Lenalidomide (Celgene Corp., Summit, NJ) was dissolved in DMSO (final concentration, 0.1%). ATO was purchased from Sigma-Aldrich (St. Louis, MO) and the stock solution (10 mM) was prepared in 0.1 N NaOH. Cell Titer 96 Aqueous One Solution Cell Proliferation Assay was purchased from Promega (Madison, WI). FITC Annexin V Apoptosis Detection Kit was purchased from BD Pharmingen (Franklin Lakes, NJ). Antibodies for Western Blotting were purchased from the following suppliers: Cleaved PARP, Caspase-3, Bcl-2, P53, P53ser15, and mouse and rabbit horseradish peroxidase (HRP)- conjugated IgG (Cell Signaling Technology, Inc., Danvers, MA); MDM2 and PP2A (Abcam, Cambridge, MA); CDC25C (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and ß-actin (Sigma-Aldrich, St. Louis, MO). Human IL-6 High Sensitivity ELISA kit was purchased from eBioscience (San Diego, CA). CycLex^® Cdc25C Protein Phosphatase Fluorometric Assay Kit was purchased from MBL International (Woburn, MA).

U266 and RPMI 8226 (8826) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). U266 and 8226 cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with penicillin (100 units/mL) and streptomycin (100 µg/mL) and 10% fetal bovine serum (v/v).

MTS assays (Cell Titer 96 Aqueous One Solution Cell Proliferation Assay, Promega) were used to quantify the percentage of metabolically active cells that were either untreated or treated with lenalidomide, ATO or both drugs. Briefly, 2 × 10⁴ cells per well were plated onto a 96-well plate. Lenalidomide, ATO, or both effects were tested at different ranges of times and concentrations and each specific time-point 20 μL of MTS reagent was added followed by incubation for an additional 4 hours at 37˚C. The plates were quantified by measuring the optical density at 490 nm (Benchmark Plus Microplate Spectrophotometer, Bio-Rad Laboratories (UK) Ltd.).

Total RNA was purified from U266 and 8226 using TRIzol-Reagent according to the manufacturer’s instructions (Life Technologies). The purity and concentration were measured using a spectrophotometer. Reverse transcription (RT) reactions were performed using iScriptTM cDNA Synthesis kit (BIO-RAD). cDNA was synthesized by adding 1 μg of total RNA, 5× iScript Reaction Mix (4 μL), and 1 μL of iScript Reverse Transcriptase to a total volume of 20 μL. The reaction was incubated at 25˚C for 5 min, 42˚C for 30 min, and 85˚C for 5 min.

Q-PCR reactions were performed by means of iQ SYBR Green Supermix of Bio-Rad. Each reaction (25 μL) contained 12.5 μL of iQ SYBR green supermix, 0.25 μL of forward primer (20 μM), 0.25 μL of reverse primer (20 μM), 11 μL of RNase-free water, and 1.0 μL of cDNA. The following cycles were performed 1 × 3 min at 95˚C, 40 amplification cycles (15 s 95˚C, 60 s 56˚C), 1 × 1 min 95˚C, 1 × 1 min 55˚C and a melting curve (80 × 10 s 55˚C with an increase of 0.5˚C per 10 s). A negative control without cDNA template was run with every assay. The optimal melting point of dsDNA I and the efficiency of the reaction were optimized beforehand. Transcript copy number per individual was calculated by normalization to GAPDH expression. Oligonucleotide primers for amplifying Cdc25C (NC_000005) forward primer: 5’-AGA GCA AGA CCC TGT CTC AA-3’, Cdc25C reverse primer: 5’-TCT CAT CCT TCC TTC ACA GC-3’. IL-6 forward primer was (5’-AGA CAG CCA CTC ACC TCT TC-3’), and IL-6 reverse primer was (5’-TCT TTG CTG CTT TCA CAC AT-3’). P53 forward primer was (5’-GTA CAT CTG GCC TTG AAA CC-3’), and p53 reverse primer was (5’-AGC TGC CCA ACT GTA GAA AC-3’). MDM2 forward primer was (5’- GTC AAT CAG CAG GAA TCA TCG-3'), and MDM2 reverse primer was (5'- CCT TTT GAT CAC TCC CAC CTT -3’). The GAPDH forward primer was (5’-CAA AAG GGT CAT CAT CTC TGC-3’), and the GAPDH reverse primer was (5’-GAG GGG CCA TCC ACA GTC TTC-3’).

