David I. Stirling

Adherence of multiple myeloma cells to bone marrow stromal cells upregulates vascular endothelial growth factor secretion: therapeutic applications.

Increased angiogenesis has recently been recognized in active multiple myeloma (MM). Since vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two key mediators of angiogenesis, we characterized the production of VEGF, b-FGF and interleukin-6 (IL-6) (a MM growth and survival factor) in MM cell lines and Epstein–Barr virus (EBV) transformed B cell lines from MM patients, patient MM cells, as well as bone marrow stromal cells (BMSCs) from normal healthy donors and MM patients. We detected secretion of VEGF, but no bFGF and IL-6, in MM cell lines (MM.1S, RPMI 8226 and U266); EBV transformed B cell lines from MM patients (IM-9, HS-Sultan and ARH77); MM cell lines resistant to doxorubicin (RPMI-DOX40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5) and dexamethasone (MM.1R); and patient MM cells (MM1 and MM2). BMSCs from MM patients and normal donors secreted VEGF, b-FGF and IL-6. Importantly, when MM cells were adhered to BMSCs, there was a significant increase in VEGF (1.5- to 3.1-fold) and IL-6 (1.9- to 56-fold) secretion. In contrast, the bFGF decreased in co-cultures of BMSCs and MM cells. Paraformaldehyde fixation of BMSCs or MM cells prior to adhesion revealed that VEGF was produced both from BMSCs and MM cells, though it may come primarily from BMSCs in some cultures. IL-6 was produced exclusively in BMSCs, rather than MM cells. Moreover, when MM cells were placed in Transwell insert chambers to allow their juxtaposition to BMSCs without cell to cell contact, induction of VEGF and IL-6 secretion persisted, suggesting the importance of humoral factors. Addition of exogenous IL-6 (10 ng/ml) increased VEGF secretion by BMSCs. Conversely, VEGF (100 ng/ml) significantly increased IL-6 secretion by BMSCs. Moreover, anti-human VEGF (1 μg/ml) and anti-human IL-6 (10 μg/ml) neutralizing antibodies reduced IL-6 and VEGF secretion, respectively, in cultures of BMSCs alone and co-cultures of BMSCs and MM cells. Finally, thalidomide (100 μM) and its immunomodulatory analog IMiD1-CC4047 (1 μM) decreased the upregulation of IL-6 and VEGF secretion in cultures of BMSCs, MM cells and co-cultures of BMSCs with MM cells. These data demonstrate the importance of stromal–MM cell interactions in regulating VEGF and IL-6 secretion, and suggest additional mechanisms whereby thalidomide and IMiD1-CC4047 act against MM cells in the BM millieu.

Amino-substituted thalidomide analogs: Potent inhibitors of TNF-α production

Abstract Thalidomide, (1), is a known inhibitor of TNF-α release in LPS stimulated human PBMC. Herein we describe the TNF-α inhibitory activity of amino substituted analogs of thalidomide (1) and its isoindolin-1-one analog, EM-12 (2). The 4-amino substituted analogs were found to be potent inhibitors of TNF-α release in LPS stimulated human PBMC.

Lenalidomide Enhances Natural Killer Cell and Monocyte-Mediated Antibody-Dependent Cellular Cytotoxicity of Rituximab-Treated CD20 + Tumor Cells

