BRCA1 Haploinsufficiency Is Masked by RNF168-Mediated Chromatin Ubiquitylation.

BRCA1 functions at two distinct steps during homologous recombination (HR). Initially, it promotes DNA end resection, and subsequently it recruits the PALB2 and BRCA2 mediator complex, which stabilizes RAD51-DNA nucleoprotein filaments. Loss of 53BP1 rescues the HR defect in BRCA1-deficient cells by increasing resection, suggesting that BRCA1's downstream role in RAD51 loading is dispensable when 53BP1 is absent. Here we show that the E3 ubiquitin ligase RNF168, in addition to its canonical role in inhibiting end resection, acts in a redundant manner with BRCA1 to load PALB2 onto damaged DNA. Loss of RNF168 negates the synthetic rescue of BRCA1 deficiency by 53BP1 deletion, and it predisposes BRCA1 heterozygous mice to cancer. BRCA1+/-RNF168-/- cells lack RAD51 foci and are hypersensitive to PARP inhibitor, whereas forced targeting of PALB2 to DNA breaks in mutant cells circumvents BRCA1 haploinsufficiency. Inhibiting the chromatin ubiquitin pathway may, therefore, be a synthetic lethality strategy for BRCA1-deficient cancers.

Discovery of a novel inhibitor of macropinocytosis with antiviral activity.

Several viruses hijack various forms of endocytosis in order to infect host cells. Here, we report the discovery of a molecule with antiviral properties that we named virapinib, which limits viral entry by macropinocytosis. The identification of virapinib derives from a chemical screen using High-Throughput Microscopy, where we identified chemical entities capable of preventing infection with a pseudotype virus expressing the spike (S) protein from SARS-CoV-2. Subsequent experiments confirmed the capacity of virapinib to inhibit infection by SARS-CoV-2, as well as by additional viruses, such as Monkeypox virus and TBEV. Mechanistic analyses revealed that the compound inhibited macropinocytosis, limiting this entry route for the viruses. Importantly, virapinib has no significant toxicity to host cells. In summary, we present the discovery of a molecule that inhibits macropinocytosis, thereby limiting the infectivity of viruses that use this entry route such as SARS-CoV2.

Distinct roles for PARP-1 and PARP-2 in c-Myc-driven B-cell lymphoma in mice.

Dysregulation of the c-Myc oncogene occurs in a wide variety of hematologic malignancies, and its overexpression has been linked with aggressive tumor progression. Here, we show that poly (ADP-ribose) polymerase 1 (PARP-1) and PARP-2 exert opposing influences on progression of c-Myc-driven B-cell lymphoma. PARP-1 and PARP-2 catalyze the synthesis and transfer of ADP-ribose units onto amino acid residues of acceptor proteins in response to DNA strand breaks, playing a central role in the response to DNA damage. Accordingly, PARP inhibitors have emerged as promising new cancer therapeutics. However, the inhibitors currently available for clinical use are not able to discriminate between individual PARP proteins. We found that genetic deletion of PARP-2 prevents c-Myc-driven B-cell lymphoma, whereas PARP-1 deficiency accelerates lymphomagenesis in the Eµ-Myc mouse model of aggressive B-cell lymphoma. Loss of PARP-2 aggravates replication stress in preleukemic Eµ-Myc B cells, resulting in accumulation of DNA damage and concomitant cell death that restricts the c-Myc-driven expansion of B cells, thereby providing protection against B-cell lymphoma. In contrast, PARP-1 deficiency induces a proinflammatory response and an increase in regulatory T cells, likely contributing to immune escape of B-cell lymphoma, resulting in an acceleration of lymphomagenesis. These findings pinpoint specific functions for PARP-1 and PARP-2 in c-Myc-driven lymphomagenesis with antagonistic consequences that may help inform the design of new PARP-centered therapeutic strategies, with selective PARP-2 inhibition potentially representing a new therapeutic approach for the treatment of c-Myc-driven tumors.

A Genome-wide CRISPR Screen Identifies CDC25A as a Determinant of Sensitivity to ATR Inhibitors.

One recurring theme in drug development is to exploit synthetic lethal properties as means to preferentially damage the DNA of cancer cells. We and others have previously developed inhibitors of the ATR kinase, shown to be particularly genotoxic for cells expressing certain oncogenes. In contrast, the mechanisms of resistance to ATR inhibitors remain unexplored. We report here on a genome-wide CRISPR-Cas9 screen that identified CDC25A as a major determinant of sensitivity to ATR inhibition. CDC25A-deficient cells resist high doses of ATR inhibitors, which we show is due to their failure to prematurely enter mitosis in response to the drugs. Forcing mitotic entry with WEE1 inhibitors restores the toxicity of ATR inhibitors in CDC25A-deficient cells. With ATR inhibitors now entering the clinic, our work provides a better understanding of the mechanisms by which these compounds kill cells and reveals genetic interactions that could be used for their rational use.

