Immunogenic cell death after combined treatment with radiation and ATR inhibitors is dually regulated by apoptotic caspases.

Introduction: Inhibitors of the ATR kinase act as radiosensitizers through abrogating the G2 checkpoint and reducing DNA repair. Recent studies suggest that ATR inhibitors can also increase radiation-induced antitumor immunity, but the underlying immunomodulating mechanisms remain poorly understood. Moreover, it is poorly known how such immune effects relate to different death pathways such as caspase-dependent apoptosis. Here we address whether ATR inhibition in combination with irradiation may increase the presentation of hallmark factors of immunogenic cell death (ICD), and to what extent caspase activation regulates this response. Methods: Human lung cancer and osteosarcoma cell lines (SW900, H1975, H460, U2OS) were treated with X-rays and ATR inhibitors (VE822; AZD6738) in the absence and presence of a pan-caspase inhibitor. The ICD hallmarks HMGB1 release, ATP secretion and calreticulin surface-presentation were assessed by immunoblotting of growth medium, the CellTiter-Glo assay and an optimized live-cell flow cytometry assay, respectively. To obtain accurate measurement of small differences in the calreticulin signal by flow cytometry, we included normalization to a barcoded control sample. Results: Extracellular release of HMGB1 was increased in all the cell lines at 72 hours after the combined treatment with radiation and ATR inhibitors, relative to mock treatment or cells treated with radiation alone. The HMGB1 release correlated largely - but not strictly - with loss of plasma membrane integrity, and was suppressed by addition of the caspase inhibitor. However, one cell line showed HMGB1 release despite caspase inhibition, and in this cell line caspase inhibition induced pMLKL, a marker for necroptosis. ATP secretion occurred already at 48 hours after the co-treatment and did clearly not correlate with loss of plasma membrane integrity. Addition of pan-caspase inhibition further increased the ATP secretion. Surface-presentation of calreticulin was increased at 24-72 hours after irradiation, but not further increased by either ATR or caspase inhibition. Conclusion: These results show that ATR inhibition can increase the presentation of two out of three ICD hallmark factors from irradiated human cancer cells. Moreover, caspase activation distinctly affects each of the hallmark factors, and therefore likely plays a dual role in tumor immunogenicity by promoting both immunostimulatory and -suppressive effects.

Analysis of RNA Polymerase II Chromatin Binding by Flow Cytometry.

RNA polymerase II (RNAPII) transcribes DNA into mRNA and thereby plays a critical role in cellular protein production. In addition, RNAPII plays a central role in DNA damage responses. Measurements of RNAPII on chromatin may thus give insight into several essential processes in eukaryotic cells. During transcription, the C-terminal domain of RNAPII becomes post-translationally modified, and phosphorylation on serine 5 and serine 2 can be used as markers for the promoter proximal and productively elongating forms of RNAPII, respectively. Here, we provide a detailed protocol for the detection of chromatin-bound RNAPII and its serine 5- and serine 2-phosphorylated forms in individual human cells through the cell cycle. We have recently shown that this method can be used to study the effects of ultraviolet DNA damage on RNAPII chromatin binding and that it can even be used to reveal new knowledge about the transcription cycle itself. Other commonly used methods to study RNAPII chromatin binding include chromatin immunoprecipitation followed by sequencing or chromatin fractionation followed by western blotting. However, such methods are frequently based on lysates made from a large number of cells, which may mask population heterogeneity, e.g., due to cell cycle phase. With strengths such as single-cell analysis, speed of use, and accurate quantitative readouts, we envision that our flow cytometry method can be widely used as a complementary approach to sequencing-based methods to study effects of different stimuli and inhibitors on RNAPII-mediated transcription. Graphical overview.

Expanding roles of cell cycle checkpoint inhibitors in radiation oncology.

