The vascular bone marrow niche influences outcome in chronic myeloid leukemia via the E-selectin - SCL/TAL1-CD44 axis.

The endosteal bone marrow niche and vascular endothelial cells provide sanctuaries for leukemic cells. In murine chronic myeloid leukemia (CML) CD44 on leukemia cells and E-selectin on bone marrow endothelium are essential mediators for the engraftment of leukemic stem cells. We hypothesized that non-adhesion of CML-initiating cells to E-selectin on the bone marrow endothelium may lead to superior eradication of leukemic stem cells in CML after treatment with imatinib than imatinib alone. Indeed, here we show that treatment with the E-selectin inhibitor GMI-1271 in combination with imatinib prolongs survival of mice with CML via decreased contact time of leukemia cells with bone marrow endothelium. Non-adhesion of BCR-ABL1(+) cells leads to an increase of cell cycle progression and an increase of expression of the hematopoietic transcription factor and proto-oncogene Scl/Tal1 in leukemia-initiating cells. We implicate SCL/TAL1 as an indirect phosphorylation target of BCR-ABL1 and as a negative transcriptional regulator of CD44 expression. We show that increased SCL/TAL1 expression is associated with improved outcome in human CML. These data demonstrate the BCR-ABL1-specific, cell-intrinsic pathways leading to altered interactions with the vascular niche via the modulation of adhesion molecules - which could be exploited therapeutically in the future

Population pharmacokinetics of mycophenolic acid in pediatric renal transplant patients using parametric and nonparametric approaches.

International audienceMycophenolic acid (MPA) is an immunosuppressive drug widely used in the prevention of acute rejection in pediatric renal transplant recipients and is characterized by a wide inter-individual variability in its pharmacokinetics. The aim of this study was to compare population pharmacokinetic modeling of MPA in pediatric renal transplant recipients given mycophenolate mofetil, the ester prodrug of MPA, using parametric and nonparametric population methods. The data from 34 pediatric renal transplants (73 full pharmacokinetic profiles obtained on day 21, months 3, 6 and 9 post-transplant) were analyzed using both the nonlinear mixed-effect modeling (NONMEM) and nonparametric adaptive grid (NPAG) approaches, based on a two-compartment model with first order lagged time absorption and first order elimination. The predictive performance of the two models was evaluated in a separate group of 32 patients. Higher mean population parameter values and ranges of individual pharmacokinetic parameters were obtained with NPAG, especially for the elimination constant ke: mean 1.16 h(-1) (0.26-4.33 h(-1)) and 0.78 h(-1) (0.66-1.15 h(-1)) with NPAG and NONMEM, respectively. With NPAG, the skewness and kurtosis values for ke (2.03 and 7.80, respectively) were far from the theoretical values expected for normal distributions. Such a non-normal distribution could explain the high value of shrinkage (35%) obtained for this parameter with the parametric NONMEM method. Bayesian forecasting of mycophenolic acid exposure using the NPAG population pharmacokinetic parameters as priors yielded a better predictive performance, with a significantly smaller bias than with the NONMEM model (-1.68% vs -9.53%, p<0.0001). In conclusion, in the present study, NPAG was found to be the most adequate population pharmacokinetic method to describe the pharmacokinetics of MPA in pediatric renal transplant recipients

A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

Regulation of protein N-glycosylation is essential in human cells. However, large-scale, accurate, and site-specific quantification of glycosylation is still technically challenging. We here introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising protein aggregation capture (PAC)-based sample preparation, multi-notch MS3 acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that enables confident identification and quantification of intact glycopeptides in complex biological samples. PAC significantly reduces sample-handling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search, which increases the identification rates of glycopeptides by 22% compared with conventional strategies. We apply SugarQuant to identify and quantify more than 5,000 unique glycoforms in Burkitt’s lymphoma cells, and determine site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence

Pretransplant assessment of human liver grafts by plasma lecithin: cholesterol acyltransferase (LCAT) activity in multiple organ donors.

In spite of the improved outcome of orthotopic liver transplantation (OLTx), primary graft nonfunction remains one of the life-threatening problems following OLTx. The purpose of this study was to evaluate plasma lecithin: cholesterol acyltransferase (LCAT) activity in multiple organ donors as a predictor of liver allograft viability prior to OLTx. Thirty-nine donors were studied during a 5-month period between April and August 1988. Allograft hepatectomy was performed using a rapid technique or its minor modification with hilar dissections, and the allografts were stored cold (4 degrees C) in University of Wisconsin (UW) solution. Early post-transplant allograft function was classified as good, fair, or poor, according to the highest SGOT, SGPT, and prothrombin time within 5 days following OLTx. Procurement records were reviewed to identify donor data, which included conventional liver function tests, duration of hospital stay, history of cardiac arrest, and graft ischemic time. Blood samples from the donors were drawn immediately prior to aortic crossclamp, and from these plasma LCAT activity was determined. Plasma LCAT activity of all donors was significantly lower than that of healthy controls (12.4 +/- 8.0 vs 39.2 +/- 13.3 micrograms/ml per hour, P less than 0.01). LCAT activity (16.4 +/- 8.3 micrograms/ml per hour) in donors of grafts with good function was significantly higher than that in those with fair (8.6 +/- 4.5 micrograms/ml per hour, P less than 0.01) or poor (7.3 +/- 2.4 micrograms/ml per hour, P less than 0.01) function.(ABSTRACT TRUNCATED AT 250 WORDS

Bioluminescence Enhanced Enzyme Immunoassay. New Ultrasensitive Detection Systems for Enzyme Immunoassays, II.

Peer Reviewe

Activation of CD44/PAK1/AKT signaling promotes resistance to FGFR1 inhibition in squamous-cell lung cancer

Lung cancer is the leading cause of cancer-related deaths worldwide. Fibroblast growth factor receptor 1 (FGFR1) gene amplification is one of the most prominent and potentially targetable genetic alterations in squamous-cell lung cancer (SQCLC). Highly selective tyrosine kinase inhibitors have been developed to target FGFR1; however, resistance mechanisms originally existing in patients or acquired during treatment have so far led to limited treatment efficiency in clinical trials. In this study we performed a wide-scale phosphoproteomic mass-spectrometry analysis to explore signaling pathways that lead to resistance toward FGFR1 inhibition in lung cancer cells that display (i) intrinsic, (ii) pharmacologically induced and (iii) mutationally induced resistance. Additionally, we correlated AKT activation to CD44 expression in 175 lung cancer patient samples. We identified a CD44/PAK1/AKT signaling axis as a commonly occurring resistance mechanism to FGFR1 inhibition in lung cancer. Co-inhibition of AKT/FGFR1, CD44/FGFR1 or PAK1/FGFR1 sensitized ‘intrinsically resistant’ and ‘induced-resistant’ lung-cancer cells synergetically to FGFR1 inhibition. Furthermore, strong CD44 expression was significantly correlated with AKT activation in SQCLC patients. Collectively, our phosphoproteomic analysis of lung-cancer cells resistant to FGFR1 inhibitor provides a large data library of resistance-associated phosphorylation patterns and leads to the proposal of a common resistance pathway comprising CD44, PAK1 and AKT activation. Examination of CD44/PAK1/AKT activation could help to predict response to FGFR1 inhibition. Moreover, combination between AKT and FGFR1 inhibitors may pave the way for an effective therapy of patients with treatment-resistant FGFR1-dependent lung cancer

Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells.

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1