Abstract PO-014: 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP): A promising radiochemotherapy for gastroenteropancreatic neuroendocrine tumors
Authors: Chow Zeta , Johnson Jeremy , Chauhan Aman , Isumi Tadahide , Anthony Lowell , Townsend Courtney M. , Evers B. Mark , Rychahou Piotr 
Published: April 2021

Abstract
Background: Peptide receptor radionuclide therapy (PRRT) is a novel radiotherapy approved for gastroenteropancreatic neuroendocrine tumors (GEP-NETs); however, PRRT has a relatively low treatment response rate of 20-30%. We hypothesize that addition of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), a ribonucleotide reductase (RnR) inhibitor that inhibits the rate-limiting enzyme of DNA synthesis and repair, can improve radiotherapy outcomes. The purpose of this study is to determine the therapeutic rationale of RnR-targeted therapy and evaluate 3-AP as a radiosensitizer in treating GEP-NET in vitro and in vivo. Methods: We utilized two human GEP-NET cell lines: BON, a pancreatic carcinoid tumor, and QGP-1, a somatostatinoma. (i) Cells were treated with 3-AP for 96h and cell proliferation assays were performed using a sulforhodamine B (SRB) kit. Next, cells were treated with 3-AP for 24, 48, 72, 96h and cell cycle analyses were performed with Beckman Coulter cell cycle kit and quantified by flow cytometry. Expression of cell cycle arrest markers (i.e., pCdk-1, cyclin D1) was assessed by immunoblotting. (ii) To determine radiosensitivity in vitro, cells were treated with 3-AP for 16h, followed by X-ray radiotherapy (XRT), and incubation in drug-free medium. Cell survival was assessed by colony formation assay and quantified by SRB kit at 14d post-XRT. Apoptosis was measured at 72 and 96h post-XRT, and expression of apoptotic proteins (i.e., cleaved PARP and cleaved CASPASE 3) was examined by immunoblotting. (iii) To determine radiosensitivity in vivo, we randomized 20 nude mice bearing QGP-1 xenografts into four groups: control; 3-AP (10mg/Kg); XRT (2 Gy); and combination (XRT immediately followed by 3-AP); each mouse received 5 consecutive treatments. Results: (i) A dose of 2500nM 3-AP arrested cells before entering G2/M phase, significantly reducing percentage of G2/M cells from 8.4% to 0.5% at 72h. Concurrently, we observed increased expression of checkpoint proteins (pRb, pCdk1, pCdk2, pCdc2) and decreased expression of cell cycle inhibitory proteins (cyclin D1, p21, p27 Kip). (ii) Exposure of QGP-1 and BON cells to 3-AP prior to XRT resulted in a synergistic decrease (i.e., 85% and 90%, respectively) in clonogenicity. Furthermore, the combination of 3-AP and XRT increased apoptosis 5-fold and cleaved PARP expression in BON cells. (iii) A significant decrease in tumor size was noted in mice with XRT alone compared to control (146mm2 vs. 280 mm2, respectively); combination treatment (3-AP + XRT) demonstrated a further 48% decrease in tumor size compared to XRT alone. There was no significant decrease in tumor size with 3-AP treatment alone. Conclusion: 3-AP treatment prior to XRT therapy resulted in significant increase in apoptosis in vitro and decrease in tumor size in vivo, suggesting synergistic cytotoxicity and radiosensitivity. Selective RNR inhibition by 3-AP provides a potent therapeutic strategy as a radiosensitizer and offers a novel approach in treating advanced GEP-NETs.
Citation Format: Zeta Chow, Jeremy Johnson, Aman Chauhan, Tadahide Isumi, Lowell Anthony, Courtney M. Townsend, B. Mark Evers, Piotr Rychahou. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP): A promising radiochemotherapy for gastroenteropancreatic neuroendocrine tumors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-014.