CDK9 Inhibitor FIT-039 Suppresses Viral Oncogenes E6 and E7 and Has a Therapeutic Effect on HPV-Induced Neoplasia.

Purpose: Cervical cancer is one of the leading causes of cancer-related deaths among women worldwide. The purpose of this study is to assess the therapeutic effect of the newly developed 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 effect on HPV gene expression in HPV+ cervical cancer cells. Primary keratinocytes monolayer and organotypic raft culture models were used to evaluate HPV viral replication and cervical intraepithelial neoplasia (CIN) phenotypes. Preclinical pharmacokinetics and toxicity tests for FIT-039 were also conducted. Finally, the anti-HPV effect of FIT-039 was further examined in vivo, using HPV+ cervical cancer xenografts.Results: FIT-039 inhibits HPV replication and expression of E6 and E7 viral oncogenes, restoring tumor suppressors p53 and pRb in HPV+ cervical cancer cells. The therapeutic effect of FIT-039 was demonstrated in CIN model of an organotypic raft culture, where FIT-039 suppressed HPV18-induced dysplasia/hyperproliferation with reduction in viral load. FIT-039 also repressed growth of HPV16+, but not HPV- cervical cancer xenografts without any significant adverse effects. Safety and pharmacokinetics of FIT-039 were confirmed for systemic and topical routes.Conclusions: The CDK9 inhibitor FIT-039 showed potent anti-HPV activity without significant toxicity in preclinical studies. Thus, FIT-039 is expected to be a novel therapeutic for CIN to prevent cervical cancer. Clin Cancer Res; 24(18); 4518-28. ©2018 AACR.


Introduction
Human 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 subsequently progresses to cervical cancer. HPV mainly remains in episomal form in CIN lesions, and spontaneous HPV clearance in cervix is accompanied by a regression of CIN, whereas years of persistent infection could result in irreversible integration of HPV genome into host cellular genome, inducing malignant progression (3). Integration of the HPV genome into the host cell genome then promotes transcription of two viral oncogenes E6 and E7, required for transformation of host keratinocytes and malignancy by targeting tumor suppressors (4)(5)(6). In the presence of E6 protein and its cofactor E6AP, p53 is targeted for ubiquitindependent degradation (4)(5)(6). The E7 protein, due to its high affinity to pRb, prevents pRb from complex formation with E2F1, inducing progression to the S-phase (5,6). Binding of E7 to pRb also subsequently induces destabilization of pRb (7,8).
Treatment of HPV-induced neoplasia and cancer represents a major unmet medical need worldwide. HPV vaccines, Gardasil and Cervarix, were introduced a decade ago, and their prophylactic effects have been demonstrated in many trials, and the reduction of infection rate of several types of HPVs is evident in countries proceeding in public HPV vaccination of teenagers (9), although the prophylactic effect of HPV vaccine is considered to be limited to approximately 90% at most, even by the 9-valent HPV vaccine (10). On the other hand, in many developing countries, HPV vaccines are still not introduced or only recently done into national immunization programs. Incidences of CIN and cervical cancer, therefore, are increasing in those countries, especially among women of reproductive ages, which leaves cervical cancer still among the leading causes of cancer-related deaths in the world. The standard care for CIN is cervical cornification, which, however, increases risk of premature birth and newborn mortality due to a compromised cervix function (11)(12)(13)(14) and nearly 10% of recurrence risk by persisting HPV infection (15,16), and noninvasive therapeutics has been demanded for CIN treatment. Thus, in this study, we examined anti-HPV compound for a therapeutic effect on HPV-induced CIN and cervical cancer.
The newly developed antiviral drug FIT-039 targets host cell factor CDK9 and suppresses transcription of various DNA viruses such as herpes simplex virus, adenovirus, cytomegalovirus, and hepatitis B virus (17)(18)(19). In the current study, we demonstrate that FIT-039 suppresses HPV early promoter activity by inhibiting CDK9 activity, leading to reduced expression of HPV oncogenes E6 and E7 in cervical cancer cells. Suppressed viral replication is also observed in keratinocytes with episomal HPV in monolayer cultures and organotypic raft tissues. Moreover, the therapeutic potentials of FIT-039 against CIN and other HPV-associated diseases are preclinically verified for further clinical investigation.

