| 1. |
- Daneshmanesh, Amir Hossein
(författare)
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ROR1 : a novel receptor tyrosine kinase with unique therapeutic potentials in chronic lymphocytic leukemia
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of small B lymphocytes in blood, bone marrow, lymph nodes and other lymphoid tissues. CLL is the most common leukemia in the Western world. Despite significant advances in understanding the pathogenesis, CLL is still a disease with no available cure. Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors participating in crucial cellular processes including proliferation, differentiation, cell-cell interaction, metabolism, signaling, migration and cell survival. More than half of the RTK families are overexpressed or mutated in different forms of human cancer. RTKs are multifunctional therapeutic targets and novel RTKs in cancer have been pursued extensively as a goal for targeted therapies. ROR1 belongs to one of twenty families of RTKs. It is a survival kinase and acts as a receptor for Wnt5a protein. Gene expression profiling studies have shown that ROR1 was upregulated in CLL patients. Characterization of ROR1 expression in CLL and the study of its functional role for possible therapeutic targeting was the driving force of this thesis. In the first study we investigated the expression pattern of ROR1 in CLL. All CLL patients (n=18) expressed ROR1 both at gene and protein levels but none of the healthy donors. CLL patients showed a ROR1 surface expression in the range of 36–92%. Western blot analysis revealed two ROR1 bands of 105 and 130 kDa. Mutation analysis of the ROR1 gene showed no major genomic aberrations. FISH analysis of PBMC from 3 CLL patients showed no rearrangement in the 1p region. The second study was conducted to examine the effects of siRNAs specifically silencing ROR1 and fibromodulin (FMOD) in CLL cells. siRNA treatment induced a specific reduction (75–95%) in FMOD and ROR1 mRNA expression. Western blot analysis for ROR1 and FMOD demonstrated that the proteins were significantly downregulated 48 h after siRNA treatment. Silencing of FMOD and ROR1 resulted in a statistically significant apoptosis of CLL cells but not of B cells from normal donors. In the third study, five ROR1 monoclonal antibodies were raised against extracellular domains of ROR1 to investigate the in vitro apoptotic effects on CLL cells. All five mAbs induced apoptosis of CLL but not of normal B cells in the absence of complement or immune effector cells. Most effective were mAbs against CRD and KNG, being superior to rituximab in vitro. Cross-linking of the anti-ROR1 mAbs using F(ab ́)2 fragments of anti-Fc antibodies significantly augmented apoptosis. Two of the mAbs induced complement-dependent cytotoxicity similar to that of rituximab. The fourth study was aimed at investigating ROR1 and ROR2 expression in hematological malignancies of lymphoid and myeloid origins. The results showed a statistically significant variation in the expression of ROR1 in various hematological malignancies. No expression of ROR2 was detected in hematological malignancies tested and PBMC of healthy donors. A statistically significant higher expression of ROR1 was detected in progressive compared to non-progressive CLL patients. ROR1 expression was shown to be stable overtime. In conclusion ROR1 was found to be ectopically expressed in CLL. Given the successful history of RTKs targeted therapies in cancer, ROR1 might be a novel potential therapeutic target structure in CLL.
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| 3. |
- Hojjat-Farsangi, Mohammad, et al.
(författare)
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RETRACTED: Inhibition of the receptor tyrosine kinase ROR1 by anti-ROR1 monoclonal antibodies and siRNA induced apoptosis of melanoma cells
- 2013
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Ingår i: PLOS ONE. - Stockholm : Karolinska Institutet, Dept of Oncology-Pathology. - 1932-6203.
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Tidskriftsartikel (refereegranskat)abstract
- RETRACTED ARTICLE. Retraction: PLoS One. 2022 May 5;17(5):e0268357. 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.
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| 4. |
- Hojjat-Farsangi, Mohammad, et al.
(författare)
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Spontaneous immunity against the receptor tyrosine kinase ROR1 in patients with chronic lymphocytic leukemia
- 2015
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Ingår i: PLOS One. - Stockholm : Karolinska Institutet, Dept of Oncology-Pathology. - 1932-6203.
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Tidskriftsartikel (refereegranskat)abstract
- 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 antiROR1 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.
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| 5. |
- Hojjat-Farsangi, Mohammad, et al.
(författare)
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The tyrosine kinase receptor ROR1 is constitutively phosphorylated in chronic lymphocytic leukemia (CLL) cells
- 2013
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Ingår i: PLOS ONE. - Stockholm : Karolinska Institutet, Dept of Oncology-Pathology. - 1932-6203.
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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