IR-induced DNA Double-strand Breaks Research Articles

Changes in repair pathways of radiation-induced DNA double-strand breaks at the midblastula transition in Xenopus embryo.

Ionizing radiation (IR) causes DNA damage, particularly DNA double-strand breaks (DSBs), which have significant implications for genome stability. The major pathways of repairing DSBs are homologous recombination (HR) and nonhomologous end joining (NHEJ). However, the repair mechanism of IR-induced DSBs in embryos is not well understood, despite extensive research in somatic cells. The externally developing aquatic organism, Xenopus tropicalis, serves as a valuable model for studying embryo development. A significant increase in zygotic transcription occurs at the midblastula transition (MBT), resulting in a longer cell cycle and asynchronous cell divisions. This study examines the impact of X-ray irradiation on Xenopus embryos before and after the MBT. The findings reveal a heightened X-ray sensitivity in embryos prior to the MBT, indicating a distinct shift in the DNA repair pathway during embryo development. Importantly, we show a transition in the dominant DSB repair pathway from NHEJ to HR before and after the MBT. These results suggest that the MBT plays a crucial role in altering DSB repair mechanisms, thereby influencing the IR sensitivity of developing embryos.

The one-carbon metabolic enzyme MTHFD2 promotes resection and homologous recombination after ionizing radiation.

The one-carbon metabolism enzyme bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase 2 (MTHFD2) is among the most overexpressed proteins across tumors and is widely recognized as a promising anticancer target. While MTHFD2 is mainly described as a mitochondrial protein, a new nuclear function is emerging. Here, we observe that nuclear MTHFD2 protein levels and association with chromatin increase following ionizing radiation (IR) in an ataxia telangiectasia mutated (ATM)- and DNA-dependent protein kinase (DNA-PK)-dependent manner. Furthermore, repair of IR-induced DNA double-strand breaks (DSBs) is delayed upon MTHFD2 knockdown, suggesting a role for MTHFD2 in DSB repair. In support of this, we observe impaired recruitment of replication protein A (RPA), reduced resection, decreased IR-induced DNA repair protein RAD51 homolog 1 (RAD51) levels and impaired homologous recombination (HR) activity in MTHFD2-depleted cells following IR. In conclusion, we identify a key role for MTHFD2 in HR repair and describe an interdependency between MTHFD2 and HR proficiency that could potentially be exploited for cancer therapy.

Different Impacts of DNA-PK and mTOR Kinase Inhibitors in Combination with Ionizing Radiation on HNSCC and Normal Tissue Cells.

Despite substantial advancements in understanding the pathomechanisms of head and neck squamous cell carcinoma (HNSCC), effective therapy remains challenging. The application of kinase inhibitors (KIs) in HNSCC, specifically mTOR and DNA-PK inhibitors, can increase radiosensitivity and therefore presents a promising strategy when used simultaneously with ionizing radiation (IR) in cancer treatment. Our study focused on the selective DNA-PK-inhibitor AZD7648; the selective mTOR-inhibitor Sapanisertib; and CC-115, a dual inhibitor targeting both mTOR and DNA-PK. The impact of these KIs on HNSCC and normal tissue cells was assessed using various analytical methods including cell death studies, cell cycle analysis, real-time microscopy, colony-forming assays and immunohistochemical staining for γH2AX and downstream mTOR protein p-S6. We detected a strong inhibition of IR-induced DNA double-strand break (DSB) repair, particularly in AZD7648-treated HNSCC, whereas normal tissue cells repaired DNA DSB more efficiently. Additionally, AZD7648 + IR treatment showed a synergistic decline in cell proliferation and clonogenicity, along with an elevated G2/M arrest and cell death in the majority of HNSCC cell lines. CC-115 + IR treatment led to an elevation in G2/M arrest, increased cell death, and a synergistic reduction in cell proliferation, though the effect was notably lower compared to the AZD7648 + IR- treated group. Sapanisertib led to a high cellular toxicity in both HNSCC and normal tissue cells, even in non-irradiated cells. Regarding cell proliferation and the induction of apoptosis and necrosis, Sapanisertib + IR was beneficial only in HPV+ HNSCC. Overall, this study highlights the potential of AZD7648 as a radiosensitizing agent in advanced-stage HPV-positive and negative HNSCC, offering a promising therapeutic strategy. However, the dual mTOR/DNA-PK-I CC-115 did not provide a distinct advantage over the use of selective KIs in our investigations, suggesting limited benefits for its application in KI + IR therapy. Notably, the selective mTOR-inhibitor Sapanisertib was only beneficial in HPV+ HNSCC and should not be applied in HPV- cases.

