A Small Molecule Targeting the Intracellular Tyrosine Kinase Domain of ROR1 (KAN0441571C) Induced Significant Apoptosis of Non-Small Cell Lung Cancer (NSCLC) Cells.

The ROR1 receptor tyrosine kinase is expressed in embryonic tissues but is absent in normal adult tissues. ROR1 is of importance in oncogenesis and is overexpressed in several cancers, such as NSCLC. In this study, we evaluated ROR1 expression in NSCLC patients (N = 287) and the cytotoxic effects of a small molecule ROR1 inhibitor (KAN0441571C) in NSCLC cell lines. ROR1 expression in tumor cells was more frequent in non-squamous (87%) than in squamous (57%) carcinomas patients, while 21% of neuroendocrine tumors expressed ROR1 (p = 0.0001). A significantly higher proportion of p53 negative patients in the ROR1+ group than in the p53 positive non-squamous NSCLC patients (p = 0.03) was noted. KAN0441571C dephosphorylated ROR1 and induced apoptosis (Annexin V/PI) in a time- and dose-dependent manner in five ROR1+ NSCLC cell lines and was superior compared to erlotinib (EGFR inhibitor). Apoptosis was confirmed by the downregulation of MCL-1 and BCL-2, as well as PARP and caspase 3 cleavage. The non-canonical Wnt pathway was involved. The combination of KAN0441571C and erlotinib showed a synergistic apoptotic effect. KAN0441571C also inhibited proliferative (cell cycle analyses, colony formation assay) and migratory (scratch wound healing assay) functions. Targeting NSCLC cells by a combination of ROR1 and EGFR inhibitors may represent a novel promising approach for the treatment of NSCLC patients.

The RNA editor ADAR2 promotes immune cell trafficking by enhancing endothelial responses to interleukin-6 during sterile inflammation.

Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.

Targeting the Receptor Tyrosine Kinase ROR1 by Small Molecules.

Receptor tyrosine kinases (RTKs) are frequently dysregulated in malignancies and important for the malignant characteristics of tumor cells. RTKs are attractive structures for drug targeting of cancer. The RTK ROR1 is of significance during embryogenesis but downregulated in post-partum tissues. However, ROR1 is overexpressed in several hematological and solid tumors and important for tumor cell proliferation, survival, migration, and metastasis. WNT5a is a main ligand for ROR1. Several clinical trials are ongoing using anti-ROR1 antibody based drugs directed against the external domain (monoclonal antibodies, BiTE, CAR-T). We have produced small molecules (KAN834/1571c) fitting to the ATP pocket of the intracellular tyrosine kinase (TK) domain of ROR1 (TK inhibitor, TKI). These inhibitors of ROR1 prevented ROR1 phosphorylation and inactivated the WNT/ß-catenin independent as well as WNT/ß-catenin dependent pathways. ROR1-TKI induced apoptosis of ROR1 positive fresh patient derived tumor cells and appropriate cell lines and a dose and time dependent tumor reduction in animal models. In combination with other clinically relevant targeting drugs as venetoclax a synergistic apoptotic effect was seen. Two other small molecules (ARI-1 and strictinin) bound also to ROR1 and inhibited tumor growth. Development of small molecule ROR1 inhibitors is warranted to include this novel therapeutic approach for cancer therapy.

ROR1 Is Expressed in Diffuse Large B-Cell Lymphoma (DLBCL) and a Small Molecule Inhibitor of ROR1 (KAN0441571C) Induced Apoptosis of Lymphoma Cells.

