Amir Hossein Daneshmanesh

Ror1, a cell surface receptor tyrosine kinase is expressed in chronic lymphocytic leukemia and may serve as a putative target for therapy

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 transcription‐polymerase chain reaction of ROR1 revealed that all patients with CLL (n = 100) spontaneously expressed ROR1 mRNA whereas enriched blood B and T cells as well as granulocytes from healthy donors (n = 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.

Monoclonal antibodies against ROR1 induce apoptosis of chronic lymphocytic leukemia (CLL) cells

ROR1 is a receptor tyrosine kinase (RTK) recently identified to be overexpressed at the gene and protein levels in chronic lymphocytic leukemia (CLL). Monoclonal antibodies (MAbs) against RTKs have been successfully applied for therapy of solid tumors. We generated five MAbs against the Ig (n=1), cysteine-rich (CRD) (n=2) and kringle (KNG) (n=2) domains, respectively, of the extracellular part of ROR1. All CLL patients (n=20) expressed ROR1 on the surface of the leukemic cells. A significantly higher frequency of ROR1 expression was found in patients with progressive versus non-progressive disease, and in those with unmutated versus mutated IgVH genes. All five MAbs alone induced apoptosis in the absence of complement or added effector cells (Annexin-V and MTT, as well as cleavage of poly-(ADP ribose)-polymerase, caspase-8 and caspase-9) of CLL cells but not of normal B cells. Most effective were MAbs against CRD and KNG, significantly superior to rituximab (P<0.005). Cross-linking of anti-ROR1 MAbs using the F(ab′)2 fragments of anti-Fc antibodies significantly augmented apoptosis. Two of the MAbs induced complement-dependent cytotoxicity (CDC) similar to that of rituximab and one anti-ROR1 MAb (KNG) (IgG1) showed killing activity by antibody-dependent cellular cytotoxicity. The identified ROR1 epitopes may provide a basis for generating human ROR1 MAbs for therapy.

Silencing of ROR1 and FMOD with siRNA results in apoptosis of CLL cells

We have previously demonstrated that ROR1 and FMOD (fibromodulin) are two genes upregulated in chronic lymphocytic leukaemia (CLL) cells compared to normal blood B cells. In this study, siRNAs were used to specifically silence ROR1 and FMOD expression in CLL cells, healthy B cells and human fibroblast cell lines. siRNA treatment induced a specific reduction (75–95%) in FMOD and ROR1 mRNA. Western blot analysis with specific antibodies for FMOD and ROR1 demonstrated that the proteins were significantly downregulated 48 h after siRNA treatment. Silencing of FMOD and ROR1 resulted in statistically significant (P ≤ 0·05–0·001) apoptosis of CLL cells but not of B cells from normal donors. Human fibroblast cell lines treated with FMOD and ROR1 siRNA did not undergo apoptosis. This is the first report demonstrating that ROR1 and FMOD may be involved in the survival of CLL cells. ROR1 in particular is further explored as potential target for therapy in CLL.

The receptor tyrosine kinase ROR1 – An oncofetal antigen for targeted cancer therapy

Targeted cancer therapies have emerged as new treatment options for various cancer types. Among targets, receptor tyrosine kinases (RTKs) are among the most promising. ROR1 is a transmembrane RTK of importance during the normal embryogenesis for the central nervous system, heart, lung and skeletal systems, but is not expressed in normal adult tissues. However, ROR1 is overexpressed in several human malignancies and may act as a survival factor for tumor cells. Its unique expression by malignant cells may provide a target for novel therapeutics including monoclonal antibodies (mAbs) and small molecule inhibitors of tyrosine kinases (TKI) for the treatment of cancer. Promising preclinical results have been reported in e.g. chronic lymphocytic leukemia, pancreatic carcinoma, lung and breast cancer. ROR1 might also be an interesting oncofetal antigen for active immunotherapy. In this review, we provide an overview of the ROR1 structure and functions in cancer and highlight emerging therapeutic options of interest for targeting ROR1 in tumor therapy.

Orphan receptor tyrosine kinases ROR1 and ROR2 in hematological malignancies

Abstract The receptor tyrosine kinase ROR1 has been shown to be overexpressed in chronic lymphocytic leukemia (CLL). The aim of this study was to further characterize the expression of ROR1 and the other member of the ROR family, ROR2, in other lymphoid and myeloid malignancies. Normal white blood cells and reactive lymph nodes were negative for ROR1 and ROR2. A significantly high and uniform surface expression of ROR1 was found in CLL/hairy cell leukemia (HCL) compared to mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), myelomas, acute lymphoblastic leukemia (ALL) and myeloid leukemias (p = 0.02 to < 0.001). The lowest proportion of ROR1+ cells was seen in FL, whereas CLL, HCL and CML had significantly higher numbers of ROR1+ cells. Longitudinal follow-up of individual patients with CLL revealed that ROR1+ cells remained stable over time in non-progressive patients, but increased when the disease progressed (p < 0.05). Thus, a variable staining pattern of ROR1 ranging from very high (CLL, HCL) and high (CML) to intermediate (myeloma and DLBCL) or low (FL) was noted. ROR2 was not detected in hematological malignancies.

