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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.

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.

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.

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.

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 NOTCH1–CCN3 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 NOTCH1–CCN3 signalling contributes to the pathogenesis of CML.

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.

// Lisa J. Crawford 1 , Gordon Anderson 1 , Cliona K. Johnston 1 , Alexandra E. Irvine 1 1 Centre for Cancer Research and Cell Biology (CCRCB), Queen’s University Belfast, Belfast, UK Correspondence to: Alexandra E. Irvine, email: s.irvine@qub.ac.uk Keywords: multiple myeloma, ubiquitin proteasome system, APC/C, FZR1, proTAME Received: April 19, 2016 Accepted: September 02, 2016 Published: September 15, 2016 ABSTRACT 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/C Fzr substrate Topoisomerase IIα (TOPIIα) or APC/C Cdc20 substrate Cyclin B. Similar effects were observed following treatment with proTAME, an inhibitor of both APC/C Fzr and APC/C Cdc20 . 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.

Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterized by the expression of the oncoprotein, Bcr-Abl kinase. CCN3 normally functions as a negative growth regulator, but it is downregulated in CML, the mechanism of which is not known. MicroRNAs (miRNAs) are small non-coding RNAs, which negatively regulate protein translation by binding to the complimentary sequences of the 3′ UTR of messenger RNAs. Deregulated miRNA expression has emerged as a hallmark of cancer. In CML, BCR-ABL upregulates oncogenic miRNAs and downregulates tumour suppressor miRNAs favouring leukaemic transformation. We report here that the downregulation of CCN3 in CML is mediated by BCR-ABL dependent miRNAs. Using the CML cell line K562, we profiled miRNAs, which are BCR-ABL dependent by transfecting K562 cells with anti-BCR-ABL siRNA. MiRNA expression levels were quantified using the Taqman Low Density miRNA array platform. From the miRNA target prediction databases we identified miRNAs that could potentially bind to CCN3 mRNA and reduce expression. Of these, miR-130a, miR-130b, miR-148a, miR-212 and miR-425-5p were significantly reduced on BCR-ABL knockdown, with both miR-130a and miR-130b decreasing the most within 24 h of siRNA treatment. Transfection of mature sequences of miR-130a and miR-130b individually into BCR-ABL negative HL60 cells resulted in a decrease of both CCN3 mRNA and protein. The reduction in CCN3 was greatest with overexpression of miR-130a whereas miR-130b overexpression resulted only in marginal repression of CCN3. This study shows that miRNAs modulate CCN3 expression. Deregulated miRNA expression initiated by BCR-ABL may be one mechanism of downregulating CCN3 whereby leukaemic cells evade negative growth regulation.

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.

Background Proteasome inhibitors have provided significant therapeutic advance in the treatment of Multiple Myeloma (MM), however resistance and dose limiting side effects remain a clinical challenge. Recent research has focused on developing strategies to target other enzymes within the ubiquitin proteasome system, with the aim of overcoming resistance and toxicity. We have previously reported deregulated expression of the E3 ligase HUWE1 in MM and demonstrated that knockdown or inhibition of HUWE1 leads to a decrease in MYC activity (Blood 2016, 128:240; 2017, 130:3077). HUWE1 is a large HECT domain E3 ligase that is involved in the regulation of key proteins such as p53, MYC and MCL-1. Mutations in HUWE1 have recently been identified in MM patients, significantly associated with the t(11;14) subgroup (blood-2018-03-840132). A recurrently mutated splice site mutation in the DUF913 (domain of unknown function 913) of HUWE1 was identified in 18% of patients, the remainder are predominantly missense mutations distributed across the coding sequence (CDS). Other studies have demonstrated that point mutations in the dimerization interface of HUWE1, which acts to regulate its activity, result in hyper-activation of HUWE1 (eLife 2017;6:e21036; Sci Rep 2017;7:15050). While 7% of the mutations reported in MM patients are found in this region, the effect of the majority of mutations on the functional significance of HUWE1 has yet to be determined. The aim of this study was to analyse HUWE1 expression and activity in HUWE1 mutant MM cell lines. Methods HUWE1 mutational status was analysed in a publically available dataset of MM cell lines (www.keatslab.org). Inhibitors of HUWE1 (BI8622, BI8626; described in EMBO Mol Med 2014, 6:1525-1541) were purchased from Syngene. The effect of the inhibitors on HUWE1 mutant MM cell lines was assessed using CellTitre-glo. HUWE1 auto-ubiquitination activity was analysed using UbiQapture-Q and Western blotting. Results HUWE1 mutations were identified in 6 out of 69 MM cell lines. HUWE1 mutational status was confirmed in 5 cell lines (U266, XG-1, XG-2, KMS-27, H1112) by Sanger sequencing. In agreement with MM patient data, HUWE1 mutations were predominantly found in cell lines expressing the t(11;14) translocation (4/6 cell lines) and are distributed in a similar manner. H1112 cells harbor the recurrent splice site mutation observed in patients, whereas the other cell lines contain missense mutations across the CDS. HUWE1 protein expression in mutant cell lines was compared with expression in 5 MM cell lines expressing wild type (WT) HUWE1 (JJN3, MM.1S, ANBL-6, KMS-18, OPM-2). No significant difference in expression was observed in the majority of HUWE1 mutant cell lines, however, HUWE1 expression was significantly lower in the H1112 cell line (p=0.002) compared to HUWE1 WT cell lines. Accordingly, HUWE1 auto-ubiquitination activity was reduced only in H1112 cells. XG-2 and U266 displayed similar sensitivity to HUWE1 inhibitors as HUWE1 WT cell lines (IC50 12-18 µM vs 9-20 µM), while XG-1, H1112 and KMS-27 were less sensitive (IC50 20-33 µM). The effect of HUWE1 on substrate proteins (e.g. MYC) varies depending on tumor type. In HUWE1 WT MM cell lines, inhibition of HUWE1 leads to significantly decreased expression of MYC and MCL-1 in (p Conclusion HUWE1 has recently been identified as a mutational driver in t(11;14) MM, however, little is known about the functional consequence of HUWE1 mutations. Here we show that the H1112 cell line, representative of the most commonly occurring HUWE1 mutation in MM, leads to reduced expression and activity of HUWE1 and is associated with low expression of HUWE1 substrates MYC and MCL-1. Conversely, expression of HUWE1 and activity against selected substrates remains unchanged in XG-2 and U266 cells. Moreover, recent studies demonstrate that certain HUWE1 mutations lead to enhanced catalytic activity. In summary, the pathogenicity of HUWE1 mutations in MM is likely to depend on the type and location of the mutation. Disclosures No relevant conflicts of interest to declare.

Transcription, translation, and secretion of adiponectin by lymphocytes is shown with receptors for adiponectin expressed on bone marrow mononuclear cells. Lymphocytes have long been established to play an important role in the regulation of hematopoiesis and produce many cytokines that act on hematopoietic progenitor cells. Previous studies by our group have shown that normal, unstimulated lymphocytes produce a protein that inhibits normal bone marrow GM colony formation. Adiponectin is an adipokine that has been demonstrated to act as a negative regulator of hematopoiesis and immune function. This study aimed to determine if the inhibitory molecule that we described previously was adiponectin. Here, we show transcription, translation, and secretion of adiponectin from lymphocytes and demonstrate that its receptors, AdipoR1 and AdipoR2, are expressed by bone marrow MNCs. We show that although the adiponectin expression is low in lymphocytes, it is sufficient to induce a significant inhibitory effect on GM precursors (CFU-GM) and activate the AMPK pathway in these cells. The regulation of adiponectin production by lymphocytes and its detailed function in suppressing GM colony formation need to be elucidated now. Our findings suggest a functional role for adiponectin as a negative regulator of granulopoiesis.

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.

Chronic Myeloid Leukaemia (CML) is characterized by expression of the constitutively active Bcr-Abl tyrosine kinase. We have shown previously that the negative growth regulator, CCN3, is down-regulated as a result of Bcr-Abl kinase activity and that CCN3 has a reciprocal relationship of expression with BCR-ABL. We now show that CCN3 confers growth regulation in CML cells by causing growth inhibition and regaining sensitivity to the induction of apoptosis. The mode of CCN3 induced growth regulation was investigated in K562 CML cells using gene transfection and treatment with recombinant CCN3. Both strategies showed CCN3 regulated CML cell growth by reducing colony formation capacity, increasing apoptosis and reducing ERK phosphorylation. K562 cells stably transfected to express CCN3 showed enhanced apoptosis in response to treatment with the tyrosine kinase inhibitor, imatinib. Whilst CCN3 expression was low or undetectable in CML stem cells, primary CD34+ CML progenitors were responsive to treatment with recombinant CCN3. This study shows that CCN3 is an important growth regulator in haematopoiesis, abrogation of CCN3 expression enhances BCR-ABL dependent leukaemogenesis. CCN3 restores growth regulation, regains sensitivity to the induction of apoptosis and enhances imatinib cell kill in CML cells. CCN3 may provide an additional therapeutic strategy in the management of CML.

Many cellular processes converge on the proteasome, and its key regulatory role is increasingly being recognized. Proteasome inhibition allows the manipulation of many cellular pathways including apoptotic and cell cycle mechanisms. The proteasome inhibitor bortezomib has enhanced responses in newly diagnosed patients with myeloma and provides a new line of therapy in relapsed and refractory patients. Malignant cells are more sensitive to proteasome inhibition than normal haematopoietic cells. Proteasome inhibition enhances many conventional therapies and its role in leukaemia is promising. Copyright © 2008 John Wiley & Sons, Ltd.

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.

Chronic myeloid leukemia (CML) is characterized by the presence of the constitutively active BCR-ABL protein tyrosine kinase. Using a multipotent hemopoietic cell line, FDCP-Mix, expressing BCR-ABL tyrosine kinase, we investigated the initial effects of this kinase in primitive hematopoietic stem cells. We identified down-regulation of a novel gene, CCN3, as a direct consequence of BCR-ABL kinase activity. CCN3 has been reported to function as a tumor suppressor gene in solid tumors. Northern and Western blotting plus immunocytochemical analysis confirmed CCN3 expression is decreased and is tyrosine-phosphorylated in BCR-ABL kinase active FDCP-Mix cells. Decreased cellular CCN3 correlated with increased CCN3 secretion in BCR-ABL kinase active cells. In vitro treatment of human CML cell lines with imatinib or siRNA directed against BCR-ABL significantly reduced BCR-ABL while increasing CCN3 expression. Cells from patients responding to imatinib showed a similar decrease in BCR-ABL and increase in CCN3. CML CD34+ cells treated with imatinib in vitro demonstrated increased CCN3 protein. Transfecting CCN3 into BCR-ABL+ cells inhibited proliferation and decreased clonogenic potential. CCN3 plays an important role in internal and external cell-signaling pathways. Thus, BCR-ABL can regulate protein levels by governing secretion, a novel mechanism for this tyrosine kinase.

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. (Cancer Res 2006; 66(12): 6379-86)

The proteasome plays a key role in regulating protein degradation in eukaryotic cells. A range of synthetic inhibitors of proteasome activity have been developed which have helped elucidate its role in the cell. These inhibitors have selectively induced apoptosis in malignant cells in vitro suggesting that the proteasome may be a novel therapeutic target. First generation proteasome inhibitors are currently showing promise in phase II/III clinical trials for patients with multiple myeloma.

Background The use of proteasome inhibitors to disrupt the ubiquitin proteasome system (UPS) has provided a significant therapeutic advance and highlights the importance of this pathway in Multiple Myeloma (MM). Selective inhibition of other enzymes that regulate the UPS may enable more specific targeting of aberrant signalling pathways with reduced side-effects. Using UPS-specific microarrays we found higher expression of the E3 ligase HUWE1 in MM cell lines compared to normal counterparts. HUWE1 is involved in the regulation of key proteins such as ARF, p53 and MYC. It has previously been reported to be deregulated in tumorigenesis and small molecule inhibitors of HUWE1 have recently shown efficacy in in vitro models of colorectal cancer. The aim of this study was to elucidate the role and therapeutic potential of HUWE1 in MM. Methods To validate our UPS microarray studies, HUWE1 expression levels were analysed in publically available datasets (GSE6477, GSE6691, GSE2113), CD138+ cells from healthy bone marrow (n=3) and MM patients (n=5) and MM cell lines [U266 (p53 mutant), OPM-2 (p53 mutant), KMS-18 (p53 WT/-), JJN3(p53 WT/-), MM.1S (p53 WT)]. shRNA-mediated gene knockdown (Source Biosciences) was used to investigate the therapeutic potential of targeting HUWE1. HUWE1 knockdown cells were analysed for viability and cell cycle status; effects on known downstream substrates were analysed by Western blotting. Snapshot Proteomics (AVM Biomed) was performed on HUWE1 knockdown cells to identify novel ubiquitinated substrates of HUWE1. Results Elevated HUWE1 expression (≥ 2-fold) was confirmed in MM cell lines and primary CD138+ MM cells compared to healthy CD138+ cells at both the gene and protein level. Analysis of publically available datasets (GSE6477, GSE6691) also demonstrated that HUWE1 was significantly increased in MM patients compared with healthy donors. Moreover, further analysis of datasets GSE6477 and GSE2113 revealed that HUWE1 expression increased significantly (p≤0.05) through a spectrum of plasma cell diseases (MGUS, smoldering MM, newly diagnosed MM, plasma cell leukemia). shRNA knockdown of HUWE1 resulted in a significant increase of cells in G2/M, an accumulation of p53 and MIZ1 and a decrease in MYC protein levels (p≤0.05). HUWE1 depletion significantly decreased (p≤0.01) the proliferation of all MM cell lines, cultured alone or in co-culture with MM patient-derived bone marrow stromal cells, regardless of p53 status. Conversely, we show that MM cell lines that carry mutant p53 were largely resistant to the MDM2 inhibitor Nutlin-3. Finally, proteomic profiling identified more than 80 putative substrates of HUWE1, confirming its known roles as an RNA binding protein and in core histone regulation. Furthermore, this analysis revealed previously unidentified involvement of HUWE1 in regulation of cell cycle (3 proteins) and MAPK signalling (4 proteins). Conclusions The proteasome is an established target in MM and there are opportunities for more specific targeting of enzymes within the UPS with reduced side-effects. Here we demonstrate that the E3 ligase HUWE1 is elevated in MM and inhibition of HUWE1 with shRNA results in growth arrest of MM cell lines, decreased expression of MYC and increased levels of p53. These effects were independent of p53 status, suggesting that inhibition of HUWE1 offers a more valuable approach than MDM2 inhibitors which show efficacy only in p53 wild type myeloma. Future studies will validate novel substrates of HUWE1 identified in this study and investigate recently identified small molecule inhibitors of HUWE1 in MM. This study demonstrates that HUWE1 is important in the growth and survival of MM cells and may provide a novel therapeutic target in MM. Disclosures No relevant conflicts of interest to declare.

Most cytotoxic drugs kill cells by instigating the process of apoptosis and it has been suggested that apoptotic markers may provide an indication of tumour chemosensitivity. The aim of this study was to determine if such a relationship exists in acute myeloid leukaemia (AML). The levels of spontaneous apoptosis, bcl-2 and bax were evaluated in 56 newly diagnosed AML patients to determine if they correlated with a response to cytotoxic therapy. Spontaneous apoptosis was lower, but bcl-2, bax and the bcl-2/bax ratio were higher in AML compared with normal individuals. AML patients with high bax expression at diagnosis had significantly better prognosis for disease-free survival, event-free survival and overall survival (P = 0.016). In the standard risk group, high bax expression was in keeping with significantly improved survival. Multivariate analysis revealed bax to be an independent predictor of survival. There was a significant reduction in bcl-2 and bax expression when AML patients entered complete remission and also in relapsed AML patients who entered a second remission. This study suggests that bax is a useful prognostic indicator in AML and may assist with therapeutic decision-making for patients in the standard risk category.

OBJECTIVE--To assess the influence of inflammatory synovial fluid (SF) on apoptosis of joint and blood neutrophils with particular reference to levels of colony stimulating factors (CSF) contained therein. METHODS--Neutrophils were separated from fresh synovial fluid and from peripheral blood by density gradient centrifugation. Apoptosis was assayed by light microscope morphology and DNA degradation. CSFs were assayed using bone marrow bioassay and enzyme linked immunosorbent assays for granulocyte (G-) and granulocyte macrophage (GM-) CSF. Separated neutrophils were cultured in vitro and exposed to: varying concentrations of SF in which CSF levels were measured, recombinant G-CSF and GM-CSF, and hyaluronic acid control solutions. Numbers of apoptotic neutrophils and CSF levels were also measured in fresh SF samples. RESULTS--The addition of autologous or heterologous inflammatory SF to blood or joint cavity neutrophils cultured in vitro caused a significant dose dependent increase in the percentage of cells becoming apoptotic with time as measured morphologically and confirmed by DNA degradation. The effect bore no relationship to levels of CSF in joint fluid, despite our finding that GM-CSF produced inhibition of neutrophil apoptosis in vitro. CONCLUSION--These data suggest that SF contains a factor or factors capable of directly or indirectly promoting neutrophil apoptosis and normally powerful enough to overcome the apoptosis inhibiting effects of cytokines such as GM-CSF at concentrations usually found in inflammatory synovial fluids.

The proteasome has been validated as a therapeutic target, with proteasome inhibitors showing particular efficacy in the treatment of Multiple Myeloma. A wide range of methods have been developed to profile proteasome activity. These include the current method of choice fluorogenic peptide substrates, as well as bioluminescent imaging, immunological methods, and more recently, site-specific fluorescent probes. The aim of this review is to evaluate the currently available methods for profiling proteasome activity and their suitability for use in translational studies. Ongoing development of techniques for profiling proteasome activity will facilitate future research into proteasome-related pathologies, thus accelerating the development of more specific drug regimes.

The Leukaemias are a group of cancers affecting the white blood cell system. Chronic myeloid leukaemia (CML) is characterised by a reciprocal translocation between chromosomes 9 and 22 resulting in the production of the BCR-ABL protein tyrosine kinase. Expression of BCR-ABL in stem cells of the myeloid blood cell population has been shown to be directly responsible for malignant transformation. We identified down-regulation of the negative growth regulator, CCN3, as a consequence of BCR-ABL expression. Investigations have shown CCN3 functions to inhibit cell growth and survival whilst promoting apoptosis; our findings suggest that BCR-ABL utilises CCN3 down-regulation to escape growth control. Further understanding of CCN3 mechanisms are required to develop new therapeutic strategies targeting this pathway.

CCN3, a founding member of the CCN family of growth regulators, was linked with hematology in 2003(1) when it was detected in human serum. CCN3 is expressed and secreted by hematopoietic progenitor cells in normal bone marrow. CCN3 acts through the core stem cell signalling pathways including Notch and Bone Morphogenic Protein, connecting CCN3 with the modulation of self-renewal and maturation of a number of cell lineages including hematopoietic, osteogenic and chondrogenic. CCN3 expression is disrupted in Chronic Myeloid Leukemia as a consequence of the BCR-ABL oncogene and allows the leukemic clone to evade growth regulation. In contrast, naive cord blood progenitors undergo enhanced clonal expansion in response to CCN3. Altered CCN3 expression is associated with numerous solid tumors including glioblastoma, melanoma, adrenocortical tumours, prostate cancer and bone malignancies including osteosarcoma. Mature CCN3 protein has five distinct modules and truncated protein variants with altered function are found in many cancers. Regulation by CCN3 is therefore cell type and isoform specific. CCN3 has emerged as a key player in stem cell regulation, hematopoiesis and a crucial component within the bone marrow microenvironment.

Objective We have previously demonstrated that proteasome activity is higher in bone marrow from patients with chronic myeloid leukemia (CML) than normal controls. This study investigates whether there is any relationship between Bcr-Abl expression and proteasome activity. Materials and Methods Fluorogenic substrate assays and an activity-based probe were used to profile proteasome activity in CML cell-line models and the effect of the proteasome inhibitor BzLLLCOCHO on these cell-line models and primary CML cells was investigated. Results We have demonstrated that oncogenic transformation by BCR-ABL is associated with an increase in proteasome proteolytic activity. Furthermore, small interfering RNA targeted against BCR-ABL reduces proteasome activity. In addition, we have found that Bcr-Abl−positive cells are more sensitive than Bcr-Abl−negative cells to induction of apoptosis by the proteasome inhibitor BzLLLCOCHO, and that sequential addition of imatinib followed by BzLLLCOCHO has an additive effect on the induction of apoptosis in Bcr-Abl−positive cells. Finally, we demonstrate that cell lines that become resistant to imatinib remain sensitive to proteasome inhibition. Conclusion This is the first time that a direct relationship has been demonstrated between BCR-ABL transformation and the enzymatic activity of the proteasome. Our results suggest that the proteasome might provide a useful therapeutic target in CML, particularly in those patients who have developed resistance to conventional treatment.

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.

On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens). This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin. pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action. pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i.e. mature neutrophils. pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.

It has been suggested that increased intramedullary apoptosis may explain the paradox between peripheral blood cytopenias and the hyper- or normo-cellular bone marrow observed in the myelodysplastic syndromes (MDS). We wished to see if culture performance could be related to the presence of apoptotic cells in a group of patients with MDS (12 patients) and other patients with peripheral blood cytopenias (six patients) which caused diagnostic difficulty. There was no correlation between LTBMC or adherent cell growth and the presence of apoptotic cells in the original marrow sample. A variable degree of apoptosis was observed in both groups of patients. LTBMC profiles correlated well with diagnosis but were unrelated to the extent of intramedullary apoptosis. This suggests that apoptosis is a much more ubiquitous process in disease than previously thought.

PEG-modified proteins have numerous advantages over their unmodified counterparts (increased half life, reduced antigenicity, improved solubility), but almost without exception, they show a modest to marked reduction in biological or enzymatic activity. However, while investigating a new protocol for the preparation of PEG-proteins, we compared PEG-modified and unmodified GM-CSF with respect to their polymorphonuclear neutrophil granulocyte (PMN) priming activities. PEG-GM-CSF was unexpectedly more active than GM-CSF in its ability to prime neutrophils to respond to the synthetic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) with an oxidative burst (assessed both by nitroblue tetrazolium reduction and ferricytochrome c reduction). These results were in contrast to the findings for colony stimulating activity and with GM-CSF induced thymidine uptake, where the biological activity was unchanged or reduced. The enhanced neutrophil priming activity of PEG-GM-CSF was confirmed using FPLC fractionated PEG-modified GM-CSF. This showed changes in the bioactivity profile consistent with both the shift in protein elution profile and enhanced activity of the PEG-modified material (reflected in the increased area under the bioactivity curve). We also excluded a neutrophil priming action for PEG-modified fetal calf serum proteins, carrier proteins and 'irrelevant' cytokine, erythropoietin. The dissociation of the two bioactivities was confirmed using individual FPLC fractions. These results suggest the presence of differences in either binding, receptor/ligand processing or signal transduction for neutrophils versus progenitors, that are differentially affected by PEG-modification of GM-CSF. The demonstration that PEG-modification can partially dissociate two biological activities suggests the feasibility of using PEG-modification to produce proteins with subtly altered spectra of biological activity and hence new ranges of clinical applications.

