Abstract Background: The PI3-kinase/AKT/mTOR pathway is a frequently dysregulated pathway in cancer and of importance for tumor cell growth, differentiation and survival. This pathway may be activated in various cancers due to aberrant activation of receptor tyrosine kinases (RTKs). The PI3-kinase/AKTmediated activation of mTOR, stimulates cell proliferation and regulation of protein translation. In pancreatic cancer, PI3-kinase and AKT are constitutively activated. ROR1 is a type I transmembrane RTK belonging to one of the twenty families of RTKs. ROR1 is overexpressed and constitutively phosphorylated in pancreatic carcinoma. A unique anti-ROR1 mAb directed against the CRD (cysteine-rich domain) of the extracellular region of ROR1 was capable of inducing apoptosis as well as dephosphorylating the ROR1 molecule in CLL cells. Aim: To investigate the apoptotic effect of the anti-ROR1 CRD mAb and effects on the downstream signaling pathways PI3-kinase/AKT/mTOR using the ROR1+ pancreatic cancer cell line PaCa-2. Methods: The MTT assay and Annexin V/PI methods were used to detect apoptosis in a 24-96 hr assay. Cell lysates were prepared from antibody treated and untreated samples and subjected to Western blot analysis for identification of the signaling molecules involved in apoptosis induced by the anti-ROR1 CRD mAb. The total and phosphorylated levels of the following signaling proteins were analysed: ROR1, p-ROR1, PI3-kinase, p-PI3-kinase, AKT, p-AKT, mTOR, p-mTOR, ERK, p-ERK, PKC and p-PKC. Phosphoproteins were measured before incubation with the mAb and after 30 min-2 h. Results: ROR1 was expressed on the surface of 40-45% of the pancreatic carcinoma cell line PaCa-2. ROR1 was constitutively phosphorylated. The frequency of apoptotic cells after incubation with the anti-ROR1 mAb ranged from 10% at 24 hr to 40% at 96 hr. Western blot analysis showed decreased levels of phosphorylated ROR1 and the PI3-kinaseδ isoform (p110δ) in treated compared to untreated samples. Phosphorylation intensity of AKT and mTOR also decreased in treated compared to untreated cells. There was no change in the phosphorylation levels of ERK and PKC proteins. Conclusion: Incubation of pancreatic cancer cells with an anti-ROR1 CRD mAb induced apoptosis. Apoptosis was preceded by dephosphorylation of ROR1, PI3-kinaseδ isoform (p110δ), AKT and mTOR proteins indicating deactivation of these signaling proteins by the anti-ROR1 mAb. Activation of mTOR molecule was related to activation of the PI3-kinase/AKT pathway. This pathway might be a survival signal in pancreatic cancer cells associated with an aberrant expression of ROR1. Further studies are warranted to better understand the signaling pathways associated with ROR1 and the downstream signaling effects of ROR1 targeting drugs, which might be a potential therapeutic option. Citation Format: Amir Hossein Daneshmanesh, Mohammad Hojjat-Farsangi, Ali Moshfegh, Eva Mikaelsson, Abdul Salam Khan, Anders Österborg, Håkan Mellstedt. Anti-ROR1 monoclonal antibodies induce apoptosis in pancreatic cancer cells via the PI3-kinase/AKT/mTOR pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4770. doi:10.1158/1538-7445.AM2014-4770