Identification of early replicating fragile sites that contribute to genome instability.

DNA double-strand breaks (DSBs) in B lymphocytes arise stochastically during replication or as a result of targeted DNA damage by activation-induced cytidine deaminase (AID). Here we identify recurrent, early replicating, and AID-independent DNA lesions, termed early replication fragile sites (ERFSs), by genome-wide localization of DNA repair proteins in B cells subjected to replication stress. ERFSs colocalize with highly expressed gene clusters and are enriched for repetitive elements and CpG dinucleotides. Although distinct from late-replicating common fragile sites (CFS), the stability of ERFSs and CFSs is similarly dependent on the replication-stress response kinase ATR. ERFSs break spontaneously during replication, but their fragility is increased by hydroxyurea, ATR inhibition, or deregulated c-Myc expression. Moreover, greater than 50% of recurrent amplifications/deletions in human diffuse large B cell lymphoma map to ERFSs. In summary, we have identified a source of spontaneous DNA lesions that drives instability at preferred genomic sites.

53BP1 mediates productive and mutagenic DNA repair through distinct phosphoprotein interactions.

The DNA damage response (DDR) protein 53BP1 protects DNA ends from excessive resection in G1, and thereby favors repair by nonhomologous end-joining (NHEJ) as opposed to homologous recombination (HR). During S phase, BRCA1 antagonizes 53BP1 to promote HR. The pro-NHEJ and antirecombinase functions of 53BP1 are mediated in part by RIF1, the only known factor that requires 53BP1 phosphorylation for its recruitment to double-strand breaks (DSBs). Here, we show that a 53BP1 phosphomutant, 53BP18A, comprising alanine substitutions of the eight most N-terminal S/TQ phosphorylation sites, mimics 53BP1 deficiency by restoring genome stability in BRCA1-deficient cells yet behaves like wild-type 53BP1 with respect to immunoglobulin class switch recombination (CSR). 53BP18A recruits RIF1 but fails to recruit the DDR protein PTIP to DSBs, and disruption of PTIP phenocopies 53BP18A. We conclude that 53BP1 promotes productive CSR and suppresses mutagenic DNA repair through distinct phosphodependent interactions with RIF1 and PTIP.

53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks.

Defective DNA repair by homologous recombination (HR) is thought to be a major contributor to tumorigenesis in individuals carrying Brca1 mutations. Here, we show that DNA breaks in Brca1-deficient cells are aberrantly joined into complex chromosome rearrangements by a process dependent on the nonhomologous end-joining (NHEJ) factors 53BP1 and DNA ligase 4. Loss of 53BP1 alleviates hypersensitivity of Brca1 mutant cells to PARP inhibition and restores error-free repair by HR. Mechanistically, 53BP1 deletion promotes ATM-dependent processing of broken DNA ends to produce recombinogenic single-stranded DNA competent for HR. In contrast, Lig4 deficiency does not rescue the HR defect in Brca1 mutant cells but prevents the joining of chromatid breaks into chromosome rearrangements. Our results illustrate that HR and NHEJ compete to process DNA breaks that arise during DNA replication and that shifting the balance between these pathways can be exploited to selectively protect or kill cells harboring Brca1 mutations.

Major pathological remissions in a patient with stage IIIA nonsmall cell lung cancer after neoadjuvant tislelizumab combined with chemotherapy: a case report and literature review.

INTRODUCTION: Currently, there are few reports of patients with locally advanced lung cancer achieving a clinical complete response by medical treatment. Preoperative neoadjuvant immunotherapy combined with chemotherapy is an option for patients with unresectable, locally advanced nonsmall cell lung cancer (NSCLC) which is of great potential, and may change traditional treatment paradigms. There are relatively few large-scale, high-quality randomized-controlled trials yet, and limitations such as short postoperative follow-up period and immature disease-free survival and overall survival data still persist. Thus, evidence-based medical evidence is urgently needed. It is worthy to explore the further treatment of patients who achieved complete response after initial treatment, though lacking of evidence by now. CASE PRESENTATION: We report a stage IIIA lung squamous cell carcinoma case who achieved a major pathologic remission after neoadjuvant treatment with tislelizumab and chemotherapy. CONCLUSION: Our case study contributes to the existing evidence on the feasibility, efficacy and safety of neoadjuvant immunotherapy combined with chemotherapy in locally advanced unresectable NSCLC.

AID is required for the chromosomal breaks in c-myc that lead to c-myc/IgH translocations.

