HIGH LEVEL OF COBLL1, A NOVEL ROR1 BINDING PARTNER, IDENTIFIES CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) PATIENTS WITH MUTATED IGHV

ABSSUB-5076

Background: Chronic lymphocytic leukemia (CLL) is a disorder with a variable clinical course and up-regulated expression of a transmembrane tyrosine-protein kinase (ROR1), a member of Wnt – planar cell polarity (PCP) pathway. This pathway has an important role in the early embryonic development and controls pathogenesis of CLL via regulation of cell migration and chemotaxis.

Aims: The main aim of the study was (i) to identify ROR1 downstream signalling by the proteomic analysis of ROR1 binding proteins in CLL and (ii) to analyse the role and clinical value of selected binding partners.

Methods: The search for ROR1 binding partners was performed using mass spectrometry. For expression analysis, a cohort of 186 unselected CLL patients was tested. B-lymphocytes were separated from peripheral blood using gradient centrifugation with non-B-cell depletion. mRNA expression was examined by qRT-PCR. The protein expression was further tested using Western Blot.  B-cells from healthy tonsills (n=4) were fasc-sorted via IgD and CD38 staining into naïve, germinal centre and memory B-cells, which were tested by qRT-PCR. Expression vectors containing COBLL1 and ROR1 were transfected into HEK293 cells to visualize their localization.

Results: We identified a novel Ror1 binding partner in CLL cells – cordon blue protein like 1 (COBLL1). In HEK293 cells, COBLL1 was localized mainly in cytoplasm but following ROR1 overexpression both proteins were strongly co-localized in the membrane filopodia. In CLL patients COBLL1 showed variable expression with significantly higher expression in the patients with mutated IGHV (n=70) compared to the patients with unmutated IGHV (n=116; p<0.0001, Mann-Whitney U test).  Accordingly, the patients with COBLL1 expression above defined threshold had longer treatment-free survival (48 months) compared to the patients with expression bellow this threshold (32 months; p=0.0004, Mantel-Cox test). Also, the expression of COBLL1 decreased with disease severity according to the cytogenetic hierarchical model (del 13q, normal karyotype vs. del 17p, del 11q; p<0.03, Mann-Whitney U test).  The difference in COBLL1 expression between the patients with mutated and unmutated IGHV was also confirmed at a protein level (n=10). Median expression in the patients with mutated IGHV was similar to the expression in healthy germinal centre B-cells, whereas median expression in the patients with unmutated IGHV corresponded to the expression in healthy naïve and memory B-cells.

Summary/Conclusion: We identified COBLL1 as a novel ROR1 interaction partner. COBLL1 expression in CLL cells correlates with patient's prognosis. Different expression in healthy tonsillar subpopulations suggests its physiological role in germinal centre transition.

Supported by MUNI/A/0830/2013, IGA NT11217-5/2010, IGA NT13493-4/2012, GACR P301/11/0747, MH CZ – DRO (FNBr, 65269705).

Keywords: Chronic lymphocytic leukemia, Quantitative RT-PCR, Wnt

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