PURPOSE: Radiation-induced activation of cell cycle checkpoints have been of long-standing interest. The WEE1, CHK1 and ATR kinases are key factors in cell cycle checkpoint regulation and are essential for the S and G2 checkpoints. Here, we review the rationale for why inhibitors of WEE1, CHK1 and ATR could be beneficial in combination with radiation. CONCLUSIONS: Combined treatment with radiation and inhibitors of these kinases results in checkpoint abrogation and subsequent mitotic catastrophe. This might selectively radiosensitize tumor cells, as they often lack the p53-dependent G1 checkpoint and therefore rely more on the G2 checkpoint to repair DNA damage. Further affecting the repair of radiation damage, inhibition of WEE1, CHK1 or ATR also specifically suppresses the homologous recombination repair pathway. Moreover, inhibition of these kinases can induce massive replication stress during S phase of the cell cycle, likely contributing to eliminate radioresistant S phase cells. Intriguingly, recent findings suggest that cell cycle checkpoint inhibitors in combination with radiation can also enhance anti-tumor immune effects. Altogether, the expanding knowledge about the functional roles of WEE1, CHK1 and ATR inhibitors support that they are promising candidates for use in combination with radiation treatment.

Caspase activation counteracts interferon signaling after G2 checkpoint abrogation by ATR inhibition in irradiated human cancer cells.

Recent studies suggest that inhibition of the ATR kinase can potentiate radiation-induced antitumor immune responses, but the extent and mechanisms of such responses in human cancers remain scarcely understood. We aimed to assess whether the ATR inhibitors VE822 and AZD6738, by abrogating the G2 checkpoint, increase cGAS-mediated type I IFN response after irradiation in human lung cancer and osteosarcoma cell lines. Supporting that the checkpoint may prevent IFN induction, radiation-induced IFN signaling declined when the G2 checkpoint arrest was prolonged at high radiation doses. G2 checkpoint abrogation after co-treatment with radiation and ATR inhibitors was accompanied by increased radiation-induced IFN signaling in four out of five cell lines tested. Consistent with the hypothesis that the cytosolic DNA sensor cGAS may detect DNA from ruptured micronuclei after G2 checkpoint abrogation, cGAS co-localized with micronuclei, and depletion of cGAS or STING abolished the IFN responses. Contrastingly, one lung cancer cell line showed no increase in IFN signaling despite irradiation and G2 checkpoint abrogation. This cell line showed a higher level of the exonuclease TREX1 than the other cell lines, but TREX1 depletion did not enhance IFN signaling. Rather, addition of a pan-caspase inhibitor restored the IFN response in this cell line and also increased the responses in the other cell lines. These results show that treatment-induced caspase activation can suppress the IFN response after co-treatment with radiation and ATR inhibitors. Caspase activation thus warrants further consideration as a possible predictive marker for lack of IFN signaling.

A novel, rapid and sensitive flow cytometry method reveals degradation of promoter proximal paused RNAPII in the presence and absence of UV.

RNA polymerase II (RNAPII) is emerging as an important factor in DNA damage responses, but how it responds to genotoxic stress is not fully understood. We have developed a rapid and sensitive flow cytometry method to study chromatin binding of RNAPII in individual human cells through the cell cycle. Indicating enhanced transcription initiation at early timepoints, levels of RNAPII were increased at 15-30min after UV-induced DNA damage. This was particularly evident for the S5 phosphorylated form of RNAPII (pRNAPII S5), which is typically associated with promoter proximal pausing. Furthermore, degradation of pRNAPII S5 frequently occurs, as its levels on chromatin were strongly enhanced by the proteasome inhibitor MG132 with and without UV. Remarkably, inhibiting pause release with 5,6-dichloro-1-beta-ribo-furanosyl benzimidazole (DRB) further promoted UV-induced degradation of pRNAPII S5, suggesting enhanced initiation may lead to a phenomenon of 'promoter proximal crowding' resulting in premature termination via degradation of RNAPII. Moreover, pRNAPII S2 levels on chromatin were more stable in S phase of the cell cycle 2h after UV, indicating cell cycle specific effects. Altogether our results demonstrate a useful new method and suggest that degradation of promoter proximal RNAPII plays an unanticipated large role both during normal transcription and after UV.

Differential Effects of Combined ATR/WEE1 Inhibition in Cancer Cells.

