ALBERT

Description: Abstract 731 Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired disorder of hemopoietic stem cells (HSCs). Affected individuals experience intravascular hemolysis and have a predisposition to thromboembolism, renal impairment and pulmonary hypertension. These symptoms vary in severity, but in general, the higher the proportion of PNH blood cells produced, the more severe the symptoms. The molecular pathogenesis in PNH is known to relate to a single gene mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A) which causes a complete or partial deficiency of glycophosphatidylinositol (GPI) anchored proteins leading to the symptoms of the disease. The recent development of eculizumab therapy in PNH has had a dramatic impact in reducing both morbidity and mortality in the disease but PNH remains incurable. A sub-optimal response to eculizumab, certainly when assessed by transfusion requirements, is often due to the underlying bone marrow failure that is considered to be universally present in PNH. The factors that lead from the development of a mutant clone to clonal expansion and symptomatic disease are poorly understood but are the key to improving responses and potentially to move towards a cure. Bone marrow failure appears to provide the environment necessary for expansion of PNH clones and small PNH clones are often detected in aplastic anemia and less commonly in myelodysplasia. There is no evidence that PNH HSCs have an intrinsic proliferative advantage compared to normal HSCs and an immune-mediated extrinsic suppression of normal hematopoiesis with a selective advantage for the PNH cells over residual normal stem cells is likely to explain the preferential development of PNH clones concurrent with bone marrow failure. To gain a better understanding of clonal expansion in PNH we have developed an in vitro PNH bone marrow culture model using a stromal cell line which allows PNH stem cells to be maintained in long term cultures and their capacity to form progenitor cells in myeloid colony forming assays to be assessed. We have evaluated bone marrow from 11 patients with PNH (median age 47 years, median granulocyte clone size 95.3%) and 10 normal controls (median age 42 years) within these long term bone marrow culture experiments. Unmanipulated bone marrow mononuclear cells (MNCs), CD34 selected cells and MNCs with their T-cell component depleted were used in this model. This in vitro model provides the environment for PNH stem cells to be maintained for up to eight weeks and, unlike previous studies, produce progenitor cells. When the patient's MNC's are used to seed the culture system there is poor growth of the culture which is only maintained for a median of 2 weeks. However if CD34 selected cells are used then the cultures are maintained for up to 8 weeks (similar to the normal controls) suggesting that there is a component within the MNC's that is responsible for suppressing the marrow culture which is removed by CD34 select. We next selectively removed the T-cells from the PNH MNC's and demonstrated that the marrow cultures now survived to the extent of the controls (see Figure). This demonstrates that the immune suppression in PNH resides in the T-cells. The progenitor cells produced in both the CD34 selected or the T-cell depleted MNCs over the course of the long term culture experiments show an increase in the proportion of normal progenitors the longer the cultures are maintained. This supports the hypothesis that PNH stem cells have no intrinsic proliferative advantage over normal HSCs and that T-cells are the critical cell suppressing the normal hematopoiesis in PNH. We are now examining the specific cell type that causes the myelosuppression in PNH and which will facilitate a targeted approach to the treatment of bone marrow failure in PNH and related disorders. In conclusion we have developed an in vitro PNH bone marrow culture model that allows the immune insult in PNH to be evaluated. Furthermore, this work confirms the important role that T-cells play in the etiology of PNH and provides a model with which to define the exact mechanism of suppression of hematopoiesis in PNH (and aplastic anemia). This information is essential to develop targeted therapies for the marrow failure seen in PNH and aplastic anemia. Disclosures: Kelly: Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Richards:Alexion Pharmaceuticals: Honoraria, Speakers Bureau. Arnold:Alexion Pharmaceuticals: Honoraria. Hill:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)–coupled collagen receptor GPVI and by the G protein–coupled receptor agonist thrombin. The glycoprotein VI (GPVI)–specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.

