Abstract 4187: Enhancing doxorubicin DNA-damaging therapy in neuroendocrine cancer via DNA-PK inhibition

Abstract Introduction: Gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) are neoplasms originating from the gastrointestinal track and pancreas. Unfortunately, most patients at the time of diagnosis have extensive metastatic disease and are not candidates for curative surgical resection of metastatic tumors. In such cases, chemotherapy can slow the progression of metastatic disease, but eventually tumors develop resistance to genotoxic agents through DNA repair pathways activation. Therefore, targeting DNA repair pathway can increase the tumor sensitivity to cancer therapies. In this study, we examined whether DNA-PK inhibition could sensitize pancreatic NETs to DNA damaging chemotherapy. Methods: DNA double-strand break damage was induced with topoisomerase II inhibitor doxorubicin. DNA-PK activity was inhibited with peposertib. Clonogenic and proliferation assays were used to evaluate the efficacy of peposertib in combination with doxorubicin in BON and QGP-1 neuroendocrine cell lines. DSB-induced DNA repair was visualized with confocal laser scanning microscopy using γ-H2AX immunofluorescence. Induction of apoptosis in chemotherapy combination groups was visualized with confocal laser scanning microscopy using cleaved caspase-3 immunofluorescence and with western blot using cleaved PARP bands. To further validate the role of DNA-PK in the response to treatments, DNA-PK knockdown cell lines were established, and confocal microscopy and western blotting were used to assess DNA damage and apoptosis. Data was analyzed using GraphPad Prism and expressed as a mean ± standard deviation. Results: DNA-PK inhibition resulted in enhancement of doxorubicin cytotoxic therapy (BON IC50 DOX = 194.73± 26.52 nM, DOX+Pep = 89.87± 17.6 nM; QGP IC50 DOX = 108.37± 23.97 nM, DOX+Pepo = 30.27± 4.80 nM) in both BON and QGP-1 cell lines. Immunofluorescence analysis detected γH2AX foci increase in the doxorubicin and peposertib combination groups. DNA-PK inhibition in combination with doxorubicin treatment significantly increased caspase-3 cleavage and PARP cleavage in BON and QGP-1 cell lines. Similar trends in DNA damage and apoptosis were observed across both drug combination and knockdown groups. Conclusions: High DNA repair activity is commonly associated with cancer drug resistance. The combination of DNA-PK inhibition with DNA-damaging therapies impaired DNA damage response and enhanced neuroendocrine cancer cells apoptosis. These findings demonstrate that selective DNA-PK inhibition provides a potent therapeutic strategy for disruption of non-homologous end joining DNA double-strand break repair and may offer a novel therapeutic approach in advanced NET chemotherapy. Citation Format: Subin Kim, Allisa Pack, Nathan L. Vanderford, Courtney M. Townsend, B. Mark Evers, Piotr Rychahou. Enhancing doxorubicin DNA-damaging therapy in neuroendocrine cancer via DNA-PK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4187.