Abstract P017: Circulating tumor DNA kinetics identify prodynorphin signaling as a target to radiosensitize non-small cell lung cancer
Authors: Wang Ziwei , Hui Angela B. , Alizadeh Ash A. , Diehn Maximilian , Moding Everett J. 
Published: January 2025

Abstract
Introduction: Definitive chemoradiation therapy (CRT) is essential for controlling locoregionally advanced non-small cell lung cancer (NSCLC); however, up to 30% of patients experience local recurrences within the radiation field. Analyzing favorable responders to treatment could enable identification of genetic alterations associated with radiosensitivity, but standard imaging cannot distinguish residual disease from inflammation and fibrosis during treatment to assess treatment response. We hypothesized that circulating tumor DNA (ctDNA) kinetics during CRT could identify genetic alterations associated with radiosensitivity that could be validated in preclinical models and harnessed to develop new combination therapies with radiotherapy (RT). Methods: We applied cancer personalized profiling by deep sequencing (CAPP-Seq) ctDNA analysis to pre-CRT and mid-treatment (mid-CRT) plasma samples from 61 patients with Stage II-III NSCLC. We identified ctDNA rapid responders as the 10 patients with the largest decrease in ctDNA concentration mid-CRT without local progression and determined the prevalence of genetic alterations in rapid responders versus slow responders using Fisher’s exact tests corrected for multiple hypothesis testing. To investigate the therapeutic potential of targeting PDYN during RT, we performed clonogenic assays on isogeneic PDYN CRISPR-Cas9 knockout and wildtype H1299 and SW1573 human NSCLC cell lines and H1299 cells treated with prodynorphin-neutralizing antibodies. Results: Mid-CRT log-fold change in ctDNA concentration was significantly associated with progression-free survival (P=0.02) in Stage II-III NSCLC. Mutations in PDYN, which encodes the opioid peptide precursor protein prodynorphin, were observed in 30% of ctDNA rapid responders and 0% of ctDNA slow responders (adjusted P=0.04). In NSCLC patients from TCGA treated with RT, the cumulative incidence of local failure was significantly lower in tumors with PDYN mutations (0% vs. 17% at 3 years, P&lt;0.001). PDYN knockout (PDYNNULL) radiosensitized human NSCLC cancer cells by clonogenic assay, and extracellular administration of prodynorphin or its cleavage product dynophin A partially restored radioresistance in PDYNNULL cells. Prodynorphin cleavage products have been reported to signal through opioid receptors and to antagonize N-methyl-D-aspartate receptor (NMDAR) signaling. Although treatment of wildtype H1299 cells with opioid receptor antagonists did not affect radiosensitivity, the NMDAR antagonist MK-801 partially restored RT resistance in PDYNNULL cells. Finally, prodynorphin-neutralizing antibodies significantly reduced clonogenic survival of human NSCLC cells. Conclusions: ctDNA kinetics enable the identification of patients responding favorably to treatment and putative targets to enhance the efficacy of RT. Targeting prodynorphin may enhance the radiosensitivity of NSCLC by increasing NMDAR signaling.
Citation Format: Ziwei Wang, Angela B. Hui, Ash A. Alizadeh, Maximilian Diehn, Everett J. Moding.Circulating tumor DNA kinetics identify prodynorphin signaling as a target to radiosensitize non-small cell lung cancer.[abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P017