RAD52: Paradigm of Synthetic Lethality and New Developments

DNA double-strand breaks and inter-strand cross-links are the most harmful types of DNA damage that cause genomic instability that lead to cancer development. The highest fidelity pathway for repairing damaged double-stranded DNA is termed Homologous recombination (HR). Rad52 is one of the key HR proteins in eukaryotes. Although it is critical for most DNA repair and recombination events in yeast, knockouts of mammalian RAD52 lack any discernable phenotypes. As a consequence, mammalian RAD52 has been long overlooked. That is changing now, as recent work has shown RAD52 to be critical for backup DNA repair pathways in HR-deficient cancer cells. Novel findings have shed light on RAD52’s biochemical activities. RAD52 promotes DNA pairing (D-loop formation), single-strand DNA and DNA:RNA annealing, and inverse strand exchange. These activities contribute to its multiple roles in DNA damage repair including HR, single-strand annealing, break-induced replication, and RNA-mediated repair of DNA. The contributions of RAD52 that are essential to the viability of HR-deficient cancer cells are currently under investigation. These new findings make RAD52 an attractive target for the development of anti-cancer therapies against BRCA-deficient cancers.

Download Full-text

Synthetic Lethality In CLL With DNA Damage Response Defect By Targeting ATR Pathway

Blood ◽

10.1182/blood.v122.21.120.120 ◽

2013 ◽

Vol 122 (21) ◽

pp. 120-120

Author(s):

Tatjana Stankovic ◽

Davies Nicholas ◽

Marwan Kwok ◽

Edward Smith ◽

Eliot Yates ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Synthetic Lethality ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Single Strand Break ◽

Strand Breaks ◽

Damage Response ◽

Repair Pathways

Abstract Ataxia Telangiectasia Mutated (ATM) protein coordinates responses to DNA double strand breaks (DSBs) and the ATM-null status caused by biallelic ATM gene inactivation in chronic lymphocytic leukemia (CLL) results in resistance to p53-dependent apoptosis. Accordingly, alternative strategies to target ATM-null CLL are needed. ATM is a serine/threonine protein kinase that synchronises rapid DNA damage response (DDR) to DNA double strand breaks (DSBs) with activation of cell cycle checkpoints, DNA repair and apoptosis via p53 activation. ATM-null cells are defective in a type of DSB repair that involves homologous recombination and rely on co-operating and compensatory DNA repair pathways for their survival. Therefore, inhibition of DNA repair pathways to which CLL cells with loss of ATM signalling become addicted could provide ‘synthetic lethality’ and induce tumour specific killing. Indeed, we have recently shown that inhibition of a single strand break protein PARP induces differential killing of ATM-null CLL tumours. Here we expand the concept of synthetic lethality in ATM-null CLL and address the question of whether ATM-null deficient CLL cells can be targeted by inhibition of the ATR protein that governs responses to post-replicative damage and co-operates with ATM. First, we addressed the status of the ATR pathway in primary CLL cells and consistent with previous findings we observed that initiation of cell cycling is required for both ATR upregulation and activation of ATR target Chk1 in response to replicating stress inducing agent hydroxyurea. We then proceeded with testing viability of the isogenic CLL cell line CII, with and without stable ATM knock down, in the presence or absence of increasing doses of ATR inhibitor AZD6738. We observed a uniform loss of cellular viability in the presence of 1 or 3 μM of inhibitor in ATM-null cells but not in the ATM-wt counterpart. Similar observation was made in primary CLL cells initiated to cycle in the presence of stimulatory oligonucleotide-ODN2006/IL2 support. To confirm the cytotoxic effect of AZD6738 in vivo we used an ATM null primary CLL xenograft model. Representative primary CLL tumour cells with 15% bialleic ATM inactivation, as assessed by percentage of 11q deletion and allelic frequency of ATM mutation 4220T>C, was engrafted in the presence of activated autologous T lymphocytes into 10 NOG mice. Upon detection of engraftment in peripheral blood, animals were treated by oral administration of either AZD6738 (50mg/kg) or vehicle alone over a 2 week period, and tumour load measured by FACS analysis of CD45+ CD19+ human cells in infiltrated spleens. We observed a reduction in tumour cell numbers in AZD6738-treated compared to vehicle-treated spleens and current investigations are underway to determine whether this difference can be attributed to the selective disappearance of CLL population with biallelic ATM loss. We suggest that targeting ATR pathway provides an attractive approach for selective killing of ATM-null CLL cells and that this approach should be considered as a future therapeutic strategy for this CLL subtype. Disclosures: Off Label Use: ATR inhibitor AZD6738 targets ATM-null phenotype inducing synthetic lethality. Jeff:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Lau:AstraZeneca Pharmaceuticals: Employment.

