Seliciclib (CYC202, R-Roscovitine)

Research Article

Seliciclib (CYC202, R-Roscovitine) Induces Cell Death in Multiple Myeloma Cells by Inhibition of RNA Polymerase II–Dependent Transcription and Down-regulation of Mcl-1

David E. MacCallum, Jean Melville, Sheelagh Frame, Kathryn Watt, Sian Anderson, Athos Gianella-Borradori, David P. Lane, and Simon R. Green

Cyclacel Ltd., Dundee Technopole, James Lindsay Place, Dundee, United Kingdom

Abstract for example, CDK2/cyclin E for entry into S phase and CDK2/cyclin Seliciclib (CYC202, R-roscovitine) is a cyclin-dependent kinase A for exit out of S phase. CDKs also regulate transcription by phosphorylating the carboxyl-terminal domain of the large subunit (CDK) inhibitor that competes for the ATP binding site on the kinase. It has greatest activity against CDK2/cyclin E, CDK7/ of RNA polymerase II (5). In humans the carboxyl-terminal domain cyclin H, and CDK9/cyclin T. Seliciclib induces apoptosis from contains 52 repeats of a heptapeptide (YSPTSPS) that can be all phases of the cell cycle in tumor cell lines, reduces tumor phosphorylated on serines, threonines, and tyrosines (5). A number growth in xenografts in nude mice and is currently in phase II of CDKs have been shown to phosphorylate these sites including: clinical trials. This study investigated the mechanism of cell CDK7/cyclin H/Mat1, part of the TFIIH complex that activates death in multiple myeloma cells treated with seliciclib. In transcriptional initiation and CDK9/cyclin T, also called P-TEFb, myeloma cells treated in vitro, seliciclib induced rapid that can activate transcriptional elongation (6). In addition, CDK8/ dephosphorylation of the carboxyl-terminal domain of the cyclin C, CDK1/cyclin B, and CDK2/cyclin E have been shown to large subunit of RNA polymerase II. Phosphorylation at these phosphorylate the carboxyl-terminal domain in vitro (6, 7). sites is crucial for RNA polymerase II–dependent transcrip- Seliciclib (CYC202, R-roscovitine) is a 2,6,9-substituted purine analogue that competes with ATP for binding to the active site tion. Inhibition of transcription would be predicted to exert on CDKs. Seliciclib has potent in vitro activity against CDK2/ its greatest effect on gene products where both mRNA and cyclin E (IC = 0.1 Amol/L), CDK7/cyclin H (IC = 0.36 Amol/L), protein have short half-lives, resulting in rapid decline of the 50 50 and CDK9/cyclin T (IC = 0.81 Amol/L; refs. 8, 9). A broad range protein levels. One such gene product is the antiapoptotic 50 of tumor cell types are inhibited by seliciclib in vitro, with an factor Mcl-1, crucial for the survival of a range of cell types average 72-hour IC value of 15 Amol/L. Treatment of cell lines including multiple myeloma. As hypothesized, following the 50 has wide-ranging effects including: accumulation of cells in G and inhibition of RNA polymerase II phosphorylation, seliciclib 1 G phases, inhibition of rRNA processing, inhibition of RNA caused rapid Mcl-1 down-regulation, which preceded the 2 polymerase II–dependent transcription, disruption of nucleoli, and induction of apoptosis. The importance of Mcl-1 was induction of apoptosis from all stages of the cell cycle (8, 10–15). confirmed by short interfering RNA, demonstrating that Human tumor xenografts in nude mice treated with seliciclib show reducing Mcl-1 levels alone was sufficient to induce apoptosis. significantly reduced growth compared with controls (8). Seliciclib These results suggest that seliciclib causes myeloma cell death is currently in phase II clinical trials in combination with by disrupting the balance between cell survival and apoptosis gemcitabine/cisplatin for non–small cell lung cancer and as a through the inhibition of transcription and down-regulation single agent for B cell malignancies including multiple myeloma. of Mcl-1. This study provides the scientific rationale for the Multiple myeloma is a disease of malignant B cells that have clinical development of seliciclib for the treatment of multiple extended survival and a low proliferation rate which accumulate in myeloma. (Cancer Res 2005; 65(12): 5399-407) the bone marrow causing osteolytic bone lesions (16). Treatment with current chemotherapeutic agents initially reduces tumor Introduction burden but the disease remains incurable, with the average patient There are more than 500 protein kinases encoded by the human survival time being 3 years. Therefore, there is an urgent need for genome and this class of enzymes has been an area of intensive novel therapies ideally based on an understanding of the biology of research for the development of novel anticancer agents (1). One the disease. Typically, the disease begins with monoclonal family of kinases that has attracted particular attention is the cyclin- gamopathies of undetermined significance that progresses to dependent kinases (CDK), which regulate two processes essential myeloma in a small percentage of cases (16, 17). Genetic changes for cancer cell survival; cell cycle progression and gene transcription that give rise to multiple myeloma can involve genes that regulate (2). CDKs control entry into each stage of the cell cycle by both the cell cycle and apoptosis. These include overexpression phosphorylating key substrates such as pRb and condensin (3, 4). of antiapoptotic Bcl-2 and down-regulation of proapoptotic CDKs usually form heterodimers with cyclins to create an active Bik (18). A recent study analyzing identical twins, one with complex and because most cyclin levels oscillate throughout the cell multiple myeloma and one without, identified overexpression of cycle, this contributes to the temporal activation of specific CDKs; Mcl-1, c-FLIP, and Dad-1 in the malignant cells of the multiple myeloma patient, emphasizing the importance of these antiapoptotic proteins in the development of this disease (19). Requests for reprints: David E. MacCallum, Cyclacel Ltd., Dundee Technopole, Mcl-1, an antiapoptotic member of the Bcl-2 family (20), was first James Lindsay Place, Dundee, DD1 5JJ, United Kingdom. Phone: 44-1382-206062; Fax: 44-1382-206067; E-mail: [email protected]. identified as a protein up-regulated in a human myeloblastic I2005 American Association for Cancer Research. leukemia cell line induced to differentiate along the monocyte www.aacrjournals.org 5399 Cancer Res 2005; 65: (12). June 15, 2005

