ALBERT

Description: The del(5q) is the most commonly reported deletion in de novo MDS and is found in 10–15% of all patients. Our group demonstrated haploinsufficiency for the ribosomal gene RPS14, which is required for the maturation of 40S ribosomal subunits and maps to the commonly deleted region in patients with the 5q- syndrome (Boultwood et al, Br J Haematol2007, 139:578–89). Haploinsufficiency of RPS14 has been shown to be the mechanism underlying the erythroid defect in this disorder (Ebert et al, Nature2008, 451:335–9). We have recently shown that haploinsufficiency of RPS14 in patients with the 5q- syndrome is associated with deregulated expression of ribosomal- and translation-related genes, suggesting that the 5q- syndrome represents a disorder of aberrant ribosome biogenesis (Pellagatti et al, Br J Haematol2008, 142:57–64). The del(5q) in the 5q-syndrome is cytogenetically indistinguishable from the del(5q) found in other MDS and in the vast majority of these patients the CDR of the 5q- syndrome will be deleted (and therefore one allele of RPS14 will be lost). We are investigating the hypothesis that haploinsufficiency of RPS14 and consequent deregulated ribosome biogenesis may also play a role in the pathogenesis of non-5q- syndrome MDS patients with del(5q). Using Affymetrix U133 Plus2.0 arrays, we have studied the expression profiles of a group of 579 ribosomal- and translation-related genes in the CD34+ cells of 21 non-5q- syndrome MDS patients with del(5q) and 95 MDS patients without del(5q). 168 of 579 ribosomal-and translation-related probe sets were found to be significantly differentially expressed between these two groups, with approximately 90% of these showing lower expression levels in patients with del(5q). Hierarchical clustering using this set of 168 genes gave a good separation between patients with and without the del(5q). RPS14 was one of the most significant differentially expressed genes, with lower expression levels in patients with del(5q) confirming its haploinsufficient status in these patients. Other significant differentially expressed genes include the ribosomal protein RPL22L1, and the translation initiation factors EIF4EBP3 and EIF4B. Interestingly, when samples from 16 patients with 5q- syndrome were included in the analysis, hierarchical clustering using significantly differentially expressed ribosomal- and translation-related genes showed that most patients with 5q- syndrome and most patients with del(5q) clustered together. We are currently using polysome profile analysis on bone marrow cells to examine the levels of the 40S ribosomal subunit in patients with del(5q) and without del(5q). Our results support the hypothesis that haploinsufficiency of RPS14 and deregulation of ribosomal- and translation-related genes contribute to disease pathogenesis in MDS patients with del(5q). An exciting possibility is that other MDS with the del(5q) and the 5q- syndrome share a related molecular basis in that they are all disorders of defective ribosomal biogenesis.

Description: Platelet factor 4 (PF4) is an abundant platelet α-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the γ-carboxyglutamic acid (Gla) domain of protein C to significantly enhance activated protein C (APC) generation by the thrombin-thrombomodulin complex on the surface of endothelial cells. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. The functional consequences of the interaction between the APC Gla domain and PF4 in relation to APC anticoagulant, anti-inflammatory and anti-apoptotic functions have not previously been fully defined. In a tissue factor-initiated thrombin generation assay, APC impaired thrombin generation as previously described. However PF4 inhibited APC anticoagulant activity in a concentration-dependent manner (IC50 for PF4 inhibition of APC anticoagulant function, 11μg/ml). In contrast, addition of two other cationic polypeptides protamine and polybrene, both significantly enhanced APC anticoagulant activity in plasma. To elucidate the mechanism through which PF4 inhibits APC anticoagulant activity, we utilized a phospholipid-dependent FVa proteolysis time course assay. In the absence of protein S, PF4 had no effect upon FVa proteolysis by APC, indicating that PF4 does not influence the ability of APC to interact with either anionic phospholipids or FVa. However, in the presence of protein S, PF4 significantly inhibited APC-mediated FVa proteolysis (3–5 fold). Collectively, these findings demonstrate that in addition to enhancing APC generation, PF4 also significantly attenuates APC anticoagulant activity in plasma by impairing critical protein S cofactor enhancement of FVa proteolysis, and suggest that PF4 contributes to the poorly-understood APC resistance phenotype associated with activated platelets. APC bound to the endothelial cell protein C receptor (EPCR) via its Gla domain can activate PAR-1 on endothelial cells, triggering complex intracellular signaling that result in anti-inflammatory and anti-apoptotic cellular responses. To ascertain whether PF4 interaction with the protein C/APC Gla domain might impair APC-EPCR-PAR-1 cytoprotective signaling, APC protection against thrombin-induced endothelial barrier permeability and staurosporine-induced apoptosis in the presence of PF4 was determined. APC significantly attenuated thrombin-induced endothelial cell barrier permeability, as expected. PF4 alone (up to 1μM) had no independent effect upon endothelial barrier permeability, and did not protect against thrombin-mediated increased permeability. In contrast to its inhibition of APC anticoagulant activity, PF4 did not significantly inhibit the endothelial barrier protective properties of APC. To determine whether PF4 might interfere with APC-mediated cytoprotection, staurosporine-induced apoptosis in EAhy926 cells was assessed by RT-PCR quantification of pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) gene expression. Pre-treatment of EAhy926 cells with APC decreased the Bax/Bcl-2 ratio close to that determined for untreated EAhy926 cells. PF4 alone, or in combination with APC, had no effect upon apoptosis-related gene expression as determined by alteration of Bax/Bcl-2 expression ratios in response to staurosporine. In summary, PF4 inhibits APC anticoagulant function via inhibition of essential protein S cofactor enhancement in plasma, whilst retaining EPCR/PAR-1 mediated cytoprotective signalling on endothelial cells. This provides a rationale for how PF4 can exert prothrombotic effects in vivo, but also mediate enhanced APC generation on the surface of endothelial cells to induce both anti-inflammatory and anti-apoptotic events. Based on these observations, we propose that PF4 acts as a critical regulator of APC generation in vivo, but also targets APC towards cytoprotective, rather than anticoagulant functions at sites of vascular injury with concurrent platelet activation.

