Abstract A041: Neil2 is a potential theratputic targert in pancreatic cancer

Abstract Background: Our previous work demonstrated that oncogenic KRAS mutations in pancreatic cancer induce aberrant transcription-replication conflicts (TRCs). TRCs arise when DNA replication forks collide with transcription complexes due to loss of spatial and temporal coordination between these processes. TRC resolution mechanisms are and adaptive dependency to KRAS-mediated oncogenic stress. Here we identify a key pathway that regulates TRCs in PDAC and performs mechanistic studies towards clinical translation. Methods: TRC factors were identified through an siRNA screen against 200 DNA repair genes in Panc1 cells using a high-throughput proximity ligation assay (HiPLA). Knock-out and complementation experiments were performed using CRISPR-Cas9 systems. Cell proliferation was measured using incucyte. Replication stress and DNA damage were measured using multiple orthogonal methods. Protein-protein interactions were assessed by co-immunoprecipitation mass spectrometry and validated by western blot. Chromatin occupancy of key factors was determined using ChIP-seq. Results: Through high-throughput proximity ligation assay (HiPLA) screening of 200 known DDR genes, we identified the BER pathway as a key mechanism for TRC resolution. Among these, Neil2 - a bifunctional glycosylase in the base excision repair (BER) pathway - emerged as one of the highest-scoring BER factors; and the only BER glycosylase involved—likely due to its transcription-coupled repair activity. Using CRISPR/Cas9-mediated knockdown, we found that loss of Neil2 led to elevated double-stranded DNA breaks (DSBs) and replication stress. Notably, this significantly impaired cell cycle progression. colony formation, and proliferation in pancreatic cancer cell lines, with minimal effects on normal pancreatic epithelial cells, highlighting Neil2’s selective oncogenic function. To elucidate the underlying mechanisms, we combined proteomic approaches—including co-immunoprecipitation and iPOND-mass spectrometry—with transcriptomic and chromatin profiling with ChIP-seq and RNAseq. Our data revealed that Neil2 plays a critical role in transcription termination, and that its dysregulation promotes RNA Polymerase II readthrough, likely contributing to R-loop accumulation, downstream TRCs, and subsequent genotoxicity. Intriguingly, deletion of Neil2’s DNA binding domain—which abolishes its DNA lesion binding capability—did not compromise its regulation of TRCs or support of cancer cell viability. This suggests that Neil2’s oncogenic role in pancreatic cancer may be DNA-binding independent. Conclusion: We uncovered a noncanonical and essential function of Neil2 in TRC regulation and oncogenic maintenance in pancreatic cancer, and we propose Neil2 as a promising therapeutic target for selective intervention in this disease. Citation Format: Fan Meng, Mustafa Raoof. Neil2 is a potential theratputic targert in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A041.