Evaluating outcomes of adult patients with acute lymphoblastic leukemia and lymphoblastic lymphoma treated on the GMALL 07/2003 protocol.

Chemotherapy-based approaches still constitute an essential feature in the treatment paradigm of adult acute lymphoblastic leukemia (ALL). The German Multicenter Study Group (GMALL) is a well-established protocol for ALL. In this study, we assessed our recent experience with the GMALL 07/2003 protocol reviewing all adult ALL patients who were treated with GMALL in three major centers in Israel during 2007-2020. The analysis comprised 127 patients with a median age of 41 years (range 17-83). Sixty-two were B-ALL (49%), 20 (16%) patients were Philadelphia chromosome positive ALL, and 45 (35%) were T-ALL. The 2-year and 5-year overall survival rates were 71% and 57%, respectively. The 2-year relapse rate was 30% with 2-year and 5-year leukemia-free survival rates of 59% and 50%, respectively. Adolescents and young adults experienced significantly longer overall survival (84 months versus 51 months; p=0.047) as well as leukemia-free survival compared with older patients (66 months versus 54 months, p=0.003; hazard ratio=0.39, 95% confidence interval, 0.19-0.79; p=0.009). T-ALL patients had longer survival compared to B-ALL patients while survival was comparable among Philadelphia chromosome positive patients and Philadelphia chromosome negative patients. An increased number of cytogenetic clones at diagnosis were tightly associated with adverse prognosis (15-month survival for ≥2 clones versus 81 months for normal karyotype; p=0.003). Positive measurable residual disease studies following consolidation were predictive for increased risk of relapse (64% versus 22%; p=0.003) and shorter leukemia-free survival (11 months versus 42 months; p=0.0003). While GMALL is an effective adult regimen, a substantial patient segment still experiences relapse.

Prognostic relevance of miR-124-3p and its target TP53INP1 in pediatric ependymoma.

Ependymoma is a malignant pediatric brain tumor, often incurable under the current treatment regimen. We aimed to evaluate the expression of microRNAs (miRs) in pediatric ependymoma tumors in an attempt to identify prognostic molecular markers which would lead to potential therapeutic targets. Following miR-array expression analysis, we focused on 9 miRs that correlated with relapse which were further validated by quantitative real-time PCR (qRT-PCR) in a cohort of 67 patients. Western blotting and immunohistochemistry were used to measure target protein expression in 20 and 34 tumor samples, respectively. High expression of miR-124-3p significantly correlated with the lower progression-free survival (PFS) of 16% compared to 67% in those expressing low levels (P = .002). Interestingly, in the group of patients with local disease (n = 56) expression levels of this miR distinguished 2 subgroups with a significantly different outcome (P = .001). miR-124-3p was identified as an independent prognostic factor of relapse in the multivariate analysis performed in the whole cohort and in the group with localized disease. In the localized group, a patient expressing high levels of miR-124-3p had a 4.1-fold increased risk for relapse (P = .005). We demonstrated the direct binding of miR-124-3p to its target TP53INP1. Negative TP53INP1 protein levels correlated with a poor outcome (P = .034). We propose miR-124-3p and TP53INP1 as new biomarkers for prognostic stratification that may be possible therapeutic targets for ependymoma.

miR expression profiling at diagnosis predicts relapse in pediatric precursor B-cell acute lymphoblastic leukemia.

Our aim was to identify miRNAs that can predict risk of relapse in pediatric patients with acute lymphoblastic leukemia (ALL). Following high-throughput miRNA expression analysis (48 samples), five miRs were selected for further confirmation performed by real time quantitative PCR on a cohort of precursor B-cell ALL patients (n = 138). The results were correlated with clinical parameters and outcome. Low expression of miR-151-5p, and miR-451, and high expression of miR-1290 or a combination of all three predicted inferior relapse free survival (P = 0.007, 0.042, 0.025, and <0.0001, respectively). Cox regression analysis identified aberrant expression of the three miRs as an independent prognostic marker with a 10.5-fold increased risk of relapse (P = 0.041) in PCR-MRD non-high risk patients. Furthermore, following exclusion of patients harboring IKZF1 deletion, the aberrant expression of all three miRs could identify patients with a 24.5-fold increased risk to relapse (P < 0.0001). The prognostic relevance of the three miRNAs was evaluated in a non-BFM treated precursor B-cell ALL cohort (n = 33). A significant correlation between an aberrant expression of at least one of the three miRs and poor outcome was maintained (P < 0.0001). Our results identify an expression profile of miR-151-5p, miR-451, and miR-1290 as a novel biomarker for outcome in pediatric precursor B-cell ALL patients, regardless of treatment protocol. The use of these markers may lead to improved risk stratification at diagnosis and allow early therapeutic interventions in an attempt to improve survival of high risk patients.

