cdk9 inhibitor fit-039 suppresses viral oncogenes e6 and e7 … · 039 on cervical neoplasia...

Translational Cancer Mechanisms and Therapy

CDK9 Inhibitor FIT-039 Suppresses ViralOncogenes E6 and E7 and Has a TherapeuticEffect on HPV-Induced NeoplasiaMasahiko Ajiro1,2, Hiroyuki Sakai3, Hiroshi Onogi4, Makoto Yamamoto2,4,Eriko Sumi5, Teruo Sawada2, Takashi Nomura6, Kenji Kabashima6,Takamitsu Hosoya7, and Masatoshi Hagiwara1,2

Abstract

Purpose: Cervical cancer is one of the leading causes ofcancer-related deaths among women worldwide. The purposeof this study is to assess the therapeutic effect of the newlydeveloped cyclin-dependent kinase 9 (CDK9) inhibitor FIT-039 on cervical neoplasia induced by human papillomavirus(HPV) infection.

Experimental Design: We examined FIT-039 for its effecton HPV gene expression in HPVþ cervical cancer cells.Primary keratinocytes monolayer and organotypic raft cul-ture models were used to evaluate HPV viral replication andcervical intraepithelial neoplasia (CIN) phenotypes. Preclin-ical pharmacokinetics and toxicity tests for FIT-039 werealso conducted. Finally, the anti-HPV effect of FIT-039 wasfurther examined in vivo, using HPVþ cervical cancerxenografts.

Results: FIT-039 inhibits HPV replication and expression ofE6 and E7 viral oncogenes, restoring tumor suppressors p53and pRb inHPVþ cervical cancer cells. The therapeutic effect ofFIT-039 was demonstrated in CIN model of an organotypicraft culture, where FIT-039 suppressed HPV18-induced dys-plasia/hyperproliferation with reduction in viral load. FIT-039also repressed growth of HPV16þ, but not HPV� cervicalcancer xenograftswithout any significant adverse effects. Safetyand pharmacokinetics of FIT-039 were confirmed for systemicand topical routes.

Conclusions: The CDK9 inhibitor FIT-039 showed potentanti-HPV activity without significant toxicity in preclinicalstudies. Thus, FIT-039 is expected to be a novel therapeuticfor CIN to prevent cervical cancer. ClinCancer Res; 24(18); 4518–28.�2018 AACR.

IntroductionHuman papillomavirus (HPV) infection is primarily associated

with various benign and malignant tumors, and more than 95%of cervical cancers are associated with the high-risk genotypes,such as HPV16 and HPV18, as well as less prevalent HPV31, 33,45, and 58 (1, 2). Persistent HPV infection induces cervical

intraepithelial neoplasia (CIN), a subset of which subsequentlyprogresses to cervical cancer. HPV mainly remains in episomalform in CIN lesions, and spontaneous HPV clearance in cervix isaccompanied by a regression of CIN, whereas years of persistentinfection could result in irreversible integration of HPV genomeinto host cellular genome, inducing malignant progression (3).Integration of the HPV genome into the host cell genome thenpromotes transcription of two viral oncogenes E6 and E7,required for transformation of host keratinocytes andmalignancyby targeting tumor suppressors (4–6). In the presence of E6protein and its cofactor E6AP, p53 is targeted for ubiquitin-dependent degradation (4–6). The E7 protein, due to its highaffinity to pRb, prevents pRb from complex formation with E2F1,inducing progression to the S-phase (5, 6). Binding of E7 to pRbalso subsequently induces destabilization of pRb (7, 8).

Treatment of HPV-induced neoplasia and cancer represents amajor unmet medical need worldwide. HPV vaccines, Gardasiland Cervarix, were introduced a decade ago, and their prophy-lactic effects have been demonstrated in many trials, and thereduction of infection rate of several types of HPVs is evident incountries proceeding in public HPV vaccination of teenagers (9),although the prophylactic effect of HPV vaccine is considered tobe limited to approximately 90% at most, even by the 9-valentHPV vaccine (10). On the other hand, in many developingcountries, HPV vaccines are still not introduced or only recentlydone into national immunization programs. Incidences of CINand cervical cancer, therefore, are increasing in those countries,especially among women of reproductive ages, which leavescervical cancer still among the leading causes of cancer-related

1Department of Drug Discovery Medicine, Graduate School of Medicine, KyotoUniversity, Kyoto, Japan. 2Department of Anatomy and Developmental Biology,Graduate School of Medicine, Kyoto University, Kyoto, Japan. 3Laboratory ofTumor Viruses, Institute for Frontier Life andMedical Sciences, Kyoto University,Kyoto, Japan. 4KinoPharma Inc., Tokyo, Japan. 5Department of Clinical Inno-vative Medicine, Institute for Advancement of Clinical and Translational Science,Kyoto University Hospital, Kyoto, Japan. 6Department of Dermatology, KyotoUniversity Graduate School of Medicine, Kyoto, Japan. 7Laboratory of ChemicalBioscience, Institute of Biomaterials and Bioengineering, Tokyo Medical andDental University, Tokyo, Japan.

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

Current address for M. Yamamoto: Japan Animal Referral Medical Center Co.,Ltd., Kawasaki, Kanagawa 213-0032, Japan.

Corresponding Author: Masatoshi Hagiwara, Department of Anatomy andDevelopmental Biology/Department of Drug Discovery Medicine, Kyoto Uni-versity Graduate School of Medicine, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 817-5753-4670; Fax: 817-5751-7529; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-17-3119

�2018 American Association for Cancer Research.

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Clin Cancer Res; 24(18) September 15, 20184518

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deaths in the world. The standard care for CIN is cervicalcornification, which, however, increases risk of premature birthand newborn mortality due to a compromised cervix function(11–14) and nearly 10% of recurrence risk by persisting HPVinfection (15, 16), and noninvasive therapeutics has beendemanded for CIN treatment. Thus, in this study, we examinedanti-HPV compound for a therapeutic effect on HPV-inducedCIN and cervical cancer.

The newly developed antiviral drug FIT-039 targets host cellfactor CDK9 and suppresses transcription of various DNA virusessuch as herpes simplex virus, adenovirus, cytomegalovirus, andhepatitis B virus (17–19). In the current study, we demonstratethat FIT-039 suppresses HPV early promoter activity by inhibitingCDK9 activity, leading to reduced expression of HPV oncogenesE6 and E7 in cervical cancer cells. Suppressed viral replication isalso observed in keratinocytes with episomal HPV in monolayercultures and organotypic raft tissues. Moreover, the therapeuticpotentials of FIT-039 against CIN and other HPV-associateddiseases are preclinically verified for further clinical investigation.

Materials and MethodsCell lines

CaSki (HPV16þ cervical cancer), C33A (HPV� cervical cancer,with mutations in p53 and pRb; ref. 20), and HaCaT (HPV�

cervical keratinocytes) cell lines were purchased fromATCC.HeLacells (HPV18þ cervical cancer) were purchased from JCRB CellBank. Primary human foreskin keratinocytes (HFK) and fibro-blasts (HFF)were purchased fromThermoFisher Scientific. CaSki,C33A, HaCaT, HeLa, and HFFs were maintained in 10%FBS/DMEM in a 5% CO2 incubator at 37�C. HFKs were maintainedin KGM EpiLife medium (Thermo Fisher Scientific) in the sameconditions. HFKs with the episomal HPV18 genome (HFK18)were prepared as follows: Full-length HPV18DNA (GenBank no.:X05015) was excised from a plasmid vector, and HPV DNA wasself-ligated with T4 DNA ligase. The circularized HPV DNA andpSV2-neo plasmids were introduced into normal HFKs withTransIT-keratinocyte (Mirus Bio LLC). After the transfection,HFKswere cultured with KGM EpiLife medium containing G418 for 96hours, and then, maintained in KGM EpiLife medium withoutG418. HFK18 was used for the experiment at 10 days after

transfection. All cell lines used in this study were verified by shorttandem repeat analysis (conducted on February 28, 2018), per-formed by Takara Bio Inc. using GenePrint 10 System (Promega)for D21S11, TH01, TPOX, vWA, Amelogenin, CSF1PO,D16S539,D7S820, D13S317, and D5S818. STR results were visualizedusing GeneMapper software (Thermo Fisher Scientific), andshown in Supplementary Fig. S1 and Supplementary Table S1.

AntibodiesThe following antibodies were used for Western blot and IHC

analysis in this study: anti-p53 (DO-1) mouse mAb (Santa CruzBiotechnology), anti-pRb mouse mAb (BD Pharmingen), anti–b-actin (AC-15) mouse mAB (Santa Cruz Biotechnology), anti-CDK9 (D-7) mouse mAB (Santa Cruz Biotechnology), anti-HPV18 L1 mouse mAB (clone 8.F.324, Abcam), goat anti-mouseIgG antibodywith Alexa 488 (Thermo Fisher Scientific), goat anti-mouse IgG with HRP (Abcam), and goat anti-mouse IgG, F(ab')with HRP (Santa Cruz Biotechnology).

WST-8 growth assayThe cells were seeded at a density of 1.0 � 104 cells into a 24-

well plate and treated with 0, 5, 10, or 20 mmol/L FIT-039 inDMEM with 10% FBS and 0.1% DMSO. The FIT-039–containingmediumwas replacedwith freshmediumafter 48hours, followedby further incubation for 48 hours. Then, the cell viability wasquantified using WST-8 growth assay as described previously(21). Briefly, the cells were incubated with 10% WST-8 solution(Nacalai Tesque) in culture medium for 30 minutes, and theabsorbance was measured at 450 nm using a photospectrometer.

RNAiRNAi was conducted with Silencer Select siRNAs (Thermo

Fisher Scientific). Lipofectamine RNAiMAX (Thermo Fisher Sci-entific) was used for the transfection of the following siRNAs:50-GGAGAAUUUUACUGUGUUUdTdT-30 and 50-AAACACAGU-AAAAUUCUCCdTdT-30 for si-CDK9, 50-GAGCUGCAAACAA-CUAUACdTdT-30 and 50-GUAUAGUUGUUUGCAGCUCdTdT-30 for si-HPV16 E6E7 (22), and 50-GGUUUAGGGUAUAUU-CUAUdTdT-30 and 50-ATAGAATATACCCTAAACC-30 for si-NS.

RT-PCRTotal RNA was extracted with TRIzol reagent (Thermo Fisher

Scientific), treated with DNase, and applied to RT-PCR. p53, pRb,and b-actin were detected by the following primer sets: oAM324(50-TGCCAACTGGCCAAGACCTG-30) and oAM325 (50-GTC-ATCCAAATACTCCACACGC-30) for p53, oAM322 (50-GATACCA-GATCATGTCAGAGAG -30) and oAM323 (50-CTGAAGAGTG-CAAACAATACATCA -30) for pRb, and oAM13 (50-CCAACCGC-GAGAAGATGACC-30) and oAM14 (50-AGCTTCTCCTTAATGT-CACG-30) for b-actin. A TaqMan system was used for HPV16E6, HPV16 E6�I/E7, HPV18 E6, and HPV18 E6�I/E7, as describedpreviously (23). TaqMan assay Hs01060665_g1 (Thermo FisherScientific) was used for human b-actin mRNA for an internalcontrol.