Chromosomal translocation requires formation of paired double-strand DNA breaks (DSBs) on heterologous chromosomes. One of the most well characterized oncogenic translocations juxtaposes c-myc and the immunoglobulin heavy-chain locus (IgH) and is found in Burkitt's lymphomas in humans and plasmacytomas in mice. DNA breaks in IgH leading to c-myc/IgH translocations are created by activation-induced cytidine deaminase (AID) during antibody class switch recombination or somatic hypermutation. However, the source of DNA breaks at c-myc is not known. Here, we provide evidence for the c-myc promoter region being required in targeting AID-mediated DNA damage to produce DSBs in c-myc that lead to c-myc/IgH translocations in primary B lymphocytes. Thus, in addition to producing somatic mutations and DNA breaks in antibody genes, AID is also responsible for the DNA lesions in oncogenes that are required for their translocation.

Weak value amplification for angular velocity measurements.

The weak-value-amplification technique has shown great importance in the measurement of tiny physical effects. Here we introduce a polarization-dependent angular velocity measurement system consisting of two Glan prisms and a true zero-order half-wave plate, where a non-Fourier-limited Gaussian pulse acts as the meter. The angular velocities measurements results agree well with theoretical predictions, and its uncertainties are bounded by the Cramér-Rao bound. We also investigate uncertainties of angular velocities for different numbers of detected photons and the smallest reliable postselection probability, which can reach ${3.42*10^{- 6}}$.

Precise analysis of the effect of basal core promoter/precore mutations on the main phenotype of chronic hepatitis B in mouse models.

High replication and mutation rates of hepatitis B virus (HBV) often lead to reduced or suppressed hepatitis B e antigen expression. The most common mutations are genomic variations in the basal core promoter (BCP) and pre-core (PC) regions. However, the effect of BCP/PC mutations on HBV phenotype in vivo remains unclear. We compared and analyzed BCP/PC mutations and BCP/PC reverse mutations in mouse models. In addition to terminating the expression of HBeAg, BCP/PC mutations also resulted in a significant decrease in HBsAg, HBV DNA, and cccDNA in the early stage, and an obvious increase in serum alanine aminotransferase throughout the transfection period. In both groups, serum HBV DNA was positively correlated with intracellular HBV DNA and cccDNA. Further, we found that interleukin-4 (IL-4) and L-10 levels were significantly lower in the BCP/PC(M) group than in the BCP/PC(R) group at 4 weeks post-injection. However, IL-1ß was significantly lower in the BCP/PC(M) group than in the BCP/PC(R) group at 26 weeks post-injection. In summary, we precisely analyzed the effect of BCP/PC mutations on the phenotype in vivo, which is important to evaluating disease progression and treatment responses of variable chronic hepatitis B patients.

DNA-damage-induced differentiation of leukaemic cells as an anti-cancer barrier.

Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL-AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4(-/-) MLL-AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL-AF9 blasts, which requires cyclin-dependent kinase inhibitor p21(Cip1) (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.

BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair.

Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice and rescues HR deficiency, as measured by hypersensitivity to polyADP-ribose polymerase (PARP) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA crosslink repair that is distinct from HR. Disruption of the nonhomologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2 provides a key activity that cannot be bypassed by ablation of 53BP1 or Ku.

Regulation of DNA end joining, resection, and immunoglobulin class switch recombination by 53BP1.

53BP1 is a DNA damage protein that forms phosphorylated H2AX (γ-H2AX) dependent foci in a 1 Mb region surrounding DNA double-strand breaks (DSBs). In addition, 53BP1 promotes genomic stability by regulating the metabolism of DNA ends. We have compared the joining rates of paired DSBs separated by 1.2 kb to 27 Mb on chromosome 12 in the presence or absence of 53BP1. 53BP1 facilitates joining of intrachromosomal DSBs but only at distances corresponding to γ-H2AX spreading. In contrast, DNA end protection by 53BP1 is distance independent. Furthermore, analysis of 53BP1 mutants shows that chromatin association, oligomerization, and N-terminal ATM phosphorylation are all required for DNA end protection and joining as measured by immunoglobulin class switch recombination. These data elucidate the molecular events that are required for 53BP1 to maintain genomic stability and point to a model wherein 53BP1 and H2AX cooperate to repress resection of DSBs.

Programming the magnitude and persistence of antibody responses with innate immunity.

