Inhibition of ATR with AZD6738 in combination with radiotherapy increases tumour growth delay without observable augmentation of late radiation induced toxicity further underpinning translation towards clinical evaluation in NSCLC.
Purpose The recent implementation of MR-Linacs has highlighted theranostic opportunities of contrast agents in both imaging and radiotherapy. There is a lack of data exploring the potential of superparamagnetic iron oxide nanoparticles (SPIONs) as radiosensitisers. Through preclinical 225 kVp exposures, this study aimed to characterise the uptake and radiobiological effects of SPIONs in tumour cell models in vitro and to provide proof-of-principle application in a xenograft tumour model. Methods SPIONs were also characterised to determine their hydrodynamic radius using dynamic light scattering and uptake was measured using ICP-MS in 6 cancer cell lines; H460, MiaPaCa2, DU145, MCF7, U87 and HEPG2. The impact of SPIONs on radiobiological response was determined by measuring DNA damage using 53BP1 immunofluorescence and cell survival. Sensitisation Enhancement Ratios (SERs) were compared with the predicted Dose Enhancement Ratios (DEFs) based on physical absorption estimations. In vivo efficacy was demonstrated using a subcutaneous H460 xenograft tumour model in SCID mice by following intra-tumoural injection of SPIONs. Results The hydrodynamic radius was found to be between 110 and 130 nm, with evidence of being monodisperse in nature. SPIONs significantly increased DNA damage in all cell lines with the exception of U87 cells at a dose of 1 Gy, 1 h post-irradiation. Levels of DNA damage correlated with the cell survival, in which all cell lines except U87 cells showed an increased sensitivity (P < 0.05) in the linear quadratic curve fit for 1 h exposure to 23.5 μg/ml SPIONs. There was also a 30.1% increase in the number of DNA damage foci found for HEPG2 cells at 2 Gy. No strong correlation was found between SPION uptake and DNA damage at any dose, yet the biological consequences of SPIONs on radiosensitisation were found to be much greater, with SERs up to 1.28 ± 0.03, compared with predicted physical dose enhancement levels of 1.0001. In vivo, intra-tumoural injection of SPIONs combined with radiation showed significant tumour growth delay compared to animals treated with radiation or SPIONs alone (P < 0.05). Conclusions SPIONs showed radiosensitising effects in 5 out of 6 cancer cell lines. No correlation was found between the cell-specific uptake of SPIONs into the cells and DNA damage levels. The in vivo study found a significant decrease in the tumour growth rate.
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Abiraterone acetate and Enzalutamide are novel anti-androgens that are key treatments to improve both progression-free survival and overall survival in patients with metastatic castration-resistant prostate cancer. In this study, we aimed to determine whether combinations of AR inhibitors with radiation are additive or synergistic, and investigated the underlying mechanisms governing this. This study also aimed to compare and investigate a biological rationale for the selection of Abiraterone versus Enzalutamide in combination with radiotherapy as currently selection is based on consideration of side effect profiles and clinical experience. We report that AR suppression with Enzalutamide produces a synergistic effect only in AR-sensitive prostate models. In contrast, Abiraterone displays synergistic effects in combination with radiation regardless of AR status, alluding to potential alternative mechanisms of action. The underlying mechanisms governing this AR-based synergy are based on the reduction of key AR linked DNA repair pathways such as NHEJ and HR, with changes in HR potentially the result of changes in cell cycle distribution, with these reductions ultimately resulting in increased cell death. These changes were also shown to be conserved in combination with radiation, with AR suppression 24 hours before radiation leading to the most significant differences. Comparison between Abiraterone and Enzalutamide highlighted Abiraterone from a mechanistic standpoint as being superior to Abiraterone for all endpoints measured. Therefore, this provides a potential rationale for the selection of Abiraterone over Enzalutamide.
IntroductionRadium-223 (223Ra) has been shown to have an overall survival benefit in metastatic castration-resistant prostate cancer (mCRPC) involving bone. Despite its increased clinical usage, relatively little is known regarding the mechanism of action of 223Ra at the cellular level.MethodsWe evaluated the effects of 223Ra irradiation in a panel of cell lines and then compared them with standard X-ray and external alpha-particle irradiation, with a particular focus on cell survival and DNA damage repair kinetics.Results223Ra exposures had very high, cell-type-dependent RBE50% ranging from 7 to 15. This was significantly greater than external alpha irradiations (RBE50% from 1.4 to 2.1). These differences were shown to be partially related to the volume of 223Ra solution added, independent of the alpha-particle dose rate, suggesting a radiation-independent mechanism of effect. Both external alpha particles and 223Ra exposure were associated with delayed DNA repair, with similar kinetics. Additionally, the greater treatment efficacy of 223Ra was associated with increased levels of residual DNA damage and cell death by mitotic catastrophe.ConclusionsThese results suggest that 223Ra exposure may be associated with greater biological effects than would be expected by direct comparison with a similar dose of external alpha particles, highlighting important challenges for future therapeutic optimization.