Southern blotTotal DNA was extracted from the HFKs, and 2 mg of the total

DNAwas separated using 0.8% agarose gel electrophoresis. Then,they were transferred onto nylon membrane (Hybond Nþ) (GEHealthcare). For the detection of HPV18-specific DNA, DIG-labeling system (Roche Diagnostics) was used. DIG-labeled

Translational Relevance

Cervical cancer accounted for 528,000 new cases and266,000 deaths of cancer worldwide in 2012. More than95% of cervical cancer cases are caused by persistent infectionof high-risk types of human papillomaviruses (HPV). Incervical intraepithelial neoplasia (CIN), a premalignant formof cervical cancer, HPV propagates mostly in episomal form,and years of persistent infection subsequently result in inte-gration of HPV genome into host keratinocyte genome, whichtriggers malignant progression. As current surgical care of CINmay impact patients' future reproductive ability, and hasapproximately 10% of recurrence rate due to persisting infec-tion, a noninvasive anti-HPV therapy has been demanded.FIT-039 is currently evaluated in the phase I/IIa clinical trialfor anti-HPV activity in skin warts. Based on the presenteddata, I/IIa clinical trial of FIT-039 for CIN will start in 2018.

CDK9 Inhibitor FIT-039 for HPV-Induced Neoplasia

www.aacrjournals.org Clin Cancer Res; 24(18) September 15, 2018 4519




probes for the LCR (7000-100 nt, GenBank no. X05015) and L1region (6137–7136 nt, GenBank no. X05015) of HPV18 wereobtained through PCR DIG Probe Synthesis Kit (Roche AppliedScience). The chemiluminescent signal was visualized by a chemi-luminescent image analyzer (LAS-3000, Fuji Film).

Organotypic raft cultureFor organotypic raft culture preparation, one part of type I

collagen, cell matrix type I-P (Nitta Gelatin Co., Ltd.) and twoparts of growth medium containing HFFs (1 � 106 cells) weremixed well and poured into a 6-cm dish, and cells were main-tained in a 5% CO2 incubator at 37�C for gel contraction. The gelwas soaked in KGM for several hours and then transferred into atranswell insert. The insert was placed in a 6-well plate and bothbottom and insert were filled with KGM. HFKs were then overlaidon the gel (day 0). Themediumwas changed to KGM/DMEM, 1:1on day 1, and 24 hours later, the medium was changed to KGM/DMEM with 1.8 mmol/L CaCl2 (day 2). On day 3, the surface ofthe collagen gelwas exposed to air, and themedium in the bottomchamber was replaced with fresh medium daily. Treatment withDMSO or FIT-039 was started on day 3 by adding the compoundto the medium. The treatment was sustained until day 10, fol-lowed by culture without compound for a week. Obtained speci-mens were embedded in optimal cutting temperature (OCT)compound and applied to cryosectioning. The sections weretransferred onto glass slides and dried. Hematoxylin and eosin(H&E) staining was carried out following a standard protocol.

Xenograft tumor assayFemale CB-17/lcr-scid/scid Jcl mice were purchased fromCLEA

Japan and used in the xenograft tumor assay. Briefly, 0.5 � 106

CaSki or C33A cells that stably expressed GLuc were suspended inHank's balanced salt solution (HBSS [þ]) and subcutaneouslyinjected into the right dorsal area of 5-week-old mice. Once thetumormass reached 200mm3, oral administration of FIT-039wasinitiated, and the tumor volume was calculated as (4/3)�p�a�b2 (a,major axis; b, minor axis) every 2 days. On the day 21, the tumormass, dorsal skin, and liver tissues were excised and fixed in 10%neutral-buffered formalin, and plasma FIT-039 concentrationwasdetermined 4 hours after the administration by high-performanceliquid chromatography (HPLC).

DNA in situ hybridizationDetection of HPV18 DNA signals in tissue sections was per-

formed with the TSA Biotin System (PerkinElmer) following themanufacturer's instructions. DIG-labeled DNA for the HPV18LCR region (7000–100 nt; GenBank no. X05015) was used asa probe, and streptavidin fluorescein (PerkinElmer) was used todetect biotin. Nuclei were counterstained using propidium iodide(Sigma-Aldrich).

IHCFor the IHC of xenograft tumor sections, formalin-fixed sec-

tions were deparaffinized with xylene, rehydrated, and treatedwith a Mouse on Mouse blocking reagent (Vector Laboratories).Next, p53was detected using an anti-p53 (DO-1) antibody (SantaCruz Biotechnology) and anti-mouse IgG Alexa-488 (ThermoFisher Scientific), and the nuclei were counterstained withHoechst 33342 (Thermo Fisher Scientific). Detection of HPV18L1 signals in the tissue sectionswas performedwith the TSABiotinSystem (PerkinElmer) following the manufacturer's instructions.

Anti-HPV18 L1 antibody (clone 8.F.324, Abcam) and goat anti-mouse IgG, F(ab')-HRP (Santa Cruz Biotechnology) were used asprimary and secondary antibodies, respectively. Nuclei werecounterstained with propidium iodide (Sigma-Aldrich). Stainedtissue sections were then analyzed by BZ-X710 fluorescencemicroscope (Keyence), and BZ-H3C cell counting software(Keyence).

Pharmacokinetics test for FIT-039[14C]-labeled FIT-039 (7.03 MBq/mg) was synthesized by

Sekisui Medical Co., Ltd. For the pharmacokinetics study byintravenous injection, [14C] FIT-039 (18 mg) and unlabeledFIT-039 (162 mg) were mixed and dissolved in 3.6 mL of DMSO,and then further mixed with 3.6 ml of Kolliphor HS15 (Sigma-Aldrich). One portion of the mixed solution was further sus-pended in 4 portions of saline, resulting in 5 mg/mL of FIT-039solution, which was used for intravenous injection of 7-week-oldmale SD rats [Charles River Laboratories; 5 mg/kg body weight(BW), 1 mL/kg BW, n ¼ 3 for each time point]. The FIT-039concentration in each tissue was determined by measuring radio-activity with a scintillation counter.

Topical administration and tissue autoradiographyof [14C] FIT-039

For cutaneous topical administration, [14C] FIT-039 (7.03MBq/mg, as described above) and unlabeled-FIT-039 (12.5 mgeach) were mixed with 74.5 mg of lactic acid, and suspended in2.4 g of polyethylene glycol tomake a 10mg/g FIT-039 ointment.FIT-039ointmentwas then topically administered ontonormal ordecornified rat skin (2.0 � 3.0 cm) at 0.5 mg/body. Normal anddecornified rats with cutaneous administration of [14C] FIT-039ointment were euthanized 2 hours after the administration, and askin tissue block was prepared withOCT compound. A 5-mmskintissue section was fixed on a nuclear emulsion–coated glass slideand exposed for 2 weeks at 4�C in the dark. Total absorption ratewas determined from the sum of radioactivity from whole body,urine, and feces. For vaginal topical administration of [14C] FIT-039 in rabbits, 10mg of [14C] FIT-039 (7.03MBq/mg) and 42mgof unlabeled-FIT-039 were dissolved with 3.3 g of polyethyleneglycol to prepare 15.5 mg/g FIT-039 ointment. A total of 8.6 mgFIT-039 (500mLof ointment)was topically administered to rabbitcervix.

FIT-039 toxicity testsFor the general toxicity test, FIT-039 in 0.5% methyl cellulose

(MC) (5 ml/(kg BW/day) was administered orally [0, 16.7, 50,and 150mg/(kg BW/day)] to male and female Beagle dogs (n¼ 3for each group, 5–7months old) or SD rats (n¼10 for each group,6 weeks old) for 4 weeks. For the genotoxicity test, CHL/IU,Chinese hamster lung-derived fibroblast-like cells were obtainedfrom ATCC. CHL/IU cells were exposed to FIT-039 in the follow-ing three conditions before chromosomal structure analysis: 1.12.5, 25, and 50 mg/mL FIT-039 for 6 hours, followed by 18 hoursin fresh media: 2. 17.5, 35, and 70 mg/mL FIT-039 for 6 hours inthe presence of S9 mix, followed by 18 in a fresh media, and 3. 5,10, and 20 mg/mL FIT-039 for 24 hours. For each condition, 200mitotic metaphase cells were analyzed by microscopy for alter-ation of chromosomal structure. Frequency of chromosomalalteration was statistically evaluated by Fisher exact test, whereP <0.05was considered statistically significant. For the respiratorytoxicity test, heart rate, tidal volume (VT), minute expiratory

Ajiro et al.

Clin Cancer Res; 24(18) September 15, 2018 Clinical Cancer Research4520




volume (VE), and enhancedpause (Penh)weremeasured at 0.5, 1,2, 4, 6, and 24 hours after oral administration of FIT039 (0, 75,150, or 300mg/kg BW) to 6 to 7 weeks oldmale SD rats (n¼ 6 ineach group). For the cardiovascular toxicity test, FIT-039 in 0.5%MC [5mL/(kg BW/day)] was orally administered [0, 16.7, 50, and150mg/(kg BW/day)] for 4 weeks or single administration (0, 75,150, and 30 mg/kg BW) to 5- to 7-month-old male and femaledogs (n ¼ 3 for each group). Cardiac function was evaluatedbefore and after the FIT-039 administration period by CardiomaxFX-3010 (Fukuda Denshi). For functional observational battery(FOB) test, FIT-039 in 0.5% MC [5 mL/(kg BW/day)] was orallyadministered (0, 250, 500, or 1,000 mg/kg) to male and femaleSD rats (n¼8 for each group, 6weeks old). FOB testwas examinedbefore (0 hour) and after (0.5, 1, 3, 6, and 24 hours) FIT-039administration. Biotoxtech conducted general toxicity, chromo-somal alteration, cardiovascular toxicity, and FOB tests.

Penetration test of FIT039 through skin tissuesSkin tissues from7- to 9-week-oldmale ratswere decornifiedby

taped stripping and resected. Resected skin tissues were thenincubated in receiver solution (40% PEG400) for 1 hour and setup for skin penetration test, so that only the inside surface of theresected skin tissuewas in contactwith the receiver solution. Then,a FIT-039 skin patch (0.1%, 0.3%, 0.5%, 1%, 3%, and 5% FIT-039) was set on the top side of the resected skin. The skin-penetrated FIT-039 present in the receiver solution at 4, 8, 12,16, 20, and 24hours after FIT-039 skin patch applicationwas thenquantified by HPLC.

ResultsFIT-039 suppresses HPV viral gene transcription in cervicalcancer cells

In oncogenic HPVs, E6 and E7 viral oncogenes are polycistro-nically transcribed fromHPV early promoter, and E6 is translatedfrom the unspliced form and E7 from the E6-spliced form (E6�I;refs. 4, 24, 25). Considering the dependence of HPV early pro-moter activity on P-TEFb (26–28), a CDK9–cyclin T complex, weexamined the effect of the CDK9-specific inhibitor FIT-039 (17–19) on HPV early gene transcription in HPV16þ CaSki andHPV18þ HeLa cells. We found that FIT-039 suppressed bothE6 and E6�I/E7 transcripts dose dependently (Fig. 1A). Consistentwith this observation, the p53 and pRb tumor suppressors,targeted by HPV E6 and E7 (29), respectively, were restoredfollowing FIT-039 treatment (Fig. 1B) without major changes inmRNA levels (Supplementary Fig. S2). I contrast, HPV� C33Acervical cancer cells and HPV�HaCaT keratinocytes did not showFIT-039–dependent changes in p53 expression (Fig. 1C). We alsoconfirmed knockdown of CDK9 resulted in increased p53 andpRb, only in HPVþ cervical cancer cells (CaSki and HeLa), con-sistent with previously reported transcriptional suppression ofHPV E6E7 following CDK9 knockdown (27), but not in HPV�

cervical cancer cells (C33A) or keratinocytes (HaCaT; Supplemen-tary Fig. S3). In addition, FIT-039 treatment showed growth-suppressive effect, comparable with E6E7 knockdown in CaSkicells (Fig. 1D and E).

FIT-039 suppresses HPV replication and amplification inhuman primary keratinocytes

We next examined whether FIT-039 treatment eliminatesepisomal HPV, a major form of the virus in premalignant CIN

lesions, in which spontaneous HPV clearance is associated withcomplete regression (30). We used primary HFKs that maintainthe episomal HPV18 genome (HFK18). HFK18 recapitulates thecell differentiation–dependent HPV lifecycle, in which HPVsreplicate under undifferentiated conditions (low calciummedia),while HPV amplification is initiated in differentiated cells (high-calcium media; ref. 31). HFK18 cells were examined followingFIT-039 treatment for 72 hours, and dose-dependent suppressionof HPV DNA was confirmed for both replication (Fig. 1F) andamplification conditions of HPVs (Fig. 1G). However, FIA-002, aFIT-039 analog with a higher IC50 against CDK9 (17), was lesseffective onHPV suppression (Fig. 1F andG). The suppressedHPVreplication is thought to be attributable to FIT-039–mediatedearly promoter inhibition, because theHPV replication regulatorsE1 and E2 (32, 33) are polycistronically transcribed together withE6 and E7 from the promoter (4).

FIT-039 treatment restores normal epithelial morphology inHPV18þ CIN model by eliminating HPV

We next investigated the therapeutic effect of FIT-039 in anHPV-associated disease model. Organotypic raft culture of HFKsserves as an experimental model of HPV-induced CIN and benigntumor, as it exhibits dysplasia and hyperplasia while maintaininga physiologic HPV lifecycle, following HPV infection (34). HFKraft cultures were constructed with normal HFKs or HFK18 andtreatedwith FIT-039 (0, 2.5, and 5mmol/L) for aweek (Fig. 2A). InHPV18þ raft cultures without FIT-039, hyperproliferation anddysplasia were evident as reported previously, resembling CIN(Fig. 2B–D, ref. 34; comparing HPV� and HPV18þ, 0 mmol/Leach). In contrast, HPV18þ raft tissues treated with FIT-039 at 2.5and5mmol/L showed recovery fromCIN-related phenotypes (Fig.2B). This included restoration of the normal thickness of thekeratinocyte layer (Fig. 2C) and dose-dependent disappearance ofdysplastic keratinocytes (Fig. 2D). Strikingly, DNA in situ hybrid-ization of the HPV18 viral genome revealed a dose-dependentsuppression of HPV18 viral load in raft cultures (Fig. 3A), con-sistent with the observations in monolayer cultures (Fig. 1F andG). Suppressed expression of L1 major capsid protein was alsoconfirmedby IHCwhen raft tissueswere treatedwith FIT-039 (Fig.3B), indicating reduced HPV viral particle production. Thus, thetherapeutic effect of FIT-039 was confirmed in a CIN model ofHPVþ organotypic raft tissue culture through inhibition of HPVgene expression and replication. From the observation that HPV-eliminated condition lasted even a week after the removal of FIT-039 from the media, we supposed that FIT-039 has a clinicalpotency to suppress recurrent HPV propagation by eliminatingepisomal HPV.