Many successful vaccines induce persistent antibody responses that can last a lifetime. The mechanisms by which they do so remain unclear, but emerging evidence indicates that they activate dendritic cells via Toll-like receptors (TLRs). For example, the yellow fever vaccine YF-17D, one of the most successful empiric vaccines ever developed, activates dendritic cells via multiple TLRs to stimulate proinflammatory cytokines. Triggering specific combinations of TLRs in dendritic cells can induce synergistic production of cytokines, which results in enhanced T-cell responses, but its impact on antibody responses remain unknown. Learning the critical parameters of innate immunity that program such antibody responses remains a major challenge in vaccinology. Here we demonstrate that immunization of mice with synthetic nanoparticles containing antigens plus ligands that signal through TLR4 and TLR7 induces synergistic increases in antigen-specific, neutralizing antibodies compared to immunization with nanoparticles containing antigens plus a single TLR ligand. Consistent with this there was enhanced persistence of germinal centres and of plasma-cell responses, which persisted in the lymph nodes for >1.5 years. Surprisingly, there was no enhancement of the early short-lived plasma-cell response relative to that observed with single TLR ligands. Molecular profiling of activated B cells, isolated 7 days after immunization, indicated that there was early programming towards B-cell memory. Antibody responses were dependent on direct triggering of both TLRs on B cells and dendritic cells, as well as on T-cell help. Immunization protected completely against lethal avian and swine influenza virus strains in mice, and induced robust immunity against pandemic H1N1 influenza in rhesus macaques.

Essential role for DNA-PKcs in DNA double-strand break repair and apoptosis in ATM-deficient lymphocytes.

The DNA double-strand break (DSB) repair protein DNA-PKcs and the signal transducer ATM are both activated by DNA breaks and phosphorylate similar substrates in vitro, yet appear to have distinct functions in vivo. Here, we show that ATM and DNA-PKcs have overlapping functions in lymphocytes. Ablation of both kinase activities in cells undergoing immunoglobulin class switch recombination leads to a compound defect in switching and a synergistic increase in chromosomal fragmentation, DNA insertions, and translocations due to aberrant processing of DSBs. These abnormalities are attributed to a compound deficiency in phosphorylation of key proteins required for DNA repair, class switching, and cell death. Notably, both kinases are required for normal levels of p53 phosphorylation in B and T cells and p53-dependent apoptosis. Our experiments reveal a DNA-PKcs-dependent pathway that regulates DNA repair and activation of p53 in the absence of ATM.

53BP1 facilitates long-range DNA end-joining during V(D)J recombination.

Variable, diversity and joining (V(D)J) recombination and class-switch recombination use overlapping but distinct non-homologous end joining pathways to repair DNA double-strand-break intermediates. 53BP1 is a DNA-damage-response protein that is rapidly recruited to sites of chromosomal double-strand breaks, where it seems to function in a subset of ataxia telangiectasia mutated (ATM) kinase-, H2A histone family member X (H2AX, also known as H2AFX)- and mediator of DNA damage checkpoint 1 (MDC1)-dependent events. A 53BP1-dependent end-joining pathway has been described that is dispensable for V(D)J recombination but essential for class-switch recombination. Here we report a previously unrecognized defect in the joining phase of V(D)J recombination in 53BP1-deficient lymphocytes that is distinct from that found in classical non-homologous-end-joining-, H2ax-, Mdc1- and Atm-deficient mice. Absence of 53BP1 leads to impairment of distal V-DJ joining with extensive degradation of unrepaired coding ends and episomal signal joint reintegration at V(D)J junctions. This results in apoptosis, loss of T-cell receptor alpha locus integrity and lymphopenia. Further impairment of the apoptotic checkpoint causes propagation of lymphocytes that have antigen receptor breaks. These data suggest a more general role for 53BP1 in maintaining genomic stability during long-range joining of DNA breaks.

Easy but Efficient: Facile Approach to Molecule with Theoretically Justified Donor-Acceptor Structure for Effective Photothermal Conversion and Intravenous Photothermal Therapy.

