Brendan Meyer

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.

T-regulatory cell modulation: the future of cancer immunotherapy?

T-regulatory cells suppress anti-tumour immunity in cancer patients and in murine tumour models. Furthermore, their activity is likely to have an effect on the effectiveness of immunotherapeutic treatments for cancer. Here we describe the current status of developing clinical strategies for modulating Treg activity in cancer patients.

Enhanced cross-priming of naive CD8+ T cells by dendritic cells treated by the IMiDs® immunomodulatory compounds lenalidomide and pomalidomide

The IMiDs® immunomodulatory compounds lenalidomide and pomalidomide are agents with anti‐inflammatory, immunomodulatory and anti‐cancer activity. An excellent success rate has been shown for multiple myeloma in phase I/II clinical trials leading to Food and Drug Administration approval of lenalidomide. One mechanism by which these drugs could enhance anti‐tumour immunity may be through enhanced dendritic cell (DC) function. Thalidomide, a compound structurally related to lenalidomide and pomalidomide, is known to enhance DC function, and we have investigated whether its analogues, pomalidomide and lenalidomide, also have functional effects on DCs. We used mouse bone marrow‐derived DCs treated with 5 or 10 μm pomalidomide, or lenalidomide from day 1 of culture. Treatment with IMiD® immunomodulatory compounds increased expression of Class I (H2‐Kb), CD86, and pomalidomide also increased Class II (I‐Ab) expression in bone marrow‐derived DCs, as measured by flow cytometry. Fluorescent bead uptake was increased by up to 45% when DCs were treated with 5 or 10 μm pomalidomide or lenalidomide compared with non‐treated DCs. Antigen presentation assays using DCs primed with ovalbumin, and syngeneic T cells from transgenic OTI and OTII mice (containing MHC restricted, ovalbumin‐specific, T cells) showed that both pomalidomide and lenalidomide effectively increased CD8+ T‐cell cross‐priming (by up to 47%) and that pomalidomide alone was effective in increasing CD4+ T‐cell priming (by 30%). Our observations suggest that pomalidomide and lenalidomide enhance tumour antigen uptake by DCs with an increased efficacy of antigen presentation, indicating a possible use of these drugs in DC vaccine therapies.

Lenalidomide enhances the anti-prostate cancer activity of docetaxel in vitro and in vivo.

In this study, we investigated the effects of combining lenalidomide and docetaxel on in vitro and in vivo models of prostate cancer as a potential strategy for treatment of castrate resistant prostate cancer (CRPC).

Inhibition of metastatic potential in colorectal carcinoma in vivo and in vitro using immunomodulatory drugs (IMiDs)

Background:Thalidomide and lenalidomide are FDA approved for the treatment of multiple myeloma and, along with pomalidomide, are being investigated in various other cancers. Although these agents display immunomodulatory, anti-angiogenic and anti-apoptotic effects, little is known about their primary mode of therapeutic action in patients with cancer.Methods:As part of a continuing research effort, we have investigated the effects of these agents on the metastatic capacity of murine colorectal cancer cell lines both in vivo and in vitro. Allied to these, we have studied their effects on the molecular pathways associated with metastasis.Results:Results indicate that thalidomide, lenalidomide and pomalidomide significantly inhibit the metastatic capability of colorectal carcinoma cells. Anchorage-independent growth, used as a coarse indicator of transformation, was significantly reduced, as were migratory capacity and invasive competence. In addition, an in vivo experimental metastasis model also showed that treatment with the drugs resulted in a significantly lower number of metastatic pulmonary nodules relative to control mice. Allied to these cellular and phenotypic changes were alterations in molecular markers of metastasis and in intracellular signalling competency.Conclusions:These results provide evidence that in addition to their immunomodulatory effects, thalidomide, lenalidomide and pomalidomide can impair the metastatic capacity of tumours, and that this mechanism may involve alterations to cell signalling functionality.