Cell lines
CaSki (HPV16 þ cervical cancer), C33A (HPV À cervical cancer, with mutations in p53 and pRb; ref. 20), and HaCaT (HPV À cervical keratinocytes) cell lines were purchased from ATCC. HeLa cells (HPV18 þ cervical cancer) were purchased from JCRB Cell Bank. Primary human foreskin keratinocytes (HFK) and fibroblasts (HFF) were purchased from Thermo Fisher Scientific. CaSki, C33A, HaCaT, HeLa, and HFFs were maintained in 10%FBS/ DMEM in a 5% CO 2 incubator at 37 C. HFKs were maintained in KGM EpiLife medium (Thermo Fisher Scientific) in the same conditions. HFKs with the episomal HPV18 genome (HFK18) were prepared as follows: Full-length HPV18 DNA (GenBank no.: X05015) was excised from a plasmid vector, and HPV DNA was self-ligated with T4 DNA ligase. The circularized HPV DNA and pSV2-neo plasmids were introduced into normal HFKs with TransIT-keratinocyte (Mirus Bio LLC). After the transfection, HFKs were cultured with KGM EpiLife medium containing G418 for 96 hours, and then, maintained in KGM EpiLife medium without G418. HFK18 was used for the experiment at 10 days after transfection. All cell lines used in this study were verified by short tandem repeat analysis (conducted on February 28, 2018), performed by Takara Bio Inc. using GenePrint 10 System (Promega)  for D21S11, TH01, TPOX, vWA, Amelogenin, CSF1PO, D16S539, D7S820, D13S317, and D5S818. STR results were visualized using GeneMapper software (Thermo Fisher Scientific), and shown in Supplementary Fig. S1 and Supplementary Table S1.

WST-8 growth assay
The cells were seeded at a density of 1.0 Â 10 4 cells into a 24well plate and treated with 0, 5, 10, or 20 mmol/L FIT-039 in DMEM with 10% FBS and 0.1% DMSO. The FIT-039-containing medium was replaced with fresh medium after 48 hours, followed by further incubation for 48 hours. Then, the cell viability was quantified 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 the absorbance was measured at 450 nm using a photospectrometer.

Southern blot
Total DNA was extracted from the HFKs, and 2 mg of the total DNA was separated using 0.8% agarose gel electrophoresis. Then, they were transferred onto nylon membrane (Hybond Nþ) (GE Healthcare). For the detection of HPV18-specific DNA, DIGlabeling system (Roche Diagnostics) was used. DIG-labeled

Translational Relevance
Cervical cancer accounted for 528,000 new cases and 266,000 deaths of cancer worldwide in 2012. More than 95% of cervical cancer cases are caused by persistent infection of high-risk types of human papillomaviruses (HPV). In cervical intraepithelial neoplasia (CIN), a premalignant form of cervical cancer, HPV propagates mostly in episomal form, and years of persistent infection subsequently result in integration of HPV genome into host keratinocyte genome, which triggers malignant progression. As current surgical care of CIN may impact patients' future reproductive ability, and has approximately 10% of recurrence rate due to persisting infection, a noninvasive anti-HPV therapy has been demanded. FIT-039 is currently evaluated in the phase I/IIa clinical trial for anti-HPV activity in skin warts. Based on the presented data, I/IIa clinical trial of FIT-039 for CIN will start in 2018.

Organotypic raft culture
For organotypic raft culture preparation, one part of type I collagen, cell matrix type I-P (Nitta Gelatin Co., Ltd.) and two parts of growth medium containing HFFs (1 Â 10 6 cells) were mixed well and poured into a 6-cm dish, and cells were maintained in a 5% CO 2 incubator at 37 C for gel contraction. The gel was soaked in KGM for several hours and then transferred into a transwell insert. The insert was placed in a 6-well plate and both bottom and insert were filled with KGM. HFKs were then overlaid on the gel (day 0). The medium was changed to KGM/DMEM, 1:1 on day 1, and 24 hours later, the medium was changed to KGM/ DMEM with 1.8 mmol/L CaCl 2 (day 2). On day 3, the surface of the collagen gel was exposed to air, and the medium in the bottom chamber was replaced with fresh medium daily. Treatment with DMSO or FIT-039 was started on day 3 by adding the compound to the medium. The treatment was sustained until day 10, followed by culture without compound for a week. Obtained specimens were embedded in optimal cutting temperature (OCT) compound and applied to cryosectioning. The sections were transferred onto glass slides and dried. Hematoxylin and eosin (H&E) staining was carried out following a standard protocol.

