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Probing the synthetic lethal effect of FANCD2 deletion in BRCA2-deficient cells reveals independent roles of FANCD2 and BRCA2 in stabilizing stalled replication forks to maintain genome stability and promote cell survival. The tumor suppressor BRCA2 plays a key role in genome integrity by promoting replication-fork stability and homologous recombination (HR) DNA repair. Here we report that human cancer cells lacking BRCA2 rely on the Fanconi anemia protein FANCD2 to limit replication-fork progression and genomic instability. Our results identify a new role of FANCD2 in limiting constitutive replication stress in BRCA2-deficient cells, thereby affecting cell survival and treatment responses.

Failure to restart replication forks stalled at genomic regions that are difficult to replicate or contain endogenous DNA lesions is a hallmark of BRCA2 deficiency. The nucleolytic activity of MUS81 endonuclease is required for replication fork restart under replication stress elicited by exogenous treatments. Here we investigate whether MUS81 could similarly facilitate DNA replication in the context of BRCA2 abrogation. Our results demonstrate that replication fork progression in BRCA2-deficient cells requires MUS81. Failure to complete genome replication and defective checkpoint surveillance enables BRCA2-deficient cells to progress through mitosis with under-replicated DNA, which elicits severe chromosome interlinking in anaphase. MUS81 nucleolytic activity is required to activate compensatory DNA synthesis during mitosis and to resolve mitotic interlinks, thus facilitating chromosome segregation. We propose that MUS81 provides a mechanism of replication stress tolerance, which sustains survival of BRCA2-deficient cells and can be exploited therapeutically through development of specific inhibitors of MUS81 nuclease activity. BRCA2-deficient cells fail to complete replication in S-phase, which results in DNA bridges and chromosome mis-segregation at mitosis. Here the authors show that MUS81 helps BRCA2-deficient cells survive replication stress by activating mitotic DNA synthesis and resolving mitotic DNA bridges.

The cells with compromised BRCA1 or BRCA2 (BRCA1/2) function accumulate stalled replication forks, which leads to replication‐associated DNA damage and genomic instability, a signature of BRCA1/2 ‐mutated tumours. Targeted therapies against BRCA1/2 ‐mutated tumours exploit this vulnerability by introducing additional DNA lesions. Because homologous recombination (HR) repair is abrogated in the absence of BRCA1 or BRCA2, these lesions are specifically lethal to tumour cells, but not to the healthy tissue. Ligands that bind and stabilise G‐quadruplexes (G4s) have recently emerged as a class of compounds that selectively eliminate the cells and tumours lacking BRCA1 or BRCA2. Pyridostatin is a small molecule that binds G4s and is specifically toxic to BRCA1/2‐deficient cells in vitro . However, its in vivo potential has not yet been evaluated. Here, we demonstrate that pyridostatin exhibits a high specific activity against BRCA1/2‐deficient tumours, including patient‐derived xenograft tumours that have acquired PARP inhibitor (PARPi) resistance. Mechanistically, we demonstrate that pyridostatin disrupts replication leading to DNA double‐stranded breaks (DSBs) that can be repaired in the absence of BRCA1/2 by canonical non‐homologous end joining (C‐NHEJ). Consistent with this, chemical inhibitors of DNA‐PKcs, a core component of C‐NHEJ kinase activity, act synergistically with pyridostatin in eliminating BRCA1/2‐deficient cells and tumours. Furthermore, we demonstrate that pyridostatin triggers cGAS/STING‐dependent innate immune responses when BRCA1 or BRCA2 is abrogated. Paclitaxel, a drug routinely used in cancer chemotherapy, potentiates the in vivo toxicity of pyridostatin. Overall, our results demonstrate that pyridostatin is a compound suitable for further therapeutic development, alone or in combination with paclitaxel and DNA‐PKcs inhibitors, for the benefit of cancer patients carrying BRCA1/2 mutations. Synopsis DNA replication and repair defects caused by loss of BRCA1/2 can be exploited therapeutically to specifically target tumours carrying mutations in these genes. Pyridostatin and its combinations with NU‐7441 and/or paclitaxel, specifically eliminate BRCA1/2‐deficient tumours in vivo . Pyridostatin, a G‐quadruplex ligand, inflicts DNA damage and exhibits anti‐tumour activity in the absence of BRCA1/2 in vivo , including against patient‐derived PARPi‐resistant tumours. DNA lesions caused by pyridostatin are repaired by C‐NHEJ. Inhibition of C‐NHEJ with the DNA‐PK inhibitor NU‐7441 sustains unrepaired double‐strand breaks in BRCA1/2‐deficient cells, even after pyridostatin removal. Combination of pyridostatin with NU‐7441 and paclitaxel is associated with long‐lasting anti‐tumour efficacy in the absence of BRCA1/2. Pyridostatin and its combinations are well tolerated in vivo and pave the way for novel targeted anti‐tumour and/or maintenance therapies. Graphical Abstract DNA replication and repair defects caused by loss of BRCA1/2 can be exploited therapeutically to specifically target tumours carrying mutations in these genes. Pyridostatin and its combinations with NU‐7441 and/or paclitaxel, specifically eliminate BRCA1/2‐deficient tumours in vivo .

