ALBERT

Description: Transferrin receptor 2 (TfR2) is a membrane glycoprotein that mediates cellular iron uptake from holotransferrin. Homozygous mutations of this gene cause one form of hereditary hemochromatosis in humans. We recently reported that homozygous TfR2(Y245X) mutant mice, which correspond to the TfR2(Y250X) mutation in humans, showed a phenotype similar to hereditary hemochromatosis. In this study, we further analyzed the phenotype as well as iron-related gene expression in these mice by comparing the TfR2-mutant and wild-type siblings. Northern blot analyses showed that the levels of expression of hepcidin mRNA in the liver were generally lower, whereas those of duodenal DMT1, the main transporter for uptake of dietary iron, were higher in the TfR2-mutant mice as compared to the wild-type siblings. Expression of hepcidin mRNA in the TfR2 mutant mice remained low even after intraperitoneal iron loading. In isolated hepatocytes from both wild-type and TfR2 mutant mice, interleukin-6 and lipopolysaccharide each induced expression of hepcidin mRNA. These results suggest that up-regulation of hepcidin expression by inflammatory stimuli is independent of TfR2 and that TfR2 is upstream of hepcidin in the regulatory pathway of body iron homeostasis. (Blood. 2005;105:376-381)

Description: Chemotherapy-resistant diffuse large B-cell lymphomas (DLBCL) pose an unresolved clinical problem and most patients die of their disease within months. The goal of this study was to test a novel, non-chemotherapy containing regimen for Non Hodgkin’s lymphomas, including DLBCL, by combining the proteasome inhibitor bortezomib (PS-341, VELCADE®) with radio-immunotherapy using ibritumomab tiuxetan (Zevalin™). Previously, we have shown that the combination of bortezomib and rituximab is an active and at least additive regimen in an in vitro and in vivo DLBCL model. These data supported an ongoing clinical trial using this combination in patients with low grade NHL. In addition, Pervan et al. identified the proteasome as a novel, sensitive target for ionizing radiation (Mol Cancer Res. 2005 (7):381–90), providing further rationale for this combination study. The combination treatment was evaluated in vivo in a SCID diffuse large B-cell lymphoma model. Bortezomib treatment did not alter the CD20 expression levels of SUDHL-16 cells in vitro. MMT assays showed an additive effect of bortezomib and gamma radiation on SUDHL-16 cell proliferation. In preliminary experiments, we identified the maximal tolerated dose of ibritumomab tiuxetan in our mouse model as 50 μCi/mouse. 2x106 SUDHL-16 cells injected s.q. in the flank resulted in local tumor growth within 4 weeks. When tumors became palpable, animals were stratified in 4 different treatment groups of 12 mice each. Mice received i.v. injections of either diluent, single agent bortezomib, single agent ibritumomab tiuxetan, or the combination of both. Ibritumomab tiuxetan was injected once i.v. at a dose of 20 μCi /mouse. Bortezomib was given i.v. as single dose of 0.01 mg/mouse. The combined treatment group received i.v. bortezomib as a radio-sensitizer 3 hrs prior to the injection of ibritumomab tiuxetan. All treatment schedules delayed tumor growth compared with controls. After 16 days of treatment, bortezomib reduced tumor progression by 52%, ibritumomab tiuxetan by 69%, and the combination treatment by 91%. Selected mice were sacrificed at day two and their tumors studied for potential immediate therapeutic effects. The Y90 content of tumors and normal tissues was measured and demonstrated targeting of Y90 to the xenograft. The tumors of the control and treatment groups did not differ by microscopic morphological appearance. In situ Tunel assays did not reveal increased apoptosis at treatment day two. In summary, bortezomib as a radio-sensitizer in combination with ibritumomab tiuxetan is an active and at least additive regimen in an in vivo DLBCL model.

