The Job or hyper-immunoglobulinemia E syndrome is a primary immunodeficiency that is usually inherited in an autosomal dominant fashion. With the discovery of mutations in the STAT3 gene in the majority of autosomal dominant cases, it is now possible to make a molecular diagnosis of hyper-IgE syndrome. Both primary and secondary immunodeficiencies, including hyper-IgE syndrome, may predispose for malignancies, especially lymphomas, mainly mature B cell lymphomas, and classical Hodgkin lymphoma. Here, we report of a 48-year-old male with hyper-IgE syndrome who developed a primary parotid gland diffuse large B cell lymphoma. Analysis for STAT3 mutations demonstrated that the causal mutation of hyper-IgE syndrome, R382Q, arose de novo in the patient and it was transmitted to three of his five children, all three of whom are clinically affected. We review the literature regarding lymphoma in hyper-IgE syndrome and the possible etiologic relationship with STAT3 mutations.
2515 Poster Board II-492 Mantle cell lymphoma is a well defined subtype of B-cell non-Hodgkin lymphoma characterized by a translocation that juxtaposes the BCL1 gene on chromosome 11q13 (which encodes cyclin D1) next to the immunoglobulin heavy chain gene promoter on chromosome 14q32. The result is constitutive overexpression of cyclin D1 (CD1) resulting in deregulation of the cell cycle and activation of cell survival mechanisms. There are no “standard” treatments for MCL. Despite response rates to many chemotherapy regimens of 50% to 70%, the disease typically progresses after treatment, with a median survival time of approximately 3-4 years. Mantle cell lymphoma represents a small portion of malignant lymphomas, but it accounts for a disproportionately large percentage of lymphoma-related mortality. Novel therapeutic approaches are needed. In 2007, Nurtjaha-Tjendraputra described how iron chelation causes post-translational degradation of cyclin D1 via von Hippel Lindau protein-independent ubiquitinization and subsequent proteasomal degradation (1). Nurtjaha-Tjendraputra demonstrated that iron chelation inhibits cell cycle progression and induces apoptosis via proteosomal degradation of cyclin D1 in various cell lines, including breast cancer, renal carcinoma, neuroepithelioma and melanoma. Our preliminary data show similar findings in mantle cell lymphoma. To establish whether iron chelation can selectively inhibit and promote apoptosis in mantle cell derived cell lines, the human MCL cell lines Jeko-1, Mino, Granta and Hb-12; the Diffuse Large B cell lymphoma line SUDHL-6; and the Burkitt's Lymphoma lines BL-41 and DG75 were grown with media only, with two different iron chelators (deferoxamine (DFO) and deferasirox) at various concentrations (10, 20, 40, 100 and 250 μM), and with DMSO as an appropriate vehicle control. Cells were harvested at 24, 48 and 72 hours. For detection of apoptotic cells, cell-surface staining was performed with FITC-labeled anti–Annexin V antibody and PI (BD Pharmingen, San Diego, CA). Cell growth was analyzed using the Promega MTS cytotoxicity assay. CD1 protein levels were assessed using standard Western blot techniques. At 24, 48 and 72 hours of incubation with iron chelators, the mantle cell lymphoma cell lines showed significantly increased rates of apoptosis compared to the non-mantle cell lymphoma cell lines (p<0.0001 for all time points). DFO and deferasirox inhibted cell growth with an IC50 of 18 and 12 μM respectively. All of the mantle cell lines had measurable cyclin D1 levels at baseline. None of the non-mantle cell lines expressed baseline measurable cyclin D1. In the mantle cell lines, cyclin D1 protein levels were no longer apparent on western blot after 24 hours of incubation with chelation. We then added ferrous ammonium sulfate (FAS) to DFO in a 1:1 molarity ratio and to deferasirox in a 2:1 ratio, and then treated the same lymphoma cell lines with the FAS/chelator mixture and with FAS alone for 72 hours. Adding iron to the chelators completely negated all the pro-apoptotic effects that were seen with iron chelation treatment. Treating with FAS alone had no effect on cell growth or apoptosis. Iron chelation therapy with both DFO and deferasirox results in decreased cell growth, increased cellular apoptosis, and decreased cyclin D1 protein levels in vitro in mantle cell lymphoma. The cytotoxic effects are prevented by coincubation with ferrous ammonium citrate, confirming that the effects are due to iron depletion. Proposed future research includes further defining the molecular basis of iron chelation effects; studying these therapies in combination with other cancer treatments both in vitro and in vivo; and studying iron chelation therapy in mantle cell lymphoma patients. 1. Nurtjahja-Tjendraputra, E., D. Fu, et al. (2007). “Iron chelation regulates cyclin D1 expression via the proteasome: a link to iron deficiency-mediated growth suppression.” Blood109(9): 4045–54. Disclosures: No relevant conflicts of interest to declare.
The effects of sucralfate ingestion on serum and specific tissue aluminum (Al) accumulation were studied in normal rats fed either a control diet or the same diet supplemented with sucralfate. Although serum Al concentrations were not significantly different between the groups, animals fed sucralfate for 8 weeks had significantly higher bone but not brain or liver Al concentrations when compared with controls. This study indicates that 8 weeks exposure to Al in sucralfate leads to an increase in bone Al concentrations, without changes in serum Al concentrations, suggesting that serum Al concentration may be a poor predictor of gastrointestinal absorption and specific tissue retention of Al.
