The Cyclin-Dependent Kinase Inhibitor SNS-032 Has Single Agent Activity in AML Cells and Is Highly Synergistic with Cytarabine

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The cyclin-dependent kinase inhibitor SNS-032 has single agent activity in AML cells and is highly synergistic with cytarabine

E Walsby1, M Lazenby2, C Pepper2 and AK Burnett2

1Cardiff Experimental Cancer Medicine Centre, Department of Haematology, School of Medicine, Cardiff University, Heath Park, Cardiff, UK and 2Department of Haematology, School of Medicine, Cardiff University, Heath Park, Cardiff, UK

SNS-032 (BMS-387032) is a selective cyclin-dependent kinase of the cell cycle.9 Deregulated progression through the cell (CDK) inhibitor. In this study, we evaluated its effects on cycle is a hallmark of malignant transformation.8,10–12 CDK7 primary acute myeloid leukemia (AML) samples (n ¼ 87). In vitro complexes with cyclin H and is involved in cell-cycle regulation exposure to SNS-032 for 48 h resulted in a mean LD50 of 139±203 nM; Cytarabine (Ara-C) was more than 35 times less as the catalytic subunit of CDK activating kinase, which potent in the same cohort. SNS-032-induced a dose-dependent activates CDKs 1, 2, 4 and 6 by phosphorylation and is essential increase in annexin V staining and caspase-3 activation. At the for mitosis.8,13–15 molecular level, SNS-032 induced a marked dephosphorylation Transcriptional CDKs, including CDK7 and 9, promote of serine 2 and 5 of RNA polymerase (RNA Pol) II and inhibited the initiation and elongation of nascent RNA transcripts.7,10 In the expression of CDK2 and CDK9 and dephosphorylated addition to its role in the cell cycle, CDK7 is an integral CDK7. Furthermore, the combination of SNS-032 and Ara-C showed remarkable synergy that was associated with reduced part of transcription factor II H (TFIIH) transcription factor, mRNA levels of the antiapoptotic genes XIAP, BCL2 and MCL1. which phosphorylates serine 5 in the carboxy terminal domain In conclusion, SNS-032 is effective as a single agent and in of RNA polymerase II (RNA Pol II) to facilitate transcription combination with Ara-C in primary AML blasts. Treatment initiation.16 CDK9, as part of elongation factor P-TEFb, with Ara-C alone significantly induced the transcription of the phosphorylates serine 2 in RNA Pol II, which is required antiapoptotic genes BCL2 and XIAP. In contrast, the combina- for elongation.16 tion of SNS-032 and Ara-C suppressed the transcription of BCL2, XIAP and MCL1. Therefore, the combination of SNS-032 Expression of the CDKs is relatively stable throughout the cell and Ara-C may increase the sensitivity of AML cells to the cycle in normal cells, although the cyclins that they are paired with cytotoxic effects of Ara-C by inhibiting the transcription of fluctuate over the cell cycle.12 Expression of CDKs is often antiapoptotic genes. perturbed in malignancy making them rational targets for therapy.17 Leukemia (2011) 25, 411–419; doi:10.1038/leu.2010.290; The aim of drugs targeting CDKs is to inhibit cell proliferation,9 but published online 7 January 2011 some of these new agents have also been shown to be potent Keywords: acute myeloid leukemia; cyclin-dependent kinases; 12 therapy inducers of apoptosis in cancer cells. New CDK inhibitors, including SNS-032, are more selective and less cytotoxic17,18 and have been shown to prolong stable disease in solid tumors.7 In this study, we investigated the in vitro effects of SNS-032 in primary AML blasts and AML cell lines both as a single agent and Introduction in combination with cytarabine (Ara-C). We demonstrated single- agent activity for SNS-032 and a remarkable degree of synergy The treatment of acute myeloid leukemia (AML) remains with Ara-C in primary cells. Furthermore, we elucidated a putative unsatisfactory and new therapeutic agents are needed. Recent mechanism of function for SNS-032 that could potentially explain developments have included the development of targeted the cytotoxic synergy seen with Ara-C. therapies designed to exploit the molecular abnormalities that frequently occur in certain AML disease subclasses. Furthermore, these targeted therapies can be used in combina- Materials and methods tion with existing therapies with the intention of achieving longer remission periods. However, to date targeted therapies, including FLT3,1,2 VEGF3,4 and aurora kinase5 inhibitors, have Cell culture Bone marrow samples were collected in ethylenediaminete- not