To accelerate the pace in the field of photothermal therapy (PTT), it is urged to develop easily accessible photothermal agents (PTAs) showing high photothermal conversion efficiency (PCE). As a proof-of-concept, hereby a conventional strategy is presented to prepare donor-acceptor (D-A) structured PTAs through cycloaddition-retroelectrocyclization (CA-RE) reaction, and the resultant PTAs give high PCE upon near-infrared (NIR) irradiation. By joint experimental-theoretical study, these PTAs exhibit prominent D-A structure with strong intramolecular charge transfer (ICT) characteristics and significantly twisting between D and A units which account for the high PCEs. Among them, the DMA-TCNQ exhibits the strongest absorption in NIR range as well as the highest PCE of 91.3% upon irradiation by 760-nm LED lamp (1.2 W cm-2). In vitro and in vivo experimental results revealed that DMA-TCNQ exhibits low dark toxicity and high phototoxicity after IR irradiation along with nude mice tumor inhibition up to 81.0% through intravenous therapy. The findings demonstrate CA-RE reaction as a convenient approach to obtain twisted D-A structured PTAs for effective PTT and probably promote the progress of cancer therapies.

The PDZ protein TIP-1 facilitates cell migration and pulmonary metastasis of human invasive breast cancer cells in athymic mice.

Tax-interacting protein 1 (TIP-1, also known as Tax1bp3) inhibited proliferation of colon cancer cells through antagonizing the transcriptional activity of beta-catenin. However, in this study, elevated TIP-1 expression levels were detected in human invasive breast cancers. Studies with two human invasive breast cancer cell lines indicated that RNAi-mediated TIP-1 knockdown suppressed the cell adhesion, proliferation, migration and invasion in vitro, and inhibited tumor growth in mammary fat pads and pulmonary metastasis in athymic mice. Biochemical studies showed that TIP-1 knockdown had moderate and differential effects on the beta-catenin-regulated gene expression, but remarkably down regulated the genes for cell adhesion and motility in breast cancer cells. The decreased expression of integrins and paxillin was accompanied with reduced cell adhesion and focal adhesion formation on fibronectin-coated surface. In conclusion, this study revealed a novel oncogenic function of TIP-1 suggesting that TIP-1 holds potential as a prognostic biomarker and a therapeutic target in the treatment of human invasive breast cancers.

Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models.

Nijmegen breakage syndrome (NBS) is a chromosomal fragility disorder that shares clinical and cellular features with ataxia telangiectasia. Here we demonstrate that Nbs1-null B cells are defective in the activation of ataxia-telangiectasia-mutated (Atm) in response to ionizing radiation, whereas ataxia-telangiectasia- and Rad3-related (Atr)-dependent signalling and Atm activation in response to ultraviolet light, inhibitors of DNA replication, or hypotonic stress are intact. Expression of the main human NBS allele rescues the lethality of Nbs1-/- mice, but leads to immunodeficiency, cancer predisposition, a defect in meiotic progression in females and cell-cycle checkpoint defects that are associated with a partial reduction in Atm activity. The Mre11 interaction domain of Nbs1 is essential for viability, whereas the Forkhead-associated (FHA) domain is required for T-cell and oocyte development and efficient DNA damage signalling. Reconstitution of Nbs1 knockout mice with various mutant isoforms demonstrates the biological impact of impaired Nbs1 function at the cellular and organismal level.

Role of genomic instability and p53 in AID-induced c-myc-Igh translocations.

Chromosomal translocations involving the immunoglobulin switch region are a hallmark feature of B-cell malignancies. However, little is known about the molecular mechanism by which primary B cells acquire or guard against these lesions. Here we find that translocations between c-myc and the IgH locus (Igh) are induced in primary B cells within hours of expression of the catalytically active form of activation-induced cytidine deaminase (AID), an enzyme that deaminates cytosine to produce uracil in DNA. Translocation also requires uracil DNA glycosylase (UNG), which removes uracil from DNA to create abasic sites that are then processed to double-strand breaks. The pathway that mediates aberrant joining of c-myc and Igh differs from intrachromosomal repair during immunoglobulin class switch recombination in that it does not require histone H2AX, p53 binding protein 1 (53BP1) or the non-homologous end-joining protein Ku80. In addition, translocations are inhibited by the tumour suppressors ATM, Nbs1, p19 (Arf) and p53, which is consistent with activation of DNA damage- and oncogenic stress-induced checkpoints during physiological class switching. Finally, we demonstrate that accumulation of AID-dependent, IgH-associated chromosomal lesions is not sufficient to enhance c-myc-Igh translocations. Our findings reveal a pathway for surveillance and protection against AID-dependent DNA damage, leading to chromosomal translocations.

DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1.

