New Facets of DNA Double Strand Break Repair: Radiation Dose as Key Determinant of HR versus c-NHEJ Engagement.

Radiation therapy is an essential component of present-day cancer management, utilizing ionizing radiation (IR) of different modalities to mitigate cancer progression. IR functions by generating ionizations in cells that induce a plethora of DNA lesions. The most detrimental among them are the DNA double strand breaks (DSBs). In the course of evolution, cells of higher eukaryotes have evolved four major DSB repair pathways: classical non-homologous end joining (c-NHEJ), homologous recombination (HR), alternative end-joining (alt-EJ), and single strand annealing (SSA). These mechanistically distinct repair pathways have different cell cycle- and homology-dependencies but, surprisingly, they operate with widely different fidelity and kinetics and therefore contribute unequally to cell survival and genome maintenance. It is therefore reasonable to anticipate tight regulation and coordination in the engagement of these DSB repair pathway to achieve the maximum possible genomic stability. Here, we provide a state-of-the-art review of the accumulated knowledge on the molecular mechanisms underpinning these repair pathways, with emphasis on c-NHEJ and HR. We discuss factors and processes that have recently come to the fore. We outline mechanisms steering DSB repair pathway choice throughout the cell cycle, and highlight the critical role of DNA end resection in this process. Most importantly, however, we point out the strong preference for HR at low DSB loads, and thus low IR doses, for cells irradiated in the G2-phase of the cell cycle. We further explore the molecular underpinnings of transitions from high fidelity to low fidelity error-prone repair pathways and analyze the coordination and consequences of this transition on cell viability and genomic stability. Finally, we elaborate on how these advances may help in the development of improved cancer treatment protocols in radiation therapy.

Strong suppression of gene conversion with increasing DNA double-strand break load delimited by 53BP1 and RAD52.

In vertebrates, genomic DNA double-strand breaks (DSBs) are removed by non-homologous end-joining processes: classical non-homologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ); or by homology-dependent processes: gene-conversion (GC) and single-strand annealing (SSA). Surprisingly, these repair pathways are not real alternative options restoring genome integrity with equal efficiency, but show instead striking differences in speed, accuracy and cell-cycle-phase dependence. As a consequence, engagement of one pathway may be associated with processing-risks for the genome absent from another pathway. Characterization of engagement-parameters and their consequences is, therefore, essential for understanding effects on the genome of DSB-inducing agents, such as ionizing-radiation (IR). Here, by addressing pathway selection in G2-phase, we discover regulatory confinements in GC with consequences for SSA- and c-NHEJ-engagement. We show pronounced suppression of GC with increasing DSB-load that is not due to RAD51 availability and which is delimited but not defined by 53BP1 and RAD52. Strikingly, at low DSB-loads, GC repairs ∼50% of DSBs, whereas at high DSB-loads its contribution is undetectable. Notably, with increasing DSB-load and the associated suppression of GC, SSA gains ground, while alt-EJ is suppressed. These observations explain earlier, apparently contradictory results and advance our understanding of logic and mechanisms underpinning the wiring between DSB repair pathways.

PTEN Loss Enhances Error-Prone DSB Processing and Tumor Cell Radiosensitivity by Suppressing RAD51 Expression and Homologous Recombination.

PTEN has been implicated in the repair of DNA double-strand breaks (DSBs), particularly through homologous recombination (HR). However, other data fail to demonstrate a direct role of PTEN in DSB repair. Therefore, here, we report experiments designed to further investigate the role of PTEN in DSB repair. We emphasize the consequences of PTEN loss in the engagement of the four DSB repair pathways-classical non-homologous end-joining (c-NHEJ), HR, alternative end-joining (alt-EJ) and single strand annealing (SSA)-and analyze the resulting dynamic changes in their utilization. We quantitate the effect of PTEN knockdown on cell radiosensitivity to killing, as well as checkpoint responses in normal and tumor cell lines. We find that disruption of PTEN sensitizes cells to ionizing radiation (IR). This radiosensitization is associated with a reduction in RAD51 expression that compromises HR and causes a marked increase in SSA engagement, an error-prone DSB repair pathway, while alt-EJ and c-NHEJ remain unchanged after PTEN knockdown. The G2-checkpoint is partially suppressed after PTEN knockdown, corroborating the associated HR suppression. Notably, PTEN deficiency radiosensitizes cells to PARP inhibitors, Olaparib and BMN673. The results show the crucial role of PTEN in DSB repair and show a molecular link between PTEN and HR through the regulation of RAD51 expression. The expected benefit from combination treatment with Olaparib or BMN673 and IR shows that PTEN status may also be useful for patient stratification in clinical treatment protocols combining IR with PARP inhibitors.

