ALBERT

Description: Background: Lenalidomide (Revlimid®) is approved for the treatment of del 5q myelodysplastic syndrome (MDS) and in combination with dexamethasone for previously treated multiple myeloma. It is currently being evaluated as treatment for other hematology and oncology conditions, including NHL as a single agent and in combination with other therapeutics. Aims: The present study evaluates the effect of lenalidomide on the proliferation of the Burkitt’s Lymphoma tumor cell line Namalwa CSN.70, as a single agent and in combination with various chemotherapeutic agents: dexamethasone, doxorubicin, vincristine, methotrexate, cytarabine, ifosfamide, cyclophosphamide, carmustine, prednisone, etoposide, rituximab, bortezomib, rapamycin, and the mixed kinase inhibitor UCN-01. Methods: Namalwa CSN.70 cells were incubated in 96-well cell culture plates with compounds for 72 hours and cell proliferation was assayed by 3H-thymidine incorporation. IC50s were calculated by nonlinear regression analyses with GraphPad Prism. Results: Namalwa cell proliferation was inhibited by lenalidomide and the chemotherapeutic agents dexamethasone, doxorubicin, vincristine, methotrexate, cytarabine, carmustine, prednisone and etoposide. Cyclophosphamide and ifosfamide had no effect, since these agents require metabolic activation by cytochrome P450 enzymes. Lenalidomide combined with dexamethasone displayed synergistic anti-proliferative effects. Lenalidomide combined with prednisone displayed partially additive anti-proliferative effects at prednisone concentrations within the range of 0.5 and 50 mM, although this was non-additive at lower concentrations of both drugs. Lenalidomide combined with etoposide displayed partially additive anti-proliferative effects at etoposide concentrations within the concentration range of 0.05 and 0.5 mM, although at lower concentrations the response became non-additive and comparable to the effect of lenalidomide alone. In contrast, lenalidomide combined with carmustine displayed antagonistic effects at low concentrations, although partially additive anti-proliferative effects were observed at higher concentrations. Lenalidomide combined with methotrexate was also antagonistic. Lenalidomide in combination with cytarabine, doxorubicin, or vincristine generated anti-proliferative responses that were equivalent to the inhibition produced by these respective chemotherapeutic agents alone. Finally, rituximab was unable to add to the anti-proliferative effect of lenalidomide, consistent with the rituximab mechanism of action being primarily antibody-dependent cell-mediated cytotoxicity (ADCC), rather than direct apoptotic signaling since there is data showing the benefit of this combination in a mouse model of disseminated NHL (Hernandez-Ilizaliturri et al. 2005 Clin Cancer Res. 11:5984). Conclusions: These results support the potential for a synergistic effect of lenalidomide in combination with dexamethasone in NHL. Furthermore, partially additive effects of lenalidomide with prednisone and etoposide suggest these may be useful combinations. Results with bortezomib, rapamycin and UCN-01 will also be discussed.

Description: Interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1) mediate growth of MM cells via activation of the mitogen-activated protein kinase (MAPK), JAK2/STAT3, and phosphatidylinositol 3′-kinase/Akt kinase (PI3-K/Akt) signaling cascades. We have previously demonstrated the in vitro and in vivo activity of Revlimid™ (CC-5013), an immunomodulatory analog (IMiD) of thalidomide, in MM. In the present study, we have examined the anti-MM activity of rapamycin, a specific mTOR inhibitor, combined with Revlimid™. This combination was highly synergistic at 0.1nmol/L of rapamycin with 0.1mmol/L of Revlimid™, and remained synergistic at higher concentrations. Based on the Chou-Talalay method, combination indices of 〈 1 were noted for all dose ranges of Revlimid™ and rapamycin, suggesting strong synergism. Importantly, this combination was able to overcome drug resistance when tested against MM cell lines resistant to conventional (doxorubicin, melphalan, dexamethasone) chemotherapy. Moreover, the combination, but not rapamycin alone, was able to overcome the growth advantage conferred on MM cells by IL-6, IGF-1, or adherence to bone marrow stromal cells (BMSCs). Cytotoxicity triggered by a combination of rapamycin with Revlimid™ resulted in apoptosis of MM cells. Furthermore, differential signaling cascades, including the MAPK and PI3-K/Akt pathways, were targeted by these drugs individually and in combination, suggesting the molecular mechanism by which they interfere with MM growth and survival. These studies therefore provide the framework for the clinical evaluation of targeted agents like mTOR inhibitors combined with immunomodulatory agents like Revlimid™ to improve patient outcome in MM.

Description: Patients with myeloproliferative disorders (MPD) have an excess of thrombotic complications which is not fully corrected by normalisation of their elevated haematocrit or thrombocytosis. The mechanisms responsible for this are not completely understood but platelets, leucocytes and endothelial cells have all been implicated. Plasma microparticles are observed to be elevated in other diseases associated with thrombotic complications. We measured platelet (PMP), endothelial (EMP), red cell (RMP) and leucocyte (LMP) microparticles in patients with MPD compared to healthy controls. Microparticles, of less than 1μm were measured by flow cytometry on platelet poor plasma obtained from citrated peripheral blood samples. Subtypes were identified using fluorescent conjugated monoclonal antibodies; EMP CD31+CD42-, PMP CD31+42+, RMP Glycophorin A + and LMP CD45+ events. Absolute concentrations of microparticles per μl plasma were calculated using Trucount beads. Results are currently available for 27 MPD patients, 9 polycythaemia vera (PV), and 18 primary thrombocythaemia and 14 healthy controls. All patients except 1 PV were on current treatment with hydroxycarbamide and all but 3 were receiving aspirin. PMP and LMP were elevated in MPD patients compared to healthy controls, PMP 12432 (1653) v 2371 (278) per μl, mean (SEM) p

Description: CC-4047 is an immunomodulatory analog of thalidomide with stronger anti-myeloma and anti-angiogenic activity than thalidomide. We could show that CC-4047 directly influences lineage commitment and differentiation of hematopoietic stem cells (Koh et al., Blood 2004 in press). We found that CC-4047 effectively inhibits erythroid cell colony formation from CD34+ cells and increases the frequency of myeloid colonies. We also demonstrated that development of both erythropoietin-independent and erythropoietin-dependent red cell progenitors was strongly inhibited by CC-4047, while terminal red cell differentiation was unaffected. However, there is little information regarding the mechanism by which CC-4047 affect hematopoiesis. Due to the fact that CC-4047 has been shown to influence secretion of proinflammatory cytokines of peripheral mononuclear cells after LPS stimulation we investigated the cytokine profile of hematopoietic progenitors treated with this drug. CD34+ cells were cultured with SCF, IL-3 and IL-6 in the presence of thalidomide (100μM) or CC-4047 (100μM) for 1, 3 or 6 days and cytokine gene expression was studied in these hematopoietic progenitor cells using gene array analyses. Furthermore, supernatants were collected and examined for IL-1b, IL-2, IL-4, IL-5, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF-a, IFN-g, GM-CSF, G-CSF, MCP-1, and MIP-1b. Our analysis revealed that cytokines supporting myelopoiesis increased very early after treatment with CC-4047. After CC-4047 stimulation, secretion of G-CSF increased within 24 hours 10-fold in comparison to control cells. MCP-1, which is known to support predominantly the granulocytic lineage and to augment the clonal expansion of hematopoietic progenitor cells, increased also up to 5-fold on day 1 under CC-4047 treatment in compared to control. Secretion of IL-10, a pro-inflammatory cytokine known to inhibit erythropoiesis, was also up regulated. In addition, IL-13, which favors the development of erythroid progenitors, decreased 3-fold by CC-4047 on day 1 compared to control. In contrast, thalidomide induced much weaker changes in cytokine secretion. This is in line with our observation that thalidomide has only weak effects on lineage commitment. Cytokine analysis after 24 hours G-CSF MCP-1 IL-10 IL-5 IL-13 pg/ml Control 162 3543 7.9 6191 2806 Thal 455 7653 10.8 3425 2563 CC-4047 1514 17734 28 968 1748 In contrast to the previous findings that CC-4047 inhibits TNF-a, IL-12 and IL-1b synthesis in activated mononuclear cells are our results showing that secretion of TNF-a, IL-12, IL-1b and also of IL-2, IL-4, IL-7, IL-8, IL-17, IFN-g, GM-CSF and MIP-1b is not significantly affected by CC-4047 and thalidomide. Analyses of cytokine gene expression confirmed our results. In conclusion, these data indicate that CC-4047 might directly influence lineage commitment of hematopoietic cells by modulation of cytokine secretion increasing the propensity of stem and/or progenitor cells to undergo myeloid cell development and concomitantly inhibiting red cell development. The influence on cytokine secretion is an early event since these changes can observed within the first 24 hours of CC-4047 treatment and depends strictly on cell type and differentiation level. CC-4047 provides a valuable tool to study the mechanisms underlying lineage commitment.

Description: Lenalidomide induces impressive response rates in patients with multiple myeloma. Unfortunately, one of the major side effects of this therapy is the increased risk of thromboembolic events (TEE) up to 17%. The reason for a higher risk of TEE associated with Lenalidomide is not known, but it has been found that aspirin is very effective in preventing TEEs induced by thalidomide and lenalidomide (Rajkumar, 2005). Cathepsin G is a serine proteinase present in the azurophilic granules of polymorphonuclear neutrophils (PMNs) and is a strong platelet aggregation agonist with potency similar to thrombin. In the current study we investigated the effect of the immunomodulatory derivatives of thalidomide (IMiDs) including Actimid and lenalidomide on cathepsin G in vitro and in vivo. To investigate the effect of IMiDs in vitro CD34+ hematopoietic progenitors were cultured in the presence of IL-3, IL-6 and SCF supporting the development of granulocytes and treated with Actimid or DMSO (control). RNA and protein were extracted for the analysis of cathepsin G. A significant up-regulation of cathepsin G (7.7 fold) was detected in oligonucleotide gene array analysis after 3 days of treatment with Actimid as compared to control group. These results were confirmed by RT-PCR, which showed a 3.7 and 7.6 fold mRNA-increase on day 6 and day 10 of culture compared to control. Next we analyzed the cathepsin G levels of patients (n=10) treated with lenalidomide before treatment, on day 15 of each cycle (1–4) as well as 1, 2, and 3 month after stop of lenalidomide treatment. We observed a continuous significant increase of the mRNA levels of PMNs over the course of treatment (baseline: 1 fold, cycle 2: 1.7 fold, cycle 3: 4.8 fold, cycle 4: 20.7 fold). These data were confirmed by measuring cathepsin G levels in patient serum by ELISA. Cathepsin G significantly increased from a baseline mean of 53 ng/ml to 77.5 ng/ml (cycle 2), 129.2 ng/ml (cycle 3) and 145.5 ng/ml (cycle 4). After stop of the lenalidomide treatment we observed a time dependent normalization to baseline levels of the cathepsin G with 61 ng/mL (1 month post treatment), 42.5 ng/mL (2 months post treatment), and 49.2 ng/mL (3 months post treatment). Our data show that IMiDs up-regulate the potent platelet aggregation activator cathepsin G in hematopoietic cells and thereby might contribute to the development of TEE in patients receiving IMiD treatment. Based on our data TEE prophylaxis should be continued for at least 1 more month after stop of therapy. For the first time our results provide an explanation why aspirin is effective in preventing TEE in patients receiving IMiDs. Further studies are needed to determine whether cathepsin G helps to predict TEE. Inhibition of cathepsin G might be a potential therapeutic target for preventing the hypercoaguable state induced by IMiD treatment.

