cyclin-dependent kinase 7/9 inhibitor sns-032 abrogates ......cancer therapy: preclinical...

Cancer Therapy: Preclinical

Cyclin-Dependent Kinase 7/9 Inhibitor SNS-032 AbrogatesFIP1-like-1 Platelet-Derived Growth Factor Receptor a andBcr-Abl Oncogene Addiction in Malignant Hematologic Cells

Yongbin Wu1,3, Chun Chen2, Xiaoyong Sun1,3, Xianping Shi1,3, Bei Jin1,3, Ke Ding4, Sai-Ching Jim Yeung5,6,and Jingxuan Pan1,3

AbstractPurpose: The "gate-keeper" mutations T674I platelet—derived growth factor receptor a (PDGFRa) in

hypereosinophilic syndrome (HES) and T315I Bcr-Abl in chronic myeloid leukemia (CML) are resistant to

imatinib and the second-generation small-molecule tyrosine kinase inhibitors (TKI). However, to combat

acquired resistance to imatinib, an alternative approach is to decrease the expression of the addicted gene to

efficiently kill resistantmalignant hematologic cells. The purpose of this studywas to evaluate the strategy of

shutting down the transcription and expression of FIP1-like-1 (FIP1L1)–PDGFRa and Bcr-Abl with SNS-

032, an inhibitor of cyclin-dependent kinase 7 (CDK7) and CDK9 in phase I clinical trials.

Experimental Design: The effects of SNS-032 on PDGFRa and Bcr-Abl signaling pathways, apoptosis,

and cell cycling were analyzed in TKI-resistant cells of HES and CML. The in vivo antitumor activity of SNS-

032 was assessed with xenografted BaF3-T674I FIP1L1-PDGFRa and KBM5-T315I Bcr-Abl cells in nude

mouse models.

Results: SNS-032 inhibited the phosphorylation on Ser5 and Ser2 of RNA polymerase II. SNS-032

decreased both themRNAandprotein levels of FIP1L1-PDGFRa andBcr-Abl and inhibited the proliferation

of malignant cells expressing FIP1L1-PDGFRa or Bcr-Abl. It also decreased the phosphorylation of

downstream molecules. It induced apoptosis by triggering both the mitochondrial pathway and the death

receptor pathway.

Conclusions: This CDK7/9 inhibitor potently inhibits FIP1L1-PDGFRa–positive HES cells and Bcr-Abl–

positive CML cells regardless of their sensitivity to imatinib. SNS-032 may have potential in treating

hematologicmalignancy by abrogating oncogene addiction.ClinCancer Res; 18(7); 1966–78.�2012AACR.

IntroductionIn "oncogene addiction", malignant cells depend highly

on the continual activation of intracellular signaling by aspecific oncogene despite a multitude of additional carcino-genic genetic changes (1–3). Thus, targeted therapy to inhibit"addicted" oncoproteins has shown some success: small-molecule tyrosine kinase inhibitors (TKI) have been used toblock Bcr-Abl, KIT, and PDGFRa in chronic myeloid leuke-mia (CML), gastrointestinal stromal tumors (GIST), andhypereosinophilic syndrome (HES), respectively (2, 4–6).

The importance of platelet-derived growth factor receptora (PDGFRa) inHES and Bcr-Abl in CML are good examplesof oncogene addiction (7, 8), but the malignant cells canadapt to the challenge of TKIs, and acquired resistance toimatinib is an emergent problem (9, 10). Mutation in thekinase domain of PDGFRa or Bcr-Abl is a common mech-anism of resistance to imatinib. Particularly, the "gate-keeper" mutations T674I PDGFRa in HES and T315I Bcr-Abl in CML, which are analogous to T670I KIT in GISTs, areresistant to the second-generation TKIs (e.g., nilotinib,dasatinib, and midostaurin; refs. 2, 7, 11). Recently, thethird-generation TKIs (e.g., AP24534, HG-7-85-1, and

Authors' Affiliations: 1Department of Pathophysiology, ZhongshanSchool of Medicine, 2Department of Pediatrics, Sun Yat-Sen Memo-rial Hospital, Sun Yat-Sen University; 3Key Laboratory of TropicalDisease Control (Sun Yat-Sen University), Ministry of Education,Guangzhou; 4Key Laboratory of Regenerative Biology and Instituteof Chemical Biology, Guangzhou Institute of Biomedicine and Health,Chinese Academy of Sciences, Beijing, PR China; and Departmentsof 5Emergency Medicine and 6Endocrine Neoplasia and HormonalDisorders, The University of Texas MD Anderson Cancer Center,Houston, Texas

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Y. Wu, C. Chen, and X. Sun contributed equally to this work.

Corresponding Authors: Jingxuan Pan, Department of Pathophysiol-ogy, Zhongshan School of Medicine, Sun Yat-Sen University, 74Zhongshan Road II, Guangzhou 510089, PR China. Phone: 86-20-87332788; Fax: 86-20-87332788; E-mail: [email protected] orSai-Ching Jim Yeung, Department of Emergency Medicine & Depart-ment of Endocrine Neoplasia and Hormonal Disorders, The Universityof Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Unit1468, Houston, TX 77030. Phone: 713-745-9911; Fax: 713-792-8743;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-1971

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(7) April 1, 20121966

on August 26, 2021. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 23, 2012; DOI: 10.1158/1078-0432.CCR-11-1971