Increased Gene Targeting in Hyper-Recombinogenic LymphoBlastoid Cell Lines Leaves Unchanged DSB Processing by Homologous Recombination.

In the cells of higher eukaryotes, sophisticated mechanisms have evolved to repair DNA double-strand breaks (DSBs). Classical nonhomologous end joining (c-NHEJ), homologous recombination (HR), alternative end joining (alt-EJ) and single-strand annealing (SSA) exploit distinct principles to repair DSBs throughout the cell cycle, resulting in repair outcomes of different fidelity. In addition to their functions in DSB repair, the same repair pathways determine how cells integrate foreign DNA or rearrange their genetic information. As a consequence, random integration of DNA fragments is dominant in somatic cells of higher eukaryotes and suppresses integration events at homologous genomic locations, leading to very low gene-targeting efficiencies. However, this response is not universal, and embryonic stem cells display increased targeting efficiency. Additionally, lymphoblastic chicken and human cell lines DT40 and NALM6 show up to a 1000-fold increased gene-targeting efficiency that is successfully harnessed to generate knockouts for a large number of genes. We inquired whether the increased gene-targeting efficiency of DT40 and NALM6 cells is linked to increased rates of HR-mediated DSB repair after exposure to ionizing radiation (IR). We analyzed IR-induced γ-H2AX foci as a marker for the total number of DSBs induced in a cell and RAD51 foci as a marker for the fraction of those DSBs undergoing repair by HR. We also evaluated RPA accretion on chromatin as evidence for ongoing DNA end resection, an important initial step for all pathways of DSB repair except c-NHEJ. We finally employed the DR-GFP reporter assay to evaluate DSB repair by HR in DT40 cells. Collectively, the results obtained, unexpectedly show that DT40 and NALM6 cells utilized HR for DSB repair at levels very similar to those of other somatic cells. These observations uncouple gene-targeting efficiency from HR contribution to DSB repair and suggest the function of additional mechanisms increasing gene-targeting efficiency. Indeed, our results show that analysis of the contribution of HR to DSB repair may not be used as a proxy for gene-targeting efficiency.

DNA Damage Clustering after Ionizing Radiation and Consequences in the Processing of Chromatin Breaks.

Charged-particle radiotherapy (CPRT) utilizing low and high linear energy transfer (low-/high-LET) ionizing radiation (IR) is a promising cancer treatment modality having unique physical energy deposition properties. CPRT enables focused delivery of a desired dose to the tumor, thus achieving a better tumor control and reduced normal tissue toxicity. It increases the overall radiation tolerance and the chances of survival for the patient. Further improvements in CPRT are expected from a better understanding of the mechanisms governing the biological effects of IR and their dependence on LET. There is increasing evidence that high-LET IR induces more complex and even clustered DNA double-strand breaks (DSBs) that are extremely consequential to cellular homeostasis, and which represent a considerable threat to genomic integrity. However, from the perspective of cancer management, the same DSB characteristics underpin the expected therapeutic benefit and are central to the rationale guiding current efforts for increased implementation of heavy ions (HI) in radiotherapy. Here, we review the specific cellular DNA damage responses (DDR) elicited by high-LET IR and compare them to those of low-LET IR. We emphasize differences in the forms of DSBs induced and their impact on DDR. Moreover, we analyze how the distinct initial forms of DSBs modulate the interplay between DSB repair pathways through the activation of DNA end resection. We postulate that at complex DSBs and DSB clusters, increased DNA end resection orchestrates an increased engagement of resection-dependent repair pathways. Furthermore, we summarize evidence that after exposure to high-LET IR, error-prone processes outcompete high fidelity homologous recombination (HR) through mechanisms that remain to be elucidated. Finally, we review the high-LET dependence of specific DDR-related post-translational modifications and the induction of apoptosis in cancer cells. We believe that in-depth characterization of the biological effects that are specific to high-LET IR will help to establish predictive and prognostic signatures for use in future individualized therapeutic strategies, and will enhance the prospects for the development of effective countermeasures for improved radiation protection during space travel.