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Cancer Research lineage (21). Mcl-1 is essential for the survival of multiple myeloma Antibodies used in this study were: RNA Polymerase II (MMR-126R, cells (22–24) as well as B cell lymphoma cells (25). Mcl-1 is thought Covance, Cambridge, United Kingdom), RNA Polymerase II carboxyl- to act by antagonizing proapoptotic proteins such as Bim (26). terminal domain phosphoserine 2 (MMR-129R, Covance), RNA Polymerase In this study, the mechanism of cell-killing by seliciclib was II carboxyl-terminal domain phosphoserine 5 (MMR-134R, Covance), PARP (H250 Santa Cruz Biotechnology, Santa Cruz, CA), Hdm2 (SMP14, Santa explored; testing the hypothesis that the ability of seliciclib to Cruz), p53 (DO1 Calbiochem, Nottingham, United Kingdom), Mcl-1 (S-19, induce myeloma cell death occurred by inhibiting transcription Santa Cruz), Bcl-2 (clone 124 Upstate, Milton Keynes, United Kingdom), XIAP and down-regulating critical proteins required for cell survival. This (610763, Becton Dickinson, Cowley, United Kingdom), survivin (NB500-201, study shows that seliciclib inhibits the kinases which phosphory- Novus Biologicals, Littleton, CO), pRb (554136, Becton Dickinson), and pRb late the carboxyl-terminal domain of RNA polymerase II resulting phospho 249/252 (44-584, Biosource, Nivelles, Belgium). in inhibition of transcription, down-regulation of the levels of Mcl-1 Terminal deoxynucleotidyltransferase dUTP nick end labeling mRNA and protein, and rapid induction of apoptosis. These data assays. Cells were seeded, treated with DMSO or 30 Amol/L seliciclib and provide scientific rationale for the clinical development of seliciclib harvested at specific time points. Cells were washed in PBS, fixed for as an agent for treating multiple myeloma. 5 minutes on ice in 1% w/v paraformaldehyde, washed in cold PBS and resuspended in 70% ethanol (À20jC), prior to storage at À20jC. Labeling was carried out using the APO-DIRECT kit (Becton Dickinson). Briefly, Materials and Methods 1 million cells were incubated for 1 hour at 37jC in the presence of terminal Cell lines and reagents. NCI-H929, LP-1, RPMI 8226, OPM2, and U266 deoxynucleotide transferase enzyme and FITC-dUTP to label DNA breaks; cells were purchased from the DSMZ (Germany). Cells were cultured at washed and then incubated in propidium iodide (5 Ag/mL) and RNase j 37 C with 5% CO2 in RPMI 1640 containing 10% FCS and 2 ng/mL of IL-6 (200 Ag/mL) for 30 minutes at room temperature to label DNA. Cells were (R&D systems, Abingdon, United Kingdom) an important survival factor analyzed using a Becton Dickinson LSR flow cytometer. The argon laser in vivo (27). Media for H929 cells was supplemented with 50 Amol/L was used as an excitation source (488 nm). Terminal deoxynucleotidyl- 2-mercaptoethanol (BDH, Poole, United Kingdom). Cells were kept at a transferase dUTP nick end labeling (TUNEL)-positive cells were designated density of between 0.2 and 1 Â 106 cells/mL. All reagents were purchased on the basis of green fluorescence (530 F 28 nm) acquired on a logarithmic from Sigma (Poole, United Kingdom) unless stated otherwise. scale. DNA content was determined using red fluorescence (575 F 26 nm) Cytotoxicity assays. Cells were seeded in 96-well plates appropriately for on a linear scale and pulse width analysis was used to exclude cell doublets their doubling time (5,000 cells per well except for H929 which were seeded and aggregates from the analysis. Cells exhibiting < 2n DNA content were at 8,000 cells per well) and incubated overnight. Stock solutions of designated as sub-G1 cells. Only cells with 2n-4n DNA content were compounds were prepared in DMSO at 100 mmol/L. A dilution series for included in the TUNEL analysis. seliciclib or 5,6-dichlorobenzimidazole riboside (DRB) was prepared in Real-time PCR. Cells were treated and harvested identically to those medium, added to cells and incubated for 72 hours. A 20% stock of alamar prepared for immunoblotting. Cell pellets were lysed and RNA extracted blue (Roche, Lewes, United Kingdom) was prepared in medium, added to using RNeasy kits (Qiagen, Crawley, United Kingdom). The RNA was each well and incubated for 2 hours. Absorbance was read at 488 to 595 nm quantified and normalized to 1 mg/mL prior to