Abstract Background: Pancreatic cancer (PCa) is a major cause of cancer related mortality in United States with < 6% survival rate. It is an aggressive and devastating disease, which is characterized by poor prognosis, invasiveness, rapid progression, profound resistance to drug treatment and recurrance after surgery. Presently, single agent based chemotherapy (e.g. Gemcitabine) is the major treatment for metastatic adenocarcinoma of pancreas but it has a tumor response rate of below 10%. Moreover combination therapy of gemcitabine and erlotinib only marginally improved survival rate. Hence, there is a dire need to identify novel ways to inhibit pancreatic cancer growth. Methods: PCa cells (MiaPaCa-2 and PanC-1) were grown to 70-80% of confluency and treated with and without prolactin (PRL) and JAK2, STAT3 and ERK phosphorylation in presence and absence of antipsychotic compound were evaluated by western blot. Growth of PCa lines (MiaPaCa-2, PanC-1, BxPC-3, AsPC-1) and normal ductal epithelial cells (HPNE) was measured by hexosaminidase and clonogenicity, respectively. Pancosphere formation was used to identify effects on stem cells. Results: We have recently identified that the receptor for the pituitary hormone prolactin is overexpressed in pancreatic cancers, and in pancreatic cancer cell lines. When prolactin (PRL) binds its cognate receptor (PRLR), it induces various downstream events including the JAK-STAT and ERK MAPK pathways. In pancreatic cancer cell lines, we observe that PRL treatment induced dose- and time-dependent JAK2, STAT3, and ERK1/2 phosphorylation. Furthermore, there was an increase in the expression of cancer stem cell (CSC) markers DCLK1 (doublecortin calmodulin like kinase 1) and CD44. In addition, PRL-induced pancosphere formation further suggesting that PRL affects stem cells. Based on these data, we conclude that PRL signaling enhances stemness in pancreatic cancers, and therefore we decided to target it for therapeutic intervention. For this, we developed a homology model for the C-terminal intracellular region of the receptor and performed a virtual screening in silico with FDA approved drugs. One compound, a first generation antipsychotic drug diphenylbutylpiperidine, also called Penfluridol was found to interact with the region of the receptor that also binds site for JAK2. The compound has a long half-life, and is used in the treatment of chronic schizophrenia and similar psychotic disorders. We have further determined that Penfluridol inhibits PRL-induced STAT3 and ERK phosphorylation. In addition, the compound reduced proliferation, colony formation, and spheroid formation. Moreover, it induced cells to undergo autophagy by activating LC3B and increasing expression of autophagy-related genes ATG5, 7 and 12. Conclusions: PRL signaling through its cognate PRLR receptor is critical for aggressive pancreatic cancer behavior, and therefore may be an effective therapeutic strategy. Citation Format: Prasad Dandawate, Gaurav Kaushik, Dharmalingam Subramaniam, Prabhu Ramamoorthy, Scott J. Weir, Roy A. Jensen, Shrikant Anant. Targetingprolactin signaling to suppress pancreatic cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1310.