Disruption of Chromatin Dynamics by Hypotonic Stress Suppresses HR and Shifts DSB Processing to Error-Prone SSA.

The processing of DNA double-strand breaks (DSBs) depends on the dynamic characteristics of chromatin. To investigate how abrupt changes in chromatin compaction alter these dynamics and affect DSB processing and repair, we exposed irradiated cells to hypotonic stress (HypoS). Densitometric and chromosome-length analyses show that HypoS transiently decompacts chromatin without inducing histone modifications known from regulated local chromatin decondensation, or changes in Micrococcal Nuclease (MNase) sensitivity. HypoS leaves undisturbed initial stages of DNA-damage-response (DDR), such as radiation-induced ATM activation and H2AX-phosphorylation. However, detection of ATM-pS1981, γ-H2AX and 53BP1 foci is reduced in a protein, cell cycle phase and cell line dependent manner; likely secondary to chromatin decompaction that disrupts the focal organization of DDR proteins. While HypoS only exerts small effects on classical nonhomologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ), it markedly suppresses homologous recombination (HR) without affecting DNA end-resection at DSBs, and clearly enhances single-strand annealing (SSA). These shifts in pathway engagement are accompanied by decreases in HR-dependent chromatid-break repair in the G2-phase, and by increases in alt-EJ and SSA-dependent chromosomal translocations. Consequently, HypoS sensitizes cells to ionizing radiation (IR)-induced killing. We conclude that HypoS-induced global chromatin decompaction compromises regulated chromatin dynamics and genomic stability by suppressing DSB-processing by HR, and allowing error-prone processing by alt-EJ and SSA.

Regulation of ABCA1-mediated cholesterol efflux by sphingosine-1-phosphate signaling in macrophages.

Sphingolipid and cholesterol metabolism are closely associated at the structural, biochemical, and functional levels. Although HDL-associated sphingosine-1-phosphate (S1P) contributes to several HDL functions, and S1P signaling regulates glucose and lipid metabolism, no study has addressed the involvement of S1P in cholesterol efflux. Here, we show that sphingosine kinase (Sphk) activity was induced by the LXR agonist 22(R)-hydroxycholesterol and required for the stimulation of ABCA1-mediated cholesterol efflux to apolipoprotein A-I. In support, pharmacological Sphk inhibition and Sphk2 but not Sphk1 deficiency abrogated efflux. The involved mechanism included stimulation of both transcriptional and functional ABCA1 regulatory pathways and depended for the latter on the S1P receptor 3 (S1P3). Accordingly, S1P3-deficient macrophages were resistant to 22(R)-hydroxycholesterol-stimulated cholesterol efflux. The inability of excess exogenous S1P to further increase efflux was consistent with tonic S1P3 signaling by a pool of constitutively generated Sphk-derived S1P dynamically regulating cholesterol efflux. In summary, we have established S1P as a previously unrecognized intermediate in LXR-stimulated ABCA1-mediated cholesterol efflux and identified S1P/S1P3 signaling as a positive-feedback regulator of cholesterol efflux. This constitutes a novel regulatory mechanism of cholesterol efflux by sphingolipids.

Abdominopelvic 1.5-T and 3.0-T MR Imaging in Healthy Volunteers: Relationship to Formation of DNA Double-Strand Breaks.

Purpose To investigate the relationship between abdominopelvic magnetic resonance (MR) imaging and formation of DNA double-strand breaks (DSBs) in peripheral blood lymphocytes among a cohort of healthy volunteers. Materials and Methods Blood samples were obtained from 40 healthy volunteers (23 women and 17 men; mean age, 27.2 years [range, 21-37 years]) directly before and 5 and 30 minutes after abdominopelvic MR imaging performed at 1.5 T (n = 20) or 3.0 T (n = 20). The number of DNA DSBs in isolated blood lymphocytes was quantified after indirect immunofluorescent staining of a generally accepted DSB marker, γ-H2AX, by means of high-throughput automated microscopy. As a positive control of DSB induction, blood lymphocytes from six volunteers were irradiated in vitro with x-rays at a dose of 1 Gy (70-90 keV). Statistical analysis was performed by using a Friedman test. Results No significant alteration in the frequency of DNA DSB induction was observed after MR imaging (before imaging: 0.22 foci per cell, interquartile range [IQR] = 0.54 foci per cell; 5 minutes after MR imaging: 0.08 foci per cell, IQR = 0.39 foci per cell; 30 minutes after MR imaging: 0.09 foci per cell, IQR = 0.63 foci per cell; P = .057). In vitro radiation of lymphocytes with 1 Gy led to a significant increase in DSBs (0.22 vs 3.43 foci per cell; P = .0312). The frequency of DSBs did not differ between imaging at 1.5 T and at 3.0 T (5 minutes after MR imaging: 0.23 vs 0.06 foci per cell, respectively [P = .57]; 30 minutes after MR imaging: 0.12 vs 0.08 foci per cell [P = .76]). Conclusion Abdominopelvic MR imaging performed at 1.5 T or 3.0 T does not affect the formation of DNA DSBs in peripheral blood lymphocytes.

Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment.

Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect.

Processing-Challenges Generated by Clusters of DNA Double-Strand Breaks Underpin Increased Effectiveness of High-LET Radiation and Chromothripsis.

Whereas most endogenous and exogenous DNA damaging agents typically generate lesions that are relatively isolated and can be repaired easily, ionizing radiation (IR) also induces clustered lesions causing DNA double strand breaks (DSBs). Moreover, forms of IR characterized by high linear energy transfer (LET) induce not only isolated DSBs but also DSB clusters - multiple DSBs in close proximity -that pose increased risks for the cell. DSB clusters can destabilize chromatin locally and compromise processing of individual DSBs within the cluster. Since the discovery of chromothripsis, a phenomenon whereby multiple DSBs locally generated by a catastrophic event causes genomic rearrangements that feed carcinogenesis, DSB clusters receive increased attention also in the field of cancer. While formation of DSB clusters after exposure to high LET is a direct and inherent consequence of the spatial distribution of the constituting energy deposition events, also called track structure, the sources of local genomic shattering underpinning chromothripsis are under investigation. Notably, many consequences of DSB clusters in the affected genome reflect processing by pathways that have evolved to repair DSBs, but which operate with widely different degrees of fidelity. The molecular underpinnings and the basis of the underlying repair pathway choices that ultimately lead to the observed consequences from DSB clusters remain unknown. We developed a tractable model of DSB clustering that allows direct analysis in cells of the consequences of certain configurations of DSB clusters. We outline the rationale for the development of this model and describe its key characteristics. We summarize results suggesting that DSB clusters compromise the first-line DSB-processing pathways of c-NHEJ and HRR, increasing as a consequence the contribution of alt-EJ, which has high propensity of generating chromosomal rearrangements. The results suggest a mechanism for the increased toxicity of high LET radiation and the extensive genomic rearrangements associated with chromothripsis.

DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferation-dependent regulation.

Eukaryotic cells respond to DNA damage by activating a comprehensive network of biochemical pathways that enable damage recognition and initiate responses leading to repair, apoptosis/autophagy or senescence. This network of responses is commonly described as the "DNA damage response" (DDR). Among the plethora of lesions generated in the DNA from various physical and chemical agents in the environment and in the cell, DNA double strand breaks (DSBs) and DNA replication stress (RS) are the most severe and induce strong DDR, as they bear high risk for cell death, or genomic alterations ultimately causing cancer. Here, we focus on DSBs and provide a state-of-the-art review of the molecular underpinnings of repair pathways that process DSBs in higher eukaryotes, their strengths and limitations, as well as aspects of repair pathway choice and hierarchy. Furthermore, we discuss the regulation of DSB repair pathways throughout the cell cycle and by processes affecting the proliferative state of the cell. We review the role of growth factors and their receptors in the regulation of each DSB repair pathway and discuss aspects of systemic regulation of DNA repair.

The imprinted NPAP1/C15orf2 gene in the Prader-Willi syndrome region encodes a nuclear pore complex associated protein.

The Prader-Willi syndrome (PWS) region in 15q11q13 harbours a cluster of imprinted genes expressed from the paternal chromosome only. Whereas loss of function of the SNORD116 genes appears to be responsible for the major features of PWS, the role of the other genes is less clear. One of these genes is C15orf2, which has no orthologues in rodents, but appears to be under strong positive selection in primates. C15orf2 encodes a 1156 amino acid protein with six nuclear localisation sequences. By protein BLAST analysis and InterProScan signature recognition search, we found sequence similarity of C15orf2 to the nuclear pore complex (NPC) protein POM121. To determine whether C15orf2 is located at nuclear pores, we generated a stable cell line that inducibly expresses FLAG-tagged C15orf2 and performed immunocytochemical studies. We found that C15orf2 is present at the nuclear periphery, where it colocalizes with NPCs and nuclear lamins. At very high expression levels, we observed invaginations of the nuclear envelope. Extending these observations to three-dimensional structured illumination microscopy, which achieves an 8-fold improved volumetric resolution over conventional imaging, we saw that C15orf2 is located at the inner face of the nuclear envelope where it strongly associates with the NPC. In nuclear envelope isolation and fractionation experiments, we detected C15orf2 in the NPC and lamina fractions. These experiments for the first time demonstrate that C15orf2 is part of the NPC or its associated molecular networks. Based on our findings, we propose 'Nuclear pore associated protein 1' as the new name for C15orf2.

Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells.

In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation.

Talazoparib enhances resection at DSBs and renders HR-proficient cancer cells susceptible to Polθ inhibition.

BACKGROUND AND PURPOSE: The PARP inhibitor (PARPi), Talazoparib (BMN673), effectively and specifically radiosensitizes cancer cells. Radiosensitization is mediated by a shift in the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) toward PARP1-independent, alternative end-joining (alt-EJ). DNA polymerase theta (Polθ) is a key component of this PARP1-independent alt-EJ pathway and we show here that its inhibition can further radiosensitize talazoparib-treated cells. The purpose of the present work is to explore mechanisms and dynamics underpinning enhanced talazoparib radiosensitization by Polθ inhibitors in HR-proficient cancer cells. METHODS AND MATERIALS: Radiosensitization to PARPis, talazoparib, olaparib, rucaparib and veliparib was assessed by clonogenic survival. Polθ-proficient and -deficient cells were treated with PARPis and/or with the Polθ inhibitors ART558 or novobiocin. The role of DNA end-resection was studied by down-regulating CtIP and MRE11 expression using siRNAs. DSB repair was assessed by scoring γH2AX foci. The formation of chromosomal abnormalities was assessed as evidence of alt-EJ function using G2-specific cytogenetic analysis. RESULTS: Talazoparib exerted pronounced radiosensitization that varied among the tested cancer cell lines; however, radiosensitization was undetectable in normal cells. Other commonly used PARPis, olaparib, veliparib, or rucaparib were ineffective radiosensitizers under our experimental conditions. Although genetic ablation or pharmacological inhibition of Polθ only mildly radiosensitized cancer cells, talazoparib-treated cells were markedly further radiosensitized. Mechanistically, talazoparib shunted DSBs to Polθ-dependent alt-EJ by enhancing DNA end-resection in a CtIP- and MRE11-dependent manner - an effect detectable at low, but not high IR doses. Chromosomal translocation analysis in talazoparib-treated cells exposed to Polθ inhibitors suggested that PARP1- and Polθ-dependent alt-EJ pathways may complement, but also back up each other. CONCLUSION: We propose that talazoparib promotes low-dose, CtIP/MRE11-dependent resection and increases the reliance of irradiated HR-proficient cancer cells, on Polθ-mediated alt-EJ. The combination of Polθ inhibitors with talazoparib suppresses this option and causes further radiosensitization. The results suggest that Polθ inhibition may be exploited to maximize talazoparib radiosensitization of HR-proficient tumors in the clinic.

Conformational transitions of proteins engaged in DNA double-strand break repair, analysed by tryptophan fluorescence emission and FRET.

We analysed protein-DNA and protein-protein interactions relevant to the repair of DNA DSBs (double-strand breaks) by NHEJ (non-homologous end-joining). Conformational transitions in mammalian DNA ligases III (LigIII) and IV (LigIV), as well as in PARP-1 [poly(ADP-ribose) polymerase-1], were analysed upon binding to double-stranded DNA by changes in tryptophan emission and FRET (Förster resonance energy transfer) from tryptophan to DNA-conjugated Alexa Fluor® 532. For LigIII, two non-equivalent high- and low-affinity DNA-binding sites are detected interacting sequentially with DNA. PARP-1 displays a single high-affinity DNA-binding site and can displace bound DNA fragments from the low-affinity site of LigIII, consistent with its mediator role in LigIII-DNA interactions. For the LX [LigIV-XRCC4 (X-ray cross-complementation group 4)] complex, a single DNA-binding site is detected. Binding of Ku to DNA was accompanied by conformational changes in the protein and intermolecular FRET from dansyl chromophores of the labelled Ku to the Alexa Fluor® chromophores of Alexa Fluor® 532-conjugated DNA. The average distance of 5.7 nm calculated from FRET data is consistent with a location of Ku at the very end of the DNA molecule. Binding of LX to Ku-DNA complexes is associated with conformational changes in Ku, translocating the protein further towards the DNA ends. The protein-protein and protein-DNA interactions detected and analysed generate a framework for the characterization of molecular interactions fundamental to the function of NHEJ pathways in higher eukaryotes.