POLD3 Is Haploinsufficient for DNA Replication in Mice.

The Pold3 gene encodes a subunit of the Polδ DNA polymerase complex. Pold3 orthologs are not essential in Saccharomyces cerevisiae or chicken DT40 cells, but the Schizosaccharomyces pombe ortholog is essential. POLD3 also has a specialized role in the repair of broken replication forks, suggesting that POLD3 activity could be particularly relevant for cancer cells enduring high levels of DNA replication stress. We report here that POLD3 is essential for mouse development and is also required for viability in adult animals. Strikingly, even Pold3(+/-) mice were born at sub-Mendelian ratios, and, of those born, some presented hydrocephaly and had a reduced lifespan. In cells, POLD3 deficiency led to replication stress and cell death, which were aggravated by the expression of activated oncogenes. Finally, we show that Pold3 deletion destabilizes all members of the Polδ complex, explaining its major role in DNA replication and the severe impact of its deficiency.

Increased Rrm2 gene dosage reduces fragile site breakage and prolongs survival of ATR mutant mice.

In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the RNR regulatory subunit RRM2 (Rrm2(TG)) present supraphysiological RNR activity and reduced chromosomal breakage at fragile sites. Moreover, increased Rrm2 gene dosage significantly extends the life span of ATR mutant mice. Our study reveals the first genetic condition in mammals that reduces fragile site expression and alleviates the severity of a progeroid disease by increasing RNR activity.

NSMCE2 suppresses cancer and aging in mice independently of its SUMO ligase activity.

The SMC5/6 complex is the least understood of SMC complexes. In yeast, smc5/6 mutants phenocopy mutations in sgs1, the BLM ortholog that is deficient in Bloom's syndrome (BS). We here show that NSMCE2 (Mms21, in Saccharomyces cerevisiae), an essential SUMO ligase of the SMC5/6 complex, suppresses cancer and aging in mice. Surprisingly, a mutation that compromises NSMCE2-dependent SUMOylation does not have a detectable impact on murine lifespan. In contrast, NSMCE2 deletion in adult mice leads to pathologies resembling those found in patients of BS. Moreover, and whereas NSMCE2 deletion does not have a detectable impact on DNA replication, NSMCE2-deficient cells also present the cellular hallmarks of BS such as increased recombination rates and an accumulation of micronuclei. Despite the similarities, NSMCE2 and BLM foci do not colocalize and concomitant deletion of Blm and Nsmce2 in B lymphocytes further increases recombination rates and is synthetic lethal due to severe chromosome mis-segregation. Our work reveals that SUMO- and BLM-independent activities of NSMCE2 limit recombination and facilitate segregation; functions of the SMC5/6 complex that are necessary to prevent cancer and aging in mice.

A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity.

The reprogramming of differentiated cells to pluripotent cells (induced pluripotent stem (iPS) cells) is known to be an inefficient process. We recently reported that cells with short telomeres cannot be reprogrammed to iPS cells despite their normal proliferation rates, probably reflecting the existence of 'reprogramming barriers' that abort the reprogramming of cells with uncapped telomeres. Here we show that p53 (also known as Trp53 in mice and TP53 in humans) is critically involved in preventing the reprogramming of cells carrying various types of DNA damage, including short telomeres, DNA repair deficiencies, or exogenously inflicted DNA damage. Reprogramming in the presence of pre-existing, but tolerated, DNA damage is aborted by the activation of a DNA damage response and p53-dependent apoptosis. Abrogation of p53 allows efficient reprogramming in the face of DNA damage and the generation of iPS cells carrying persistent DNA damage and chromosomal aberrations. These observations indicate that during reprogramming cells increase their intolerance to different types of DNA damage and that p53 is critical in preventing the generation of human and mouse pluripotent cells from suboptimal parental cells.

Correction: Limited Role of Murine ATM in Oncogene-Induced Senescence and p53-Dependent Tumor Suppression.

[This corrects the article DOI: 10.1371/journal.pone.0005475.].

PD-L1ATTAC mice reveal the potential of depleting PD-L1 expressing cells in cancer therapy.