The Impact of NAD Bioavailability on DNA Double-Strand Break Repair Capacity in Human Dermal Fibroblasts after Ionizing Radiation.

Nicotinamide adenine dinucleotide (NAD) serves as a substrate for protein deacetylases sirtuins and poly(ADP-ribose) polymerases, which are involved in the regulation of DNA double-strand break (DSB) repair molecular machinery by various mechanisms. However, the impact of NAD bioavailability on DSB repair remains poorly characterized. Herein, using immunocytochemical analysis of γH2AX, a marker for DSB, we investigated the effect of the pharmacological modulation of NAD levels on DSB repair capacity in human dermal fibroblasts exposed to moderate doses of ionizing radiation (IR). We demonstrated that NAD boosting with nicotinamide riboside did not affect the efficiency of DSB elimination after the exposure of cells to IR at 1 Gy. Moreover, even after irradiation at 5 Gy, we did not observe any decrease in intracellular NAD content. We also showed that, when the NAD pool was almost completely depleted by inhibition of its biosynthesis from nicotinamide, cells were still able to eliminate IR-induced DSB, though the activation of ATM kinase, its colocalization with γH2AX and DSB repair capacity were reduced in comparison to cells with normal NAD levels. Our results suggest that NAD-dependent processes, such as protein deacetylation and ADP-ribosylation, are important but not indispensable for DSB repair induced by moderate doses of IR.

Modeling of ionizing radiation-induced chromosome aberration and tumor prevalence based on two classes of DNA double-strand breaks clustering in chromatin domains

There has been some controversy over the use of radiobiological models when modeling the dose-response curves of ionizing radiation (IR)-induced chromosome aberration and tumor prevalence, as those curves usually show obvious non-targeted effects (NTEs) at low doses of high linear energy transfer (LET) radiation. The lack of understanding the contribution of NTEs to IR-induced carcinogenesis can lead to distinct deviations of relative biological effectiveness (RBE) estimations of carcinogenic potential, which are widely used in radiation risk assessment and radiation protection. In this work, based on the initial pattern of two classes of IR-induced DNA double-strand breaks (DSBs) clustering in chromatin domains and the subsequent incorrect repair processes, we proposed a novel radiobiological model to describe the dose-response curves of two carcinogenic-related endpoints within the same theoretical framework. The representative experimental data was used to verify the consistency and validity of the present model. The fitting results indicated that, compared with targeted effect (TE) and NTE models, the current model has better fitting ability when dealing with the experimental data of chromosome aberration and tumor prevalence induced by multiple types of IR with different LETs. Notably, the present model without introducing an NTE term was adequate to describe the dose-response curves of IR-induced chromosome aberration and tumor prevalence with NTEs in low-dose regions. Based on the fitting parameters, the LET-dependent RBE values were calculated for three given low doses. Our results showed that the RBE values predicted by the current model gradually decrease with the increase of doses for the endpoints of chromosome aberration and tumor prevalence. In addition, the calculated RBE was also compared with those evaluated from other models. These analyses show that the proposed model can be used as an alternative tool to well describe dose-response curves of multiple carcinogenic-related endpoints and effectively estimate RBE in low-dose regions.

Screen identifies fasudil as a radioprotector on human fibroblasts.

