Abstract 5430: Inhibition of de novo and salvage pathways for dNTP synthesis enhances sensitivity to ionizing radiation in pancreatic neuroendocrine tumor cells
Authors: Castle Jennifer , McCorkle J. Robert , Johnson Jeremy , Chauhan Aman , Ivy Percy , Arnold Susanne , Carson William , Evers B. Mark , Rychahou Piotr , Kolesar Jill 
Published: June 2022

Introduction: The treatment of gastroenteropancreatic neuroendocrine tumors (GEP-NET) with ionizing radiation (IR) is a promising treatment modality for a disease that carries a poor prognosis. However, these tumors confer high levels of radioresistance based on their ability to repair DNA damaged by IR. Deoxynucleoside triphosphates (dNTPs) are needed for DNA repair and are produced by two pathways: de novo and salvage, which depend on ribonucleotide reductase (RNR) and deoxycytidine kinase (dCK), respectively. Ataxia telangiectasia and Rad3-related protein (ATR) activates dCK and can be targeted with inhibitors. We hypothesized that blocking both pathways for dNTP synthesis with an ATR inhibitor (ATRi) and the RNR inhibitor (RNRi), triapine, will sensitize GEP-NETs to IR in vitro.

Methods: (i) The CellTiter-Glo (Promega) luminescent cell viability assay established drug sensitivities of two pancreatic NET cell lines (BON and QGP-1) for triapine and three ATRi (AZD6738, VX-970, BAY1895344). Assays were performed with at least three replicates. Data were fit using a four-parameter log-logistic model, and IC50 values were calculated with R statistical software. (ii) Loewe synergy models were used to calculate the two-drug combination effects in both cell lines with synergy scores greater than 0 indicating synergistic effects and scores less than 0 representative of antagonistic effects. (iii) Clonogenic assays were performed and analyzed via colorimetry with sulforhodamine B to assess the effect of each drug alone and in combination with IR. (iv) Immunoblots were used to assess ATR activation following IR, RNRi, and ATRi treatments.

Results: (i) Cell viability assays showed lower IC50 values in the BON cell line compared to the QGP-1 cell line (BON cell IC50 : triapine 2.6µM ± 0.6µM, AZD6738 1.0µM ± 0.2µM, VX-970 204nM ± 20nM, BAY1895344 139nM ± 15nM; IC50 in QGP-1 cells: triapine 6.1µM ± 2.7µM, AZD6738 7.9µM ± 2.9µM, VX-970 1.7µM ± 0.5µM, BAY1895344 1.1µM ± 2.8µM). (ii) Loewe synergy models estimated the combination of BAY1895344 and triapine to be synergistic with scores greater than 0 in both cell lines (BON 9.89, p-value 4.07e-11; QGP-1 15.91, p-value 1.96e-7). (iii) Clonogenic assays showed an increase in efficacy of BAY1895344 when combined with IR in both BON (IC50 s: 150nM ± 8nM without radiation versus 18nM ± 35nM with 2Gy) and QGP-1 cells (IC50 values: 277nM ± 48nM without radiation versus 93nM ± 4nM with 2Gy). (iv) Western blot analysis showed strong activation of the ATR pathway with IR and triapine alone with subsequent inhibition by an ATRi.

Conclusion: Our findings show that treatment of GEP-NET cell lines with inhibitors that block the de novo and salvage pathways for dNTP production markedly sensitize these cells to subsequent IR. The combination of IR with both an ATRi and an RNRi has the potential to be an effective treatment modality for GEP-NETs.

Citation Format: Jennifer Castle, J. Robert McCorkle, Jeremy Johnson, Aman Chauhan, Percy Ivy, Susanne Arnold, William Carson, B. Mark Evers, Piotr Rychahou, Jill Kolesar. Inhibition of de novo and salvage pathways for dNTP synthesis enhances sensitivity to ionizing radiation in pancreatic neuroendocrine tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5430.