Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR-1) Is Expressed in Low Grade NHL and B-CLL and Activates the Non Canonical Wnt Pathway

Abstract

ROR-1, an orphan receptor tyrosine kinase (RTK) carrying an extracellular WNT binding motif, is highly expressed in many tissues during development. Expression of ROR-1 in lymphoid cells has first been noted in a gene expression profiling study of B-cell chronic lymphocytic leukemia (B-CLL), where ROR-1 was expressed in CLL cells but not in diffuse large B-cell lymphoma or in normal B-cells (Rosenwald, 2001). B-CLL is the most common type of leukemia in western countries and is characterized by an accumulation of mature B lymphocytes in the blood and lymphoid tissues. The prolonged survival of the malignant cells in-vivo is dependent on signals derived from the microenvironment. ROR-1 recently has been implicated in mediating stroma dependent survival signals, with WNT5a being a putative ligand for ROR-1 (Fukuda, 2008). In addition, an RNAi screen identified ROR-1 as a tyrosine kinase with anti-apoptotic effects in Hela cells (MacKeigan, 2005). With this background, we have further examined the role of ROR-1 in the survival of B-cell malignancies. We first characterized ROR-1 surface expression by flow cytometry on various lymphoid cell lines and primary cells. We found ROR-1 was highly expressed in mantle cell lymphoma (MCL) cell lines and moderately expressed in the CLL derived EBV positive cell line MEC-1. In contrast, cell lines derived from other B-cell (BJAB, SUDHL-4) and T-cell (Jurkat) malignancies had no ROR-1 expression. Moreover, ROR-1 was highly expressed in primary MCL cells (n= 5) and in follicular lymphoma (n=1). Comparable levels of expression were also detected in CLL cells independent of IgVH mutation status (IgVH unmutated: n=11; IgVH mutated: n= 8). To investigate whether and how ROR-1 could activate intracellular signaling pathways, we chose a monoclonal antibody to induce ROR-1 activation through receptor dimerization. In response to antibody binding, ROR-1 was tyrosine phosphorylated within minutes in a dose dependent manner in MCL cell lines and in primary CLL and MCL cells. Intriguingly, the MCL cell line UPN-1, bearing high ROR-1 surface expression (specific MFI ratio of 19), showed constitutive ROR-1 phosphorylation that further increased after crosslinking. Several studies on ROR-2, the closest ROR-1 homolog, reported activation of the non-canonical WNT signaling pathway and c-Jun N-terminal kinase (JNK) activation. We therefore investigated whether ROR-1 cross-linking could activate JNK and/or lead to stabilization of beta-catenin, the main mediator of canonical WNT signaling. Indeed, we detected JNK phosphorylation in MCL cell lines and in primary CLL cells, while we could not detect beta-catenin activation. In conclusion, ROR-1 surface expression is high in MCL cell lines and primary cells from CLL, MCL and FL patients. ROR-1 crosslinking leads to ROR-1 phosphorylation in both cell lines and primary cells followed by activation of the non-canonical WNT/JNK pathway. Our findings suggest that ROR-1, which plays important roles in embryonic development and organogenesis, could play an important role in the pathogenesis of low grade lymphomas and B-CLL.