Xenograft tumor assay
Female CB-17/lcr-scid/scid Jcl mice were purchased from CLEA Japan and used in the xenograft tumor assay. Briefly, 0.5 Â 10 6 CaSki or C33A cells that stably expressed GLuc were suspended in Hank's balanced salt solution (HBSS [þ]) and subcutaneously injected into the right dorsal area of 5-week-old mice. Once the tumor mass reached 200 mm 3 , oral administration of FIT-039 was initiated, and the tumor volume was calculated as (4/3)ÁpÁaÁb 2 (a, major axis; b, minor axis) every 2 days. On the day 21, the tumor mass, dorsal skin, and liver tissues were excised and fixed in 10% neutral-buffered formalin, and plasma FIT-039 concentration was determined 4 hours after the administration by high-performance liquid chromatography (HPLC).

DNA in situ hybridization
Detection of HPV18 DNA signals in tissue sections was performed with the TSA Biotin System (PerkinElmer) following the manufacturer's instructions. DIG-labeled DNA for the HPV18 LCR region (7000-100 nt; GenBank no. X05015) was used as a probe, and streptavidin fluorescein (PerkinElmer) was used to detect biotin. Nuclei were counterstained using propidium iodide (Sigma-Aldrich).

IHC
For the IHC of xenograft tumor sections, formalin-fixed sections were deparaffinized with xylene, rehydrated, and treated with a Mouse on Mouse blocking reagent (Vector Laboratories). Next, p53 was detected using an anti-p53 (DO-1) antibody (Santa Cruz Biotechnology) and anti-mouse IgG Alexa-488 (Thermo Fisher Scientific), and the nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific). Detection of HPV18 L1 signals in the tissue sections was performed with the TSA Biotin System (PerkinElmer) following the manufacturer's instructions.

Pharmacokinetics test for FIT-039
[ 14 C]-labeled FIT-039 (7.03 MBq/mg) was synthesized by Sekisui Medical Co., Ltd. For the pharmacokinetics study by intravenous injection, [ 14 C] FIT-039 (18 mg) and unlabeled FIT-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 suspended in 4 portions of saline, resulting in 5 mg/mL of FIT-039 solution, which was used for intravenous injection of 7-week-old male SD rats [Charles River Laboratories; 5 mg/kg body weight (BW), 1 mL/kg BW, n ¼ 3 for each time point]. The FIT-039 concentration in each tissue was determined by measuring radioactivity with a scintillation counter.

Topical administration and tissue autoradiography of [ 14 C] FIT-039
For cutaneous topical administration, [ 14 C] FIT-039 (7.03 MBq/mg, as described above) and unlabeled-FIT-039 (12.5 mg each) were mixed with 74.5 mg of lactic acid, and suspended in 2.4 g of polyethylene glycol to make a 10 mg/g FIT-039 ointment. FIT-039 ointment was then topically administered onto normal or decornified rat skin (2.0 Â 3.0 cm) at 0.5 mg/body. Normal and decornified rats with cutaneous administration of [ 14 C] FIT-039 ointment were euthanized 2 hours after the administration, and a skin tissue block was prepared with OCT compound. A 5-mm skin tissue section was fixed on a nuclear emulsion-coated glass slide and exposed for 2 weeks at 4 C in the dark. Total absorption rate was determined from the sum of radioactivity from whole body, urine, and feces. For vaginal topical administration of [ 14 C] FIT-039 in rabbits, 10 mg of [ 14 C] FIT-039 (7.03 MBq/mg) and 42 mg of unlabeled-FIT-039 were dissolved with 3.3 g of polyethylene glycol to prepare 15.5 mg/g FIT-039 ointment. A total of 8.6 mg FIT-039 (500 mL of ointment) was topically administered to rabbit cervix.

Penetration test of FIT039 through skin tissues
Skin tissues from 7-to 9-week-old male rats were decornified by taped stripping and resected. Resected skin tissues were then incubated in receiver solution (40% PEG400) for 1 hour and set up for skin penetration test, so that only the inside surface of the resected skin tissue was in contact with 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 skinpenetrated FIT-039 present in the receiver solution at 4, 8, 12, 16, 20, and 24 hours after FIT-039 skin patch application was then quantified by HPLC.