delivered a survival advantage in this condition. traacetic acid from newly diagnosed, previously untreated, The cyclin-dependent kinases (CDKs) are a family of highly AML patients with the patients’ informed consent. In some related serine/threonine kinases that require binding to cyclin instances peripheral blood samples were collected instead of partners to become active heterodimeric complexes composed 6 bone marrow. Patient characteristics are shown in Table 1. AML of a catalytic subunit coupled to a regulatory subunit. Some blasts were enriched by density gradient centrifugation using CDKs (CDK2, 4, 6) have a role in cell-cycle regulation by Histopaque (Sigma, Poole, UK) and were subsequently main- regulating the transition between G1/S phase, S-phase progres- 7,8 tained in Roswell Park Memorial Institute medium (RPMI) sion and G2/M transition. In combination with extracellular supplemented with 10% fetal bovine serum. All cultures were signals and checkpoint pathways CDKs enforce tight regulation maintained at 37 1C in a 5% CO2 humidified atmosphere. Cell viability was measured on a Vi-Cell XR (Beckman Coulter, Correspondence: Dr E Walsby, Department of Haematology, School of High Wycombe, UK) and cell numbers in each culture were Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. E-mail: [email protected] monitored by cell counts using the same machine. AML cell 5 Received 22 June 2010; revised 6 October 2010; accepted 15 lines (HL-60, NB4) were maintained between 2 Â 10 and November 2010; published online 7 January 2011 1 Â 106 cells/ml using the culture media described above. The cyclin-dependent kinase inhibitor SNS-032 has single agent activity in AML E Walsby et al 412 Table 1 Patient characteristics assay. LD50 values were calculated using Calcusyn software (Biosoft, Cambridge, UK). Characteristic Number Mean P (%) IC50(nM) Annexin V positivity Sex 0.12 Cells were treated with SNS-032 at concentrations between Male 55 (63) 118.1 (±127.0) 33 and 526 nM, and incubated for 48 h. Annexin V positivity was Female 30 (34) 188.9 (±296.3) F using the Annexin V Apoptosis Detection Kit (Axxora (UK) Ltd., Not available 2 (2) Nottingham, UK) according to the manufacturer’s instructions. Briefly, cells were washed in phosphate-buffered saline (PBS) FAB group and resuspended in the supplied binding buffer containing M0 0 (F) FFcalcium chloride and incubated with fluorescein-labeled M1 23 (26) 211.0 (±283.0) F Annexin V in the dark for 10 min. Untreated samples were also ± F M2 10 (11) 84.4 ( 75.5) prepared. Cells were rewashed with PBS and resuspended in M3 1 (1) 21.8 (±n/a) F M4 11 (13) 91.0 (±108.7) F binding buffer with 1 mg/ml propidium iodide (PI). Data on the M5 13 (15) 37.9 (±36.1) F annexin V and PI positivity and forward and side scatter of the M6 1 (1) 11.3 (±n/a) F cells was collected on a FACS Calibur flow cytometer (Becton M7 0 (0) F Dickinson, Oxford, UK) and analysed using WinMDI Unknown 29 (33) 168.6 (±210.7) F software (http://www.methods.info/software/flow/winmdi.html). LD50 values were calculated using Calcusyn software (Biosoft). Type of AML All experiments were performed in triplicate. De novo 72 (81) F Secondary 12 (15) F MDS 3 (2 RAEB-t) (3) F Caspase-3 activation AML cell lines were incubated in the presence of SNS-032 at FLT3 status 66, 132, 263 and 526 nM for 48 h. Cells were harvested by WT 55 (63) 125.9 (±172.2) 0.27 centrifugation and washed in PBS and incubated for 1 h at 37 1C ITD 25 (29) 182.0 (±274.4) in the presence of PhiPhiLux G1D2 substrate (Calbiochem, Unknown 8 (9) Nottingham, UK). The substrate contains two fluorophores TKD wt 74 (85) 139.9 (±193.7) 0.41 separated by a quenching linker sequence that was cleaved ± TKD mutant 4 (5) 59.68 ( 18.94) by active caspase-3. Once cleaved, the resulting products TKD unknown 9 (10) fluoresced green and were quantified using a FACS Calibur flow cytometer. NPM1 status 0.85 WT 59 (69) 134.6 (±181.1) 0.85 Mutant 26 (30) 143.5 (±253.9) Unknown 1 (1) Cell cycle Following 24 h of treatment with SNS-032 (33, 66 and 132 nM), 6 m 2 1 Â 10 AML cell lines NB4 and HL-60 were harvested in 200 l Years R of cold PBS, resuspended in 1 ml of ice-cold 70% ethanol and 1 Age stored at À20 C. Before analysis by flow cytometry, the stored Median 54.5 F cells were pelleted and resuspended in 40 mg/ml of PI and Mean 53.6 F 100 mg/ml of RNase (DNase-free) in PBS and incubated at 37 1C Range 17–85 for 30 min. Each cell line was tested in triplicate.