The phosphatase assay was performed as per manufacturer’s instructions. Briefly, 2.5 µL of rh-Cdc25C (0.1 µg/µL) was incubated for 30 min at 37˚C with the indicated concentrations of either Cdc25˚C Phosphatase inhibitor (CDC25 Phosphatase Inhibitor II, NSC 663284 (Calbiochem) or treatment with lenalidomide, ATO or both in 1× assay buffer. After incubation, samples were aliquoted into 96-well plates and 5 µL of the 10× fluorophospho substrate, OMFP, was added. After 15 min of incubation at room temperature, 25 μL of stop solution was added to each well. The sample was measured on a synergy fluorescent plate reader with excitation at 485 nm and emission at 528 nm. Percentage activity was calculated according to the equation (OD of test sample/OD of control) × 100.

U266 and 8226 at a concentration of 2.0 × 105 cells per well were seeded in a 12-well plate. The cells were either untreated or treated with 20 μM of lenalidomide, 1 μM ATO or both for an additional 48 h. The plates were incubated for 4 d and analyzed for apoptosis using the Annexin V apoptosis kit (BD Pharmingen). Each well was resuspended in 1× binding buffer at a concentration of 1.0 × 106 cells per milliliter and stained with AnnexinFITC and propidium iodide. All samples were analyzed by flow cytometry using a FACScalibur within 30 min of staining. For cell-cycle analysis, cells were treated with lenalidomide, ATO, or both for 48 h at 37˚C, then washed and resuspended in BD Stain Buffer (106/ml). Cold ethanol (3 ml) was added while vortexing, and the cells were then incubated overnight at 4˚C and washed 2 times in stain buffer. One mL of PI staining solution (3.8 mM Na citrate, 50 μg/ml PI in PBS) was added to each cell pellet along with 50 μL of RNase A solution (10 μg/ml). Cell suspensions were incubated for 4 h at 4˚C before analysis on BD FACScalibur (BD BioSciences).

The cells were solubilized with lysis buffer (50mM TrisHCl (pH 7.4), 0.5% (v/v) Nonidet P-40 (Nonidet P-40), 150mM NaCl, 5mM EDTA, 50mM NaF, 1mM Na3VO4, 1mM phenylmethylsulfonyl fluoride, 10μg/ml leupeptin, and 10μg/ml aprotinin), and whole cell lysates (WCLs) were prepared by centrifugation at 12,000 g for 15 min at 4˚C. Proteins from WCLs were separated by SDS-PAGE (10% PAGE) and transferred onto PVDF membrane filters (Immobilon; Millipore). The membranes were immunoblotted with the respective antibodies, and bound antibodies were visualized with horseradish peroxidaseconjugated antibodies against mouse or rabbit IgGs (BioRad) using chemiluminescence reagents (Western Lightning; PerkinElmer Life Sciences).

Data are presented as mean ± SD. Differences between two groups were evaluated by Student’s t-test; p < 0.05 was considered to be statistically significant.