FIT-039 suppresses HPV replication and amplification in human primary keratinocytes
We next examined whether FIT-039 treatment eliminates episomal HPV, a major form of the virus in premalignant CIN lesions, in which spontaneous HPV clearance is associated with complete regression (30). We used primary HFKs that maintain the episomal HPV18 genome (HFK18). HFK18 recapitulates the cell differentiation-dependent HPV lifecycle, in which HPVs replicate under undifferentiated conditions (low calcium media), while HPV amplification is initiated in differentiated cells (highcalcium media; ref. 31). HFK18 cells were examined following FIT-039 treatment for 72 hours, and dose-dependent suppression of HPV DNA was confirmed for both replication (Fig. 1F) and amplification conditions of HPVs (Fig. 1G). However, FIA-002, a FIT-039 analog with a higher IC 50 against CDK9 (17), was less effective on HPV suppression ( Fig. 1F and G). The suppressed HPV replication is thought to be attributable to FIT-039-mediated early promoter inhibition, because the HPV replication regulators E1 and E2 (32,33) are polycistronically transcribed together with E6 and E7 from the promoter (4).

FIT-039 treatment restores normal epithelial morphology in HPV18 þ CIN model by eliminating HPV
We next investigated the therapeutic effect of FIT-039 in an HPV-associated disease model. Organotypic raft culture of HFKs serves as an experimental model of HPV-induced CIN and benign tumor, as it exhibits dysplasia and hyperplasia while maintaining a physiologic HPV lifecycle, following HPV infection (34). HFK raft cultures were constructed with normal HFKs or HFK18 and treated with FIT-039 (0, 2.5, and 5 mmol/L) for a week ( Fig. 2A). In HPV18 þ raft cultures without FIT-039, hyperproliferation and dysplasia were evident as reported previously, resembling CIN ( Fig. 2B-D, ref. 34; comparing HPV À and HPV18 þ , 0 mmol/L each). In contrast, HPV18 þ raft tissues treated with FIT-039 at 2.5 and 5 mmol/L showed recovery from CIN-related phenotypes (Fig.  2B). This included restoration of the normal thickness of the keratinocyte layer (Fig. 2C) and dose-dependent disappearance of dysplastic keratinocytes (Fig. 2D). Strikingly, DNA in situ hybridization of the HPV18 viral genome revealed a dose-dependent suppression of HPV18 viral load in raft cultures (Fig. 3A), consistent with the observations in monolayer cultures ( Fig. 1F and G). Suppressed expression of L1 major capsid protein was also confirmed by IHC when raft tissues were treated with FIT-039 (Fig.  3B), indicating reduced HPV viral particle production. Thus, the therapeutic effect of FIT-039 was confirmed in a CIN model of HPV þ organotypic raft tissue culture through inhibition of HPV gene expression and replication. From the observation that HPVeliminated condition lasted even a week after the removal of FIT-039 from the media, we supposed that FIT-039 has a clinical potency to suppress recurrent HPV propagation by eliminating episomal HPV.