Radioprotectors safeguard biological system exposed to ionizing radiation (IR) by protecting normal cells from radiation damage during radiotherapy. Due to the toxicity and limited clinical utility of the present radioprotectors, it prompts us to identify novel radioprotectors that could alleviate IR-induced cytotoxicity of normal tissues. To identify new radioprotectors, we screened a chemical molecular library comprising 253 compounds in normal human fibroblasts (HFs) or 16HBE cells upon IR by CCK-8 assays and clonogenic survival assays. Fasudil was identified as a potential effective radioprotector. The results indicated that Fasudil exerts radioprotective effects on HFs against IR-induced DNA double-strand breaks (DSBs) through the regulation of DSB repair. Fasudil increased homologous recombination (HR) repair by 45.24% and decreased non-homologous end-joining (NHEJ) by 63.88% compared with untreated cells, without affecting changes to cell cycle profile. We further found that fasudil significantly facilitated the expression and foci formation of HR core proteins such as Rad51 and BRCA1 upon IR, and decreased the expression of NHEJ-associated proteins such as DNA-PKcs at 24 h post-IR. Our study identified fasudil as a novel radioprotector that exert radioprotective effects on normal cells through regulation of DSB repair by promoting HR repair.

Heterogeneity of Organization of Subcompartments in DSB Repair Foci.

Cells assemble compartments around DNA double-strand breaks (DSBs). The assembly of this compartment is dependent on the phosphorylation of histone H2AX, the binding of MDC1 to phosphorylated H2AX, and the assembly of downstream signaling and repair components. The decision on whether to use homologous recombination or nonhomologous end-joining repair depends on competition between 53BP1 and BRCA1. A major point of control appears to be DNA replication and associated changes in the epigenetic state. This includes dilution of histone H4 dimethylation and an increase in acetylation of lysine residues on H2A and H4 that impair 53BP1 binding. In this article, we examined more closely the spatial relationship between 53BP1 and BRCA1 within the cell cycle. We find that 53BP1 can associate with early S-phase replicated chromatin and that the relative concentration of BRCA1 in DSB-associated compartments correlates with increased BRCA1 nuclear abundance as cells progress into and through S phase. In most cases during S phase, both BRCA1 and 53BP1 are recruited to these compartments. This occurs for both IR-induced DSBs and breaks targeted to an integrated LacO array through a LacI-Fok1-mCherry fusion protein. Having established that the array system replicates this heterogeneity, we further examined the spatial relationship between DNA repair components. This enabled us to precisely locate the DNA containing the break and map other proteins relative to that DNA. We find evidence for at least three subcompartments. The damaged DNA, single-stranded DNA generated from end resection of the array, and nuclease CtIP all localized to the center of the compartment. BRCA1 and 53BP1 largely occupied discrete regions of the focus. One of BRCA1 or 53BP1 overlaps with the array, while the other is more peripherally located. The array-overlapping protein occupied a larger volume than the array, CtIP, or single-stranded DNA (ssDNA). Rad51 often occupied a much larger volume than the array itself and was sometimes observed to be depleted in the array volume where the ssDNA exclusively localizes. These results highlight the complexity of molecular compartmentalization within DSB repair compartments.

Black Phosphorus Quantum Dots Enhance the Radiosensitivity of Human Renal Cell Carcinoma Cells through Inhibition of DNA-PKcs Kinase.

Renal cell carcinoma (RCC) is one of the most aggressive urological malignancies and has a poor prognosis, especially in patients with metastasis. Although RCC is traditionally considered to be radioresistant, radiotherapy (RT) is still a common treatment for palliative management of metastatic RCC. Novel approaches are urgently needed to overcome radioresistance of RCC. Black phosphorus quantum dots (BPQDs) have recently received great attention due to their unique physicochemical properties and good biocompatibility. In the present study, we found that BPQDs enhance ionizing radiation (IR)-induced apoptotic cell death of RCC cells. BPQDs treatment significantly increases IR-induced DNA double-strand breaks (DSBs), as indicated by the neutral comet assay and the DSBs biomarkers γH2AX and 53BP1. Mechanistically, BPQDs can interact with purified DNA–protein kinase catalytic subunit (DNA-PKcs) and promote its kinase activity in vitro. BPQDs impair the autophosphorylation of DNA-PKcs at S2056, and this site phosphorylation is essential for efficient DNA DSBs repair and the release of DNA-PKcs from the damage sites. Consistent with this, BPQDs suppress nonhomologous end-joining (NHEJ) repair and lead to sustained high levels of autophosphorylated DNA-PKcs on the damaged sites. Moreover, animal experiments indicate that the combined approach with both BPQDs and IR displays better efficacy than monotreatment. These findings demonstrate that BPQDs have potential applications in radiosensitizing RCC cells.

