Novel antibodies directed against the human erythropoietin receptor: creating a basis for clinical implementation.

Recombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been controversial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that several of these specifically recognize EPOR by Western blot, immunoprecipitation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment.

The matricellular protein CCN3 regulates NOTCH1 signalling in chronic myeloid leukaemia.

Deregulated NOTCH1 has been reported in lymphoid leukaemia, although its role in chronic myeloid leukaemia (CML) is not well established. We previously reported BCR-ABL down-regulation of a novel haematopoietic regulator, CCN3, in CML; CCN3 is a non-canonical NOTCH1 ligand. This study characterizes the NOTCH1­CCN3 signalling axis in CML. In K562 cells, BCR-ABL silencing reduced full-length NOTCH1 (NOTCH1-FL) and inhibited the cleavage of NOTCH1 intracellular domain (NOTCH1-ICD), resulting in decreased expression of the NOTCH1 targets c-MYC and HES1. K562 cells stably overexpressing CCN3 (K562/CCN3) or treated with recombinant CCN3(rCCN3) showed a significant reduction in NOTCH1 signalling (> 50% reduction in NOTCH1-ICD, p < 0.05).Gamma secretase inhibitor (GSI), which blocks NOTCH1 signalling, reduced K562/CCN3 colony formation but increased that of K562/control cells. GSI combined with either rCCN3 or imatinib reduced K562 colony formation with enhanced reduction of NOTCH1 signalling observed with combination treatments. We demonstrate an oncogenic role for NOTCH1 in CML and suggest that BCR-ABL disruption of NOTCH1­CCN3 signalling contributes to the pathogenesis of CML.

Bortezomib induces apoptosis in primitive chronic myeloid leukemia cells including LTC-IC and NOD/SCID repopulating cells.

Chronic myeloid leukemia (CML) is treated effectively with tyrosine kinase inhibitors (TKIs); however, 2 key problems remain-the insensitivity of CML stem and progenitor cells to TKIs and the emergence of TKI-resistant BCR-ABL mutations. BCR-ABL activity is associated with increased proteasome activity and proteasome inhibitors (PIs) are cytotoxic against CML cell lines. We demonstrate that bortezomib is antiproliferative and induces apoptosis in chronic phase (CP) CD34+ CML cells at clinically achievable concentrations. We also show that bortezomib targets primitive CML cells, with effects on CD34+38(-), long-term culture-initiating (LTC-IC) and nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells. Bortezomib is not selective for CML cells and induces apoptosis in normal CD34+38(-) cells. The effects against CML cells are seen when bortezomib is used alone and in combination with dasatinib. Bortezomib causes proteasome but not BCR-ABL inhibition and is also effective in inhibiting proteasome activity and inducing apoptosis in cell lines expressing BCR-ABL mutations, including T315I. By targeting both TKI-insensitive stem and progenitor cells and TKI-resistant BCR-ABL mutations, we believe that bortezomib offers a potential therapeutic option in CML. Because of known toxicities, including myelosuppression, the likely initial clinical application of bortezomib in CML would be in resistant and advanced disease.

Guidelines for the measurement of BCR-ABL1 transcripts in chronic myeloid leukaemia.

Molecular testing for the BCR-ABL1 fusion gene by real time quantitative polymerase chain reaction (RT-qPCR) is the most sensitive routine approach for monitoring the response to therapy of patients with chronic myeloid leukaemia. In the context of tyrosine kinase inhibitor (TKI) therapy, the technique is most appropriate for patients who have achieved complete cytogenetic remission and can be used to define specific therapeutic milestones. To achieve this effectively, standardization of the laboratory procedures and the interpretation of results are essential. We present here consensus best practice guidelines for RT-qPCR testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 21 testing laboratories in the United Kingdom and Ireland in accordance with the procedures of the UK Clinical Molecular Genetics Society.

The E3 ligase HUWE1 inhibition as a therapeutic strategy to target MYC in multiple myeloma.