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DCC-2036; refs. 12–15) targeting these "gate-keeper"mutants have been developed, but their efficacy and safetyremain to be validated in clinical trials.An alternative strategy to treat malignancies with onco-

gene addiction and TKI resistance has shown promisewithout significant host toxicity: the expression of theoncoprotein PDGFRa and Bcr-Abl suppressed by HSP inhi-bitors (e.g., 17-AAG, IPI-504, and celastrol; refs. 16–19),transcription inhibitors (e.g., flavopiridol and triptolide;refs. 11, 20, 21), or translation inhibitors (e.g., homohar-ringtonine; ref. 22). Despite the cell-wide impact of stop-ping transcription or translation, the relatively fast proteinturnover rate of the "addicted" oncoproteins provides atherapeutic window to selectively kill malignant cells bypulse treatments with these inhibitors (22–24).Cyclin-dependent kinases (CDK) regulate cell-cycle pro-

gression, and a potent inhibitor of CDK2, 7, and 9 beinginvestigated for treating hematologic malignancies is SNS-032 (also known as BMS-387032; ref. 25). SNS-032 inhibitsCDK7 and 9, thereby blocking the phosphorylation of Ser5and Ser2 of the C-terminal domain of RNA pol II, therebyinhibiting transcriptional initiation and elongation (26,27).Both HES and CML are myeloproliferative diseases

(9, 28). HES is characterized by symptomatic persistenteosinophilia (>1,500 eosinophils/mL in the peripheralblood for >6months) with target organ damage (e.g., heart,lung, and skin; refs. 29, 30), and CML is characterized byexpansion of immature granulocyte filled in peripheralblood, bone marrow, and spleen (15, 31). In this study,we aimed to evaluate the antineoplastic activity of SNS-032in HES and CML cells and examine whether it abrogates

oncogene addiction by inhibiting RNA synthesis. SNS-032,at nanomolar concentrations, inhibited the expression ofFIP1-like-1 (FIP1L1)–PDGFRa and Bcr-Abl. SNS-032inhibited cell cycling and growth and induced apoptosisboth in wild-type andmutant malignant hematologic cells.We assessed the in vivo effect of SNS-032 in nude mousexenograft models. Abrogating oncogene addiction by inhi-biting RNA synthesis is at least one mechanism of action ofSNS-032 in addition to inhibiting cell cycling in the FIP1L1-PDGFRa–positive HES cells and the Bcr-Abl–positive CMLcells, regardless of resistance to imatinib.

Materials and MethodsReagents

SNS-032 synthesized in our laboratory was dissolved indimethyl sulfoxide (DMSO) for a 20 mmol/L stock solu-tion, which was frozen in aliquots and stored at �20�C.Imatinib was a product of Novartis Pharmaceuticals (22).Caspase-3 inhibitor benzyloxycarbonyl-Asp (OMe)-Glu(OMe)-Val-Asp (OMe)-fluoromethylketone (z-DEVD-fmk)was obtained from BD Biosciences. MG132 was purchasedfrom EMD Biosciences. Antibodies against PARP, Bcl-2,X-linked inhibitor of apoptosis protein (XIAP), active cas-pase-3, and cytochrome c (clone 6H2.B4) were from BDBiosciences Pharmingen; rabbit polyclonal antibodiesagainst c-Abl (C-19), apoptosis-inducing factor (AIF), Bax,Bcl-XL, and Mcl-1 (S-19) were from Santa Cruz Biotechnol-ogy; anti-Bim was from Stressgen; antibodies against phos-pho-PDGFRa (Y1018), phospho-c-Abl (Y245), caspase-3,-8, -9, phospho-Erk1/2 (T202/Y204), extracellular signal—regulated kinase (Erk) 1/2, phospho-Akt (Ser473), and Aktwere from Cell Signaling Technology; anti-survivin wasfrom Novus Biologicals; rabbit anti-human antibodiesagainst RNA polymerase II (pol II), phospho-RNA Pol II(Ser2), and phospho-RNA Pol II (Ser5) were from BethylLaboratories; antibodies against phospho-STAT3 (Y705),anti-STAT3, phospho-STAT5 (Y694/Y699; clone 8-5-2),anti-STAT5, and anti-PDGFRa were from Upstate Technol-ogy; cycloheximide and anti-actin were from Sigma-Aldrich; and anti-mouse immunoglobulin G and anti-rab-bit immunoglobulin G horseradish peroxidase–conjugatedantibodies were from Pierce Biotechnology (20, 22, 32).

Cell cultureThe EOL-1 cell line, which carries the FIP1L1-PDGFRa

fusion oncogene (33), and BaF3 cells expressing wild-typeor T674I FIP1L1-PDGFRa were previously described(8, 20). Cells were cultured in RPMI-1640 medium (Invi-trogen) supplemented with 10% fetal calf serum (KibbutzBeit). The KBM5 cells expressing 210-kDa wild-type(KBM5) or T315I Bcr-Abl (KBM5-T315I) were cultured aspreviously described (32). Cells are incubated at 37�C andin water vapor–saturated air with 5% CO2 at one atmo-spheric pressure.

Cell viability assayThe MTS assay (CellTiter 96Aqueous One Solution

reagent; Promega) was used to evaluate cell viability as

Translational RelevanceResistance to tyrosine kinase inhibitors (TKI) such as

imatinib is an emerging problem for molecular targetedtherapy. The second-generation TKIs (e.g., nilotinib anddasatinib) can overcome resistance caused by mostmutations except the "gate-keeper" mutations [e.g.,T674I platelet-derived growth factor receptor a(PDGFRa) in hypereosinophilic syndrome (HES),T315I Bcr-Abl in chronic myelogenous leukemia(CML)]. An alternative approach for many of theseTKI-resistant mutants is to decrease the expression of an"addicted" oncogene, which ensures continual activa-tion of intracellular signaling, to kill the malignant cells.Here, we report that SNS-032 [an inhibitor of cyclin-dependent kinase 7 (CDK7) and CDK9] decreased boththe mRNA and protein levels of FIP1-like 1 (FIP1L1)–PDGFRa and Bcr-Abl, abrogated their downstream sig-naling, respectively, and inhibited the growth of HESand CML cells. The CDK inhibitor SNS-032 may havepotential for treating FIP1L1-PDGFRa–positive HES orCML regardless of their sensitivity to imatinib by abro-gating oncogene addiction.

