The tyrosine kinase receptor ROR1 is constitutively phosphorylated in chronic lymphocytic leukemia (CLL) cells

PLoS One. 2013 Oct 24;8(10):e78339. doi: 10.1371/journal.pone.0078339. eCollection 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / immunology
  • Apoptosis / genetics
  • Cell Line, Tumor
  • Glycosylation
  • Humans
  • Leukemia, Lymphocytic, Chronic, B-Cell / genetics*
  • Phosphorylation / genetics*
  • Protein Isoforms / genetics
  • Receptor Protein-Tyrosine Kinases / genetics*
  • Receptor Tyrosine Kinase-like Orphan Receptors / genetics*

Substances

  • Antibodies, Monoclonal
  • Protein Isoforms
  • ROR1 protein, human
  • Receptor Protein-Tyrosine Kinases
  • Receptor Tyrosine Kinase-like Orphan Receptors

Grant support

This study was supported by grants from VINNOVA (http://www.vinnova.se/sv/), Proj. No: 2009-00116, P36147-1. The CLL Global Research Foundation (http://www.cllglobal.org/), Proj No: 10501300. EUCAAD 200755 project funded under the auspices of the EU Seventh Framework Programme (http://ki.se/ki/jsp/polopoly.jsp?d=9877&a=128974&dispd=40337&l=sv). Grant Agreement No: 200755. The Swedish Research Council (http://www.vr.se/), Avtals-ID: C0235101, Proj. No: K2013-64X-21464-04-3. The Swedish Cancer Society (http://www.cancerfonden.se/), Contract No: 12 0842. The Cancer Society in Stockholm (http://www.uicc.org/membership/cancer-society-stockholm), Contract No: 121273. The King Gustaf Vth Jubilee Fund (http://www.rahfo.se/Om-Cancer/), Contract No: 124272. The Stockholm County Council (http://sll.se/), Proj No: 20120051. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.