Synergistic effects of proteasome inhibitor carfilzomib in combination with tyrosine kinase inhibitors in imatinib-sensitive and -resistant chronic myeloid leukemia models.

The tyrosine kinase inhibitor (TKI) imatinib has transformed the treatment and outlook of chronic myeloid leukemia (CML); however, the development of drug resistance and the persistence of TKI-resistant stem cells remain obstacles to eradicating the disease. Inhibition of proteasome activity with bortezomib has been shown to effectively induce apoptosis in TKI-resistant cells. In this study, we show that exposure to the next generation proteasome inhibitor carfilzomib is associated with a decrease in ERK signaling and increased expression of Abelson interactor proteins 1 and 2 (ABI-1/2). We also investigate the effect of carfilzomib in models of imatinib-sensitive and -resistant CML and demonstrate a potent reduction in proliferation and induction of apoptosis in a variety of models of imatinib-resistant CML, including primitive CML stem cells. Carfilzomib acts synergistically with the TKIs imatinib and nilotinib, even in imatinib-resistant cell lines. In addition, we found that the presence of immunoproteasome subunits is associated with an increased sensitivity to carfilzomib. The present findings provide a rational basis to examine the potential of carfilzomib in combination with TKIs as a potential therapy for CML, particularly in imatinib-resistant disease.

Enhanced lymphocyte interferon (IFN)-γ responses in a PTEN mutation-negative Cowden disease kindred.

Identification of immune modifiers of inherited cancer syndromes may provide a rationale for preventive therapy. Cowden disease (CD) is a genetically heterogeneous inherited cancer syndrome that arises predominantly from germline phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation and increased phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signalling. However, many patients with classic CD diagnostic features are mutation-negative for PTEN (PTEN M-Neg). Interferon (IFN)-γ can modulate the PI3K/mTOR pathway, but its association with PTEN M-Neg CD remains unclear. This study assessed IFN-γ secretion by multi-colour flow cytometry in a CD kindred that was mutation-negative for PTEN and other known susceptibility genes. Because IFN-γ responses may be regulated by killer cell immunoglobulin-like receptors (KIR) and respective human leucocyte antigen (HLA) ligands, KIR/HLA genotypes were also assessed. Activating treatments induced greater IFN-γ secretion in PTEN M-Neg CD peripheral blood lymphocytes versus healthy controls. Increased frequency of activating KIR genes, potentially activating KIR/HLA compound genotypes and reduced frequency of inhibitory genotypes, were found in the PTEN M-Neg CD kindred. Differences of IFN-γ secretion were observed among PTEN M-Neg CD patients with distinct KIR/HLA compound genotypes. Taken together, these findings show enhanced lymphocyte secretion of IFN-γ that may influence the PI3K/mTOR CD causal molecular pathway in a PTEN mutation-negative CD kindred.

Methods for measuring proteasome activity: current limitations and future developments.

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.

CCN3--a key regulator of the hematopoietic compartment.

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, naïve 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.

Use of imatinib mesylate in elderly patients in Northern Ireland: evidence of comparable haematological and molecular responses to younger patients.

Advanced age is an indicator of poor prognosis in chronic myeloid leukaemia (CML). Since obtaining its UK licence in 2001, the tyrosine kinase inhibitor imatinib mesylate has effected a paradigm shift in the treatment of CML. We compared survival and molecular response rates in elderly patients to younger patients presenting with CML since the introduction of imatinib. Twenty-five patients aged >60 years were identified. No significant survival difference was found when this group was compared with younger patients. In the elderly group, 53% of those with molecular data (36% of all elderly patients) had a major molecular response as assessed by real time quantitative PCR (RT-PCR). The advent of imatinib therapy appears to have ameliorated much of the negative impact of advancing age on survival in patients with CML.

Report on the fifth international workshop on the CCN family of genes.

The Fifth International Workshop on the CCN Family of Genes was held in Toronto October 18-22, 2008. This bi-annual workshop provides a unique opportunity for the presentation and discussion of cutting edge research in the CCN field. The CCN family members have emerged as extracellular matrix associated proteins which play a crucial role in cardiovascular and skeletal development, fibrosis and cancer. Significant progress has been made in the development of model systems to tease apart the CCN signalling pathways in these systems. Results presented at the conference suggest that targeting these pathways now shows real promise as a therapeutic strategy.

Peptide leucine arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin of the Northern Leopard frog, Rana pipiens.

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.