Preclinical assessment of FIT-039 for HPV-associated benign tumors
As FIT-039 showed a promising therapeutic effect by targeting HPV in a raft culture CIN model, we next assessed the pharmacokinetics and the toxicity of FIT-039 to evaluate its suitability as an anti-HPV drug candidate. A pharmacokinetic assay following intravenous injection of FIT-039 revealed its accumulation in several tissues (Supplementary Table S2). Oral gavage of FIT-039 showed more sustainable blood concentrations than intravenous or intraperitoneal routes (Supplementary Fig. S4). Toxicity profiling was also conducted for FIT-039, including blood and urea tests, genotoxicity, respiratory, cardiovascular, sensory, and motor functions (Supplementary Table S3), and we did not find any toxicity attributable to FIT-039 administration up to the highest doses we examined in each test (Supplementary Table  S3). Although FIT-039 is expected to be available for oral administration, we prepared FIT-039 ointment and evaluated its distribution following topical administration considering the potential application of FIT-039 in HPV-associated cutaneous and anogenital tumors, such as condyloma, and CIN. The distribution test using [ 14 C]-labeled FIT-039 was confirmed in vivo in both intact ( Fig. 4A) and decornified (Fig. 4B) skin tissues (9% and 28% of total absorption rate in normal and decornified skin, respectively, at 48 hours after the administration). Sustainable retention of FIT-039, administered in ointment, for more than 24 hours in rabbit vaginal mucosa was also confirmed (Fig. 4C and D). In addition, plasma uptake rate of FIT-039 by topical application was much lower than that by intravenous administration (Supplementary  Table S4), minimizing the potential risks of an adverse effect. Moreover, we formulated a FIT-039 skin patch to achieve a more sustainable topical administration of FIT-039. The FIT-039 skin patch 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 HPV16 þ CaSki, but not HPV À C33A cervical cancer cells We next investigated potential anti-HPV effect of FIT-039 in vivo using cervical cancer xenografts with an integrated HPV genome. HPV16 þ CaSki and HPV À C33A cervical cancer cells were transplanted subcutaneously, and tumor volume was monitored for 3 weeks with daily oral administration of FIT-039 (150 or 300 mg/kg BW). We confirmed a significant retardation of HPV16 þ CaSki xenograft tumor growth in mice treated with 300 mg/kg BW FIT-039 (Fig. 5A), whereas the HPV À C33A xenograft was unresponsive to FIT-039 (Fig. 5B), indicating HPV integration dependency of FIT-039 responsivity. No apparent adverse effects or body weight changes occurred during the observation period ( Fig. 5C and D). Consistently, immunohistostaining of HPV16 þ 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 was accompanied by reduced mRNA levels of E6 and E6 Ã I/E7 (Fig. 5G). Such difference was not evident in HPV À C33A xenograft tumors ( Fig. 5H and I). Eight to 12 mmol/L of plasma FIT-039 was confirmed in mice with 300 mg/kg BW administration (Fig. 5J). In addition, liver and skin tissues, which showed FIT-039 retention (Supplementary Table S2), did not indicate histopathologic alterations ( Supplementary Fig. S6) or p53 upregulation (Supplementary Fig. S7) following FIT-039 administration, further confirming that the effect of FIT-039 is specific to HPV-positive cells.

