Clinical studies involving patients with myelodysplastic syndromes or multiple myeloma have shown the efficacy of lenalidomide by reducing and often eliminating malignant cells while restoring the bone marrow function. To better understand these clinical observations, we investigated and compared the effects of lenalidomide and a structurally related analogue, CC-4047, on the proliferation of two different human hematopoietic cell models: the Namalwa cancer cell line and normal CD34 + progenitor cells. Both compounds had antiproliferative effects on Namalwa cells and pro-proliferative effects on CD34 + cells, whereas p21WAF-1 expression was up-regulated in both cell types. In Namalwa cells, the up-regulation of p21 WAF-1 correlated well with the inhibition of cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 activity leading to pRb hypophosphorylation and cell cycle arrest, whereas in CD34 + progenitor cells the increase of p21 WAF-1 did not inhibit proliferation. Similarly, antiproliferation results were observed in two B lymphoma cell lines (LP-1 and U266) but interestingly not in normal B cells where a protection of apoptosis was found. Finally, CC-4047 and lenalidomide had synergistic effects with valproic acid [a histone deacetylase (HDAC) inhibitor] by increasing the apoptosis of Namalwa cells and enhancing CD34 + cell expansion. Our results indicate that lenalidomide and CC-4047 have opposite effects in tumor cells versus normal cells and could explain, at least in part, the reduction of malignant cells and the restoration of bone marrow observed in patients undergoing lenalidomide treatment. Moreover, this study provides new insights on the cellular pathways affected by lenalidomide and CC-4047, proposes new potential clinical uses, such as bone marrow regeneration, and suggests that the combination of lenalidomide or CC-4047 with certain HDAC inhibitors may elevate the therapeutic index in the treatment of hematologic malignancies. [Cancer Res 2007;67(2):746-55]

Sickle-cell disease (SCD) and β thalassemia constitute worldwide public health problems. New therapies, including hydroxyurea, have attempted to augment the synthesis of fetal hemoglobin (HbF) and improve current treatment. Lenalidomide and pomalidomide are members of a class of immunomodulators used as anticancer agents. Because clinical trials have demonstrated that lenalidomide reduces or eliminates the need for transfusions in some patients with disrupted blood cell production, we investigated the effects of lenalidomide and pomalidomide on erythropoiesis and hemoglobin synthesis. We used an in vitro erythropoiesis model derived from human CD34 + progenitor cells from normal and SCD donors. We found that both compounds slowed erythroid maturation, increased proliferation of immature erythroid cells, and regulated hemoglobin transcription, resulting in potent induction of HbF without the cytotoxicity associated with other HbF inducers. When combined with hydroxyurea, pomalidomide and, to a lesser extent, lenalidomide were found to have synergistic effects on HbF upregulation. Our results elucidate what we believe to be a new mechanism of action of pomalidomide and lenalidomide and support the hypothesis that pomalidomide, used alone or in combination with hydroxyurea, may improve erythropoiesis and increase the ratio of fetal to adult hemoglobin. These findings support the evaluation of pomalidomide as an innovative new therapy for β-hemoglobinopathies.

No abstract

A systematic comparison of transcriptional activation by papillomavirus E2 proteins revealed that the E2 proteins from high-risk human papillomaviruses (human papillomavirus type 16 [HPV-16] and HPV-18) are much more active than are the E2 proteins from low-risk HPVs (HPV-6b and HPV-11). Despite the tropism of HPVs for particular epithelial cell types, this difference in transcriptional activation was observed in a number of different epithelial and nonepithelial cells. The enhanced activities of the E2 proteins from high-risk HPVs did not result from higher steady-state levels of protein in vivo, and in vitro DNA-binding assays revealed similar binding properties for these two classes of E2 proteins. These results demonstrate that the E2 proteins from high-risk HPVs have an intrinsically enhanced potential to activate transcription from promoters with E2-responsive elements. We found that there are also substantial differences between the activation properties of the bovine papillomavirus type 1 E2 protein and those of either of the two classes of HPV E2 proteins, especially with regard to requirements for particular configurations of E2 binding sites in the target promoter. Our results indicate that there are at least three distinct functional classes of E2 proteins and that these classes of E2 proteins may perform different roles during the respective viral life cycles.