Imatinib-Resistant Cells Are Responsive to SNS-032

www.aacrjournals.org Clin Cancer Res; 18(7) April 1, 2012 1967




describedpreviously (22, 32). Briefly, 2�105 cells permL in100 mL were exposed to various concentrations of SNS-032for 72 hours. Control cells received DMSO for a finalconcentration the same as the highest concentration ofSNS-032 but less than 0.1% v/v. Twenty microliters of theMTS solution per well was added 4 hours before culturetermination. The absorbance was read on a 96-well platereader at wavelength 490 nm.

Cell-cycle analysis by flow cytometryAfter drug treatment, cells were collected, fixed, and

stained with propidium iodide (PI; Sigma-Aldrich) accord-ing to standard protocols. Cell-cycle distribution was deter-mined by use of a FACSCalibur flow cytometer equippedwith CellQuest Pro software (Becton Dickinson;refs. 22, 32).

Measurement of apoptosis by flow cytometryApoptosis was measured by use of Annexin V-FITC (fluo-

rescein isothiocyanate; for EOL-1, KBM5) or Annexin V-phycoerythrin (PE; for PDGFRa-positive BaF3 cells) apo-ptosis detection kits (BD Biosciences Pharmingen) andanalyzed with use of a FACSCalibur flow cytometer andCellQuest Pro software as described (22, 32).

RNA isolation and real-time quantitative RT-PCRFive micrograms of total RNA extracted with TRIzol

(Invitrogen) was used for reverse transcription with theRNA PCR first-strand SuperScript Kit (Invitrogen). Then,50 ng of total cDNAwas used for real-time quantitative PCR(qRT-PCR) with the SYBR Premix Ex Taq Kit (TaKaRa). EachPCR reactionwas carried out in a 20-mL volumeon a96-welloptical reaction plate in Roche LightCycler 480. The relativegene expression was analyzed by the comparative Ct meth-od with 18S ribosomal RNA used as an endogenous controlafter confirming that the efficiencies of the target and theendogenous control amplifications were approximatelyequal.

The specific primers for real-time PCR were FIP1L1-PDGFRa forward, 50-GGG CAA ATG AGA ACA GCAACA-30 and reverse, 50-CAA GAC CCG ACC AAG CAC TAGT-30; Bcr-Abl forward, 50-AAGCGCAACAAGCCCACTGTCTAT-30 and reverse, 50-CTT CGT CTG AGA TAC TGG ATTCCT-30;Mcl-1 forward, 50-GAAAGCTGCATCGAACCATT-30 and reverse, 50-ACA TTC CTG ATG CCA CCT TC-30; and18S forward, 50-AAA CGG CTA CCA CAT CCA AG-30 andreverse, 50-CCT CCA ATG GAT CCT CGT TA-30.

Western blot analysisWestern blot analysis, except for AIF and cytochrome c

release detection, involved whole-cell lysates prepared inradioimmunoprecipitation assay (RIPA) buffer (1 � PBS,1% w/v NP-40, 0.5% w/v sodium deoxycholate, 0.1% w/vSDS) supplemented with stock solutions to final concen-trations of 10mmol/L b-glycerophosphate, 1mmol/L sodi-um orthovanadate, 10 mmol/L NaF, 1 mmol/L phenyl-methylsulfonyl fluoride, and 1� Roche Complete ProteaseInhibitor Cocktail (Roche; ref. 32). The cytosolic fraction

was prepared with digitonin extraction buffer [10 mmol/LPIPES (pH 6.8), 0.015% (w/v) digitonin, 300 mmol/Lsucrose, 100 mmol/L NaCl, 3 mmol/L MgCl2, 5 mmol/LEDTA, and 1 mmol/L phenylmethylsulfonyl fluoride] asdescribed previously to evaluate the level of AIF and cyto-chrome c in the cytosol (34).

Clonogenicity assayBaF3 cells expressingwild-type or T674I FIP1L1-PDGFRa

(2 � 105/mL) or KBM5 cells expressing wild-type or T315IBcr-Abl were treated with increasing concentrations of SNS-032 or diluent (DMSO, control) for 24 hours and washedwith PBS, then seeded in methylcellulose media (Cat#03231; Stem Cell Technologies) for mouse cells (BaF3) orIscove’s medium containing 0.3% agar and 20% fetal calfserum (KBM5) in the absence of drug treatment, respec-tively. After incubation for approximately 7days at 37�C in atissue culture chamber, colonies of 50 or more cells werecounted under an inverted phase-contrast microscope.

siRNA transfectionON-TARGET plus SMART pool siRNA duplexes against

humanMcl-1 andON-TARGET plus Non-Targeting siRNAs(mock siRNA; cat# D-001810-0X) were synthesized byDharmacon RNA Tech. Transfection of anti-Mcl-1 or thecontrol siRNA duplexes into EOL-1 cells involved the CellLine Nucleofector Kit T (Amaxa) and programO-17 accord-ing to the manufacturer’s instructions (20).

Nude mouse xenograft modelNude nu/nu BALB/c mice were bred at the animal facility

of Sun Yat-Sen University, Guangzhou, China. The micewere housed in barrier facilities with a 12-hour light–darkcycle, with food and water available ad libitum. A mixture of1 � 107 of BaF3-T674I cells with Matrigel (BD BiosciencesPharmingen) or KBM5-T315I cells (3 � 107) were inocu-lated subcutaneously on the flanks of 4- to 6-week-oldmalenude mice (17). Tumors were measured every other daywith use of calipers. Tumor volumes were calculated by thefollowing formula: a2 � b � 0.4, where a is the smallestdiameter and b is the diameter perpendicular to a. Four daysafter subcutaneous inoculation, when tumors were palpa-ble (�100 mm3), mice were randomized to receive treat-ment with vehicle (tissue culture medium containingDMSO 0.1% v/v) or SNS-032 (15 mg/kg injected intraper-itoneally every 2 days) for about 2 weeks. SNS-032 wasdissolved in tissue culture gradeDMSObefore dilution. Thebody weight, feeding behavior, and motor activity of eachanimal were monitored as indicators of general health. Theanimals were then euthanized, and tumor xenografts wereimmediately removed, weighed, stored, and fixed. All ani-mal studies were conducted with the approval of the SunYat-Sen University Institutional Animal Care and UseCommittee.

