ROR1-a novel receptor tyrosine kinase with unique therapeutic potentials in chronic lymphocytic leukemia

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

Wnt5a induces ROR1 to complex with HS1 to enhance migration of chronic lymphocytic leukemia cells.

ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a conserved, oncoembryonic surface antigen expressed in chronic lymphocytic leukemia (CLL). We found that ROR1 associates with hematopoietic-lineage-cell-specific protein 1 (HS1) in freshly isolated CLL cells or in CLL cells cultured with exogenous Wnt5a. Wnt5a also induced HS1 tyrosine phosphorylation, recruitment of ARHGEF1, activation of RhoA and enhanced chemokine-directed migration; such effects could be inhibited by cirmtuzumab, a humanized anti-ROR1 mAb. We generated truncated forms of ROR1 and found its extracellular cysteine-rich domain or kringle domain was necessary for Wnt5a-induced HS1 phosphorylation. Moreover, the cytoplamic, and more specifically the proline-rich domain (PRD), of ROR1 was required for it to associate with HS1 and allow for F-actin polymerization in response to Wnt5a. Accordingly, we introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1 PRD at positions 784, 808, 826, 841 or 850 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1P→︀A mutants, ROR1P(841)A had impaired capacity to recruit HS1 and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate HS1 or activate ARHGEF1, and was unable to enhance CLL-cell motility. Collectively, these studies indicate HS1 plays an important role in ROR1-dependent Wnt5a-enhanced chemokine-directed leukemia-cell migration

Small-molecule inhibitors of the receptor tyrosine kinases : promising tools for targeted cancer therapies

Chemotherapeutic and cytotoxic drugs are widely used in the treatment of cancer. In spite of the improvements in the life quality of patients, their effectiveness is compromised by several disadvantages. This represents a demand for developing new effective strategies with focusing on tumor cells and minimum side effects. Targeted cancer therapies and personalized medicine have been defined as a new type of emerging treatments. Small molecule inhibitors (SMIs) are among the most effective drugs for targeted cancer therapy. The growing number of approved SMIs of receptor tyrosine kinases (RTKs) i.e., tyrosine kinase inhibitors (TKIs) in the clinical oncology imply the increasing attention and application of these therapeutic tools. Most of the current approved RTK–TKIs in preclinical and clinical settings are multi-targeted inhibitors with several side effects. Only a few specific/selective RTK–TKIs have been developed for the treatment of cancer patients. Specific/selective RTK–TKIs have shown less deleterious effects compared to multi-targeted inhibitors. This review intends to highlight the importance of specific/selective TKIs for future development with less side effects and more manageable agents. This article provides an overview of: (1) the characteristics and function of RTKs and TKIs; (2) the recent advances in the improvement of specific/selective RTK–TKIs in preclinical or clinical settings; and (3) emerging RTKs for targeted cancer therapies by TKIs.Felix Mindus foundationPublishe

Comparative protein interaction network analysis identifies shared and distinct functions for the human ROCO proteins

Signal transduction cascades governed by kinases and GTPases are a critical component of the command and control of cellular processes, with the precise outcome partly determined by direct protein‐protein interactions (PPIs). Here, we use the human ROCO proteins as a model for investigating PPI signalling events – taking advantage of the unique dual kinase/GTPase activities and scaffolding properties of these multidomain proteins. We report PPI networks that encompasses the human ROCO proteins, developed using two complementary approaches. First, using our recently developed weighted PPI network analysis (WPPINA) pipeline, a confidence‐weighted overview of validated ROCO protein interactors was obtained from peer‐reviewed literature. Second, novel ROCO PPIs were assessed experimentally via protein microarray screens. We compared the networks derived from these orthologous approaches to identify common elements within the ROCO protein interactome; functional enrichment analysis of this common core of the network identified stress response and cell projection organisation as shared functions within this protein family. Despite the presence of these commonalities, our results suggest that many unique interactors and therefore some specialised cellular roles have evolved for different members of the ROCO proteins. Overall, this multi‐approach strategy to increase the resolution of protein interaction networks represents a prototype for the utility of PPI data integration in understanding signalling biology

Therapeutic Potential and Challenges of Targeting Receptor Tyrosine Kinase ROR1 with Monoclonal Antibodies in B-Cell Malignancies