The receptor tyrosine kinase ROR1 is absent in most normal adult tissues, but overexpressed in several malignancies. In this study, we explored clinical and functional inhibitory aspects of ROR1 in diffuse large B-cell lymphoma (DLBCL). ROR1 expression in tumor cells was more often observed in primary refractory DLBCL, Richter's syndrome and transformed follicular lymphoma than in relapsed and non-relapsed DLBCL patients (p < 0.001). A survival effect of ROR1 expression was preliminarily observed in relapsed/refractory patients independent of gender and stage but not of age, cell of origin and international prognostic index. A second generation small molecule ROR1 inhibitor (KAN0441571C) induced apoptosis of ROR1+ DLBCL cell lines, similar to venetoclax (BCL-2 inhibitor) but superior to ibrutinib (BTK inhibitor). The combination of KAN0441571C and venetoclax at EC50 concentrations induced almost complete killing of DLBCL cell lines. Apoptosis was accompanied by the downregulation of BCL-2 and MCL-1 and confirmed by the cleavage of PARP and caspases 3, 8, 9. PI3Kδ/AKT/mTOR (non-canonical Wnt pathway) as well as ß-catenin and CK1δ (canonical pathway) were inactivated. In zebra fishes transplanted with a ROR1+ DLBCL cell line, KAN0441571C induced a significant tumor reduction. New drugs with mechanisms of action other than those available for DLBCL are warranted. ROR1 inhibitors might represent a novel promising approach.

A receptor tyrosine kinase ROR1 inhibitor (KAN0439834) induced significant apoptosis of pancreatic cells which was enhanced by erlotinib and ibrutinib.

There is a great unmet medical need in pancreatic carcinoma (PC) for novel drugs with other mechanisms of action than existing. PC cells express the onco-fetal RTK ROR1, absent on most normal post-partem cells. ROR1 is involved in proliferation, survival, EMT and metastasis of tumor cells in various malignancies. A small molecule inhibitor (KAN0439834) (530 Da) targeting the TK domain of ROR1 was developed and the activity in ROR1 expressing human PC cell lines (n = 8) evaluated. The effects were compared to a murine mAb against the external part of ROR1, gemcitabine, erlotinib and ibrutinib. KAN0439834 induced significant apoptosis of the tumor cells. EC50 values for KAN0439834 varied between 250-650 nM depending on the cell line. The corresponding values for erlotinib and ibrutinib were 10-40 folds higher. KAN0439834 was much more effective in inducing tumor cell death than the ROR1 mAb although both inhibited ROR1 phosphorylation and downstream non-canonical Wnt pathway molecules. Combination of KAN0439834 with erlotinib or ibrutinib had significant additive effects on tumor cell death. A first-in-class small molecule ROR1 inhibitor (KAN0439834) showed promising in vitro activity against a number of human PC cell lines. Interesting is the additive effects of erlotinib and ibrutinib which warrants further studies as both these agents are in clinical trials for pancreatic carcinoma.

Autologous T cells expressing the oncogenic transcription factor KLF6-SV1 prevent apoptosis of chronic lymphocytic leukemia cells.

Crosstalk between leukemic cells and the tumor microenvironment is of importance in chronic lymphocytic leukemia (CLL). T cells seem to sustain the survival of CLL cells by various mechanisms. The Krüppel-like family of transcription factors (KLFs) are identified as regulators of proliferation and cell death. In the present study, we analyzed the expression of the wild type (WT) gene KLF6 and the oncogenic splice variant 1 (KLF6-SV1) at the mRNA level in subsets of T cells from CLL patients (n = 29), multiple myeloma patients (n = 6) and normal donors (n = 10). RNA Silencing was used for wtKLF6 and KLF6-SV1. Tumor cell apoptosis was measured. A significant overexpression of wtKLF6 and KLF6-SV1 in T cells of CLL patients compared to normal donors and myeloma patients was noted (p<0.002). Western blot showed that both wtKLF6 and KLF6-SV1 were expressed in purified T cells from CLL patients. KLF6-SV1 siRNA transfection induced a significant down-regulation of KLF6-SV1 in CLL T cells, which lost the capability to sustain the growth of leukemic cells. However, no such a significant effect was seen after wtKLF6 transfection of the autologous T cells. The results suggest that KLF6-SV1 may play a role in the regulation of survival CLL cells.