storage at À70jC. and data were analyzed (Excel Fit v4.0) to determine the IC50 (concentration Quantitative real-time PCR was done on a Roche LightCycler using equal of compound that inhibited cell growth by 50%) for each compound. amounts of total RNA. The sequence of the primers were as follows: To test the effect of exposure time to seliciclib on viability, cells were 5 seeded in flasks at 0.3 Â 10 cells/mL, treated with DMSO or seliciclib, and Mcl-1 (5V-GGTGGCATCAGGAATGT-3V) aliquots removed at specific time points. These cells were centrifuged at (5V-ATCAATGGGGAGCACT-3V) 800 Â g for 5 minutes, washed and replated into 96-well plates in the 28S rRNA (5V-AAGCAGGAGGTGTCAGAAA-3V) absence of drug. Cell viability was determined, using alamar blue, 72 hours V V after the start of the experiment. (5 -GTAAAACTAACCTGTCTCACG-3 ) Preparation and analysis of cell lysates by immunoblotting. Cells were seeded at 0.4 Â 106 cells/mL in T25 flasks and treated with either The RNA Master SYBR Green I kit (Roche) was used to determine the 30 Amol/L seliciclib, 60 Amol/L DRB, 300 nmol/L MG132, or DMSO. Cells relative amount of each mRNA. Samples were prepared in duplicate and were removed at a variety of time points, washed twice with PBS, and the PCR reactions were also run in duplicate. The change in expression in the cell pellets snap-frozen in liquid nitrogen prior to storage at À70jC. Cells presence of compound compared with the DMSO control was calculated were resuspended in high salt buffer [50 mmol/L Tris-HCl (pH 7.6), 1% using the formula: fold change in expression = 2DCt, where 2 is the NP40, 0.4 mol/L NaCl, 5 mmol/L EDTA, 5 mmol/L DTT, 1 mmol/L maximum efficiency of the PCR reaction and DCt is the change in crossing phenylmethylsulfonyl fluoride, 2 mmol/L NaF, 1 mmol/L Na3VO4, 20 mmol/L point values (sample Ct À DMSO control Ct). h-glycerol phosphate, 5 mmol/L sodium pyrophosphate, and a protease Transfer of short interfering RNA by reversible cell permeabiliza- inhibitor cocktail] and allowed to lyse for 15 minutes prior to sonication for tion. Streptolysin O was used to reversibly permeabilize H929 cells using a 5 seconds at 5 amp (Sanyo soniprep 150). The protein concentration of each modification of a published protocol (28). In brief, streptolysin O was lysate was determined using a Bradford reagent assay (Bio-Rad, Hemel suspended at 1,000 units/mL in Mg2+- and Ca2+-free PBS containing 0.01% Hempstead, United Kingdom). bovine serum albumin and activated by adding DTT to 5 mmol/L prior to Lysate (30 Ag) was mixed with gel loading buffer containing reducing incubating for 2 hours at 37jC. Cells were washed twice in RPMI 1640 and agent and separated in either 3% to 8% or 10% polyacrylamide gels using resuspended at 25 Â 106 cells/mL. Activated streptolysin O was added to denaturing electrophoretic conditions (Invitrogen, Glasgow, United King- the wells of a 48-well plate at 14 units/106 cells. The relevant short dom). Proteins were transferred to nitrocellulose membranes (Hybond ECL, interfering RNAs (siRNAs) were added to each well (final concentration 6 Amersham, Chalfont St. Giles, United Kingdom) using wet electrophoretic 10 Amol/L) followed by 5 Â 10 cells and incubated at 37jC5%CO2 with transfer. Membranes were stained with Ponceau S to confirm equal loading occasional mixing. After 10 minutes, growth medium containing 10% FCS before blocking in 5% nonfat milk in PBS with 0.1% Tween 20 (PBSTM) for (1 mL) was added to each well and the cells were returned to 37jC. After 1 hour. Membranes were incubated overnight at 4jC with primary antibody, 30 minutes, growth medium containing 10% FCS (9 mL) was added and the diluted in PBSTM. Membranes were washed in PBS and 0.1% Tween 20 cells were incubated for 24 hours before harvesting. The Mcl-1 siRNA was (PBST) and incubated for 1 hour in PBSTM containing horseradish a smart pool consisting of four independent siRNAs designed using cDNA peroxidase-conjugated secondary antibody. Membranes were washed and sequence: GI33519457 (Dharmacon, Lafayette, CO). incubated with enhanced chemiluminescence solution (Amersham) and Annexin V staining. Cells were centrifuged at 500 Â g for 5 minutes, exposed to X-ray film (Amersham). washed twice in PBS and once in annexin buffer [10 mmol/L Hepes (pH 7.4),