Download Full-text

Synthetic lethality: exploiting the addiction of cancer to DNA repair

Blood ◽

10.1182/blood-2011-01-313734 ◽

2011 ◽

Vol 117 (23) ◽

pp. 6074-6082 ◽

Cited By ~ 116

Author(s):

Montaser Shaheen ◽

Christopher Allen ◽

Jac A. Nickoloff ◽

Robert Hromas

Keyword(s):

Dna Repair ◽

Cancer Cells ◽

Cancer Cell ◽

Synthetic Lethality ◽

Double Strand Breaks ◽

Repair Pathway ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Repair Pathways ◽

Cancer Multiple

Abstract Because cancer at its origin must acquire permanent genomic mutations, it is by definition a disease of DNA repair. Yet for cancer cells to replicate their DNA and divide, which is the fundamental phenotype of cancer, multiple DNA repair pathways are required. This produces a paradox for the cancer cell, where its origin is at the same time its weakness. To overcome this difficulty, a cancer cell often becomes addicted to DNA repair pathways other than the one that led to its initial mutability. The best example of this is in breast or ovarian cancers with mutated BRCA1 or 2, essential components of a repair pathway for repairing DNA double-strand breaks. Because replicating DNA requires repair of DNA double-strand breaks, these cancers have become reliant on another DNA repair component, PARP1, for replication fork progression. The inhibition of PARP1 in these cells results in catastrophic double-strand breaks during replication, and ultimately cell death. The exploitation of the addiction of cancer cells to a DNA repair pathway is based on synthetic lethality and has wide applicability to the treatment of many types of malignancies, including those of hematologic origin. There is a large number of novel compounds in clinical trials that use this mechanism for their antineoplastic activity, making synthetic lethality one of the most important new concepts in recent drug development.

Download Full-text

Single-Strand Annealing in Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms22042167 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2167

Author(s):

Janusz Blasiak

Keyword(s):

Dna Repair ◽

Synthetic Lethality ◽

Single Strand ◽

Repair System ◽

Dna Double Strand Breaks ◽

End Joining ◽

Cancer Pathogenesis ◽

Single Strand Annealing ◽

Non Homologous End Joining ◽

Strand Annealing

DNA double-strand breaks (DSBs) are among the most serious forms of DNA damage. In humans, DSBs are repaired mainly by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). Single-strand annealing (SSA), another DSB repair system, uses homologous repeats flanking a DSB to join DNA ends and is error-prone, as it removes DNA fragments between repeats along with one repeat. Many DNA deletions observed in cancer cells display homology at breakpoint junctions, suggesting the involvement of SSA. When multiple DSBs occur in different chromosomes, SSA may result in chromosomal translocations, essential in the pathogenesis of many cancers. Inhibition of RAD52 (RAD52 Homolog, DNA Repair Protein), the master regulator of SSA, results in decreased proliferation of BRCA1/2 (BRCA1/2 DNA Repair Associated)-deficient cells, occurring in many hereditary breast and ovarian cancer cases. Therefore, RAD52 may be targeted in synthetic lethality in cancer. SSA may modulate the response to platinum-based anticancer drugs and radiation. SSA may increase the efficacy of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated 9) genome editing and reduce its off-target effect. Several basic problems associated with SSA, including its evolutionary role, interplay with HRR and NHEJ and should be addressed to better understand its role in cancer pathogenesis and therapy.