Description: Despite major advances, multiple myeloma (MM) remains an incurable malignancy. Recently we have found that disease stabilization was achieved in 64% of patients with advanced MM treated with the farnesyltransferase inhibitor R115777 (Zarnestra) in a phase 2 clinical trial. In order to enhance R115777 antitumor activity in MM, we examined the combination of this novel agent with other anticancer drugs in MM cell lines. In this study, R115777 was found to synergize with paclitaxel and docetaxel, but not with other chemotherapy agents, including doxorubicin, 5-fluorouracil, cisplastin, melphalan, mitoxantrone, and dexamethasone. R115777 synergized with paclitaxel to inhibit MM cell proliferation and to induce apoptosis. Synergism in the induction of apoptosis was accompanied by increase in cytochrome c release and caspase-3 activation. Furthermore, flow cytometry analysis also showed that paclitaxel and R115777 synergized to induce G2/M cell-cycle arrest. Importantly, synergism was observed in taxane- and R115777-resistant MM cells. In the human severe combined immunodeficient (SCID-hu) bone model of myeloma growth, the ability of paclitaxel to inhibit tumor growth in vivo was enhanced by R115777. Combination of paclitaxel or docetaxel with R115777 in the treatment of MM cells from patients with multiple myeloma was more beneficial than treatment with single agents. Our results provide the basis for combination therapy clinical trials with paclitaxel or docetaxel with R115777 in MM patients. (Blood. 2005;105:4759-4766)

Description: HIV infection is associated with an increased risk of Non-Hodgkin’s B cell lymphoma (AIDS-NHL). AIDS-NHL may arise, in part, because the patients may be immunocompromised and tumor escape takes place. The standard treatment for NHL is chemotherapy, however, many patients become refractory to such treatments. Alternative treatment modalities include immunotherapy, though, even in the presence of an effective anti-tumor response, the tumor may develop mechanisms of resistance to immune-mediated cytotoxicity (e.g., Fas-ligand, TRAIL) and resistance to apoptosis. We have shown that overexpression of the transcription factor Yin-Yang 1 (YY1) is involved in the regulation of tumor cell resistance to FasL-induced apoptosis. The direct role of YY1 was demonstrated in cells transfected with siRNA YY1 which were sensitized to Fas-induced apoptosis (Vega, et al., 2005, Journal of Immunology (In Press)). In addition, we have also shown that overexpression of YY1 and X-linked inhibitor of apoptosis (XIAP) regulate the resistance of tumor cells to TRAIL-induced apoptosis (Ng and Bonavida, 2002, Molecular Cancer Therapeutics1:1051–1058, Huerta-Yepez, et al., 2004, Oncogene23:4993–5003). Hence, we hypothesized that one mechanism of AIDS-NHL immune escape may be due to overexpression of YY1 and XIAP. Tissue arrays containing formalin fixed, paraffin embedded sections from AIDS lymphoma were obtained from the AIDS and Cancer Specimen Resource (ACSR) of the National Cancer Institute (NCI). These arrays consisted of 21 Burkitt, 29 Large Cell Lymphoma, and 6 Small Cell Lymphoma. Immunohistochemistry was performed for the expression of YY1, and XIAP. The arrays were scored and both the percent of positive cells and the intensity were recorded and the data were analyzed statistically. The findings reveal that YY1 and XIAP were overexpressed in the majority of the AIDS-NHL patient specimens. In addition, there was a significant correlation between YY1 and XIAP expression in all 3 types of lymphoma. These studies and studies based on in vitro findings with AIDS-NHL cell lines suggest that overexpression of YY1 and XIAP may be involved in the pathogenesis of AIDS-NHL and are potential markers for tumor unresponsiveness to immune-mediated cytotoxic therapies. Furthermore, inhibitors of YY1 and XIAP expression/activity may be targets for therapeutic intervention when combined with immunotherapy.

Description: Pleiotrophin (PTN) is a secreted angiogenic protein. We have recently shown that malignant plasma cells express high amounts of PTN and that it is elevated in MM patient serum. In this study, we show that this protein leads to angiogenesis through a novel mechanism - transdifferentiation of monocytes into endothelial cells. First, we isolated human monocytes (CD14+ cells) from peripheral blood using immunomagnetic bead isolation. We excluded the presence of endothelial cells in these CD14-expressing cells using RT-PCR on 106 monocytes with primers to genes expressed by endothelial cells including Flk-1, Tie2, CD144, and von Willebrand factor (vWF). We cultured these purified monocytes on collagen I for one week in the presence of PTN, mCSF and VEGF. Cells cultured with the combination of mCSF and PTN developed Flk-1-expressing tube-like structures, and the addition of VEGF increased tube formation. Next, we performed RT-PCR analysis with primers to these endothelial genes on monocytes cultured with PTN, mCSF and VEGF following serial dilution in cells (T or B lymphocytes) that lack monocyte or endothelial cell gene expression. Many of the cells expressed Tie2 RNA (〉10%), and a smaller proportion (0.1–1%) also expressed Flk-1, CD144 and vWF RNA. In contrast, purified monocytes incubated with mCSF, VEGF, or PTN alone or the combinations of mCSF and VEGF or PTN and VEGF lacked Flk-1 staining, did not form tubes and failed to express endothelial cell RNA. We also assessed these monocytes in three dimensional matrices using Matrigel. Cells treated with mCSF and PTN invaded the matrix and began to form tube-like structures in the three dimensional gels as early as 7 days following culture whereas monocytes treated with mCSF, VEGF, or PTN alone did not form these structures. Next, we transduced human monocytic THP-1 cells that lack PTN expression with PTN-sense or -anti-sense constructs. Using RT-PCR, THP-1 cells transduced with PTN expressed endothelial cell genes and lost expression of the monocyte genes c-fms and CD68. In contrast, endothelial cell RNA was not detected in either THP-1 cells infected with anti-sense or the GFP control vectors. We used Transwell plates to co-culture THP-1 monocytes with human MM RPMI8226 or U266 cells or cell lines lacking PTN expression. We also added serum from MM patients with high levels of PTN or normal controls lacking PTN to THP-1 cells. THP-1 cells cultured with the MM cell lines or MM serum expressed endothelial genes and lost expression of monocyte RNA. Endothelial gene expression was blocked by the addition of an anti-PTN antibody but not by a control antibody. Control serum and cell lines lacking PTN did not induce endothelial gene expression or changes in monocyte RNA expression in the THP-1 cells. These experiments define a previously unrecognized novel mechanism leading to angiogenesis in cancer patients - the transdifferentiation of monocytes into endothelial cells by a factor highly produced by the malignant cells in MM. These findings also suggest a potential new specific target, PTN, to inhibit angiogenesis in cancer patients and should have profound clinical implications.

Description: The potential cardiotoxicity of chemotherapic drugs is well known. For example anthracycline-based regimens are extremely effective for various hematological malignancies. The main disadvantage is cardiotoxicity particularly, in elderly patients who are frequently treated with a consequent dose reduction. The diagnosis and prognosis in patients with suspected heart failure needs a specific monitoring by echocardiography during and after chemotherapy regimens. We tested the interest of NT-proBNP as alternative marker for the detection of left ventricular dysfunction. Brain or B-type natriuretic peptide (BNP) and N-terminal fragment of B-type natriuretic peptide (NT-proBNP) are considered to be valuable biomarkers for the detection of disease state in patients with suspected heart failure. Methods During 1 year, blood samples of 31 patients with hematological malignancies, treated with usual chemotherapy were selected on a routine basis. Patients had the diagnosis of acute leukemia (AL), B-chronic lymphocytic leukemia (B-CLL), multiple myeloma (MM) and non Hodgkin lymphoma (NHL). Venous blood was drawn in the early morning and centrifuged at 2000 g for 15 minutes. The obtained clear plasma fraction was stored at −20°C until the assay. All plasma samples were analyzed for NT-proBNP using an electro chemiluminescence immuno assay (proBNP kit Roche Diagnostics, Mannheim, Germany) on Elecsys 2010 analyser. All assays were performed blind to clinical informations on the patients. Results The mean age of the patients was 72 (range: 36–88). There were 15 men (48 %) and 16 women (52 %). Five patients were smokers (16 %) and 7 (22.6%) had cardiovascular diseases (4 hypertension, 2 heart failure, 1 pace maker). Only 3 patients had a subnormal renal function. There were 6 patients with AL, 6 with B-CLL, 11 with MM and 8 with NHL. The administered medications were divided in 3 cardio-toxicity stages: 10 (32.25 %) patients received stage 3 cardiotoxicity regimens, 10 (32.25 %) stage 2 and 11 (35.5 %) stage 1. Fourteen patients (45 %) died in relation with hematological malignancies and none in relation with heart failure. But treatment regimens have been reduced, discontinued, modified or stopped in 7 patients after heart failure diagnosis with echocardiography. All these patients received stage 2 or 3 cardiotoxicity chemotherapy regimens and 4 had prior cardiovascular diseases. The mean age was 74 (range: 66–82). Only one patient is alive in this subgroup. Considering the age and the heart state of our 31 patients, chemotherapeutic treatments need or not to be adjust. The cardiac risk at diagnosis was assessed by left-ventricular ejection fraction (VEF) measurement. We shows that NT-proBNP brings reliable results to assess that risk, with a positive correlation to the VEF. Figure Figure Conclusion Despite the limitations of this preliminary study the measurement of the NT-proBNP concentration at baseline and during cardiotoxic regimens in patients with hematological malignancies seems to be a promising method to identify patients with an increased risk of cardiovascular adverse effects for it evolves earlier than VEF and is very well correlate to VEF loss and cardiotoxicity.

Description: Background: APRIL (A Proliferation-Inducing Ligand) and BLyS (B Lymphocyte Stimulator) are potent survival factors for normal B cells and are over-expressed in plasma cell malignancies. BLyS binds to 3 members of the TNF-R family of receptors, TACI, BCMA and BAFF-R, whereas APRIL binds to TACI and BCMA and also to heparan sulfate proteoglycans such as syndecan-1, which is expressed by most plasma cells. APRIL and BLyS are produced by the malignant myeloma cells themselves, as well as by cells within the tumor environment, resulting in the enhanced survival of the malignant cells via both an autocrine and paracrine loop. In vitro, a blockade of BLyS and APRIL has been shown to induce myeloma-cell apoptosis. In the present clinical trial we have used a soluble receptor fusion protein comprised of the extracellular domain of TACI and the Fc portion of a human IgG (TACI-Ig) to neutralize both APRIL and BLyS in patients with multiple myeloma (MM) or Waldenström’s macroglobulinemia (WM). The aim was to determine the tolerability, the pharmacokinetics (PK), the pharmacodynamics and the biological activity of TACI-Ig. Methods: The trial is an open-label, dose-escalation study followed by a classical Simon 2-stage trial designed to determine the maximum tolerated dose as well as the optimal biologic dose of TACI-Ig, in patients with refractory or relapsed MM or active WM. Eligible patients are enrolled in sequential cohorts to receive five weekly subcutaneous injections of TACI-Ig at 2, 4, 7 or 10 mg/kg. Patients who demonstrate at least stable disease after the first cycle are allowed to receive 2 additional treatment cycles. PK is assessed after the 1st and 5th dosing. Usual safety parameters are assessed, including measurement of potential anti-TACI-Ig antibodies. The biological activity assessment comprises M-protein, beta 2-microglobulin, soluble syndecan-1, lymphocyte subpopulation counts (by flow cytometric analysis), polyclonal immunoglobulins, serum and urinary free light chains and CRP. Evaluation of response is assessed using modified Bladé criteria at the end of cycles 1 and 3. Results: Preliminary results of the first 3 cohorts of the dose-escalation study are reported. Six MM patients and 3 WM patients have entered the trial. No dose limiting toxicity has been observed and no SAE related to study drug has been reported to date. Mild injection site erythema (1 patient) is the only drug-related toxicity reported to date. Two MM patients and 1 WM patient demonstrated a stabilization of disease through the end of the third cycle, 3 MM patients and 1 WM patient demonstrated progressive disease after the first cycle and 1 MM and 1 WM patient have not been fully evaluated yet. Polyclonal immunoglobulins in 6/9 (5 MM and 1 WM) patients and soluble syndecan-1 in 2/5 MM patients show a decrease during treatment, while CRP levels are not affected. Conclusions: Treatment with TACI-Ig was well tolerated at the dose levels tested so far. A biological response in accordance with the expected TACI-Ig mode of action is observed in this heavily treated refractory population. Accrual of patients at higher dose levels is ongoing and will be presented.