Description: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of hemopoietic stem cells (HSC) that is characterized by intravascular hemolysis and venous thrombosis. The PNH HSC’s (and their progeny) have a somatic mutation of the X-linked phosphatidylinositol glycan class A gene (PIG-A) which causes a complete or partial deficiency of glycophosphatidylinositol (GPI) anchored proteins, which in turn produces the symptoms of the disease. PIG-A mutations account for all of classical acquired PNH cases thus far reported as a single acquired PIG-A mutation on the only active X-chromosome results in GPI-deficiency. A novel mutation in the promoter of the phosphatidylinositol glycan class M gene (PIG-M) has recently been reported in two unrelated families exhibiting autosomal recessive inheritance of congenital GPI-deficiency. This point mutation reduces PIG-M transcription and causes partial deficiency of GPI-anchored proteins. It is possible that this point mutation in PIG-M occurs at low frequency in the population but predisposes to mutation of the other PIG-M allele and could result in acquired PNH. To determine the prevalence of the PIG-M in classical acquired PNH the PIG-M promoter region spanning the proposed mutation was sequenced to look for heterozygotes with the C → G substitution at position −270. This mutation was not identified in 36 patients with PNH. There is convincing evidence that GPI-deficient HSC’s have no intrinsic proliferative advantage over normal HSC’s suggesting that other factors underlie the clonal expansion of PNH cells. The most likely explanation appears to be due to factors which are extrinsic to the PNH clone such as immune attack on normal HSC. Recently secondary molecular events within the PNH clone have been suggested to provide the growth advantage allowing expansion of the PNH clone. One such proposed event is a translocation affecting the HMGA2 gene, which has been described in 2 cases of PNH. HMGA2 is a member of the high motility group of proteins and acts as an architectural transcription factor. HMGA2 proteins are associated with gene activation and are mainly expressed during embryonic development. Rearrangements of HMGA2 commonly occur in benign mesenchymal tumours, but have also been identified as infrequent events in myeloid malignancies. To gain insight into the potential role of HMGA2 deregulation in the pathogenesis of PNH we evaluated the expression of HMGA2 mRNA by means of quantitative RT-PCR in peripheral blood samples of 42 PNH patients (median age: 42, range: 18–81) and ten normal controls. All PNH samples had large granulocyte clones (median size 97.81%) but showed no increase in HMGA2 mRNA. In nine of the 42 PNH patients blood samples underwent additional enrichment for CD15 positive cells to maximize the proportion of PNH granulocytes present. This again showed no aberrant expression of HMGA2 mRNA. This is the largest reported group with PNH evaluated for HMGA2 expression. Despite two case reports suggesting that the deregulation of HMGA2 may be a pathophysiological factor in occasional cases of PNH our data indicates that this mechanism accounts for the growth advantage of PNH cells in, at most, only a small minority of patients. Similarly, the PIG-M promoter mutation observed in congenital GPI-deficiency was not found in any of our patients with classical acquired PNH indicating that it has no or little role in the pathophysiology of PNH.

Description: Key Points Paroxysmal nocturnal hemoglobinuria identifies a role for GPI-linked proteins in the homeostasis of human NK cell subsets. GPI-deficient NK cells exhibit impaired chemotactic responses.