Disulfiram Alone Functions as a Radiosensitizer for Pancreatic Cancer Both In Vitro and In Vivo.

The prognosis of pancreatic cancer remains very poor worldwide, partly due to the lack of specificity of early symptoms and innate resistance to chemo-/radiotherapy. Disulfiram (DSF), an anti-alcoholism drug widely used in the clinic, has been known for decades for its antitumor effects when simultaneously applied with copper ions, including pancreatic cancer. However, controversy still exists in the context of the antitumor effects of DSF alone in pancreatic cancer and related mechanisms, especially in its potential roles as a sensitizer for cancer radiotherapy. In the present study, we focused on whether and how DSF could facilitate ionizing radiation (IR) to eliminate pancreatic cancer. DSF alone significantly suppressed the survival of pancreatic cancer cells after exposure to IR, both in vitro and in vivo. Additionally, DSF treatment alone caused DNA double-strand breaks (DSBs) and further enhanced IR-induced DSBs in pancreatic cancer cells. In addition, DSF alone boosted IR-induced cell cycle G2/M phase arrest and apoptosis in pancreatic cancer exposed to IR. RNA sequencing and bioinformatics analysis results suggested that DSF could trigger cell adhesion molecule (CAM) signaling, which might be involved in its function in regulating the radiosensitivity of pancreatic cancer cells. In conclusion, we suggest that DSF alone may function as a radiosensitizer for pancreatic cancer, probably by regulating IR-induced DNA damage, cell cycle arrest and apoptosis, at least partially through the CAM signaling pathway.

Abstract 3065: Modeling common aspects of the metabolic response of cancer cells to ionizing radiation

Abstract Flexibility and reprogramming of cancer metabolism supports cancer progression and therapy resistance. In previous work we described opportunities for overcoming environment-induced resistance to ionizing radiation (IR) by pharmacologic inhibition of metabolic processes [1,2,3]. Here, we hypothesized that certain aspects of cancer metabolism will support the repair of radiation-induced DNA double-strand breaks (DSBs) and that the identification of pathways critical to the repair of radiation-induced DNA damage and cell survival will allow to enhance sensitivity of cancer cells to IR by interfering with such metabolic liabilities. Yet little is known about metabolic aspects of the cancer cell response to IR. We therefore systematically screened a panel of cancer cell lines with distinct genetic backgrounds for time-dependent changes in their acute metabolic response to IR and recorded the kinetics of IR-induced DSB in parallel. Mathematical modelling of the obtained metabolic and radiobiological data revealed a common metabolic response of irradiated cancer cells with impact on the kinetics of DSB repair. These were characterized by a rapid but transient downregulation of glycolysis and mitochondrial respiration, and gradual reactivation of glycolysis prior to mitochondrial recovery, presumably to compensate for disturbed metabolic functions of impaired mitochondria. Application of fractionated IR doses retarded mitochondrial recovery, highlighting the inhibitory effects of IR on mitochondrial functions. Furthermore, combining IR with inhibitors of compensatory glycolysis slowed mitochondrial recovery and DNA repair kinetics, supporting the functional relevance of compensatory glycolysis for DNA repair. Mathematical modeling of the metabolic response to IR allowed us to generate new hypotheses on the metabolic requirements of irradiated cancer cells and potential associated metabolic vulnerabilities. Further mechanistic studies will reveal the role of oncogenic drivers and co-mutations in metabolic enzymes for metabolic flexibility and metabolic liabilities of irradiated cancer cells in support of personalized approaches for combinatorial treatments. [1] Matschke J, et al., Antioxid Redox Signal 2016; 25: 89-107. [2] Hlouschek J et al., Front Oncol 2018; 8: 170. [3] Hlouschek J et al., Cancer Lett 2018; 439: 24-38. Supported by grants of the DFG (GRK1739/2 to V.J. and MA 8970/1-1 to J.M.), the Deutsche Krebshilfe/Mildred Scheel-Stiftung (70112711 to V.J.). Citation Format: Adam Krysztofiak, Klaudia A. Szymonowicz, Julian Hlouschek, Christoph Waterkamp, Kexu Xiang, Daniel Hoffmann, Verena Jendrossek, Johann Matschke. Modeling common aspects of the metabolic response of cancer cells to ionizing radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3065.