Chronic Myeloid Leukaemia (CML) is characterized by expression of the constitutively active BCR-ABL tyrosine kinase. Previously, we identified down-regulation of the negative growth regulator, CCN3, as a result of BCR-ABL kinase activity. Reduced CCN3 expression is a prominent feature in both primary human CML cells and cell lines. We now show that CCN3 is growth inhibitory and enhances imatinib induced growth inhibition. To evaluate the biological consequence of CCN3 expression in CML, K562 cells were stably transfected with a construct containing CCN3 (pCMV82-23) and growth characteristics and activation of signaling pathways were compared to cells transfected with empty vector (control). CCN3 expression was undetected by Real-time PCR in control cells whilst pCMV82-23 cells expressed 2.25 × 106 copies per 50ng of cDNA. pCMV82-23 cells showed reduced colony formation capacity (p=0.003) and reduced cell growth over a period of five days (p=0.005). Investigation of cellular signaling showed CCN3 expression resulted in significant down-regulation of three major signaling pathways and demonstrated reduced phosphorylation of ERK (p=0.002), pAKT (p=0.017) and pSTAT5 (p=0.005) compared to control cells. Protein levels for total ERK, AKT and STAT5 were unaffected by CCN3 expression. Flow cytometry showed that sustained CCN3 expression resulted in an accumulation of cells within the subG0 stage of the cell cycle (11.4% ± 3 (p=0.040)). To determine if CCN3 expression could influence sensitivity to the BCR-ABL kinase inhibitor, imatinib, pCMV82-23 cells and control cells were treated with imatinib (5uM) for 48h. Control cells treated with imatinib showed moderate growth inihibition (19.6% ± 2.5) compared to untreated control. pCMV82-23 cells showed a significant increase in the magnitude of imatinib induced growth inhibition (63.3% ± 10.5 (p=0.043)). This was associated with an increased accumulation of cells in the subG0 area of the cell cycle, 34.6% ± 5 for pCMV82-23 cells compared to control cells (21.7% ± 8) in response to imatinib treatment (p=0.006). To then determine if these effects could be reproduced using recombinant CCN3 (rCCN3), K562 cells were treated with imatinib (5uM) alone or in combination with rCCN3 (10nM) for 48h. K562 cells treated with the combination of rCCN3 and imatinib showed enhanced growth inhibition (71.8% ± 7.9) compared to cells treated with imatinib alone (81.1% ± 9.2 (p=0.008)). Loss of CCN3 is consistent with properties associated with the CML phenotype. Sustained expression of CCN3 in K562 cells restores growth control and re-establishes induction of apoptosis. Both increased expression of CCN3 or addition of the recombinant protein provide additional benefit for imatinib induced growth inhibition thus providing a novel avenue for therapeutic intervention.

Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell, characterised by the constitutively active tyrosine kinase BCR-ABL. The current first-line therapy for CML is the tyrosine kinase inhibitor imatinib. Although imatinib induces durable responses, a number of patients develop resistance to this treatment, highlighting the need to identify new molecular targets in this disease. Proteasome inhibition has recently emerged as a novel anti-cancer therapy. There is evidence to suggest that the proteasome is a valid target in CML. We have previously reported that proteasome activity is higher in bone marrow from patients with CML than normal controls. Furthermore, we demonstrated using a cell line model that Bcr-Abl positive cells were more sensitive to induction of apoptosis by proteasome inhibition than Bcr-Abl negative cells. The present study investigates the relationship between Bcr-Abl expression and proteasome activity and the effect of proteasome inhibition on primary human CML cells. Conventional fluorogenic substrate assays for all three catalytic activities of the proteasome [chymotrypsin-like (CT-L), trypsin-like (T-L), post glutamyl peptide hydrolysing (PGPH)] and proteasome activesite label DansylAhx3L3VS were used to profile proteasome activity levels in ts-Bcr-Abl FDCP-Mix cells and mock transfected FDCP-Mix cells. Both methods confirmed that Bcr-Abl positive cells have higher levels of proteasome activity than Bcr-Abl negative cells (p ≤ 0.04; Figure 1a). Conversely, downregulation of BCR-ABL using si-RNA was associated with a significant decrease in proteasome activity (p < 0.05; Figure 1b). The ability of the proteasome inhibitor BzLLLCOCHO to induce apoptosis in primary human CML cells was evaluated using Mitosensor™ and Hoescht/Propidium Iodide staining. Treatment with BzLLLCOCHO (1μM), selectively induced apoptosis in primary CML cells compared to normal mononuclear cells (39 ± 9.6 % vs 18.1 ± 4.02 %, 72 hrs, p = 0.01). Drug combination experiments were performed with BzLLLCOCHO (1 μM) and imatinib (1 μM) in ts-Bcr-Abl FDCP-mix cells and primary CML cells. Using Calcusyn software to generate the median effect of Chou-Talalay, the sequential addition of imatinib followed by BzLLLCOCHO was found to synergistically enhance the induction of apoptosis in ts-Bcr-Abl FDCP-Mix cells and resulted in additive effects in primary CML cells (n=4). The effect of the compounds on Bcr-Abl kinase activity was assessed by immunoblotting for phosphorylated Crkl. No effect on Bcr-Abl activity was seen following treatment of ts-Bcr-Abl FDCP-Mix cells and primary CML cells with BzLLLCOCHO alone, however, the combination of BzLLLCOCHO and imatinib resulted in a greater reduction of Bcr- Abl activity (59.8 ± 9.9 %) than imatinib alone (34.1 ± 9.6 %). Finally, we investigated the effect of BzLLLCOCHO on two human CML cell lines which are resistant to imatinib (KCL22-r, LAMA84-r). Imatinib resistant cells were found to be equally as sensitive to induction of apoptosis by BzLLLCOCHO as their imatinib sensitive counterparts (KCL22-s 30.7 ± 5.7 % vs KCL22-r 32 ± 1.7 %; LAMA84-s 56.3 ± 3.2 % vs Lama84-r 57.2 ± 7.9 %; 72 hrs). The present findings suggest that higher proteasome activity in Bcr- Abl positive cells may render these cells more susceptible to induction of apoptosis by proteasome inhibition and provide a rational basis to examine the potential of proteasome inhibitors as a therapeutic target in CML, particularly in imatinib resistant disease. Figure 1a. Bcr-Abl+ cells contain significantly greater levels of proteasome activity than Bcr-Abl cells siRNA directed against BCR-ABL decreases proteasome activity. Figure 1a. Bcr-Abl+ cells contain significantly greater levels of proteasome activity than Bcr-Abl cells . / siRNA directed against BCR-ABL decreases proteasome activity.