In Chuvash polycythemia, homozygosity for the 598 C->T mutation in the von Hippel-Lindau gene (VHL) leads to upregulation of hypoxia inducible factor-1a (HIF1a), a transcription factor that mediates cellular responses to hypoxia. This defect in the oxygen-sensing pathway causes increased expression of a broad range of hypoxia-regulated genes. Clinically, Chuvash polycythemia (CP) patients display not only erythrocytosis, but also premature mortality related to cerebrovascular and peripheral thrombotic events. As it is not clear that the thrombophilic nature of CP correlates with elevated hematocrit (Gordeuk et al, Blood103: 3924, 2004), we postulated that homocysteine may be a contributive factor, as preliminary data suggests that CP homozygotes have elevated plasma homocysteine levels (Sergueva, in preparation). Levels of homocysteine depend on its synthesis, involving S-adenosylmethionine, and its metabolism, either via remethylation to methionine, involving methylenetetrahydrofolate reductase (MTHFR), or via degradation by transsulfuration, involving cystathionine beta-synthase (CBS). Severe MTHFR and CBS deficiencies due to rare homozygous mutations lead to extremely high levels of serum homocysteine and are characterized clinically by a high incidence of thromboembolic complications, in addition to a wide range of other clinical symptoms. A recent microarray analysis that looked at the regulation of gene transcription by HIF-1a revealed that CBS and MTHFR gene expressions appear to be down regulated by hypoxia in endothelial cells (Manalo et al, Blood 105: 659, 2005). Downregulation of the genes responsible for homocysteine metabolism may therefore explain the elevated plasma homocysteine concentrations in CP. As hypoxia-regulated genes are often cell-type specific, we studied several types of easily accessible cells and detected expression of CBS and MTHFR in platelets, granulocytes, and EBV-immortalized lymphocytes in normal controls. In order to quantitate this expression, we used real-time RT-PCR and found no quantitative difference between EBV-immortalized lymphocytes in 4 homozygous CP patients, 3 heterozygote CP patients and 1 control. We then examined the peripheral blood from one CP patient and three controls. Although the numbers were small, the CP granulocytes and platelets showed decreased expression of MTHFR compared to controls, with decreased CBS expression seen in the CP granulocytes. These results suggest that the upregulation of HIF1a seen in CP patients might lead to decreased metabolism of homocysteine, which in turn, might contribute to the increased thromboembolic risk seen in CP. As these findings will need to be confirmed with a larger number of patients, we are currently in the process of collecting all accessible CP samples from the U.S. and from Chuvashia and the Italian island of Ischia (Perrotta et al, Blood 107: 514, 2006). Using the peripheral blood cells and in vitro expanded endothelial cells from these patients (Ingram et al, Blood104:2752, 2004), we hope to analyze the transcripts and enzyme activity of the genes involved in homocysteine synthesis and metabolism and to correlate these findings with CP plasma homocysteine levels.
Cell proliferation is dependent upon iron, and numerous studies have shown that iron limitation arrests cells in the G1 phase of the cell cycle. A recent study of the molecular basis of these observations (Richardson, et al. Blood2007;109:4045) examined the ability of iron chelators to inhibit cell proliferation and to induce apoptosis, focusing on the role of iron chelation on cyclin D1. Cyclin D1 assembles with cdk-4 or cdk-6, generating an active holo-enzyme that catalyzes a rate limiting step in G1/S progression. This complex phosphorylates substrates, including the retinoblastoma protein, which regulate S phase entrance. Richardson’s group demonstrated that the G1/S arrest after Fe depletion is mediated, in part, by a decrease in cyclin D1 via ubiquitin-independent proteasomal degradation. Studies looking specifically at mantle cell lymphoma cell lines, however, have not yet been reported. Mantle Cell lymphoma is an interesting target for potential iron chelation as it is associated with a balanced translocation (t11;14) which leads to upregulation of BCL1 and to the constitutive overproduction of cyclin D1. We studied five different cell lines - JeKo (Mantle Cell Lymphoma), BL-41 (Burkitt Cell Lymphoma), DG-75 (Burkitt Cell Lymphoma), SUDHL-6 (Diffuse Large B cell Lymphoma) and EBV-immortalized lymphocytes from normal controls - and incubated them with four different iron chelators - deferoxamine (DFO), 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), Pyridoxal Isonicotinoyl Hydrazone (PIH), and Salicylaldehyde Isocotinoyl Hydrazone (SIH). We then measured and compared cell cycle proliferation (using the Cellometer Auto T4, an instrument that measures cell count, cell viability, and cell size) and the rate of apoptosis (via propidium iodide FACS analysis). At 24 hours incubation, the mantle cell lymphoma lines showed significantly increased rates of apoptosis compared with non-chelated mantle cell controls (5% vs. 48%, p=0.04). The diffuse large B cell lymphoma line showed a lesser increase in apoptosis that did not reach statistical significant (6.5% vs. 14%, p=0.07), while the Burkitt’s lymphoma lines and the EBV immortalized lymphocytes showed no significant difference (BL-41, 3.4% vs. 4.1%, p=0.50; DG-75, 6% vs. 5.9%, p=0.99; EBV lymphocytes, 12.5% vs. 12.7%, p=0.96). At 72 hours of incubation with chelators, the EBV lymphocytes showed increased apoptosis compared to untreated controls (2.5% vs. 44.5%, p=0.002), while the apoptotic rate increased in the diffuse large B cell lymphoma line (3.8% vs. 48%, p=0.001) and even more dramatically in the mantle cell lymphoma line (1.5% vs. 64%, p=0.0006). The two Burkitt’s lymphoma lines were affected to a lesser degree at 72 hours by the presence of iron chelators (BL-41, 0.9% vs, 3.9%, p=0.02; DG-75, 5.5% vs. 8.9%, p=0.11). Although iron chelation, especially at longer incubation times, did affect all cell lines to various degrees, the chelator-mediated effects do appear to be specific for cell type, with mantle cell lymphoma cells displaying higher rates of apoptosis compared with other lymphomas and normal lymphocytes. These initial results will now be followed by examination of cyclin D1 expression after iron chelation. If overexpression of cyclin D1 in mantle cell lymphoma releases cells from their normal controls and acts as an oncogene, then a decrease in cyclin D1 levels via iron chelation could be added to the therapeutic armamentarium of mantle cell lymphoma.