Description: CC-4047, an immunomodulatory analog of thalidomide, inhibits multiple myeloma with unknown effects on the human osteoclast lineage. Early osteoclast progenitors are of hematopoietic origin and differentiate into mature bone resorbing multinucleated osteoclasts. We investigated the effects of CC-4047 and thalidomide on human osteoclastogenesis, using in vitro receptor activator of NFκ-B ligand/macrophage colony-stimulating factor–stimulated bone marrow cell cultures. Treating bone marrow cultures with CC-4047 for 3 weeks decreased osteoclast formation accompanied by complete inhibition of bone resorption. The inhibitory effect was similar when cultures were treated for 3 weeks or for only the first week (90% inhibition), indicating that CC-4047 inhibits early stages of osteoclast formation. Inhibition of osteoclastogenesis by CC-4047 was mediated by a shift of lineage commitment to granulocyte colony-forming units at the expense of granulocyte-macrophage colony-forming units. Further studies revealed that this shift in lineage commitment was mediated through down-regulation of PU.1. Treatment with thalidomide resulted in significantly less potent inhibition of osteoclast formation and bone resorption. These results provide evidence that CC-4047 blocks osteoclast differentiation during early phases of osteoclastogenesis. Therefore, CC-4047 might be a valuable drug for targeting both tumors and osteoclastic activity in patients with multiple myeloma and other diseases associated with osteolytic lesions.

Description: Thalidomide (Thal) and its immunomodulatory analog, Revlimid (Rev) have demonstrated anti-myeloma activity in phase II and III studies and part of their activity is immune-mediated. We have previously demonstrated co-stimulatory effects of Thal and Rev on T cells mediated via B7-CD28 activation pathway. To understand the molecular events involved in T cell co-stimulation, we investigated the effects of Thal and Rev on the genome-wide gene expression profile of T cells with or without anti-CD3 activation. Normal PBMC were incubated with Thal or Rev in the presence or absence of anti-CD3 antibodies (200ng/ml) for 3 or 24 hours at 37oC and total RNA was analyzed using U133 GeneChip (Affymetrix). Although Thal and Rev do not show significant effect on T cell proliferation without CD3 stimulation, we observed up-regulation (〉2-folds) of 56 genes and down-regulation of 42 genes at 3 hours, and up-regulation of 41genes and down-regulation of 35 genes at 24 hours with Rev. Significant up-regulation of IFN-α (10-folds) was observed at 3 hours. Up-regulation of chemokine CXC5 (9 fold); IFN-β (5 fold), IL-7 (5 fold) and IL-2 (3 fold); and signaling genes like JK 3 (10 folds) and MAPKK5 (5 folds) was observed at 24 hours with Rev. Following anti-CD3 activation, Rev induced significant upregulation of 59 and 48 genes and down-regulation of 126 and 61 genes at 3 and 24 hours respectively. These include down-regulation of Th2 cytokine genes IL-5(5 folds) and TGF-β (2 folds), and apoptotsis related genes, TNFSF12 (4 folds) and TNFRSF 6b (4 folds). Genes involved in B7-CD28 activation pathway and up-regulated include CXC 5 (9 folds), IL-8 (5 folds) and IL-3 (4 folds). Interestingly, p52, which is a key molecule in CD28 signaling, was elevated 18 folds. Additionally less than 2 fold increase was observed in other CD28 signaling genes like IKK, Vov, and RelA (p65). Rev also down-regulated, at 24 hours, the expression of IL-10 (6 folds), IL-17 (3 folds) and TGF-β (3 folds) which allows sustained co-stimulation. Similar changes with lesser magnitude were observed with Thal. These results provide insight into early molecular changes induced by Thal and Rev that is important for co-stimulation and suggest new molecular targets to generate effective immune responses. Moreover, these observations facilitate the pre-clinical rationale for the use of these immunomodulatory compounds to improve clinical outcome in myeloma.

Description: Previous studies have demonstrated the in vitro and in vivo activity of CC-5013 (Revlimid), an immunomodulatory analog (IMiD) of thalidomide, in multiple myeloma (MM). In the present study, we have examined the anti-MM activity of rapamycin (Rapamune), a specific mTOR inhibitor, combined with CC-5013. Based on the Chou-Talalay method, combination indices of less than 1 were obtained for all dose ranges of CC-5013 when combined with rapamycin, suggesting strong synergism. Importantly, this combination was able to overcome drug resistance when tested against MM cell lines resistant to conventional chemotherapy. Moreover, the combination, but not rapamycin alone, was able to overcome the growth advantage conferred on MM cells by interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), or adherence to bone marrow stromal cells (BMSCs). Combining rapamycin and CC-5013 induced apoptosis of MM cells. Differential signaling cascades, including the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3′-kinase/Akt kinase (PI3K/Akt) pathways, were targeted by these drugs individually and in combination, suggesting the molecular mechanism by which they interfere with MM growth and survival. These studies, therefore, provide the framework for clinical evaluation of mTOR inhibitors combined with IMiDs to improve patient outcome in MM.

Description: Lenalidomide is an oral anti-angiogenic, anti-proliferative and immunomodulatory drug approved for the treatment of Low- or Intermediate-risk myelodysplastic syndrome associated with a del(5q) cytogenetic abnormality, and in combination with dexamethasone for previously treated multiple myeloma. Early clinical results suggest potential clinical efficacy in B-cell non-Hodgkin lymphoma (NHL). In this study, we investigated lenalidomide-mediated inhibition of cell proliferation and angiogenic factors in the following NHL subtypes: mantle cell lymphoma (MCL), diffuse large-B-cell lymphoma, and follicular lymphoma (FL). The effect of lenalidomide on these tumor cells was determined after 1–3 days of treatment by 3H-thymidine incorporation, Luminex-based microbead array, and real-time qRT-PCR. We also assessed the expression of tumor suppressor genes such as p21cip1 and secreted protein acidic and rich in cysteine (SPARC). We found that lenalidomide induced direct anti-proliferative effects on each NHL subtype, with MCL cells being the most sensitive. Pro-angiogenic factors such as vascular endothelial growth factor (VEGF) were expressed at a high level in all lymphoma cell lines. Lenalidomide inhibited VEGF production at much lower concentrations than required for anti-proliferative effects, particularly in MCL and FL cell lines. Addition of recombinant human VEGF or neutralizing anti-VEGF antibody had no effect on MCL cell proliferation, suggesting that these effects are independent. Mechanistic studies indicated that lenalidomide strongly increased the gene expression of the tumor suppressor genes p21cip1 and SPARC to varying degrees. Elevation of SPARC mRNA significantly correlated with both the anti-proliferative and the VEGF-suppressive effects of lenalidomide on MCL cells (p 〈 0.05). Transfection of tumor cells with SPARC siRNA led to significant resistance to lenalidomide suggesting that this effect is mediated at least in part through the up-regulation of SPARC. In conclusion, lenalidomide demonstrates anti-proliferative activity against multiple NHL cell subtypes with greatest potency against MCL. The potent anti-VEGF activity of lenalidomide supports the anti-angiogenic potential of the drug. Thus, lenalidomide-induced up-regulation of SPARC mRNA correlates with MCL sensitivity and may have biomarker potential.

Description: Although thalidomide (Thal) was initially used to treat multiple myeloma (MM) because of its known antiangiogenic effects, the mechanism of its anti-MM activity is unclear. These studies demonstrate clinical activity of Thal against MM that is refractory to conventional therapy and delineate mechanisms of anti-tumor activity of Thal and its potent analogs (immunomodulatory drugs [IMiDs]). Importantly, these agents act directly, by inducing apoptosis or G1 growth arrest, in MM cell lines and in patient MM cells that are resistant to melphalan, doxorubicin, and dexamethasone (Dex). Moreover, Thal and the IMiDs enhance the anti-MM activity of Dex and, conversely, are inhibited by interleukin 6. As for Dex, apoptotic signaling triggered by Thal and the IMiDs is associated with activation of related adhesion focal tyrosine kinase. These studies establish the framework for the development and testing of Thal and the IMiDs in a new treatment paradigm to target both the tumor cell and the microenvironment, overcome classical drug resistance, and achieve improved outcome in this presently incurable disease.