WBC 0.001 Median 32.0 Immunoblotting Mean 53.5 P ¼ 0.76 Range 1–254 AML cell lines treated with 0, 131, 263 and 526 nM SNS-032 for 6 h were washed three times in ice-cold PBS, then lyzed by resuspension in lysis buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 1% Igepal (Sigma Aldrich, Poole, UK), 10% glycerol, 10 mM ethylenediaminetetraacetic acid, 20 mM NaF and 3 mM NaVO4) Cytotoxicity assays plus complete protease inhibitors (MiniComplete ethylenedia- In vitro toxicity assays were performed on primary material from minetetraacetic acid-free; Roche, Burgess Hill, UK) for 30 min 87 AML patients over a 48-h period using a (3-(4,5-dimethylthia- at 4 1C followed by centrifugation at 16 000 Â g. The clarified zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- protein lyzates were quantified and equal aliquots were tetrazolium) (MTS) cell proliferation assay (Promega UK Ltd, subjected to electrophoresis using NuPage precast 4–12% Southampton, UK). Only samples with a viability of 490% at Bis-Tris gels (Invitrogen, Paisley, UK) followed by transfer to the start of the assay were included in the analysis. LD50 values nitrocellulose membranes (Invitrogen). Immunoblotting was (the concentration of drug required to kill 50% of the cells) performed with antibodies to CDK2, CDK7, CDK9, phospho- were calculated from the sigmoidal dose-response curves. Cells CDK2, phospho-CDK7 (Abcam, Cambridge, UK), phopsho- 5 (1 Â 10 /well) were treated with SNS-032 (33–4200 nM) and CDK9 (Cell Signaling Technology Inc., New England Biolabs, Ara-C (63–8000 nM) by serial dilution and incubated for 48 h in Hitchin, UK), tubulin (Abcam) and RNA pol II carboxy terminal a final volume of 90 ml. Following the culture period, 20 mlof domain phospho-serine 2 and phospho-serine 5 (Active Motif, MTS reagent was added and incubated for a further 4 h. Rixensart, Belgium). Blots were visualized by chemilumines- The absorbance of the reaction after this time was read by cence (ECL Advance; Amersham Biosciences, Piscataway, NJ, spectrophotometry at 490 nm and the percentage of viable USA), with the resultant images being analyzed using a cells calculated relative to untreated control cells in the same LAS-3000 dark box (Fujifilm UK Ltd, Bedford, UK).