High bax expression is a good prognostic indicator in acute myeloid leukaemia.

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.

Discrimination of granulocyte colony-stimulating factor isoforms by high-performance capillary electrophoresis.

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which acts primarily to stimulate the proliferation, differentiation and activation of committed progenitor cells of the neutrophil-granulocyte lineage into functionally mature neutrophils. The traditional biological assays employed to detect G-CSF are a myeloid bone marrow colony assay, a factor-dependent cell line specific for G-CSF and commercially available immunoassays. However, these methods will not distinguish between glycosylated and non-glycosylated forms of the molecule. In this study high-performance capillary electrophoresis (HPCE) was used to analyse glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (r-met-hG-CSF). Glycosylated G-CSF preparations contained human serum albumin (HSA), added as a protein carrier. Glycosylated and non-glycosylated G-CSFs were prepared in 40 mM Na2HPO4 buffer, pH 2.5, containing hydroxypropylmethylcellulose (HPMC) or 50 mM Na2HPO4 buffer, pH 9.0. Glycosylated G-CSF could be separated into two distinct glycoform populations at the lower pH studied. Differences in migration time and peak shape between glycosylated and non-glycosylated G-CSF were demonstrated. HPCE analysis of G-CSF produced using a baculovirus expression vector system revealed a further distinct G-CSF glycoform and demonstrated the resolving power of the technique.

Increased apoptotic peripheral blood neutrophils in systemic lupus erythematosus: relations with disease activity, antibodies to double stranded DNA, and neutropenia.

OBJECTIVE: To quantify the percentage of apoptotic peripheral blood neutrophils in systemic lupus erythematosus (SLE) and to determine the relations with disease activity and neutropenia. METHODS: Neutrophil apoptosis in SLE patients (n =50) was assessed by flow cytometry using annexin V binding and fluorescent labelled anti-fas. Rheumatoid arthritis (RA, n =20) and inflammatory bowel disease patients (IBD, n =20) were studied as disease controls. RESULTS: The percentage of apoptotic neutrophils, determined by annexin V binding, was increased in peripheral blood of SLE patients (median = 3.25%) compared with normal healthy donors (n =20, median = 1.20%) and disease controls (RA: median = 1.15%) (IBD: median = 1.15%). SLE neutrophil apoptosis correlated positively with lupus disease activity measured by SLAM score. SLE patients with increased antibodies to dsDNA (>10 mg/ml) had increased apoptotic neutrophils. Eight of 14 neutropenic SLE patients had increased apoptotic neutrophils. Increased neutrophil fas expression compared with normal controls was observed in SLE, RA, and IBD. CONCLUSION: Neutrophil fas expression is increased non-specifically in inflammatory disease. Increased circulating apoptotic neutrophils in SLE correlate positively with disease activity (SLAM) and may contribute to autoantigen excess including dsDNA.

Long-term bone marrow culture profiles in patients with myelodysplastic syndromes are not explicable by defective apoptosis.

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.

Spontaneous intramedullary apoptosis is present in disorders other than myelodysplasia.

Myelodysplastic syndrome (MDS) is a group of hematopoietic disorders characterized by peripheral cytopenias in the presence of normo- or hypercellular dysplastic marrow. It has been suggested that premature intramedullary apoptosis may contribute to this phenomenon. We used terminal dUTP nick-end labeling (TUNEL) of bone marrow biopsy specimens and cytocentrifuge preparations from patients with MDS and a variety of other hematopoietic disorders to determine whether there is increased intramedullary apoptosis in MDS and whether any such effect is specific to MDS. TUNEL labeling of bone marrow from 24 patients with MDS revealed significant positivity in 10 of 11 patients with refractory anemia (RA), five of seven with RA and excess of blasts (RAEB), all three patients with RAEB in transformation (RAEB-t), and all three patients with RA with ring sideroblasts (RARS). The percent of positive cells ranged from 5 to 50% but showed no apparent correlation with morphological subtype. In a series of 29 patients with acute leukemia, 17 showed significant positivity (13 of 13 with myeloid disease: three M1, seven M2, one M3, two M4; four of 16 patients with lymphoid disease: one Burkitt-type lymphoma, two null acute leukemia, and one common acute lymphoid leukemia). Intramedullary apoptosis was associated with myeloid or early committed progenitor cells and was highest in secondary acute myeloid leukemia (AML). Normal bone marrow samples from 12 individuals showed no evidence of apoptosis. Our results suggest that an increased level of intramedullary apoptosis is apparent in both patients with MDS and those with AML; those with secondary AML have the highest levels. The relative absence of such findings in lymphoid malignancy suggests that the apoptotic pathways are different in this lineage.