Proteasome inhibitors have provided a significant advance in the treatment of multiple myeloma (MM). Consequently, there is increasing interest in developing strategies to target E3 ligases, de-ubiquitinases, and/or ubiquitin receptors within the ubiquitin proteasome pathway, with an aim to achieve more specificity and reduced side-effects. Previous studies have shown a role for the E3 ligase HUWE1 in modulating c-MYC, an oncogene frequently dysregulated in MM. Here we investigated HUWE1 in MM. We identified elevated expression of HUWE1 in MM compared with normal cells. Small molecule-mediated inhibition of HUWE1 resulted in growth arrest of MM cell lines without significantly effecting the growth of normal bone marrow cells, suggesting a favorable therapeutic index. Studies using a HUWE1 knockdown model showed similar growth inhibition. HUWE1 expression positively correlated with MYC expression in MM bone marrow cells and correspondingly, genetic knockdown and biochemical inhibition of HUWE1 reduced MYC expression in MM cell lines. Proteomic identification of HUWE1 substrates revealed a strong association of HUWE1 with metabolic processes in MM cells. Intracellular glutamine levels are decreased in the absence of HUWE1 and may contribute to MYC degradation. Finally, HUWE1 depletion in combination with lenalidomide resulted in synergistic anti-MM activity in both in vitro and in vivo models. Taken together, our data demonstrate an important role of HUWE1 in MM cell growth and provides preclinical rationale for therapeutic strategies targeting HUWE1 in MM.

Proteasome inhibitors: a therapeutic strategy for haematological malignancy.

The proteasome is a multicatalytic enzyme complex responsible for the regulated degradation of intracellular proteins. In recent years, inhibition of proteasome function has emerged as a novel anti-cancer therapy. Proteasome inhibition is now established as an effective treatment for relapsed and refractory multiple myeloma and offers great promise for the treatment of other haematological malignancies, when used in combination with conventional therapeutic agents. Bortezomib is the first proteasome inhibitor to be used clinically and a second generation of proteasome inhibitors with differential pharmacological properties are currently in early clinical trials. This review summarises the development of proteasome inhibitors as therapeutic agents and describes how novel assays for measuring proteasome activity and inhibition may help to further delineate the mechanisms of action of different proteasome inhibitors. This will allow for the optimized use of proteasome inhibitors in combination therapies and provide the opportunity to design more potent and therapeutically efficacious proteasome inhibitors.

Comparative selectivity and specificity of the proteasome inhibitors BzLLLCOCHO, PS-341, and MG-132.

The 26S proteasome is a multicatalytic protease responsible for regulated intracellular protein degradation. Its function is mediated by three main catalytic activities: (a) chymotrypsin-like (CT-L), (b) trypsin-like, and (c) peptidylglutamyl peptide hydrolysing (PGPH). Proteasome inhibition is an emerging therapy for many cancers and is a novel treatment for multiple myeloma. Here, we profile the contributions of the three catalytic activities in multiple myeloma cell lines and compare the specificity and cytotoxicity of the novel proteasome inhibitor BzLLLCOCHO and inhibitors PS-341 (Velcade, bortezomib) and MG-132. Using fluorogenic substrates and an active site-directed probe specific for proteasome catalytic subunits, we show differential subunit specificity for each of the inhibitors. Addition of BzLLLCOCHO strongly inhibited all three catalytic activities, treatment with PS-341 completely inhibited CT-L and PGPH activities, and treatment with MG-132 resulted in weak inhibition of the CT-L and PGPH activities. Multiple myeloma cells were more sensitive to induction of apoptosis by PS-341 and MG-132 than BzLLLCOCHO. This study emphasizes the need for further investigation of the effects of these compounds on gene and protein expression in the cell to allow for the development of more specific and targeted inhibitors.

TRIM proteins in blood cancers.

Post-translational modification of proteins with ubiquitin plays a central role in regulating numerous cellular processes. E3 ligases determine the specificity of ubiquitination by mediating the transfer of ubiquitin to substrate proteins. The family of tripartite motif (TRIM) proteins make up one of the largest subfamilies of E3 ligases. Accumulating evidence suggests that dysregulation of TRIM proteins is associated with a variety of diseases. In this review we focus on the involvement of TRIM proteins in blood cancers.

