ARP2/3- and resection-coupled genome reorganization facilitates translocations [preprint]

DNA end-resection and nuclear actin-based movements orchestrate clustering of double-strand breaks (DSBs) into homology-directed repair (HDR) domains. Here, we analyze how actin nucleation by ARP2/3 affects damage-dependent and -independent 3D genome reorganization and facilitates pathologic repair. We observe that DNA damage, followed by ARP2/3-dependent establishment of repair domains enhances local chromatin insulation at a set of damage-proximal boundaries and affects compartment organization genome-wide. Nuclear actin polymerization also promotes interactions between DSBs, which in turn facilitates aberrant intra- and inter-chromosomal rearrangements. Notably, BRCA1 deficiency, which decreases end-resection, DSB mobility, and subsequent HDR, nearly abrogates recurrent translocations between AsiSI DSBs. In contrast, loss of functional BRCA1 yields unique translocations genome-wide, reflecting a critical role in preventing spontaneous genome instability and subsequent rearrangements. Our work establishes that the assembly of DSB repair domains is coordinated with multiscale alterations in genome architecture that enable HDR despite increased risk of translocations with pathologic potential

Chimeric IgH-TCR/ translocations in T lymphocytes mediated by RAG

Translocations involving the T cell receptor alpha/delta (TCRα/δ) chain locus, which bring oncogenes in the proximity of the TCRα enhancer, are one of the hallmark features of human T cell malignancies from ataxia telangiectasia (AT) and non-AT patients. These lesions are frequently generated by the fusion of DNA breaks at the TCRα/δ locus to a disperse region centromeric of the immunoglobulin heavy chain (IgH) locus. Aberrant VDJ joining accounts for TCRα/δ associated DNA cleavage, but the molecular mechanism that leads to generation of the "oncogene partner" DNA break is unclear. Here we show that in ATM deficient primary mouse T cells, IgH/TCRα/δ fusions arise at a remarkably similar frequency as in human AT lymphocytes. Recombinase-activating gene (RAG) is responsible for both TCRα/δ as well as IgH associated breaks on chromosome 12 (Chr12), which are subject to varying degrees of chromosomal degradation. We suggest a new model for how oncogenic translocations can arise from two non-concerted physiological DSBs

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.We would want to thank Enrique de Alava for providing ES lines. Work in O.F. laboratory was supported by Fundación Botín, by Banco Santander through its Santander Universities Global Division and by grants from MINECO (SAF2014-57791-REDC and SAF2014-59498-R), Fundació La Marato de TV3, Howard Hughes Medical Institute and the European Research Council (ERC-617840). The A.N. laboratory was supported by the Intramural Research Program of the NIH, the National Cancer Institute, the Center for Cancer Research, an Ellison Medical Foundation Senior Scholar in Aging, and the Alex Lemonade Stand Foundation Award. J.A. laboratory is supported by Asociación Pablo Ugarte, ASION-La Hucha de Tomás, Fundación La Sonrisa de Alex and Instituto de Salud Carlos III (PI12/00816 and Spanish Cancer Network RTICC RD12/0036/0027). A.L. laboratory was supported by the Danish National Research Foundation (DNRF115), Danish Council for Independent Research (Sapere Aude, DFF-Starting Grant 2014) and Danish Cancer Society (KBVU-2014).S

Plasmodium Infection Promotes Genomic Instability and AID-Dependent B Cell Lymphoma

SummaryChronic infection with Plasmodium falciparum was epidemiologically associated with endemic Burkitt’s lymphoma, a mature B cell cancer characterized by chromosome translocation between the c-myc oncogene and Igh, over 50 years ago. Whether infection promotes B cell lymphoma, and if so by which mechanism, remains unknown. To investigate the relationship between parasitic disease and lymphomagenesis, we used Plasmodium chabaudi (Pc) to produce chronic malaria infection in mice. Pc induces prolonged expansion of germinal centers (GCs), unique compartments in which B cells undergo rapid clonal expansion and express activation-induced cytidine deaminase (AID), a DNA mutator. GC B cells elicited during Pc infection suffer widespread DNA damage, leading to chromosome translocations. Although infection does not change the overall rate, it modifies lymphomagenesis to favor mature B cell lymphomas that are AID dependent and show chromosome translocations. Thus, malaria infection favors mature B cell cancers by eliciting protracted AID expression in GC B cells.PaperCli

53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination.

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.We thank Anthony Tubbs for comments on the paper; Jennifer Mehalko and Dom Esposito (Protein Expression Laboratory, Frederick National Laboratory for Cancer Research) for transgenic constructs; Karim Baktiar, Diana Haines, and Elijah Edmonson (Pathology/Histotechnology Laboratory, Frederick National Laboratory for Cancer Research) for rodent necropsy, pathology analysis, and imaging; Joseph Kalen and Nimit Patel (Small Animal Imaging Program, Frederick National Laboratory for Cancer Research) for X-ray computed tomography (CT) scan imaging; Jennifer Wise and Kelly Smith for assistance with animal work; Davide Robbiani and Kai Ge for antibodies; Dan Durocher for shieldin constructs; David Goldstein and the CCR Genomics core for sequencing support; and Neil Johnson for discussions. Research in the J.M.S. laboratory is supported by NIH grant R01CA197506. Research in the N.M. laboratory is supported by NIH grant R01 227001. The A.N. laboratory is supported by the Intramural Research Program of the NIH, an Ellison Medical Foundation Senior Scholar in Aging Award (AG-SS-2633-11), the Department of Defense Idea Expansion (W81XWH-15-2-006) and Breakthrough (W81XWH-16-1-599) Awards, the Alex's Lemonade Stand Foundation Award, and an NIH Intramural FLEX Award.S

