nk cell speciﬁc cdk8 deletion enhances antitumor responses · nk cell–speciﬁc cdk8 deletion...

Research Article

NK Cell–Specific CDK8 Deletion EnhancesAntitumor ResponsesAgnieszka Witalisz-Siepracka, Dagmar Gotthardt, Michaela Prchal-Murphy,Zrinka Didara, Ingeborg Menzl, Daniela Prinz, Leo Edlinger, Eva Maria Putz,and Veronika Sexl

Abstract

Cyclin-dependent kinase 8 (CDK8) is a member of thetranscription-regulating CDK family. CDK8 activates orrepresses transcription by associating with the mediator com-plex or by regulating transcription factors. Oncogenic activity ofCDK8 has been demonstrated in several cancer types. TargetingCDK8 represents a potential therapeutic strategy. Becauseknockdown of CDK8 in a natural killer (NK) cell line enhancescytotoxicity and NK cells provide the first line of immunedefense against transformed cells, we asked whether inhibitingCDK8 would improve NK-cell antitumor responses. In thisstudy, we investigated the role of CDK8 in NK-cell functionin vivo using mice with conditional ablation of CDK8 in

NKp46þ cells (Cdk8fl/flNcr1Cre). Regardless of CDK8 expres-sion, NK cells develop and mature normally in bone marrowand spleen. However, CDK8 deletion increased expression ofthe lytic molecule perforin, which correlated with enhancedNK-cell cytotoxicity in vitro. This translates into improved NKcell–mediated tumor surveillance in vivo in three independentmodels: B16F10 melanoma, v-ablþ lymphoma, and a slowlydeveloping oncogene-driven leukemia. Our results therebydefine a suppressive effect of CDK8 on NK-cell activity. Ther-apies that target CDK8 in cancer patients may enhance NK-cellresponses against tumor cells. Cancer Immunol Res; 6(4); 458–66.�2018 AACR.

IntroductionCyclin-dependent kinase 8 (CDK8) belongs to the family of

transcription regulating CDKs, and its serine/threonine kinaseactivity is activated by binding to cyclin C (1). CDK8 associateswith cyclin C, MED12, and MED13 to form the kinase module ofthe mediator complex, which acts as a bridge between transcrip-tion factors and RNA polymerase II (1–5). In addition, CDK8contributes to the activation or repression of transcription byphosphorylating multiple transcription factors. CDK8 has beenimplicated in the regulation of oncogenic pathways such as thep53, Wnt–b-catenin, or TGFb pathways (4, 6–11). Overexpres-sion or amplification of theCDK8 gene has been detected in coloncancer and leads to b-catenin hyperactivity (7).High expression ofCDK8 correlateswith poor prognosis of colon, breast, andovariancancer (9, 12, 13). These findings classified CDK8 as a bona fideoncogene, and inhibition of CDK8 is considered a promisingtarget in antitumor therapy (14). CDK8 inhibitors are underdevelopment and several preclinical studies show encouragingeffects (13, 15–18).

CDK8 regulates transcription downstream of cytokines utiliz-ing the JAK–STAT pathway, thereby also interfering with andregulating immune responses (19). Upon binding of a ligand to acognate receptor, JAK kinases phosphorylate tyrosine residues onSTAT proteins, which then translocate to the nucleus to induceligand–specific gene transcription (20). Once bound to DNA,STATs undergo an additional phosphorylation event on serineresidues to acquire full transcriptional activity (21, 22). CDK8 isan upstream kinase of serine phosphorylation for several STATs,including STAT1–S727 (19, 23). We found a function of theCDK8-STAT1 axis in natural killer (NK) cells (23), innate lym-phocytes that provide the first line of defense against transformedand infected cells. We showed that NK cells harbor a constitutive,CDK8-mediated phosphorylation of STAT1–S727, which dam-pens NK-cell functions. Mice lacking the S727 phosphorylationsite in STAT1 (Stat1S727A) display enhanced NK cell–mediatedcytotoxicity and tumor surveillance. The finding that knockdownof CDK8 in an NK-cell line enhanced cytotoxicity and phenocop-ied the hyperactivity of Stat1S727A NK cells in vitro (23) promptedus to investigate the role of CDK8 in NK cells in vivo. Here,we used two mouse models to do so; we deleted Cdk8 selectivelyin NKp46þ cells (Cdk8fl/fl Ncr1Cre) or deleted it induciblyin all interferon-responsive cells present in multiple organs(Cdk8fl/flMx1Cre; ref. 24). Evidence obtained in these mousemodels led us to conclude that NK cells lacking CDK8 developand mature normally but exhibit hyperactivity in vitro andenhanced tumor surveillance in vivo. The function of CDK8appears to be NK cell–intrinsic, as the effects of CDK8 deletionwere comparable between the two mouse models. Thus, reduc-ing CDK8 expression or activity in cancer patients may bothimpair tumor cell viability and also increase NK cell–mediatedtumor immune surveillance.

