Therapeutic Targeting of Oncogene-induced Transcription-Replication Conflicts in Pancreatic Ductal Adenocarcinoma

Purpose Transcription-replication conflicts (TRCs) are a major source of endogenous replication stress in cancer. We previously discovered that pancreatic ductal adenocarcinoma (PDAC) demonstrates uniquely high levels of TRCs compared to other common solid tumors. Here, we characterize the mechanism of action, oncogene-dependency, PDAC subtype-specificity, and preclinical activity of a TRC-targeting small molecule – AOH1996 – in a spectrum of PDAC models. We also provide first clinical evidence of the activity of AOH1996 in a PDAC patient. Experimental Design The oncogene-dependent toxicity of AOH1996 was examined in KRAS(G12D) inducible systems. Next, the effect of AOH1996 was evaluated on replication fork progression, TRCs, DNA damage response, cell cycle progression, and apoptosis in PDAC cell lines. PDAC subtype-specific responses were evaluated in organoid cultures, and in vivo efficacy was evaluated in murine and patient-derived xenografts. Efficacy in a PDAC patient was evaluated by radiographic response assessment and progression-free survival. Results AOH1996 demonstrated dose-dependent cytotoxicity that was exquisitely dependent on oncogenic KRAS(G12D) induction. Cytotoxicity of AOH1996 was evident in several human and murine PDAC cell lines (Average IC50 across cell lines 0.72μM). Mechanistically, AOH1996 inhibited replication fork progression and promoted TRCs through enhanced interaction between RNA Polymerase II and Proliferating cell nuclear antigen which resulted in transcription-dependent DNA damage and global transcription shutdown. AOH1996 demonstrated activity in all organoid lines tested with varying potency (IC50 406nM – 2μM). Gene expression analysis demonstrated that organoids with replication stress high or very strongly basal signature were most vulnerable to AOH1996. In PDAC mouse model studies, AOH1996 reduced tumor growth rate, enhanced tumor-selective DNA damage and prolonged survival (Median 14 days vs. 21 days, P=0.04) without observable toxicity. The first patient with chemotherapy-refractory PDAC who was treated with AOH1996 monotherapy demonstrated early evidence of efficacy (49% shrinkage of the two hepatic metastases with stabilization of disease at other sites). Conclusions Therapeutic targeting of TRCs using small molecule inhibition is safe and effective in preclinical models. Pre-clinical data along with proof-of-concept activity in a patient with chemotherapy-refractory PDAC provides rationale for further clinical development of TRC targeting strategies. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial NCT05227326 ### Funding Statement As listed in the manuscript title page ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: City of Hope Institutional Review Board I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors