J. Blake Bartlett

The anti-cancer agents lenalidomide and pomalidomide inhibit the proliferation and function of T regulatory cells

Lenalidomide (Revlimid®; CC-5013) and pomalidomide (CC-4047) are IMiDs® proprietary drugs having immunomodulatory properties that have both shown activity in cancer clinical trials; lenalidomide is approved in the United States for a subset of MDS patients and for treatment of patients with multiple myeloma when used in combination with dexamethasone. These drugs exhibit a range of interesting clinical properties, including anti-angiogenic, anti-proliferative, and pro-erythropoietic activities although exact cellular target(s) remain unclear. Also, anti-inflammatory effects on LPS-stimulated monocytes (TNF-α is decreased) and costimulatory effects on anti-CD3 stimulated T cells, (enhanced T cell proliferation and proinflammatory cytokine production) are observed These drugs also cause augmentation of NK-cell cytotoxic activity against tumour-cell targets. Having shown that pomalidomide confers T cell-dependant adjuvant-like protection in a preclinical whole tumour-cell vaccine-model, we now show that lenalidomide and pomalidomide strongly inhibit T-regulatory cell proliferation and suppressor-function. Both drugs inhibit IL-2-mediated generation of FOXP3 positive CTLA-4 positive CD25high CD4+ T regulatory cells from PBMCs by upto 50%. Furthermore, suppressor function of pre-treated T regulatory cells against autologous responder-cells is abolished or markedly inhibited without drug related cytotoxicity. Also, Balb/C mice exhibit 25% reduction of lymph-node T regulatory cells after pomalidomide treatment. Inhibition of T regulatory cell function was not due to changes in TGF-β or IL-10 production but was associated with decreased T regulatory cell FOXP3 expression. In conclusion, our data provide one explanation for adjuvant properties of lenalidomide and pomalidomide and suggest that they may help overcome an important barrier to tumour-specific immunity in cancer patients.

The anti-cancer drug lenalidomide inhibits angiogenesis and metastasis via multiple inhibitory effects on endothelial cell function in normoxic and hypoxic conditions

Lenalidomide (Revlimid) is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk Myelodysplastic Syndromes (MDS) associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone for the treatment of multiple myeloma patients who have received at least one prior therapy. Previous reports suggest that lenalidomide is anti-angiogenic and this property appears to be related to efficacy in patients with MDS. We have investigated the effect of lenalidomide on the formation of microvessels in a novel in vitro angiogenesis assay utilizing human umbilical arterial rings and in a capillary-like cord formation assay using cultured primary endothelial cells. We found that lenalidomide consistently inhibits both sprout formation by arterial rings and cord formation by endothelial cells in a dose-dependent manner. We also found an inhibitory effect of lenalidomide on the associations between cadherin 5, beta-catenin and CD31, adherens junction proteins whose interaction is critical for endothelial cell cord formation. Furthermore, lenalidomide inhibited VEGF-induced PI3K-Akt pathway signaling, which is known to regulate adherens junction formation. We also found a strong inhibitory effect of lenalidomide on hypoxia-induced endothelial cell formation of cords and HIF-1 alpha expression, the main mediator of hypoxia-mediated effects and a key driver of angiogenesis and metastasis. Anti-metastatic activity of lenalidomide in vivo was confirmed in the B16-F10 mouse melanoma model by a >40% reduction in melanoma lung colony counts versus untreated mice. Our results suggest that inhibitory effects on microvessel formation, in particular adherens junction formation and inhibition of hypoxia-induced processes support a potential anti-angiogenic and anti-metastatic mechanism for this clinically active drug.

Synergistic antitumor effects of lenalidomide and rituximab on mantle cell lymphoma in vitro and in vivo.

Rituximab (RTX), a chimeric anti‐CD20 antibody, is associated with direct induction of apoptosis and antibody‐dependent cell‐mediated cytotoxicity (ADCC) with clinical efficacy in mantle cell lymphoma (MCL). Lenalidomide (LEN), a novel immunomodulatory agent, sensitizes tumor cells and enhances ADCC. Our study attempted to elucidate the mechanism of LEN‐enhanced RTX‐mediated cytotoxicity of MCL cells. We found that LEN and RTX induced growth inhibition of both cultured and fresh primary MCL cells. LEN enhanced RTX‐induced apoptosis via upregulating phosphorylation of c‐Jun N‐terminal protein kinases (JNK), Bcl‐2, Bad; increasing release of cytochrome‐c; enhancing activation of caspase‐3, ‐8, ‐9 and cleavage of PARP. Meanwhile, LEN activated NK cells and increased CD16 expression on CD56lowCD16+ NK cells. Whole PBMCs but not NK cell‐depleted PBMCs treated with LEN augmented 30% of RTX‐dependent cytotoxicity. Daily treatment with LEN increased NK cells by 10‐folds in SCID mice, and combination of LEN and RTX decreased tumor burden and prolonged survival of MCL‐bearing SCID mice. Taken together, our study demonstrates that LEN plus RTX provides a synergistically therapeutic effect on MCL cells by enhancing apoptosis and RTX‐dependent NK cell‐mediated cytotoxicity and may be an optimal combination in the clinical trial of relapsed or refractory MCL. Am. J. Hematol. 2009.

