ALBERT

Description: Dasatinib (BMS-354825) is a novel, oral, multi-targeted kinase inhibitor of BCR-ABL and SRC kinases. Data from a phase I study suggest that dasatinib exhibits potent activity with high hematologic and cytogenetic response rates in CML patients with myeloid blast crisis (MBC) who were imatinib (IM)-resistant (IM-R) or -intolerant (IM-I). Here we report the preliminary results from one Phase II trial (Study CA180006 or ‘START-B’) in MBC, which was initiated in December 2004. This open-label study was carried out in 37 centers worldwide between December 2004 and May 2005. A total of 74 IM-R or IM-I MBC pts were accrued (41 male, median age 56 years [range 21–71]). Preliminary data are currently available on the first 34 pts (29 IM-R and 5 IM-I). Dasatinib was administered orally, at a dose of 70 mg twice daily (BID) in a continuous daily dosing schedule; dose escalation to 100 mg BID was permitted for patients who did not achieve hematologic response and dose reduction to 50 mg and 40 mg BID was allowed in the presence of persistent toxicity. Complete blood counts were performed weekly and bone marrow assessment, including cytogenetic analysis, was performed monthly. Mutations in the BCR-ABL domain were assessed in all pts. Pretreatment characteristics of these 34 pts included: 71% male, median age 54 years (range 21 – 71). Median duration of CML from first diagnosis was 49.3 months (range 5.6 – 215.5). Prior therapy included bone marrow transplant (5 pts, 15%) and interferon (18 pts, 53%). In 44% of pts, the highest IM dose was 〉600 mg/day and 41% of pts received IM for 〉3 years. Best responses to IM were complete hematologic response (CHR) in 82% of pts and major cytogenetic response in 39% of pts (complete in 27%, and partial in 12%). At baseline, 35% of pts had a WBC count ≥20 x 103/mm3, 71% had a platelet count

Description: Abstract 1923 Poster Board I-946 We recently characterized CD148 as a potential marker for mantle cell lymphoma using mass spectrometry analysis of cell-derived microvesicles in a restricted set of patients (ASH Annual Meeting Abstracts, Nov 2008; 112: 1766). CD148 is a plasma membrane receptor with phosphatase activity related to CD45, composed of an extracellular domain containing 8 fibronectin type II-like domains, a transmembrane region and a single intracellular phosphatase domain. Interestingly, it was recently shown that deletion of the phosphatase domain of CD148 in mice blocks B cell development at the transitional stage, with a dramatic increase of marginal zone B cells. BCR signalling events are also substantially altered in CD148/CD45 doubly deficient mice. In the present study, we analyzed the expression of CD148 using flow cytometry in a larger group of controls and patients with circulating pathologic B-cells, including 93 chronic lymphocytic leukemia (CLL), 46 small lymphocytic lymphomas with Matutes score inferior or equal to 3 (SLL), 35 MCL, all harboring (11;14) translocation and/or cyclin D1 overexpression, 5 marginal zone lymphomas (MZL), 5 splenic lymphoma with villous lymphocytes (SLVL), as well as 30 controls. Mean fluorescence intensity of direct CD148 staining with phycoerythrin conjugated 143−41 clone was used for expression comparison. CD148 MFI of the 30 control cells was weak and very homogeneous (mean = 168, SD = 31), as well as in the 93 CLL (mean = 199, SD = 84). SLL cases (n=46) were stained slightly higher (mean = 297, SD = 138) revealing a slight but significant difference with CLL (p

Description: Hematopoietic and nervous systems are linked via innervation of bone marrow (BM) niche cells. Hematopoietic stem/progenitor cells (HSPCs) express neurotransmitter receptors, such as the g-aminobutyric acid (GABA) type B receptor subunit 1 (GABBR1), suggesting that HSPCs could be directly regulated by neurotransmitters like GABA that directly bind to GABBR1. We performed imaging mass spectrometry (IMS) and found that endogenous GABA molecule is regionally localized and concentrated near the endosteum of the BM niche. To better understand the role of GABBR1 in regulating HSPCs, we generated a constitutive Gabbr1 knockout mouse model. Analysis revealed that HSPC numbers were significantly reduced in the BM compared to wild-type littermates. Moreover, Gabbr1 null hematopoietic stem cells (HSCs) had diminished capacity to reconstitute irradiated recipients in a competitive transplantation model. Gabbr1 null HSPCs were less proliferative under steady-state conditions and upon stress. Colony assays demonstrated that almost all Gabbr1 null HSPCs were in a slow or non-cycling state. In vitro differentiation of Gabbr1 null HSPCs in co-cultures, produced fewer overall cell numbers with significant defects in differentiation and expansion of the B cell lineage. To determine if GABBR1 agonist could stimulate human umbilical cord blood (UCB) HSPCs, we performed brief ex vivo treatment prior to transplant into immunodeficient mice, with significant increases in long-term engraftment of HSPCs compared to GABBR1 antagonist or vehicle treatments. Our results indicate a direct role for GABBR1 in HSPC proliferation, and identify a potential target to improve HSPC engraftment in clinical transplantation.

