Ror1, a cell surface receptor tyrosine kinase is ... - BPS - Wiley

Int. J. Cancer: 123, 1190–1195 (2008) ' 2008 Wiley-Liss, Inc.

Ror1, a cell surface receptor tyrosine kinase is expressed in chronic lymphocytic leukemia and may serve as a putative target for therapy Amir H. DaneshManesh1, Eva Mikaelsson1, Mahmood Jeddi-Tehrani1,2, Ali Ahmad Bayat2, Roya Ghods2, 1,5 € Mahyar Ostadkarampour2, Mehdi Akhondi3, Svetlana Lagercrantz4, Catharina Larsson4, Anders Osterborg , 2,6 1,5* 1 Fazel Shokri , Hakan and Hodjattallah Rabbani ˚ Mellstedt 1 Immune and Gene Therapy Lab, CCK, Karolinska Institutet and Karolinska University Hospital Solna, Stockholm, Sweden 2 Monoclonal Antibody Research Center, Avesina Research Institute, Tehran, Iran 3 Department of Genetics and Embryology, Reproductive Biotechnology Research Center, Avesina Research Institute, Tehran, Iran 4 Departments of Molecular Medicine and Surgery, CMM, Karolinska University Hospital Solna, Stockholm, Sweden 5 Departments of Oncology and Hematology, Karolinska University Hospital Solna, Stockholm, Sweden 6 Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Gene profiling studies of patients with chronic lymphocytic leukemia (CLL) has revealed increased expression of Ror1, a cell surface receptor tyrosine kinase. The aim of present study was to analyze gene and protein expression of Ror1 in CLL cells and normal blood leukocytes. Gene expression analysis reverse transcriptionpolymerase chain reaction of ROR1 revealed that all patients with CLL (n 5 100) spontaneously expressed ROR1 mRNA whereas enriched blood B and T cells as well as granulocytes from healthy donors (n 5 10) were negative. A strong nonphysiological activation signal (PMA/ionomycin) was required to induce expression in vitro in normal lymphocytes. Major genomic aberrations (mutations or truncation) of ROR1 were not observed. Protein expression was analyzed by Western blot using a panel of polyclonal anti-Ror antibodies as well as flow cytometry. Blood lymphocytes from 18/18 CLL patients, but none of the 10 healthy donors, expressed surface Ror1. The majority of CLL cells exhibited Ror1 surface expression (71% mean; range 36–92%) with a mean fluorescence intensity (MFI) of 20 (range 10–45). The corresponding MFI of CD19 on CLL cells was 26 (range 9–48). There was no difference in the Ror1 protein expression comparing IgVH mutated and unmutated cases as well as progressive and nonprogressive CLL patients. Two different variants of the Ror1 protein, 105 and 130 kDa, were identified. The Ror1 protein expression in patients with CLL but not in normal leukocytes merits further studies of its role in the pathobiology of CLL, which may provide a basis for development of Ror1 directed targeted therapy. ' 2008 Wiley-Liss, Inc. Key words: B-CLL; tyrosine kinase receptors; Ror1

Chronic lymphocytic leukemia (CLL) originates from B lymphocytes, which differ in activation and maturation stage and are derived from antigen experienced B cells with different immunoglobulin heavy chain variable (IgVH) gene mutations.1 Patients with mutated IgVH genes have a better prognosis compared to patients with unmutated genes.2,3 Global gene expression profiling studies have revealed partly distinguishing but in general overlapping expression profiles in mutated and unmutated leukemic B cells, suggesting a common phenotype.4,5 Gene expression profiling studies showed a 43.8-fold increase of the orphan receptor tyrosine kinase (RTK) ROR1 in CLL cells.4 Ror1 is a member of the RTK family of orphan receptors related to muscle specific kinase and Trk neurotrophin receptors.6–8 Ror receptors are cell surface receptors participating in signal transduction, cell–cell interaction, regulation of cell proliferation, differentiation, cell metabolism and survival.7,9 They are evolutionarily highly conserved between different species e.g., human, mouse, Drosophila and C. elegans, suggesting important biological functions. The human ROR1 gene has a coding region of 2814 bp with a predicted 937 amino acids sequence and 105 kDa protein size including an Ig-like domain, cysteine-rich domain, kringle domain, tyrosine kinase domain and proline-rich domain (Fig. 1).9 Publication of the International Union Against Cancer