Simultaneous Targeting of RSK and AKT Efficiently Inhibits YB-1-Mediated Repair of Ionizing Radiation-Induced DNA Double-Strand Breaks in Breast Cancer Cells

Y-box binding protein 1 (YB-1) overexpression is associated with chemotherapy- and radiation therapy resistance. Ionizing radiation (IR), receptor tyrosine kinase ligands, and mutation in KRAS gene stimulate activation of YB-1. YB-1 accelerates the repair of IR-induced DNA double-strand breaks (DSBs). Ribosomal S6 kinase (RSK) is the main kinase inducing YB-1 phosphorylation. We investigated the impact of RSK targeting on DSB repair and radiosensitivity. The triple negative breast cancer (TNBC) cell lines MDA-MB-231, MDA-MB-468, and Hs 578T, in addition to non-TNBC cell lines MCF7, HBL-100, and SKBR3, were used. MCF-10A cells were included as normal breast epithelial cells. The RSK inhibitor LJI308 was used to investigate the role of RSK activity in S102 phosphorylation of YB-1 and YB-1-associated signaling pathways. The activation status of the underlying pathways was investigated by Western blotting after treatment with pharmacologic inhibitors or transfection with siRNA. The impact of LJI308 on DSB repair and postirradiation cell survival was tested by the γH2AX foci and the standard clonogenic assays, respectively. LJI308 inhibited the phosphorylation of RSK (T359/S363) and YB-1 (S102) after irradiation, treatment with EGF, and in cells expressing a KRAS mutation. LJI308 treatment slightly inhibited DSB repair only in some of the cell lines tested. This was shown to be due to PI3K-dependent stimulation of AKT or constitutive AKT activity mainly in cancer cells but not in normal breast epithelial MCF-10A cells. Simultaneous targeting of AKT and RSK strongly blocked DSB repair in all cancer cell lines, independent of TNBC status or KRAS mutation, with a minor effect in MCF-10A cells. Cotargeting of RSK- and AKT-induced radiation sensitivity in TNBC MDA-MB-231 and non-TNBC MCF7 cells but not in MCF-10A cells. Simultaneous targeting of RSK and AKT might be an efficient approach to block the repair of DSBs after irradiation and to induce radiosensitization of breast cancer cells.

The Determinant of DNA Repair Pathway Choices in Ionising Radiation-Induced DNA Double-Strand Breaks.

Ionising radiation- (IR-) induced DNA double-strand breaks (DSBs) are considered to be the deleterious DNA lesions that pose a serious threat to genomic stability. The major DNA repair pathways, including classical nonhomologous end joining, homologous recombination, single-strand annealing, and alternative end joining, play critical roles in countering and eliciting IR-induced DSBs to ensure genome integrity. If the IR-induced DNA DSBs are not repaired correctly, the residual or incorrectly repaired DSBs can result in genomic instability that is associated with certain human diseases. Although many efforts have been made in investigating the major mechanisms of IR-induced DNA DSB repair, it is still unclear what determines the choices of IR-induced DNA DSB repair pathways. In this review, we discuss how the mechanisms of IR-induced DSB repair pathway choices can operate in irradiated cells. We first briefly describe the main mechanisms of the major DNA DSB repair pathways and the related key repair proteins. Based on our understanding of the characteristics of IR-induced DNA DSBs and the regulatory mechanisms of DSB repair pathways in irradiated cells and recent advances in this field, We then highlight the main factors and associated challenges to determine the IR-induced DSB repair pathway choices. We conclude that the type and distribution of IR-induced DSBs, chromatin state, DNA-end structure, and DNA-end resection are the main determinants of the choice of the IR-induced DNA DSB repair pathway.