Immunohistochemical stainingFormalin-fixed xenografts were embedded in paraffin

and sectioned. Tumor xenograft sections (4 mm) were

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Clin Cancer Res; 18(7) April 1, 2012 Clinical Cancer Research1968




immunostained by use of the MaxVision Kit (Maixin Bio).After staining with the primary antibodies of rabbit anti-human Ki67 or c-Abl, MaxVision reagent (50 mL) wasapplied to each slide (17). Color was developed with0.05% diaminobenzidine and 0.03% H2O2 in 50 mmol/L Tris-HCl (pH7.6), and the slideswere counterstainedwithhematoxylin. A negative control for Ki67 was included foreach xenograft specimen by substituting the primary anti-body with pre-immune goat serum.

Statistical analysisAll experiments were carried out at least 3 times, and

results are expressed as mean � 95% confidence intervals(CI) unless otherwise stated. Comparisons between 2groups were analyzed by t test and between more than 2groups by ANOVA with post hoc comparison by Tukey test.GraphPad Prism 5.0 (GraphPad Software) was used forstatistical analysis. P < 0.05 was considered statisticallysignificant.

Figure 1. SNS-032 inhibits theexpression of PDGFRa andBcr-Abl at transcriptional level. A,EOL-1 cells and BaF3 cellsexpressing wild-type (WT) or T674IFIP1L1-PDGFRa were incubatedwith indicated concentrations ofSNS-032 for 24 hours (top) or a fixedconcentration for different durations(bottom); the phosphorylated andtotal levels of RNA pol II, PDGFRa,CDK7, and 9 were analyzed byimmunoblotting. B, KBM5 or KBM5-T315I cells were incubated withindicated concentrations of SNS-032 for 24 hours; the phosphorylatedand total levels of indicated proteinswere analyzed by immunoblotting. C,SNS-032 decreases mRNA levels ofPDGFRa and Bcr-Abl concentrationdependently. EOL-1, KBM5, andKBM5-T315I cells were incubatedwith SNS-032 at the indicatedconcentrations for 12 hours,respectively; quantitative RT-PCRanalysis of mRNA level of PDGFRaand Bcr-Abl was conducted. D,quantitative RT-PCR analysis ofmRNA level of PDGFRa in EOL-1cells treated with SNS-032 at 75nmol/L for different durations. ��,P<0.0001 compared with control. Datawere analyzed by ANOVA with posthoc comparison by Tukey test. Errorbars, SD.






ResultsSNS-032 downregulates mRNA of PDGFRa and Bcr-Abl

Because SNS-032 is a selective inhibitor of CDK7 and 9,consequently disabling RNA pol II and gene transcription(35, 36), we first examined its effect on the expression of theaddicted genes FIP1L1-PDGFRa (HES) and Bcr-Abl (CML).ExposingHES cells to increasing concentrations of SNS-032or a fixed concentration of SNS-032 for various durationsled to a concentration- and time-dependent decrease inphosphorylated RNA pol II at Ser2 and Ser5 (Fig. 1A) butnot total protein. Similar results were obtained in CML cells(Fig. 1B). These findings are in accordance with the mech-anism of action of SNS-032 inhibiting the activity of RNApol II (27, 35, 37) by competingwithATP-binding pocket toCDK7 and 9 (35, 36). The levels of total and phosphory-lated PDGFRa and Bcr-Abl were decreased concentration

and time dependently (Fig. 1A and B). qRT-PCR revealedthat SNS-032 decreased the mRNA levels of PDGFRa andBcr-Abl concentration dependently in EOL-1, KBM5, andKBM5-T315I cells (Fig. 1C). Time course experimentsshowed that PDGFRa mRNA decreased after 3 hours andcontinued to decrease with 75 nmol/L SNS-032 (Fig. 1D).Therefore, SNS-032 treatment led to downregulated mRNAof PDGFRa and Bcr-Abl.

SNS-032 blocks FIP1L1-PDGFRa and Bcr-Abldownstream signaling

Because of the appreciably decreased levels of phosphor-ylated and total FIP1L1-PDGFRa and Bcr-Abl after SNS-032treatment, we further examined their downstream signal-ing. SNS-032 concentration and time dependentlydecreased the basal phosphorylation levels of STAT3,

Figure 2. SNS-032 inhibits thedownstream signaling of PDGFRaand Bcr-Abl. Western blot analysisof phosphorylated and total STAT,Erk1/2, and Akt in lysates of EOL-1and BaF3 cells expressing wild-type (WT) or T674I FIP1L1-PDGFRa, KBM5 cells with wild-type or T315I Bcr-Abl treated withincreasing concentrations of SNS-032 for 24 hours (A andC) or a fixedconcentration of SNS-032 fordifferent durations (B).

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Figure3. SNS-032 inhibits thegrowthofwild-type (WT)ormutant cells ofHESandCML.A,SNS-032 inhibits thecell viability ofHESandCMLcells. HESorCMLcells were exposed to increasing concentrations of SNS-032 for 72 hours and then underwent MTS assay. Data are mean� 95% confidence intervals from 3independent experiments. B, clonogenicity of BaF3 cells expressingWT or T674I FIP1L1-PDGFRa in methylcellulose and KBM5 cells with WT or T315I Bcr-Abl in soft agar was inhibited by SNS-032 in a concentration-dependent manner. Data are mean � 95% confidence intervals. C, effect of SNS-032 on thedistribution of cell-cycle phases in HES and CML cells. The indicated malignant hematologic cells were exposed to SNS-032 for 12 (EOL-1 cells) or 24 hours(BaF3 and KBM5 cells), respectively, and then analyzed by flow cytometry after staining with propidium iodide.






