e16329 Background: Chemoresistance is the main reason for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Thus, there is an urgent need to identify new targets and compounds to overcome chemotherapeutic resistance. Methods: The high-throughput virtual drug screening, molecular docking analysis and organoid biobank-based drug screening were performed to identify the candidates to efficiently overcome chemotherapy resistance. In vitro and in vivo PDOs, PDXs and KPC GEMM models were used to evaluate the effects of the compounds. Spatial single cell RNA-sequence, spatial metabolomics and proteomics were employed to elucidate the mechanism. Additionally, an ongoing open-label, randomized, single-arm, single-center phase 2 investigator-initiated clinical trial (NCT05947825) is investigating the efficacy and safety of the candidate in sensitizing gemcitabine plus nab-paclitaxel (GnP) in previously untreated advanced PDAC patients. Objective tumor responses were assessed according to RECIST1.1 criteria and any adverse events were graded using CTCAE 5.0. The primary endpoint was progression-free survival (PFS), and secondary endpoints included objective response rate (ORR), disease control rate (DCR), overall survival (OS), and treatment-emergent adverse events (TEAEs). Results: High-throughput drug screening of chemotherapy-resistant PDOs identified Sitagliptin that could significantly induce the degradation of RRM1/RRM2 in a ubiquitin proteasome-dependent manner and further result in unrestrained pyrimidine biosynthesis and alleviation of replication stress. In vitro and in vivo assays confirmed that Sitagliptin could synergistically enhance the ability of GnP regimen to suppress PDAC progression. In the phase 2 study (NCT05947825), previously untreated advanced PDAC received Sitagliptin, 100mg/qd and gemcitabine (G, 1000mg/m2 )plus nab-paclitaxel (nP, 125 mg/m2 )on Days 1 and 8 in a 21-day cycle. Till February 1, 2024, 12 patients with ECOG 0 to 2 have been enrolled in the study. The primary endpoint has not been reached. Among the 8 evaluable patients, 3 showed partial responses and 5 had stable diseases. The objective response rate (ORR) and disease control rate (DCR) were 37.5% and 100% respectively. There were no instances of treatment-related death in this study. The most common treatment-emergent adverse events (TEAEs) were decreased white blood cells and fatigue. One patient experience skin rash. Conclusions: Sitagliptin could effectively improve efficacy of GnP regimen by suppressing RRM1/RRM2-mediated pyrimidine metabolism. Sitagliptin plus GnP regimen is a feasible regimen with a manageable safety profile. Sitagliptin in combination with GnP regimen shows encouraging antitumor activity in PDAC, meriting continued development. Clinical trial information: NCT05947825 .