Discussion
Approximately 11% of human cancers worldwide are attributable to viral carcinogenesis, and in most cases, viral gene expression functions as a direct contributor of host cellular transformation and/or malignancy (1,2). Although an obvious strategy to avert virus-induced carcinogenesis is to inhibit viral oncogene functions, such successful inhibitors remain unavailable, in part because of their largely nonenzymatic natures. An alternative  strategy is to suppress viral oncogene expression in host cells, by targeting viral or host cell factors responsible for their expression. CDK inhibitors are of interest for their antiviral effect against a broad spectrum of viral species, as exemplified by the pan-specific CDK inhibitor flavopiridol and oligo-specific roscovitine (35)(36)(37). However, their practical development as antivirals has been hampered by cytotoxicity due to a cross-inhibition of cell-cycle CDKs (CDK1-6 and 14-18; ref. 38). Conversely, we have demonstrated that FIT-039 shows selective inhibition of CDK9 by targeting the ATP-binding pocket, achieving antiviral activity against multiple viruses without major toxicity to host cells (17)(18)(19). These data indicate that, as seen for HPV (26)(27)(28), the P-TEFb complex (CDK9 and cyclin T) plays a major role in viral promoter activity in multiple viral species (39)(40)(41)(42)(43)(44)(45). The ability of FIT-039 to target viral promoter activities prompted us to test its effect on a tumor virus, in which expression of specific viral genes per se is an inducer of transformation and/or malignant progression (19). In our current study, we demonstrated promising antiviral activity of FIT-039 against HPV. The anti-HPV effect of FIT-039 was achieved by suppressing the viral early promoter for E6 and E7 viral oncogene expression and viral replication, and therapeutic 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 genomic DNA, and red for propidium iodide staining of nuclei; A) and immunohistostaining of HPV18 L1 major capsid protein (green for HPV18 L1 capsid, and red for propidium iodide staining of nuclei; B) for organotypic raft culture tissues with or without HPV18 genome. Raft tissue cultures were incubated with 0.1% DMSO or FIT-039 for 1 week and further cultured for 1 week without compound before fixation (Fig. 2B). Dotted line indicates basal membrane. mx, matrix/fibroblasts layer; scale bar, 100 mm. effect was further confirmed in experimental models of CIN and cervical cancer. We also found that HPV suppression lasts up to a week after the removal of FIT-039 from organotypic raft culture medium, following FIT-039 treatment (Fig. 3). This observation indicates that recurrent propagation of HPV will be prevented once the viral load of HPV reaches to a sufficiently lower level in this condition. It is also suggested that benign tumors with episomal HPVs (e.g., CIN, condyloma, and common warts) are FIT-039 suppresses xenograft tumor growth of HPV16 þ CaSki but not HPV À C33A cervical cancer cells. A and B, Tumor volume was plotted for specimens from mice with HPV16 þ CaSki (A) and HPV À C33A xenograft tumors (B). ÃÃÃ , P < 0.001 by Student t test between mice treated with 0 and 300 mg/kg BW FIT-039 on day 21. Mean AE SD are shown. C and D, Body weight plot for mice with HPV16 þ CaSki (C) and HPV À C33A xenograft tumors (D). Mean AE SD are shown. E-G, Immunohistostaining image of p53 (E), plot for p53 nuclear accumulation rates (F), and HPV16 E6 and E6 Ã I/E7 transcripts (G) for xenograft tissues of HPV16 þ 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 nuclear accumulation rates (I) for HPV À C33A xenograft tumors following FIT-039 oral administration for 21 days. Tissue sections were counterstained with Hoechst 33342 for nuclear DNA. J, Plasma concentration of FIT-039 in mice with CaSki or C33A xenograft tumors, at 4 hours after oral administration [0 or 300 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 AE SD in F-I; mean AE SE in J; n.s., P ! 0.05; Ã , P < 0.05; ÃÃ , P < 0.01 by Student t test in F and G and I and J. primarily targeted by FIT-039 by eliminating HPVs, in contrast to malignant tumors in which HPV genome is mostly integrated irreversibly into host cellular genome. Therefore, a phase I/IIa clinical trial of FIT-039 skin patch for HPV þ common warts was conducted 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 application of FIT-039 skin patch in the trial, and thus, anti-HPV effect by repeated administration of FIT-039 through skin patches will be further studied. In addition, we are currently preparing a cervical tablet of FIT-039 for a topical administration to CIN lesion as the phase I/IIa clinical trial.
Our proposed therapeutics with FIT-039 provides the following benefits over the other previous anti-HPV strategies, including therapeutic vaccines (46), antiestrogen therapy (47,48), and viral entry inhibition (49,50): (i) The emergence of drug-escape mutant strains is expected to be reduced, as FIT-039 targets host cell factor CDK9. (ii) An effective dose without systemic adverse effects is easily achieved by topical administration. (iii) A therapeutic effect is expected on both episomal and integrated HPV forms by inhibiting HPV early promoter activity. (iv) HPV variation does not limit the efficacy of FIT-039, which is crucial for an effective anti-HPV agent because numerous genotypes are usually associated with a single disease (e.g., approximately 15 HPV genotypes are implicated in cervical cancer), and infection with multiple genotypes is also common. Furthermore, the antiviral effect of FIT-039 has been confirmed for a wide spectrum of viruses other than HPVs (17)(18)(19). Past studies indicated CDK9, over the other CDKs, is prone to be recruited to viral promoters, in association and/or cooperation with viral proteins (39)(40)(41)(42)(43)(44)(45), which we consider the mechanical background of the common antiviral effect of FIT-039. For instance, ICP22 of human simplex herpesvirus (HSV) forms complex with CDK9 and SPT5 to activate HSV-1 late gene promoter (40,41), as well as upregulating other HSV-1 transcripts (42). Regarding EB virus (EBV), P-TEFb is required for EBNA2-dependent transcripts, and CDK9 inhibition by 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole has been previously reported to suppress those transcripts (44,45). Although mechanism of viral transcriptional regulation by CDK9 is less characterized for hepatitis B virus (HBV), the recent report by Francisco and colleagues demonstrated that HBV transcription from cccDNA employs superelongation complex, including CDK9, as well as P-TEFb activator BRD4, and inhibition of BRD4 or CDK9 results in suppressed HBV transcription from cccDNA (19,43). In retrovirus HIV-1, CDK9 in complex with Cyclin T and HIV-1 Tat proteins, activates HIV-1 LTR to promote viral transcription from HIV-1 proviral DNA (39). Therefore, our current preclinical data of FIT-039 provide a rationale for further clinical investigation, not only for HPV-induced cervical neoplasia, but also for other malignant diseases caused by CDK9-dependent viruses.

Disclosure of Potential Conflicts of Interest
M. Hagiwara is a consultant/advisory board member for KinoPharma, Inc. No potential conflicts of interest were disclosed by the other authors.