Figure 4. SNS-032 induces apoptosis in HES cells expressing FIP1L1-PDGFRa. A, EOL-1 cells (for 12 hours, top) and BaF3 cells expressing WT or T674IFIP1L1-PDGFRa (for 24 hours) were treated with SNS-032 at increasing concentrations (middle) or with 800 nmol/L SNS-032 for various durations (bottom),then underwent Annexin V staining for flow cytometry of cell death. Data are mean� 95% confidence intervals from 3 independent experiments. ��, P < 0.01;��, P < 0.0001, ANOVA with post hoc comparison with control by Tukey test. B, immunoblotting of PARP cleavage and caspase-3 activation in whole-celllysates of HES cells treated with SNS-032 at different concentrations and for different durations. C, time course measurement of AIF and cytochromec in cytosolic fraction of EOL-1 (150 nmol/L), BaF3-WT and BaF3-T674I (800 nmol/L) cells treated with SNS-032. D, effect of SNS-032 on apoptosis-relatedproteins. EOL-1 cells and BaF3 cells were treated with the indicated concentrations for 24 hours (top) or for various durations with a fixed concentration ofSNS-032 (150 nmol/L for EOL-1, 800 nmol/L for BaF3, bottom); levels of XIAP, Mcl-1, Bax, Bim, and survivin were examined by immunoblotting inwhole-cell extracts.

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Erk1/2, and Akt inHES cells (Fig. 2A and B). The decrease inphosphorylated STAT3, Erk1/2, and Akt preceded thedecrease in their total protein levels, suggesting that adecrease in the upstream signals from phosphorylatedPDGFRa contributed to the decrease in the downstreamsignals of STAT3, Erk1/2, and Akt. Similarly, we discoveredthat the levels of phosphorylated downstream targets of Bcr-Ablwere downregulated in SNS-032–treatedCML cells (Fig.2C). Thus, SNS-032 abolished the kinase activity of both

PDGFRa and Bcr-Abl and activation of their downstreamtargets.

SNS-032 inhibits the growth of PDGFRa- or Bcr-Abl–positive cells

We next evaluated the effect of SNS-032 on viabilityof HES and CML cells. EOL-1, BaF3-WT, or T674IFIP1L1-PDGFRa cells were incubated with or withoutvarious concentrations of SNS-032 for 72 hours, and cell

Figure 5. SNS-032 induces caspase-3–dependent cleavage of Mcl-1. A, ectopic Mcl-1 expression abrogates SNS-032–induced apoptosis. At 24 hoursafter transfection with pCMV5-flag empty vector or construct encoding Mcl-1, EOL-1 cells were exposed to various concentrations of SNS-032 foranother 12 hours, then Mcl-1 and PARP levels were detected by immunoblotting (left top). Cell viability was evaluated by trypan blue exclusion(left bottom). At 24 hours after siRNA knockdown of human Mcl-1 or mock, EOL-1 cells were exposed to SNS-032 for 3 hours, then underwent Westernblot analysis (right top) and cell death assay (trypan blue exclusion, right bottom). �, P < 0.05; ��, P < 0.01; ��, P < 0.0001, Student t test. B, SNS-032decreases mRNA of Mcl-1 concentration and time dependently. Quantitative RT-PCR of Mcl-1 mRNA levels in EOL-1 cells treated with SNS-032 atthe indicated concentrations for 12 hours (left) or 75 nmol/L for various durations (right). ��, P < 0.0001, ANOVA with post hoc comparison byTukey test. Data are mean � SD. C, SNS-032 accelerated the turnover rate of Mcl-1 protein. EOL-1 cells were exposed to 150 nmol/L SNS-032 for1 hour, treated with cycloheximide (CHX), 5 mg/mL, and then underwent Western blot analysis. D, SNS-032 elicits a proteosome-independentbut caspase-3–dependent cleavage of Mcl-1. EOL-1 cells were treated with 50 nmol/L SNS-032 in the presence (plus pretreatment for 30 minutes)or absence of 500 nmol/L MG132 (left) or 20 mmol/L z-DEVD-fmk (right) for 12 hours, and then underwent Western blot analysis of Mcl-1 proteinlevels.






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viability, as measured by MTS assay, was potently de-creased by SNS-032 in EOL-1 cells, wild-type- and T674IFIP1L1-PDGFRa–expressing BaF3 cells (IC50 ¼ 135, 508,and 417 nmol/L, respectively; Fig. 3A, left). In KBM5or KBM5-T315I cells, MTS assay showed that the IC50

values were 231 and 97 nmol/L, respectively (Fig. 3A,right). In independent experiments, we also assessed theeffect of SNS-032 on clonogenicity of BaF3-WT or T674IFIP1L1-PDGFRa and KBM5 or KBM5-T315I cells. Thesecells were exposed to increasing concentrations of SNS-032 for 24 hours and then assayed for colony formationin the absence of drugs with the methods described inMaterials and Methods. SNS-032 potently decreased thenumber of surviving clonogenic BaF3 and KBM5 cells in adose-dependent manner [IC50 ¼ 617 nmol/L (BaF3-WT),508 nmol/L (BaF3-T674I), 58 nmol/L (KBM5), and 39nmol/L (KBM5-T315I); Fig. 3B].Flow cytometric analysis revealed no significant alter-

ation in cell-cycle distribution in EOL-1, BaF3, and KBM5cells treated with various concentrations of SNS-032 for 24hours, except for the appearance of a sub-G1 apoptoticpopulation (Fig. 3C). Therefore, SNS-032 potently inhib-ited the growth of EOL-1, T674I FIP1L1-PDGFRa–expres-sing BaF3, and KBM5-T315I cells without significantlyaffecting the cell cycle.