The ability to transmit genetic information through generations depends on the preservation of genome integrity. Genetic abnormalities affect cell differentiation, causing tissue specification defects and cancer. We addressed genomic instability in individuals with Differences of Sex Development (DSD), characterized by gonadal dysgenesis, infertility, high susceptibility for different types of cancer, especially Germ Cell Tumors (GCT), and in men with testicular GCTs. Whole proteome analysis of leukocytes, supported by specific gene expression assessment, and dysgenic gonads characterization, uncovered DNA damage phenotypes with altered innate immune response and autophagy. Further examination of DNA damage response revealed a reliance on deltaTP53, which was compromised by mutations in the transactivation domain in DSD-individuals with GCT. Accordingly, drug-induced rescue of DNA damage was achieved by autophagy inhibition but not by stabilization of TP53 in DSD-individuals’ blood in vitro. This study elucidates possibilities for prophylactic treatments of DSD-individuals, as well as new diagnostic approaches of GCT.

Nature Communications 8: Article number: 15983 (2017); Published: 17 July 2017; Updated: 26 October 2017 In this Article, there are errors in the labelling of the y axis in Fig. 1a and Supplementary Fig. 1b. The labels ‘20’, ‘40’, ‘60’, ‘80’ and ‘100’ should have been ‘0.5’, ‘1.0’ and ‘1.5’ in Fig. 1a and the labels ‘50’, ‘100’, ‘150’ and ‘200’ should have been ‘1’, ‘2’ and ‘3’ in Supplementary Fig.

Crystallographic analysis depicting the interaction of the kinase inhibitor SCH772984 with ERK1/2 reveals a unique binding pocket distinct from off-targets such as haspin and is associated with slow binding kinetics and prolonged inhibitory activity. Activation of the ERK pathway is a hallmark of cancer, and targeting of upstream signaling partners led to the development of approved drugs. Recently, SCH772984 has been shown to be a selective and potent ERK1/2 inhibitor. Here we report the structural mechanism for its remarkable selectivity. In ERK1/2, SCH772984 induces a so-far-unknown binding pocket that accommodates the piperazine-phenyl-pyrimidine decoration. This new binding pocket was created by an inactive conformation of the phosphate-binding loop and an outward tilt of helix αC. In contrast, structure determination of SCH772984 with the off-target haspin and JNK1 revealed two canonical but distinct type I binding modes. Notably, the new binding mode with ERK1/2 was associated with slow binding kinetics in vitro as well as in cell-based assay systems. The described binding mode of SCH772984 with ERK1/2 enables the design of a new type of specific kinase inhibitors with prolonged on-target activity.

This corrects the article DOI: 10.1038/ncomms15983.