The p38/MK2 Pathway Functions as Chk1-Backup Downstream of ATM/ATR in G2-Checkpoint Activation in Cells Exposed to Ionizing Radiation.

We have recently reported that in G2-phase cells (but not S-phase cells) sustaining low loads of DNA double-strand break (DSBs), ATM and ATR regulate the G2-checkpoint epistatically, with ATR at the output-node, interfacing with the cell cycle through Chk1. However, although inhibition of ATR nearly completely abrogated the checkpoint, inhibition of Chk1 using UCN-01 generated only partial responses. This suggested that additional kinases downstream of ATR were involved in the transmission of the signal to the cell cycle engine. Additionally, the broad spectrum of kinases inhibited by UCN-01 pointed to uncertainties in the interpretation that warranted further investigations. Here, we show that more specific Chk1 inhibitors exert an even weaker effect on G2-checkpoint, as compared to ATR inhibitors and UCN-01, and identify the MAPK p38α and its downstream target MK2 as checkpoint effectors operating as backup to Chk1. These observations further expand the spectrum of p38/MK2 signaling to G2-checkpoint activation, extend similar studies in cells exposed to other DNA damaging agents and consolidate a role of p38/MK2 as a backup kinase module, adding to similar backup functions exerted in p53 deficient cells. The results extend the spectrum of actionable strategies and targets in current efforts to enhance the radiosensitivity in tumor cells.

Low CDK Activity and Enhanced Degradation by APC/CCDH1 Abolishes CtIP Activity and Alt-EJ in Quiescent Cells.

Alt-EJ is an error-prone DNA double-strand break (DSBs) repair pathway coming to the fore when first-line repair pathways, c-NHEJ and HR, are defective or fail. It is thought to benefit from DNA end-resection-a process whereby 3' single-stranded DNA-tails are generated-initiated by the CtIP/MRE11-RAD50-NBS1 (MRN) complex and extended by EXO1 or the BLM/DNA2 complex. The connection between alt-EJ and resection remains incompletely characterized. Alt-EJ depends on the cell cycle phase, is at maximum in G2-phase, substantially reduced in G1-phase and almost undetectable in quiescent, G0-phase cells. The mechanism underpinning this regulation remains uncharacterized. Here, we compare alt-EJ in G1- and G0-phase cells exposed to ionizing radiation (IR) and identify CtIP-dependent resection as the key regulator. Low levels of CtIP in G1-phase cells allow modest resection and alt-EJ, as compared to G2-phase cells. Strikingly, CtIP is undetectable in G0-phase cells owing to APC/C-mediated degradation. The suppression of CtIP degradation with bortezomib or CDH1-depletion rescues CtIP and alt-EJ in G0-phase cells. CtIP activation in G0-phase cells also requires CDK-dependent phosphorylation by any available CDK but is restricted to CDK4/6 at the early stages of the normal cell cycle. We suggest that suppression of mutagenic alt-EJ in G0-phase is a mechanism by which cells of higher eukaryotes maintain genomic stability in a large fraction of non-cycling cells in their organisms.

The high toxicity of DSB-clusters modelling high-LET-DNA damage derives from inhibition of c-NHEJ and promotion of alt-EJ and SSA despite increases in HR.