Separation and Enrichment of Hematopoietic Stem Cells for CCN Studies.

The regulation of blood cell production (hematopoiesis) by CCN proteins is an area of increasing interest to hematologists. There is some discordance in the literature in this area due to the use of mixed or ill-defined cell populations for experiments. Expression of, and response to, CCN proteins is specific to both cell type and differentiation status. Here, we describe methods to prepare defined hematopoietic cell populations and associated functional assays.

The role of the CCN family of proteins in blood cancers.

Haematopoiesis is the term used to describe the production of blood cells. This is a tightly regulated hierarchical system in which mature circulating blood cells develop from a small population of haematopoietic stem (HSC) and progenitor cells within the microenvironment of the bone marrow. Molecular and genetic abnormalities arising in these stem cells lead to a block in the normal programme of proliferation and differentiation and result in the development of the blood cancers known as the leukaemias and lymphomas. Recently the regulatory role of the bone marrow microenvironment or niche has also become increasingly recognised. The interface between the bone and bone marrow (endosteum) and the region surrounding the blood vessels (perivascular) provide distinct niches harbouring quiescent HSC or proliferative HSC respectively. Current chemotherapeutic regimes can often successfully target the proliferative HSC but disease relapse occurs due to residual quiescent HSC. Understanding these developmental and regulatory processes and the associated cell communication mechanisms are thus crucial to the development of new treatment strategies. The CCN family of proteins have been recognised to play a key role in all aspects of haematopoiesis.

Identification of the APC/C co-factor FZR1 as a novel therapeutic target for multiple myeloma.

Multiple Myeloma (MM) is a haematological neoplasm characterised by the clonal proliferation of malignant plasma cells in the bone marrow. The success of proteasome inhibitors in the treatment of MM has highlighted the importance of the ubiquitin proteasome system (UPS) in the pathogenesis of this disease. In this study, we analysed gene expression of UPS components to identify novel therapeutic targets within this pathway in MM. Here we demonstrate how this approach identified previously validated and novel therapeutic targets. In addition we show that FZR1 (Fzr), a cofactor of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C), represents a novel therapeutic target in myeloma. The APC/C associates independently with two cofactors, Fzr and Cdc20, to control cell cycle progression. We found high levels of FZR1 in MM primary cells and cell lines and demonstrate that expression is further increased on adhesion to bone marrow stromal cells (BMSCs). Specific knockdown of either FZR1 or CDC20 reduced viability and induced growth arrest of MM cell lines, and resulted in accumulation of APC/CFzr substrate Topoisomerase IIα (TOPIIα) or APC/CCdc20 substrate Cyclin B. Similar effects were observed following treatment with proTAME, an inhibitor of both APC/CFzr and APC/CCdc20. Combinations of proTAME with topoisomerase inhibitors, etoposide and doxorubicin, significantly increased cell death in MM cell lines and primary cells, particularly if TOPIIα levels were first increased through pre-treatment with proTAME. Similarly, combinations of proTAME with the microtubule inhibitor vincristine resulted in enhanced cell death. This study demonstrates the potential of targeting the APC/C and its cofactors as a therapeutic approach in MM.

Peptide Tyrosine Arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin secretions of the dusky gopher frog, Rana sevosa.

An octadecapeptide was isolated from the skin secretions of the dusky gopher frog (Rana sevosa) on the basis of histamine release from rat peritoneal mast cells. This peptide was purified to homogeneity by HPLC and found to have the following primary structure, YLKGCWTKSYPPKPCFSR, using both Edman degradation chemistry and peptide sequencing using high-resolution mass spectrometry (Q-TOF MS). The peptide, named peptide Tyrosine Arginine (pYR) shares 77.8% homology with peptide Leucine Arginine (pLR). The effects of the natural amidated peptide, non-amidated peptide and C-loop region of pYR on granulopoiesis and neutrophil apoptosis were investigated. All three analogues inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, the mature neutrophil. Thus, pYR is a novel member of an important and emerging new class of amphibian peptides with hemopoietic actions.