Leukemia The cyclin-dependent kinase inhibitor SNS-032 has single agent activity in AML E Walsby et al 413 Quantitative PCR HL-60 had LD50 values of 191 and 153 nM, respectively, when AML cell lines NB4 and HL-60 treated for 6 h with 131, 263 and tested under the same conditions. There was only a weak 526 nM SNS-032 were harvested for RNA extraction. Untreated correlation between the sensitivity to SNS-032 and Ara-C in the 2 cells and cells treated with Ara-C (250, 500, 1000 nM) and primary AML samples (r ¼ 0.1532; Figure 1b). Furthermore, SNS-032 and Ara-C in combination (ratio 1:1.9) were collected. there was no significant difference between the response to RNA was extracted using the RNeasy kit (Qiagen, Crawley, UK) SNS-032 and FAB group (P ¼ 0.27), age (P ¼ 0.50) or previous including the on column DNase I treatment according to the treatment (P ¼ 0.80), sex (P ¼ 0.12), FLT3 mutation status manufacturers protocol. A measure of 1 mg RNA was reverse (ITD P ¼ 0.27, TKD P ¼ 0.41) or NPM1 status (P ¼ 0.85). In vitro transcribed into complementary DNA in reactions in 5 mM response to SNS-032 did not correlate with white blood count 2 MgCl2,1mM dGTP, 1 mM dATP, 1 mM dTTP, 1 mM dCTP, (WBC) at diagnosis (r ¼ 0.0001, Table 1). The karyotype of 1U/ml RNase inhibitor, 2.5 U/ml MuLV reverse transcriptase, samples is shown in Supplementary Table 1 together with the 2.5 mM random hexamers and 1 Â PCR buffer II (ABI). The respective in vitro LD50 value for SNS-032. reaction conditions for the reverse transcription were 25 1C for 10 min, 42 1C for 30 min and 95 1C for 5 min. Quantitative PCR was performed using a Light Cycler and FastStart DNA Master SNS-032 induces apoptosis in both primary AML SYBR Green I (Roche) according to the manufacturer’s instruc- cells and myeloid cell lines tion using 1 ml complementary DNA per reaction and specific In this study, apoptosis was measured by annexin V and primers for CDK2, CDK7, CDK9, XIAP, MCL1 and BCL2 propidium iodide labeling and was shown to increase in a dose- The expression levels were compared with ABL expression in dependent manner in both primary AML cells and the myeloid the same sample performed concurrently. The primer sequences cell lines NB4 and HL-60 following exposure to SNS-032 for used were: 48 h (Figures 1c and d respectively). Over the same timeframe, ABL forward 50-CGGCTCTCGGAGGAGACGTAGA-30 caspase-3 activation was significantly increased when com- reverse 50-CCCAACCTTTTCGTTGCACTGT-30 pared with untreated cell populations (Figure 1e; Po0.05). CDK2 forward 50-TTGTCAAGCTGCTGGATGTC-30 reverse 50-TGATGAGGGGAAGAGGAATG-30 CDK7 forward 50-GGCCGGACATGTGTAGTCTT-30 SNS-032 results in cell-cycle arrest reverse 50-CATTTTCAGTGCCTGTGTGG-30 Cell-cycle analysis was performed on NB4 and HL-60 cells treated CDK9 forward 50-AAATACGAGAAGCTCGCCAA-30 with SNS-032 for 24 h (Figure 2). NB4 cells showed increased reverse 50-AAGGTCATGCTCGCAGAAGT-30 percentages of cells in the G1 and S phases of the cell cycle while XIAP forward 50-CCATATACCCGAGGAACCCT-30 HL-60 cells showed significant accumulations of cells in G1 phase reverse 50-TTTCCACCACAACAAAAGCA-30 only. When tested by ANOVA the changes in cell-cycle MCL1 forward 50-AAAAGCAAGTGGCAAGAGGA-30 distribution were significant in both cell lines (NB4, P ¼ 0.026; reverse 50-TTAATGAATTCGGCGGGTAA-30 HL-60, P ¼ 0.0002) suggesting that SNS-032 induces a cell-cycle BCL2 forward 50-AAGATTGATGGGATCGTTGC-30 arrest before the induction of apoptosis in these cell lines. It was reverse 50-TGTGCTTTGCATTCTTGGAC-30 impossible to assess the cell-cycle effects of SNS-032 on primary AML blasts, as all the ex vivo samples tested showed greater than 90% of the cells in G0/G1. Synergy determination with Ara-C Synergy between SNS-032 and Ara-C was assessed in primary AML samples (n ¼ 25) using an experimentally determined SNS-032 inhibits the expression of CDK2, and constant molar ratio of SNS-032 and Ara-C (1:1.9). To establish 9 and dephosphorylates CDK7 the most effective concentration range for each agent, dose- The protein levels of CDK2, 7 and 9 were determined by response curves were generated and LD50 values were immunoblotting at time 0 and after 6 h of treatment of NB4 and calculated using non-linear regression analysis. In subsequent HL-60 cell lines with SNS-032. This timepoint was chosen to experiments, cells were treated with serial dilutions of each drug assess whether changes in CDK expression and/or activation individually and with both drugs simultaneously in a fixed molar preceded apoptosis induction in the cell lines. Levels of both ratio. Calcusyn software was used to determine whether CDK2 and CDK9 were decreased following exposure to any synergy existed between the agents using the Chou and SNS-032 relative to the expression of the a-tubulin loading Talalay method.19 It should be noted that the determination of control (Figures 3a and c). In contrast, CDK7 expression was not the optimal molar ratio was constrained by the fact that we altered by treatment of the myeloid cell lines with SNS-032 only considered clinically relevant doses of Ara-C in these relative to the expression of a-tubulin (Figure 3b). Additionally, experiments. the levels of phosphorylated CDK2, 7 and 9 were determined at time 0 and after 6 h of treatment with SNS-032. Phosphorylated CDK7 and CDK9 levels were reduced with increasing doses Results of SNS-032, whereas there was no change in the levels of phosphorylated CDK2. The transcription of CDK2, 7 and 9 in SNS-032 is cytotoxic in myeloid cell lines and NB4 and HL-60 cell lines was also assessed following treatment primary AML samples with SNS-032 for 6 h using quantitative PCR. In keeping The cytotoxic effects of SNS-032 and Ara-C were assessed in with the protein data, CDK2 and CDK9 mRNA expression primary mononuclear cells isolated from AML patients at levels were significantly reduced relative to the housekeeping diagnosis (n ¼ 87) using an MTS assay. Patient characteristics control gene ABL following exposure to SNS-032 (P ¼ 0.022 are shown in Table 1. SNS-032 induced a mean LD50 of and P ¼ 0.015, respectively). In contrast, mRNA expression of 139±203 nM following 48 h exposure (Figure 1a). By compar- CDK7 was not significantly altered (P ¼ 0.94), confirming the ison, Ara-C was more than 35 times less potent in the same post-translational effects of SNS-032 on this target protein cohort (mean LD50 ¼ 4.9±5.0 mM). Myeloid cell lines NB4 and (Figure 3d).