Antibodies targeting the PD-1 receptor and its ligand PD-L1 have shown impressive responses in some tumors of bad prognosis. We hypothesized that, since immunosuppressive cells might present several immune checkpoints on their surface, the selective elimination of PD-L1 expressing cells could be efficacious in enabling the activation of antitumoral immune responses. To address this question, we developed an inducible suicidal knock-in mouse allele of Pd-l1 (PD-L1ATTAC) which allows for the tracking and specific elimination of PD-L1-expressing cells in adult tissues. Consistent with our hypothesis, elimination of PD-L1 expressing cells from the mouse peritoneum increased the septic response to lipopolysaccharide (LPS), due to an exacerbated inflammatory response to the endotoxin. In addition, mice depleted of PD-L1+ cells were resistant to colon cancer peritoneal allografts, which was associated with a loss of immunosuppressive B cells and macrophages, concomitant with an increase in activated cytotoxic CD8 T cells. Collectively, these results illustrate the usefulness of PD-L1ATTAC mice for research in immunotherapy and provide genetic support to the concept of targeting PD-L1 expressing cells in cancer.

ATM regulates ATR chromatin loading in response to DNA double-strand breaks.

DNA double-strand breaks (DSBs) are among the most deleterious lesions that can challenge genomic integrity. Concomitant to the repair of the breaks, a rapid signaling cascade must be coordinated at the lesion site that leads to the activation of cell cycle checkpoints and/or apoptosis. In this context, ataxia telangiectasia mutated (ATM) and ATM and Rad-3-related (ATR) protein kinases are the earliest signaling molecules that are known to initiate the transduction cascade at damage sites. The current model places ATM and ATR in separate molecular routes that orchestrate distinct pathways of the checkpoint responses. Whereas ATM signals DSBs arising from ionizing radiation (IR) through a Chk2-dependent pathway, ATR is activated in a variety of replication-linked DSBs and leads to activation of the checkpoints in a Chk1 kinase-dependent manner. However, activation of the G2/M checkpoint in response to IR escapes this accepted paradigm because it is dependent on both ATM and ATR but independent of Chk2. Our data provides an explanation for this observation and places ATM activity upstream of ATR recruitment to IR-damaged chromatin. These data provide experimental evidence of an active cross talk between ATM and ATR signaling pathways in response to DNA damage.

Global chromatin compaction limits the strength of the DNA damage response.

In response to DNA damage, chromatin undergoes a global decondensation process that has been proposed to facilitate genome surveillance. However, the impact that chromatin compaction has on the DNA damage response (DDR) has not directly been tested and thus remains speculative. We apply two independent approaches (one based on murine embryonic stem cells with reduced amounts of the linker histone H1 and the second making use of histone deacetylase inhibitors) to show that the strength of the DDR is amplified in the context of "open" chromatin. H1-depleted cells are hyperresistant to DNA damage and present hypersensitive checkpoints, phenotypes that we show are explained by an increase in the amount of signaling generated at each DNA break. Furthermore, the decrease in H1 leads to a general increase in telomere length, an as of yet unrecognized role for H1 in the regulation of chromosome structure. We propose that slight differences in the epigenetic configuration might account for the cell-to-cell variation in the strength of the DDR observed when groups of cells are challenged with DNA breaks.

Activation of the integrated stress response is a vulnerability for multidrug-resistant FBXW7-deficient cells.

FBXW7 is one of the most frequently mutated tumor suppressors, deficiency of which has been associated with resistance to some anticancer therapies. Through bioinformatics and genome-wide CRISPR screens, we here reveal that FBXW7 deficiency leads to multidrug resistance (MDR). Proteomic analyses found an upregulation of mitochondrial factors as a hallmark of FBXW7 deficiency, which has been previously linked to chemotherapy resistance. Despite this increased expression of mitochondrial factors, functional analyses revealed that mitochondria are under stress, and genetic or chemical targeting of mitochondria is preferentially toxic for FBXW7-deficient cells. Mechanistically, the toxicity of therapies targeting mitochondrial translation such as the antibiotic tigecycline relates to the activation of the integrated stress response (ISR) in a GCN2 kinase-dependent manner. Furthermore, the discovery of additional drugs that are toxic for FBXW7-deficient cells showed that all of them unexpectedly activate a GCN2-dependent ISR regardless of their accepted mechanism of action. Our study reveals that while one of the most frequent mutations in cancer reduces the sensitivity to the vast majority of available therapies, it renders cells vulnerable to ISR-activating drugs.

A Chemical Screen Identifies Compounds Capable of Selecting for Haploidy in Mammalian Cells.

The recent availability of somatic haploid cell lines has provided a unique tool for genetic studies in mammals. However, the percentage of haploid cells rapidly decreases in these cell lines, which we recently showed is due to their overgrowth by diploid cells present in the cultures. Based on this property, we have now performed a phenotypic chemical screen in human haploid HAP1 cells aiming to identify compounds that facilitate the maintenance of haploid cells. Our top hit was 10-Deacetyl-baccatin-III (DAB), a chemical precursor in the synthesis of Taxol, which selects for haploid cells in HAP1 and mouse haploid embryonic stem cultures. Interestingly, DAB also enriches for diploid cells in mixed cultures of diploid and tetraploid cells, including in the colon cancer cell line DLD-1, revealing a general strategy for selecting cells with lower ploidy in mixed populations of mammalian cells.

Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.

E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.

USP7 is a SUMO deubiquitinase essential for DNA replication.

Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates DNA replication. We have previously shown that chromatin around replisomes is rich in SUMO and poor in Ub, whereas mature chromatin exhibits an opposite pattern. How this SUMO-rich, Ub-poor environment is maintained at sites of DNA replication in mammalian cells remains unexplored. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Inhibition or genetic deletion of USP7 leads to the accumulation of Ub on SUMOylated proteins, which are displaced away from replisomes. Our findings provide a model explaining the differential accumulation of SUMO and Ub at replication forks and identify an essential role of USP7 in DNA replication that should be considered in the development of USP7 inhibitors as anticancer agents.

Efficacy of ATR inhibitors as single agents in Ewing sarcoma.

Ewing sarcomas (ES) are pediatric bone tumors that arise from a driver translocation, most frequently EWS/FLI1. Current ES treatment involves DNA damaging agents, yet the basis for the sensitivity to these therapies remains unknown. Oncogene-induced replication stress (RS) is a known source of endogenous DNA damage in cancer, which is suppressed by ATR and CHK1 kinases. We here show that ES suffer from high endogenous levels of RS, rendering them particularly dependent on the ATR pathway. Accordingly, two independent ATR inhibitors show in vitro toxicity in ES cell lines as well as in vivo efficacy in ES xenografts as single agents. Expression of EWS/FLI1 or EWS/ERG oncogenic translocations sensitizes non-ES cells to ATR inhibitors. Our data shed light onto the sensitivity of ES to genotoxic agents, and identify ATR inhibitors as a potential therapy for Ewing Sarcomas.

Targeting the kinase activities of ATR and ATM exhibits antitumoral activity in mouse models of MLL-rearranged AML.

Among the various subtypes of acute myeloid leukemia (AML), those with chromosomal rearrangements of the MLL oncogene (AML-MLL) have a poor prognosis. AML-MLL tumor cells are resistant to current genotoxic therapies because of an attenuated response by p53, a protein that induces cell cycle arrest and apoptosis in response to DNA damage. In addition to chemicals that damage DNA, efforts have focused on targeting DNA repair enzymes as a general chemotherapeutic approach to cancer treatment. Here, we found that inhibition of the kinase ATR, which is the primary sensor of DNA replication stress, induced chromosomal breakage and death of mouse AML(MLL) cells (with an MLL-ENL fusion and a constitutively active N-RAS independently of p53. Moreover, ATR inhibition as a single agent exhibited antitumoral activity, both reducing tumor burden after establishment and preventing tumors from growing, in an immunocompetent allograft mouse model of AML(MLL) and in xenografts of a human AML-MLL cell line. We also found that inhibition of ATM, a kinase that senses DNA double-strand breaks, also promoted the survival of the AML(MLL) mice. Collectively, these data indicated that ATR or ATM inhibition represent potential therapeutic strategies for the treatment of AML, especially MLL-driven leukemias.

Activation of ARF by oncogenic stress in mouse fibroblasts is independent of E2F1 and E2F2.

The ARF tumour suppressor protein (p14(ARF) in human and p19(ARF) in mouse) is a major mediator of the activation of p53 in response to oncogenic stress. Little is known about the signalling pathways connecting oncogenic stimuli to the activation of ARF. Regulation of ARF occurs primarily at the transcriptional level and several modulators of ARF transcription have been identified. Notably, ectopic expression of E2F1 upregulates ARF transcriptionally, and both E2F1 and ARF have been implicated in apoptosis and cell-cycle arrest. We have used primary mouse fibroblasts deficient for E2F1, E2F2, or both to determine the possible role of these E2F proteins as upstream regulators of ARF in response to oncogenic stimuli and other stresses. In particular, we have studied the effects of oncogenic Ras and the viral oncoprotein E1A on ARF levels, neoplastic transformation, and sensitization to apoptosis. We have also examined the behaviour of the E2F-deficient MEFs with respect to immortalization and sensitivity to DNA damage. None of the ARF-mediated responses that we have analysed is significantly affected in E2F1(-/-), E2F2(-/-) or E2F1/2(-/-) MEFs, and ARF is upregulated normally in all cases. Taken together, our results indicate that the activation of ARF in response to oncogenic stress can occur by E2F1- and E2F2-independent mechanisms. This challenges previous suggestions implicating E2F factors as key mediators in the activation of ARF by oncogenic stress.