Thalidomide-derived immunomodulatory drugs as therapeutic agents.

Thalidomide, a drug originally used to treat morning sickness, was removed from the market place in the early 1960s after it was found to cause serious congenital birth defects. However, thalidomide has recently been investigated in a new light following its activity in a number of chronic diseases. Moreover, like thalidomide itself, its second-generation immunomodulatory drug (IMiD®) analogues have been shown to act as powerful anticancer agents and are clearly active in the treatment of patients with relapsed multiple myeloma. These new drugs, in particular the second-generation IMiDs, lenalidomide (CC-5013, REVLIMID™; Celgene Corp., NJ, USA) and CC-4047 (ACTIMID™; Celgene Corp.), offer improvements over thalidomide (a first-generation IMiD) in terms of efficacy and safety in human studies. The key to the therapeutic potential of IMiDs lies in the fact that the drugs have multiple mechanisms of action, which may produce both anti-inflammatory and antitumour effects. These effects are probably contextual, depending both on the cell type and the stimulus involved. Mechanisms associated with IMiD activity include TNF-α-inhibitory, T cell costimulatory and antiangiogenic activities. Studies of the mechanisms of action of these drugs are ongoing and will facilitate the continued development of this class of compound in a number of diseases.

The Synthetic Compound CC-5079 Is a Potent Inhibitor of Tubulin Polymerization and Tumor Necrosis Factor-α Production with Antitumor Activity

We have found that the synthetic compound CC-5079 potently inhibits cancer cell growth in vitro and in vivo by a novel combination of molecular mechanisms. CC-5079 inhibits proliferation of cancer cell lines from various organs and tissues at nanomolar concentrations. Its IC(50) value ranges from 4.1 to 50 nmol/L. The effect of CC-5079 on cell growth is associated with cell cycle arrest in G(2)-M phase, increased phosphorylation of G(2)-M checkpoint proteins, and apoptosis. CC-5079 prevents polymerization of purified tubulin in a concentration-dependent manner in vitro and depolymerizes microtubules in cultured cancer cells. In competitive binding assays, CC-5079 competes with [(3)H]colchicine for binding to tubulin; however, it does not compete with [(3)H]paclitaxel (Taxol) or [(3)H]vinblastine. Our data indicate that CC-5079 inhibits cancer cell growth with a mechanism of action similar to that of other tubulin inhibitors. However, CC-5079 remains active against multidrug-resistant cancer cells unlike other tubulin-interacting drugs, such as Taxol and colchicine. Interestingly, CC-5079 also inhibits tumor necrosis factor-alpha (TNF-alpha) secretion from lipopolysaccharide-stimulated human peripheral blood mononuclear cells (IC(50), 270 nmol/L). This inhibitory effect on TNF-alpha production is related to its inhibition of phosphodiesterase type 4 enzymatic activity. Moreover, in a mouse xenograft model using HCT-116 human colorectal tumor cells, CC-5079 significantly inhibits tumor growth in vivo. In conclusion, our data indicate that CC-5079 represents a new chemotype with novel mechanisms of action and that it has the potential to be developed for neoplastic and inflammatory disease therapy.

Lenalidomide enhances antibody-dependent cellular cytotoxicity of solid tumor cells in vitro: influence of host immune and tumor markers

We evaluated the effect of combining lenalidomide with therapeutic antibodies on antibody-dependant cell-mediated cytotoxicity (ADCC) of solid tumor cells, and the requirement for expression of natural killer (NK) cell-activating receptors and their solid tumor surface ligands. Twenty-three human tumor cell lines (colon, breast, lung, head and neck, ovary, and bone sarcoma) were analyzed. NK effector cells were isolated from healthy donors, pre-treated with and without lenalidomide, and incubated with antibody-coated tumor cells to determine ADCC. In blocking experiments, NK cells were pre-incubated with anti-DNAM-1 or anti-NKG2D antibodies, and target colorectal cells were pre-incubated with anti-CD155 (PVR), anti-MIC-A/B, or anti-ULBP 3 antibodies. Differences between groups were assessed using unpaired and paired Student’s t test and one-way ANOVA. Lenalidomide enhanced NK cell-mediated ADCC of trastuzumab- and cetuximab-coated tumor cells. Activity against colorectal cancer cells was dependent on target antigen expression, but independent of KRAS status and FcγRIIIa genotype. The extent of ADCC and its enhancement by lenalidomide correlated with NK cell expression of NKG2D and DNAM-1, and tumor cell expression of PVR and MIC-A. Blocking of NKG2D and, to a lesser extent, DNAM-1 inhibited ADCC. Anti-MIC-A/B monoclonal antibody blocked natural cytotoxicity, but not ADCC. Lenalidomide enhances the ability of IgG1-isotype antibodies to mediate ADCC of solid tumor cells, the extent of which is largely dependent on NKG2D–NKG2D ligand interactions, but appears to be independent of MIC-A/B. This provides a rationale for exploratory clinical studies and an assessment of potential biomarkers predictive of clinical benefit.