Activation of the ataxia telangiectasia mutated (ATM) kinase triggers diverse cellular responses to ionizing radiation (IR), including the initiation of cell cycle checkpoints. Histone H2AX, p53 binding-protein 1 (53BP1) and Chk2 are targets of ATM-mediated phosphorylation, but little is known about their roles in signalling the presence of DNA damage. Here, we show that mice lacking either H2AX or 53BP1, but not Chk2, manifest a G2-M checkpoint defect close to that observed in ATM(-/-) cells after exposure to low, but not high, doses of IR. Moreover, H2AX regulates the ability of 53BP1 to efficiently accumulate into IR-induced foci. We propose that at threshold levels of DNA damage, H2AX-mediated concentration of 53BP1 at double-strand breaks is essential for the amplification of signals that might otherwise be insufficient to prevent entry of damaged cells into mitosis.

Comparison of reduced intensity and myeloablative conditioning regimens for stem cell transplantation in patients with malignancies: a meta-analysis.

OBJECTIVE: The reduced intensity conditioning (RIC) stem cell transplantation is widely employed for the treatment of many hematologic malignancies, but the survival effectiveness is still unclear. This study conducted an updated meta-analysis to determine whether any significant difference could be found by using RIC vs. myeloablative conditioning (MAC) regimen for transplantation in patients with malignancies. METHODS: We electronically searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, and relevant articles (1987.01-2009.12). Comparative studies were carried out on clinical therapeutic effect of RIC and MAC on the survival outcomes and the transplantation-related complications. RESULTS: We obtained 1776 records, and 29 studies totaling 6235 patients have been assessed. Compared with MAC regimen, the RIC regimen had a higher overall survival (OS) at one-yr and no difference at two-yr later after transplantation. RIC regimen had significantly lower rates of disease-free survival (DFS) after two-yr follow-up, lower incidences of ≥ II degree acute graft-versus-host disease (aGVHD), and lower TRM [OR, 0.61, 95% CI (0.53, 0.69)], but with a higher relapse rate [OR, 1.88(1.41, 2.51)]. No significant difference was found in rates of cytomegalovirus (CMV) infection and chronic GVHD between the regimens. CONCLUSIONS: This meta-analysis confirmed that compared with MAC condition regimen, the RIC regimen had a consistently equivalent or even better rate in OS, but with lower DFS at longer follow-up.

Ablation Therapy Combined with EGFR TKIs in the Treatment of Advanced Non-Small Cell Lung Cancer: A Meta-Analysis of Randomized Controlled Trials.

OBJECTIVE: Systematically evaluate the efficacy of physical ablation combined with TKI in the treatment of advanced non-small cell lung cancer (NSCLC). METHODS: We performed a comprehensive search of databases including OVID, PubMed, EMBASE, the Cochrane Library, and three Chinese databases (China National Knowledge Infrastructure, Wanfang Database, and Chongqing Weipu Database). The aim was to identify randomized controlled trials (RCT) investigating physical ablation as the treatment for advanced NSCLC. We also evaluated the methodological quality of the included studies and summarized the data extracted for meta-analysis with Review Manager 5.3. RESULTS: A total of 9 studies, including 752 patients, were evaluable. The meta-analysis results show that the complete response rate (CRR) (RR: 2.23, 95% CI: 1. 46 to 3.40, P 0.01), partial response rate (PRR) (RR: -2.25, 95% CI: 1.41 to 3.59, P 0.01), and disease control rate (DCR) (RR: -2.80, 95% CI: 1.64 to 4.80, P< 0.01) of patients with advanced NSCLC who received physical ablation combined with TKI therapy were higher than those who did not receive physical ablation therapy. The control groups from seven of the studies had a total of 606 patients with targeted therapies and chemotherapy. The complete response rate was (CRR) (RR: 2.48, 2.4895% CI: 1.55 to 2.47, P 0.01), partial response rate (PRR) (RR: -1.66, 95% CI: 1.20 to 2.31, P< 0.01), and disease control rate (DCR) (RR: -2.68, 95% CI: 1.41 to 5.06, P< 0.01) for patients with advanced NSCLC who had received physical ablation combined with targeted therapies and chemotherapy, compared to patients who had not received physical ablation therapy. This difference was statistically significant. Above all, these results showed that the clinical efficacy of physical ablation combined EGFR-TKIs therapy (regardless of whether it was combined with chemotherapy) was better than that of EGFR-TKIs therapy alone. CONCLUSION: Physical ablation combined with TKI treatment in patients with advanced NSCLC can improve efficacy.