A pilot study of single nucleotide polymorphisms in the interleukin-6 receptor and their effects on pre- and post-transplant serum mediator level and outcome after allogeneic stem cell transplantation.

Interleukin (IL)-6 is an important regulator of immunity and inflammation in many diseases. Single nucleotide polymorphisms (SNPs) in the IL-6 gene influence outcome after allogeneic stem cell transplantation (ASCT), but the possible importance of SNPs in the IL-6 receptor has not been examined. We therefore investigated whether SNPs in the IL-6R gene influenced biochemical characteristics and clinical outcomes after ASCT. We examined the IL-6 promoter variant rs1800975 and the IL-6R SNPs rs4453032, rs2228145, rs4129267, rs4845374, rs4329505, rs4845617, rs12083537, rs4845618, rs6698040 and rs4379670 in a 101 population-based cohort of allotransplant recipients and their family donors. Patients being homozygous for the major alleles of the IL-6R SNPs rs2228145 and rs4845618 showed high pretransplant CRP serum levels together with decreased sIL-6R levels; the decreased IL-6R levels persisted 6 months post-transplant. In contrast, patients being homozygous for the minor allele of the IL-6R SNP rs4379670 showed decreased pretransplant CRP levels. Furthermore, the IL-6R rs4845618 donor genotype showed an association with severe acute graft-versus-host disease (GVHD), whereas the donor genotype of the IL-6 SNP rs1800795 was associated with decreased survival 100 days post-transplant. Finally, the recipient genotype of the IL-6R SNP rs4329505 showed a strong association with 2-years non-relapse mortality, and this effect was also highly significant in multivariate analysis. IL-6 and IL-6R SNPs influence the clinical outcome after allogeneic stem cell transplantation.

Differences in proliferative capacity of primary human acute myelogenous leukaemia cells are associated with altered gene expression profiles and can be used for subclassification of patients.

OBJECTIVES: Proliferative capacity of acute myelogenous leukaemia (AML) blasts is important for leukaemogenesis, and we have investigated whether proliferative capacity of primary human AML cells could be used for subclassification of patients. MATERIALS AND METHODS: In vitro proliferative capacity of AML cells derived from two independent groups was investigated. Cells were cultured under highly standardized conditions and proliferation assayed by (3) H-thymidine incorporation after seven days culture. Patients were subclassified by clustering models, and gene expression profile was examined by microarray analyses. RESULTS: Based on proliferative capacity of the AML cells, three different patient clusters were identified: (i) autocrine proliferation that was increased by exogenous cytokines; (ii) detectable proliferation only in presence of exogenous cytokines; and (iii) low or undetectable proliferation even in presence of exogenous cytokines. Patients with highest proliferative capacity cells had no favourable prognostic impact by NPM-1 mutation. Analysis of gene expression profiles showed that the most proliferative cells generally had altered expression of genes involved in regulation of transcription/RNA functions, whereas patients with high proliferative capacity and internal tandem duplications (ITDs) in the FLT3 cytokine receptor gene had altered expression of several molecules involved in cytoplasmic signal transduction. CONCLUSIONS: In vitro proliferative capacity of primary human AML cells was considerably variable between patients and could be used to identify biologically distinct patient subsets.

Impact of malignant stem cell burden on therapy outcome in newly diagnosed chronic myeloid leukemia patients.

Chronic myeloid leukemia (CML) stem cells appear resistant to tyrosine kinase inhibitors (TKIs) in vitro, but their impact and drug sensitivity in vivo has not been systematically assessed. We prospectively analyzed the proportion of Philadelphia chromosome-positive leukemic stem cells (LSCs, Ph+CD34+CD38-) and progenitor cells (LPCs, Ph+CD34+CD38+) from 46 newly diagnosed CML patients both at the diagnosis and during imatinib or dasatinib therapy (ClinicalTrials.gov NCT00852566). At diagnosis, the proportion of LSCs varied markedly (1-100%) between individual patients with a significantly lower median value as compared with LPCs (79% vs 96%, respectively, P=0.0001). The LSC burden correlated with leukocyte count, spleen size, hemoglobin and blast percentage. A low initial LSC percentage was associated with less therapy-related hematological toxicity and superior cytogenetic and molecular responses. After initiation of TKI therapy, the LPCs and LSCs rapidly decreased in both therapy groups, but at 3 months time point the median LPC level was significantly lower in dasatinib group compared with imatinib patients (0.05% vs 0.68%, P=0.032). These data detail for the first time the prognostic significance of the LSC burden at diagnosis and show that in contrast to in vitro data, TKI therapy rapidly eradicates the majority of LSCs in patients.

Loss-of-Function CNKSR2 Mutation Is a Likely Cause of Non-Syndromic X-Linked Intellectual Disability.