Inhibitors of WEE1 and ATR kinases are considered promising for cancer treatment, either as monotherapy or in combination with chemo- or radiotherapy. Here, we addressed whether simultaneous inhibition of WEE1 and ATR might be advantageous. Effects of the WEE1 inhibitor MK1775 and ATR inhibitor VE822 were investigated in U2OS osteosarcoma cells and in four lung cancer cell lines, H460, A549, H1975, and SW900, with different sensitivities to the WEE1 inhibitor. Despite the differences in cytotoxic effects, the WEE1 inhibitor reduced the inhibitory phosphorylation of CDK, leading to increased CDK activity accompanied by ATR activation in all cell lines. However, combining ATR inhibition with WEE1 inhibition could not fully compensate for cell resistance to the WEE1 inhibitor and reduced cell viability to a variable extent. The decreased cell viability upon the combined treatment correlated with a synergistic induction of DNA damage in S-phase in U2OS cells but not in the lung cancer cells. Moreover, less synergy was found between ATR and WEE1 inhibitors upon co-treatment with radiation, suggesting that single inhibitors may be preferable together with radiotherapy. Altogether, our results support that combining WEE1 and ATR inhibitors may be beneficial for cancer treatment in some cases, but also highlight that the effects vary between cancer cell lines.

New link between the RNA polymerase II-CTD and replication stress.

Conflicts between transcription and replication are a major source of replication stress. Our recent findings show that proper dephosphorylation of Serine 5 in the carboxy-terminal domain (CTD) of DNA-directed RNA polymerase II subunit RPB1 is needed to prevent such conflicts in human cells.

WDR82/PNUTS-PP1 Prevents Transcription-Replication Conflicts by Promoting RNA Polymerase II Degradation on Chromatin.

Transcription-replication (T-R) conflicts cause replication stress and loss of genome integrity. However, the transcription-related processes that restrain such conflicts are poorly understood. Here, we demonstrate that the RNA polymerase II (RNAPII) C-terminal domain (CTD) phosphatase protein phosphatase 1 (PP1) nuclear targeting subunit (PNUTS)-PP1 inhibits replication stress. Depletion of PNUTS causes lower EdU uptake, S phase accumulation, and slower replication fork rates. In addition, the PNUTS binding partner WDR82 also promotes RNAPII-CTD dephosphorylation and suppresses replication stress. RNAPII has a longer residence time on chromatin after depletion of PNUTS or WDR82. Furthermore, the RNAPII residence time is greatly enhanced by proteasome inhibition in control cells but less so in PNUTS- or WDR82-depleted cells, indicating that PNUTS and WDR82 promote degradation of RNAPII on chromatin. Notably, reduced replication is dependent on transcription and the phospho-CTD binding protein CDC73 after depletion of PNUTS/WDR82. Altogether, our results suggest that RNAPII-CTD dephosphorylation is required for the continuous turnover of RNAPII on chromatin, thereby preventing T-R conflicts.

The Dual Cell Cycle Kinase Inhibitor JNJ-7706621 Reverses Resistance to CD37-Targeted Radioimmunotherapy in Activated B Cell Like Diffuse Large B Cell Lymphoma Cell Lines.

The CD37 targeting radioimmunoconjugate 177Lu-lilotomab satetraxetan (Betalutin) is currently being evaluated in a clinical phase 2b trial for patients with follicular lymphoma (FL) and in a phase 1 trial for patients with diffuse large B-cell lymphoma (DLBCL). Herein we have investigated the effect of 177Lu-lilotomab satetraxetan in seven activated B-cell like (ABC) DLBCL cell lines. Although the radioimmunoconjugate showed anti-tumor activity, primary resistance was observed in a subset of cell lines. Thus, we set out to identify drugs able to overcome the resistance to 177Lu-lilotomab satetraxetan in two resistant ABC-DLBCL cell lines. We performed a viability-based screen combining 177Lu-lilotomab satetraxetan with the 384-compound Cambridge Cancer Compound Library. Drug combinations were scored using Bliss and Chou-Talalay algorithms. We identified and characterized the dual-specific CDK1/2 and AURA/B kinase inhibitor JNJ-7706621 as compound able to revert the resistance to RIT, alongside topoisomerase and histone deacetylases (HDAC) inhibitors.

p21 limits S phase DNA damage caused by the Wee1 inhibitor MK1775.

