Phosphorylation of Receptor Tyrosine Kinase ROR1 At Tyrosine 641, 646 and Serine 652 residues Might Be of Importance for the Survival of CLL Leukemic Cells
Authors: Hojjat-Farsangi Mohammad , Daneshmanesh Amir Hossein , Norin Martin , Sandin Åsa , Khan Abdul Salam , Moshfegh Ali , Rossmann Eva , Osterborg Anders , Mellstedt Hakan 
Published: October 2019

Abstract
Abstract 3885

Background:
Receptor tyrosine kinases (RTK) play crucial roles for different normal cellular processes as cell proliferation/differentiation, apoptosis and survival, as well as for the malignant phenotype of many types of cancer. ROR1, as a member of twenty RTKs families, has important functions during normal embryogenesis. ROR1 has shown to be overexpressed in chronic lymphocytic leukemia (CLL), mantle cell lymphoma and other hematological malignancies, as well as in solid tumors. ROR1 inhibition in CLL cells and cell lines with high expression of ROR1 induced specific apoptosis of the cells. In this study, we investigated the effects of an anti-ROR1 mAb against the extracellular CRD domain for specific dephosphorylation at the tyrosine kinase domain of ROR1 in CLL cells. The CRD domain contains a frizzle receptor, which is considered to be the ligand-binding region for e.g. Wnt5a. Wnt5a has been suggested to stimulate growth of CLL cells.

Aims:
To investigate phosphorylation of tyrosine and serine residues, within the tyrosine kinase domain of ROR1, of importance for survival of CLL cells.

Methods:
Bioinformatic analysis of the ROR1 structure revealed that three amino acid residues in the tyrosine kinase domain might be critically phosphorylated. Based on this prediction, a 19 amino acid long peptide, phosphorylated at two tyrosine (tyrosine 641, 646) and one serine (serine 652) residues was designed and used for immunization of rabbits. An anti-phospho-ROR1 (pROR1) polyclonal antibody (pAb) with high titers of anti-pROR1 pAb was purified, using phospho-peptide affinity chromatography. The specificity of anti-pROR1 pAb was determined by ELISA, immunoprecipitation (IP) and western blot experiments. An anti-ROR1 mAb (IgG) (CRD 1D8 clone) was used to analyse the effects on ROR1 phosphorylation in CLL cells at tyrosine 641, 646 and serine 652 residues preceding apoptosis. ROR1 phosphorylation was investigated by western blot and IP of ROR1 probed with anti-pROR1 pAb, from untreated and CLL cells treated with the anti-CRD 1D8 mAb. Quantitative intracellular staining of ROR1 by flowcytometry in time kinetics experiment after treatment with anti-CRD 1D8 mAb was also used to check phosphorylation of ROR1. Annexin V/PI staining (flowcytometry), MTT assay, PARP and caspase 8 cleavage as well as MCL-1 protein (western blot) were used for detection of apoptosis. To investigate phosphorylation and localization of 64–130 kDa ROR1 isoforms in various compartments of CLL cells, lysates were prepared from the nucleus and cytoplasmic proteins of CLL cells.

Results:
Two tyrosine (641, 646) residues and one serine (652) residue of the tyrosine kinase domain were phosphorylated in CLL cells. As previously described (Mellstedt et al, Abstract No: 1771, 53th ASH annals meeting, 2011), the 64, 105 and 130 kDa ROR1 isoforms were shown to be constitutively phosphorylated at tyrosine and serine residues in CLL leukemic cells. Treatment of CLL cells with an anti-ROR1 mAb against the CRD domain induced rapid dephosphorylation of ROR1 at tyrosine 641, 646 and serine 652 residues within 20 min and gradually increased up to 4 hours. The phosphorylated 64 kDa ROR1 isoform was localized to the nucleus of CLL cells and probably represents an intracellular part of ROR1, while the ROR1 130 kDa isoform was presented both in cytoplasm and nucleus of CLL cells.

Conclusion:
Our data show that the ROR1 molecule is phosphorylated at tyrosine 641, 646 and serine 652 residues. The presence of 64 and 130 kDa ROR1 isoforms in the nucleus of CLL cells may suggest a role of these isoforms as transcription factors. Collectively, the data might suggest that phosphorylated ROR1 may be an important protein for the growth of CLL cells as well as an interesting structure to target in a therapeutic intervention.

Disclosures:
No relevant conflicts of interest to declare.