Description: Abstract 1466 Poster Board I-489 B-lymphocyte induced maturation protein 1 (BLIMP-1) has been defined as a key driver of the genetic reprogramming during differentiation of B-cells to plasma cells. Frequent inactivation of PRDM1, the BLIMP-1 gene, in diffuse large B-cell lymphoma (DLBCL) indicates that loss of function is an important event in lymphomagenesis. Only a limited set of direct BLIMP-1 target genes have been defined. In order to better understand the function of human BLIMP-1 in differentiation and malignancy we have established a more comprehensive set of occupied promoters. These data provide an extended view of the regulatory network controlled by BLIMP-1, and identify novel sets of targets involved in transcription and immune response. The composition of occupied promoters identifies complexity in BLIMP-1 binding motif selection, and substantial overlap between BLIMP-1 sites and Interferon regulatory factor (IRF) elements. Consistent with active competition between BLIMP-1 and IRFs, target genes associated with such overlapping motifs are found to be preferentially induced in response to BLIMP-1 knockdown. Finally BLIMP-1 targets are found to include key components of DLBCL gene expression signatures. This map of BLIMP-1 occupied promoters thus illuminates key aspects of function in normal and malignant cell biology. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3899 Introduction: Combination chemo-immunotherapy has improved the survival in previously untreated CLL, but the treatment of relapsed and fludarabine refractory CLL continues to be challenging. Inhibiting the B-cell receptor (BCR) signalling pathway appears to be beneficial in the majority of patients but is not sufficient to eradicate disease and combination approaches are already being investigated. We have previously undertaken large scale protein expression profiling in CLL and identified several molecules involved in neurotransmission that are highly expressed in CLL. Aim: To explore signalling mechanisms in neuronal markers that are aberrantly expressed in CLL, and to determine whether they have therapeutic potential in combination with BCR inhibitors. Methods: RNA expression data (Haslinger et al; Journal of Clinical Oncology. 2004) was used to identify candidate antigens that could be screened for protein level expression. Screening was restricted to antigens expressed on plasma membrane with an extracellular epitope and a commercially available antibody suitable for testing in combination with mouse monoclonal gating antibodies (i.e. fluorochrome-conjugated monoclonal, non-conjugated mouse monoclonal labelled using the zenon technique, or rabbit polyclonal detected using fluorochrome-conjugated secondary goat F(ab')2 anti-rabbit antibody). In vitro viability experiments were performed using either mononuclear cells, separated using lymphoprep, or purified CLL cells isolated with a B-cell isolation kit (Miltenyi Biotec), cultured with or without the presence of CD40L transfected fibroblasts as a supporting stromal layer. Viability was assessed using Annexin-V, 7-AAD and counting beads. To study pathway interactions, the B-cell receptor was stimulated by crosslinking using goat F(ab')2anti-human IgM or IgD and signalling responses were assessed by Syk phosphorylation status (Syk pY348 PE, BD Phosflow) and calcium flux (Fluo-3/Fura red, Invitrogen). Results: 84 target antigens were assessed of which 11 showed binding on all mononuclear cells and 9/84 showed binding on CLL cells and/or normal B-cells but not other leucocytes. Of the B-cell targets, 4/9 molecules have a role in neurotransmission, such as the nicotinic acetyl choline receptor subunit β4 (CHRNB4) and dopamine receptor D4 (DRD4). CHRNB4 had the highest level of expression on B-cell and CLL surface membranes and therefore other subunits of nicotinic acetyl choline receptor were further evaluated using RT-PCR. Of the 15 subunits tested 10 were expressed on CLL cells suggesting the possibility of an important role in the pathophysiology of CLL. However, in vitro experiments did not show a significant effect on viability of CLL cells with either acetyl choline or its antagonist mecamylamine. Unlike acetyl choline, dopamine was found to be effective in reducing CLL cell survival in vitro, in a dose dependent manner with an LC50 of 8.2μM (95%CI- 5.2 to 12.9, n=6). Assessment of dopamine receptor agonists and agonists demonstrated that the D2-like family specific antagonist domperidone enhances the activity of dopamine and also has single agent activity in reducing in vitro CLL cell viability. To determine the effect of dopamine on BCR pathway signalling, syk phosphorylation and calcium flux after IgD crosslinking were measured. Stimulation of the BCR pathway by crosslinking using goat F(ab')2 anti human IgD increased the mean fluorescent intensity of syk phosphorylation by 1.9 fold (p=0.0024) compared to 1.5 fold (p=0.0186) when pre-incubated with dopamine and 1.2 fold (p=0.0059) with a syk inhibitor. Similarly the mean percentage rise in CLL cells fluxing calcium has fallen from 20.1% to 8.2% (p=〈 0.0001) when treated with dopamine and to 5.6% (p=〈 0.0001) when treated with syk inhibitor. In vitro testing demonstrated a synergistic effect in blocking of the D2 and the BCR pathways as a combination of PI3 kinase δ inhibitor, GS-1101, along with domperidone reduced CLL cell viability more efficiently than either agent alone. Conclusion: Systematic screening has identified several new targets in CLL, in particular several neuronal markers which are highly expressed and functionally active. Biological investigations suggest that these neuronal receptors could play an important role in the pathophysiology of CLL and show potential synergies with therapeutically active BCR inhibitors. Disclosures: Hillmen: Alexion Pharmaceuticals, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.