Previous studies by our group have shown that normal unstimulated lymphocytes produce a protein which inhibits colony formation of granulopoietic progenitors, but has no effect on erythroid progenitors. Therefore, this inhibitor was initially designated GIA (granulopoietic inhibitory activity). GIA was identified as a glycoprotein of approximately 30 kDa, with a pI of 7.9 - 8.4. Futhermore, we demonstrated that this inhibitor may have physiological significance in that its production is altered in patients with neutropenia. GIA has proved difficult to characterise to date since it is produced in relatively low amounts although it has a high specific biological activity. Adiponectin is an adipokine reported to share many of the inhibitory characteristics of GIA and has been demonstrated to act as a negative regulator of hematopoiesis and immune response. This study aimed to determine whether GIA is adiponectin or if it represents an adiponectin-like molecule. Lymphocyte conditioned medium (LCM) from lymhocytes cultured at 1 X 106 cells/ml in HL-1 minimal medium was used as a source of GIA. Inclusion of LCM as 10% of the top layer of agar in a myeloid colony assay inhibited growth of CFU-GM by 52 ± 11 % (n=3), confirming the presence of the inhibitory activity. RNA and protein from lymphocytes and LCM harvested over a 7 day culture period were subsequently investigated for adiponectin expression. Western blot analysis demonstrated a distinct banding pattern in days 3-7 LCM corresponding to monomers, dimers, trimers and greater. This is consistent with adiponectin which circulates as a multimer of trimers. Characterisation of GIA at the transcript level confirmed that GIA is in fact adiponectin. The N-terminal collagenous domain, C-terminal globular domain and full length adiponectin were amplified by RT-PCR analysis. Adiponectin is thought to be secreted exclusively from adipocytes and much of our current knowledge of this molecule relates to its metabolic functions. Our study provides evidence that adiponectin is also produced by lymphocytes and may play a role in the pathogenesis of neutropenia.

Chronic Myeloid Leukemia (CML) is characterized by expression of the constitutively active BCR-ABL tyrosine kinase. Previously, we have identified downregulation of the negative growth regulator, CCN3, as a result of BCR-ABL kinase activity and detected reduced CCN3 expression in human CML cell lines and primary human CML cells. We now report a reciprocal relationship of BCR-ABL and CCN3 expression and the functional consequence of expressing CCN3 in BCR-ABL+ cells. Real-time PCR was used to examine the relationship between BCR-ABL and CCN3 expression in human K562 cells. Parental K562 cells showed high expression of BCR-ABL (4.68 x104 transcripts in 5 μL of cDNA) whilst CCN3 expression was not detected. Treatment with siRNA directed against BCR-ABL (0.5 μg per106 cells) for 72 hours resulted in a 3.7 fold decrease in BCR-ABL and 6.1 fold increase in CCN3 expression (mean Ct change 1.9 ± 0.2 and 2.6 ± 0.5 for BCR-ABL and CCN3 respectively, n=3, p=0.001). Similarly, K562 cells treated with imatinib (1 micromolar) for 96 hours showed a 5.9 fold decrease in BCR-ABL expression and a 4.2 fold increase in CCN3 expression (mean Ct change 2.5 ± 0.1 and 2.1± 0.2 for BCR-ABL and CCN3 respectively, n=3, p=0.001). To investigate CCN3 function, we expressed CCN3 in BCR-ABL+ cells using Nucleofector technology (Amaxa, GmbH). K562 cells were transfected with either the pCb6+ vector (Invitrogen,UK) or pCb6+ vector containing the CCN3 construct. Cells were analysed 24 hours post-transfection by flow cytometry and also after 7 days in methyl cellulose culture to determine clonogenicity. Cell cycle analysis was performed on 20,000 events using the winMDI software. CCN3 expression in BCR-ABL+ cells resulted in an accumulation of cells in the subG0 phase of the cell cycle (mean for subG0 9.9% ± 4.6 and 21.8% ± 0.7 for the pCb6+ vector alone and pCb6+ vector containing CCN3 construct respectively). CCN3 expression significantly increased the number of cells within the subG0 area of the cell cycle (n=3, p=0.027). In addition, CCN3 expression reduced the clonogenic capacity of BCR-ABL+ cells. K562 cells transfected with the pCb6+ vector containing CCN3 construct formed significantly fewer colonies on methyl cellulose in comparison to cells that had been transfected with the pCb6+ vector alone (n=3, p=0.027). This study demonstrates a reciprocal relationship between CCN3 and BCR-ABL expression. CCN3 is known to be a negative growth regulator and increased expression of CCN3 in BCR-ABL+ cells inhibits proliferation and decreases clonogenic potential. Thus CCN3 down-regulation mediated by BCR-ABL offers growth advantage to hematopoietic cells.