Purpose: Lenalidomide has significant activity in myelodysplastic syndromes, multiple myeloma, and non-Hodgkins lymphoma (NHL). In previous studies, natural killer (NK) cell expansion by lenalidomide was shown to enhance the cytotoxic effect of rituximab. This study assessed the ability of lenalidomide to enhance antibody-dependent cellular cytotoxicity (ADCC) in rituximab-treated NHL cell lines and primary tumor cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) in vitro. Experimental Design: An in vitro ADCC system was used to assess the ability of lenalidomide to enhance human NK cell and monocyte function in response to rituximab. Results: Lenalidomide directly enhanced IFN-γ production via Fc-γ receptor-mediated signaling in response to IgG. It was also a potent enhancer of NK cell-mediated and monocyte-mediated tumor cell ADCC for a variety of rituximab-treated NHL cell lines in vitro, an effect that was dependent on the presence of antibody and either interleukin-2 or interleukin-12. Lenalidomide also enhanced the ability of NK cells to kill primary tumor cells derived from three patients with B-CLL who have been treated previously with fludarabine plus cyclophosphamide. Enhanced NK cell ADCC was associated with enhanced granzyme B and Fas ligand expression and could be inhibited by a granzyme B inhibitor and partially inhibited by antibody to FasL. Enhanced NK cell Fc-γ receptor signaling is associated with enhanced phosphorylated extracellular signal-related kinase levels leading to enhanced effector function. Conclusions: These findings suggest that lenalidomide has the potential to enhance the rituximab-induced killing of NHL cell lines and primary B-cell chronic lymphocytic leukemia cells via a NK cell-mediated and monocyte-mediated ADCC mechanism in vitro, providing a strong rationale for the combination of lenalidomide with IgG1 antibodies to target tumor-specific antigens in patients with cancer.

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.

Apremilast, a cAMP phosphodiesterase-4 inhibitor, demonstrates anti-inflammatory activity in vitro and in a model of psoriasis

Background and purpose:  Apremilast is an orally administered phosphodiesterase‐4 inhibitor, currently in phase 2 clinical studies of psoriasis and other chronic inflammatory diseases. The inhibitory effects of apremilast on pro‐inflammatory responses of human primary peripheral blood mononuclear cells (PBMC), polymorphonuclear cells, natural killer (NK) cells and epidermal keratinocytes were explored in vitro, and in a preclinical model of psoriasis.

Immunomodulatory analogs of thalidomide inhibit growth of Hs Sultan cells and angiogenesis in vivo

We have previously shown that thalidomide and its potent immunomodulatory derivatives (IMiDs) inhibit the in vitro growth of multiple myeloma (MM) cell lines and patient MM cells that are resistant to conventional therapy. In this study, we further characterize the effect of these drugs on growth of B cell malignancies and angiogenesis. We established a beige-nude-xid (BNX) mouse model to allow for simultaneous in vivo measurement of both anti-tumor and anti-angiogenic effects of thalidomide and its analogs. Daily treatment (50 mg/kg/d) with thalidomide or IMiDs was nontoxic. The IMiDs were significantly more potent than thalidomide in vivo in suppressing tumor growth, evidenced by decreased tumor volume and prolonged survival, as well as mediating anti-angiogenic effects, as determined by decreased microvessel density. Our results therefore show that the IMiDs have more potent direct anti-tumor and anti-angiogenic effects than thalidomide in vivo, providing the framework for clinical protocols evaluating these agents in MM and other B cell neoplasms.

Protective antitumor immunity induced by a costimulatory thalidomide analog in conjunction with whole tumor cell vaccination is mediated by increased Th1-type immunity.

Thalidomide and its novel T cell costimulatory analogs (immunomodulatory drugs) are currently being assessed in the treatment of patients with advanced cancer. However, neither tumor-specific T cell costimulation nor effective antitumor activity has been demonstrated in vivo. In this study, we assessed the ability of an immunomodulatory drug (CC-4047/ACTIMID) to prime a tumor-specific immune response following tumor cell vaccination. We found that the presence of CC-4047 during the priming phase strongly enhanced antitumor immunity in the vaccinated group, and this correlated with protection from subsequent live tumor challenge. Protection was associated with tumor-specific production of IFN-γ and was still observed following a second challenge with live tumor cells 60 days later. Furthermore, CD8+ and CD4+ splenocyte fractions from treated groups secreted increased IFN-γ and IL-2 in response to tumor cells in vitro. Coculture of naive splenocytes with anti-CD3 mAb in the presence of CC-4047 directly costimulated T cells and increased Th1-type cytokines. Our results are the first to demonstrate that a costimulatory thalidomide analog can prime protective, long-lasting, tumor-specific, Th1-type responses in vivo and further support their ongoing clinical development as novel anti-cancer agents.