Abstract 994: Combining ruthenium metallo-intercalators and targeted radionuclide therapy for EGFR-overexpressing oesophageal cancer

Abstract Targeted radionuclide therapy (TRT) combines the specificity of molecular targeting with the potent cytotoxicity of ionising radiation. An emerging question is how to most effectively integrate TRT with radiosensitizing small molecules [1]. Auger electron-emitting radionuclides such as 111In are well-suited as the therapeutic radionuclide as their short-range reduces “cross fire” damage to neighbouring cells. As a novel chemical class of radiosensitizing agent, we have found ruthenium polypyridyl complexes (RPCs) that intercalate between DNA base pairs such as Ru(phen)2(tpphz)2+ (phen = 1,10 phenanthroline, tpphz = tetrapyridophenazine) act to stall replication forks and impede mitotic progression in human oesophageal cancer cells [2,3]. This dual mechanism of action results in preferential growth inhibition of rapidly-proliferating oesophageal cancer cells with elevated mitotic indices. In contrast to cisplatin and other DNA-damaging agents, relatively low DSB levels are generated in response to RPC metallo-intercalation and reduced cytotoxicity towards non-cancer cells is observed. In addition to this, treatment is able to enhance cancer cell sensitivity to IR by increasing IR-induced DNA double-strand break (DSB) damage, decreasing cell survival in a synergistic manner. Based on these discoveries, we explore how nanoparticle-mediated delivery of Ru(phen)2(tpphz)2+ and the Auger electron emitting radionuclide 111In can achieve localised cytotoxicity enhancement specifically in EGFR-overexpressing oesophageal cancer cells, thereby demonstrating both a combinational effect alongside molecular targeting for this novel combination [4].

A common Chk1-dependent phenotype of DNA double-strand break suppression in two distinct radioresistant cancer types.

Triple-negative breast cancers (TNBC) are often resistant to treatment with ionizing radiation (IR). We sought to investigate whether pharmacologic inhibition of Chk1 kinase, which is commonly overexpressed in TNBC, preferentially sensitizes TNBC cells to IR. Ten breast cancer cell lines were screened with small molecule inhibitors against Chk1 and other kinases. Chk1 inhibition was also tested in isogenic KRAS mutant or wild-type cancer cells. Cellular radiosensitization was measured by short-term and clonogenic survival assays and by staining for the DNA double-strand break (DSB) marker γ-H2AX. Radiosensitization was also assessed in breast cancer biopsies using an ex vivo assay. Aurora B kinase-dependent mitosis-like chromatin condensation, a marker of radioresistance, was detected using a specific antibody against co-localized phosphorylation of serine 10 and trimethylation of lysine 9 on histone 3 (H3K9me3/S10p). Expression of CHEK1 and associated genes was evaluated in TNBC and lung adenocarcinoma. Inhibition of Chk1 kinase preferentially radiosensitized TNBC cells in vitro and in patient biopsies. Interestingly, TNBC cells displayed lower numbers of IR-induced DSBs than non-TNBC cells, correlating with their observed radioresistance. We found that Chk1 suppressed IR-induced DSBs in these cells, which was dependent on H3K9me3/S10p-a chromatin mark previously found to indicate radioresistance in KRAS mutant cancers. Accordingly, the effects of Chk1 inhibition in TNBC were reproduced in KRAS mutant but not wild-type cells. We also observed co-expression of genes in this Chk1 chromatin pathway in TNBC and KRAS mutant lung cancers. Chk1 promotes an unexpected, common phenotype of chromatin-dependent DSB suppression in radioresistant TNBC and KRAS mutant cancer cells, providing a direction for future investigations into overcoming the treatment resistance of TNBC.