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2.5 mmol/L CaCl2, and 140 mmol/L NaCl]. Cells were resuspended at 1 Â 106/mL and 100 AL was transferred to a 5 mL tube prior to incubation for 10 minutes in the dark at room temperature with 5 AL of annexin stain (Becton Dickinson) and 10 AL of propidium iodide (50 mg/mL). Annexin buffer (1 mL) was added and the cells were analyzed by flow cytometry. Annexin V–positive cells were designated on the basis of green fluorescence and propidium iodide–positive cells were designated on the basis of red fluorescence.

Results The effect of seliciclib and DRB on multiple myeloma cell viability. The IC50 for seliciclib was determined in five myeloma cell lines. Cells were seeded in triplicate in 96-well plates, treated with serial dilutions of seliciclib and tested for viability after 72 hours. The average IC50 after seliciclib treatment was 15.5 Amol/L (Table 1) with the most sensitive cell lines being H929 (8.84 Amol/L) and LP-1 (12.68 Amol/L), and the most resistant being RPMI 8226 (19.5 Amol/L). The IC50 was also determined following 24 hours exposure to seliciclib, these values were equivalent to the 72-hour IC50, indicating that a relatively short exposure to seliciclib was sufficient to kill cells (data not shown). In addition to the seliciclib IC50 for these myeloma cell lines, the IC50 of DRB was also determined (Table 1). DRB is a well- characterized inhibitor of transcription, that principally inhibits CDK9 (29), and in this respect, DRB has a similar mode-of-action to another established CDK inhibitor, flavopiridol (30–32). In general, the IC50 values for DRB were higher than those for seliciclib but a similar profile of sensitivities between the cell lines was detected (Table 1). Seliciclib induces rapid apoptosis in multiple myeloma cells. H929, LP-1, and RPMI 8226 cells were treated with either DMSO (control) or 30 Amol/L seliciclib (twice the average IC50) and harvested at specific time points. Cells were fixed and stained using TUNEL to identify the proportion of cells containing fragmented DNA, a late stage marker of apoptosis (33). Induction of TUNEL was increased following 3 hours of seliciclib treatment and continued to accumulate to z60% by 24 hours (Fig. 1). As reported previously (8), apoptosis occurred from all phases of the cell cycle (data not shown). Exposure time required to reduce myeloma cell viability. To Figure 1. Time course of induction of apoptosis by seliciclib in multiple determine the minimum exposure time required to induce myeloma cells detected by TUNEL. Cells were treated with DMSO or 30 Amol/L maximum cytotoxicity, cells were treated with DMSO or seliciclib; seliciclib. At the time points indicated, cells were collected, fixed, stained using TUNEL, and analyzed by flow cytometry. The extent of apoptosis is expressed as a percentage of cells positively stained using TUNEL. A, H929; B, LP-1; C, RPMI 8226. Results represent the average and SD for three independent Table 1. IC50 (Amol/L) for seliciclib and DRB in a range of experiments. multiple myeloma cell lines at specific time points, cells were washed and returned to fresh Seliciclib DRB medium in the absence of seliciclib. After a total of 72 hours of growth, cell viability was determined. An 8-hour treatment with A A Cell line IC50 ( mol/L) SD IC50 ( mol/L) SD either 20 or 30 Amol/L seliciclib reduced the 72-hour viability by at least 50% in all three cells lines (Fig. 2). Treatment of RPMI 8226 H929 8.84 1.05 20.13 2.67 for 8 hours induced a maximal effect at both concentrations LP-1 12.68 1.89 29.81 4.84 U266 17.93 4.22 41.02 3.95 (Fig. 2C) but longer exposure was required to induce a maximal OPM2 18.46 5.98 29.52 4.101 cytotoxicity in H929 and LP-1 cells, 16 and 24 hours, respectively RPMI 8226 19.5 1.37 46.79 1.7 (Fig. 2A and B). These data, in addition to the previous results, Average 15.48 2.12 33.45 1.25 indicate that exposure to seliciclib for z8 hours markedly reduces cell viability by inducing apoptosis. Molecular changes in seliciclib-treated multiple myeloma NOTE: Results represent the average and SD for three independent cells. Because both seliciclib and DRB can inhibit CDKs experiments. responsible for RNA polymerase II phosphorylation, experiments were done to determine whether the mechanism by which seliciclib www.aacrjournals.org 5401 Cancer Res 2005; 65: (12). June 15, 2005