ROR1 is located on chromosomal region 1p31.3 (http://www. ensembl.org), a region where chromosomal aberrations are not frequently seen in hematological malignancies. The human ROR1 is expressed in heart, lung and kidney but less in placenta, pancreas and skeletal muscles.10 ROR1 was originally cloned from a neuroblastoma cell line7 and subsequently a shorter form lacking the entire extracellular domain but containing the transmembrane domain was isolated from a fetal brain library. Truncated Ror1(tRor1) gene has been reported in fetal and adult human central nervous system, in human leukemias, lymphoma cell lines, and in a variety of human cancers derived from neuroectoderm.10 A shorter transcript from exons 1–7 including a short part of intron 7 has also been described with a predicted length of 393 amino acids and a molecular weight of 44 kDa (Ensembl ID; ENSG00000185483). In our study, we present data demonstrating that Ror1 is uniformly expressed at the gene and protein levels in the leukemic cells of all patients with CLL, but not in normal lymphocytes unless a strong activation signal was provided. Material and methods Patients The WHO Classification of Neoplasms of the Hematopoetic and Lymphoid Tissues was applied.11 The diagnosis of CLL (n 5 100) was based on immunophenotyping (CD51/CD191/CD231/ IgM1) and the presence of >5.0 3 109/l lymphocytes in peripheral blood. Patients with CLL were considered to have progressive disease according to a modification of the criteria of the NCI committee, if there was a progression during the preceding 3 months in disease-related anaemia (hemoglobin 98%.

FIGURE 2 – Specificity control of the polyclonal antibodies. Upper panel: Western blot analyses using a commercially available antiRor1 polyclonal antibody (N-Ror1com) and own produced rabbit polyclonal antibody (N-Ror1-46). Both antibodies reacted with the same 37 kDa band. The recombinant extracellular part of the Ror1 protein was expressed in E.coli and supernatant concentrated 30X. (The NRor1-46 antibody did not react with the recombinant cytoplasmic Ror1 protein (data not shown)). Lower panel: Western blot using a serially diluted commercially available recombinant Ror1 protein representing a cytoplasmic region and probed with our rabbit polyclonal antibody C-Ror1-904. [The C-Ror1-904 antibody did not react with the recombinant extracellular part of the Ror1 protein (data not shown)].

ating the polyclonal antibodies. The polyclonal antibodies were purified by affinity purification. A recombinant protein representing an intracellular region of Ror1 with a molecular weight of around 70 kD (Carna Biosciences, Chuo-ku, Kobe, Japan) was used for specificity control of the C-Ror1-904 polyclonal antibody (Fig. 2, lower panel). The specificity of the N-Ror1-46 antibody, was checked against a recombinant protein of the extracellular domain of Ror1 spanning aa 33 to 458 expressed in bacteria given an expected molecular weight of around 40 kD. Briefly, the region was PCR amplified using a human full-length cDNA clone EN1031_D08 Ror1 gene (Origene Technologies, Rockville, MD) as template. The PCR products were cloned into pGEM-T easy vector and subcloned into pcDNA3.11 vector (Invitrogen) and transformed into E.coli strain Origami (Invitrogen). The integrity of the insert was verified by DNA sequencing. After selecting an in-frame clone, the supernatant of 24 hr cultured bacteria was collected and concentrated 30 times using Amicon Ultra-15 Centrifugal Filter Units (separation of polypeptides >10 kDa) (Millipore Corporation, Bedford, MA). The concentrated recombinant was subjected to Western blot and probed with N-Ror1-46 and a commercially available anti-Ror1 antibody [goat anti-Ror1 polyclonal antibody (N-Ror1com) (R&D systems, Minneapolis, MN)] to determine the specific reactivity (Fig. 2). Western blot The goat anti-Ror1 polyclonal antibody (N-Ror1com) (R&D systems) as well as the antibodies produced in our lab (N-Ror1-46 and C-Ror1-904) were used in Western blot. Cells were lysed in a buffer containing 1% Triton X-100, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA and 1% protease inhibitor cocktail (Sigma). Protein concentration was measured by Thermo Scientific BCA Protein Assay Kit (Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions. Fifty micrograms of cell lysates were run on a 10% Bis-Tris SDS-PAGE gel (Invitrogen) at 120 V for 3 hr under reducing conditions. After electrophoresis, resolved proteins were transferred onto ImmobilonPVDF membranes (Millipore Corporation) in a mini Transblot cell (Invitrogen). The membranes were blocked for 1.5 hr at room temperature with 5% nonfat milk in PBS plus 0.05% Tween 20