Flavin-containing monooxygenase 3 (FMO3) role in busulphan metabolic pathway.

Busulphan (Bu) is an alkylating agent used in the conditioning regimen prior to hematopoietic stem cell transplantation (HSCT). Bu is extensively metabolized in the liver via conjugations with glutathione to form the intermediate metabolite (sulfonium ion) which subsequently is degraded to tetrahydrothiophene (THT). THT was reported to be oxidized forming THT-1-oxide that is further oxidized to sulfolane and finally 3-hydroxysulfolane. However, the underlying mechanisms for the formation of these metabolites remain poorly understood. In the present study, we performed in vitro and in vivo investigations to elucidate the involvement of flavin-containing monooxygenase-3 (FMO3) and cytochrome P450 enzymes (CYPs) in Bu metabolic pathway. Rapid clearance of THT was observed when incubated with human liver microsomes. Furthermore, among different recombinant microsomal enzymes, the highest intrinsic clearance for THT was obtained via FMO3 followed by several CYPs including 2B6, 2C8, 2C9, 2C19, 2E1 and 3A4. In Bu- or THT-treated mice, inhibition of FMO3 by phenylthiourea significantly suppressed the clearance of both Bu and THT. Moreover, the simultaneous administration of a high dose of THT (200µmol/kg) to Bu-treated mice reduced the clearance of Bu. Consistently, in patients undergoing HSCT, repeated administration of Bu resulted in a significant up-regulation of FMO3 and glutathione-S-transfrase -1 (GSTA1) genes. Finally, in a Bu-treated patient, additional treatment with voriconazole (an antimycotic drug known as an FMO3-substrate) significantly altered the Bu clearance. In conclusion, we demonstrate for the first time that FMO3 along with CYPs contribute a major part in busulphan metabolic pathway and certainly can affect its kinetics. The present results have high clinical impact. Furthermore, these findings might be important for reducing the treatment-related toxicity of Bu, through avoiding interaction with other concomitant used drugs during conditioning and hence improving the clinical outcomes of HSCT.

Vitamin D receptor activation reduces inflammatory cytokines and plasma MicroRNAs in moderate chronic kidney disease - a randomized trial.

BACKGROUND: Chronic kidney disease (CKD) is a major risk factor for cardiovascular disease (CVD), partly due to endothelial dysfunction and chronic inflammation. Vitamin D treatment in end stage renal disease is suggested to modulate the immune system and lead to improved outcomes. We and others have demonstrated that treatment with vitamin D or activated vitamin D analogues protects the endothelial function in less severe renal disease as well. Since the endothelial protection might be mediated by vitamin D effects on inflammation, we assessed levels of pro-inflammatory cytokines and micro RNAs (miRs) in patients with moderate CKD, treated with an active vitamin D analogue (paricalcitol). METHODS: Thirty-six patients with moderate CKD were randomized to 12 weeks treatment with placebo, 1 µg, or 2 µg paricalcitol daily. Cytokines were measured by Milliplex 26-plex. Total RNA was isolated from plasma and miRs were determined by quantitative reverse transcription PCR analysis. RESULTS: Selected pro-inflammatory cytokines decreased significantly following treatment, while no change was observed in the placebo group. The micro RNAs; miR 432-5p, miR 495-3p, and miR 576-5p were significantly downregulated in the active treated groups, compared to the placebo group. CONCLUSION: Paricalcitol treatment for 12 weeks in patients with moderate CKD reduces cytokines and micro RNAs involved in atherosclerosis and inflammation. The potentially protective role of vitamin D receptor activation in the inflammatory processes regarding the long-term outcomes in CKD patients warrants further studies. TRIAL REGISTRATION: SOLID study; NCT01204528 , April 27, 2010.

Mono-epoxy-tocotrienol-α enhances wound healing in diabetic mice and stimulates in vitro angiogenesis and cell migration.

