Abstract A047: Investigating NKAPL, a potential marker for platinum resistance, in high-grade serous ovarian cancer

Abstract Understanding mechanisms of acquired chemoresistance in high-grade serous ovarian cancer (HGSOC) is a critical unmet question, with potential to improve patient outcome. Previously, we found NKAPL (NF- KB activating protein-like) to be hypermethylated and reduced gene expression was seen in chemoresistant HGSOC tumors. We hypothesize that NKAPL methylation and expression may be an indicator of therapeutic resistance. We aim to confirm the correlation between NKAPL methylation and expression and establish its role as a predictor of therapeutic resistance in-vitro and in-vivo. We first characterized a panel of HGSOC cell lines for chemosensitivity, NKAPL methylation and gene expression. DNA methylation was established through quantitative methylation specific PCR in platinum-sensitive: OVCAR4, CAOV3, PEO1, PEO4 and UWB1.289-Parental and resistant cell lines: COV318, Kuramochi and UWB1.289-BRCA1. NKAPL expression was determined by RT-qPCR and protein expression was measured by Western blotting. To investigate the role of NKAPL in chemoresistance, plasmid overexpression was performed. An NKAPL overexpression plasmid was designed through VectorBuilder and transfection was optimized in HEK293T cells using Lipofectamine2000. Since NKAPL has been shown to intervene with the NOTCH signaling pathway previously, changes in expression of NOTCH target genes were determined through RT-qPCR. Gene expression of NKAPL was detected at low levels in multiple cell lines apart from the platinum-resistant COV318 cells which showed a high level of expression. DNA methylation of NKAPL was detected in all eight HGSOC cell lines at different levels with no significant difference between sensitive and resistant cell lines. A significant negative correlation between NKAPL methylation and expression was seen across the HGSOC cell line panel. Cell lines from the same patient before and after receiving platinum treatment (PEO1 & PEO4) showed a significant increase in NKAPL methylation. Western blotting is ongoing. Overexpression was optimized in HEK293T cells resulting in an average ∼396,700-fold (log2FC ≈ 18.6) increase in NKAPL expression across three technical replicates, indicating strong transcriptional upregulation. Gene expression results analysis of seven downstream NOTCH target genes is ongoing. Previous work showed the hypermethylation of the NKAPL gene to correspond with decreased gene expression potentially indicating poor prognosis and platinum-resistance. We confirmed the negative correlation between gene methylation and expression in a HGSOC cell line panel. The paired cell lines, PEO1 and PEO4, further supported the hypothesis of NKAPL methylation affecting platinum sensitivity. Further data analysis and investigation into the mechanism of NKAPL is needed. As a next step, NKAPL overexpression will be performed in HGSOC cells to investigate effects on downstream targets and chemosensitivity. After completion of the in-vitro work NKAPL methylation and expression will be determined in a larger cohort of HGSOC patient samples. Citation Format: Lea Schäfer, Romina Silva, Gavin Stewart, Sharon O'Toole, Antoinette S. Perry. Investigating NKAPL, a potential marker for platinum resistance, in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr A047.