The Immunomodulatory Drug (IMID®) CC-4047 Enhances the Proliferation and Anti-Tumor Function of Gamma Delta T Cells

The Immunomodulatory Drug (IMID ) CC-4047 Enhances the Proliferation and Anti-Tumor Function of Gamma Delta T Cells Christine Galustian, Debbie Klaschka, Marie-Christine Labarthe, J Blake Bartlett, Angus G Dalgleish. Department of Oncology, St Georges Hospital, Tooting, London, United Kingdom; Celgene Corporation, Warren, NJ. The immunomodulatory thalidomide derivatives (IMiDs ) lenalidomide (CC-5013, REVLIMID), and CC-4047 (ACTIMID) are currently in clinical development for the treatment of a variety of inflammatory and oncological diseases with particularly successful results seen in multiple myeloma (MM) patients. These compounds are less toxic than thalidomide and have been shown to enhance tumor-specific immunity and to exert antiangiogenic effects in-vivo. The mechanisms of IMiD-induced enhancement of anti-tumor immunity are not well understood, but are thought to involve enhancement of both innate and adaptive immunity. In this study we have investigated the potential for the potent costimulatory IMiD , CC-4047, to influence gamma delta (gd) TCR+ T cell mediated anti-tumor immunity. Gamma delta (gd) T cells have been shown to play a role in innate surveillance function against tumors and are directly cytotoxic for a variety of tumor cell lines in vitro. We have now demonstrated that CC-4047 strongly costimulates both aCD3-activated fresh gd T cells and purified gd T cell lines expanded and activated with the physiological gd T cell ligand, isopentenyl pyrophosphate (IPP). Costimulation with CC-4047 favors production of IFN-g and TNF-a, but not the Th2 cytokine IL-4, and leads to early and enhanced production of pro-inflammatory cytokines in response to co-culture with a variety of tumor cells. The proliferation of gd T cells in response to IPP is strongly enhanced (by ;40%) in CC-4047 treated PBMC cultures over a period of 8–12 days. This is most likely due to a protective effect of CC-4047 on the gd T cells, since treatment results in an increase in the percentage of live gd T cells by 40% over a period of 6 or 7 days compared to gd T cells treated with IPP alone. Furthermore, we also demonstrate that CC-4047 can increase the cytotoxic activity of gd T cells against the MM cell target RPMI-8226 by up to 50%. The increase in MM cell killing by gd T cells is associated with an increase in NKG2D expression. The optimization and further investigation of the effects of IMiDs on gd T cells and other cells of the innate immune system may provide an important contribution to understanding the anti-tumor activities associated with successful treatment of cancer with this class of compound.

Abstract A5: Lenalidomide inhibits hypoxia-induced HIF-1{alpha} production and prevents the invasive phenotype in epithelial solid tumor cells

Background: In hematological malignancies lenalidomide (len) exerts tumoricidal and immunomodulatory effects and may also influence the tumor cell microenvironment by blocking the proangiogenic and proinvasive effects of growth factors and/or hypoxia. In solid tumor models len is antimetastatic but lacks overt tumoricidal activity. These observations led us to explore the effect of len on hypoxic solid tumor cells, in particular the expression of HIF-α and its upstream regulators p53 and HDM-2. Methods: Eighteen solid tumor cell lines were tested for the effect of len on the response to hypoxia (1 % O 2 , 5% CO 2 , 94% N 2 ; Invivo2 400 hypoxic workstation). Cell lysates from three cell lines (HCT-15, MDA-MB-231 and SKOV-2) were assessed for HIF-1α, p53 and HDM-2 proteins by immunoblot. We also performed immunoprecipitation studies to explore the effect of len on the interaction of these proteins. The effect of len on HIF-1α was assessed in p53 siRNA transfected HCT-15 cells and on HIF-1 α mRNA in transfected Luc-HIF NIH 3T3 cells. Len effects on hypoxia-induced trans-well invasion of three cell lines were also assessed. Results: Len (1 µM) significantly inhibited hypoxia-induced HIF-1 a protein levels dose-dependently in the majority of tumor cell lines tested; breast (by 68%, MDA-MB-231; 65%, MCF-7), colorectal (76%, HCT-116; 65%, HCT-15; 82% HT-29), ovarian (71%, OVCAR-3; 66%, SKOV-3), renal (83%, 786-O; 57%, HEK293), prostate (54%, DU- 145) and pancreatic (45%, MiaPaca-3)(all p Conclusions: These results suggest that len inhibits the proinvasive hypoxic response in multiple, diverse tumor lines and may exert antimetastatic effects within the hypoxic solid tumor microenvironment. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A5.