Exploiting Transcription-Replication Collisions to Treat Pancreatic Cancer: AOH1996 as a Precision Vulnerability Drug

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest forms of cancer, marked by late diagnosis, aggressive progression, and a dismal five-year survival rate of less than 11%. Despite advances in surgery, chemotherapy, and radiation, most patients present with unresectable or metastatic disease, and therapeutic responses are typically short-lived. The tumor’s dense stroma, complex mutational landscape, and intrinsic resistance to DNA-damaging agents make PDAC uniquely difficult to treat. While the discovery of BRCA-related homologous recombination (HR) deficiencies has opened the door to PARP inhibitors in a minority of patients, the overwhelming majority lack targetable genomic vulnerabilities. In this therapeutic void, the scientific community continues to search for new mechanisms that could be exploited to improve patient outcomes. One of the more recent conceptual breakthroughs in oncology has been the recognition that replication stress—particularly stress caused by transcription-replication conflicts (TRCs)—is a critical driver of genomic instability in many cancers, including PDAC. These TRCs arise when the DNA replication machinery collides with active transcription complexes, often triggered by high levels of oncogene activity. In PDAC, the near-universal presence of KRAS mutations creates a hyperactive cellular environment that overloads both transcription and replication pathways. These opposing forces on the DNA strand generate physical blockages, strand breaks, and stalled forks—eventually cascading into DNA damage and chromosomal instability. Yet, paradoxically, cancer cells adapt to this chaos and continue to proliferate, often by rewiring their DNA repair and cell cycle checkpoints.

This unique reliance on TRC tolerance mechanisms in PDAC suggested an unexploited vulnerability. If researchers could selectively disrupt the cancer cell’s ability to cope with transcription-induced replication stress—without harming normal cells—this could represent a new therapeutic frontier. In a new research paper published in Gastroenterology Journal and led by Professor Linda Malkas and Assistant Professor Mustafa Raoof and colleagues at City of Hope in California, the researchers focused on AOH1996, a novel small-molecule inhibitor of proliferating cell nuclear antigen (PCNA), a key coordinator of DNA replication and repair. What sets this study apart is its ambition to bridge molecular insight with translational relevance. Rather than merely describing a new mechanism, the researchers rigorously tested AOH1996 in diverse models—ranging from engineered cell lines to patient-derived organoids and murine xenografts. They also included early human clinical evidence, making it one of the few studies to trace a complete arc from mechanistic rationale to clinical applicability. The central hypothesis was elegant yet bold: by amplifying TRCs beyond a tolerable threshold through PCNA inhibition, AOH1996 would selectively collapse the cancer cell’s replication program—inducing lethal DNA damage while sparing healthy tissues. In doing so, the team hoped to carve a path forward for treating a disease that has long resisted meaningful progress.

To uncover whether targeting transcription-replication conflicts could genuinely weaken pancreatic cancer cells, the researchers began by testing AOH1996 in engineered human pancreatic cells with inducible KRAS mutations. When KRAS was activated, these cells experienced heightened replication stress, mirroring what is typically seen in pancreatic tumors. Treatment with AOH1996 in this stressed state triggered a marked increase in DNA damage, evident through elevated levels of γH2AX, a well-established marker for DNA breaks. In contrast, the same cells without KRAS activation showed little to no damage, underscoring the selective toxicity of AOH1996 toward oncogene-driven stress.

This early indication of specificity prompted the team to widen their analysis. Using real-time proliferation assays and dose-response studies, they observed that AOH1996 reduced viability in a range of pancreatic cancer cell lines in a KRAS-dependent manner. Intriguingly, the compound wasn’t limited to KRAS-mutant cells; those with alternate oncogenic drivers like BRAF deletions also responded, suggesting that the key vulnerability lay in the level of replication stress, not necessarily the exact mutation. Across these experiments, the half-maximal inhibitory concentration (IC50) values varied but consistently fell in the micromolar to sub-micromolar range, a promising profile for a candidate drug.

The authors performed DNA fiber assays to visualize the replication machinery in real time. AOH1996 caused replication forks to stall without any compensatory increase in new origin firing, which meant that the drug was not only halting progress but preventing backup systems from kicking in. This stalling was accompanied by cell cycle arrest and a dose-dependent rise in apoptosis, confirmed through TUNEL staining and flow cytometry. Essentially, the drug pushed cells to a point of no return—unable to complete replication, unable to divide, and eventually, forced into death. But the most illuminating set of findings emerged from proximity ligation assays and transcription quantification. AOH1996 increased the physical interaction between RNA Polymerase II and PCNA, intensifying transcription-replication collisions. This wasn’t just theoretical: the elevated interaction directly correlated with DNA damage, and interestingly, blocking transcription with DRB almost completely blunted the effect of AOH1996. In short, the damage required ongoing transcription—validating that TRCs were the true Achilles’ heel. Further, AOH1996 led to the degradation of RNA polymerases and reduced global transcription, silencing the very engine that sustains tumor cell proliferation.

What makes the implications even more powerful is that AOH1996 doesn’t require BRCA mutations, mismatch repair deficiency, or any of the usual biomarkers that stratify eligibility for existing targeted therapies. Instead, it capitalizes on the inherent chaos driven by oncogenes like KRAS and MYC—chaos that until now, cancer cells have skillfully managed to exploit. AOH1996 doesn’t just increase replication stress; it weaponizes it. By forcing transcription machinery into dangerous collisions with replication forks, the drug overwhelms the cell’s ability to patch the damage, leading to selective, irreversible breakdown. This specificity—attacking only cells already burdened by high transcriptional and replicative tension—has significant clinical implications. In both organoid and animal models, AOH1996 showed minimal toxicity, offering a therapeutic index that is rarely seen in such aggressive cancers. That it was able to shrink tumors even in patients with advanced, chemotherapy-resistant disease hints at its potential to serve as a backbone for future combination therapies, or even as a standalone option in select biomarker-positive populations. Perhaps most compelling is how the study reintroduces functional transcriptional stress as a tractable biomarker for therapeutic prediction. Instead of relying solely on static DNA mutations, we may be moving toward dynamic, transcriptionally-driven signatures that tell us how cells behave—not just what mutations they carry. If validated in larger cohorts, the “replication stress high” signature could guide precision treatment strategies, particularly in subtypes like basal PDAC, which currently carry the worst prognosis and fewest options.