Genomic instability is a hallmark of cancer. The tumour suppressors BRCA1 and BRCA2 play key roles in genome integrity by promoting homologous recombination DNA repair and replication fork stability. Deficiency in these functions has been described as the Achilles heel of BRCA-defective tumours. This provides an important rationale for further exploring the roles of BRCA1 and BRCA2 in DNA replication and genome stability. G-quadruplexes, alternative DNA structures formed by guanine-rich single- stranded DNA, represent natural replication fork barriers. This study demonstrates that treatment with the G-quadruplex-stabilising compound pyridostatin selectively decreases viability of BRCA1- and BRCA2-deficient cells by inducing replication stress and DNA damage. Furthermore, this work identifies a new role of the Fanconi anaemia protein FANCD2 in limiting replication fork progression and genomic instability in human cancer cells lacking BRCA2. This function of FANCD2 is vital for BRCA2-deficient cell survival and affects treatment responses. Finally, the data presented here reveal a synthetic lethal interaction between MRE11 nuclease and BRCA2. Characterisation of a novel chemical MRE11 nuclease inhibitor with activity on 2D and 3D cell culture models for BRCA2 deficiency highlights the clinical relevance for the use of MRE11 inhibitors for targeting BRCA2-deficient tumours. In summary, this report describes novel roles of BRCA1 and BRCA2 relevant for selective targeting of BRCA-deficient tumour cells.

The ability to transmit genetic information through generations depends on preservation of genome integrity. Genetic abnormalities affect cell differentiation, causing tissue specification defects and cancer. We addressed genomic instability in individuals with Differences of Sex Development (DSD), characterized by gonadal dysgenesis, infertility, high susceptibility for different types of cancer, especially Germ Cell Tumors (GCT), and in men with testicular GCTs. Whole proteome analysis of leukocytes, supported by specific gene expression assessment, and dysgenic gonads characterization, uncovered DNA damage phenotypes with altered innate immune response and autophagy. Further examination of DNA damage response revealed a reliance on deltaTP53, which was compromised by mutations in the transactivation domain in DSD-patients with GCT. Accordingly, drug-induced rescue of DNA damage was achieved by autophagy inhibition but not by stabilization of TP53 in DSD-patients blood in vitro. This study elucidates possibilities for prophylactic treatments of DSD patients as well as new diagnostic approaches of GCT. Competing Interest Statement The authors have declared no competing interest.

The AZFa locus functional in human spermatogenesis has been mapped to proximal Yq11 by molecular deletion analysis. Infertile men with complete aplasia of germ cells and only Sertoli cells in their testis tubules (SCO syndrome) are frequently found to have a genomic DNA deletion in this Y region, caused by the recombination of two homologous HERV15 sequence blocks. This AZFa deletion extends 792 kb and includes two functional Y genes, USP9Y and DBY. Its frequency of deletion might be different in distinct Y chromosomal haplogroups (D2* and J*). Mutation analyses and positional expression analyses in human testis tissue sections have indicated that probably both genes are translationally controlled in the male germ line. Accordingly, their male-specific cellular functions are different from that of their functional homologs on the X chromosome, USP9X and DBX, explaining the AZFa patients' pathology. Comparative sequence analysis of the X–Y homologous genome region in proximal Yq11 revealed that the AZFa locus might also include the UTY gene mapped distal to DBY, like UTX is mapped distal to DBX in Xp11.4. This chromatin domain has been evolutionarily conserved on the sex chromosomes for 100 million years and has probably evolved before mammalian radiation. Molecular deletion mapping also points to an overlap of the AZFa locus with the HY antigen (HYA) locus in proximal Yq11, and with the gonadoblastoma Y (GBY) locus in proximal Yp11 and Yq11. Each AZFa gene expresses distinct HY antigens, the presence of which might be hazardous in the gonad cells of women with dysgenetic gonads and a Y chromosome in the karyotype 46, XY/45, XX by contributing to the expression of gonadoblastoma cells. The different expression profiles of the human DBY and its mouse Dby homolog suggest that the evolutionary pressure on Y genes functional for spermatogenesis in mouse and man might be different when considering the single genes, but more comparable after setting the focus on the complete gene content of the mouse and human Y chromosomes, because they are involved in the same genetic networks controlling the spermatogenic germ cell maturation process.