title: The tyrosine kinase receptor ROR1 is constitutively phosphorylated in chronic lymphocytic leukemia (CLL) cells.
abstract: Phosphorylation of receptor tyrosine kinases (RTKs) has a key role in cellular functions contributing to the malignant phenotype of tumor cells. We and others have previously demonstrated that RTK ROR1 is overexpressed in chronic lymphocytic leukemia (CLL). Silencing siRNA downregulated ROR1 and induced apoptosis of CLL cells. In the present study we analysed ROR1 isoforms and the phosphorylation pattern in CLL cells (n=38) applying western blot and flow-cytometry using anti-ROR1 antibodies and an anti-phospho-ROR1 antibody against the TK domain. Two major ROR1 bands with the size of 105 and 130 kDa respectively were identified, presumably representing unglycosylated (immature) and glycosylated (mature) ROR1 respectively as well as a 260 kDa band which may represent dimerized ROR1. A ROR1 band of 64 kDa that may correspond to a C-terminal fragment was also noted, present only in the nucleus. The 105 kDa ROR1 isoform was more frequently expressed in non-progressive as compared to progressive CLL patients (p=0.03). The 64, 105, 130 and 260 kDa bands were constitutively phosphorylated both at tyrosine and serine residues. Phosphorylation intensity of the mature (130 kDa) isoform was significantly higher in progressive than in non-progressive disease (p<0.001). Incubation of CLL cells with a mouse anti-ROR1 KNG or an anti-ROR1 CRD mAb respectively induced dephosphorylation of ROR1 before entering apoptosis. In conclusion CLL cells expressed different isoforms of ROR1 which were constitutively phosphorylated. The mature, phosphorylated ROR1 isoform was associated with a progressive disease stage. Targeting ROR1 by mAbs induced specific dephosphorylation and leukemic cell death. ROR1 might be an interesting therapeutic target.
authors.affiliation: Department of Oncology-Pathology, Immune and Gene therapy Lab, Cancer Center Karolinska (CCK), Karolinska University Hospital Solna and Karolinska Institute, Stockholm, Sweden.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Hojjat-Farsangi
authors.forename: Abdul Salam
authors.initials: AS
authors.lastname: Khan
authors.forename: Amir Hossein
authors.initials: AH
authors.lastname: Daneshmanesh
authors.forename: Ali
authors.initials: A
authors.lastname: Moshfegh
authors.forename: Asa
authors.initials: A
authors.lastname: Sandin
authors.forename: Ladan
authors.initials: L
authors.lastname: Mansouri
authors.forename: Marzia
authors.initials: M
authors.lastname: Palma
authors.forename: Jeanette
authors.initials: J
authors.lastname: Lundin
authors.forename: Anders
authors.initials: A
authors.lastname: Österborg
authors.forename: Håkan
authors.initials: H
authors.lastname: Mellstedt
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D017209:Apoptosis / Q000235:genetics; D045744:Cell Line, Tumor; D006031:Glycosylation; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics*; D010766:Phosphorylation / Q000235:genetics*; D020033:Protein Isoforms / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D057050:Receptor Tyrosine Kinase-like Orphan Receptors / Q000235:genetics*
chemical_list: D000911:Antibodies, Monoclonal; D020033:Protein Isoforms; C078716:ROR1 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D057050:Receptor Tyrosine Kinase-like Orphan Receptors