Diabetes mellitus is characterized by hyperglycemia and capillary hypoxia that causes excessive production of free radicals and impaired antioxidant defense, resulting in oxidative stress and diabetes complications such as impaired wound healing. We have previously shown that modified forms of tocotrienols possess beneficial effects on the biosynthesis of the mevalonate pathway lipids including increase in mitochondrial CoQ. The aim of this study is to investigate the effects of mono-epoxy-tocotrienol-α on in vitro and in vivo wound healing models as well as its effects on mitochondrial function. Gene profiling analysis and gene expression studies on HepG2 cells and human dermal fibroblasts were performed by microarray and qPCR, respectively. In vitro wound healing using human fibroblasts was studied by scratch assay and in vitro angiogenesis using human dermal microvascular endothelial cells was studied by the tube formation assay. In vivo wound healing was performed in the diabetic db/db mouse model. For the study of mitochondrial functions and oxygen consumption rate Seahorse XF-24 was employed. In vitro, significant increase in wound closure and cell migration (p<0.05) both in normal and high glucose and in endothelial tube formation (angiogenesis) (p<0.005) were observed. Microarray profiling analysis showed a 20-fold increase of KIF26A gene expression and 11-fold decrease of lanosterol synthase expression. Expression analysis by qPCR showed significant increase of the growth factors VEGFA and PDGFB. The epoxidated compound induced a significantly higher basal and reserve mitochondrial capacity in both HDF and HepG2 cells. Additionally, in vivo wound healing in db/db mice, demonstrated a small but significant enhancement on wound healing upon local application of the compound compared to treatment with vehicle alone. Mono-epoxy-tocotrienol-α seems to possess beneficial effects on wound healing by increasing the expression of genes involved in cell growth, motility and angiogenes as well as on mitochondrial function.

Differential expression of viral agents in lymphoma tissues of patients with ABC diffuse large B-cell lymphoma from high and low endemic infectious disease regions.

Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin's lymphoma (NHL) in adults, accounts for approximately 30-40% of newly diagnosed lymphomas worldwide. Environmental factors, such as viruses and bacteria, may contribute to cancer development through chronic inflammation and the integration of oncogenes, and have previously been indicated in cervical cancer, hepatocellular carcinoma, gastric cancer and lymphoproliferative disorders. In the present study, the presence of microbial agents was analyzed in the lymphoma tissue of patients with activated B-cell like (ABC) DLBCL. The present study compared two groups of patients from geographically varied regions that possess a difference in the prevalence of viral and other microbial agents. The patient populations were from Sweden (a low endemic infectious disease region) and Egypt (a high endemic infectious disease region). A differential expression of several viruses in lymphoma tissues was noted when comparing Swedish and Egyptian patients. JC polyomavirus (JCV) was detected in Swedish and Egyptian patients and, uniquely, the complete hepatitis B virus (HBV) genome was detected only in Egyptian lymphoma patients. None of these viruses were detected in control lymph tissues from Sweden or Egypt. In total, 38% of the Egyptian patients were found to have HBV surface antigens (HBsAgs) in their serum; however, HBsAgs were not found in any of the Swedish patients. The percentage of serum HBsAgs in Egyptian patients with ABC DLBCL was significantly increased compared with the general Egyptian population (P<0.05). The present study may support a notion that viral agents, including JCV and HBV, may be involved in the tumorigenesis of DLBCL in regions of high infectious disease.

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-3ß, ß-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.

Cytochrome P450 Oxidoreductase Influences CYP2B6 Activity in Cyclophosphamide Bioactivation.