Preclinical assessment of FIT-039 for HPV-associated benigntumors

As FIT-039 showed a promising therapeutic effect by targetingHPV in a raft culture CIN model, we next assessed the pharma-cokinetics and the toxicity of FIT-039 to evaluate its suitability asan anti-HPV drug candidate. A pharmacokinetic assay followingintravenous injection of FIT-039 revealed its accumulation inseveral tissues (Supplementary Table S2). Oral gavage of FIT-039 showed more sustainable blood concentrations than intra-venous or intraperitoneal routes (Supplementary Fig. S4). Toxic-ity profiling was also conducted for FIT-039, including blood andurea tests, genotoxicity, respiratory, cardiovascular, sensory, andmotor functions (Supplementary Table S3), and we did not find






any toxicity attributable to FIT-039 administration up to thehighest doses we examined in each test (Supplementary TableS3). Although FIT-039 is expected to be available for oral admin-istration, we prepared FIT-039 ointment and evaluated its distri-bution following topical administration considering the potentialapplication of FIT-039 in HPV-associated cutaneous and anogen-ital tumors, such as condyloma, and CIN. The distribution testusing [14C]-labeled FIT-039 was confirmed in vivo in both intact

(Fig. 4A) and decornified (Fig. 4B) skin tissues (9% and 28% oftotal absorption rate in normal and decornified skin, respectively,at 48hours after the administration). Sustainable retention of FIT-039, administered in ointment, for more than 24 hours in rabbitvaginal mucosa was also confirmed (Fig. 4C and D). In addition,plasma uptake rate of FIT-039 by topical application was muchlower than that by intravenous administration (SupplementaryTable S4), minimizing the potential risks of an adverse effect.

Figure 1.

FIT-039 suppresses HPV early viral transcripts and replication. A, HPV16þ CaSki and HPV18þ HeLa cells were treated with FIT-039 (5 or 10 mmol/L) or DMSO(0 mmol/L) for 24 hours prior to RNA extraction, and E6 (unspliced) and E7 (E6 intron-spliced E6�I) mRNAswere quantified using TaqMan qRT-PCR. Fold changes inb-actin–normalized mRNA copy number are shown. Mean � SD; �� , P < 0.01 compared with FIT-039 0 mmol/L, Student t test. B and C, Western blot analysisof p53 and pRb in HPV16þ CaSki and HPV18þ HeLa (B), as well as HPV� C33A and HaCaT cells (C), treated with FIT-039 (5 or 10 mmol/L) or DMSO (0 mmol/L) for48 hours. � , Truncated and unphosphorylated mutant pRb in C33A (20). D and E, Knockdown effect of HPV16 E6E7 on cell proliferation of HPV16þ CaSkicells. Cell proliferation was quantified with WST-8 assay at 96 hours after 10 mmol/L FIT-039 treatment or siRNA-mediated knockdown of HPV16 E6E7. Mean� SD;si-NS, nonspecific siRNA control; �� , P < 0.01 compared with FIT-039 0 mmol/L or si-NS, Student t test. F and G, Two micrograms of DNA was extracted fromundifferentiated (low Ca2þ medium) (F) or differentiated (1.8 mmol/L Ca2þ medium) HFK18 (G) to be analyzed by Southern blotting. Cells were treatedwith FIT-039 (2.5, 5, or 10 mmol/L), FIT-039 analogue FIA-002 (2.5, 5, or 10 mmol/L), or DMSO (0 mmol/L) for 72 hours prior to DNA extraction. HPV18 genomicDNA was detected using DIG-labeled antisense probe for HPV18 LCR. Blank arrowheads, nick forms; filled arrowhead, supercoiled form of HPV18 DNA.

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Moreover, we formulated a FIT-039 skin patch to achieve a moresustainable topical administration of FIT-039. The FIT-039 skinpatch showed dose-dependent penetration in rat skin tissue(Supplementary Fig. S5), as well as low level of plasma uptake(Supplementary Table S5).

FIT-039 suppresses xenograft tumor growth of HPV16þ CaSki,but not HPV� C33A cervical cancer cells

Wenext investigated potential anti-HPV effect of FIT-039 in vivousing cervical cancer xenografts with an integrated HPV genome.HPV16þ CaSki and HPV� C33A cervical cancer cells were trans-planted subcutaneously, and tumor volume was monitoredfor 3 weeks with daily oral administration of FIT-039 (150 or300 mg/kg BW). We confirmed a significant retardation ofHPV16þ CaSki xenograft tumor growth in mice treated with300 mg/kg BW FIT-039 (Fig. 5A), whereas the HPV� C33Axenograft was unresponsive to FIT-039 (Fig. 5B), indicating HPVintegration dependency of FIT-039 responsivity. No apparentadverse effects or body weight changes occurred during theobservation period (Fig. 5C and D). Consistently, immunohis-tostaining of HPV16þ CaSki xenograft tissue confirmed increased

rate of p53 nuclear expression in FIT-039–treated tumor cells,compared with that of untreated cells (Fig. 5E and F), which wasaccompaniedby reducedmRNA levels of E6 andE6�I/E7 (Fig. 5G).Such difference was not evident in HPV� C33A xenograft tumors(Fig. 5H and I). Eight to 12 mmol/L of plasma FIT-039 wasconfirmed in mice with 300 mg/kg BW administration (Fig. 5J).In addition, liver and skin tissues, which showedFIT-039 retention(Supplementary Table S2), did not indicate histopathologic altera-tions (Supplementary Fig. S6) or p53 upregulation (Supplemen-tary Fig. S7) following FIT-039 administration, further confirmingthat the effect of FIT-039 is specific to HPV-positive cells.

DiscussionApproximately 11% of human cancers worldwide are attribut-

able to viral carcinogenesis, and in most cases, viral gene expres-sion functions as a direct contributor of host cellular transforma-tion and/or malignancy (1, 2). Although an obvious strategy toavert virus-induced carcinogenesis is to inhibit viral oncogenefunctions, such successful inhibitors remain unavailable, in partbecause of their largely nonenzymatic natures. An alternative

Figure 2.