(PBS-T). All immunostainings were performed in PBS-T supplemented with 5% nonfat milk. Filters were incubated with appropriate dilutions of the anti-Ror1 antibodies over night at 14
C. After extensive washing with PBS-T, the filters were incubated with peroxidase-conjugated goat anti-rabbit immunoglobulins (DakoCytomation, Glostrup, Denmark) for 1.5 hr at room temperature followed by washing and developing with ECL chemiluminescence detection system (GE Healthcare). Surface staining and flow cytometry Cells were analyzed by flow cytometry (FACSCalibur BD Biosciences) using N-Ror1com (primary antibody), PE conjugated anti-CD3 (BD Biosciences, San Jose, CA), PE-Cy5-conjugated anti-CD19 (e-Bioscience, San Diego, CA), FITC-conjugated antiCD19 (BioLegend, San Diego, CA), FITC-conjugated swine anti-goat IgG antibody (secondary antibody) (Southern biotech, Birmingham, AL) and mouse serum (blocking serum) (DakoCytomation). Surface staining of CLL cells and normal PBMC was performed as described.16 Briefly, 2 3 106 cells were washed in PBS and pre-incubated with serum-free RPMI (Invitrogen), at 37
C for 1 hr followed by 3 washings with RPMI. One microgram of the antiRor1 antibody (N-Ror1com) (R&D systems) was added and incubated at 14
C for 30 min. The cells were washed twice with FACS buffer (PBS, 0.1% sodium azide and 0.5% FBS). FITC swine anti-goat IgG (1:100) (Southern biotech) was added and incubated at 14
C for further 30 min. Blocking was performed by adding 10 ll of 10% mouse serum followed by incubation at 14
C for 20 min. Both CD3 and CD19 antibodies were then added to the cells and incubated at 14
C for 30 min. The cells were finally washed twice with FACS buffer and fixed by adding 1% paraformaldehyde in PBS. The CellQuest software program (BD) was used to determine the percentage of Ror11 cells of the CD19 population. Sequencing of the ROR1 gene PBMC of CLL patients were isolated, total RNA prepared and cDNA synthesized as described above. ROR1 specific primers were designed to amplify truncated t-Ror1 (primers P9 and P10), the extracellular domains including Ig, CRD, and kringle domains (primers P5 and P6), as well as the kinase domain (primers P7 and P8) (Fig. 1). The PCR products were cloned into pGEM-T easy vector (Promega) and subjected to sequencing using T7, Sp6 and gene specific primers (Table I). Dual color fluorescence in situ hybridization Fluorescence in situ hybridization (FISH) was carried out on nuclei isolated from blood or bone marrow cells of patients with CLL using 3 BAC clones spanning the ROR1 locus in 1p31.3. BAC DNA was isolated with QIAGEN Plasmid Mini Kit (QIAGEN GmbH, Hilden, Germany) and labeled by nick translation (Roche Diagnostics GmbH, Mannheim, Germany) according to the recommendations of the manufacturers. RP11-265C4 was

Ror1 IN B-CLL

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FIGURE 3 – ROR1 gene expression (RT-PCR) in leukemic cells of CLL patients and a healthy control donor. Positive control represents the PCR product cloned into pGEM-T easy vector. Negative control is the reaction mixture without template. Marker is a 100 bp DNA ladder. The beta-actin gene was used to verify the integrity of synthesized cDNA.