Leukemia The cyclin-dependent kinase inhibitor SNS-032 has single agent activity in AML E Walsby et al 414

Figure 1 Toxicity of SNS-032 to primary AML cells in vitro and myeloid cell lines. Primary AML cells isolated from newly diagnosed patients were treated with (a) SNS-032 or Ara-C for 48 h over a range of concentrations in an MTS assay. The LD50 of each of the 87 patient samples tested is shown. SNS-032 has a mean LD50 of 139.6 nM±202.7 while the mean LD50 for Ara-C was 4880 nM±5020. (b) A small amount of correlation was seen between primary AML samples that responded to Ara-C and SNS-032 in vitro over 48 h. (c) Primary AML cells treated with SNS-032 for 48 h showed dose-dependent increases in annexin V and PI positivity. Myeloid cell lines were also treated with SNS-032 over 48 h and showed increases in (d) annexin V and propidium iodide positivity and (e) activated caspase-3 detected by ﬂow cytometry, which demonstrate that SNS-032 induces apoptosis in myeloid cell lines NB4 and HL-60 in a dose-dependent manner over 48 h.

Figure 2 Cell-cycle analysis was performed by ﬂow cytometry after 24 h treatment of (a) NB4 and (b) HL-60 cell lines with SNS-032 at a range of concentrations. NB4 cells showed that SNS-032 resulted in a G1/S phase block in cell cycle and HL-60 cells became blocked in G1 phase when tested by ANOVA (NB4 P ¼ 0.026, HL-60 P ¼ 0.0002).

SNS-032 dephosphorylates serine 2 and serine 5 of phosphorylation events are dependent on CDK7 and CDK9, RNA polymerase II respectively so we assessed the phosphorylation status of these RNA pol II is phosphorylated at serine 5 by the transcription serine residues following exposure to SNS-032. Primary AML factor TFIIH and at serine 2 integral to P-TEFb.16 These two cells, as well as NB4 and HL-60 cells were treated with SNS-032

Leukemia The cyclin-dependent kinase inhibitor SNS-032 has single agent activity in AML E Walsby et al 415

Figure 3 Immunoblotting was performed on protein extracted from NB4 and HL-60 cells treated with SNS-032 for 6 h. (a) The levels of CDK2, (b) CDK7 and (c) CDK9 expressed in the cells were detected, as well the levels of phopshorylated CDK2, 7 and 9. Expression of a-tubulin was used as a loading control. The expression of CDK2 and 9 decreased with increasing SNS-032 concentration, whereas CDK7 expression was not affected at the transcriptional level. When phosphorylated CDK2, 7 and 9 were examined increasing SNS-032 concentrations decreased the levels of phosphorylated CDK7 and 9 detectable but not phosphorylated CDK2. Expression was quantitated using AIDA Image Analyser v4.19 (Fujiﬁlm) and is shown relative to the tublin control and the untreated control sample. (d) Expression of CDK2, 7 and 9 at the mRNA level was also investigated using quantitative PCR following a 6 h exposure to SNS-032. The level of mRNA detected was compared with the level of expression of the housekeeping gene ABL and with untreated control samples. Expression of CDK 2 and 9 was found to be decreased relative to untreated samples on treatment with SNS-032 but CDK7 mRNA expression was not affected.