3729 Poster Board III-665 Mantle cell lymphoma (MCL) is a well defined B-cell non-Hodgkin lymphoma characterized by a translocation that juxtaposes the BCL1 gene on chromosome 11q13, which encodes cyclin D1 (CD1), next to the immunoglobulin heavy chain gene promoter on chromosome 14. The resulting constitutive overexpression of CD1 leads to a deregulated cell cycle and activation of cell survival mechanisms. In addition, the gene which encodes GST-n, an enzyme that has been implicated in the development of cancer resistance to chemotherapy, is also located on chromosome 11q13 and is often coamplified along with the BCL1 gene in MCL (1). These two unique biological features of MCL - the overproduction of cyclin D1 and GST-n – may be involved in the carcinogenesis, tumor growth and poor response of this disease to treatment, and they offer potential mechanisms for targeted anti-cancer therapy. Nitric oxide (NO) is a biologic effector molecule that contributes to a host's immune defense against microbial and tumor cell growth. Indeed, NO is potently cytotoxic to tumor cells in vitro (2–4). However, NO is also a potent vasodilator and induces hypotension, making the in vivo administration of NO very difficult. To use NO in vivo requires agents that selectively deliver NO to the targeted malignant cells. A new compound has recently been developed that releases NO upon interaction with glutathione in a reaction catalyzed by GST-n. JS-K seeks to exploit known GST-n upregulation in malignant cells by generating NO directly in cancer cells, and it has been shown to decrease the growth and increase apoptosis in vitro in AML cell lines, AML cells freshly isolated from patients, multiple myeloma cell lines, hepatoma cells and prostate cancer cell lines (3, 5–7). JS-K also decreases tumor burden in NOD/SCID mice xenografted with AML and multiple myeloma cells (5, 7). Importantly, JS-K has been used in cytotoxic doses in the mouse model without significant hypotension. To evaluate whether JS-K treatment has anti-tumor activity in MCL, the human MCL cell lines Jeko1, Mino, Granta and Hb-12 were grown with media only, with JS-K at varying concentrations and with DMSO as an appropriate vehicle control. For detection of apoptotic cells, cell-surface staining was performed with FITC-labeled anti–Annexin V and PI. Cell growth was evaluated using the Promega MTS cytotoxicity assay. Results show that JS-K (at concentrations up to 10 μM) inhibits the growth of MCL lines compared to untreated controls, with an average IC50 of 1 μM. At 48 hours of incubation, all cell lines showed a significantly greater rate of apoptosis than untreated controls. A human MCL xenograft model was then created by subcutaneously injecting two NOD/SCID IL2Rnnull mice with luciferase-transfected Hb12 cells. Seven days post-injection, one of the mice was treated with JS-K at a dose of 4 μmol/kg (expected to give peak blood levels of around 17 mM in a 20 g mouse). Injections of JS-K were given intravenously through the lateral tail vein 3 times a week. The control mouse was injected with an equivalent volume of micellar formulation (vehicle) without active drug. The Xenogen bioluminescence imaging clearly showed a difference in tumor viability, with a significantly decreased signal in the JS-K treated mouse. Our studies demonstrate that JS-K markedly decreases cell proliferation and increases apoptosis in a concentration- and time-dependent manner in mantle cells in vitro. In a xenograft model of mantle cell lymphoma, treatment with JS-K results in decreased tumor viability. Proposed future research includes further defining the molecular basis of these treatment effects; using this therapy in combination with other cancer treatments both in vitro and in vivo; and studying JS-K treatment in MCL patients. Disclosures: Shami: JSK Therapeutics: Founder, Chief Medical Officer, Stockholder.
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