activity is lost in cells that recover from the G1 arrest, pointing to the involvement of a phosphatase with fast turnover., Conclusion: Our data indicate that G1 checkpoint maintenance relies on CHK2 and that its negative regulation is crucial for G1 checkpoint recovery after DSB induction.

The antihelmintic drug ABZ and its metabolites belong to the chemical family of benzimidazoles (BZM) that act as potent tubulin polymerization inhibitors, suggesting a potential re-direction of BZMs for cancer therapy. Applying UV-Vis spectrometry we here demonstrate ABZ as a DNA intercalator. This insight led us to determine the primary mode of ABZ action in mammalian cells. As revealed by RNA sequencing, ABZ did neither grossly affect replication as analyzed by survival and replication stress signaling, nor the transcriptome. Actually, unbiased transcriptome analysis revealed a marked cell cycle signature in ABZ exposed cells. Indeed, short-term exposure to ABZ arrested mammalian cells in G2/M cell cycle stages associated with frequent gains and losses of chromatin. Cellular analyses revealed ABZ as a potent mammalian spindle poison for normal and malignant cells, explaining the serious chromosome segregation defects. Since chromosomal aberrations promote both cancer development and cell death, we determined if besides its general cytotoxicity, ABZ could predispose to tumor development. As measured by loss of heterozygosity (LOH) in vitro and in vivo ABZ was found as a potent inducer of LOH and accelerator of chromosomal missegregation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Will Castro, Pieters, Alemdehy, Aslam, Buoninfante, Raaijmakers, Pilzecker, van den Berk, te Riele, Medema, Pedrosa and Jacobs.)

Accurate control of centrosome number is essential for proper chromosome segregation, and it is well established that centrosome abnormalities can trigger a p53-dependent cell cycle arrest. Two new studies published in The EMBO Journal demonstrate how PIDD1 is recruited to centrosomes and that the localization of PIDD1 to distal appendages of centrosomes is required for PIDDosome activation at clustered supernumerary centrosomes., (© 2021 The Authors.)

DNA replication is challenged by numerous exogenous and endogenous factors that can interfere with the progression of replication forks. Substantial accumulation of single-stranded DNA during DNA replication activates the DNA replication stress checkpoint response that slows progression from S/G2 to M phase to protect genomic integrity. Whether and how mild replication stress restricts proliferation remains controversial. Here, we identify a cell cycle exit mechanism that prevents S/G2 phase arrested cells from undergoing mitosis after exposure to mild replication stress through premature activation of the anaphase promoting complex/cyclosome (APC/C CDH1 ). We find that replication stress causes a gradual decrease of the levels of the APC/C CDH1 inhibitor EMI1/FBXO5 through Forkhead box O (FOXO)-mediated inhibition of its transcription factor E2F1. By doing so, FOXOs limit the time during which the replication stress checkpoint is reversible and thereby play an important role in maintaining genomic stability., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

There has been an increase in the use of machine learning and artificial intelligence (AI) for the analysis of image-based cellular screens. The accuracy of these analyses, however, is greatly dependent on the quality of the training sets used for building the machine learning models. We propose that unsupervised exploratory methods should first be applied to the data set to gain a better insight into the quality of the data. This improves the selection and labeling of data for creating training sets before the application of machine learning. We demonstrate this using a high-content genome-wide small interfering RNA screen. We perform an unsupervised exploratory data analysis to facilitate the identification of four robust phenotypes, which we subsequently use as a training set for building a high-quality random forest machine learning model to differentiate four phenotypes with an accuracy of 91.1% and a kappa of 0.85. Our approach enhanced our ability to extract new knowledge from the screen when compared with the use of unsupervised methods alone.