Description: Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells which differentiate to maintain and replenish blood lineages throughout life. Due to these characteristics, HSPC transplants represent a cure for patients with a variety of hematological disorders. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. The nervous system has been shown to indirectly influence hematopoiesis by innervating the niche; however, we present a direct route of HSPC regulation via expression of neurotransmitter receptors on HSPC surface. We have identified Gamma Aminobutyric acid (GABA) receptor B subunit 1 (Gabbr1), a hitherto unknown hematopoietic player, as a regulator of HSPC function. GABBR1 is known to be expressed on human HSPCs (Steidl et al., Blood 2004), however its function in their regulation remains unknown. Based on published RNA-seq data (Nestorowa et al., Blood 2016), we discovered that Gabbr1 is expressed on a subset of HSPCs. We confirmed this expression using RT-qPCR to assay hematopoietic populations in the bone marrow (BM). Surface receptor expression analysis showed that Gabbr1 protein is expressed on a subset of BM HSPCs. To detect GABA, the ligand for Gabbr1 in the BM microenvironment, we utilized imaging mass spectrometry (IMS). We detected regionally specific GABA signal in the endosteal region of the BM. We further identified B cells as a cellular source of GABA in the BM. To understand the role of Gabbr1 in hematopoiesis, we generated CRISPR-Cas9 Gabbr1 null mutants on a C57/BL6 background suitable for hematopoietic studies and studied their hematopoietic phenotype. We discovered a decrease in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but the long-term hematopoietic stem cells (LT-HSCs) remain unaffected. Further analysis of peripheral blood of Gabbr1 null mutants showed decreased white blood cells due to reduced B220+ cells. This differentiation defect was confirmed in an in vitro differentiation assay where Gabbr1 null HSPCs displayed an impaired ability to produce B cells. We show that Gabbr1 null HSCs show diminished reconstitution ability when transplanted in a competitive setting. Reduced Gabbr1 null HSC reconstitution persisted in secondary transplant recipients indicating a cell autonomous role for Gabbr1 in regulating reconstitution of HSCs in transplant recipients. Our results show a crucial role for Gabbr1 in HSPC regulation and may translate to human health as a rare human SNP within the GABBR1 locus that correlates with altered leukocyte counts has been reported (Astle et al., Cell 2016). Our studies indicate an important role for Gabbr1 in HSPC reconstitution and differentiation into B cell lineages. Disclosures No relevant conflicts of interest to declare.

Description: Patients with isolated pulmonary embolism (PE) have a distinct clinical profile from those with deep vein thrombosis (DVT)-associated PE, with more pulmonary conditions and atherosclerosis. These findings suggest a distinct molecular pathophysiology and the potential involvement of alternative pathways in isolated PE. To test this hypothesis, data from 532 individuals from the Genotyping and Molecular Phenotyping of Venous ThromboEmbolism (GMP-VTE) Project, a multi-center prospective cohort study with extensive biobanking, were analyzed. Targeted, high-throughput proteomics, machine learning, and bioinformatic methods were applied to contrast the acute-phase plasma proteomes of isolated PE patients (n=96) against those of patients with DVT-associated PE (n=276) or isolated DVT (n=160). This resulted in the identification of shared molecular processes between PE phenotypes, as well as an isolated PE-specific protein signature. Shared processes included upregulation of inflammation, response to oxidative stress, and the loss of pulmonary surfactant. The isolated PE-specific signature consisted of five proteins: interferon-γ (IFNG), glial cell line-derived neurotrophic growth factor (GDNF), polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3), peptidyl arginine deiminase type-2 (PADI2) and interleukin-15 receptor subunit α (IL-15Rα). These proteins were orthogonally validated using cis protein quantitative trait loci (cis pQTLs). External replication in an independent population-based cohort (n=5,778) further validated the proteomic results, and showed that they were prognostic for incident primary isolated PE in individuals without history of VTE (median time to event: 2.9 years, interquartile range: 1.6 - 4.2 years), supporting their possible involvement in the early pathogenesis. This study has identified molecular overlaps and differences between VTE phenotypes. In particular, the results implicate non-canonical pathways more commonly associated with respiratory and atherosclerotic disease in the acute pathophysiology of isolated PE.