ESTRO 2022 - Abstract Book

S371

Abstract book

ESTRO 2022

A.S. Köseer 1

1 National Center for Tumor Diseases (NCT), Dresden, Germany: German Cancer Research Center (DKFZ), Heidelberg; Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, and Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Dresden, Germany. , OncoRay – National Center for Radiation Research in Oncology , Dresden, Germany Purpose or Objective Head and neck squamous cell carcinoma (HNSCC) is the eight most common cancer in Europe. Patients with HNSCC receive primary radiochemotherapy (RCTx) and postoperative radiochemotherapy (PORT-C) as standard treatments. Patients show diverse responses due to tumor heterogeneity highlighting the importance of patient stratification. Tumor relapses can be attributed to cancer stem cells (CSCs). In HNSCC, CSCs are identified by several markers, including CD98hc and high aldehyde dehydrogenase (ALDH) activity contributed by ALDH1A1 and ALDH1A3 isoforms producing retinoic acid (RA) (1,2,3). CD98hc and CD98hc-related amino acid transporters (CD98hc-AATs) function as stress response and stemness regulators (4). Our previous study showed that CD98hc-associated signaling mechanisms play a critical role in HNSCC radioresistance. High CD98hc expression level is a prognostic marker of poor locoregional control in locally advanced HNSCC patients treated with PORT-C or primary RCTx (5,6). Our study aims to decipher the interplay between CD98hc-AATs and CSC phenotype. Materials and Methods To analyze the relationship between CD98hc-AATs and ALDH isoforms, we treated HNSCC cell lines with all-trans retinoic acid (ATRA). We performed siRNA-mediated knockdown of ALDH1A1 or ALDH1A3 isoforms and RA receptors RARA and RXRA to investigate the influence of ALDH activity on CD98hc-AATs. The expression levels of CD98hc-AATs and ALDH isoforms were measured by RT-qPCR and Western blot. Sphere-forming capacity of HNSCC cell lines were investigated upon depletion of CD98hc-AATs. TCGA data mining was used to correlate RARA/RXRA expression with patient outcome. Results Our data show that ATRA treatment significantly increased CD98hc and LAT1 expression levels and resulted in HNSCC radiosensitization in a dose-dependent manner. Knockdown of ALDH1A1 , ALDH1A3 , RARA or RXRA lead to CD98hc and LAT1 downregulation. Expression levels of RARA/RXRA correlate with HNSCC radiosensitivity and patient outcomes. We identified putative RARA/RXRA binding sites on SLC3A2 and SLC7A5 promoters. Downregulation of ALDH1A1 and ALDH1A3 upon LAT1 knockdown suggests an interplay between CD98hc-AATs and ALDH isoforms. Additionally, knockdown of LAT2 and xCT affects size and numbers of spheres, respectively, in Cal33 cells. Conclusion These results indicate that the expression of CD98hc-AATs are is associated with the regulation of HNSCC CSC properties, and are affected by ALDH activity. This interplay can be critical for tumor growth and radioresistance. Thus, evaluation of RARA and RXRA expression in samples of patients treated with PORT-C and RCTx will be performed to validate the clinical relevance of our findings. References: 1. Kurth I, et al., Oncotarget . 2015. R. Prevo 1 , M. Ranzani 2 , R. Puliyadi 3 , G. Rodriguez-Berriguete 1 , H. Robinson 2 , E. Rajendra 2 , V. Grinkevich 2 , M. Boursier 2 , A. Cicconi 2 , D. Piscitello 2 , A. Cerutti 2 , J. Majithiya 2 , R. Heald 2 , M. Stockley 2 , G. Smith 2 , G. Higgins 3 1 University of Oxford, CRUK RadNet Oxford, Department of Oncology, Oxford, United Kingdom; 2 Artios Pharma, Babraham Research Campus, Cambridge, United Kingdom; 3 University of Oxford, Department of Oncology, Oxford, United Kingdom Purpose or Objective DNA Polymerase theta (POLQ) is a DNA repair enzyme that has low or absent expression in most normal tissues but is frequently overexpressed in many cancer types, particularly in tumours with homologous recombination (HR) deficiency, which rely on POLQ for repair of replication-associated DNA double strand breaks. As a radiosensitisation target, POLQ inhibition is expected to be effective in a wide range of tumours irrespective of HR status. We previously showed that depleting POLQ through siRNA radiosensitises tumour but not normal cells (Higgins et al., 2010, Cancer Res . 70: 2984). Recently Artios developed novel and specific POLQ inhibitors (including ART558 and ART812) that showed synthetic lethality against BRCA and also Shieldin deficiency (Zatreaunu et al., 2021 Nat. Commun . 12:3636). The purpose of this work was to test these inhibitors for their ability to induce tumour specific radiosensitisation in vitro and in vivo. We also aimed to define the cellular characteristics that predispose to POLQi-mediated radiosensitisation. Materials and Methods Cell line panels for head & neck, colorectal and lung cancer were screened for radiosensitisation by POLQ inhibition in colony formation assays. Imaging of DNA damage foci and flow cytometry analysis of G2M arrest and BrdU incorporation were used to study the DNA damage response and cell cycle progression. Colorectal HCT116 subcutaneous xenografts were given daily doses of POLQ inhibitor combined with a fractionated regime of 10 x 2 Gy. 2. Krause M, et al., Adv Drug Deliv Rev . 2017. 3. Peitzsch C, et al., Cancers (Basel) . 2019. 4. Kahya U, et al., Cancers (Basel) . 2021. 5. Linge A, et al., Clin Cancer Res . 2016. 6. Digomann D, et al., Clin Cancer Res . 2019. OC-0428 Development of DNA polymerase theta inhibitors as tumour specific radiosensitisers