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Lenalidomide (Revlimid®) is approved for the treatment of previously treated multiple myeloma (MM) in combination with dexamethasone. Single agent lenalidomide has direct antiproliferative activity against MM cells and direct immunomodulatory activity via costimulation of T cell and natural killer (NK) cell responses. The ECOG Phase III trial in newly diagnosed MM (E4A03) reported that low dose (40 mg/d on days 1, 8, 15 & 22 of a 28-day cycle) dexamethasone in combination with lenalidomide showed an overall survival advantage when compared with the higher, standard-dose (40 mg/d on days 1-,4, 9–12 & 17–20 of a 28-day cycle) of dexamethasone that is used in combination with lenalidomide. We have examined the pharmacological activity of lenalidomide in combination with dexamethasone with respect to direct MM antiproliferative and T/NK cell stimulation in vitro. Antiproliferative activity against various MM cell lines was associated with increased expression of tumor suppressor genes, including Egr1, Egr3, p21, and p27. The specific tumor suppressor genes induced varied among the MM cell lines, even among those that were sensitive to lenalidomide. Lenalidomide and dexamethasone induced cell cycle arrest in G0/G1 phase, activation of caspases 3/7, and apoptosis in the sensitive cell lines. The combination of lenalidomide and dexamethasone was partially additive in most MM cell lines, while the most dramatic combination effect observed was induction of p21 gene expression and apoptosis in LP-1 MM cells. In contrast to its synergistic effect on MM cell proliferation, dexamethasone was strongly antagonistic to the T and NK cell costimulatory effects of lenalidomide. Specifically, in primary human T cells dexamethasone inhibited lenalidomide-enhanced IL-2 production. Furthermore, in primary human NK cells dexamethasone inhibited lenalidomide-enhanced IFN-γ production. These findings suggest that the lenalidomide plus low-dose dexamethasone regimen may be able to facilitate the MM cell killing via induction of tumor suppressor gene expression, cell cycle arrest, and apoptosis, while allowing lenalidomide to enhance T and NK cell function to a greater magnitude. The enhanced survival with the low-dose dexamethasone schedule in MM patients suggests exploring further dosing schedules of dexamethasone in combination with lenalidomide in this population, with measurement of biomarkers of T and NK cell function.

1α,25-Dihydroxyvitamin D3, (1,25 D3) the biologically active form of vitamin D, is well established as an inhibitor of cancer cell growth in addition to its primary role in maintaining bone mineralization. In breast cancer cell lines, inhibitory effects on cell cycle arrest, angiogenesis, invasion and metastasis have been observed in addition to pro-apoptotic effects. 1,25 D3 also inhibits and prevents breast cancer growth in several mouse models, and a correlation between vitamin D receptor expression on breast cancer cells and disease free survival of breast cancer patients has also been observed. However, resistance to vitamin D and hypercalcaemia at higher doses are key limiting factors in clinical use. The drug Revlimid ® (lenalidomide) which has shown great promise in multiple myeloma, can also modulate signalling in apoptotic and cell growth pathways leading to inhibition of cell growth, inhibition of metastasis and invasion. Our study aimed to determine whether lenalidomide treatment of breast cancer cells resistant to vitamin D would result in an acquisition of sensitivity to vitamin D and a resultant inhibition of cell growth. The cell lines MCF-12A, MCF-7 and MDA-MB-231,representing non-tumorigenic, tumorigenic and metastatic breast lines respectively were used. The latter line was also vitamin D resistant. Cells were treated with lenalidomide and/or 1,25 D3 using a dose of 100 nM 1,25 D3 (a clinically tolerable dose giving IC50 inhibition of MCF-7 and MCF-12A cells). Results showed that whereas lenalidomide had no effect on the growth of the vitamin D sensitive lines, and gave only 20% inhibition of growth of MDA-MB-231 at 10 µM, a 50% inhibition of cell growth by 1,25 D3 (equivalent to that seen with the sensitive lines) was achieved in the presence of lenalidomide at a concentration of 1 µM. Further investigation revealed that the mechanism of this effect was an increase in apoptosis of the cell line, shown by an increase in parp cleavage and annexin V expression. An array measuring proteins associated with several signalling cascades showed that the combination of 1,25 D3 and lenalidomide resulted in an increase in proapoptotic proteins (phosphorylated P53, P21 and claspin, in addition to a decrease in BCL-2. Although both drugs had individual effects on pro-apototic proteins, and anti-apoptotic proteins, the combination resulted in an overall increase of pro-apoptotic protein expression leading to the observed inhibition of cell viability and growth. These results demonstrate the potential for the use of lenalidomide and 1,25 D3 in combination for tumours that are refractory to vitamin D. 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 5418.