Figure 4 Cell extracts from NB4 and HL-60 cells treated for 6 h with SNS-032 were immunoblotted. (a) RNA pol II phosphorylation at serine 2 was eradicated on treatment with SNS-032. (b) RNA pol II phosphorylation at serine 5 was reduced in a concentration-dependent manner by treatment with SNS-032. (c) Tubulin was used as a loading control. These results suggest that SNS-032 via its affects on CDK 7 and CDK9 affects TFIIH function and elongation of nascent RNA transcription. (d) Primary AML samples showing decreases in RNA pol II phosphorylation at serine 2 and serine 5. Protein detection was quantitated and shown relative to tubulin and the untreated control sample. for 6 h and the level of phosphorylation of RNA pol II at the phosphorylation of RNA pol II suggests that SNS-032 effects serine 2 and 5 residues was measured by immunoblotting elongation of transcription of nascent RNA species through the and compared with untreated controls. In NB4 and HL-60 cell downregulation of CDK9 expression. The decrease in RNA pol II lines serine 2 phosphorylation of RNA pol II was completely serine 5 phosphorylation also indicates that SNS-032 affects the inhibited by SNS-032 at all the concentrations used (Figure 4a) function of TFIIH through the dephosphorylation of CDK7. while serine 5 phosphorylation was decreased in a dose- These results are in agreement with a previously published dependent manner (Figure 4b). Importantly, we also demon- report that showed decreased phosphorylation of serine 2 and strated dephosphorylation of RNA pol II at the serine 2 and 5 in serine 5 of RNA pol II after 6 h of treatment with SNS-032 in primary AML cells (Figure 4c). The inhibition of serine 2 primary CLL cells.16

Leukemia The cyclin-dependent kinase inhibitor SNS-032 has single agent activity in AML E Walsby et al 416 SNS-032 shows strong synergy with Ara-C in primary to investigate the effects of these agents both alone and AML cells in combination at the molecular level. In the first instance, Given the putative mechanism of function of SNS-032, and the we assessed the mRNA expression of CDK2, CDK7 and CDK9 lack of strong correlation between SNS-032 and Ara-C, we next following exposure to SNS-032, Ara-C and the combination explored the potential for these agents to synergize with one thereof in the NB4 and HL-60 cell lines. CDK2 and CDK9 another. The optimal molar ratio for the combination of these mRNA expression were significantly downregulated in response agents was determined experimentally using the MTS assay. To to SNS-032 alone and in combination with Ara-C. However, establish the most effective concentration range for each agent, Ara-C alone had no significant effect on CDK2 or CDK9 dose-response curves were generated and LD50 values (the transcription (Figures 6a and c). In contrast, CDK7 mRNA concentration required to kill 50% of the cells) were calculated expression was not significantly altered following treatment with using non-linear (sigmoidal) regression analysis. In subsequent SNS-032 or the combination but was increased by treatment experiments cells were treated with serial dilutions of each drug with Ara-C alone (Figure 6b). Taken together, these data suggest individually and with both drugs simultaneously in a fixed ratio. that Ara-C does not interfere with the mechanisms by which Analysis of the combined drug effects was made using the SNS-032 exerts its cytotoxic effects through inhibition of CDK2 median effect method. Briefly, this involved plotting the dose- and 9 at the mRNA level. However, this does not seem to response curves for each drug and for multiple diluted fixed- provide a rationale for the synergy seen between the two agents. ratio combinations and then calculating the combination index We next focused our attention on genes that are transcrip- (CI). Both agents were used at therapeutically relevant con- tionally regulated by CDK7 and CDK9 in particular we centrations in all of the in vitro assays that is, maximum investigated the transcription of the antiapoptotic genes MCL1, concentrations were below the maximum tolerated/therapeutic XIAP and BCL2. Using quantitative PCR following 6 h of dose. SNS-032 plasma concentrations of 0.3 mM have been treatment of NB4 and HL-60 cells, we measured the level of achieved in B-cell malignancies in phase 1 trials16 while plasma mRNA expression of MCL1, XIAP and BCL2 in response to the concentrations between 1.0 and 50 mM, depending on the agents both alone and in combination. MCL1 expression was dosing regime used, have been previously reported.20 On the variable when treated with SNS-032 (Figure 6d), but we did not basis of an assessment of the CI values, the optimal molar ratio observe the large decreases in expression that other groups for SNS-032:Ara-C was 1:1.9. All 25 primary AML samples have reported in response to CDK inhibitors in CLL and tested showed a very strong synergistic interaction between the osteosarcoma cells.7,8,16,21 However, SNS-032 treatment of two agents with a mean CI of 0.25±0.23 (Figure 5). Synergy the cell lines resulted in significant decreases in both XIAP was also seen in both the NB4 and HL-60 cell lines when (Figure 6e; Po0.0001) and BCL2 (Figure 6f; Po0.0001), which SNS-032 and Ara-C were combined at the same ratio (mean CI is consistent with previous reports of CDK inhibitors in other of NB4 cells 0.56±0.08, mean CI of HL-60 cells 0.08±0.09). diseases.7,8,16,21–23 Importantly, treatment with Ara-C alone significantly increased the transcription of BCL2 and XIAP (P ¼ 0.0002 and P ¼ 0.0028, respectively). Both of these Molecular determination of mechanisms of synergy genes have been implicated in drug resistance mechanisms in Given the strong synergistic interaction between SNS-032 and AML24–27 and so their transcriptional activation may limit the Ara-C, it seemed likely that their primary mechanisms of apoptotic response to Ara-C. Remarkably, the combination of function were distinct from one another. We therefore, set out SNS-032 and Ara-C resulted in decreased MCL1 expression

