RAD51C facilitates checkpoint signaling by promoting CHK2 phosphorylation

The RAD51 paralogues act in the homologous recombination (HR) pathway of DNA repair. Human RAD51C (hRAD51C) participates in branch migration and Holliday junction resolution and thus is important for processing HR intermediates late in the DNA repair process. Evidence for early involvement of RAD51 during DNA repair also exists, but its function in this context is not understood. In this study, we demonstrate that RAD51C accumulates at DNA damage sites concomitantly with the RAD51 recombinase and is retained after RAD51 disassembly, which is consistent with both an early and a late function for RAD51C. RAD51C recruitment depends on ataxia telangiectasia mutated, NBS1, and replication protein A, indicating it functions after DNA end resection but before RAD51 assembly. Furthermore, we find that RAD51C is required for activation of the checkpoint kinase CHK2 and cell cycle arrest in response to DNA damage. This suggests that hRAD51C contributes to the protection of genome integrity by transducing DNA damage signals in addition to engaging the HR machinery

Oligonucleotide-Functionalized Gold Nanoparticles for Synchronous Telomerase Inhibition, Radiosensitization, and Delivery of Theranostic Radionuclides

Telomerase represents an attractive target in oncology as it is expressed in cancer but not in normal tissues. The oligonucleotide inhibitors of telomerase represent a promising anticancer strategy, although poor cellular uptake can restrict their efficacy. In this study, gold nanoparticles (AuNPs) were used to enhance oligonucleotide uptake. “match” oligonucleotides complementary to the telomerase RNA template subunit (hTR) and “scramble” (control) oligonucleotides were conjugated to diethylenetriamine pentaacetate (DTPA) for 111In-labeling. AuNPs (15.5 nm) were decorated with a monofunctional layer of oligonucleotides (ON–AuNP) or a multifunctional layer of oligonucleotides, PEG(polethylene glycol)800-SH (to reduce AuNP aggregation) and the cell-penetrating peptide Tat (ON–AuNP–Tat). Match–AuNP enhanced the cellular uptake of radiolabeled oligonucleotides while retaining the ability to inhibit telomerase activity. The addition of Tat to AuNPs increased nuclear localization. 111In–Match–AuNP–Tat induced DNA double-strand breaks and caused a dose-dependent reduction in clonogenic survival of telomerase-positive cells but not telomerase-negative cells. hTR inhibition has been reported to sensitize cancer cells to ionizing radiation, and 111In–Match–AuNP–Tat therefore holds promise as a vector for delivery of radionuclides into cancer cells while simultaneously sensitizing them to the effects of the emitted radiation

Radiolabeled oligonucleotides targeting the RNA subunit of telomerase inhibit telomerase and induce DNA damage in telomerase-positive cancer cells

Telomerase is expressed in the majority (>85%) of tumours, but has restricted expression in normal tissues. Long-term telomerase inhibition in malignant cells results in progressive telomere shortening and reduction in cell proliferation. Here we report the synthesis and characterisation of radiolabeled oligonucleotides that target the RNA subunit of telomerase, hTR, simultaneously inhibiting enzymatic activity and delivering radiation intracellularly. Oligonucleotides complementary (match) and non-complementary (scramble or mismatch) to hTR were conjugated to diethylenetriaminepentaacetic dianhydride (DTPA), allowing radiolabeling with the Auger electron-emitting radionuclide indium-111 (111In). Match oligonucleotides inhibited telomerase activity with high potency which was not observed with scramble or mismatch oligonucleotides. DTPA-conjugation and 111In-labeling did not change telomerase inhibition. In telomerase-positive cancer cells, unlabeled match oligonucleotides had no effect on survival, however, 111In-labeled match oligonucleotides significantly reduced clonogenic survival and upregulated the DNA damage marker γH2AX. Minimal radiotoxicity and DNA damage was observed in telomerase-negative cells exposed to 111In-match oligonucleotides. Match oligonucleotides localised in close proximity to nuclear Cajal bodies in telomerase-positive cells. In comparison to match oligonucleotides, 111In-scramble or 111In-mismatch oligonucleotides demonstrated reduced retention and negligible impact on cell survival. This study indicates the therapeutic activity of radiolabeled oligonucleotides that specifically target hTR through potent telomerase inhibition and DNA damage induction in telomerase-expressing cancer cells, and paves way for the development of novel oligonucleotide radiotherapeutics targeting telomerase-positive cancers

Chlorambucil targets BRCA1/2-deficient tumours and counteracts PARP inhibitor resistance.

Due to compromised homologous recombination (HR) repair, BRCA1- and BRCA2-mutated tumours accumulate DNA damage and genomic rearrangements conducive of tumour progression. To identify drugs that target specifically BRCA2-deficient cells, we screened a chemical library containing compounds in clinical use. The top hit was chlorambucil, a bifunctional alkylating agent used for the treatment of chronic lymphocytic leukaemia (CLL). We establish that chlorambucil is specifically toxic to BRCA1/2-deficient cells, including olaparib-resistant and cisplatin-resistant ones, suggesting the potential clinical use of chlorambucil against disease which has become resistant to these drugs. Additionally, chlorambucil eradicates BRCA2-deficient xenografts and inhibits growth of olaparib-resistant patient-derived tumour xenografts (PDTXs). We demonstrate that chlorambucil inflicts replication-associated DNA double-strand breaks (DSBs), similarly to cisplatin, and we identify ATR, FANCD2 and the SNM1A nuclease as determinants of sensitivity to both drugs. Importantly, chlorambucil is substantially less toxic to normal cells and tissues in vitro and in vivo relative to cisplatin. Because chlorambucil and cisplatin are equally effective inhibitors of BRCA2-compromised tumours, our results indicate that chlorambucil has a higher therapeutic index than cisplatin in targeting BRCA-deficient tumours.This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska‐Curie grant agreement No. 722729. Research in M.T. laboratory is supported by Cancer Research UK, Medical Research Council and University of Oxford