FIT-039 restores normal epithelial morphology in HPV-infected organotypic raft tissue cultures. A, Experimental diagram of FIT-039 treatment of organotypicraft culture tissue. Normal HFKs and HFK18 were used for organotypic raft culture. Following epithelial layer formation, raft tissues were incubated withFIT-039 (2.5 or 5 mmol/L) or DMSO (0 mmol/L) for 1 week and further cultured for 1 weekwithout compounds. B, Brightfield images of hematoxylin and eosin (H&E)–stained raft tissues in individual conditions. Scale bar, 50 mm. Boxed areas are magnified (� 2.5) in D. C, Thickness of keratinocyte layer (without cornified layer)was measured in randomly selected fields (n ¼ 4). Mean � SD; �� , P < 0.001 by Student t test compared with HPV� DMSO-treated condition. D, Magnifiedimage of upper part of keratinocyte layer from B. Dotted line depicts border of cornified layer (cr). Arrowheads indicate dysplasia-like cells. Scale bar, 10 mm.






strategy is to suppress viral oncogene expression in host cells, bytargeting viral or host cell factors responsible for their expression.CDK inhibitors are of interest for their antiviral effect against abroad spectrum of viral species, as exemplified by the pan-specificCDK inhibitor flavopiridol and oligo-specific roscovitine (35–37). However, their practical development as antivirals has beenhampered by cytotoxicity due to a cross-inhibition of cell-cycleCDKs (CDK1-6 and 14-18; ref. 38). Conversely, we have dem-onstrated that FIT-039 shows selective inhibition of CDK9 bytargeting the ATP-binding pocket, achieving antiviral activityagainst multiple viruses without major toxicity to host cells

(17–19). These data indicate that, as seen for HPV (26–28), theP-TEFb complex (CDK9 and cyclin T) plays a major role in viralpromoter activity in multiple viral species (39–45). The ability ofFIT-039 to target viral promoter activities prompted us to test itseffect on a tumor virus, in which expression of specific viral genesper se is an inducer of transformation and/or malignant progres-sion (19).

In our current study, we demonstrated promising antiviralactivity of FIT-039 against HPV. The anti-HPV effect of FIT-039was achievedby suppressing the viral early promoter for E6 andE7viral oncogene expression and viral replication, and therapeutic

Figure 3.

FIT-039 suppresses viral load of HPV18 in organotypic raft tissue culture CIN model. A and B, DNA in situ hybridization of HPV18 (green for HPV18 genomicDNA, and red for propidium iodide staining of nuclei; A) and immunohistostaining of HPV18 L1 major capsid protein (green for HPV18 L1 capsid, and red forpropidium iodide staining of nuclei; B) for organotypic raft culture tissues with or without HPV18 genome. Raft tissue cultures were incubated with 0.1% DMSOor FIT-039 for 1 week and further cultured for 1 week without compound before fixation (Fig. 2B). Dotted line indicates basal membrane. mx, matrix/fibroblastslayer; scale bar, 100 mm.

Ajiro et al.





Figure 4.

Preclinical evaluation of FIT-039, topically administered in skin and cervical mucosa. A and B, Distribution of [14C] FIT-039 in untreated (A) or decornified (B) dorsalskin of SD rats 2 hours after treatment with FIT-039 ointment (0.5 mg/body, 1.76 MBq/body of [14C] FIT-039). Silver particles [black in bright fields (top) or whitein dark field (bottom)] represent presence of [14C] FIT-039. Scale bar, 100 mm (20 mm for insets on the right) in A and B; co, corium; ep, epidermis; hf, hairfollicles; sc, stratumcorneum.C andD,Distributionof [14C] FIT-039 following topical administrationwith ointment in rabbit cervix. Shownarewholemount radiographs at4 hours (C) and 24 hours (D) after [14C] FIT-039 ointment treatment; co, colon; lv, liver; st, stomach; ut, uterus; va, vagina. CPM scale is shown in C and D.






effect was further confirmed in experimental models of CIN andcervical cancer. We also found that HPV suppression lasts up to aweek after the removal of FIT-039 from organotypic raft culturemedium, following FIT-039 treatment (Fig. 3). This observation

indicates that recurrent propagation of HPV will be preventedonce the viral load of HPV reaches to a sufficiently lower level inthis condition. It is also suggested that benign tumors withepisomal HPVs (e.g., CIN, condyloma, and common warts) are

Figure 5.

FIT-039 suppresses xenograft tumor growth of HPV16þ CaSki but not HPV� C33A cervical cancer cells. A and B, Tumor volume was plotted for specimensfrommice with HPV16þCaSki (A) and HPV�C33A xenograft tumors (B). �� , P <0.001 by Student t test betweenmice treatedwith 0 and 300mg/kg BWFIT-039 onday 21. Mean � SD are shown. C and D, Body weight plot for mice with HPV16þ CaSki (C) and HPV� C33A xenograft tumors (D). Mean � SD are shown. E–G,Immunohistostaining image of p53 (E), plot for p53 nuclear accumulation rates (F), and HPV16 E6 and E6�I/E7 transcripts (G) for xenograft tissues ofHPV16þ CaSki treated with mock or FIT-039 [300 mg/(kg BW/day)] for 21 days. H and I, Immunohistostaining image of p53 (H) and plot for p53 nuclearaccumulation rates (I) for HPV� C33A xenograft tumors following FIT-039 oral administration for 21 days. Tissue sections were counterstained with Hoechst33342 for nuclear DNA. J, Plasma concentration of FIT-039 in mice with CaSki or C33A xenograft tumors, at 4 hours after oral administration [0 or300 mg/(kg BW/day)] on day 21 [n ¼ 6 for 0 mg/(kg BW/day) and n ¼ 6 for 300 mg/(kg BW/day)]. Scale bar, 50 mm in E and H; mean � SD in F–I; mean � SEin J; n.s., P � 0.05; � , P < 0.05; �� , P < 0.01 by Student t test in F and G and I and J.