3441 Poster Board III-329 Background CLL is characterized by the progressive accumulation of monoclonal B lymphocytes. One theory to explain how CLL cells avoid elimination through immune surveillance mechanisms is through a defect in the ability of T-cells to form immunological synapses with antigen-presenting tumor B-cells (Ramsay et al JCI 2008). Lenalidomide is an immunomodulatory agent with clinical activity in the treatment of B-cell malignancies. Recent laboratory studies showed that lenalidomide not only stimulates T- and natural killer (NK)-cell-mediated ADCC, it also restores the T-cell-mediated ability to form immunological synapses with CLL tumor cells. Since NK cells also exert cytotoxicity through immune synapse formation, here we explore how lenalidomide affects NK-cell-mediated cytotoxicity mechanisms and whether this activity is altered in the presence of rituximab since published studies showed that lenalidomide-pretreated B-cells have a down-regulated surface CD20 expression. Further, we investigated the molecular events associated with immune synapse formation and the effect of lenalidomide. Methods Immune synapse formation was assessed in NK cells (from healthy donors PBMCs) co-cultured with either B-CLL cells derived from pts or with K562 cells (positive control). Cells were fixed and the ability to form synapses was assessed via immunohistochemisty co-staining for either F-actin and CD2, or F-actin and perforin (a cytolytic protein found in NK cells). Synapse formation was visualized by microscopy and measured via relative mean fluorescent intensity. Activity of RhoA, Rac1, Cdc42 were measured using Rho GTPases assay kits. Inhibition of lenalidomide-mediated immune synapse activity was assayed using the cell permeable Rho inhibitor C3 (0.5 mM). Flow cytometry was used to measure changes in surface CD20 and CD54 (ICAM-1) expression in B-CLL samples from 3 pts after treatment with lenalidomide. Results Lenalidomide induced the formation of immunological synapses between NK cells and primary B-CLL cells (p<.01) or the K562 cell line. Lenalidomide activated NK cells regardless of the presence of target cells, as measured by F-actin and perforin staining. RhoA and Rac1 were activated at the immunological synapse in the presence of lenalidomide. Inhibition of RhoA by the C3 inhibitor blocked F-actin localization, as well as perforin accumulation induced by lenalidomide at cell-cell contact sites, indicating inhibition of immune synapses and the associated cytolytic activity. This was also observed with Rac1 inhibition, but to a lesser degree than with RhoA inhibition. Functionality of formed synapses was confirmed by co-localization of F-actin and perforin at the synapse sites. 3 CLL pt samples treated ex vivo with lenalidomide demonstrated variable changes in CD20 expression: a 20-30% decrease in CD20 expression was observed in 2 B-CLL pt samples, whereas CD20 levels remained unchanged in the third. In the presence of rituximab, lenalidomide-induced synapse formation between NK cells and B-cells from CLL patients was further enhanced. This was accompanied by upregulation of costimulatory and adhesion molecule CD54 on B-CLL cells suggesting increased antigen presentation, which might contribute to the increased synapse formation. Conclusion Lenalidomide can directly activate NK-cell-mediated anti-tumor activity through enhanced formation of immune synapses via the regulation of Rho and Rac1 GTPases and the cytoskeleton. Despite some down-modulation of CD20 expression in lenalidomide-pretreated B-CLL cells, the immune synapse activity increases when lenalidomide is combined with rituximab suggesting that combining lenalidomide and anti-CD20 antibodies warrants exploration in the CLL clinical setting. Disclosures Gaidarova: Celgene: Employment, Equity Ownership. Li:Celgene: Employment. Corral:Celgene: Employment. Glezer:Celgene: Employment, Equity Ownership. Schafer:Celgene: Employment. Xie:Celgene: Employment. Lopez-Girona:Celgene: Employment.