Analysis of the effect of prior therapy on progenitor cell yield: use of a chemotherapy scoring system.

A quantitative analysis of peripheral blood stem cell (PBSC) yield, measuring absolute numbers of CD34+ cells x 10(6)/kg and CFU-C x 10(4)/kg was performed in 74 consecutive patients. The interval or 'gap' from the end of previous chemotherapy to the date of priming was recorded in weeks. Geometric mean CD34 and CFU-C values were significantly higher in patients with a score of < or = 60 compared to those with score > 60 (P = 0.003 and 0.02, respectively) and a significant difference in CD34 values was also found when scores of < or = 38 were compared with scores > 38 (P = 0.003), with the difference in CFU-C values approaching significance (P = 0.08). Patients exposed to toxicity factor 4 drugs had significant lowering of both CD34 and CFU-C values (P < 0.001 and P = 0.038) and this emerged as the only independent factor when analysed using linear regression. No significant difference in the geometric mean CD34 or CFU-C values of patients was found in any of the gap categories analysed. Prior exposure to toxicity factor 4 drugs had a significant adverse effect on stem cell yield and should be avoided or minimized prior to stem cell harvesting.

Human blood and synovial fluid neutrophils cultured in vitro undergo programmed cell death which is promoted by the addition of synovial fluid.

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.

Measurement of colony-stimulating factors in synovial fluid: potential clinical value.

In this study, 100 synovial fluid (SF) samples from patients with a variety of arthritides were assayed for levels of colony-stimulating factors (CSFs) using a human bone-marrow bioassay and enzyme immunoassays for granulocyte (G-) and granulocyte-macrophage (GM-) CSFs. GM-CSF was found more frequently in samples from rheumatoid arthritis (RA) subjects (49%) than in non-RA samples (29%). Absence of GM- but not G- or bioassay CSFs characterised samples from subjects with psoriatic arthritis and ankylosing spondylitis (n = 14). There was strong evidence of an antagonistic relationship between levels of G- and GM-CSFs in samples from RA patients, an effect independent of drug treatment. However, treatment with non-steroidal anti-inflammatory agents (NSAIDs) may affect reported CSF concentrations: G-CSF levels were significantly lower in samples from subjects not taking NSAIDs. These results suggest that SF-CSF estimations using commercially available assays could provide useful diagnostic clues for clinicians, but careful interpretation is warranted particularly in patients on long-term NSAID treatment.

Granulocyte and granulocyte-macrophage colony-stimulating factors in cord and maternal serum at delivery.

Impaired neutrophil responses contribute to the neonate's increased susceptibility to infection. Because granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhance granulocyte and macrophage number and function, their use in the management of neonatal sepsis may be beneficial. Little is known about the endogenous levels of G-CSF and GM-CSF. In adults, raised values for G-CSF, but not GM-CSF, have been demonstrated in patients with infection, and conflicting data has emerged regarding CSF levels in neonates. We have used an ELISA to measure maternal and cord serum G-CSF and GM-CSF at the time of delivery, with gestational age between 25 and 42 wk. In mothers, an inverse linear relationship between gestational age and GM-CSF levels (p = 0.049) was found, but no association with G-CSF levels was observed. In neonates, a quadratic association was found between GM-CSF levels and gestational age (p = 0.019), whereas G-CSF levels showed an inverse linear association (p = 0.015). In addition, an association was found between maternal and cord GM-CSF (p = 0.007) but not G-CSF levels in paired samples. The effect of gestational age on the cytokine levels could not be explained by the white cell count, the absolute neutrophil count, pregnancy-induced hypertension, or the presence of infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug-induced neutropenia due to direct effects on CFU-C--ten years of culture experience.

Seventeen patients with suspected drug-induced neutropenia were referred to our laboratory for investigation within a 10-year period. In each case, the suspected drugs were incorporated separately into in vitro cultures of the patients' bone marrow. The cultures were performed in triplicate, using multiple controls. In 10 of these patients a drug-induced inhibition of CFU-C was demonstrated in vitro. The in vitro culture technique is a valuable investigation in patients with suspected drug-induced neutropenia, as it can help identify the causative agent, especially in cases of multidrug administration. It also has a useful application in allowing the clinician to predict drugs to which a patient may be unduly sensitive, and prescribe accordingly.