Description: Activated protein C (APC) plays a critical anticoagulant role by inactivating factor Va (FVa) and factor VIIIa (FVIIIa) and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor (EPCR) can initiate PAR-1 mediated cytoprotective signalling. Although protein S constitutes a critical cofactor for APC anticoagulant function, the molecular basis through which protein S interacts with APC is not fully understood. In this study, we employed a site-directed mutagenesis strategy to characterise the effects of four single amino acid substitutions (D35T, D36A, L38D and A39V) within a region of the APC Gla domain important for protein S cofactor enhancement. To maintain Gla domain structural integrity, each residue was substituted with the corresponding residue of the human prothrombin Gla domain. Protein C variants were expressed in HEK 293 cells and purified by ion-exchange chromatography. Upon activation, the amidolytic activity of each recombinant APC variant was identical to that of wild type APC. The anticoagulant function of recombinant wild type and variant APC was compared in a tissue factor-initiated thrombin generation assay using protein C-deficient plasma. Wild type APC diminished thrombin generation in a concentration-dependent manner as expected. Variants APC-D35T, APC-D36A and APC-A39V exhibited only mildly impaired (

Description: Protamine sulphate is a positively-charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, protamine also possesses intrinsic anticoagulant properties. Furthermore, administration of excess protamine in the neutralization of UFH is associated with increased bleeding, particularly following cardiothoracic surgery. In this study we have investigated the molecular mechanisms underlying the anticoagulant properties of protamine. In pooled normal plasma, we observed a dose-dependent prolongation of both PT and APTT assays with increasing protamine concentrations (0–30μg/ml). The anticoagulant effects of protamine in normal plasma were also examined using a tissue factor-initiated thrombin generation assay. 30μg/ml protamine resulted in a two-fold prolongation of lag-time, a two-fold reduction in peak thrombin generation, and a 41±17% (p=0.047) decrease in endogenous thrombin potential (ETP). In heparinised plasma (0.3U/ml), addition of increasing protamine concentration initially reversed the anticoagulant effect of heparin, resulting in a progressive increase in ETP to that of normal plasma. However, further increases in protamine resulted in a dose-dependent reduction in ETP to a minimum of 61±16%. Recent studies have shown that platelet factor 4 (PF4), another cationic protein used to reverse heparin, can bind to the anionic Gla domain of protein C, thereby enhancing APC generation (up to 25-fold). Consequently, we investigated potential interaction(s) between protamine and the protein C anticoagulant pathway. As expected, in normal plasma, APC (0–20nM) caused a concentration-dependent prolongation in APTT to 180±8%, and a parallel reduction in ETP. However in the presence of 30μg/ml protamine, the effects of APC on both the APTT and ETP were markedly enhanced compared to the effect of either substance alone. As APC down-regulates thrombin generation by inactivating FVa and FVIIIa, we used a phospholipid-dependent FVa proteolysis assay to elucidate the mechanism responsible for the synergistic interaction between APC and protamine. The ability of APC to reduce FVa cofactor activity in this assay (in the presence or absence of protein S) was not significantly affected by the presence of protamine. However, a potent synergistic anticoagulant interaction between APC and protamine was also observed in plasma from patients with homozygous FV Leiden, suggesting protamine enhances APC cleavage of FVa at position Arg-306. To determine whether protamine influences the rate of FVIIIa proteolysis by APC, we expressed and purified an APC-resistant FVIII variant (R336Q/R562Q). FVIII-deficient plasma was spiked with physiological concentrations of wild type or variant FVIII, and the anticoagulant effects of protamine ± APC studied using plasma thrombin generation assays. Similar synergistic anticoagulant effects of APC in combination with protamine were observed for both wildtype and variant FVIII. To assess whether protamine also influences procoagulant processes, the rate of factor V activation by thrombin was analysed by SDS-PAGE. In the presence of protamine (30μg/ml), FVa generation was significantly reduced. In addition to inhibiting the rate of FVa generation, we also observed that protamine significantly impaired the functional activity of the prothrombinase complex in a concentration dependent manner. In contrast, cationic polybrene had no significant effect on either rate of FVa generation or prothrombinase complex activity. In conclusion, we have shown a novel and profound anticoagulant synergy between protamine sulphate and APC. Moreover, we demonstrate that this synergistic effect is mediated by independent effects on FVa generation and proteolysis respectively. These novel findings provide further insights into the molecular mechanism underlying the anticoagulant effect of excess protamine in human plasma.

