Apoptosis induction mediated through PI3-kinase/AKT/mTOR pathway using anti-ROR1 monoclonal antibody in chronic lymphocytic leukemia cells.

Abstract

7087 Background: The PI3K/AKT/mTOR is a central pathway activated in many types of cancer. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase regulating cell growth, proliferation and survival. In CLL cells PI3K pathway is constitutively activated leading to AKT activation with subsequent phosphorylation of other downstream signaling molecules. ROR1 is a type I transmembrane RTK, overexpressed and constitutively phosphorylated in CLL. A unique anti-ROR1 mAb directed against CRD region of ROR1 was capable of inducing direct apoptosis as well as dephosphorylating the ROR1 molecule. Here, we investigated the apoptotic effect of the anti-ROR1 mAb and effects on the PI3K/AKT/mTOR pathway using primary CLL cells. Methods: Apoptosis was detected by the MTT assay and Annexin V/PI methods in a 24 h assay. Antibody untreated and treated cell lysates were prepared and subjected to Western blot analysis for identification of the signaling molecules involved in apoptosis induced by the ROR1 mAb. We analysed total and phosphorylated levels of the following signaling proteins: AKT, p-AKT, PI3K, p-PI3K, mTOR, p-mTOR, ERK, p-ERK, PKC and p-PKC. Phosphoproteins were measured before incubation with the mAb and after 20 min-24 h. Results: ROR1 detection on surface of the CLL cells was 80-85% and apoptotic frequency 45-50%. Western blot analysis showed decreased levels of p-AKT, p85 isoform of p-PI3K and p-mTOR in treated compared to untreated samples. No changes in the phosphorylation levels of ERK and PKC proteins were seen. Conclusions: Incubation of CLL cells with the anti-ROR1 mAb induced apoptosis of CLL cells. Apoptosis was preceded by dephosphorylation of PI3K, AKT and mTOR proteins indicating deactivation of these proteins by the ROR1 mAb. In untreated CLL cells no effect was noted. Furthermore no dephosphorylation of PKC or ERK was seen. We suggest that activation of mTOR might occur via the PI3K/AKT pathway and may be a survival signal in CLL cells associated with the aberrant expression of ROR1. Further studies are warranted to understand better the signaling pathways associated with ROR1 and the downstream signaling effects of ROR1 targeting drugs.