Leukemia (2011) 25, 411–419 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu ORIGINAL ARTICLE 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.