SNS-032 induces apoptosis in HES and CML cellsTo confirm the capability of SNS-032 to induce

apoptosis, control and SNS-032–treated HES andCML cells were assessed by flow cytometry after stainingwith Annexin V and PI or 7-AAD. After 12 hours of 150nmol/L SNS-032 treatment, the population of apop-totic (Annexin V–positive) EOL-1 cells reached to about85% (Fig. 4A, top). SNS-032 induced a substantialapoptosis in BaF3-WT and BaF3-T674I in a concentra-tion- and time-dependent manner (Fig. 4A, middle andbottom).Similarly, the population of apoptotic cells was

about 55% (KBM5) and 61% (KBM5-T315I) after in-cubating with 250 nmol/L SNS-032 for 24 hours(Supplementary Fig. S1A). As well, SNS-032 induced aconcentration- and time-dependent specific cleavage ofPARP (Fig. 4B) and caspase-8, -9, and -3 in HES cells.Similar results were also obtained in CML cells (Sup-plementary Fig. S1B). These data supported the occur-rence of apoptosis in these malignant hematologic cellsafter SNS-032 treatment.

SNS-032 leads to release of cytochrome c anddownregulation of Mcl-1

Mitochondrial depolarization is an early event during theonset of apoptosis.We therefore tested the release ofAIF andcytochrome c from mitochondria into the cytosol. In bothHES and CML cells exposed to SNS-032 for various dura-tions, SNS-032 induced a substantial time-dependentrelease of AIF and cytochrome c into cytosol (Fig. 4C andSupplementary Fig. S1C), which implies that SNS-032triggered the intrinsic (mitochondrial) pathway ofapoptosis.

To elucidate the mechanism of SNS-032–induced apo-ptosis, we evaluated the expression of apoptosis-relatedproteins. Western blot analysis in HES cells revealed asubstantial decrease in levels of Mcl-1 in a concentration-and time-dependent manner, with no alteration in levels ofBax, Bim, and survivin (Fig. 4D). The Mcl-1 protein levelswere downregulated in CML cells as well (SupplementaryFig. S1D). An intriguing feature of SNS-032–inducedchange in Mcl-1 was the conversion of its precursor (42kDa) to a28-kDaproapoptotic fragment associatedwith theonset of apoptosis in EOL-1 cells (Fig. 4D, left).

Mcl-1 is a critical regulator in SNS-032–mediatedapoptosis in HES cells

To examine the relative contribution of Mcl-1 in SNS-032–induced apoptosis, EOL-1 cells transfected with emptyvector or the Mcl-1 plasmid were exposed to various con-centrations of SNS-032 for 12 hours. The cells transfectedwith empty vector underwent extensive apoptosiswith SNS-032 exposure, whereas Mcl-1–transfected cells showed noincrease in apoptosis with SNS-032 exposure, as evaluatedby specific cleavage of PARP and trypan blue dye exclusion(Fig. 5A, left). On the other hand, siRNA silencing of Mcl-1significantly potentiated the capability of SNS-032 toinduce apoptosis (Fig. 5A, right). Of note, silencing Mcl-1 in the absence of SNS-032only slightly induced apoptosis.Therefore, Mcl-1 is a critical factor in SNS-032–mediatedapoptosis in HES cells.

SNS-032 leads to Mcl-1 downregulation by inhibitingits transcription and inducing its cleavage by caspase-3activation

We next investigated the mechanism of Mcl-1 down-regulation in response to SNS-032 treatment by qRT-PCR.The mRNA level of Mcl-1 was decreased concentration andtime dependently (Fig. 5B), which suggests that SNS-032

Figure 6. SNS-032 abrogates the growth of tumors transplanted in nudemicewith downregulation of T674I PDGFRa and T315I-Bcr-Abl. A, nudemice bearingBaF3-T674I FIP1L1-PDGFRa or KBM5-T315I xenograft tumors were treated with vehicle or SNS-032 (15 mg/kg intraperitoneally every 2 days; BaF3), orvehicle, imatinib (50mg/kg, administered by gavage daily, served as a control) or SNS-032 (15mg/kg intraperitoneally every 2 days; KBM5-T315I) for about 2weeks after inoculation. The estimated tumor sizes were plotted against the number of days after SNS-032 treatment began. ��, P < 0.01; ��, P < 0.0001,Student t test, compared between vehicle-treated animals andSNS-032–treated ones at the same time points. Data aremean� 95%confidence intervals. B,after SNS-032 treatment, xenografts were dissected, photographed, andweighed. Representative tumors removed frommice of each group are shown (top).Data (bottom) show the mean weight of tumors from each group (n ¼ 8 for BaF3-T674I xenografts, n ¼ 6 for KBM5-T315I xenografts). ��, P < 0.0001,Student t test (left) or ANOVA with post hoc comparison by Tukey test (right). Data are mean � 95% confidence intervals. C, immunoblotting ofphosphorylation of RNA pol II, PDGFRa, Bcr-Abl, and the downstream molecules in xenograft tissues after inoculation and SNS-032 treatment. D,immunohistochemical analysis of Ki67 in BaF3-T674I xenograft tissues from mice was conducted (left). Both Ki67 and anti-c-Abl in KBM5-T315I xenografttissues were analyzed (right). Hematoxylin and eosin (H&E)–stained serial sections of the same xenografts are shown.






inhibitsMcl-1 at the transcription level. Because the half-lifeof Mcl-1 protein is relatively short (8), we assessed whetherSNS-032 affected the turnover rate of Mcl-1 protein. Weincubated EOL-1 cells with 100 nmol/L SNS-032 for 1 hourand then added the translation inhibitor cycloheximide.Western blot analysis revealed a progressive decrease inMcl-1 protein levelwith cycloheximide treatment in control cellsnot treated with SNS-032 (Fig. 5C), and treatment withSNS-032 accelerated the decrease in Mcl-1 level, whichsuggests that SNS-032 accelerated the turnover of Mcl-1protein in the absence of de novo protein synthesis.