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Cancer Research induced apoptosis was through an inhibition of transcription, in an S249/T252 phosphorylation, although by 8 hours, dephosphoryla- analogous manner to DRB. H929 cells were treated with either tion of S249/T252 was detected (Fig. 3). These data again reflect the DMSO, seliciclib (30 Amol/L), or DRB (60 Amol/L) and collected at differences between kinases inhibited by seliciclib and DRB. specific time points; these compound concentrations represent The ability of seliciclib to inhibit phosphorylation of the levels of equal potency, that is, twice the average IC50 value. To carboxyl-terminal domain of RNA polymerase II in a similar examine the molecular changes in seliciclib-treated multiple manner to DRB suggests that seliciclib could also be inhibiting myeloma cells, the level and phosphorylation state of proteins transcription. To explore this further, the effects of seliciclib and important for RNA polymerase II–dependent transcription and cell DRB on the levels of key cell survival proteins were investigated. survival were studied. The use of low percentage acrylamide gels The levels of Mcl-1 protein rapidly decreased after treatment for enabled the detection of two main forms of the RNA polymerase II 3 hours with either compound (Fig. 3, lanes 7 and 11), whereas large subunit. In DMSO-treated cells, the total RNA polymerase II little change was detected in the levels of XIAP, survivin, or Bcl-2, antibody detected both the slow migrating hyperphosphorylated consistent with Mcl-1 having a very short-lived mRNA and protein. IIO form and the fast migrating hypophosphorylated IIA form, The decrease in Mcl-1 preceded an increase in the level of cleaved along with other intermediates as previously reported (34). PARP, a well-characterized marker of apoptosis (37) that was In contrast, phosphoserine 2 and phosphoserine 5 were only detectable following 5 hours of treatment with either seliciclib or detected in the hyperphosphorylated IIO form (Fig. 3, lanes 1-5). DRB (Fig. 3, lanes 8 and 12). Hdm2, like Mcl-1, has a very short- Treatment for 1.5 hours with either seliciclib or DRB resulted in the lived mRNA and protein (38), and similarly, levels of Hdm2 rapidly rapid reduction in the levels of phosphoserine 2 and phosphoserine declined after either seliciclib or DRB treatment (Fig. 3, lanes 7 5 in the absence of changes in the total RNA polymerase II levels and 11). Because Hdm2 is an E3 ubiquitin ligase for p53 (39, 40), (Fig. 3, lanes 6 and 10). Careful analysis of the changes in mobility the reduction in the level of Hdm2 was followed by the expected detected with these antibodies showed subtle differences between accumulation in p53 levels (Fig. 3, lanes 7 and 11). the effects of seliciclib and DRB on the mobility of RNA polymerase To determine whether these were general effects of seliciclib on II. The faster migrating hypophosphorylated IIA form was detected myeloma cells, the studies were expanded to evaluate the cellular with the anti-phosphoserine 5 antibody after treatment with DRB effects of seliciclib on LP-1 and RPMI 8226 cells. Cells were treated but not seliciclib. Furthermore, the total RNA polymerase II with 30 Amol/L seliciclib and harvested at various time points. antibody detected less protein in the hyperphosphorylated IIO As seen in H929 cells, treatment with seliciclib induced rapid position after treatment with DRB compared with seliciclib. This dephosphorylation of serine 2 on the carboxyl-terminal domain of may reflect the differences in kinase specificities, suggesting that RNA polymerase II in both cell lines (Fig. 4A and B, lane 7). DRB exerts greater effects than seliciclib, leading to a more Dephosphorylation of serine 2 was followed by a reduction in the significant increase in the hypophosphorylated IIA form. levels of Mcl-1 that began to decrease after 1.5 hours of treatment The phosphorylation status of pRb was analyzed at the S249/T252 (Fig. 4A and B, lane 7). The loss of Mcl-1 again preceded the onset sites, which are reported to be sites for CDK phosphorylation (35, 36). of apoptosis that was detected by PARP cleavage at 3 to 5 hours Dephosphorylation at S249/T252 occurred after 1.5 hours of seliciclib (Fig. 4A and B, lane 8). Taken together, these results suggest that in treatment in the absence of changes in total pRb levels (Fig. 3, lane 9). myeloma cells, seliciclib like DRB, rapidly inhibits phosphorylation Dephosphorylation of pRb occurred with similar kinetics to that of of the carboxyl-terminal domain of RNA polymerase II, resulting in RNA polymerase II after seliciclib treatment. This suggests that down-regulation of Mcl-1 prior to the induction of apoptosis. S249/T252 and RNA polymerase II may be phosphorylated by the Seliciclib inhibits transcription of Mcl-1. To confirm that the same kinase(s). DRB treatment did not result in rapid changes in loss of Mcl-1 protein was due to a block in transcription, not an

Figure 2. Effect of exposure time to seliciclib on multiple myeloma cell viability. Cells were treated with DMSO, 20, or 30 Amol/L seliciclib. At the indicated time points, cells were collected, washed, replated, and tested for cell viability using alamar blue at 72 hours. Viability is expressed as a percentage of control cell viability. A, H929; B, LP-1; C, RPMI 8226. Results are the average and SD for three independent experiments.

