Allogeneic hematopoietic cell transplantation in intermediate risk acute myeloid leukemia negative for FLT3-ITD, NPM1- or biallelic CEBPA mutations.

BACKGROUND: The value of allogeneic hematopoietic cell transplantation (alloHCT) as postremission treatment is not well defined for patients with intermediate-risk acute myeloid leukemia (AML) without FLT3-ITD, biallelic CEBPA-, or NPM1 mutations (here referred to as NPM1mut-neg/CEBPAdm-neg/FLT3-ITDneg AML) in first complete remission (CR1). PATIENTS AND METHODS: We addressed this question using data from two prospective randomized controlled trials on intensive induction- and risk-stratified postremission therapy. The NPM1mut-neg/CEBPAdm-neg/FLT3-ITDneg AML subgroup comprised 497 patients, aged 18-60 years. RESULTS: In donor versus no-donor analyses, patients with a matched related donor had a longer relapse-free survival (HR 0.5; 95% CI 0.3-0.9, P = 0.02) and a trend toward better overall survival (HR 0.6, 95% CI 0.3-1.1, P = 0.08) compared with patients who received postremission chemotherapy. Notably, only 58% of patients in the donor group were transplanted in CR1. We therefore complemented the donor versus no-donor analysis with multivariable Cox regression analyses, where alloHCT was tested as a time-dependent covariate: overall survival (HR 0.58, 95% CI 0.37-0.9, P = 0.02) and relapse-free survival (HR 0.51, 95% CI 0.34-0.76; P = 0.001) for patients who received alloHCT compared with chemotherapy in CR1 were significantly longer. CONCLUSION: Outside clinical trials, alloHCT should be the preferred postremission treatment of patients with intermediate risk NPM1mut-neg/CEBPAdm-neg/FLT3-ITDneg AML in CR1. CINICALTRIALS.GOV IDENTIFIER: NCT00180115, NCT00180102.

MicroRNA-21 targets tumor suppressor genes ANP32A and SMARCA4.

MicroRNA-21 (miR-21) is a key regulator of oncogenic processes. It is significantly elevated in the majority of human tumors and functionally linked to cellular proliferation, survival and migration. In this study, we used two experimental-based strategies to search for novel miR-21 targets. On the one hand, we performed a proteomic approach using two-dimensional differential gel electrophoresis (2D-DIGE) to identify proteins suppressed upon enhanced miR-21 expression in LNCaP human prostate carcinoma cells. The tumor suppressor acidic nuclear phosphoprotein 32 family, member A (ANP32A) (alias pp32 or LANP) emerged as the most strongly downregulated protein. On the other hand, we applied a mathematical approach to select correlated gene sets that are negatively correlated with primary-miR-21 (pri-miR-21) expression in published transcriptome data from 114 B-cell lymphoma cases. Among these candidates, we found tumor suppressor SMARCA4 (alias BRG1) together with the already validated miR-21 target, PDCD4. ANP32A and SMARCA4, which are both involved in chromatin remodeling processes, were confirmed as direct miR-21 targets by immunoblot analysis and reporter gene assays. Furthermore, knock down of ANP32A mimicked the effect of enforced miR-21 expression by enhancing LNCaP cell viability, whereas overexpression of ANP32A in the presence of high miR-21 levels abrogated the miR-21-mediated effect. In A172 glioblastoma cells, enhanced ANP32A expression compensated for the effects of anti-miR-21 treatment on cell viability and apoptosis. In addition, miR-21 expression clearly increased the invasiveness of LNCaP cells, an effect also seen in part upon downregulation of ANP32A. In conclusion, these results suggest that downregulation of ANP32A contributes to the oncogenic function of miR-21.

Cyclophilins contribute to Stat3 signaling and survival of multiple myeloma cells.

Signal transducer and activator of transcription 3 (Stat3) is the major mediator of interleukin-6 (IL-6) family cytokines. In addition, Stat3 is known to be involved in the pathophysiology of many malignancies. Here, we show that the cis-trans peptidyl-prolyl isomerase cyclophilin (Cyp) B specifically interacts with Stat3, whereas the highly related CypA does not. CypB knockdown inhibited the IL-6-induced transactivation potential but not the tyrosine phosphorylation of Stat3. Binding of CypB to Stat3 target promoters and alteration of the intranuclear localization of Stat3 on CypB depletion suggested a nuclear function of Stat3/CypB interaction. By contrast, CypA knockdown inhibited Stat3 IL-6-induced tyrosine phosphorylation and nuclear translocation. The Cyp inhibitor cyclosporine A (CsA) caused similar effects. However, Stat1 activation in response to IL-6 or interferon-gamma was not affected by Cyp silencing or CsA treatment. As a result, Cyp knockdown shifted IL-6 signaling to a Stat1-dominated pathway. Furthermore, Cyp depletion or treatment with CsA induced apoptosis in IL-6-dependent multiple myeloma cells, whereas an IL-6-independent line was not affected. Thus, Cyps support the anti-apoptotic action of Stat3. Taken together, CypA and CypB both play pivotal roles, yet at different signaling levels, for Stat3 activation and function. These data also suggest a novel mechanism of CsA action.

BCL3 is induced by IL-6 via Stat3 binding to intronic enhancer HS4 and represses its own transcription.

