Abstract A050: NEIL2 regulates RNA polymerase II dynamics to suppress transcription–replication conflicts in pancreatic cancer
Fan Meng, Tony Fernandez, Changfei Luan, Marc Attiyeh, Qianhua Feng, Alexander Lee, Haihua Feng, Tiane Li, Tingting Zhou, Yun Li, Rockne Russel, Terence Williams, David Dawson, Binghui Shen, Jen Jen Yeh, Yilun Liu, Maria Ninova, Mustafa RaoofAbstract
Background:
Transcription–replication conflicts (TRCs), arising from spatiotemporal interference between transcription and replication machinery, are major sources of genome instability in cell. In pancreatic ductal adenocarcinoma (PDAC), oncogenic KRAS promotes TRC formation, and pharmacological exacerbation of endogenous TRCs exhibits antitumor activity. However, the clinical significance and regulatory mechanisms of TRCs in PDAC remain poorly understood. Here, we identify Nei-like DNA glycosylase 2 (NEIL2), a bifunctional DNA glycosylase involved in transcription-coupled base excision repair (TC-BER), as an essential suppressor of TRCs through regulation of RNA polymerase II (RNAPII) dynamics in PADC cells.
Methods:
TRCs were quantified in human PDAC tissue microarrays (TMAs) and pancreatic cell models using proximity ligation assays (PLA). Mechanistic studies were performed in CRISPR/Cas9-mediated NEIL2 knockout (N2KO) and siRNA-mediated knockdown models using immunostaining, DNA fiber assays, flow cytometry, real-time cell analysis (RTCA), genomic analyses, R-loop profiling, RNA polymerase II (RNAPII) chromatin immunoprecipitation sequencing (ChIP-seq), and co-immunoprecipitation (Co-IP)-based proteomics.
Results:
Using PLA-based TRC detection in patient samples, we provide direct evidence that TRCs are enriched in tumor cells relative to stroma and adjacent normal ductal epithelium. TRCs positively correlate with tumor size, and grade. We orthogonally validate the critical role of NEIL2 in regulating TRCs using CRISPR-mediated gene knockout. Mechanistically, we found that NEIL2-regulates TRCs independent of its DNA binding domain which is critical for aberrant base detection in BER. Loss of NEIL2 results in aberrant RNAPII occupancy at both the transcription start (TSS) and end sites (TES) through unique mechanisms, collectively promoting TRC formation and DNA damage. Functionally, Loss of NEIL2 selectively impairs PDAC cell viability and proliferation in both 2D and 3D in-vitro models but has no effect on normal cells. Collectively, our study defines an essential non-canonical and adaptive function of NEIL2 in mitigating TRCs in PDAC.
Conclusions:
Our findings establish TRCs as clinically relevant features of PDAC and identify NEIL2 as a critical regulator of TRC suppression and RNAPII dynamics. These results uncover a novel oncogenic role for NEIL2 and support therapeutic targeting of NEIL2 and TRCs in PDAC.
Citation Format:
Fan Meng, Tony Fernandez, Changfei Luan, Marc Attiyeh, Qianhua Feng, Alexander Lee, Haihua Feng, Tiane Li, Tingting Zhou, Yun Li, Rockne Russel, Terence Williams, David Dawson, Binghui Shen, Jen Jen Yeh, Yilun Liu, Maria Ninova, Mustafa Raoof. NEIL2 regulates RNA polymerase II dynamics to suppress transcription–replication conflicts in pancreatic cancer [abstract]. In: Proceedings of the AACR Conference on Pancreatic Cancer: New Frontiers in Biology and Therapeutic Development; 2026 Sep 25-28; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(18_Suppl_2):Abstract nr A050.