The Wee1 inhibitor MK1775 (AZD1775) is currently being tested in clinical trials for cancer treatment. Here, we show that the p53 target and CDK inhibitor p21 protects against MK1775-induced DNA damage during S-phase. Cancer and normal cells deficient for p21 (HCT116 p21-/-, RPE p21-/-, and U2OS transfected with p21 siRNA) showed higher induction of the DNA damage marker γH2AX in S-phase in response to MK1775 compared to the respective parental cells. Furthermore, upon MK1775 treatment the levels of phospho-DNA PKcs S2056 and phospho-RPA S4/S8 were higher in the p21 deficient cells, consistent with increased DNA breakage. Cell cycle analysis revealed that these effects were due to an S-phase function of p21, but MK1775-induced S-phase CDK activity was not altered as measured by CDK-dependent phosphorylations. In the p21 deficient cancer cells MK1775-induced cell death was also increased. Moreover, p21 deficiency sensitized to combined treatment of MK1775 and the CHK1-inhibitor AZD6772, and to the combination of MK1775 with ionizing radiation. These results show that p21 protects cancer cells against Wee1 inhibition and suggest that S-phase functions of p21 contribute to mediate such protection. As p21 can be epigenetically downregulated in human cancer, we propose that p21 levels may be considered during future applications of Wee1 inhibitors.

Regulation of ATR activity via the RNA polymerase II associated factors CDC73 and PNUTS-PP1.

Ataxia telangiectasia mutated and Rad3-related (ATR) kinase is a key factor activated by DNA damage and replication stress. An alternative pathway for ATR activation has been proposed to occur via stalled RNA polymerase II (RNAPII). However, how RNAPII might signal to activate ATR remains unknown. Here, we show that ATR signaling is increased after depletion of the RNAPII phosphatase PNUTS-PP1, which dephosphorylates RNAPII in its carboxy-terminal domain (CTD). High ATR signaling was observed in the absence and presence of ionizing radiation, replication stress and even in G1, but did not correlate with DNA damage or RPA chromatin loading. R-loops were enhanced, but overexpression of EGFP-RNaseH1 only slightly reduced ATR signaling after PNUTS depletion. However, CDC73, which interacted with RNAPII in a phospho-CTD dependent manner, was required for the high ATR signaling, R-loop formation and for activation of the endogenous G2 checkpoint after depletion of PNUTS. In addition, ATR, RNAPII and CDC73 co-immunoprecipitated. Our results suggest a novel pathway involving RNAPII, CDC73 and PNUTS-PP1 in ATR signaling and give new insight into the diverse functions of ATR.

A novel role for ATR/Rad3 in G1 phase.

Checkpoint kinases are important in cellular surveillance pathways that help cells to cope with DNA damage and protect their genomes. In cycling cells, DNA replication is one of the most sensitive processes and therefore all organisms carefully regulate replication initiation and progression. The checkpoint kinase ATR plays important roles both in response to DNA damage and replication stress, and ATR inhibitors are currently in clinical trials for cancer treatment. Therefore, it is important to understand the roles of ATR in detail. Here we show that the fission yeast homologue Rad3 and the human ATR regulate events also in G1 phase in an unperturbed cell cycle. Rad3Δ mutants or human cells exposed to ATR inhibitor in G1 enter S phase prematurely, which results in increased DNA damage. Furthermore, ATR inhibition in a single G1 reduces clonogenic survival, demonstrating that long-term effects of ATR inhibition during G1 are deleterious for the cell. Interestingly, ATR inhibition through G1 and S phase reduces survival in an additive manner, strongly arguing that different functions of ATR are targeted in the different cell-cycle phases. We propose that potential effects of ATR inhibitors in G1 should be considered when designing future treatment protocols with such inhibitors.

Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading.

Recent studies have shown synergistic cytotoxic effects of simultaneous Chk1- and Wee1-inhibition. However, the mechanisms behind this synergy are not known. Here, we present a flow cytometry-based screen for compounds that cause increased DNA damage in S-phase when combined with the Wee1-inhibitor MK1775. Strikingly, the Chk1-inhibitors AZD7762 and LY2603618 were among the top candidate hits of 1664 tested compounds, suggesting that the synergistic cytotoxic effects are due to increased S-phase DNA damage. Combined Wee1- and Chk1-inhibition caused a strong synergy in induction of S-phase DNA damage and reduction of clonogenic survival. To address the underlying mechanisms, we developed a novel assay measuring CDK-dependent phosphorylations in single S-phase cells. Surprisingly, while Wee1-inhibition alone induced less DNA damage compared to Chk1-inhibition, Wee1-inhibition caused a bigger increase in S-phase CDK-activity. However, the loading of replication initiation factor CDC45 was more increased after Chk1- than Wee1-inhibition and further increased by the combined treatment, and thus correlated well with DNA damage. Therefore, when Wee1 alone is inhibited, Chk1 suppresses CDC45 loading and thereby limits the extent of unscheduled replication initiation and subsequent S-phase DNA damage, despite very high CDK-activity. These results can explain why combined treatment with Wee1- and Chk1-inhibitors gives synergistic anti-cancer effects.