A 90-day oral gavage toxicity study of D-methylphenidate and D, L-methylphenidate in Sprague-Dawley rats

D-methylphenidate is an enantiomer of D,L-methylphenidate and was developed as an improved treatment for attention deficit hyperactivity disorder in children. The current study was performed to determine and compare the toxicity of 2-50 mg/kg per day D-MPH and 100 mg/kg per day D,L-MPH for 90 days in rats with the top D-MPH dose being equimolar to 100 mg/kg D,L-MPH. The top D-MPH and D,L-MPH doses were at least 67 times that of the human dose and produced systemic exposures that were over 10 times higher than those typically achieved in children. During the course of the study, one male each from the 50 mg/kg per day and D,L-MPH groups and one female from the 50 mg/kg group died. Incidences of material around nose/eyes, scabbing, foot swelling, alopecia and abrasions were evident at 50 mg/kg per day D-MPH and 100 mg/kg per day D,L-MPH doses. Body weight and its changes decreased in a dose-dependent manner for D-MPH males. There were significant changes in some clinical chemistry measurements at the terminal bleed in the high dose groups of both sexes although most of these changes were resolved by the recovery bleed. Differences in absolute and relative body and certain organ weights for high dose D-MPH and D,L-MPH groups were seen at terminal necropsy with the differences no longer present after the recovery period. No abnormal or gross histopathological changes were associated with any of these organ weight changes reported for the terminal and recovery periods. Based on body weight changes, the no observed adverse effect level for D-MPH in rats was 20 mg/kg. Overall, the toxicity profile observed in rats with 50 mg/kg per day D-MPH was comparable to that of an equimolar dose of D,L-MPH (100 mg/kg per day) when given repeatedly for 90 days using a twice a day dosing regimen.

Thalidomide and its analogues have distinct and opposing effects on TNF-α and TNFR2 during co-stimulation of both CD4+ and CD8+ T cells

Thalidomide (Thd) is clinically useful in a number of conditions where its efficacy is probably related to its anti‐TNF‐α activity. More recently, Thd has also been shown to co‐stimulate T cells and second generation co‐stimulatory (IMiD™) analogues are currently being assessed in the treatment of cancer patients. However, in contrast to their known suppressive effects during inflammatory stimuli, the effects of Thd/IMiDs on TNF‐α and TNF receptors (TNFRs) during T cell co‐stimulation are not known. We sought to determine the effect of Thd, two clinically relevant IMiDs (CC‐4047, ACTIMID™ and CC‐5013, REVIMID™) and a non‐stimulatory SelCID analogue (CC‐3052) on TNF‐α production and on the expression and shedding of TNFRs during co‐stimulation. We found that co‐stimulation of PBMC with Thd/IMiDs, but not CC‐3052, prevented αCD3‐induced T cell surface expression of TNFR2 and thereby reduced soluble TNFR2 (sTNFR2) levels. However, there was no effect on total (surface/intracellular) TNFR2 protein expression, suggesting inhibition of trafficking to the cell membrane. The extent of co‐stimulation by Thd/IMiDs (assessed by CD69/CD25 expression and IL‐2/sIL‐2Rα production) was similar for CD4+ and CD8+ T lymphocytes and correlated with TNFR2 inhibition. Co‐stimulation, but not the early inhibitory effect on TNFR2, was IL‐2‐dependent and led to increased TNF‐α production by both CD4+ and CD8+ T lymphocytes. The clinical relevance of this observation was confirmed by the elevation of serum TNF‐α during REVIMID™ treatment of patients with advanced cancer. Together, these results suggest a possible role for TNF‐mediated events during co‐stimulation and contrast with the TNF inhibitory effects of Thd and its analogues during inflammatory stimuli.

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.