Download Full-text

UAF1 DNA Binding Activity Is Critical for RAD51-Mediated Homologous DNA Pairing

Blood ◽

10.1182/blood-2019-130435 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2497-2497

Author(s):

Fengshan Liang ◽

Adam S Miller ◽

Carolilne Tang ◽

Patrick Sung ◽

Gary M. Kupfer

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Binding ◽

Complex Formation ◽

Binding Activity ◽

Loop Formation ◽

Dna Binding Activity ◽

D Loop ◽

In Cells ◽

Dna Pairing

Background: In the Fanconi anemia (FA) DNA repair pathway, DNA damage induces the mono-ubiquitination of the FANCI-FANCD2 (ID2) heterodimer by the FA core complex through its inherent E3 ligase activity. The timely deubiquitination of ID2 by USP1-UAF1 deubiquitinase complex is also critically important for the FA DNA repair. UAF1 has a DNA binding activity, which is required for FANCD2 deubiquitination. UAF1 also enhances RAD51-mediated homologous DNA pairing in a manner that is dependent on complex formation with RAD51AP1. UAF1 deficient cells are impaired for DNA repair by homologous recombination (HR).The biochemical and cellular functions of UAF1 DNA binding activity in HR remain elusive. Methods:UAF1 wild type and DNA binding mutant proteins were purified and used to define its biochemical properties in HR. In vitroD-loop formation and synaptic complex assembly assay were performed to discover the DNA binding of UAF1 in RAD51 recombinase enhancement. U2OS-DR-GFP cell lines with impaired UAF1 or RAD51AP1DNA binding were generated to examine HR efficiency and DNA damage resistance. Results:UAF1 preferentially binds an HR-intermediate-like DNA substrate (D-loop, Fig.1). The DNA binding deficient mutant of UAF1 is unable to stimulate RAD51AP1 promotion of RAD51-mediated D-loop (Fig. 2) and the ability to recruit homologous DNA to form the presynaptic complex formation in HR (Fig. 3). In cells, the UAF1 DNA-binding mutant is compromised for the ability to repair DNA damage and to implement HR (Fig. 4). Such activity correlates with the ability to confer resistance to DNA cross linking agents such as mitomycin C (Fig. 4). The DNA binding of UAF1 and RAD51AP1 have a coordinated role in HR-directed DNA damage repair (Fig. 5). Conclusions: UAF1 DNA binding activity is indispensable for its function in enhancing RAD51-mediated homologous DNA pairing within the context of the UAF1-RAD51AP1 complex. UAF1 DNA binding deficiency causes DNA damage sensitivity and impairs HR efficiency in cells. Translational Applicability:Our findings reveal a critical role of UAF1 DNA binding in DNA repair and genome maintenance. The identification of UAF1's role in repair will enable targeted efforts to improve molecular approaches for FA therapy. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Exploiting DNA Damage Repair in Precision Cancer Therapy: BRCA1 as a Prime Therapeutic Target

Cancers ◽

10.3390/cancers13143438 ◽

2021 ◽

Vol 13 (14) ◽

pp. 3438

Author(s):

Liliana Raimundo ◽

Juliana Calheiros ◽

Lucília Saraiva

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Targeted Therapy ◽

Cancer Cells ◽

Synthetic Lethality ◽

Dna Damage Repair ◽

Driver Mutations ◽

Molecular Alterations ◽

Damage Repair ◽

Repair Activity

Precision medicine aims to identify specific molecular alterations, such as driver mutations, allowing tailored and effective anticancer therapies. Poly(ADP)-ribose polymerase inhibitors (PARPi) are the prototypical example of targeted therapy, exploiting the inability of cancer cells to repair DNA damage. Following the concept of synthetic lethality, PARPi have gained great relevance, particularly in BRCA1 dysfunctional cancer cells. In fact, BRCA1 mutations culminate in DNA repair defects that can render cancer cells more vulnerable to therapy. However, the efficacy of these drugs has been greatly affected by the occurrence of resistance due to multi-connected DNA repair pathways that may compensate for each other. Hence, the search for additional effective agents targeting DNA damage repair (DDR) is of crucial importance. In this context, BRCA1 has assumed a central role in developing drugs aimed at inhibiting DNA repair activity. Collectively, this review provides an in-depth understanding of the biology and regulatory mechanisms of DDR pathways, highlighting the potential of DDR-associated molecules, particularly BRCA1 and its interconnected partners, in precision cancer medicine. It also affords an overview about what we have achieved and a reflection on how much remains to be done in this field, further addressing encouraging clues for the advance of DDR targeted therapy.

