ROR1 Isoforms Are Constitutively Phosphorylated in Chronic Lymphocytic Leukemia (CLL) - a Survival Factor for CLL Cells

Phosphorylation of receptor tyrosine kinases (RTK) plays an important role for many aspects of cell life, as cell-cycle progression, differentiation, apoptosis, intercellular communication and cell survival. RTKs as e.g. EGFR, HER2/neu, VEGFR, IGFR etc. are important structures contributing to the malignant phenotype of many tumors. Targeting RTKs by monoclonal antibodies (MAb) or small inhibitory molecules has been successful for the treatment of various cancers. ROR is one of the twenty RTK families and consists of two members, ROR1 and ROR2. ROR1 and ROR2 have important functions during embryogenesis. ROR1 is uniquely expressed in CLL cells compared to normal tissues. ROR1 siRNA transfection of CLL cells induced specific apoptosis of the leukemic cells.

To study ROR1 protein variants in CLL, ROR1 phosphorylation and relation to clinical activity of the disease.

Phosphorylation of primary human CLL cells from patients with non-progressive and progressive disease, and a series of cell lines were studied applying immunoprecipitation (IP) of the ROR1 molecule, using anti ROR1 and irrelevant MAbs by Western blotting as well as in intracytoplasmic staining of the cells by flowcytometry, using an anti-phospho ROR1 (pROR1) MAb (a MAb specifically recognizing a phospho-peptide of the cytoplasmic TK domain of ROR1) and semi quantification of the staining intensity as well as by p-tyrosine and p-serine MAbs.

IP of the ROR1 molecule of fresh leukemic cells using ROR1 specific antibodies and subsequent Western blot with anti ROR1 MAbs, showed various protein bands. Two bands had the size of 105 and 130 KDa probably representing unglycosylated or partially glycosylated ROR1 and the fully glycosylated glycoform respectively. One of the two bands dominated in individual patients. A band of 260 KDa could also be detected in a large number of patients probably representing dimerized monomers. Finally a band of 64 KDa could also be noted which may represent an intracellular part of ROR1, as has been described in fetal and adult human CNS, leukemia and lymphoma cell lines (Reddy UR et al, Oncogene. 1996; 13:1555–9). The 64, 105 and 130 KDa bands were constitutively phosphorylated in all patients both at tyrosine and serine residues. The intensity of phosphorylation of the 130 KDa band was significantly higher in patients with progressive disease vs. non-progressive disease (p=0.0001). There was no relation between the pattern of the different ROR1 protein bands and disease activity.

Using 9 cell lines of different hematologic malignancies including CLL, similar protein bands and phosphorylation status as for fresh CLL cells were noted.

We have also produced specific mouse monoclonal antibodies against defined epitopes of the extracellular parts of ROR1. One of those MAbs (anti-KNG, IgG1) induced a high degree of specific apoptosis of the CLL cells (ASH Annual Meeting Abstracts, Nov 2010; 116: 916) and could also be shown to induce a specific dephosphorylation of the cytoplasmic TK domain which was noted already after 20 min and gradually increased up to 4h. No effects were noted using irrelevant MAbs or healthy donor lymphocytes.

Our results showed that the ROR1 molecule in CLL cells are expressed in various protein variants probably representing different glycosylation patterns (105–130 KDa), dimerized monomers (260 KDa) and a truncated protein variant (64 KDa). ROR1 was constitutively phosphorylated (64, 105 and 130 KDa) both at serine and tyrosine residues. Treatment of CLL cells in vitro with a ROR1 specific antibody induced specific apoptosis as well as rapid dephosphorylation of ROR1. Collectively the data suggest that phosphorylated ROR1 might be an important structure for the growth potential of CLL cells and an interesting structure to target in a therapeutic intervention.

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