Stem cell quest.

Chronic Myeloid Leukaemia has been a valuable model system for experimental haematologists for many years. Virtually all patients (>95 %) have the same genetic change which has driven the development of the first targeted therapies, tyrosine kinase inhibitors (TKIs). Since the introduction of TKIs in 2000 it has become clear that this approach has significantly improved the outcome for these patients. Nevertheless drug resistance inevitably develops and it is clear that the disease is controlled rather than eradicated. The recent publication by Herrmann et al. has defined a sub-population of leukaemic stem cells which are responsible for propagating the disease. CD26 now provides a new specific target for the malignant stem cells and offers the possibility of true curative therapy.

The NOTCH signaling pathway in normal and malignant blood cell production.

The NOTCH pathway is an evolutionarily conserved signalling network, which is fundamental in regulating developmental processes in invertebrates and vertebrates (Gazave et al. in BMC Evol Biol 9:249, 2009). It regulates self-renewal (Butler et al. in Cell Stem Cell 6:251-264, 2010), differentiation (Auderset et al. in Curr Top Microbiol Immunol 360:115-134, 2012), proliferation (VanDussen et al. in Development 139:488-497, 2012) and apoptosis (Cao et al. in APMIS 120:441-450, 2012) of diverse cell types at various stages of their development. NOTCH signalling governs cell-cell interactions and the outcome of such responses is highly context specific. This makes it impossible to generalize about NOTCH functions as it stimulates survival and differentiation of certain cell types, whereas inhibiting these processes in others (Meier-Stiegen et al. in PLoS One 5:e11481, 2010). NOTCH was first identified in 1914 in Drosophila and was named after the indentations (notches) present in the wings of the mutant flies (Bigas et al. in Int J Dev Biol 54:1175-1188, 2010). Homologs of NOTCH in vertebrates were initially identified in Xenopus (Coffman et al. in Science 249:1438-1441, 1990) and in humans NOTCH was first identified in T-Acute Lymphoblastic Leukaemia (T-ALL) (Ellisen et al. in Cell 66:649-61, 1991). NOTCH signalling is integral in neurogenesis (Mead and Yutzey in Dev Dyn 241:376-389, 2012), myogenesis (Schuster-Gossler et al. in Proc Natl Acad Sci U S A 104:537-542, 2007), haematopoiesis (Bigas et al. in Int J Dev Biol 54:1175-1188, 2010), oogenesis (Xu and Gridley in Genet Res Int 2012:648207, 2012), differentiation of intestinal cells (Okamoto et al. in Am J Physiol Gastrointest Liver Physiol 296:G23-35, 2009) and pancreatic cells (Apelqvist et al. in Nature 400:877-881, 1999). The current review will focus on NOTCH signalling in normal and malignant blood cell production or haematopoiesis.

Proteasome proteolytic activity in hematopoietic cells from patients with chronic myeloid leukemia and multiple myeloma.

BACKGROUND AND OBJECTIVES: The proteasome is a multicatalytic complex found in all eukaryotic cells; it is responsible for the degradation of key regulatory proteins associated with the cell cycle and apoptosis. In vitro, proteasome inhibitors can induce selective apoptosis in some malignant cell types as opposed to in their normal counterparts and first generation compounds are currently in clinical trials for the treatment of multiple myeloma. The objective of our study was to develop a method to extract and measure functional proteasome activity in primary human cells so that this method could then be used to determine whether patients might benefit from proteasome inhibitor therapy. DESIGN AND METHODS: Optimal proteasome extraction and assay conditions were established with myeloma and leukemic cell lines. These conditions were then applied to primary human cells from patients. Proteasome was extracted using lysis buffer and activity measured as turnover of a peptide fluorescent substrate. RESULTS: Cells expressing bcr-abl showed significantly higher proteasome levels (372+/-16 AFU/1x10(6) cells/min) than did bcr-abl-negative cells (151+/- 8 AFU/1x10(6) cells/min) and were more sensitive to induction of apoptosis by proteasome inhibitor. Human myeloid leukemia cell lines showed higher levels of activity than those representing myeloma (eg HL-60 cells 947+/-25 AFU/1x10(6) cells/min; U266 177+/-6 AFU/1x10(6) cells/min). Primary cells from patients had similar levels of activity to those of the comparable cell line model. INTERPRETATION AND CONCLUSIONS: This simple method measures functional proteasome activity in primary leukemic cells and demonstrates for the first time that this activity is higher in myeloid leukemia than in myeloma cells.