Download Full-text

DNA Double Strand Breaks Repair Inhibitors: Relevance as Potential New Anticancer Therapeutics

Current Medicinal Chemistry ◽

10.2174/0929867325666180214113154 ◽

2019 ◽

Vol 26 (8) ◽

pp. 1483-1493 ◽

Cited By ~ 7

Author(s):

Paulina Kopa ◽

Anna Macieja ◽

Grzegorz Galita ◽

Zbigniew J. Witczak ◽

Tomasz Poplawski

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Cancer Cells ◽

Clinical Success ◽

Alkylating Agents ◽

Parp Inhibitors ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Replication Process ◽

Strand Breaks

DNA double-strand breaks are considered one of the most lethal forms of DNA damage. Many effective anticancer therapeutic approaches used chemical and physical methods to generate DNA double-strand breaks in the cancer cells. They include: IR and drugs which mimetic its action, topoisomerase poisons, some alkylating agents or drugs which affected DNA replication process. On the other hand, cancer cells are mostly characterized by highly effective systems of DNA damage repair. There are two main DNA repair pathways used to fix double-strand breaks: NHEJ and HRR. Their activity leads to a decreased effect of chemotherapy. Targeting directly or indirectly the DNA double-strand breaks response by inhibitors seems to be an exciting option for anticancer therapy and is a part of novel trends that arise after the clinical success of PARP inhibitors. These trends will provide great opportunities for the development of DNA repair inhibitors as new potential anticancer drugs. The main objective of this article is to address these new promising advances.

Download Full-text

RAD52 as a Potential Target for Synthetic Lethality-Based Anticancer Therapies

Cancers ◽

10.3390/cancers11101561 ◽

2019 ◽

Vol 11 (10) ◽

pp. 1561 ◽

Cited By ~ 12

Author(s):

Toma ◽

Sullivan-Reed ◽

Śliwiński ◽

Skorski

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Cancer Cells ◽

Tumor Cells ◽

Synthetic Lethality ◽

Lethal Effect ◽

Drug Induced ◽

Synthetic Lethal ◽

Repair Mechanisms ◽

Cancer Tumor

Alterations in DNA repair systems play a key role in the induction and progression of cancer. Tumor-specific defects in DNA repair mechanisms and activation of alternative repair routes create the opportunity to employ a phenomenon called “synthetic lethality” to eliminate cancer cells. Targeting the backup pathways may amplify endogenous and drug-induced DNA damage and lead to specific eradication of cancer cells. So far, the synthetic lethal interaction between BRCA1/2 and PARP1 has been successfully applied as an anticancer treatment. Although PARP1 constitutes a promising target in the treatment of tumors harboring deficiencies in BRCA1/2—mediated homologous recombination (HR), some tumor cells survive, resulting in disease relapse. It has been suggested that alternative RAD52-mediated HR can protect BRCA1/2-deficient cells from the accumulation of DNA damage and the synthetic lethal effect of PARPi. Thus, simultaneous inhibition of RAD52 and PARP1 might result in a robust dual synthetic lethality, effectively eradicating BRCA1/2-deficient tumor cells. In this review, we will discuss the role of RAD52 and its potential application in synthetic lethality-based anticancer therapies.

Download Full-text

RepID-deficient cancer cells are sensitized to a drug targeting p97/VCP segregase

Molecular & Cellular Toxicology ◽

10.1007/s13273-021-00121-0 ◽

2021 ◽

Author(s):

Sang-Min Jang ◽

Christophe E. Redon ◽

Haiqing Fu ◽

Fred E. Indig ◽

Mirit I. Aladjem

Keyword(s):

Dna Damage ◽

Cancer Cells ◽

Cell Sorting ◽

Clonogenic Survival ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Ubiquitinated Proteins ◽

Damage Response ◽

Survival Assay

Abstract Background The p97/valosin-containing protein (VCP) complex is a crucial factor for the segregation of ubiquitinated proteins in the DNA damage response and repair pathway. Objective We investigated whether blocking the p97/VCP function can inhibit the proliferation of RepID-deficient cancer cells using immunofluorescence, clonogenic survival assay, fluorescence-activated cell sorting, and immunoblotting. Result p97/VCP was recruited to chromatin and colocalized with DNA double-strand breaks in RepID-deficient cancer cells that undergo spontaneous DNA damage. Inhibition of p97/VCP induced death of RepID-depleted cancer cells. This study highlights the potential of targeting p97/VCP complex as an anticancer therapeutic approach. Conclusion Our results show that RepID is required to prevent excessive DNA damage at the endogenous levels. Localization of p97/VCP to DSB sites was induced based on spontaneous DNA damage in RepID-depleted cancer cells. Anticancer drugs targeting p97/VCP may be highly potent in RepID-deficient cells. Therefore, we suggest that p97/VCP inhibitors synergize with RepID depletion to kill cancer cells.