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Figure 3. Molecular events in H929 cells treated with seliciclib or DRB. H929 cells were treated with DMSO (lanes 1-5), 30 Amol/L seliciclib (lanes 6-9), or 60 Amol/L DRB (lanes 10-13). At the indicated time points, cells were collected and whole cell lysates were prepared. Equal amounts of lysate were separated by denaturing gel electrophoresis and analyzed by immunoblotting using the antibodies indicated. The position of the hyperphosphorylated RNA polymerase II (IIO) and hypophosphorylated RNA polymerase II (IIA) is indicated. These results are representative of at least three independent experiments. Arrow, a fragment of cleaved PARP.

activation of a proteolytic pathway or changes in translation bition of Mcl-1 transcription due to the inhibition of the carboxyl- rates, the levels of Mcl-1 mRNA following seliciclib treatment terminal domain phosphorylation by seliciclib. were measured by real-time PCR. Total RNA was prepared from Loss of Mcl-1 is specific to seliciclib treatment and not a cells treated with DMSO or 30 Amol/L seliciclib. Previously, it has general prerequisite for the induction of apoptosis. Following been shown that roscovitine does not affect the production of seliciclib treatment, the decrease in Mcl-1 protein prior to PARP rRNA (13), therefore 28S rRNA was used as a control for total cleavage suggested that this might be part of the mechanism by RNA levels. Seliciclib caused a rapid reduction by 2- to 5-fold which seliciclib induces apoptosis. To show that the loss of Mcl-1 of Mcl-1 mRNA in H929 cells and LP-1 cells with the levels was not just a function of the apoptotic process and thereby not remaining decreased for several hours (Fig. 5). These results directly related to seliciclib’s mode of action, the impact of a second confirm that the loss of Mcl-1 protein correlated with an inhi- apoptotic-inducing agent on Mcl-1 levels was studied. MG132 is

Figure 4. Molecular events in multiple myeloma cells treated with seliciclib. Cells were treated with DMSO (lanes 1-5)or30Amol/L seliciclib (lanes 6-10). At the indicated time points, cells were collected and whole cell lysates were prepared. Equal amounts of lysate were separated by denaturing gel electrophoresis and analyzed by immunoblotting using the antibodies indicated. A, LP-1; B, RPMI 8226. These results are representative of at least three independent experiments. Arrow, a fragment of cleaved PARP. www.aacrjournals.org 5403 Cancer Res 2005; 65: (12). June 15, 2005

seliciclib acts by inhibiting kinases that phosphorylate the carboxyl-terminal domain of the large subunit of RNA polymerase II resulting in the inhibition of transcription. This leads to the rapid down-regulation of Mcl-1 mRNA and protein which is required for myeloma cell survival (Figs. 3-6). The targeting of cyclin-dependent kinases by seliciclib in myeloma cells. Accumulating lines of evidence suggest that CDK7 and CDK9 are implicated in the activation of transcriptional initiation and elongation, respectively, via direct phosphorylation of the carboxyl-terminal domain of RNA polymerase II (6). CDK7 can phosphorylate the serine 5 position whereas CDK9 phosphorylates serines at both the 2 and 5 positions. Phosphorylation of the carboxyl-terminal domain of RNA polymerase II at these sites is Figure 5. Effect of seliciclib on the levels of Mcl-1 mRNA measured by real-time central not only to transcription but also mRNA capping, splicing, PCR. H929 and LP-1 cells were treated with DMSO as a control or 30 Amol/L seliciclib and collected at the indicated time points. Total RNA was prepared, and cleavage, and polyadenylation. This is because the large flexible levels of Mcl-1 mRNA were quantified using real-time PCR and compared with carboxyl-terminal domain acts as a landing pad for docking of a control 28S rRNA. Values are expressed as fold decrease compared with controls. Results are representative of three independent experiments. range of factors required for these processes and it is believed that phosphorylation regulates factor binding (44). The in vitro kinase specificity for seliciclib shows that it principally inhibits CDK2/ a proteosome inhibitor, which as a class of compounds are known cyclin E, CDK7/cyclin H, and CDK9/cyclin T (8, 9). In seliciclib- to effectively induce apoptosis in myeloma cells (41, 42). MG132, as treated myeloma cells, the phosphorylation of the serine 2 and 5 a proteosome inhibitor, therefore has a very distinct mode-of- positions of the carboxyl-terminal domain of RNA polymerase II action from seliciclib, thus if both compounds caused a decrease in was rapidly