We next examined whether the ubiquitin/proteasomepathwaywas involved in the increased turnover ofMcl-1 afterSNS-032 treatment. Pharmacologic inhibition of protea-somes by MG132 pretreatment (30 minutes) and continualtreatment did not abrogate the SNS-032–induced decreasein Mcl-1 level (Fig. 5D, left), indicating that downregulationof Mcl-1 does not depend on proteasome pathway.

Early reports showed that activation of caspase-3 bychemotherapeutic agents led to cleavage ofMcl-1 atAsp127,thus resulting in 2 fragments, 28-kDa Mcl-1128–350 and 17-kDaMcl-11–127 (38). In contrast to the intact form ofMcl-1,Mcl-1128–350 has a proapoptotic function during apoptosis.Because of the time-dependent conversion of intact Mcl-1into the proapoptotic form (Mcl-1128–350, 28 kDa; Fig. 4D,left), we suspected that Mcl-1 might be cleaved by theactivated caspase-3. Pretreatment (30 minutes) and contin-ual treatment with the caspase-3 inhibitor z-DEVD-fmkabrogated the conversion of intact Mcl-1 into Mcl-1128–350 induced by SNS-032 (Fig. 5D, right). The results sug-gested that caspase-3 may be the main effector of theaccelerated decrease in Mcl-1 level in the absence of de novoprotein synthesis with SNS-032 exposure.

SNS-032 inhibits the growthof xenograftedBaF3-T674IFIP1L1-PDGFRa or KBM5-T315I cells in nude mice

We assessed the in vivo effect of SNS-032 with a nudemouse xenograft model. Four days after subcutaneous inoc-ulation of BaF3-T674I cells or KBM5-T315I cells in mice,when tumors were palpable (�100 mm3), the mice wererandomized to receive treatments with vehicle or SNS-032for approximately 2 weeks. The growth curve (the estimatedtumor size calculated by tumor dimension vs. time) wassignificantly inhibited by SNS-032 (Fig. 6A). The meanweight of tumors in the treated groupwas significantly lowerthan that in the control group(P<0.0001; Fig. 6B). Thebodyweights of the mice were stable, with no significant differ-ences between treated and control mice (data not shown).Motor activity and feeding behavior were all normal.

The levels of RNA pol II, PDGFRa, Bcr-Abl, and down-stream molecules were significantly lower in tumor tissuesfrom mice treated with SNS-032 than in control treatment(Fig. 6C), so SNS-032 may abolish the kinase activity ofPDGFRa and Bcr-Abl and activation of its downstreamtargets in vivo. Immunohistochemical analysis with ananti-Ki67 antibody (to measure cell proliferation) revealedthat SNS-032 decreased the proliferation of xenografted

BaF3-T674I and KBM5-T315I cells (Fig. 6D). Immunohis-tochemical analysis with an anti–c-Abl antibody revealedthat the c-Abl immunoreactivity was appreciably decreasedafter treatment with SNS-032 (Fig. 6D, right). Therefore,SNS-032 inhibited both xenografted BaF3-T674I cells andKBM5-T315I cells in vivo.

DiscussionSome cases ofHES feature oncogene addiction of FIP1L1-

PDGFRa, a chimera formed by deletion of an 800-kb regionon chromosome 4q12 (9). Most patients with CML weregenerated by Bcr-Abl, a fusion gene formed by a reciprocalchromosomal translocation t(9;22)(q34;q11) (39–41).Both patients with HES with FIP1L1-PDGFRa addictionand patients with CML generally respond to imatinib.However, acquired resistance to multiple TKIs that is medi-ated by the gatekeeper point mutations (e.g., T674I inFIP1L1-PDGFRa, T315I in Bcr-Abl) presents a challengefor therapy for HES and CML (7, 15).

New generations of TKIs have been designed (i.e., nilo-tinib, PKC412, dasatinib, sunitinib, and sorafenib). Theseinhibitors may have activity against the gatekeeper T674Imutant FIP1L1-PDGFRa (7, 42–45). For nilotinib, its effecton T674I FIP1L1-PDGFRa mutant is controversial. Niloti-nib was found to inhibit BaF3 cells carrying T674I FIP1L1-PDGFRa (IC50 ¼ 376 nmol/L; ref. 46), but another reportsuggested that nilotinib was not active (42). Whether suni-tinib has activity against the T674I mutation is alsounknown. In CML treatment, nilotinib and dasatinib caneffectively inhibit the activity of most point mutations (e.g.,E255K, M351T) but T315I in Bcr-Abl (15, 28, 31).

To search for an alternative therapy for TKI-resistant HESand CML, particularly those harboring T674I and T315I(gatekeeper mutations), we evaluated SNS-032, a potentinhibitor of CDK7 and 9. SNS-032 blocked transcription-enabling phosphorylation of RNA pol II by CDK7 and 9(27). SNS-032was originally designed as a selective, specificCDK inhibitor against CDK2, 7, and 9. The enzymaticactivity assay showed that the IC50 values against CDK2,7, 9 were 38 to 48, 62, and 4 nmol/L, respectively, whereasthe IC50 values against other kinases were more than 1,000nmol/L (36). Kinase-binding profiling analysis showed thatSNS-032 binds with tyrosine kinases PDGFRa and c-Ablwith IC50 value more than 10 mmol/L (44, 47). Takentogether, these reports support that SNS-032 does notdirectly inhibit the kinase activities of PDGFRa and Bcr-Abl. We confirmed the inhibitory effect of SNS-032 ontranscription. In both HES and CML cells, SNS-032 signif-icantly inhibited RNA polymerase level, thus decreasinglevels of FIP1L1-PDGFRa and Bcr-Abl mRNA and proteinregardless of the T674I or T315I mutation status (Fig. 1C).SNS-032 inhibited transcription at the endogenous FIP1L1-PDGFRapromoter inEOL-1 cells and the artificial promoterin the expression plasmid for FIP1L1-PDGFRa in BaF3 cellswith equal potency. SNS-032 decreased the PDGFRa andBcr-Abl protein levels, resulting in loss of kinase activities, asassayed by the phosphorylation of downstream targets.

