ABSTRACT Introduction T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive cancer arising from T-cell progenitors. Although current treatments, including chemotherapy and glucocorticoids, have significantly improved survival, T-ALL remains a fatal disease and new treatment options are needed. Since more than 60% of T-ALL cases bear oncogenic NOTCH1 mutations, inhibitors of NOTCH1 signalling are being actively investigated for the treatment of T-ALL. However, they have limited efficacy in tumours due to dose-limiting toxicities. Recent evidence indicates that γ-secretase activity not only occurs at the cell surface, but also in the acidic environment of lysosomes and endosomes. In addition, endosomes have been shown to play a key role in the NOTCH signal relay. Therefore, we hypothesised that chloroquine (CQ), a clinically used antimalarial drug that inhibits lysosomal function and autophagy, may inhibit T-ALL growth and survival. Material and methods T-ALL cell lines were treated with γ-secretase inhibitor (GSI), CQ or a combination treatment. Cell viability was determined by trypan blue exclusion. Propidium Iodide (PI) staining was combined with Annexin V staining or Edu incorporation to measure apoptosis and perform cell cycle analysis, respectively. Flow cytometry was used to determine cytoplasmic ROS levels and yH2AX expression. To address the effect of CQ on NOTCH1 signalling, cleaved NOTCH1 was assessed by immunoblotting, NOTCH1 target gene expression by qPCR and NOTCH1 expression at the cell surface by flow cytometry. In vivo T-ALL xenografts were grown and treated with different doses of GSI, CQ or a combination treatment. Tumour growth and gastro-intestinal toxicity were assessed. Results and discussions CQ decreases T-ALL cell viability and proliferation and reduces GSI sensitive T-ALL xenograft growth, which was not observed in GSI-resistant T-ALL cell lines or xenografts. Mechanistically, CQ impairs the redox balance and induces DNA damage with a subsequent DNA damage response. In addition, we observed that CQ interferes with intracellular trafficking and processing of oncogenic NOTCH1. Interestingly, we show for the first time that the addition of CQ to γ-secretase inhibition has an additive therapeutic effect on T-ALL and reduces the concentration of GSI required to obtain tumour control. Conclusion Overall, our results suggest that CQ may be a promising alternative as a repurposed drug in the treatment of T-ALL, either as a single treatment or in combination with NOTCH1 inhibition, increasing the therapeutic ratio.

S107 ______________________________________________________________________________________________________not able to form new tumors and have high expression of CD127 on their T cells, a marker for immunological memory.This new treatment will be further investigated in a Phase I study for patients with an oligometastatic solid tumor (NCT02086721).

Purpose/Objective:Conformal radiotherapy involves irradiation of large volume of normal tissue with low and medium doses, which biological relevance is not clear yet.Here we aimed to compare effects associated with local body irradiation during treatment of two differently located solid tumors: head and neck cancer (HNC) and prostate cancer.Serum proteome features were used as a surrogate for general reaction of whole patient's organism to radiation.Materials and Methods: Here we compared effects associated with local body irradiation during RT of two solid tumors: head &neck cancer (HNC) and prostate cancer (PC).About 120 HNC and 120 PC patients were enrolled into the study.All patients were subjected to radical IMRT with maximum GTV doses in the range 50-73.8Gy and 74-76 Gy, respectively.Acute mucosal toxicity and gastrointestinal/genitourinary toxicity was assessed.Three consecutive blood samples were collected before, during and after RT.The endogenous serum peptidome was profiled using MALDI mass spectrometry.Furthermore, serum proteins were analyzed using label-free LC-MS/MS 'shotgun' approach in complete sets of samples representing 20 patients from each cancer group.Results: In case of HNC patients radiation-induced changes in serum peptidome accumulated constantly during the treatment and their highest level was detected soon after the end of RT (~60% of components changed their abundance at significance level p<0.0001 between pre-and postexposure samples).Moreover, changes in serum peptidome correlated significantly with intensity of acute mucosal reactions and volume of normal tissue irradiated with low-tomedium doses.In contrast, in case of PC patients majority of radiation-induced changes were detected 2-3 weeks after start of RT and their extend was less significant (~30% of components changed their abundance at significance level p<0.05).Furthermore, correlations between changes in serum peptidome and escalation of radiation toxicity or volume of tissues irradiated at low/medium doses were not detected.About 200 serum proteins were identified and quantified: ~10% and ~2% of identified proteins changed their abundance between pre-and post-exposure samples from HNC and PC patients, respectively.Conclusions: The effects of local irradiation were documented at the level of serum proteome, which is an apparent indicator of the patient's whole body response.Significant differences were noted between patients irradiated because of HNC and PC in spite of the fact that similar volumes of tissues were irradiated with similar doses in both groups.This observation presumably reflected differences in radiosensitivity and corresponding early radiation toxicity in normal tissues/organs adjacent to cancer target and exposed to low-to-medium doses during IMRT.