Description: Antithymocyte/antilymphocyte globulins are polyclonal antihuman T-cell antibodies used clinically to treat acute transplant rejection. These reagents deplete T cells, but a rabbit antihuman thymocyte globulin has also been shown to induce regulatory T cells in vitro. To examine whether antithymocyte globulin–induced regulatory cells might be functional in vivo, we generated a corresponding rabbit antimurine thymocyte globulin (mATG) and tested its ability to induce regulatory cells in vitro and whether those cells can inhibit acute graft-versus-host disease (GVHD) in vivo upon adoptive transfer. In vitro, mATG induces a population of CD4+CD25+ T cells that express several cell surface molecules representative of regulatory T cells. These cells do not express Foxp3 at either the protein or mRNA level, but do show suppressive function both in vitro and in vivo when adoptively transferred into a model of GVHD. These results demonstrate that in a murine system, antithymocyte globulin induces cells with suppressive activity that also function in vivo to protect against acute GVHD. Thus, in both murine and human systems, antithymocyte globulins not only deplete T cells, but also appear to generate regulatory cells. The in vitro generation of regulatory cells by anti-thymocyte globulins could provide ad-ditional therapeutic modalities for immune-mediated disease.

Description: Vorinostat (suberoylanilide hydroxamic acid, SAHA) is a histone deacetylase inhibitor active clinically in cutaneous T-cell lymphoma and preclinically in leukemia. A phase 1 study was conducted to evaluate the safety and activity of oral vorinostat 100 to 300 mg twice or thrice daily for 14 days followed by 1-week rest. Patients with relapsed or refractory leukemias or myelodysplastic syndromes (MDS) and untreated patients who were not candidates for chemotherapy were eligible. Of 41 patients, 31 had acute myeloid leukemia (AML), 4 chronic lymphocytic leukemia, 3 MDS, 2 acute lymphoblastic leukemia, and 1 chronic myelocytic leukemia. The maximum tolerated dose (MTD) was 200 mg twice daily or 250 mg thrice daily. Dose-limiting toxicities were fatigue, nausea, vomiting, and diarrhea. Common drug-related adverse experiences were diarrhea, nausea, fatigue, and anorexia and were mild/moderate in severity. Grade 3/4 drug–related adverse experiences included fatigue (27%), thrombocytopenia (12%), and diarrhea (10%). There were no drug-related deaths; 7 patients had hematologic improvement response, including 2 complete responses and 2 complete responses with incomplete blood count recovery (all with AML treated at/below MTD). Increased histone acetylation was observed at all doses. Antioxidant gene expression may confer vorinostat resistance. Further evaluation of vorinostat in AML/MDS is warranted.

Description: The Y chromosome encodes male-specific minor histocompatibility (H-Y) antigens that stimulate T- and B-lymphocyte responses after sex-mismatched allogeneic hematopoietic cell transplantation (HCT). A CD8+ cytotoxic T lymphocyte (CTL) clone that recognizes a novel HLA-B*2705–restricted H-Y antigen encoded by the DDX3Y gene was isolated from a male who had received a hematopoietic cell graft from his human leukocyte antigen (HLA)–identical sister. The antigenic peptide is a decamer that differs from the homologous DDX3X-encoded peptide at 4 positions. Expression of DDX3Y and of the H-Y epitope that it encodes was examined by quantitative polymerase chain reaction (PCR) and by CTL recognition assays. Expression of DDX3Y is detected in all myeloid and lymphoid leukemic cells that carry an intact Y chromosome. Moreover, the DDX3Y-encoded H-Y epitope is presented on the surface of both myeloid and lymphoid leukemic cells from male HLA-B*2705+ patients. DDX3Y-specific CTLs prevent engraftment of human acute leukemia in nonobese diabetic/severe combined immune deficient mice, demonstrating that the DDX3Y-encoded H-Y antigen is also expressed in leukemic stem cells. These results demonstrate that CD8+ T-cell responses against DDX3Y have the potential to contribute to graft-versus-leukemia (GVL) activity after female into male allogeneic HCT. This study is registered at http://clinicaltrials.gov as NCT00107354.