Wu et al.





Our in vitro and the mouse model results showed thatSNS-032 significantly inhibits the growth of imatinib-resis-tant BaF3-T674I and KBM5-T315I cells. PDGFRa in thesubset of PDGFRa-positive HES and Bcr-Abl in CML aretypical models of oncogene addiction, and our previousreports have shown that silencing PDGFRa in both EOL-1cells and BaF3 cells expressing FIP1L1-PDGFRa after trans-fection with siRNA duplexes induces apoptosis (20). Sim-ilar to triptolide (20), flavopiridol (11), and IPI-504 (18),SNS-032 may impact multiple molecules, but downregula-tion of PDGFRa and Bcr-Abl is likely the major factor toinduce apoptosis in PDGFRa-positive HES cells and CMLcells, respectively.We identified a substantial role of Mcl-1 in SNS-032–

induced apoptosis.Mcl-1 is often overexpressed in leukemiccells, and it protects malignant cells against apoptosisinduced by chemotherapeutic agents such as flavopiridol,triptolide, and sorafenib (11, 20, 24, 40). Lowering Mcl-1level by antisense oligonucleotides or siRNA can increasethe sensitivity of leukemia cells to chemotherapy (41). OurqRT-PCR data showed that SNS-032 inhibited Mcl-1 attranscription levels. Time chase experiments showed thatthe turnover of Mcl-1 was only slightly accelerated andwas not responsible for the substantial decrease in Mcl-1level with SNS-032 treatment. Analysis of posttranslationalprotein stability showed that the decrease in Mcl-1 leveldepended on activated caspase-3 but not the proteasomes.SNS-032 may induce the cleavage of intact Mcl-1(42 kDa) at Asp127, thereby generating the Mcl-1128–350

(28 kDa) fragment to potentiate apoptosis (38, 48). Thus,Mcl-1128–350 generated by activated caspase-3 after SNS-032treatment may provide positive feedback to apoptosis.Downregulation of Mcl-1 by SNS-032 likely contributes toapoptosis of EOL-1 cells. Silencing Mcl-1 by siRNA sensi-tized EOL-1 cells to SNS-032 (Fig. 5A, right); conversely,EOL-1 cells ectopically expressing Mcl-1 cDNA were resis-tant to SNS-032–induced apoptosis (Fig. 5A, left). Takentogether, our results and those of others support a signif-icant role of Mcl-1 in SNS-032–mediated apoptosis inleukemia cells (27). Together, our findings suggested thatMcl-1 mRNA level decreased by SNS-032 treatment may bethe primary reason for the reduction of Mcl-1 protein, andthe caspase-3–mediated Mcl-1 cleavage triggered by apo-

ptosis may accelerate the process by offering a positivefeedback.

In summary, SNS-032, a potent inhibitor of CDK7 and 9,which leads to inhibition of RNA pol II function, potentlyinhibits cell proliferation and induces apoptosis in resis-tant malignant cells expressing PDGFRa or Bcr-Abl. SNS-032 decreases both PDGFRa and Bcr-Abl mRNA levelsand subsequent downstream signaling. It downregulatesMcl-1 and triggers the intrinsic apoptosis pathway. Cas-pase-3–mediated Mcl-1 cleavage may potentiate the SNS-032–induced apoptosis. SNS-032 inhibited the growth ofxenografted cells expressing T674I FIP1L1-PDGFRa andthe xenografted KBM5-T315I cells in nude mice. There-fore, inhibiting transcription with SNS-032 is a strategy totreat HES or CML, including TKI-resistant patients, thatwarrants further clinical investigation.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Wu, J. PanDevelopment of methodology: X. ShiAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): Y. Wu, X. Sun, K. Ding, J. PanAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): C. Chen, S.-C. J. Yeung, J. PanWriting, review, and/or revision of themanuscript: Y.Wu, S.-C. J. Yeung,J. PanStudy supervision: J. PanCarried out experiments: Y. Wu, X. Shi, B. JinReviewed data: C. Chen

Grant SupportThis study was supported by grants from the National Natural Science

Fund for Distinguished Young Scholars (no. 81025021 to J. Pan), theNational Basic Research Program of China (973 Program grant no.2009CB825506 to J. Pan), the Research Foundation of Education Bureauof Guangdong Province, China (grant cxzd1103 to J. Pan), the ResearchFoundation of Guangzhou Bureau of Science and Technology, China (grantto J. Pan), the National Natural Science Fund of China (no. 90713036 toJ. Pan, no. 30772367 to C. Chen), the National Hi-Tech Research andDevelopment Program of China (863 Program grant no. 2008AA02Z420to J. Pan), and the Fundamental Research Funds for the Central Universities(to J. Pan).

The costs of publicationof this articlewere defrayed inpart by thepaymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 1, 2011; revisedDecember 28, 2011; accepted January 26,2012; published OnlineFirst March 23, 2012.

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Wu et al.





2012;18:1966-1978. Published OnlineFirst March 23, 2012.Clin Cancer Res Yongbin Wu, Chun Chen, Xiaoyong Sun, et al. Oncogene Addiction in Malignant Hematologic Cells

and Bcr-AblαFIP1-like-1 Platelet-Derived Growth Factor Receptor Cyclin-Dependent Kinase 7/9 Inhibitor SNS-032 Abrogates

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