FIGURE 5 – Western blot. All 3 antibodies showed a 105 kDa band. C-Ror1-904 also detected an estimated 130 kDa variant of Ror1. The blots were stripped and stained with a beta-actin antibody to show the integrity of the loaded samples.

FIGURE 4 – A representative example of ROR1 expression in activated (PMA/ionomycin) normal B- and T-lymphocytes, tonsil B-cells, and leukemic CLL cells after 48 hr of culture. The expression was determined by quantitative real-time PCR. Fold increase was related to the level observed at time zero. CLL cells and tonsil B cells which constitutively expressed Ror1 mRNA could not be further activated, while the strong activation signal induced gene expression of ROR1 in normal B and T cells.

labeled with fluorescein-12-dUTP and cohybridized with either RP11-30A5 or RP11-91B5 labeled with tetramethylrhodamine-5dUPT (Roche) using standard methods. For each case 200 nuclei were scored in a Zeiss Axioskop epifluorescence microscope (Carl Zeiss Jena GmbH, Jena, Germany). Fusion, touching or close location within 1–3 probe sizes of the 2 probes were scored as positive for colocalisation. Results ROR1 gene expression in CLL patients and healthy donors PBMC of all CLL patients (n 5 100) as well as BMMC (n 5 2) expressed ROR1 at the mRNA level. ROR1 was weakly expressed also on normal tonsil B cells (2/2) but not in healthy donor PBMC (0/10), isolated normal B cells (0/6) and T cells (0/3) or enriched blood granulocytes (0/10) (Table II). Representative RT-PCR experiments of healthy donors and CLL patients are shown in Figure 3. Mutation analysis of cloned extracellular and cytoplasmic kinase domains of the ROR1 gene was analyzed in 10 CLL patients and showed no major genomic aberrations. Only few point mutations (silent mutations) were found (data not shown). PCR amplification to detect a truncated ROR1 (t-Ror1) using primers P9 and P10 did not give rise to any amplicon (data not shown).

FIGURE 6 – Cell surface staining for Ror1 (N-Ror1com) and CD19 of leukemic cells from 2 progressive and nonprogressive CLL patients as well as PBMC of a healthy control donor.

Expression of ROR1 in activated cells Next, we analyzed whether ROR1 might be induced after in vitro activation. CLL cells, normal B- and T-lymphocytes and tonsil B cells were cultured with PMA/ionomycin for 48 hr to provide a strong activation signal. CLL cells and normal tonsil B cells, which constitutively expressed Ror1 mRNA, could not be further activated. In contrast, a 15 to 25-fold increase in the ROR1 mRNA expression was observed in in vitro activated normal B and T cells. A representative experiment is shown in Figure 4. The activated normal B cells also expressed Ror1 at the protein level (Western blot) (data not shown).

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DANESHMANESH ET AL. TABLE III – PROTEIN EXPRESSION OF ROR1 IN CLL PATIENTS (n 5 18) IN RELATION TO IgVH MUTATIONAL STATUS AND CLINICAL PHASE Patients

CLL-1 CLL-2 CLL-3 CLL-4 CLL-5 CLL-6 CLL-7 CLL-8 CLL-9 CLL-10 CLL-11 CLL-12 CLL-13 CLL-14 CLL-15 CLL-16 CLL-17 CLL-18 Mean 6 SEM Healthy controls doners (n 5 10)

Disease phase

Nonprogressive Nonprogressive Nonprogressive Nonprogressive Nonprogressive Nonprogressive Nonprogressive Nonprogressive Nonprogressive Progressive Progressive Progressive Progressive Progressive Progressive Progressive Progressive Progressive

IgVH mutation status

M UM UM M UM M UM M M M M UM M UM M M M UM

Western blot N-Ror1com (kDa)

N-Ror1-46 (kDa)

C-Ror1-904 (kDa)

105 105 105 105 105 105 105 105 105 105 105 105 105 105 105 105 105 105

105 105 105 105 105 105 105 105 105 105 105 105 105 105 105 105 105 105

105, 130 105, 130 105, 130 105, 130 105, 130 105, 130 105, 130 105, 130 105, 130 105, 130 105, 130 105, 130 105, 130 105, 130 105, 130 105, 130 105, 130 105, 130