inhibited, suggesting that seliciclib directly inhibits the Mcl-1 levels prior to the induction of apoptosis, this would suggest kinases responsible for these phosphorylation events. The potent that the loss of Mcl-1 is a fundamental part of the apoptotic in vitro inhibition of CDK7 and CDK9 by seliciclib, together with process rather than specifically related to seliciclib’s mode-of- the critical involvement of these kinases in carboxyl-terminal action. LP-1 cells were treated with DMSO or 300 nmol/L MG132 domain phosphorylation, suggests that CDK7 and CDK9 are both and collected at various time points. As an indicator of apoptotic cellular targets of seliciclib. Inhibition of carboxyl-terminal domain induction, PARP cleavage was clearly detected following 16 hours phosphorylation provides a link between kinase activity and the of MG132 treatment without any apparent change in the levels of general transcriptional inhibition by roscovitine previously Mcl-1 (Fig. 6A). Thus, following MG132 treatment, the induction of reported (11, 31). In these reports, measurement of the levels PARP cleavage occurred before a decrease in Mcl-1 levels that was of newly synthesized mRNA showed a 70% decrease after 2 hours in distinct contrast to seliciclib treatment where the Mcl-1 decrease of treatment with 25 Amol/L roscovitine (11); whereas gene preceded PARP cleavage (Figs. 3 and 4). Correspondingly the loss of expression analysis showed that roscovitine inhibited the tran- Mcl-1 prior to the onset of apoptosis is characteristic for seliciclib; scription of a subset of genes but the effect did not seem to be and because Mcl-1 is reported to be essential for myeloma cell as widespread as other transcriptional inhibitors, including DRB survival (22, 23), this could cause the onset of apoptosis seen in and flavopiridol, at doses that gave equivalent cellular effects (31). seliciclib-treated myeloma cells. The latter result is of particular interest because both DRB and Mcl-1 is required for survival of multiple myeloma cells. To flavopiridol are primarily CDK9 inhibitors, unlike seliciclib, and confirm that Mcl-1 is vital for survival of multiple myeloma cells, both have broader effects on transcription (29–31, 45). In vitro Mcl-1 expression was inhibited using siRNAs. H929 cells were used characterization of the kinases inhibited by DRB shows a 25-fold in these experiments because the importance of Mcl-1 in the selectivity for CDK9 over both CDK7 and CDK2 (29, 46).1 Seliciclib survival of this cell line has not been determined previously unlike on the other hand, whereas approximately equipotent as DRB LP-1 and RPMI 8226 cells where Mcl-1 antisense has been used to against CDK9 (IC = 0.81 Amol/L) is significantly more active show that it is essential (22, 23). Transfection was accomplished 50 against CDK7 (IC = 0.36 Amol/L) and even more so against CDK2 using streptolysin O and transfection efficiency was judged to be 50 (IC = 0.1 Amol/L; refs. 8, 9). The differences in kinase specificity 85% using FITC dye penetration (data not shown). Mcl-1 siRNA, 50 between seliciclib and DRB would explain the subtle, but distinct but not control gl3 siRNA (43) caused a dramatic reduction in the differences seen in the levels of hyperphosphorylated IIO and levels of Mcl-1 protein (Fig. 6B) but no change in the levels of the hypophosphorylated IIA forms of RNA polymerase II following related protein Bcl-2. Furthermore, following 24 hours of treatment, treatment with these compounds (Fig. 3). the Mcl-1 siRNA induced apoptosis detectable by PARP cleavage The rapid dephosphorylation of pRb at S249/T252 after seliciclib (Fig. 6) and quantified by flow cytometry using TUNEL (f70% of but not DRB treatment identifies this phosphorylation site as cells) and annexin V staining (f80% of cells). These results show a marker of seliciclib activity (Fig. 3). The S249/T252 sites became the importance of Mcl-1 for the survival of multiple myeloma cells. dephosphorylated at the same rate as the carboxyl-terminal Therefore, the loss of Mcl-1 caused by seliciclib’s inhibition of domain of RNA polymerase II suggesting the same kinase(s) maybe transcription would be sufficient to induce apoptosis in these cells. responsible. The cellular kinase(s) responsible for these phosphor- ylations have not been determined yet, although in vitro kinase Discussion data show that these sites can be phosphorylated by CDK4/cyclin This study reports that the CDK inhibitor, seliciclib (CYC202, R-roscovitine), inhibits myeloma cell growth in a time- and dose- dependent manner (Figs. 1 and 2). Evidence is provided that 1 W. Jackson, personal communication.