Heavy-ion radiotherapy utilizing high linear energy transfer (high-LET) ionizing radiation (IR) is a promising cancer treatment modality owing to advantageous physical properties of energy deposition and associated toxicity over X-rays. Therapies utilizing high-LET radiation will benefit from a better understanding of the molecular mechanisms underpinning their increased biological efficacy. Towards this goal, we investigate here the biological consequences of well-defined clusters of DNA double-strand breaks (DSBs), a form of DNA damage, which on theoretical counts, has often been considered central to the enhanced toxicity of high-LET IR. We test clonal cell lines harboring in their genomes constructs with appropriately engineered I-SceI recognition sites that convert upon I-SceI expression to individual DSBs, or DSB-clusters comprising known numbers of DSBs with defined DNA-ends. We find that, similarly to high-LET IR, DSB-clusters of increasing complexity, i.e. increasing numbers of DSBs, with compatible or incompatible ends, compromise classical non-homologous end-joining, favor DNA end-resection and promote resection-dependent DSB-processing. Analysis of RAD51 foci shows increased engagement of error-free homologous recombination on DSB-clusters. Multicolor fluorescence in situ hybridization analysis shows that complex DSB-clusters markedly increase the incidence of structural chromosomal abnormalities (SCAs). Since RAD51-knockdown further increases SCAs-incidence, we conclude that homologous recombination suppresses SCAs-formation. Strikingly, CtIP-depletion inhibits SCAs-formation, suggesting that it relies on alternative end-joining or single-strand annealing. Indeed, ablation of RAD52 causes a marked reduction in SCAs, as does also inhibition of PARP1. We conclude that increased DSB-cluster formation that accompanies LET-increases, enhances IR-effectiveness by promoting DNA end-resection, which suppresses c-NHEJ and enhances utilization of alt-EJ or SSA. Although increased resection also favors HR, on balance, error-prone processing dominates, causing the generally observed increased toxicity of high-LET radiation. These findings offer new mechanistic insights into high-LET IR-toxicity and have translational potential in the clinical setting that may be harnessed by combining high-LET IR with inhibitors of PARP1 or RAD52.

BMN673 Is a PARP Inhibitor with Unique Radiosensitizing Properties: Mechanisms and Potential in Radiation Therapy.

BMN673 is a relatively new PARP inhibitor (PARPi) that exhibits superior efficacy in vitro compared to olaparib and other clinically relevant PARPi. BMN673, similar to most clinical PARPi, inhibits the catalytic activities of PARP-1 and PARP-2 and shows impressive anticancer potential as monotherapy in several pre-clinical and clinical studies. Tumor resistance to PARPi poses a significant challenge in the clinic. Thus, combining PARPi with other treatment modalities, such as radiotherapy (RT), is being actively pursued to overcome such resistance. However, the modest to intermediate radiosensitization exerted by olaparib, rucaparib, and veliparib, limits the rationale and the scope of such combinations. The recently reported strong radiosensitizing potential of BMN673 forecasts a paradigm shift on this front. Evidence accumulates that BMN673 may radiosensitize via unique mechanisms causing profound shifts in the balance among DNA double-strand break (DSB) repair pathways. According to one of the emerging models, BMN673 strongly inhibits classical non-homologous end-joining (c-NHEJ) and increases reciprocally and profoundly DSB end-resection, enhancing error-prone DSB processing that robustly potentiates cell killing. In this review, we outline and summarize the work that helped to formulate this model of BMN673 action on DSB repair, analyze the causes of radiosensitization and discuss its potential as a radiosensitizer in the clinic. Finally, we highlight strategies for combining BMN673 with other inhibitors of DNA damage response for further improvements.

Selective vulnerability of ARID1A deficient colon cancer cells to combined radiation and ATR-inhibitor therapy.

ARID1A is frequently mutated in colorectal cancer (CRC) cells. Loss of ARID1A function compromises DNA damage repair and increases the reliance of tumor cells on ATR-dependent DNA repair pathways. Here, we investigated the effect of ionizing radiation (IR), in combination with ATR inhibitors (ATRi) in CRC cell lines with proficient and deficient ARID1A. The concept of selective vulnerability of ARID1A deficient CRC cells to ATRi was further tested in an ex vivo system by using the ATP-tumor chemosensitivity assay (ATP-TCA) in cells from untreated CRC patients, with and without ARID1A expression. We found selective sensitization upon ATRi treatment as well as after combined treatment with IR (P<0.001), especially in ARID1A deficient CRC cells (P <0.01). Knock-down of ARID1B further increased the selective radiosensitivity effect of ATRi in ARID1A negative cells (P<0.01). Mechanistically, ATRi abrogates the G2 checkpoint (P<0.01) and homologous recombination repair (P<0.01) in ARID1A deficient cells. Most importantly, ex-vivo experiments showed that ATRi had the highest radiosensitizing effect in ARID1A negative cells from CRC patients. Collectively, our results generate pre-clinical and clinical mechanistic rationale for assessing ARID1A defects as a biomarker for ATR inhibitor response as a single agent, or in a synthetic lethal approach in combination with IR.