Potential role of WSB1 isoforms in growth and survival of neuroblastoma cells.

BACKGROUND: WD repeat and SOCS box containing protein 1 (WSB1) generates three isoforms that were found to play a role in cancer cell growth and tumor progression. We have studied their expression in neuroblastoma (NB). METHODS: The behavior of the expression levels of the WSB1 isoforms was analyzed in NB cell lines, in an in vivo NB xenograft mouse model, and in primary NB tumors using real-time PCR. Effective WSB1 small interfering RNAs were transfected into cultured NB cell lines, and cell viability was analyzed using XTT assay and flow cytometry. RESULTS: A significant predominance of the WSB1 isoform 3 (WSB1(3)) expression level was demonstrated in all NB systems examined. Correspondingly, combination of WSB1(3) silencing together with WSB1 isoforms 1+2 silencing in NB cells showed reduced growth, enhanced apoptosis rate, and increased sensitivity to chemotherapeutic agents, specifically related to low expression of WSB1(3). CONCLUSION: Our results point to a possible differential role of WSB1 isoforms in NB and suggest WSB1(3) as a target for therapy in NB.

MiR-192 directly binds and regulates Dicer1 expression in neuroblastoma.

Neuroblastoma (NB) arises from the embryonic neural crest and is the most common extracranial solid tumor in children under 5 years of age. Reduced expression of Dicer1 has recently been shown to be in correlation with poor prognosis in NB patients. This study aimed to investigate the mechanisms that could lead to the down-regulation of Dicer1 in neuroblastoma. We used computational prediction to identify potential miRs down-regulating Dicer1 in neuroblastoma. One of the miRs that were predicted to target Dicer1 was miR-192. We measured the levels of miR-192 in 43 primary tumors using real time PCR. Following the silencing of miR-192, the levels of dicer1 cell viability, cell proliferation and migration capability were analyzed. Multivariate analysis identified miR-192 as an independent prognostic marker for relapse in neuroblastoma patients (p=0.04). We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3' UTR of Dicer1 on positions 1232-1238 and 2282-2288. An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic. Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression.

Functional analysis of Arabidopsis postprenylation CaaX processing enzymes and their function in subcellular protein targeting.

Prenylation is a posttranslational protein modification essential for developmental processes and response to abscisic acid. Following prenylation, the three C-terminal residues are proteoliticaly removed and in turn the free carboxyl group of the isoprenyl cysteine is methylated. The proteolysis and methylation, collectively referred to as CaaX processing, are catalyzed by Ste24 endoprotease or Rce1 endoprotease and by an isoprenyl cysteine methyltransferase (ICMT). Arabidopsis (Arabidopsis thaliana) contains single STE24 and RCE1 and two ICMT homologs. Here we show that in yeast (Saccharomyces cerevisiae) AtRCE1 promoted a-mating factor secretion and membrane localization of a ROP GTPase. Furthermore, green fluorescent protein fusion proteins of AtSTE24, AtRCE1, AtICMTA, and AtICMTB are colocalized in the endoplasmic reticulum, indicating that prenylated proteins reach this compartment and that CaaX processing is likely required for subcellular targeting. AtICMTB can process yeast a-factor more efficiently than AtICMTA. Sequence and mutational analyses revealed that the higher activity AtICMTB is conferred by five residues, which are conserved between yeast Ste14p, human ICMT, and AtICMTB but not in AtICMTA. Quantitative real-time reverse transcription-polymerase chain reaction and microarray data show that AtICMTA expression is significantly lower compared to AtICMTB. AtICMTA null mutants have a wild-type phenotype, indicating that its function is redundant. However, AtICMT RNAi lines had fasciated inflorescence stems, altered phylotaxis, and developed multiple buds without stem elongation. The phenotype of the ICMT RNAi lines is similar to farnesyltransferase beta-subunit mutant enhanced response to abscisic acid2 but is more subtle. Collectively, the data suggest that AtICMTB is likely the major ICMT and that methylation modulates activity of prenylated proteins.