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Figure 6. The role of Mcl-1 in cell survival in myeloma cells. A, LP-1 cells were treated with DMSO (lanes 1-7) or 300 nmol/L MG132 (lanes 8-13) collected at various time points and analyzed by immunoblotting using the antibodies indicated. This concentration was equivalent to twice the IC50 of MG132 and represents an equal cellular potency to that used previously for seliciclib. Arrow, a fragment of cleaved PARP. B, 929 cells were transfected with siRNA using streptolysin O as the transfection reagent. Cells were left untreated (lane 1), mock-treated with streptolysin O (lane 2), treated with streptolysin O in the presence of 10 Amol/L siRNA: gl3 control (lane 3), FITC (lane 4), or Mcl-1 (lane 5). After 24 hours, cells were collected and whole cell lysates were prepared. Equal amounts of lysate were separated by denaturing gel electrophoresis and analyzed by immunoblotting using the antibodies indicated. C, cells were transfected, collected after 24 hours, fixed and stained by TUNEL and analyzed by flow cytometry. Results show the percentage of cells positively stained by TUNEL and SD for three independent experiments. D, cells were transfected and collected after 24 hours, stained for annexin V and analyzed by flow cytometry. Results show the percentage of annexin V–stained cells and SD for three independent experiments.

D (36). It would seem unlikely that S249/T252 are phosphorylated it is likely that S780, S807/S811, and T821 are targeted by different by CDK4/cyclin D in cells, due to the lack of potency of seliciclib kinases to the one that phosphorylates S249/T252, the explanation towards CDK4/cyclin D (8), especially considering that the original that the kinase is the same but the rate of dephosphorylation of in vitro work did not examine the potential of CDK7 or CDK9 to S780, S807/S811, and T821 may be slower, cannot be excluded phosphorylate these sites (36). Seliciclib has also been shown to completely. down-regulate other phosphorylation sites on pRb including S780, The mechanism of seliciclib-induced apoptosis in myeloma S807/S811, and T821 (15).2 Significantly longer exposure was cells. The effects of seliciclib on RNA polymerase II suggest that required, however, to reduce the levels of phosphorylation at these one potential mechanism for seliciclib-induced cell death may be sites. Considering the rapid decrease in phosphorylation state of through the inhibition of transcription. Cell survival factors are the S249/S252 sites in this study (1.5 hours), these data suggest that often highly regulated at both the mRNA and protein level, the decrease in the phosphorylation of the other pRb sites may be therefore, potentially representing key candidates to be affected by caused by subsequent effects on downstream kinases. For example, a transcriptional block. Investigations into the effects of seliciclib CDK7 as part of the CDK-activating kinase complex, phosphor- on a number of short half-life antiapoptotic proteins found that ylates and activates a range of kinases (47), therefore, inhibition of rapid changes in Mcl-1 mRNA and protein were detected (Figs. 3-5). CDK7 by seliciclib could indirectly inhibit the activities of other Mcl-1 protein has a reported half-life of 30 minutes (48) potentially CDKs that phosphorylate pRb. Alternatively, the inhibition of as a consequence of having proteolytic degradation motifs that are transcription by seliciclib could lead to the down-regulation of the found in highly regulated proteins (49). In myeloma cells, the loss of levels of cyclins or CDKs that regulate these sites. Finally, although Mcl-1 was followed rapidly by the onset of apoptosis as detected by PARP cleavage and TUNEL analysis. Mcl-1 degradation was not a general prerequisite for the initiation of the apoptotic process, 2 D.E. MacCallum and S.R. Green, unpublished results. because treatment of myeloma cells with the proteosome inhibitor, www.aacrjournals.org 5405 Cancer Res 2005; 65: (12). June 15, 2005

MG132, induced apoptosis in the absence of changes in Mcl-1 seliciclib kills cells with similar potency regardless of whether or protein levels (Fig. 6A). It should be noted that at later time points not they contain wild-type p53. Therefore, although the activation following MG132 treatment, Mcl-1 levels were decreased, but these of the p53 pathway is a potent mechanism of cell-killing, it does not changes occurred several hours after the induction of apoptosis. seem to be the primary contributor to the overall effects of Mcl-1 degradation at this late stage was probably because Mcl-1 is seliciclib. a target for proteolytic cleavage by caspases (25). Previously, The characteristics of multiple myeloma cells—slow growth and antisense RNA against Mcl-1 has been used to show that Mcl-1 is evasion of apoptosis—make myeloma a particularly difficult essential for the survival of some myeloma cell lines including disease to treat. The seliciclib-induced down-regulation of the RPMI 8226 and LP-1 (22, 23). In this study, siRNAs were used to antiapoptotic protein Mcl-1 seems to overcome the evasion of show the essential role of Mcl-1 for the survival of H929 myeloma programmed cell death in multiple myeloma cells by switching the cells and that specific down-regulation of Mcl-1 induced apoptosis balance towards apoptosis. The use of seliciclib to treat myeloma in the majority of cells. Therefore, the cytotoxic effect of seliciclib in has intriguing possibilities not only as a single agent but also in myeloma cells can be explained by the rapid loss of Mcl-1 following combination with other compounds. This study provides the the inhibition of RNA polymerase II phosphorylation. scientific rationale for a clinical study to investigate the potential Seliciclib treatment lead to an induction of p53 in H929 cells effects of seliciclib for the treatment of multiple myeloma. In (Fig. 3), whereas no such effect was seen in LP-1 and RPMI 8226 addition, this work also identifies antibodies against a number of cells; probably due to the presence of mutant p53 in these cell lines phosphoproteins as potential biomarkers of seliciclib activity that (50). The p53 protein is central to the apoptotic pathway induced directly relate to its mechanism of action. Such biomarkers may be by cellular stress and is tightly regulated by Hdm2 (51), which useful to show the mode of action of seliciclib in vivo using patient serves as an E3 ubiquitin ligase, targeting p53 for proteolytic biopsy samples. degradation. Both Hdm2 mRNA and protein are known to have short half-lives, and like Mcl-1, Hdm2 protein levels were reduced Acknowledgments rapidly by seliciclib treatment. As would be expected, p53 started to accumulate shortly after the loss of Hdm2 (Fig. 3). Roscovitine Received 1/31/2005; revised 3/14/2005; accepted 4/10/2005. The costs of publication of this article were defrayed in part by the payment of page treatment of other cancer cell lines also causes a decrease in Hdm2 charges. This article must therefore be hereby marked advertisement in accordance levels and induction of p53 (11, 12). Furthermore, a decrease in with 18 U.S.C. Section 1734 solely to indicate this fact. We are grateful to Wayne Jackson for in vitro kinase assay results and to Ian Hdm2 and induction of p53 also occurs after flavopiridol (52) or Fleming and other Cyclacel colleagues for insightful discussions and critical reading of DRB treatment (this study). This work and others (8) show that the manuscript.

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David E. MacCallum, Jean Melville, Sheelagh Frame, et al.

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