53BP1 is a well-known mediator of the cellular response to DNA damage. Two alternative mechanisms have been proposed to explain 53BP1's interaction with DNA double-strand breaks (DSBs), one by binding to methylated histones and the other via an RNF8 E3 ligase-dependent ubiquitylation pathway. The formation of RNF8 and 53BP1 irradiation-induced foci are both dependent on histone H2AX. To evaluate the contribution of the RNF8-dependent pathway to 53BP1 function, we generated RNF8 knockout mice. We report that RNF8 deficiency results in defective class switch recombination (CSR) and accumulation of unresolved immunoglobulin heavy chain-associated DSBs. The CSR DSB repair defect is milder than that observed in the absence of 53BP1 but similar to that found in H2AX(-/-) mice. Moreover, similar to H2AX but different from 53BP1 deficiency, RNF8(-/-) males are sterile, and this is associated with defective ubiquitylation of the XY chromatin. Combined loss of H2AX and RNF8 does not cause further impairment in CSR, demonstrating that the two genes function epistatically. Importantly, although 53BP1 foci formation is RNF8 dependent, its binding to chromatin is preserved in the absence of RNF8. This suggests a two-step mechanism for 53BP1 association with chromatin in which constitutive loading is dependent on interactions with methylated histones, whereas DNA damage-inducible RNF8-dependent ubiquitylation allows its accumulation at damaged chromatin.

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.

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.

In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT.

SAR445088 is an anti-C1s humanized monoclonal antibody that inhibits activated C1s in the proximal portion of the classical complement system and has the potential to provide clinical benefit in the treatment of complement-mediated diseases. A phase I, first-in-human, double-blind, randomized, placebo-controlled, dose-escalation trial of single and multiple doses of SAR445088 was conducted in 93 healthy participants to evaluate the safety, tolerability, and pharmacokinetic (PK) and pharmacodynamic (PD) profiles. Single (intravenous [i.v.] and subcutaneous [s.c.]) ascending doses (SAD) and multiple (s.c.) ascending doses (MAD) of SAR445088 were well-tolerated. The PK of SAR445088 was characterized by slow absorption after the s.c. dose and a long half-life (mean terminal half-life [t1/2 ] 8-15 weeks). Two PD assays were used to measure inhibition of the classical complement pathway (CP): Wieslab CP and complement mediated hemolytic capacity (CH50). The estimated half-maximal inhibitory concentration (IC50 ) and 90% inhibitory concentration (IC90 ) for the Wieslab CP assay were 96.4 and 458 μg/ml, respectively, and 16.6 and 57.0 μg/ml, respectively, for the CH50 assay. In summary, SAR445088 was well-tolerated and had favorable PK and PD profiles after SAD (i.v. or s.c.) and MAD (s.c.) in humans. These findings warrant further clinical investigations in patients with classical complement-mediated disorders.

The Shieldin complex shields double-strand DNA breaks (DSBs) from nucleolytic resection. Curiously, the penultimate Shieldin component, SHLD1, is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1, and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1, thereby ensuring a proper balance between end protection and resection during DSB repair. The loss of THAP1-dependent SHLD1 expression confers cross-resistance to poly (ADP-ribose) polymerase (PARP) inhibitor and cisplatin in BRCA1-deficient cells and shorter progression-free survival in ovarian cancer patients. Moreover, the embryonic lethality and PARPi sensitivity of BRCA1-deficient mice is rescued by ablation of SHLD1. Our study uncovers a transcriptional network that directly controls DSB repair choice and suggests a potential link between DNA damage and pathogenic THAP1 mutations, found in patients with the neurodevelopmental movement disorder adult-onset torsion dystonia type 6.

Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib has been approved for treatment of advanced ovarian cancer associated with BRCA1 and BRCA2 mutations. BRCA1- and BRCA2-mutated cells, which are homologous recombination (HR) deficient, are hypersensitive to PARPi through the mechanism of synthetic lethality. Here we examine the effect of PARPi on HR-proficient cells. Olaparib pretreatment, PARP1 knockdown or Parp1 heterozygosity of Brca2(cko/ko) mouse embryonic stem cells (mESCs), carrying a null (ko) and a conditional (cko) allele of Brca2, results in viable Brca2(ko/ko) cells. PARP1 deficiency does not restore HR in Brca2(ko/ko) cells, but protects stalled replication forks from MRE11-mediated degradation through its impaired recruitment. The functional consequence of Parp1 heterozygosity on BRCA2 loss is demonstrated by a significant increase in tumorigenesis in Brca2(cko/cko) mice. Thus, while olaparib efficiently kills BRCA2-deficient cells, we demonstrate that it can also contribute to the synthetic viability if PARP is inhibited before BRCA2 loss.

Topoisomerase II (TOP2) relieves topological stress in DNA by introducing double-strand breaks (DSBs) via a transient, covalently linked TOP2 DNA-protein intermediate, termed TOP2 cleavage complex (TOP2cc). TOP2ccs are normally rapidly reversible, but can be stabilized by TOP2 poisons, such as the chemotherapeutic agent etoposide (ETO). TOP2 poisons have shown significant variability in their therapeutic effectiveness across different cancers for reasons that remain to be determined. One potential explanation for the differential cellular response to these drugs is in the manner by which cells process TOP2ccs. Cells are thought to remove TOP2ccs primarily by proteolytic degradation followed by DNA DSB repair. Here, we show that proteasome-mediated repair of TOP2cc is highly error-prone. Pre-treating primary splenic mouse B-cells with proteasome inhibitors prevented the proteolytic processing of trapped TOP2ccs, suppressed the DNA damage response (DDR) and completely protected cells from ETO-induced genome instability, thereby preserving cellular viability. When degradation of TOP2cc was suppressed, the TOP2 enzyme uncoupled itself from the DNA following ETO washout, in an error-free manner. This suggests a potential mechanism of developing resistance to topoisomerase poisons by ensuring rapid TOP2cc reversal.

Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.

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.

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN, knowledge that would be helpful for informing clinical development of WRN-targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system wherein the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We find that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we find no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provided the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggested a potential therapeutical rationale for dual targeting of WRN and ATR.

Sutimlimab, a first-in-class humanized immunoglobulin G4 (IgG4) monoclonal antibody that selectively inhibits the classical complement pathway at C1s, rapidly halted hemolysis in the single-arm CARDINAL study in recently transfused patients with cold agglutinin disease (CAD). CADENZA was a 26-week randomized, placebo-controlled phase 3 study to assess safety and efficacy of sutimlimab in patients with CAD without recent (within 6 months prior to enrollment) transfusion history. Forty-two patients with screening hemoglobin ≤10 g/dL, elevated bilirubin, and ≥1 CAD symptom received sutimlimab (n = 22) or placebo (n = 20) on days 0 and 7 and then biweekly. Composite primary endpoint criteria (hemoglobin increase ≥1.5 g/dL at treatment assessment timepoint [mean of weeks 23, 25, 26], avoidance of transfusion, and study-prohibited CAD therapy [weeks 5-26]) were met by 16 patients (73%) on sutimlimab, and 3 patients (15%) on placebo (odds ratio, 15.9 [95% confidence interval, 2.9, 88.0; P < .001]). Sutimlimab, but not placebo, significantly increased mean hemoglobin and FACIT-Fatigue scores at treatment assessment timepoint. Sutimlimab normalized mean bilirubin by week 1. Improvements correlated with near-complete inhibition of the classical complement pathway (2.3% mean activity at week 1) and C4 normalization. Twenty-one (96%) sutimlimab patients and 20 (100%) placebo patients experienced ≥1 treatment-emergent adverse event. Headache, hypertension, rhinitis, Raynaud phenomenon, and acrocyanosis were more frequent with sutimlimab vs placebo, with a difference of ≥3 patients between groups. Three sutimlimab patients discontinued owing to adverse events; no placebo patients discontinued. These data demonstrate that sutimlimab has potential to be an important advancement in the treatment of CAD. This trial was registered at www.clinicaltrials.gov as #NCT03347422.

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.