Based on its selective cell surface expression in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), receptor tyrosine kinase ROR1 has recently emerged as a promising target for therapeutic monoclonal antibodies (mAbs). To further assess the suitability of ROR1 for targeted therapy of CLL and MCL, a panel of mAbs was generated and its therapeutic utility was investigated.A chimeric rabbit/human Fab library was generated from immunized rabbits and selected by phage display. Chimeric rabbit/human Fab and IgG1 were investigated for their capability to bind to human and mouse ROR1, to mediate antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and internalization, and to agonize or antagonize apoptosis using primary CLL cells from untreated patients as well as MCL cell lines. A panel of mAbs demonstrated high affinity and specificity for a diverse set of epitopes that involve all three extracellular domains of ROR1, are accessible on the cell surface, and mediate internalization. The mAb with the highest affinity and slowest rate of internalization was found to be the only mAb that mediated significant, albeit weak, ADCC. None of the mAbs mediated CDC. Alone, they did not enhance or inhibit apoptosis.Owing to its relatively low cell surface density, ROR1 may be a preferred target for armed rather than naked mAbs. Provided is a panel of fully sequenced and thoroughly characterized anti-ROR1 mAbs suitable for conversion to antibody-drug conjugates, immunotoxins, chimeric antigen receptors, and other armed mAb entities for preclinical and clinical studies

ROR1 Is Expressed in Human Breast Cancer and Associated with Enhanced Tumor-Cell Growth

Receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is expressed during embryogenesis and by certain leukemias, but not by normal adult tissues. Here we show that the neoplastic cells of many human breast cancers express the ROR1 protein and high-level expression of ROR1 in breast adenocarcinoma was associated with aggressive disease. Silencing expression of ROR1 in human breast cancer cell lines found to express this protein impaired their growth in vitro and also in immune-deficient mice. We found that ROR1 could interact with casein kinase 1 epsilon (CK1ε) to activate phosphoinositide 3-kinase-mediated AKT phosphorylation and cAMP-response-element-binding protein (CREB), which was associated with enhanced tumor-cell growth. Wnt5a, a ligand of ROR1, could induce ROR1-dependent signaling and enhance cell growth. This study demonstrates that ROR1 is expressed in human breast cancers and has biological and clinical significance, indicating that it may be a potential target for breast cancer therapy

Stat3 Activates the Receptor Tyrosine Kinase Like Orphan Receptor-1 Gene in Chronic Lymphocytic Leukemia Cells

BACKGROUND: The receptor tyrosine kinase like orphan receptor (ROR)-1 gene is overexpressed in chronic lymphocytic leukemia (CLL). Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors gamma-interferon activation sequence-like elements typically activated by Stat3, we hypothesized that Stat3 activates ROR1. METHODOLOGY/PRINCIPAL FINDINGS: Because IL-6 induced Stat3 phosphorylation and upregulated Ror1 protein levels in MM1 cells, we used these cells as a model. We transfected MM1 cells with truncated ROR1 promoter luciferase reporter constructs and found that IL-6 induced luciferase activity of ROR1-195 and upstream constructs. Co-transfection with Stat3 siRNA reduced the IL-6-induced luciferase activity, suggesting that IL-6 induced luciferase activity by activating Stat3. EMSA and the ChIP assay confirmed that Stat3 binds ROR1, and EMSA studies identified two Stat3 binding sites. In CLL cells, EMSA and ChIP studies determined that phosphorylated Stat3 bound to the ROR1 promoter at those two ROR1 promoter sites, and ChIP analysis showed that Stat3 co-immunoprecipitated DNA of STAT3, ROR1, and several Stat3-regulated genes. Finally, like STAT3-siRNA in MM1 cells, STAT3-shRNA downregulated STAT3, ROR1, and STAT3-regulated genes and Stat3 and Ror1 protein levels in CLL cells. CONCLUSION/SIGNIFICANCE: Our data suggest that constitutively activated Stat3 binds to the ROR1 promoter and activates ROR1 in CLL cells

INVESTIGATION OF TRIBOLOGICAL CHARACTERISTICS OF AL/NANO SiO2NANOCOMPOSITES PRODUCED BY ACCUMULATIVE ROLL BONDING (ARB) PROCESS

Accumulative roll-bonding process (ARB) is an important severe plastic deformation technique for production of the ultrafine grained, nanostructured and nanocomposite materials in the form of plates and sheets. In the present work, this process used for manufacturing Al/SiO 2 nanocomposites by using Aluminum 1050 alloy sheets and nano sized SiO 2 particles, at ambient temperature. After 8 cycles of ARB process, the tribological properties and wear resistance of produced nanocomposites were investigated. The wear tests by abrasion were performed in a pinon-disc tribometer. Results show that by increasing ARB cycles and the amount of nano powders, the friction coefficient of produced nanocomposites decreases