MtDNA depleted PC3 cells exhibit Warburg effect and cancer stem cell features.

Reducing mtDNA content was considered as a critical step in the metabolism restructuring for cell stemness restoration and further neoplastic development. However, the connections between mtDNA depletion and metabolism reprograming-based cancer cell stemness in prostate cancers are still lack of studies. Here, we demonstrated that human CRPC cell line PC3 tolerated high concentration of the mtDNA replication inhibitor ethidium bromide (EtBr) and the mtDNA depletion triggered a universal metabolic remodeling process. Failure in completing that process caused lethal consequences. The mtDNA depleted (MtDP) PC3 cells could be steadily maintained in the special medium in slow cycling status. The MtDP PC3 cells contained immature mitochondria and exhibited Warburg effect. Furthermore, the MtDP PC3 cells were resistant to therapeutic treatments and contained greater cancer stem cell-like subpopulations: CD44+, ABCG2+, side-population and ALDHbright. In conclusion, these results highlight the association of mtDNA content, mitochondrial function and cancer cell stemness features.

Hypoxia-induced alterations of G2 checkpoint regulators.

Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, genome stability is also surveilled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can influence the DNA damage-induced cell cycle checkpoints. Here, we show that hypoxia (24 h 0.2% O2) alters the expression of several G2 checkpoint regulators, as examined by microarray gene expression analysis and immunoblotting of U2OS cells. While some of the changes reflected hypoxia-induced inhibition of cell cycle progression, the levels of several G2 checkpoint regulators, in particular Cyclin B, were reduced in G2 phase cells after hypoxic exposure, as shown by flow cytometric barcoding analysis of individual cells. These effects were accompanied by decreased phosphorylation of a Cyclin dependent kinase (CDK) target in G2 phase cells after hypoxia, suggesting decreased CDK activity. Furthermore, cells pre-exposed to hypoxia showed increased G2 checkpoint arrest upon treatment with ionizing radiation. Similar results were found following other hypoxic conditions (∼0.03% O2 20 h and 0.2% O2 72 h). These results demonstrate that the DNA damage-induced G2 checkpoint can be altered as a consequence of hypoxia, and we propose that such alterations may influence the genome stability of hypoxic tumors.

Targeting lung cancer through inhibition of checkpoint kinases.

Inhibitors of checkpoint kinases ATR, Chk1, and Wee1 are currently being tested in preclinical and clinical trials. Here, we review the basic principles behind the use of such inhibitors as anticancer agents, and particularly discuss their potential for treatment of lung cancer. As lung cancer is one of the most deadly cancers, new treatment strategies are highly needed. We discuss how checkpoint kinase inhibition in principle can lead to selective killing of lung cancer cells while sparing the surrounding normal tissues. Several features of lung cancer may potentially be exploited for targeting through inhibition of checkpoint kinases, including mutated p53, low ERCC1 levels, amplified Myc, tumor hypoxia and presence of lung cancer stem cells. Synergistic effects have also been reported between inhibitors of ATR/Chk1/Wee1 and conventional lung cancer treatments, such as gemcitabine, cisplatin, or radiation. Altogether, inhibitors of ATR, Chk1, and Wee1 are emerging as new cancer treatment agents, likely to be useful in lung cancer treatment. However, as lung tumors are very diverse, the inhibitors are unlikely to be effective in all patients, and more work is needed to determine how such inhibitors can be utilized in the most optimal ways.

Differential DNA methylation analysis of breast cancer reveals the impact of immune signaling in radiation therapy.