A Roberts Syndrome Individual With Differential Genotoxin Sensitivity and a DNA Damage Response Defect

Roberts syndrome (RBS) is a rare, recessively transmitted developmental disorder characterized by growth retardation, craniofacial abnormalities, and truncation of limbs. All affected individuals to date have mutations in the ESCO2 (establishment of cohesion 2) gene, a key regulator of the cohesin complex, which is involved in sister chromatid cohesion and DNA double-strand break (DSB) repair. Here we characterize DNA damage responses (DDRs) for the first time in an RBS-affected family. Lymphoblastoid cell lines were established from an RBS family, including the proband and parents carrying ESCO2 mutations. Various DDR assays were performed on these cells, including cell survival, chromosome break, and apoptosis assays; checkpoint activation indicators; and measures of DNA breakage and repair. Cells derived from the RBS-affected individual showed sensitivity to ionizing radiation (IR) and mitomycin C-induced DNA damage. In this ESCO2 compound heterozygote, other DDRs were also defective, including enhanced IR-induced clastogenicity and apoptosis; increased DNA DSB induction; and a reduced capacity for repairing IR-induced DNA DSBs, as measured by γ-H2AX foci and the comet assay. In addition to its developmental features, RBS can be, like ataxia telangiectasia, considered a DDR-defective syndrome, which contributes to its cellular, molecular, and clinical phenotype.

A Common Chk1-Dependent Phenotype of DNA Double-Strand Break Suppression in Two Distinct Radioresistant Cancer Types

Triple-negative breast cancers (TNBC) are often resistant to treatment with ionizing radiation (IR). We sought to investigate whether pharmacologic inhibition of Chk1 kinase, which is commonly overexpressed in TNBC, preferentially sensitizes TNBC cells to IR. In a panel of 10 breast cancer cell lines with/ without TN receptor status, we screened with small molecule inhibitors against Chk1 and related kinases. Chk1 inhibition was also tested in isogenic KRAS mutant or wild-type cancer cells. Cellular radiosensitization was measured by short-term and clonogenic survival assays and by staining for the DNA double-strand break (DSB) marker γ-H2AX. Radiosensitization was also assessed in breast cancer biopsies using a previously established ex vivo assay. Aurora B kinase-dependent mitosis-like chromatin condensation, a marker of radioresistance, was detected using a specific antibody against co-localized phosphorylation of serine 10 and trimethylation of lysine 9 on histone 3 (H3K9me3/S10p). Expression of CHEK1 and associated genes was explored in TNBC and lung adenocarcinoma patients. In vitro and patient biopsy studies identified a positive association between TN hormone receptor status and radiosensitization by Chk1 inhibition. Dose enhancement factors (SF10%) were 1.3-1.7. Interestingly, lower numbers of IR-induced DSBs were observed in TNBC when compared to non-TNBC cells, correlating with their observed radioresistance. Unexpectedly, we found that Chk1 suppressed IR-induced DSBs in these cells by ∼1.5-fold, which was dependent on the H3K9me3/S10p mark, previously found to indicate radioresistance in KRAS mutant lung cancer. The effects of Chk1 inhibition seen in TNBC were reproduced in KRAS mutant but not wild-type cells. Consistent with our functional studies we observed co-expression of genes in this Chk1 pathway in TNBC and KRAS mutant lung cancers. Chk1 promotes an unexpected, common phenotype of chromatin-dependent DSB suppression in radioresistant TNBC and KRAS mutant cancer cells, providing a direction for future investigations into overcoming the treatment resistance of these challenging tumor types.

Sinomenine hydrochloride sensitizes cervical cancer cells to ionizing radiation by impairing DNA damage response.

The use of plant-based compounds derived from traditional medicine to improve human diseases has been gaining momentum, due to their high bioavailability and moderate adverse effects. Sinomenine is one such biomonomer alkali compound derived from Sinomenium acutum and is known for its anti-inflammatory and antitumor effects. However, the molecular mechanism(s) of its antitumor properties are not fully characterized. In the present study, we evaluated the radiosensitizing effects of the water-soluble sinomenine, sinomenine hydrochloride (SH) in human cervical cancer cell line (HeLa). SH sensitized HeLa cells to ionizing radiation (IR) by promoting accumulation of IR-induced DNA double-strand breaks (DSBs) and also by interfering with DNA damage checkpoint activation. We then investigated the molecular mechanisms underlying the SH-mediated cellular sensitization to IR and found that SH inhibited the expression of DNA damage response (DDR) factors Ku80 and Rad51 at the transcription level. Finally, the radiosensitizing activity of SH was confirmed in a cervical cancer mouse xenograft model. The combinatorial treatment of SH and IR significantly slowed the tumor growth rate compared with IR alone. Collectively, our study not only provides molecular insights into the novel role of SH in cellular response to IR, but also suggests a therapeutic potential of SH as a radiosensitizer in cervical cancer therapy.