T regulatory cells, the evolution of targeted immunotherapy

T regulatory cells are able to suppress anti-tumour immunity in pre-clinical models and in patients. This review highlights the important discoveries in Treg immunology critical to the evolution of targeted immunotherapy. We also describe the therapeutic applications that are currently being assessed and their future potential.

Persistently high levels of immunosuppressive cytokines in patients after radical prostatectomy

A significant proportion of men undergoing ‘curative’ radical prostatectomy (RP) for organ-confined prostate cancer relapse within 5 years. A number of adverse risk factors have been identified, but to date no adjuvant treatment as improved the outlook for these men. We proposed that these patients, despite small tumour burdens, may be immunosuppressed from their cancer, which may be amenable to immune modulation. We investigated their immune profile using sensitive functional cytokine assays, both pre- and post-surgery. In comparison with controls, RP patients expressed higher levels of both T helper type 1 (Th1) (interleukin (IL)-2 and tumour necrosis factor-α) and Th2 cytokines (IL-4, -5 and -10) with little change after removal of tumour. Further analysis based on known poor-prognostic factors indicated a trend to expression of higher levels of Th2 cytokines IL-4 and IL-5 in worse prognosis patients rather than the mixed Th1/2 found across the whole cohort. Persistently high levels of both Th1 and Th2 cytokines were detected in RP compared to control patients, despite the removal of relatively small tumour burdens. Cytokine expression studies may be useful as surrogate marker of potential disease progression, and could be used to identify patients who may benefit from immune modulation post-surgery.

Activation and Genetic Modification of Human Monocyte-Derived Dendritic Cells using Attenuated Salmonella typhimurium

Live attenuated bacterial vectors, such as Salmonella typhimurium, have shown promise as delivery vehicles for DNA. We have examined two new strains of S. typhimurium and their impact on dendritic cell maturation (CD12-sifA/aroC mutant and WT05-ssaV/aroC, both in TML background). Strain WT05 matured dendritic cells in a more efficient way; caused higher release of cytokines TNF-α, IL-12, IL-1β; and was efficient for gene transfer. These findings suggest that the genetic background of the attenuation can influence the pattern of inflammatory immune response to Salmonella infection.

Abstract A43: Lenalidomide enhances the anti-proliferative activity of docetaxel in in vivo and in vitro models of prostate cancer

Introduction: Prostate cancer is the most commonly diagnosed form of cancer in men in the United Sates, with the second highest rate of mortality. In 2004, the FDA approved the use of docetaxel in combination with prednisone for the treatment of castrate resistant prostate cancer (CRPC). However, it commonly elicits toxic reactions at effective doses. Thus combination of docetaxel with agents exhibiting nonoverlapping toxicities may be an effective way of improving treatment options for patients. Docetaxel and thalidomide have been reported to show efficacy in the treatment of patients with metastatic CRPC. Lenalidomide (Revlimid ® ), a thalidomide analogue, is FDA approved for the treatment of previously treated multiple myeloma (MM) in combination with dexamethasone and in the del 5q subset of myelodysplastic syndromes (MDS). Reports from a phase I study suggest that docetaxel and lenalidomide is an active combination in CRPC and a phase III trial is now underway. Lenalidomide is known to exert immunomodulatory and anti-angiogenic effects but is generally considered to exert direct tumoricidal effects only against subsets of haematological tumor cells. However, there are reports of multiple effects on signalling pathways related to cell survival and proliferation, including TNF-α, IL-6, ERK, PI3K, VEGF, p27, and P21 which may also effect solid tumor biology. Aims: We have evaluated direct single agent lenalidomide activity as well as potential synergistic effects of combining docetaxel and lenalidomide using in vitro and in vivo models of prostate cancer. Methods: The effect of lenalidomide alone (up to 1 µM) or in combination with docetaxel (0.32-200 nM) was assessed on the proliferation of EGF-stimulated prostate cancer cells. Annexin V and TUNEL assays were used to investigate the apoptotic effects of combining lenalidomide and docetaxel on EGF-stimulated prostate cancer cells. in vitro transwell invasion assays were also performed to assess the ability of lenalidomide to inhibit EGF-induced PC3 and DU145 cell invasion. The effect of combining docetaxel and lenalidomide in an in vivo model of tumor growth was studied by injecting 5×10 6 PC3 cells into the flanks of nude mice. Mice were treated with docetaxel (8 mg/kg, biweekly) and lenalidomide (50 mg/kg, daily) until tumors reached 1400 mm 3 . Results: Single agent lenalidomide did not significantly inhibit EGF-induced PC3 proliferation; however at 1 µM (PC3) and 0.01 µM (DU145), lenalidomide reduced the IC50 of docetaxel by approximately 40%. These combinations increased apoptosis by upto 50% (p Conclusions: These results demonstrate that lenalidomide alone displays direct effects against prostate cancer cells by inhibiting growth factor induced invasion. Lenalidomide enhances the antiproliferative effects of docetaxel in vitro through an enhancement of apoptosis of prostate cancer cells. Combinatorial activity was further confirmed in a xenograft model showing that combination of lenalidomide and docetaxel produced a hyperadditive effect that slows tumor progression leading to enhanced median overall survival. These results support the use of lenalidomide in combination with docetaxel as an effective treatment for patients with CRPC. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A43.