In a non-dysmorphic 5-year-old boy with developmental delay, well-controlled epilepsy, and microcephaly, a 234-kb deletion of Xp22.12 was detected by copy number analysis. The maternally inherited deletion removed the initial 15 of the 21 exons of the connector enhancer of KSR-2 gene called CNKSR2 or CNK2. Our finding suggests that loss of CNKSR2 is a novel cause of non-syndromic X-linked mental retardation, an assumption supported by high gene expression in the brain, localization to the post-synaptic density, and a role in RAS/MAPK-dependent signal transduction.

Correlation analysis of p53 protein isoforms with NPM1/FLT3 mutations and therapy response in acute myeloid leukemia.

The wild-type tumor-suppressor gene TP53 encodes several isoforms of the p53 protein. However, while the role of p53 in controlling normal cell cycle progression and tumor suppression is well established, the clinical significance of p53 isoform expression is unknown. A novel bioinformatic analysis of p53 isoform expression in 68 patients with acute myeloid leukemia revealed distinct p53 protein biosignatures correlating with clinical outcome. Furthermore, we show that mutated FLT3, a prognostic marker for short survival in AML, is associated with expression of full-length p53. In contrast, mutated NPM1, a prognostic marker for long-term survival, correlated with p53 isoforms ß and γ expression. In conclusion, p53 biosignatures contain useful information for cancer evaluation and prognostication.

PHF6 Deletions May Cause Borjeson-Forssman-Lehmann Syndrome in Females.

In a 16-year-old girl with intellectual disability, irregular teeth, slight body asymmetry, and striated skin pigmentation, highly skewed X-inactivation increased the likelihood of an X-linked cause of her condition. Among these, prominent supraorbital ridges and hearing loss suggested a filaminopathy, but no filamin A mutation was found. The correct diagnosis, Borjeson-Forssman-Lehmann syndrome (BFLS, MIM#301900), was first made when a copy number array identified a de novo 15-kb deletion of the terminal 3 exons of the PHF6 gene. In retrospect, her phenotype resembled that of males with BFLS. Such deletions of PHF6 have not been reported previously. This might be because PHF6 mutations are rarely looked for in females since classical BFLS so far has been thought to be a male-specific syndrome, and large PHF6 deletions might be incompatible with male fetal survival. If this is the case, sporadic BFLS could be more frequent in females than in males.

A novel brain metastases model developed in immunodeficient rats closely mimics the growth of metastatic brain tumours in patients.

AIMS: brain metastasis is a common cause of mortality in cancer patients, and associated with poor prognosis. Our objective was to develop a clinically relevant animal model by transplanting human biopsy spheroids derived from metastatic lesions into brains of immunodeficient rats. METHODS: nine different patient brain metastases from four different primary cancers were implanted into brains of immunodeficient rats. The xenografts were compared with patient tumours by magnetic resonance imaging, histochemistry, immunohistochemistry and DNA copy number analysis. RESULTS: after transplantation, tumour growth was achieved in seven out of nine human brain metastases. Spheroids derived from four of the metastases initiated in the rat brains were further serially transplanted into new animals and a 100% tumour take was observed during second passage. Three of the biopsies were implanted subcutaneously, where no tumour take was observed. The animal brain metastases exhibited similar radiological features as observed clinically. Histological comparisons between the primary tumours from the patients, the patient brain metastases and the derived xenografts showed striking similarities in histology and growth patterns. Also, immunohistochemistry showed a strong marker expression similarity between the patient tumours and the corresponding xenografts. DNA copy number analysis between the brain metastases, and the corresponding xenografts revealed strong similarities in gains and losses of chromosomal content. CONCLUSION: we have developed a representative in vivo model for studying the growth of human metastatic brain cancers. The model described represents an important tool to assess responses to new treatment modalities and for studying mechanisms behind metastatic growth in the central nervous system.

Specific cellular signal-transduction responses to in vivo combination therapy with ATRA, valproic acid and theophylline in acute myeloid leukemia.

Acute myeloid leukemia (AML) frequently comprises mutations in genes that cause perturbation in intracellular signaling pathways, thereby altering normal responses to growth factors and cytokines. Such oncogenic cellular signal transduction may be therapeutic if targeted directly or through epigenetic regulation. We treated 24 selected elderly AML patients with all-trans retinoic acid for 2 days before adding theophylline and the histone deacetylase inhibitor valproic acid (ClinicalTrials.gov NCT00175812; EudraCT no. 2004-001663-22), and sampled 11 patients for peripheral blood at day 0, 2 and 7 for single-cell analysis of basal level and signal-transduction responses to relevant myeloid growth factors (granulocyte-colony-stimulating factor, granulocyte/macrophage-colony-stimulating factor, interleukin-3, Flt3L, stem cell factor, erythropoietin, CXCL-12) on 10 signaling molecules (CREB, STAT1/3/5, p38, Erk1/2, Akt, c-Cbl, ZAP70/Syk and rpS6). Pretreatment analysis by unsupervised clustering and principal component analysis divided the patients into three distinguishable signaling clusters (non-potentiated, potentiated basal and potentiated signaling). Signal-transduction pathways were modulated during therapy and patients moved between the clusters. Patients with multiple leukemic clones demonstrated distinct stimulation responses and therapy-induced modulation. Individual signaling profiles together with clinical and hematological information may be used to early identify AML patients in whom epigenetic and signal-transduction targeted therapy is beneficial.