Inhibition of the receptor tyrosine kinase ROR1 by anti-ROR1 monoclonal antibodies and siRNA induced apoptosis of melanoma cells

The receptor tyrosine kinase (RTK) ROR1 is overexpressed and of importance for the survival of various malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL). There is limited information however on ROR1 in melanoma. In the present study we analysed in seven melanoma cell lines ROR1 expression and phosphorylation as well as the effects of anti-ROR1 monoclonal antibodies (mAbs) and ROR1 suppressing siRNA on cell survival. ROR1 was overexpressed at the protein level to a varying degree and phosphorylated at tyrosine and serine residues. Three of our four self-produced anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8) induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). The ESTDAB081 and 094 cell lines respectively were resistant to direct apoptosis of the four anti-ROR1 mAbs alone but not in CDC or ADCC. ROR1 siRNA transfection induced downregulation of ROR1 expression both at mRNA and protein levels proceeded by apoptosis of the melanoma cells (ESTDAB049, ESTDAB112, DFW and A375) including ESTDAB081, which was resistant to the direct apoptotic effect of the mAbs. The results indicate that ROR1 may play a role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable target for mAb therapy.

The Tyrosine Kinase Receptor ROR1 Is Constitutively Phosphorylated in Chronic Lymphocytic Leukemia (CLL) Cells

Phosphorylation of receptor tyrosine kinases (RTKs) has a key role in cellular functions contributing to the malignant phenotype of tumor cells. We and others have previously demonstrated that RTK ROR1 is overexpressed in chronic lymphocytic leukemia (CLL). Silencing siRNA downregulated ROR1 and induced apoptosis of CLL cells. In the present study we analysed ROR1 isoforms and the phosphorylation pattern in CLL cells (n=38) applying western blot and flow-cytometry using anti-ROR1 antibodies and an anti-phospho-ROR1 antibody against the TK domain. Two major ROR1 bands with the size of 105 and 130 kDa respectively were identified, presumably representing unglycosylated (immature) and glycosylated (mature) ROR1 respectively as well as a 260 kDa band which may represent dimerized ROR1. A ROR1 band of 64 kDa that may correspond to a C-terminal fragment was also noted, present only in the nucleus. The 105 kDa ROR1 isoform was more frequently expressed in non-progressive as compared to progressive CLL patients (p=0.03). The 64, 105, 130 and 260 kDa bands were constitutively phosphorylated both at tyrosine and serine residues. Phosphorylation intensity of the mature (130 kDa) isoform was significantly higher in progressive than in non-progressive disease (p<0.001). Incubation of CLL cells with a mouse anti-ROR1 KNG or an anti-ROR1 CRD mAb respectively induced dephosphorylation of ROR1 before entering apoptosis. In conclusion CLL cells expressed different isoforms of ROR1 which were constitutively phosphorylated. The mature, phosphorylated ROR1 isoform was associated with a progressive disease stage. Targeting ROR1 by mAbs induced specific dephosphorylation and leukemic cell death. ROR1 might be an interesting therapeutic target.

The PI3K/AKT/mTOR pathway is involved in direct apoptosis of CLL cells induced by ROR1 monoclonal antibodies.

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Dishevelled proteins are significantly upregulated in chronic lymphocytic leukaemia

Dishevelled (DVL) proteins are components of the Wnt signalling pathways, and increased expression is associated with various malignancies. Information on DVLs in chronic lymphatic leukaemia (CLL) is limited. The aim of the present study was to investigate the role of DVLs in CLL cells and association with Wnt pathways downstream of ROR1. DVL1, 2 and 3 were exclusively expressed in CLL cells as compared to normal peripheral blood mononuclear cells (PBMCs). The expression of DVL1 and DVL3 proteins was significantly more pronounced in progressive than in non-progressive disease (p < 0.01), whereas the level of DVL2 was significantly higher in non-progressive as compared to progressive disease (p < 0.001). Treatment of CLL cells with anti-ROR1 specific monoclonal antibodies induced dephosphorylation of ROR1 as well as of tyrosine and serine residues of both DVL2 and DVL3. However, gene silencing of DVLs in the CLL cell line (EHEB) did not induce detectable apoptosis. Non-progressive CLL patients had a different protein activity pattern with regard to Wnt signalling pathway proteins as GSK-3β, β-catenin and AKT as compared to progressive disease. The DVL2 protein may play a role in the activation of signalling pathways in CLL during early stages of the disease, while DVL1 and 3 may have a role in later phases of the leukaemia.

Spontaneous Immunity Against the Receptor Tyrosine Kinase ROR1 in Patients with Chronic Lymphocytic Leukemia

Background ROR1 is a receptor tyrosine kinase expressed in chronic lymphocytic leukemia (CLL) and several other malignancies but absent in most adult normal tissues. ROR1 is considered an onco-fetal antigen. In the present study we analysed spontaneous humoral and cellular immunity against ROR1 in CLL patients. Materials and Methods Antibodies against ROR1 were analysed in 23 patients and 20 healthy donors by ELISA and Western blot. Purified serum IgG from patients was tested for cytotoxicity against CLL cells using the MTT viability assay. A cellular immune response against ROR1 derived HLA-A2 restricted 9 aa and 16 aa long peptides were analysed using peptide loaded dendritic cells co-cultured with autologous T cells from CLL patients (n = 9) and healthy donors (n = 6). IFN-γ, IL-5 and IL-17A-secreting T cells were assessed by ELISPOT and a proliferative response using a H3-thymidine incorporation assay. Results The majority of CLL patients had antibodies against ROR1. Significantly higher titers of anti-ROR1 antibodies were noted in patients with non-progressive as compared to progressive disease. The extracellular membrane-close ROR1 KNG domain seemed to be an immunodominant epitope. Ten patients with high titers of anti-ROR1 binding antibodies were tested for cytotoxicity. Five of those had cytotoxic anti-ROR1 antibodies against CLL cells. ROR1-specific IFN-γ and IL-17A producing T cells could be detected in CLL patients, preferentially in non-progressive as compared to patients with progressive disease (p<0.05). Conclusion ROR1 seemed to spontaneously induce a humoral as well as a T cell response in CLL patients. The data support the notion that ROR1 might be a specific neo-antigen and may serve as a target for immunotherapy.