Stress-Induced Phosphorylation of Nuclear YB-1 Depends on Nuclear Trafficking of p90 Ribosomal S6 Kinase.

Ionizing radiation (IR) and epidermal growth factor (EGF) stimulate Y-box binding protein-1 (YB-1) phosphorylation at Ser-102 in KRAS wild-type (KRASwt) cells, whereas in KRAS mutated (KRASmut) cells, YB-1 is constitutively phosphorylated, independent of IR or EGF. YB-1 activity stimulates the repair of IR-induced DNA double-strand breaks (DSBs) in the nucleus. Thus far, the YB-1 nuclear translocation pattern after cell exposure to various cellular stressors is not clear. In the present study, we investigated the pattern of YB-1 phosphorylation and its possible translocation to the nucleus in KRASwt cells after exposure to IR, EGF treatment, and conditional expression of mutated KRAS(G12V). IR, EGF, and conditional KRAS(G12V) expression induced YB-1 phosphorylation in both the cytoplasmic and nuclear fractions of KRASwt cells. None of the stimuli induced YB-1 nuclear translocation, while p90 ribosomal s6 kinase (RSK) translocation was enhanced in KRASwt cells after any of the stimuli. EGF-induced RSK translocation to the nucleus and nuclear YB-1 phosphorylation were completely blocked by the EGF receptor kinase inhibitor erlotinib. Likewise, RSK inhibition blocked RSK nuclear translocation and nuclear YB-1 phosphorylation after irradiation and KRAS(G12V) overexpression. In summary, acute stimulation of YB-1 phosphorylation does not lead to YB-1 translocation from the cytoplasm to the nucleus. Rather, irradiation, EGF treatment, or KRAS(G12V) overexpression induces RSK activation, leading to its translocation to the nucleus, where it activates already-existing nuclear YB-1. Our novel finding illuminates the signaling pathways involved in nuclear YB-1 phosphorylation and provides a rationale for designing appropriate targeting strategies to block YB-1 in oncology as well as in radiation oncology.

Loss of PTEN-assisted G2/M checkpoint impedes homologous recombination repair and enhances radio-curability and PARP inhibitor treatment response in prostate cancer

Here we report that PTEN contributes to DNA double-strand break (DSB) repair via homologous recombination (HR), as evidenced by (i) inhibition of HR in a reporter plasmid assay, (ii) enhanced sensitivity to mitomycin-C or olaparib and (iii) reduced RAD51 loading at IR-induced DSBs upon PTEN knockdown. No association was observed between PTEN-status and RAD51 expression either in-vitro or in-vivo in a tissue microarray of 1500 PTEN-deficient prostate cancer (PC) samples. PTEN depletion and sustained activation of AKT sequestered CHK1 in the cytoplasm, thus impairing the G2/M-checkpoint after irradiation. Consistently, AKT inhibition recovered the G2/M-checkpoint and restored HR efficiency in PTEN-depleted cells. We show that, although PTEN loss correlates with a worse prognosis, it may predict for improved response of PC patients to radiotherapy. Further, we provide evidence for the use of PTEN as a biomarker for predicting the response to PARP inhibitors as radiosensitizing agents in prostate cancer. Collectively, these data implicate PTEN in maintaining genomic stability by delaying G2/M-phase progression of damaged cells, thus allowing time for DSB repair by HR. Furthermore, we identify PTEN-status in PC as a putative predictor of (i) radiotherapy response and (ii) response to treatment with PARP inhibitor alone or combined with radiotherapy.

Refining Radiation for the Next Century.

![][1] Wilhelm Roentgen's report more than a century ago describing X-ray imaging fostered in the application of ionizing radiation (IR) for cancer therapy. Today, about half of all the patients with cancer are treated with radiotherapy and it remains one of our most effective