Congenital bone marrow failure in DNA-PKcs mutant mice associated with deficiencies in DNA repair

The nonhomologous end-joining (NHEJ) pathway is essential for radioresistance and lymphocyte-specific V(D)J (variable [diversity] joining) recombination. Defects in NHEJ also impair hematopoietic stem cell (HSC) activity with age but do not affect the initial establishment of HSC reserves. In this paper, we report that, in contrast to deoxyribonucleic acid (DNA)–dependent protein kinase catalytic subunit (DNA-PKcs)–null mice, knockin mice with the DNA-PKcs(3A/3A) allele, which codes for three alanine substitutions at the mouse Thr2605 phosphorylation cluster, die prematurely because of congenital bone marrow failure. Impaired proliferation of DNA-PKcs(3A/3A) HSCs is caused by excessive DNA damage and p53-dependent apoptosis. In addition, increased apoptosis in the intestinal crypt and epidermal hyperpigmentation indicate the presence of elevated genotoxic stress and p53 activation. Analysis of embryonic fibroblasts further reveals that DNA-PKcs(3A/3A) cells are hypersensitive to DNA cross-linking agents and are defective in both homologous recombination and the Fanconi anemia DNA damage response pathways. We conclude that phosphorylation of DNA-PKcs is essential for the normal activation of multiple DNA repair pathways, which in turn is critical for the maintenance of diverse populations of tissue stem cells in mice

EXO1 protects BRCA1-deficient cells against toxic DNA lesions

Inactivating mutations in the BRCA1 and BRCA2 genes impair DNA double-strand break (DSB) repair by homologous recombination (HR), leading to chromosomal instability and cancer. Importantly, BRCA1/2 deficiency also causes therapeutically targetable vulnerabilities. Here, we identify the dependency on the end resection factor EXO1 as a key vulnerability of BRCA1-deficient cells. EXO1 deficiency generates poly(ADP-ribose)-decorated DNA lesions during S phase that associate with unresolved DSBs and genomic instability in BRCA1-deficient but not in wild-type or BRCA2-deficient cells. Our data indicate that BRCA1/EXO1 double-deficient cells accumulate DSBs due to impaired repair by single-strand annealing (SSA) on top of their HR defect. In contrast, BRCA2-deficient cells retain SSA activity in the absence of EXO1 and hence tolerate EXO1 loss. Consistent with a dependency on EXO1-mediated SSA, we find that BRCA1-mutated tumors show elevated EXO1 expression and increased SSA-associated genomic scars compared with BRCA1-proficient tumors. Overall, our findings uncover EXO1 as a promising therapeutic target for BRCA1-deficient tumors

Replication Fork Stability Confers Chemoresistance in BRCA-deficient Cells

Brca1- and Brca2-deficient cells have reduced capacity to repair DNA double-strand breaks (DSBs) by homologous recombination (HR) and consequently are hypersensitive to DNA damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore HR activity at DSBs. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARPi and cisplatin resistance is associated with replication fork (RF) protection in Brca2-deficient tumor cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of RF protection, highlighting the complexities by which tumor cells evade chemotherapeutic interventions and acquire drug resistance

ATM and PRDM9 regulate SPO11-bound recombination intermediates during meiosis

Meiotic recombination is initiated by SPO11-induced double-strand breaks (DSBs). In most mammals, the methyltransferase PRDM9 guides SPO11 targeting, and the ATM kinase controls meiotic DSB numbers. Following MRE11 nuclease removal of SPO11, the DSB is resected and loaded with DMC1 filaments for homolog invasion. Here, we demonstrate the direct detection of meiotic DSBs and resection using END-seq on mouse spermatocytes with low sample input. We find that DMC1 limits both minimum and maximum resection lengths, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles. Through enzymatic modifications to END-seq, we identify a SPO11-bound meiotic recombination intermediate (SPO11-RI) present at all hotspots. We propose that SPO11-RI forms because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm–/– spermatocytes, trapped SPO11 cleavage complexes accumulate due to defective MRE11 initiation of resection. Thus, in addition to governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing

The hSSB1 orthologue Obfc2b is essential for skeletogenesis but dispensable for the DNA damage response in vivo.

Human single-stranded DNA-binding protein 1 (hSSB1), encoded by OBFC2B, was recently characterized as an essential factor for the initiation of DNA damage checkpoints and the maintenance of genomic stability. Here, we report that loss of Obfc2b in mice results in perinatal lethality characterized by growth delay and skeletal abnormalities. These abnormalities are associated with accumulation of γH2ax, apoptosis and defective pre-cartilage condensation, which is essential for normal bone formation. However, deficiency of Obfc2b does not affect the initiation of DNA damage checkpoints, Atm activation, or the maintenance of genomic stability in B lymphocytes and primary fibroblasts. Loss of Obfc2b results in increased expression of its homologue Obfc2a (hSSB2). In contrast to Obfc2b deficiency, depletion of Obfc2a in fibroblasts results in impaired proliferation, accumulation of γH2ax and increased genomic instability. Thus, the hSSB1 orthologue Obfc2b has a unique function during embryogenesis limited to cell types that contribute to bone formation. While being dispensable in most other cell lineages, its absence leads to a compensatory increase in Obfc2a protein, a homologue required for the maintenance of genomic integrity