Ajiro et al.




primarily targeted by FIT-039 by eliminating HPVs, in contrast tomalignant tumors in which HPV genome is mostly integratedirreversibly into host cellular genome. Therefore, a phase I/IIaclinical trial of FIT-039 skin patch for HPVþ common warts wasconducted between 2016 and 2017 at Kyoto University Hospital(Kyoto, Japan; UMIN-CTR (http://www.umin.ac.jp/ctr/index.htm; unique identifier: UMIN000019866). No adverse effect,including contact dermatitis, was observed by single applicationof FIT-039 skin patch in the trial, and thus, anti-HPV effect byrepeated administration of FIT-039 through skin patches will befurther studied. In addition, we are currently preparing a cervicaltablet of FIT-039 for a topical administration to CIN lesion as thephase I/IIa clinical trial.

Our proposed therapeuticswith FIT-039provides the followingbenefits over the other previous anti-HPV strategies, includingtherapeutic vaccines (46), antiestrogen therapy (47, 48), and viralentry inhibition (49, 50): (i) The emergence of drug-escapemutant strains is expected to be reduced, as FIT-039 targets hostcell factor CDK9. (ii) An effective dose without systemic adverseeffects is easily achieved by topical administration. (iii) A thera-peutic effect is expected on both episomal and integrated HPVforms by inhibiting HPV early promoter activity. (iv) HPV vari-ation does not limit the efficacy of FIT-039, which is crucial for aneffective anti-HPV agent because numerous genotypes are usuallyassociated with a single disease (e.g., approximately 15 HPVgenotypes are implicated in cervical cancer), and infection withmultiple genotypes is also common. Furthermore, the antiviraleffect of FIT-039 has been confirmed for a wide spectrum ofviruses other than HPVs (17–19). Past studies indicated CDK9,over the other CDKs, is prone to be recruited to viral promoters, inassociation and/or cooperation with viral proteins (39–45),which we consider the mechanical background of the commonantiviral effect of FIT-039. For instance, ICP22 of human simplexherpesvirus (HSV) forms complex with CDK9 and SPT5 to acti-vate HSV-1 late gene promoter (40, 41), as well as upregulatingother HSV-1 transcripts (42). Regarding EB virus (EBV), P-TEFb isrequired for EBNA2-dependent transcripts, and CDK9 inhibitionby 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole has beenpreviously reported to suppress those transcripts (44, 45).Althoughmechanism of viral transcriptional regulation by CDK9is less characterized for hepatitis B virus (HBV), the recent reportby Francisco and colleagues demonstrated that HBV transcriptionfrom cccDNA employs superelongation complex, including

CDK9, as well as P-TEFb activator BRD4, and inhibition of BRD4or CDK9 results in suppressed HBV transcription from cccDNA(19, 43). In retrovirus HIV-1, CDK9 in complex with Cyclin T andHIV-1 Tat proteins, activates HIV-1 LTR to promote viral tran-scription from HIV-1 proviral DNA (39). Therefore, our currentpreclinical data of FIT-039 provide a rationale for further clinicalinvestigation, not only for HPV-induced cervical neoplasia, butalso for other malignant diseases caused by CDK9-dependentviruses.

Disclosure of Potential Conflicts of InterestM. Hagiwara is a consultant/advisory board member for KinoPharma, Inc.

No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: M. Yamamoto, E. Sumi, M. HagiwaraDevelopment of methodology: H. SakaiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Ajiro, H. Sakai, H. OnogiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Ajiro, H. Sakai, H. Onogi, T. Sawada, K. Kaba-shima, M. HagiwaraWriting, review, and/or revision of themanuscript:M.Ajiro,H. Sakai, E. Sumi,T. Nomura, K. Kabashima, M. HagiwaraAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E. Sumi, T. SawadaStudy supervision: K. Kabashima, M. HagiwaraOther (synthesized FIT-039 and FIA-002): T. Hosoya

AcknowledgmentsWe wish to acknowledge Shuh Narumiya of Kyoto University Graduate

School of Medicine for his critical suggestions and encouragements of thisstudy. We also thank Miki Tsutsui for technical support, and members of theHagiwara laboratory for their assistance and discussion. This study wassupported by the following grants: Translational Research Network Program(JP161m0103006j0005(c-6-2)), Program for Basic and Clinical Research onHepatitis (JP18fk0210209h1003), and Basis for Supporting Innovative DrugDiscovery, and Life Science Research (BINDS; JP18am0101092) of the JapanAgency for Medical Research and Development (AMED).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 22, 2017; revised March 14, 2018; accepted April 25, 2018;published first April 30, 2018.

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2018;24:4518-4528. Published OnlineFirst April 30, 2018.Clin Cancer Res Masahiko Ajiro, Hiroyuki Sakai, Hiroshi Onogi, et al. Has a Therapeutic Effect on HPV-Induced NeoplasiaCDK9 Inhibitor FIT-039 Suppresses Viral Oncogenes E6 and E7 and

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