Description: Phosphatidylethanolamine (PE) and glucosylceramide (GlyCer) are cell surface lipids that preferentially enhance anticoagulant, rather than procoagulant pathways. In particular, both PE and GlyCer enhance the anticoagulant activity of activated protein C (APC). Previous studies have indicated that specific APC Gla domain residues may mediate APC interaction with PE and GlyCer. To investigate whether specific APC residues mediate PE and GlyCer enhanced APC anticoagulant activity, we expressed a series of APC variants in which APC Gla domain residues not shared with the human prothrombin Gla domain were substituted with their prothrombin amino acid equivalent. The anticoagulant activity of each APC Gla domain variant was assessed in a tissue factor-initiated thrombin generation assay containing phospholipid vesicles of differing composition (80% PC/20% PS); or PC/PS/PE (60%/20%/20%); or PC/PS/GlyCer (60%/20%/20%). For each of these lipid mixtures, thrombin generation (endogenous thrombin potential, ETP) was not significantly different in the absence of APC. In the presence of PC/PS vesicles, APC reduced thrombin generation by 63±3% at the highest APC concentration tested (6nM). However, APC impairment of thrombin generation was enhanced 3-fold in the presence of PC/PS/PE compared with vesicles containing PC/PS alone, and in the presence of PC/PS/GlyCer was enhanced 4.3-fold. Enhancement of anticoagulant function by PE and GlyCer was similar for the majority of the APC Gla domain variants tested. Interestingly, one APC variant (APC-I18V) exhibited similar anticoagulant activity to that of wild type APC with PC/PS vesicles, but was not enhanced by the presence of PE- or GlyCer-containing vesicles. Phospholipid vesicles containing PE or GlyCer have been previously described to enhance protein S cofactor enhancement of APC. Therefore, to further characterize APC-I18V, we assessed the ability of wild type APC and APC-I18V to be enhanced by protein S in the presence of PC/PS, PC/PS/PE or PC/PS/GlyCer using a protein S-sensitive thrombin generation assay. In the presence of PC/PS, increasing protein S concentration in protein S-deficient plasma resulted in an APC-mediated slow decrease in thrombin generation, irrespective of whether wild type or APC-I18V was used (IC50 for protein S-mediated APC inhibition of thrombin generation with PC/PS, 130nM). However, in the presence of PC/PS/PE or PC/PS/GlyCer, thrombin generation was impaired by wild type APC at 3–4-fold lower protein S concentration than that observed when PC/PS vesicles alone were used (IC50, PC/PS/PE=31.5nM and PC/PS/GlyCer=37.5nM). APC-I18V, however, did not exhibit a similarly increased sensitivity to protein S in the presence of PE or GlyCer, as the anticoagulant activity of this variant was the same as when only PC/PS was included. To investigate whether the loss of specific neutral lipid enhancement in APC-I18V affected its ability to initiate cytoprotective signaling via EPCR-PAR-1 on endothelial cells, the capacity of APC-I18V to inhibit thrombin-induced endothelial cell barrier permeability was assessed. When cells were pre-treated with either wild type APC or APC-I18V, there was a significant enhancement in barrier integrity and attenuation of thrombin-induced permeability (P

Description: Clonal heterogeneity has not been described in patients with myelodysplastic syndrome (MDS) of the 5q- syndrome subtype, for which lenalidomide has emerged as a highly potent treatment. Interestingly, transformation to acute myeloid leukemia (AML) occurs more frequently in patients without a cytogenetic response to lenalidomide. We describe two patients with classical 5q- syndrome with complete erythroid and partial cytogenetic response to lenalidomide, who evolved to high-risk MDS and AML, respectively, with complex karyotypes including del(5q). Immunohistochemistry of pre-treatment marrow biopsies revealed small fractions of progenitors with aberrant cytoplasmic nucleophosmin (NPMc+) expression and in one patient also a TP53 mutation. Both lesions may induce a state of genomic instability, and represent novel findings in 5q- syndrome. These subclones remained stable during response, but expanded at transformation, suggesting that pre-existing cells with more malignant genotype may be insensitive to lenalidomide and responsible for disease progression. We are currently investigating a larger patient material for the presence of subclones with molecular lesions in order to assess the potential impact on the risk of leukemic evolution.