In order to compare in vitro the individual or combined effect of these drugs’ on MM cells we initially investigated the effects of ATO, lenalidomide of their combination on the viability the MM cell lines U266 and 8226. We treated cultured cells with either 1 μM ATO, 20 μM lenalidomide or both for 48 hours and tested the cellular viability in an MTS assay (Figures 1(a) and (b)). We observed that while lenalidomide only modestly inhibited cell growth ATO, with or without lenalidomide, had a profound effect on the viability of both cell lines. Interestingly, while together they increased the apoptotic effect it was in an additive manner, slightly increasing ATO’s apoptotic effects on these cells. Similarly, different concentrations of lenalidomide alone did not induce apoptosis of MM myeloma cells while ATO demonstrated a clear threshold for cytotoxicity of these cell lines (Figures 1(c) and (d)). To corroborate these results and also to understand the contribution of lenalidomide to apoptotic effects on these MM cells we repeated our experiments with ATO, lenalidomide or both at the same concentrations mentioned above and analyzed cells at either 24 or 48 hours by AnnexinV/PI expression in flow cytometry. Indeed, just as we observed for viability, the highest increases in apoptotic cells were in ATO treated cells either as a single agent or in combination (Figures 2(a) and (b)). In order to understand the apoptotic mechanisms of each agent or their combination we performed western blot on the lysates of MM cells treated with each one of them and measured for PARP, caspase 3 and the antiapoptotic molecule Bcl-2. Strong PARP cleavage was induced by the presence of ATO in the culture on both cell lines but lenalidomide had differential effects on both cell lines. Lenalidomide induced a slight increase in PARP cleavage in U266 cell but no caspase 3 cleavage while the reverse was seen in 8226 cells, no PARP cleavage but caspase 3 instead. In both lenalidomide had no effect on the expression of Bcl2. Therefore, the slight decrease in cellular viability induced by lenalidomide can be due to these apoptotic effects although it is clear that they do not add much to ATO as a cytotoxic agent.

Use One of the main cytokines affected by lenalidomide is IL-6. We therefore wanted to understand if ATO can provide the same effects or if the combination of ATO and lenalidomide could maintain the beneficial effects seen with lenalidomide. Cells were cultured with ATO (1 μM), lenalidomide (20 μM) or both and the total RNA and cell culture supernatants were collected. IL-6 mRNA expression, as measured by real time RT-PCR, demonstrates that while ATO significantly downregulates IL-6 in myeloma cells, lenalidomide’s effect on IL-6 inhibition is much stronger. Furthermore, the combination of lenalidomide and ATO has an additive effect, demonstrating the highest downregulation of IL-6 expression on both cell lines (Figures 3(a) and (b)). Conversely, while U266 IL-6 secretion levels were downregulated throughout, we did not observe a significant difference with either compound on the secretion of IL-6 by 8226 cells indicating that there is still a difference between those cell lines that prevents the downregulation of IL-6 secretion even if its production is reduced (Figures 3(c) and (d)).

Next we tested the effect these compounds had on P53 and MDM2. Western blot analysis revealed that total protein levels of P53 were unaffected. Additionally, P53 activity was also unaltered by treatment as measured by the phosphorylation of P53 at the serine 15 residue. Both

lenalidomide and ATO were able to downregulate the expression of MDM2. Not surprisingly, MDM2 was also downregulated when the drugs were combined (Figure 4(a)). Gene expression experiments confirmed our protein results where there were no apparent changes in P53 but downregulation of MDM2 was observed after any of the treatments (Figures 4(b) and (c)). These effects were observed in both cell lines tested, but in the case of 8226 cells, the combination of lenalidomide and ATO resulted in undetectable MDM2 protein levels.

As discussed in the introduction, we recently reported that lenalidomide’s action is enhanced in MDS patients with 5(q) deletion, and this is proposed to be due to the haplodeficiency of PP2A and Cdc25c. Considering this, we propose that downregulation of these molecules by other methods could open the possibilities to use lenalidomide to treat other diseases. We next tested whether treatment with ATO could downregulate Cdc25C, PP2A, or both. We found that Cdc25C, but not PP2A was down-regulated in the presence of lenalidomide, ATO, or both compounds (Figures 5(a) and (d)). Although there only was a modest decrease in Cdc25C’ protein levels with treatment of lenalidomide or ATO alone, this effect was much more profound when the cells were treated with both drugs (Figures 5(a), (b), (d) and (e)). We also measured the phosphatase activity of Cdc25C, and similar to what we saw with protein expression, Cdc25C phosphatase activity was inhibited only slightly by lenalidomide, but when the cells were treated with both lenalidomide and ATO, inhibition was similar to what we

saw when the cells were treated with a specific Cdc25c inhibitor (Figures 5(c) and (f)).