ATM/ATR-dependent responses to dysfunctional telomeres at the G2/M transition

Mammalian telomeres are nucleoprotein complexes at the end of chromosomes containing a specific protein complex, called shelterin. Shelterin protects chromosome ends from the DNA damage response (DDR), by facilitating the formation of a telomeric capping structure, called the T-loop. During their elongation in S phase, telomeres become transiently uncapped and can be sensed as DNA damage in G2 phase. This leads to the recruitment of DDR factors, such as phosphorylated histone H2AX (γH2AX), to the telomeres forming the so-called, telomere dysfunction-induced foci (TIFs). My PhD work described here, indicates that DNA damage occurring during interphase can persist after entry into mitosis, indicated by the detection of γH2AX at a subset of mitotic telomeres in human and mouse cells. This accumulation of γH2AX to mitotic telomeres is ATM-dependent and the γH2AX-labelled uncapped telomeres that persist, are shorter than the average telomere length for the entire cell population. Most importantly, my work suggests that telomere uncapping, naturally occurring or artificially induced, is detected by two parallel ATM/ATR-dependent pathways at the G2/M transition: a p53/p21-dependent pathway through the ATM/ATR-mediated phosphorylation of p53 at Ser15 and a CHK1/CHK2-dependent pathway that acts through negative regulation of CDC25 phosphatases. In particular, telomere uncapping triggered by TRF2 depletion leads to CHK2-dependent CDC25A degradation, while POT1 depletion results in CHK1-mediated CDC25A and CDC25C degradation. Both pathways act as sensors of unprotected telomeres at the G2/M transition and block cell cycle progression through inhibition of CDK1/Cyclin B complex, allowing telomere re-capping before entry into mitosis. This mechanism protects telomere integrity by the maintenance of a cell cycle stage conducive for capping reactions and thereby prevents genomic instability induced by telomere dysfunction. Finally, I studied the cellular functions of 3 poorly characterised shelterin components, TRF1, RAP1 and TPP1, in telomere protection. TRF1 and to a lesser extent RAP1 were shown to be important for telomere protection by suppressing DDR at the telomeres, while TPP1 was shown to be mainly responsible for the recruitment of the catalytic subunit of telomerase, TERT , to the chromatin, contributing to telomere maintenance. In conclusion, my work on both human and mouse models, reveals an important part of the DDR pathways activated by dysfunctional telomeres, as well as the molecular mechanisms underlying the cell cycle specific regulation of telomere capping, which ensures that only cells with intact telomeres enter mitosis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Selective targeting of homologous recombination deficiency

Homologous recombination (HR) is a key DNA repair pathway essential for cell viability. Counter-intuitively, HR deficiency can trigger carcinogenesis. Understanding the mechanisms that allow the rampant proliferation of HR-deficient tumour cells is crucial for the development of improved therapeutic modalities to selectively inhibit the outgrowth of these cells. Recently, we identified extracellular signal-regulated kinase 1 (ERK1) as a factor required for the proliferation of BRCA2-deficient cells regardless of their p53 status (Carlos et al., 2013). Here, we report the therapeutic potential of two chemical ERK1/2 inhibitors, SCH772984 and VTX-11e, for selective targeting of HR-deficient tumours due to their ability to specifically obstruct proliferation of HR-deficient cells. G-quadruplexes (G4s), secondary DNA structures formed by guanine-rich (G-rich) single-stranded DNA (ssDNA), represent natural barriers to replication fork progression. In this study, we demonstrate that treatment with G4 stabilisers selectively decreases viability of BRCA2- and RAD51-deficient cells. We identify DNA damage response activation and acute replication stress as main sources for the cellular toxicity of G4 stabilisers specifically in the context of HR deficiency. Taken together, the results presented here indicate that HR is required for replication of genomic regions with G4-forming potential to prevent genomic instability stemming from inefficient replication of these sites. Persistent G4 structures lead to DNA damage accumulation, which enables selective killing of cells whose HR-mediated repair has been compromised. This is an important finding with profound implications for the therapeutic exploitation of HR deficiency in the clinic.This thesis is not currently available in OR

RAD51 localization and activation following DNA damage.

The efficient repair of double-strand breaks in DNA is critical for the maintenance of genome stability. In response to ionizing radiation and other DNA-damaging agents, the RAD51 protein, which is essential for homologous recombination, relocalizes within the nucleus to form distinct foci that can be visualized by microscopy and are thought to represent sites where repair reactions take place. The formation of RAD51 foci in response to DNA damage is dependent upon BRCA2 and a series of proteins known as the RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), indicating that the components present within foci assemble in a carefully orchestrated and ordered manner. By contrast, RAD51 foci that form spontaneously as cells undergo DNA replication at S phase occur without the need for BRCA2 or the RAD51 paralogues. It is known that BRCA2 interacts directly with RAD51 through a series of degenerative motifs known as the BRC repeats. These interactions modulate the ability of RAD51 to bind DNA. Taken together, these observations indicate that BRCA2 plays a critical role in controlling the actions of RAD51 at both the microscopic (focus formation) and molecular (DNA binding) level