Release of angiopoietin-1 by primary human acute myelogenous leukemia cells is associated with mutations of nucleophosmin, increased by bone marrow stromal cells and possibly antagonized by high systemic angiopoietin-2 levels.

The balance between proangiogenic Angiopoietin-1 (Ang-1) and the antagonistic Ang-2 is important both for leukemogenesis and chemosensitivity in human acute myelogenous leukemia (AML). We examined the release of Ang-1 and Ang-2 by AML cells cultured alone and in cocultures with stromal cells. Detectable Ang-1 release from AML cells was observed for most patients (62/91), whereas Ang-2 release was detected only for a minority (23/91). Coculture of AML and stromal cells led to increased Ang-1 levels. Furthermore, the role of the angiopoietin system was investigated by characterizing whether the differences in angiopoietin expression in AML patients can be related to nucleophosmin (NPM1) mutations. We compared the gene expression profiles of AML cells derived from 19 patients with FLT3 mutations and normal cytogenetics with and without NPM1 mutations and observed increased expression of Ang-1 in patients with NPM1 mutations. Finally, we found significantly higher Ang-2 levels in serum of AML patients compared with healthy controls. Our results suggest that AML cells are a major source of Ang-1 in leukemic bone marrow, especially in patients with NPM1 mutations, but the local levels are also influenced by stromal cells. Local Ang-2 release from AML cells is less common, but high systemic levels of Ang-2 may affect bone marrow angioregulation.

Tiling resolution array CGH of dic(7;9)(p11 approximately 13;p11 approximately 13) in B-cell precursor acute lymphoblastic leukemia reveals clustered breakpoints at 7p11.2 approximately 12.1 and 9p13.1.

The dic(7;9)(p11 approximately 13;p11 approximately 13) is a recurrent chromosomal abnormality in acute lymphoblastic leukemia (ALL), mainly of B-lineage. Although more than 20 dic(7;9)-positive ALLs have been reported to date, the molecular genetic consequences of this aberration are unknown. We performed tiling resolution (32K) genome-wide array-based comparative genomic hybridization (array CGH) analysis of three cases with dic(7;9) in order to characterize the breakpoints on 7p and 9p. The analysis showed a clustering of breakpoints within 9p13.1 in all three cases and within 7p11.2 in two cases; the array CGH revealed two different breakpoints - 7p12.1 and 7p14.1 - in the remaining case. Based on these findings the abnormality should hence be designated dic(7;9)(p11.2 approximately 12.1;p13.1). Locus-specific fluorescence in situhybridization analysis of one of the cases narrowed down the 7p11.2 breakpoint to a <500-kb segment in this sub-band, a region containing three known genes. Unfortunately, lack of material precluded further molecular genetic studies, and it thus remains unknown whether the pathogenetically important outcome of the dic(7;9) is formation of a chimeric gene or loss of 7p and/or 9p material.

Ultrarapid caspase-3 dependent apoptosis induction by serine/threonine phosphatase inhibitors.

The protein phosphatase (PP) inhibitors nodularin and microcystin-LR induced apoptosis with unprecedented rapidity, more than 50% of primary hepatocytes showing extensive surface budding and shrinkage of cytoplasm and nucleoplasm within 2 min. The apoptosis was retarded by the general caspase inhibitor Z-VAD.fmk. To circumvent the inefficient uptake of microcystin and nodularin into nonhepatocytes, toxins were microinjected into 293 cells, Swiss 3T3 fibroblasts, promyelocytic IPC-81 cells, and NRK cells. All cells started to undergo budding typical of apoptosis within 0.5 - 3 min after injection. This was accompanied by cytoplasmic and nuclear shrinkage and externalization of phosphatidylserine. Overexpression of Bcl-2 did not delay apoptosis. Apoptosis induction was slower and Z-VAD.fmk independent in caspase-3 deficient MCF-7 cells. MCF-7 cells stably transfected with caspase-3 showed a more rapid and Z-VAD.fmk dependent apoptotic response to nodularin. Rapid apoptosis induction required inhibition of both PP1 and PP2A, and the apoptosis was preceded by increased phosphorylation of several proteins, including myosin light chain. The protein phosphorylation occurred even in the presence of apoptosis-blocking concentrations of Z-VAD.fmk, indicating that it occurred upstream of caspase activation. It is suggested that phosphatase-inhibiting toxins can induce caspase-3 dependent apoptosis in an ultrarapid manner by altering protein phosphorylation.

cAMP induces co-translational modification of proteins in IPC-81 cells.