Abstract 5386: Lenalidomide enhances the anti-prostate cancer activity of docetaxel in vitro and in vivo

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Prostate cancer accounts for approximately a third of all cancers in men. The FDA has approved the use of docetaxel in combination with prednisone for the treatment of Castrate Resistant Prostate Cancer (CRPC). However, it commonly elicits toxic reactions at effective doses. Thus combination of docetaxel with agents exhibiting non-overlapping toxicities is required. Combined, docetaxel and thalidomide are effective in treating patients with metastatic CRPC. Lenalidomide (Revlimid®), a thalidomide analogue, is FDA approved for multiple myeloma (MM) in combination with dexamethasone and in del 5q myelodysplastic Syndrome (MDS). Phase I study results suggest that docetaxel and lenalidomide actively combine in HRPC and a phase III trial is underway. Lenalidomide is known to exert direct tumoricidal effects only against subsets of hematological tumors. However, there are multiple effects on signalling pathways related to cell survival and proliferation which may also affect solid tumor biology. Aims: We have evaluated direct single agent lenalidomide activity as well as potential synergistic effects of combining docetaxel and lenalidomide using in vitro and in vivo models of prostate cancer. Methods: The effect of lenalidomide alone (up to 1 microM) or in combination with docetaxel (0.32-200nM) was assessed on the proliferation of EGF-stimulated prostate cancer cells. In vitro transwell invasion assays were also performed to assess the ability of lenalidomide to inhibit EGF-induced PC3 and DU145 cell invasion. The effect of combining docetaxel and lenalidomide in an in vivo model of tumor growth was studied by injecting 5×106 PC3 cells into the flanks of nude mice. Mice were treated with docetaxel (8mg/kg, bi-weekly) and lenalidomide (50mg/kg, daily) until tumors reached 1400mm3. Results: Single agent lenalidomide did not significantly inhibit EGF-induced PC3 proliferation; however at 1 microM, lenalidomide reduced the IC50 of docetaxel by an average of 42%. Lenalidomide displayed potent single agent activity by completely inhibiting EGF-induced PC3 and DU145 cell invasion (p<0.001). In mice treated with a combination of lenalidomide and docetaxel, median survival increased to 59 days vs 48 days (lenalidomide alone) and 41 days (docetaxel alone) with a corresponding reduction (p<0.05) in tumor growth. Conclusions: These results demonstrate that lenalidomide alone displays direct effects against prostate cancer cells by inhibiting growth factor induced invasion. Lenalidomide enhances the anti-proliferative effect of docetaxel in vitro. Combinatorial activity was further confimed in a xenograft model showing that combination of lenalidomide and docetaxel produced a hyperadditive effect that slows tumor progression leading to enhanced median overall survival. These results support the use of lenalidomide in combination with docetaxel as an effective treatment for patients with CRPC. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5386.