BCL3 is a proto-oncogene affected by chromosomal translocations in some patients with chronic lymphocytic leukemia. It is an IkappaB family protein that is involved in transcriptional regulation of a number of NF-kappaB target genes. In this study, interleukin (IL)-6-induced BCL3 expression and its effect on survival of multiple myeloma (MM) cells were examined. We demonstrate the upregulation of BCL3 by IL-6 in INA-6 and other MM cell lines. Sequence analysis of the BCL3 gene locus revealed four potential signal transducer and activator of transcription (Stat) binding sites within two conserved intronic enhancers regions: one located within enhancer HS3 and three within HS4. Chromatin immunoprecipitation experiments showed increased Stat3 binding to both enhancers upon IL-6 stimulation. Silencing Stat3 expression by small interfering RNA (siRNA) abrogated BCL3 expression by IL-6. Using reporter gene assays, we demonstrate that BCL3 transcription depends on HS4. Mutation of the Stat motifs within HS4 abolished IL-6-dependent BCL3 induction. Furthermore, BCL3 transcription was inhibited by its own gene product. This repressive feedback is mediated by NF-kappaB sites within the promoter and HS3. Finally, we show that overexpression of BCL3 increases apoptosis, whereas BCL3-specific siRNA does not affect the viability of INA-6 cells suggesting that BCL3 is not essential for the survival of these cells.

Interleukin-6 signal transduction and lymphocyte function.

Interleukin-6 (IL-6) is a pleiotropic cytokine that acts on a wide variety of cell types. It has important regulatory functions in the immune system, is a mediator of the acute-phase response, and is involved in the regulation of differentiation, proliferation, and survival of target cells. A major signal transduction pathway for IL-6 involves activation of JAK kinases and the transcription factor Stat3. In addition, a great many of other signalling pathways are induced. Stat3 has been shown to be a central player of IL-6 signalling in many systems whereas the functions of most other IL-6-activated pathways are not yet understood. In this review, we discuss the current knowledge on IL-6 functions in the immune system, IL-6 signal transduction, and its significance for lymphocyte function.

Different expression of the alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein in human keratinocytes and fibroblasts.

We compared, using a combination of different immunological methods and by competitive PCR, the expression of the alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2-M-R/LRP) in human keratinocytes and fibroblasts. This receptor has previously been found in skin only in dermal cells associated with fibroblasts and dendritic cells. For immunodetection we used mouse monoclonal antibodies against the two subunits of the receptor and against the receptor-associated protein (RAP), known as the regulatory protein of the receptor activity. The alpha2-M-R/LRP was found to be predominantly located intracellularly in keratinocytes whereas a distinct labelling of the outer membrane surface was found in fibroblasts. RAP is abundant in fibroblasts but is less expressed in keratinocytes. In frozen skin sections receptor immunoreactivity was detected in the epidermis with increased reactivity of basal keratinocytes, as well as in the dermis in association with dermal fibroblasts. By immunoprecipitation of biotinylated cell extracts, polypeptides were identified corresponding to the alpha-subunit and beta-subunit of the receptor as well as to the coprecipitating RAP. Competitive PCR revealed the presence of 67.9 and 2049.7 ag of alpha2-M-R/LRP mRNA per cell in keratinocytes and fibroblasts, respectively. The results demonstrate that both cell types express alpha-M-R/LRP mRNA and contain receptor protein as well as RAP but in different quantities and subcellular localizations.

Hexokinase 2 from Saccharomyces cerevisiae: regulation of oligomeric structure by in vivo phosphorylation at serine-14.

Homodimeric hexokinase 2 from Saccharomyces cerevisiae is known to have two sites of phosphorylation: for serine-14 the modification in vivo increases with glucose exhaustion [Kriegel et al. (1994) Biochemistry 33, 148-152], while for serine-157 it occurs in vitro with ATP in the presence of nonphosphorylateable five-carbon analogues of glucose [Heidrich et al. (1997) Biochemistry 36, 1960-1964]. We show now by site-directed mutagenesis and sedimentation analysis that serine-14 phosphorylation affects the oligomeric state of hexokinase, its substitution by glutamate causing complete dissociation; glutamate exchange for serine-157 does not. Phosphorylation of wild-type hexokinase at serine-14 likewise causes dissociation in vitro. In view of the higher glucose affinity of monomeric hexokinase and the high hexokinase concentration in yeast [Womack, F., and Colowick, S. P. (1978) Arch. Biochem. Biophys. 191, 742-747; Mayes, E. L., Hoggett, J. G., and Kellett, G. L. (1983) Eur. J. Biochem. 133, 127-134], we speculate that the in vivo phosphorylation at serine-14 as transiently occurring in glucose derepression might provide a mechanism to improve glucose utilization from low level and/or that nuclear localization of the monomer might be involved in the signal transduction whereby glucose causes catabolite repression.

Autophosphorylation-inactivation site of hexokinase 2 in Saccharomyces cerevisiae.

Hexokinase 2 from Saccharomyces cerevisiae is phosphorylated in vivo at serine-15 [Kriegel et al. (1994) Biochemistry 33, 148-152] and undergoes ATP-dependent autophosphorylation-inactivation in vitro when incubated in the presence of D-xylose [Fernandez et al. (1988) J. Gen. Microbiol. 134, 2493-2498]. This study identifies the site of inactivation by autophosphorylation as serine-158 by observation of a single tryptic peptide difference, peptide sequencing, and size determination by mass spectrometry. Mutation of serine-158 to alanine and cysteine, respectively, prevents autophosphorylation and causes a drastic decrease of the catalytic activity while mutational change to glutamate results in a complete loss of enzyme activity. The catalytically active mutant enzymes display an increased affinity for glucose and exhibit higher K(M) with respect to MgATP. Phosphoserine/phosphothreonine-specific protein phosphatase-2A completely reverses the autophosphorylative inactivation of the wild-type enzyme.