Description: Bone resorption leading to osteolytic bone disease is characteristic of multiple myeloma (MM). Recent studies show the presence of bone-resorbing osteoclasts and bone-forming osteoclasts in the circulation, and these cells may correlate with bone disease and change with anti-bone resorptive therapies. We have investigated whether there is an imbalance in the expression of osteoblast and osteoclast genes in the peripheral blood mononuclear cells (PBMCs) from MM patients relative to normal age-matched controls and the effect of bisphosphonate treatment on the expression of these genes. We analyzed the expression of a panel of osteoblast-related (bone alkaline phosphatase [bone AP], bone morphogenic protein 2 [BMP2], collagen I and osteocalcin) and osteoclast-related (b3 integrin, calcitonin, receptor for activation of nuclear factor kappa B [RANK] and tartrate-resistant alkaline phosphatase [TRAP]) genes by semi-quantitative RT-PCR on total RNA isolated from PBMCs obtained following density gradient separation. We demonstrated that the expression of the osteoblast-related gene BMP2 was reduced in eight of nine MM patients when compared with normal donors. In marked contrast, three osteoclast-related genes, b3 integrin, RANK and TRAP, were more highly expressed in all nine MM patients compared to the normal donors; only calcitonin expression was similar to the control subjects. Interestingly, patients receiving bisphosphonate treatment appeared to show increased osteoblast gene expression with higher amounts of bone AP, BMP2 and osteocalcin RNA compared to the patients not receiving anti-bone resorptive therapy. However, there was no alteration in the level of the RNA in any of the four osteoclast genes compared to patients not receiving anti-bone resorptive therapy. We are extending our analysis to a larger panel of MM patients in order to determine the relationship between these circulating cells and bone disease, overall clinical status and change in their levels with anti-bone resorptive therapy. In addition, we are also investigating whether there exist larger and smaller numbers of circulating osteoclasts and osteoblasts, respectively, in MM patients, or whether these circulating cells show alteration of their expression of these genes. Our semi-quantitative RT-PCR results are being correlated with immunohistochemical staining results from osteoblast and osteoclast markers obtained on PBMCs from MM and normal subjects. These studies provide evidence that the number of circulating osteoblasts and osteoclasts is altered in patients with MM, and also may suggest that bisphosphonate therapy may also be associated with changes in these cell populations.

Description: Pleiotrophin (PTN) is a heparin-binding growth factor that binds CD138 and stimulates angiogenesis, tumor growth and metastasis in some solid tumors. Recently, we have shown that this factor is highly produced by multiple myeloma (MM) cell lines including RPMI8226 and U266 and fresh malignant plasma cells, and is secreted into the culture medium following short-term culture of bone marrow from MM patients. We investigated the effects of PTN on MM growth in vitro and in vivo using a SCID-hu murine MM model. We determined the anti-proliferative effects of suppressing PTN by cloning a whole PTN sense or anti-sense cDNA construct containing the green fluorescent protein (GFP) gene into the MM cell lines RPMI8226 and U266. Cells transduced with sense PTN showed markedly increased proliferation compared to cells transduced with vector alone whereas the anti-sense-containing MM cells showed reduced cell numbers. In addition, we treated RPMI8226 and U266 cells with a polyclonal anti-PTN antibody and evaluated its effect on MM growth. These cells were cultured for 48 hours in the presence of the anti-PTN antibody at a concentration of 100 micrograms/ml or a control antibody, and effects on cell growth assessed with an MTT assay. Marked anti-MM effects were observed with the anti-PTN antibody compared to the control antibody in both cell lines [RPMI8226 (p 〈 0.01) and U266 (p 〈 0.001)]. In order to further define the importance of PTN in the growth of MM in a more