An elevated cAMP concentration results in growth arrest and protein synthesis-dependent apoptosis in the promyelocytic leukaemia cell line IPC-81. A comparison of two-dimensional gels of extracts from these cells labelled with [(35)S]methionine revealed that five distinct protein spots were induced by cAMP in a protein-synthesis-dependent manner. The spots seemed to result from the acidic shift of a precursor protein. The most abundant spot was phospho-actin. The spots induced by cAMP in intact cells were induced by cAMP-dependent protein kinase (cAPK) during the translation in vitro of mRNA from the leukaemia cells. The effect of cAPK was strictly co-translational, none of the spots being induced when cAPK was added after translation. This suggested that the protein spots arose by co-translational phosphorylation catalysed by cAPK. Two of the protein spots, phospho-actin and a protein with a molecular mass of 30 kDa and an isoelectric point of 4.5, were studied further with respect to expression. They were produced during the whole pre-apoptotic period, had cellular half-lives of several hours and were induced by the same concentrations of cAMP analogue that induced apoptosis. It is suggested that the accumulation of co-translationally modified proteins could be important for long-term cAMP signalling.

cAMP inhibits translation by inducing Ca2+/calmodulin-independent elongation factor 2 kinase activity in IPC-81 cells.

Treatment of IPC-81 cells led to inhibition of protein synthesis, which was accompanied by an increase in the average size of polysomes and a decreased rate of elongation, indicating that it involved inhibition of peptide chain elongation. This inhibition was also associated with increased phosphorylation of elongation factor eEF2 (which inhibits its activity) and enhanced Ca2+/calmodulin-independent activity of eEF2 kinase. Previous work has shown that phosphorylation of eEF2 kinase by cAMP-dependent protein kinase (cAPK) in vitro induces such activator-independent activity, and the present data show that such a mechanism can occur in intact cells to link physiological levels of cAPK activation with inhibition of protein synthesis.

The compartmentalization of protein synthesis: importance of cytoskeleton and role in mRNA targeting.

Following the synthesis of mRNA molecules in eukaryotic cells, the transcripts are processed in the nucleus and subsequently transported through the nuclear membrane into the cytoplasm before being sequestered into polysomes where the information contained in the RNA molecule is translated into an amino acid sequence. Recent evidence suggests that an association of mRNAs with the cytoskeleton might be important in targeting mechanisms and, furthermore, in the transport of mRNA from the nucleus to its correct location in the cytoplasm. Until recently, polysomes have been considered to exist in two classes, namely free or membrane-bound. There is now compelling evidence, however, that ribosomes, in addition to being associated with endoplasmic reticulum membranes, also are associated with components of the cytoskeleton. Thus, a large number of morphological and biochemical studies have shown that mRNA, polysomes and translational factors are associated with cytoskeletal structures. Although the actual nature and significance of the interaction between components of the translational apparatus and the cytoskeleton is not yet understood in detail, it would seem evident that such interactions are important in both the spatial organization and control of protein synthesis. Recent work has shown that a subcellular fraction, enriched in cytoskeletal components, contains polysomes and these (cytoskeletal-bound) polysomes have been shown to contain specific mRNA species. Thus, a population of cytoskeletal-bound polysomes may provide a specialized mechanism for the sorting, targeting and topographical segregation of mRNAs. In this review, current knowledge of the subcellular compartmentalization of mRNAs is discussed.

The mRNAs for cyclin A, c-myc and ribosomal proteins L4 and S6 are associated with cytoskeletal-bound polysomes in HepG2 cells.

Cytosolic, cytoskeleton and membrane fractions were extracted from HepG2 cells by a sequential detergent/salt extraction procedure. The cytosolic fraction contained 93% of the lactic dehydrogenase activity while the cytoskeleton fraction was enriched in actin and vimentin. The distribution of mRNAs for c-myc, glucose transporter 1, ribosomal proteins L4 and S6 and cyclin A were investigated by Northern hybridization of total RNA extracted from polysomes isolated from cytosolic, cytoskeleton and membrane fractions. The membrane-bound polysomes were enriched in the glucose transporter 1 mRNA and the cytoskeleton-bound polysomes were enriched in the mRNAs for the two ribosomal proteins, c-myc and cyclin A. The results suggest that the mRNAs for nuclear proteins are one class of mRNAs which are translated on polysomes associated with the cytoskeleton; this may be related to the requirement to transport the newly synthesized protein to the nucleus.