Targeting the ubiquitin proteasome system in haematological malignancies.

The ubiquitin proteasome system (UPS) plays a central role in cellular protein homeostasis through the targeted destruction of damaged/misfolded proteins and regulatory proteins that control critical cellular functions. The UPS comprises a sequential series of enzymatic activities to covalently attach ubiquitin to proteins to target them for degradation through the proteasome. Aberrancies within this system have been associated with transformation and tumourigenesis and thus, the UPS represents an attractive target for the development of anti-cancer therapies. The use of the first-in-class proteasome inhibitor, bortezomib, in the treatment of Plasma Cell Myeloma and Mantle Cell Lymphoma has validated the UPS as a therapeutic target. Following on its success, efforts are focused on the development of second-generation proteasome inhibitors and small molecule inhibitors of other components of the UPS. This review will provide an overview of the UPS and discuss current and novel therapies targeting the UPS.

CCN3 suppresses mitogenic signalling and reinstates growth control mechanisms in Chronic Myeloid Leukaemia.

CCN3, a tumour suppressor gene, is down-regulated as a result of BCR-ABL tyrosine kinase activity in Chronic Myeloid Leukaemia (CML). We have established a stable CCN3 expression model in the human K562 CML cell line and have further validated the role for CCN3 in the leukaemogenic process. K562 cells stably transfected with CCN3 (K562/CCN3; 2.25 × 10(6) copies per 50 ng cDNA) demonstrated over 50% reduction in cell growth in comparison to cells stably transfected with empty vector (K562/control; p = 0.005). K562/CCN3 cells had reduced colony formation capacity (reduced by 29.7%, p = 0.03) and reduced mitogenic signalling in comparison to K562/control cells (reduced by 29.5% (p = 0.002) and 37.4% (p = 0.017) for phosphorylation levels of ERK and AKT respectively). K562/CCN3 cells showed an accumulation of events within the subG(0) phase of the cell cycle and increased apoptosis was confirmed by a three-fold increase in annexin V binding (p < 0.05). K562/CCN3 cells exposed to Imatinib (1 µM and 5 µM) showed an increase in events within the subG(0) phase of cell cycle over 96 h and mirrored the enhanced cell kill demonstrated by Annexin staining. Wild type K562 cells treated with recombinant human Ccn3 (10 nM) in combination with Imatinib (5 µM) also displayed enhanced cell kill (p = 0.008). K562/CCN3 cells displayed increased adhesion to matrigel™ (2.92 ± 0.52 fold increase compared to K562/control) which was commensurate with increased expression of the alpha 6 and beta 4 integrins (6.53 ± 0.47 and 1.94 ± 0.07 fold increase in gene expression respectively (n = 3, p < 0.05)). CCN3 restores cellular growth regulatory properties that are absent in CML and sensitises CML cells to imatinib induced apoptosis. CCN3 may provide novel avenues for the development of alternate therapeutic strategies.

Proteasome inhibitors in cancer therapy.

The ubiquitin proteasome pathway plays a critical role in regulating many processes in the cell which are important for tumour cell growth and survival. Inhibition of proteasome function has emerged as a powerful strategy for anti-cancer therapy. Clinical validation of the proteasome as a therapeutic target was achieved with bortezomib and has prompted the development of a second generation of proteasome inhibitors with improved pharmacological properties. This review summarises the main mechanisms of action of proteasome inhibitors in cancer, the development of proteasome inhibitors as therapeutic agents and the properties and progress of next generation proteasome inhibitors in the clinic.