clinically relevant in vivo setting, we determined whether this polyclonal anti-PTN antibody could suppress tumor growth and human paraprotein secretion using our SCID-hu murine model of human myeloma LAGλ-1. LAGλ-1 has been previously shown by our group to produce large amounts of PTN as measured in mouse serum by ELISA and by RT- PCR analysis on freshly isolated LAGλ-1 tumor cells. Thirty SCID mice (n = 5 mice/group) were implanted with a 0.4 – 0.6 cm3 LAGλ-1 tumor fragment into the left hind limb muscle. Fourteen days following implantation, mice were randomized into treatment groups, and received treatment intraperitoneally (IP) with anti-PTN antibody at doses of 0.1, 0.3, 1.0, 3.0 or 10 mg/kg or vehicle alone twice weekly. Mice receiving anti-PTN antibody at the highest doses (3.0 and 10 mg/kg) showed marked inhibition of tumor growth [3.0 mg/kg (p 〈 0.03), 10 mg/kg (p 〈 0.008)] as well as decreases in levels of human paraprotein [3.0 mg/kg (p 〈 0.004), 10 mg/kg (p 〈 0.003)]. Notably, immunohistochemical staining with an anti-CD138 antibody showed a marked reduction in cells with CD138 positivity in the LAGλ-1 tumors from animals treated with anti-PTN antibody compared to mice treated with vehicle alone. These in vitro and in vivo results demonstrate that PTN may be a potential new target for the treatment of MM. The effects of this therapy on angiogenesis and cell signaling are currently under investigation.

Description: Signal transducer and activator of transcription 3 (Stat3) is a key mediator of several cytokines and growth factors signaling pathways. On myeloid cells, activation of Stat3 to its phosphorylated form (pStat3) has been shown to negatively regulate inflammatory responses. Recently, we have unambiguously demonstrated that Stat3 signaling in APCs also play a central role in the decision leading to immune activation versus immune tolerance of antigen-specific T-cells1. In spite of these advances, there is however a paucity of therapeutic strategies targeting this signaling pathway in immune cells. Using a high throughput cytoblot screening for phospho-Stat3 inhibition, we have recently identified a family of natural compounds known as Cucurbitacins that effectively disrupt Stat3 signaling at different levels2. Three compounds have been identified, Cucurbitacin A (CuA) that inhibits phospho-JAK-2, Cucurbitacin I (CuI) a dual inhibitor of p-JAK2 and p-Stat3 and Cucurbitacin Q (CuQ) a selective inhibitor of p-Stat3. In vitro treatment of peritoneal elicited macrophages (PEM) and bone marrow-derived dendritic cells (DCs) with increasing concentrations of CuA or CuI resulted in inhibition of p-Stat3 and enhanced antigen presentation to naive CD4+ T cells specific for a MHC class II restricted epitope of influenza hemagglutinin (HA). Indeed, these clonotypic T cells displayed increased antigen-specific proliferation and IL-2 production as compared to clonotypic T cells encountering cognate antigen on untreated APCs. Furthermore, unlike untreated PEM or DCs, which are unable to trigger IFN-gamma production by CD4+ T-cells, Cucurbitacin-treated APCs efficiently trigger the production of this cytokine by naïve CD4+ T-cells in response to cognate antigen. Given the above results, we explored next whether inhibition of Stat3 signaling in B-cell lymphomas by Cucurbitacins might increase the intrinsic antigen-presenting capabilities of these malignant B-cells. Reminiscent of our findings with bone marrow derived APCs, Cucurbitacin-treated A20 lymphoma cells also display enhanced antigen-presenting cell function leading to increased proliferation, IL-2 and IFN-gamma by naive antigen-specific CD4+ T-cells. More importantly, tolerant CD4+ T-cells (isolated from lymphoma bearing mice) exposed to Cucurbitacin-treated A20 B-cells regained their ability to proliferate and produce significant amounts of IL-2 and IFN-gamma in response to cognate antigen stimulation. Taken together, the ability of Cucurbitacins to inhibit p-Stat3 in normal APCs as well as in malignant B-cells make these natural compounds a promising agents to overcome the remarkable barrier that tolerance to tumor antigens has imposed to cancer immunotherapeutic strategies.