Radical radiotherapy, often in combination with hormone ablation, is a safe and effective treatment option for localised or locally-advanced prostate cancer. However, up to 30% of patients with locally advanced PCa will go on to develop biochemical failure, within 5 years, following initial radiotherapy. Improving radiotherapy response is clinically important since patients exhibiting biochemical failure develop castrate-resistant metastatic disease for which there is no curative therapy and median survival is 8–18 months. The aim of this research was to determine if loss of PTEN (highly prevalent in advanced prostate cancer) is a novel therapeutic target in the treatment of advanced prostate cancer. Previous work has demonstrated PTEN-deficient cells are sensitised to inhibitors of ATM, a key regulator in the response to DSBs. Here, we have shown the role of PTEN in cellular response to IR was both complex and context-dependent. Secondly, we have confirmed ATM inhibition in PTEN-depleted cell models, enhances ionising radiation-induced cell killing with minimal toxicity to normal prostate RWPE-1 cells. Furthermore, combined treatment significantly inhibited PTEN-deficient tumour growth compared to PTEN-expressing counterparts, with minimal toxicity observed. We have further shown PTEN loss is accompanied by increased endogenous levels of ROS and DNA damage. Taken together, these findings provide pre-clinical data for future clinical evaluation of ATM inhibitors as a neoadjuvant/adjuvant in combination with radiation therapy in prostate cancer patients harbouring PTEN mutations.
This study sought to determine the therapeutic efficacy of AZD6738, a small molecule inhibitor of ataxia telangiectasia related 3 kinase (ATR), in combination with ionising radiation (IR) for the treatment of PTEN deficient non-small cell lung cancer (NSCLC). An isogenic PTEN deficient cell model were generated in H460 and A549 cell lines by HuSH PTEN shRNA constructs in pGFP-V-RS vectors from Origene. Radiosensitivity was determined at various concentrations of AZD6738 by clonogenic assay following IR with 225 kV X-rays. Target validation and mechanistic evaluation of the ATR radiation treatments was performed by western blotting. Cell cycle distribution was obtained using propidium iodine flow cytometry analysis which was performed 48 hours following treatment with AZD6738 alone or in combination with IR. In vivo efficacy was determined using cell line derived xenografts in female SCID mice treated with AZD6738 at a concentration of 25 mg/kg per day by oral gavage for 28 days. Animals were exposed to size fraction and hypofractionated radiation dose under CBCT image guidance using the small animal radiotherapy research platform (SARRP). Radiation induced toxicity in normal lung tissue effects was investigated in a model of radiation induced fibrosis in C57BL6/J mice. In-vitro, combined treatment with AZD6738 and IR selectively targets PTEN-deficient tumour cells causing a significant reduction in clonogenic survival for both H460- and A549-PTEN KO cell models (p= <0.05 at 8Gy). Following 2 Gy IR exposure there were significantly higher mean foci per cell in both H460-KO and A549-KO cells in comparison to WT cells (p= <0.05 at 2, 4 and 8 hours following treatment). Both PTEN-KO cell lines exhibited increased sub G0/G1 and G2/M populations in comparison to PTEN-WT cell lines when treated with AZD6738 and IR. Minimal toxicity was observed in both normal HBE and BEAS-2B cell lines. Currently in vivo tumour response experiments are underway to investigate IR and ATR kinase inhibitor AZD6738 using H460-WT and H460-PTEN KO cell models as well as determining the effects of AZD6738 in a radiation induced model of lung fibrosis. Approximately 10% of NSCLC patients have mutations in PTEN. This research therefore elucidates PTEN loss as a therapeutic target for combined IR with pharmacological inhibition of ATR in NSCLC. Targeting ATR inhibition in PTEN-deficient NSCLC may serve to increase radiation sensitivity in an in vivo model thus highlighting its clinical relevance as a potential personalised medicine approach for patients with PTEN deficient NSCLC. Citation Format: Victoria Louise Dunne, Kelly Redmond, Caroline Coffey, Karl Butterworth, Kevin Prise, Gerard Hanna. Sensitivity of PTEN deficient non-small cell lung cancer to ionising radiation through inhibition of ataxia terangiectasia related 3 kinase (ATR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 835. doi:10.1158/1538-7445.AM2017-835
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