Download Full-text

Platinum Complexes in Colorectal Cancer and Other Solid Tumors

Cancers ◽

10.3390/cancers13092073 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2073

Author(s):

Beate Köberle ◽

Sarah Schoch

Keyword(s):

Colorectal Cancer ◽

Dna Damage ◽

Dna Repair ◽

Cancer Cells ◽

Cisplatin Resistance ◽

Platinum Complexes ◽

Dna Lesions ◽

Dna Damage Signaling ◽

Repair Mechanisms ◽

P53 Inactivation

Cisplatin is one of the most commonly used drugs for the treatment of various solid neoplasms, including testicular, lung, ovarian, head and neck, and bladder cancers. Unfortunately, the therapeutic efficacy of cisplatin against colorectal cancer is poor. Various mechanisms appear to contribute to cisplatin resistance in cancer cells, including reduced drug accumulation, enhanced drug detoxification, modulation of DNA repair mechanisms, and finally alterations in cisplatin DNA damage signaling preventing apoptosis in cancer cells. Regarding colorectal cancer, defects in mismatch repair and altered p53-mediated DNA damage signaling are the main factors controlling the resistance phenotype. In particular, p53 inactivation appears to be associated with chemoresistance and poor prognosis. To overcome resistance in cancers, several strategies can be envisaged. Improved cisplatin analogues, which retain activity in resistant cancer, might be applied. Targeting p53-mediated DNA damage signaling provides another therapeutic strategy to circumvent cisplatin resistance. This review provides an overview on the DNA repair pathways involved in the processing of cisplatin damage and will describe signal transduction from cisplatin DNA lesions, with special attention given to colorectal cancer cells. Furthermore, examples for improved platinum compounds and biochemical modulators of cisplatin DNA damage signaling will be presented in the context of colon cancer therapy.

Download Full-text

MiR223-3p promotes synthetic lethality in BRCA1-deficient cancers

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903150116 ◽

2019 ◽

Vol 116 (35) ◽

pp. 17438-17443 ◽

Cited By ~ 6

Author(s):

Gayathri Srinivasan ◽

Elizabeth A. Williamson ◽

Kimi Kong ◽

Aruna S. Jaiswal ◽

Guangcun Huang ◽

...

Keyword(s):

Dna Repair ◽

Cancer Cells ◽

Synthetic Lethality ◽

Negative Regulator ◽

Nonhomologous End Joining ◽

Chromosomal Translocations ◽

Replication Forks ◽

Repair Pathway ◽

End Joining ◽

Parp1 Inhibitors

Defects in DNA repair give rise to genomic instability, leading to neoplasia. Cancer cells defective in one DNA repair pathway can become reliant on remaining repair pathways for survival and proliferation. This attribute of cancer cells can be exploited therapeutically, by inhibiting the remaining repair pathway, a process termed synthetic lethality. This process underlies the mechanism of the Poly-ADP ribose polymerase-1 (PARP1) inhibitors in clinical use, which target BRCA1 deficient cancers, which is indispensable for homologous recombination (HR) DNA repair. HR is the major repair pathway for stressed replication forks, but when BRCA1 is deficient, stressed forks are repaired by back-up pathways such as alternative nonhomologous end-joining (aNHEJ). Unlike HR, aNHEJ is nonconservative, and can mediate chromosomal translocations. In this study we have found that miR223-3p decreases expression of PARP1, CtIP, and Pso4, each of which are aNHEJ components. In most cells, high levels of microRNA (miR) 223–3p repress aNHEJ, decreasing the risk of chromosomal translocations. Deletion of the miR223 locus in mice increases PARP1 levels in hematopoietic cells and enhances their risk of unprovoked chromosomal translocations. We also discovered that cancer cells deficient in BRCA1 or its obligate partner BRCA1-Associated Protein-1 (BAP1) routinely repress miR223-3p to permit repair of stressed replication forks via aNHEJ. Reconstituting the expression of miR223-3p in BRCA1- and BAP1-deficient cancer cells results in reduced repair of stressed replication forks and synthetic lethality. Thus, miR223-3p is a negative regulator of the aNHEJ DNA repair and represents a therapeutic pathway for BRCA1- or BAP1-deficient cancers.

Download Full-text