ESTRO 2020 Abstract Book

S248 ESTRO 2020

eliminate all malignant cells with clonogenic potential cellular plasticity and intratumor heterogeneity reducing local tumor control rates and patient survival. Within the present study, we investigated cellular plasticity and epigenetic modulation in head and neck squamous cell carcinoma (HNSCC) upon conventional irradiation (IR) with 220 kV photons and 150 MeV protons. Material and Methods We used 3D colony-formation assay to determine radiosensitivity, γH2AX assay to evaluate DNA damage repair capacity and tumor sphere formation to investigate the self-renewal capacity of several HNSCC cell lines. IR- induced lineage switch from non-CSCs into aldehyde dehydrogenase (ALDH) positive CSCs was demonstrated using color-coded HNSCC cells. These radiobiological and stem cell read-outs were correlated with epigenetic remodeling and metabolic adaptations upon IR. Therefore, we used western blot analysis to illustrate differential histone methylation pattern, histone demethylase activity was determined using EpiQuik/Epigenase assay, and EPIC array to analyze global DNA methylation. Mass spectrometry was applied to analyze tricarboxylic cycle (TCA) metabolites important as co-factors for the regulation of enzymes regulating chromatin modifications. Using a chemical library, we screen > 150 epigenetic targeting compounds for their radiosensitizing, DNA damaging and CSC-targeting potential. The clinical potential of identified and validated chromatin modulators was investigated via gene expression analysis from tumor tissues of patients with locally advanced HNSCC treated with primary and postoperative radiochemotherapy (RCTx) and retrospectively correlated to loco-regional tumor control (LRC), overall survival and distant metastases. Results Our data demonstrated that IR is leading to an acquisition of stem-like features in HNSCC cell lines. We identified the histone H3K27 demethylase JMJD3/KDM6B and UTX/KDM6A as key regulator of IR-induced cellular plasticity and demonstrated radiosensitizing and CSC eliminating potential for the corresponding chemical inhibitor GSK-J1. Moreover, both genes are differential expressed in HNSCC patients and have the potential to discriminate a poor prognosis subgroup after RCTx. Conclusion Epigenetic biomarkers may have prognostic and therapeutic potential for patients with HNSCC treated with RT. Further functional studies are necessary to characterize the molecular mechanisms for a future clinical translation. OC-0449 Pre-clinical evaluation of combining radiation with Notch signalling inhibition in melanoma K. Thippu Jayaprakash 1,2 , M. Hussein 3 , A. Nisbet 3,4 , R. Shaffer 2 , A. Michael 1,2 , M. Ajaz 1,2 1 University of Surrey, Department of Clinical and Experimental Medicine- Faculty of Health and Medical Sciences, Guildford, United Kingdom ; 2 Royal Surrey County Hospital, Department of Oncology, Guildford, United Kingdom ; 3 Royal Surrey County Hospital, Department of Medical Physics, Guildford, United Kingdom ; 4 University of Surrey, Department of Physics- Faculty of Engineering and Physical Sciences, Guildford, United Kingdom Purpose or Objective Notch signalling plays a key role in cellular response to radiation. We previously demonstrated synergistic effect with a strategy of combining radiation and Notch signalling inhibition with a gamma secretase inhibitor, RO4929097,

and also showed impairment of cell migration in melanoma using in-vitro models. In this study we further investigate the effects of this promising strategy on cellular proliferation, quantified impairment of cell migration and explored potential biological mechanisms underpinning this approach using in-vitro melanoma models. Material and Methods Melanoma cells, A375, SKMEL28 and G361 were grown using standard tissue culture methods and were exposed to various combinations of radiation doses (1, 2, 4, and 8 Gy) and RO4929097 concentrations (1, 3, 10, 30 and 100 µM). Clonogenic and trans-well migration assays were performed to assess cellular proliferation and to quantify impairment of cell migration respectively. Melanospheres grown in serum-free media were exposed to this combination. Melanosphere size was regarded as the measure of ability of cells to adopt a stem cell phenotype to evaluate phenotype plasticity. Quantitative In-Cell Western assay was used to evaluate changes in gammaH2AX foci signal intensity as a measure of double- strand DNA damage response. GraphPad Prism was used for statistical analysis. Results Combining radiation with Notch signalling inhibition significantly reduced colony numbers in clonogenic assays (Two-way ANOVA, for A375 p<0.0001 for radiation effect and p=0.0015 for RO4929097 effect and for SKMEL28 p<0.0001 for both effects) (Figure 1, A & B) . Notch signalling inhibition not only impaired cell migration but also attenuated radiation induced enhancement in cell migration (Two-way ANOVA, for all three cells, A375, SKMEL28 and G361, p<0.0001 for RO4929097 effect, and p=0.62, p=0.49, p=0.51 for radiation effect respectively) (Figure 1, C & D) , confirming our earlier observations of impairment of cell migration with this approach.

There were statistically significant reductions in melanospheres size (Two-way ANOVA, for A375 p<0.0001 for both effects and for SKMEL28 p=0.77 for radiation effect and p<0.0001 for RO4929097 effect) while Notch signalling inhibition, at higher doses reduced the levels of radiation induced gammaH2AX foci signal intensity (Two- way ANOVA, A375 p=0.02, SKMEL28 p=0.005) (Figure 2) .