Figure 5 The existence of synergy between SNS-032 and Ara-C was determined using a ratio of 1 SNS-032 to 1.9 Ara-C and treating 25 primary AML samples to these two agents separately and in combination for 48 h. (a) Dose-response curve for primary AML cells treated with Ara-C, SNS-032 or Ara-C and SNS-032. (b) Median-effect plot for treated AML cells where Fa ¼ fraction affected and Fu ¼ fraction unaffected. (c) A representative example of the CI plot showing CI values of o1 occurred over a wide range of drug concentrations. (d) In all of the samples tested the combination index at the LD50 was less than one, which is indicative of synergy between the agents tested (mean CI ¼ 0.13).

Leukemia The cyclin-dependent kinase inhibitor SNS-032 has single agent activity in AML E Walsby et al 417

Figure 6 The molecular mechanism by which Ara-C and SNS-032 function in synergy was investigated at the mRNA level by quantitative PCR following 6-h treatment of NB4 an HL-60 cells. Expression levels of (a) CDK2, (b) CDK7 and (c) CDK9 showed that Ara-C in combination with SNS-032 reduced the expression of CDK2 and CDK9 but not CDK7. This suggests that Ara-C does not interfere with primary effects that SNS-032 exerts on these myeloid cell lines. Additionally the expression of antiapoptotic genes (d) MCL1,(e) XIAP and (f) BCL2 were analysed. All three genes were signiﬁcantly inhibited by the combination of Ara-C and SNS-032 suggesting that suppression antiapoptotic genes may contribute to the high levels of synergy seen between these two agents.