Radiotherapy (RT) is a central treatment modality for breast cancer patients. The purpose of our study was to investigate the DNA methylation changes in tumors following RT, and to identify epigenetic markers predicting treatment outcome. Paired biopsies from patients with inoperable breast cancer were collected both before irradiation (n = 20) and after receiving 10-24 Gray (Gy) (n = 19). DNA methylation analysis was performed by using Illumina Infinium 27K arrays. Fourteen genes were selected for technical validation by pyrosequencing. Eighty-two differentially methylated genes were identified in irradiated (n = 11) versus nonirradiated (n = 19) samples (false discovery rate, FDR = 1.1%). Methylation levels in pathways belonging to the immune system were most altered after RT. Based on methylation levels before irradiation, a panel of five genes (H2AFY, CTSA, LTC4S, IL5RA and RB1) were significantly associated with clinical response (p = 0.041). Furthermore, the degree of methylation changes for 2,516 probes correlated with the given radiation dose. Within the 2,516 probes, an enrichment for pathways involved in cellular immune response, proliferation and apoptosis was identified (FDR < 5%). Here, we observed clear differences in methylation levels induced by radiation, some associated with response to treatment. Our study adds knowledge on the molecular mechanisms behind radiation response.

PLK1-inhibition can cause radiosensitization or radioresistance dependent on the treatment schedule.

BACKGROUND AND PURPOSE: PLK1-inhibitors are emerging as new potential anticancer agents. It is therefore important to explore the combined effects of PLK1-inhibitors with conventional therapies. Based on the functional roles of PLK1 in both mitosis and the G2 checkpoint, we hypothesized that the treatment schedule might influence the combined effects of PLK1-inhibiton and radiation. MATERIALS AND METHODS: Human osteosarcoma U2OS and colorectal cancer HT29 and SW620 cells were treated with the PLK1-inhibitor BI2536 before or after X-ray irradiation (0-6 Gy). Clonogenic assays, flow cytometry, immunofluorescence and mCherry-53BP1 time-lapse imaging were used to assay cell survival, cell cycle progression and DNA damage repair. RESULTS: Treatment with the PLK1-inhibitor for 24h before radiation caused cells to accumulate in G2/M and resulted in increased radiosensitivity. In contrast, the cytotoxic effects of the two treatments were less-than-additive when cells were treated with the PLK1-inhibitor for 24h after radiation. This resistance was associated with a prolonged G2 checkpoint causing enhanced repair of the radiation-induced damage and decreased BI2536-mediated mitotic damage. CONCLUSIONS: PLK1-inhibitors need to be administrated several hours before radiation to achieve radiosensitization. If PLK1-inhibitors are given after radiation, cell killing is reduced due to the prolonged G2 checkpoint.

The efficacy of CHK1 inhibitors is not altered by hypoxia, but is enhanced after reoxygenation.

Inhibitors of CHK1 are in clinical trials for cancer treatment in combination with DNA-damaging agents. Importantly, it was previously suggested that hypoxic cancer cells may be particularly sensitive to CHK1 inhibition. However, this suggestion was based on studies in severe, toxic levels of hypoxia (anoxia). The influence of less severe hypoxia on the efficacy of CHK1 inhibitors, administered either as single agents or in combination with other treatments, remains to be investigated. Here, we have assayed the effects of the CHK1 inhibitors, AZD7762 and UCN-01, during various hypoxic conditions and after reoxygenation in the absence and presence of ionizing radiation. Treatment with CHK1 inhibitors during acute or prolonged hypoxia (< 0.03%, 0.2%, and 1% O2; 3 h or 20-24 h) gave similar effects on cell survival as treatment with these inhibitors during normoxia. CHK1 inhibitors combined with ionizing radiation showed similar radiosensitization in hypoxic and normoxic cells. However, when the inhibitors were administered after reoxygenation following prolonged hypoxia (< 0.03% and 0.2%; 20-24 h), we observed decreased cell survival and stronger induction of the DNA damage marker, γH2AX, in S-phase cells. This was accompanied by enhanced phosphorylation of the single-stranded DNA-binding replication protein A. These results suggest that the cytotoxic effects of CHK1 inhibitors are enhanced after reoxygenation following prolonged hypoxia, most likely due to the increased replication-associated DNA damage. Combining CHK1 inhibitors with other treatments that cause increased reoxygenation, such as fractionated radiotherapy, might therefore be beneficial.