INTRODUCTION: Cyclophosphamide is commonly used as an important component in conditioning prior to hematopoietic stem cell transplantation, a curative treatment for several hematological diseases. Cyclophosphamide is a prodrug activated mainly by cytochrome P450 2B6 (CYP2B6) in the liver. A high degree of inter- and intra-individual variation in cyclophosphamide kinetics has been reported in several studies. MATERIALS AND METHODS: Hydroxylation of cyclophosphamide was investigated in vitro using three microsomal batches of CYP2B6*1 with different ratios of POR/CYP expression levels. Twenty patients undergoing hematopoietic stem cell transplantation were also included in the study. All patients received an i.v. infusion of cyclophosphamide (60 mg/kg/day, for two days) as a part of their conditioning. Blood samples were collected from each patient before cyclophosphamide infusion, 6 h after the first dose and before and 6 h after the second dose. POR gene expression was measured by mRNA analysis and the pharmacokinetics of cyclophosphamide and its active metabolite were determined. RESULTS: A strong correlation between the in vitro intrinsic clearance of cyclophosphamide and the POR/CYP ratio was found. The apparent Km for CYP2B6.1 was almost constant (3-4 mM), while the CLint values were proportional to the POR/CYP ratio (3-34 µL/min/nmol CYP). In patients, the average expression of the POR gene in blood was significantly (P <0.001) up-regulated after cyclophosphamide infusion, with high inter-individual variations and significant correlation with the concentration ratio of the active metabolite 4-hydroxy-cyclophosphamide/cyclophosphamide. Nine patients were carriers for POR*28; four patients had relatively high POR expression. CONCLUSIONS: This investigation shows for the first time that POR besides CYP2B6 can influence cyclophosphamide metabolism. Our results indicate that not only CYPs are important, but also POR expression and/or activity may influence cyclophosphamide bioactivation, affecting therapeutic efficacy and treatment related toxicity and hence on clinical outcome. Thus, both POR and CYP genotype and expression levels may have to be taken into account when personalizing treatment schedules to achieve optimal therapeutic drug plasma concentrations of cyclophosphamide.

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.

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.

Cyclophosphamide alters the gene expression profile in patients treated with high doses prior to stem cell transplantation.

BACKGROUND: Hematopoietic stem cell transplantation is a curative treatment for several haematological malignancies. However, treatment related morbidity and mortality still is a limiting factor. Cyclophosphamide is widely used in condition regimens either in combination with other chemotherapy or with total body irradiation. METHODS: We present the gene expression profile during cyclophosphamide treatment in 11 patients conditioned with cyclophosphamide for 2 days followed by total body irradiation prior to hematopoietic stem cell transplantation. 299 genes were identified as specific for cyclophosphamide treatment and were arranged into 4 clusters highly down-regulated genes, highly up-regulated genes, early up-regulated but later normalized genes and moderately up-regulated genes. RESULTS: Cyclophosphamide treatment down-regulated expression of several genes mapped to immune/autoimmune activation and graft rejection including CD3, CD28, CTLA4, MHC II, PRF1, GZMB and IL-2R, and up-regulated immune-related receptor genes, e.g. IL1R2, IL18R1, and FLT3. Moreover, a high and significant expression of ANGPTL1 and c-JUN genes was observed independent of cyclophosphamide treatment. CONCLUSION: This is the first investigation to provide significant information about alterations in gene expression following cyclophosphamide treatment that may increase our understanding of the cyclophosphamide mechanism of action and hence, in part, avoid its toxicity. Furthermore, ANGPTL1 remained highly expressed throughout the treatment and, in contrast to several other alkylating agents, cyclophosphamide did not influence c-JUN expression.

Expression of microRNA-1234 related signal transducer and activator of transcription 3 in patients with diffuse large B-cell lymphoma of activated B-cell like type from high and low infectious disease areas.

Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, and infectious agents are suspected to be involved in the tumorigenesis of DLBCL. MicroRNAs (miRNAs) are non-coding RNAs modulating protein expression. We compared miRNA expression profiles in lymph node tissues of patients with DLBCL of the activated B-cell like (ABC) type from two geographical areas with different background exposures, Sweden and Egypt. We showed previously that DLBCL tissues of the ABC-type in Swedish patients had a higher expression of signal transducer and activator of transcription 3 (STAT3) compared to Egyptian patients. Here, we analyzed the involvement of miRNAs in STAT3 regulation. miR-1234 was significantly up-regulated in Egyptian patients with DLBCL compared to Swedish patients (p < 0.03). The miR-1234 expression level correlated inversely with the expression of STAT3. The Stat3 protein was down-regulated in cells transfected with miR-1234, suggesting that STAT3 might be a potential target for miR-1234. miR-1234 and STAT3 might be involved in the tumorigenesis of DLBCL of ABC type and possibly associated with environmental background exposure.

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.

Leukocyte proliferation and immune modulator production in patients with chronic kidney disease.

INTRODUCTION: In Chronic Kidney Disease (CKD), immune cells are affected by uremic retention toxins. Given this effect, we analyzed lymphocyte proliferative response and immune modulators production following in vitro stimulation. METHODS: Whole blood was drawn from healthy controls, patients with eGFR <20 ml/min/1.73 m(2) (Pre-dialysis, CKD stages 4 and 5) and hemodialysis patients (stage 5D). Peripheral cells were incubated for six days with pokeweed mitogen, concanavalin A, Staphylococcus enterotoxin A or influenza A vaccine. Peripheral lymphocyte proliferation was then analyzed by the "Flow-cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood" (FASCIA) method, and cytokine profile in the cell supernatants was analyzed by the Milliplex multi-array method. RESULTS: The absolute number of lymphoblasts in response to mitogenic stimulation and the number of cells in each CD4+ and CD8+ subpopulation were similar comparing the three groups, except for a single decline in number of lymphoblasts after stimulation with Staphylococcus enterotoxin A, comparing dialysis patients with healthy controls. Levels of interleukin (IL)-2 (p=0.026), -10 (p=0.019) and -15 (p=0.027) in the Staphylococcus enterotoxin A-stimulated supernatant were lower in hemodialysis patients compared to healthy controls. Levels of IL-15 (p=0.017) from pre-dialysis patients and levels of IL-5 (p=0.019) from hemodialysis patients in influenza A vaccine-stimulated supernatants were also lower compared to controls. In pokeweed mitogen-stimulated supernatant, IL-2 levels (p=0.013) were lower in hemodialysis patients compared to pre-dialysis patients. TNF-α, IL-10, IL-12, IL-15, IL-8, MCP-1, IP-10, IFN-α2, IL-1α and eotaxin levels were all significantly higher in plasma obtained from CKD patients. CONCLUSION: Our results suggest that T-cells from CKD patients have similar proliferative response to stimulation compared with healthy individuals. Moreover, however the immune cells show inability to produce selected cytokines, most likely due to the uremic milieu or dialysis procedure.

Activation of Wnt/ß-catenin pathway in monocytes derived from chronic kidney disease patients.

Patients with chronic kidney disease (CKD) have significantly increased morbidity and mortality resulting from infections and cardiovascular diseases. Since monocytes play an essential role in host immunity, this study was directed to explore the gene expression profile in order to identify differences in activated pathways in monocytes relevant to the pathophysiology of atherosclerosis and increased susceptibility to infections. Monocytes from CKD patients (stages 4 and 5, estimated GFR <20 ml/min/1.73 m(2)) and healthy donors were collected from peripheral blood. Microarray gene expression profile was performed and data were interpreted by GeneSpring software and by PANTHER tool. Western blot was done to validate the pathway members. The results demonstrated that 600 and 272 genes were differentially up- and down regulated respectively in the patient group. Pathways involved in the inflammatory response were highly expressed and the Wnt/ß-catenin signaling pathway was the most significant pathway expressed in the patient group. Since this pathway has been attributed to a variety of inflammatory manifestations, the current findings may contribute to dysfunctional monocytes in CKD patients. Strategies to interfere with this pathway may improve host immunity and prevent cardiovascular complications in CKD patients.