(P ¼ 0.021) and decreased expression of XIAP (Po0.0001) In this study, we showed that SNS-032 modulates the effects and BCL2 (P ¼ 0.0002), providing a rationale for at least one of CDKs at both the transcriptional and post-translational levels. mechanism for the synergy seen with these agents. We CDK2 and CDK9 were inhibited by SNS-032 at the transcrip- hypothesize that SNS-032-mediated inhibition of antiapoptotic tional level while CDK7 was prevented from being activated genes would increase the sensitivity of AML cells to the effects of through the inhibition of protein phosphorylation. These results Ara-C in the combination thereby inducing cytotoxic synergy. are similar to those previously reported data in CLL cells.16 Furthermore, we investigated the downstream effects of CDK7 and CDK9 inhibition on phosphorylation of RNA pol II at serine Discussion 5 and 2, respectively. We showed dephosphorylation of serine 2 at all the drug concentrations used and a concentration- AML remains a difficult disease to treat and survival times dependent dephosphorylation at serine 5 using the same remain disappointingly short.28–30 CDKs have an essential role concentrations. Crucially, these results were recapitulated in in cell proliferation and their potential as molecular targets for primary AML cells demonstrating that these effects were not anti-cancer therapies has becoming increasingly recognised.31 confined to the two cell lines used in this study. As the development of the pan CDK inhibitor flavopiridol Importantly, the combination of SNS-032 with Ara-C demon- more specific CDK inhibitors have been developed32–34 with strated a remarkable degree of cytotoxic synergy. CDK encouraging results. In this study, we present our findings on the inhibitors, specifically flavopiridol, have previously been CDK inhibitor SNS-032 in primary AML cells both alone and in suggested to have the capacity to be enhancers of cytotoxic combination with Ara-C. chemotherapy, potentially through CDK9 inhibition resulting SNS-032 resulted in in vitro cell killing, at a sub-micromolar in inhibition of RAD51, a DNA repair protein involved in level, in samples derived from 87 newly diagnosed AML homologous recombination.17 The same study suggested that patients. Additionally, we used the myeloid cell lines NB4 and hematological malignancies might be more sensitive to cell- HL-60 to demonstrate that SNS-032 results in apoptosis as cycle blockade and apoptosis induction than solid tumors. It is evidenced by increased annexin V and PI staining and elevated worthy of note here that the vast majority of primary AML cells levels of caspase-3 activation. Although these cell lines do not show little evidence of cell-cycle activity in vitro so the principal reflect the heterogeneity of the AML subgroups, they are cytotoxic effects of SNS-032 are likely to be caused by informative about many aspects of the responses of AML cells transcriptional repression rather than cell-cycle arrest in our in to new agents.35 Previously SNS-032 has been shown to vitro assays. In this regard, the antiapoptotic genes BCL2 and have cell killing activity in glioblastoma18 and CLL.16 In the XIAP were significantly downregulated by SNS-032 alone and CLL study SNS-032 activity was found to be selective for CLL in combination with Ara-C, under which conditions MCL1 cells compared with non-malignant cells and its efficacy was expression was also inhibited. This pattern of inhibition of not affected by patient characteristics, p53 status or fluda- antiapoptotic genes has also been observed in response rabine resistance making it potentially useful in refractory AML to flavopiridol, seliciclib and SNS-032 in CLL cells7,16 and in patients too. osteosarcoma cell lines.8 Reduction of antiapoptotic proteins

Leukemia The cyclin-dependent kinase inhibitor SNS-032 has single agent activity in AML E Walsby et al 418 in response to treatment with CDK inhibitors may be adequate 9 Maude SL, Enders GH. Cdk inhibition in human cells compromises to induce significant cell death in some instances. Furthermore, chk1 function and activates a DNA damage response. Cancer Res their inhibition may sensitize cancer cells to other apoptotic 2005; 65: 780–786. stimuli resulting from DNA damage.7 Previous studies have 10 Cai D, Latham Jr VM, Zhang X, Shapiro GI. Combined depletion of cell cycle and transcriptional cyclin-dependent kinase activities shown that these elevated expression of these proteins can induces apoptosis in cancer cells. Cancer Res 2006; 66: 9270–9280. inhibit cytotoxic responses to chemotherapeutic agents like 11 Yu C, Rahmani M, Dai Y, Conrad D, Krystal G, Dent P et al. 36–38 Ara-C. In this regard, our data suggests that exposure to The lethal effects of pharmacological cyclin-dependent kinase Ara-C increases the transcription of antiapoptotic genes and this inhibitors in human leukemia cells proceed through a phospha- may be important in the development of drug resistance to this tidylinositol 3-kinase/Akt-dependent process. Cancer Res 2003; agent.38–40 In contrast, the combination of SNS-032 and Ara-C 63: 1822–1833. 12 Grant S, Roberts JD. The use of cyclin-dependent kinase inhibitors significantly suppressed the transcription of these same anti- alone or in combination with established cytotoxic drugs in cancer apoptotic genes. Undoubtedly, the mechanisms of synergy chemotherapy. Drug Resist Updat 2003; 6: 15–26. between these agents are complex. However, the level of 13 Lolli G, Johnson LN. CAK-Cyclin-dependent activating kinase: a suppression of antiapoptotic genes observed with the combina- key kinase in cell cycle control and a target for drugs? 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