In vivo direct molecular imaging of early tumorigenesis and malignant progression induced by transgenic expression of GFP-Met.

The tyrosine kinase receptor Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), play an important role in normal developmental processes, as well as in tumorigenicity and metastasis. We constructed a green fluorescent protein (GFP) Met chimeric molecule that functions similarly to the wild-type Met receptor and generated GFP-Met transgenic mice. These mice ubiquitously expressed GFP-Met in specific epithelial and endothelial cells and displayed enhanced GFP-Met fluorescence in sebaceous glands. Thirty-two percent of males spontaneously developed adenomas, adenocarcinomas, and angiosarcomas in their lower abdominal sebaceous glands. Approximately 70% of adenocarcinoma tumors metastasized to the kidneys, lungs, or liver. Quantitative subcellular-resolution intravital imaging revealed very high levels of GFP-Met in tumor lesions and in single isolated cells surrounding them, relative to normal sebaceous glands. These single cells preceded the formation of local and distal metastases. Higher GFP-Met levels correlated with earlier tumor onset and aggressiveness, further demonstrating the role of Met-HGF/SF signaling in cellular transformation and acquisition of invasive and metastatic phenotypes. Our novel mouse model and high-resolution intravital molecular imaging create a powerful tool that enables direct real-time molecular imaging of receptor expression and localization during primary events of tumorigenicity and metastasis at single-cell resolution.

Turning ON the switch--RhoGEFs in plants.

Plant ROPs (or RACs) are Ras-related, small GTP-binding proteins that function as molecular switches in numerous signaling cascades. ROPs (RACs) cycle between a GDP-bound, inactive state and a GTP-bound, active state. Activation requires guanine-nucleotide exchange factors (GEFs), which were not identified in plants until recently. Alfred Wittinghofer and co-workers' description of a group of plant-specific RhoGEFs opens the way for the elucidation of ROP signaling cascades from membrane receptors to responses.

Detection of protein-protein interactions in plants using bimolecular fluorescence complementation.

Protein function is often mediated via formation of stable or transient complexes. Here we report the determination of protein-protein interactions in plants using bimolecular fluorescence complementation (BiFC). The yellow fluorescent protein (YFP) was split into two non-overlapping N-terminal (YN) and C-terminal (YC) fragments. Each fragment was cloned in-frame to a gene of interest, enabling expression of fusion proteins. To demonstrate the feasibility of BiFC in plants, two pairs of interacting proteins were utilized: (i) the alpha and beta subunits of the Arabidopsis protein farnesyltransferase (PFT), and (ii) the polycomb proteins, FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Members of each protein pair were transiently co-expressed in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis. Reconstitution of a fluorescing YFP chromophore occurred only when the inquest proteins interacted. No fluorescence was detected following co-expression of free non-fused YN and YC or non-interacting protein pairs. Yellow fluorescence was detected in the cytoplasm of cells that expressed PFT alpha and beta subunits, or in nuclei and cytoplasm of cells that expressed FIE and MEA. In vivo measurements of fluorescence spectra emitted from reconstituted YFPs were identical to that of a non-split YFP, confirming reconstitution of the chromophore. Expression of the inquest proteins was verified by immunoblot analysis using monoclonal antibodies directed against tags within the hybrid proteins. In addition, protein interactions were confirmed by immunoprecipitations. These results demonstrate that plant BiFC is a simple, reliable and relatively fast method for determining protein-protein interactions in plants.