Michowski and colleagues (2020) engineered analog-sensitive Cdk1 knockin mice to identify Cdk1 targets in embryonic stem cells, which led them to discover a novel function for Cdk1 in shaping the epigenetic landscape by direct regulation of epigenetic modulators., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The cyclin-dependent kinase 5 (CDK5), originally described as a neuronal-specific kinase, is also frequently activated in human cancers. Using conditional CDK5 knockout mice and a mouse model of highly metastatic melanoma, we found that CDK5 is dispensable for the growth of primary tumors. However, we observed that ablation of CDK5 completely abrogated the metastasis, revealing that CDK5 is essential for the metastatic spread. In mouse and human melanoma cells CDK5 promotes cell invasiveness by directly phosphorylating an intermediate filament protein, vimentin, thereby inhibiting assembly of vimentin filaments. Chemical inhibition of CDK5 blocks the metastatic spread of patient-derived melanomas in patient-derived xenograft (PDX) mouse models. Hence, inhibition of CDK5 might represent a very potent therapeutic strategy to impede the metastatic dissemination of malignant cells., Competing Interests: Competing interest statement: P.S. has been a consultant at Novartis, Genovis, Guidepoint, The Planning Shop, ORIC Pharmaceuticals, and Exo Therapeutics; his laboratory receives research funding from Novartis. W.M. is currently an employee of Cedilla Therapeutics.

Most human cancers are aneuploid, due to a chromosomal instability (CIN) phenotype. Despite being hallmarks of cancer, however, the roles of CIN and aneuploidy in tumor formation have not unequivocally emerged from animal studies and are thus still unclear. Using a conditional mouse model for diverse degrees of CIN, we find that a particular range is sufficient to drive very early onset spontaneous adenoma formation in the intestine. In mice predisposed to intestinal cancer (Apc Min/+ ), moderate CIN causes a remarkable increase in adenoma burden in the entire intestinal tract and especially in the distal colon, which resembles human disease. Strikingly, a higher level of CIN promotes adenoma formation in the distal colon even more than moderate CIN does, but has no effect in the small intestine. Our results thus show that CIN can be potently oncogenic, but that certain levels of CIN can have contrasting effects in distinct tissues.

The cellular response to DNA breaks is influenced by chromatin compaction. To identify chromatin regulators involved in the DNA damage response, we screened for genes that affect recovery following DNA damage using an RNAi library of chromatin regulators. We identified genes involved in chromatin remodeling, sister chromatid cohesion, and histone acetylation not previously associated with checkpoint recovery. Among these is the PHD finger protein 6 (PHF6), a gene mutated in Börjeson-Forssman-Lehmann syndrome and leukemic cancers. We find that loss of PHF6 dramatically compromises checkpoint recovery in G2 phase cells. Moreover, PHF6 is rapidly recruited to sites of DNA lesions in a PARP-dependent manner and required for efficient DNA repair through classical non-homologous end joining. These results indicate that PHF6 is a novel DNA damage response regulator that promotes end joining-mediated repair, thereby stimulating timely recovery from the G2 checkpoint., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

In order to legally conduct clinical research into new cancer treatments with medicinal products based on genetically modified organisms (GMOs) and treat cancer patients with these products, Dutch hospitals must obtain an environmental permit from the Minister of Infrastructure & Water Management. In the Netherlands, permit applications are assessed more strictly than in other EU member states, even if the products do not pose any real risk to the population and the environment. As a result, Dutch patients have to wait longer before the clinical trial or therapy with these GMO products can commence. This makes current Dutch environmental policy a serious obstacle to public health. Recently physicians and researchers have therefore pleaded with Dutch MPs for an adjustment of the lower national environmental regulations. Unfortunately, these pleas were not supported by said elected members of parliament, and the Ministry of Infrastructure & Water Management seems to be holding on to a pointless environmental permit for clinical research and treatment of cancer patients with these GMO medicinal products.

Condensin is a conserved SMC complex that uses its ATPase machinery to structure genomes, but how it does so is largely unknown. We show that condensin's ATPase has a dual role in chromosome condensation. Mutation of one ATPase site impairs condensation, while mutating the second site results in hyperactive condensin that compacts DNA faster than wild-type, both in vivo and in vitro. Whereas one site drives loop formation, the second site is involved in the formation of more stable higher-order Z loop structures. Using hyperactive condensin I, we reveal that condensin II is not intrinsically needed for the shortening of mitotic chromosomes. Condensin II rather is required for a straight chromosomal axis and enables faithful chromosome segregation by counteracting the formation of ultrafine DNA bridges. SMC complexes with distinct roles for each ATPase site likely reflect a universal principle that enables these molecular machines to intricately control chromosome architecture., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)