The proteasome is a mutlicatalytic protease with three main catalytic activities - chymotrypsin-like (CT-L), trypsin-like (T-L) and peptidylglutamyl peptide hydrolising (PGPH). Proteasome inhibition is an emerging therapy for many cancers and is a novel treatment for multiple myeloma (MM). The CT-L activity, considered to be the rate-limiting step in protein degradation, is the primary target of many proteasome inhibitors. We have compared the specificity and potency of the novel proteasome inhibitor BzLLLCOCHO to the previously characterised inhibitors PS-341 (Velcade, bortezomib) and MG-132. Specific fluorogenic substrates were used to measure proteasome proteolytic activity in the presence and absence of the inhibitory compounds. An active site directed probe with a dansyl-sulfonamidohexanoyl hapten tag was used in conjuction with immunoblotting to determine the subunit specificity of the proteasome inhibitors (Nature Methods2005;2:357–362). MM cell lines (U266, OPM-2, KMS-11, KMS-18) were incubated with 10 μM BzLLLCOCHO, 5 nM PS-341 or 1 μM MG-132 for 24 hrs and proteasome activity was measured. Addition of BzLLLCOCHO reduced CT-L activity by 83 ± 13 % in the fluorogenic assay, and T-L and PGPH activities were reduced by 93 ± 6 % and 92 ± 2 % respectively. Immunoblot results revealed a similar pattern, the T-L and PGPH subunits were completely inhibited by BzLLLCOCHO and there was only weak labeling of the CT-L subunit with the active site probe. In contrast, treatment with PS-341 completely inhibited the CT-L and PGPH activities and incubation with MG-132 resulted in weak inhibition of the CT-L and PGPH activities, neither inihibitor significantly affected T-L activity. The ability of the different inihibitors to induce apoptosis in MM cell lines was then evaluated. All three inhibitors were demonstrated to act through both the caspase-8 and caspase-9 signalling pathways. Using Mitosensor™ and Hoescht/Propidium Iodide staining we found that MM cells were more sensitive to the induction of apoptosis by PS-341 and MG-132 than BzLLLCOCHO (U266 cells treated for 72 hrs with BzLLLCOCHO 51 % apoptosis, PS-341 79 % apoptosis and MG-132 84 % apoptosis). BzLLLCOCHO is a cell permeable and potent inhibitor of all three proteolytic activities of the proteasome. PS-341 and MG-132 inhibited only two of the three proteasome activities but were more efficient than BzLLLCOCHO at inducing apoptosis in MM cell lines. MG-132 is known to inhibit non proteasomal proteases such as Cathepsin B and Calpain 1 which may contribute to its potency. Further investigation on the effects of these inhibitors on gene and protein expression in the cell may lead to the development of more specific and targeted inhibitors.

Summary Peripheral blood lymphocytes from 20 patients with neutropenia not consistent with aplastic anaemia were tested for their ability to inhibit the proliferation of normal granulopoietic precursor cells (CFU–GM) in agar culture. Two patients, both with features of an autoimmune disorder, had lymphocytes which were more inhibitory than normal lymphocytes to both normal and their own CFU–GM. Two other patients had lymphaocytes which were more inhibitory than normal lymphocytes to either their own CFU-GM or normal CFU–GM but not both. Eight patients had lymphocytes which were significantly less inhibitory than normal lymphocytes to either normal or their own CFU-GM, but only one showed this feature against both normal and their own CFU-GM. One patient had a highly potent plasma inhibitor of CFU-GM — this patient had received multiple transfusions and had a leucocyte antibody of a broad specificity. No clinical or haematological features were common to any of these groups of patients which reflects the heterogeneity of patients studied and stresses the importance of controls.

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.