Institute of Pharmacology and Toxicology, University of Veterinary Medicine,Vienna, Austria.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

A. Witalisz-Siepracka and D. Gotthardt contributed equally to this article.

Corresponding Author: Veronika Sexl, University of Veterinary MedicineVienna, Veterin€arplatz 1, A-1210 Vienna, Austria. Phone: 43-1-25077-2910; Fax:43-1-25077-2990; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-17-0183

�2018 American Association for Cancer Research.

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Materials and MethodsMice and cell lines

Conditional C57BL/6N-Cdk8fl/fl (Cdk8tm1c(EUCOMM)Hmgu) micewere provided by Yann Herault (IGMBC). The Cdk8tm1c allele ofthe mutant was generated frommice with the Cdk8tm1a knockoutfirst allele (described by International Mouse Phenotyping Con-sortium https://www.mousephenotype.org) by excision of thelacZ-neo cassette via Flp-recombination. The conditional poten-tial of Cdk8fl/fl mice was activated by Cre-recombination andexcision of the loxP-flanked exon 5 of Cdk8 resulting in a frame-shift and the translation of a truncated CDK8 protein, which issubjected to nonsense-mediated decay. Tissue-specific recombi-nation was induced by cross breeding of Cdk8fl/fl with B6N-Tg(Mx1Cre);(24) and B6N-Tg(Ncr1Cre);(25)mice. All animals wereonC57BL/6Nbackground, age andgendermatched (6–12weeks)and maintained at the University of Veterinary Medicine Viennaunder specific pathogen-free conditions according to Federationfor Laboratory Animal Science Associations (FELASA) guidlines(2014). The animal experiments were approved by the Ethics andAnimal Welfare Committee of the University of Veterinary Med-icine Vienna and granted by the national authority (AustrianFederal Ministry of Science and Research) according to Section8ff of Law for Animal Experiments under license BMWF-68.205/0218-II/3b/2012 and were conducted according to the guidelinesof FELASA and ARRIVE. To inducibly delete Cdk8 in vivo, Cdk8fl/fl

andCdk8fl/flMx1Cremice were injected intraperitoneally with 200mg of poly(I:C) (InvivoGen) on days 0, 3, 6, and 9. On day 12, thedeletion of Cdk8 was confirmed by PCR of DNA isolated fromperipheral blood lymphocytes. Poly(I:C)-treated mice were ana-lyzed or used for tumor challenge 7 days after the last treatment(day 16).

Themouse lymphoma cell lines YAC-1 (26) andRMA-S (kindlyprovided by Prof. A. Cerwenka; ref. 27) and the v-ablþ leukemiccell line (B6-4; generated in the laboratory of Prof. Veronika Sexl)were cultured in RPMI1640 (Sigma) complete medium andmouse melanoma cell line B16F10 (kindly provided by ThomasFelzmann; ref. 28) was cultured in DMEM (Sigma) completemedium, both containing 10% FCS (Bio & Sell), 100 U/mLpenicillin, 100 mg/mL streptomycin (Sigma) and 50 mmol/L 2-mercaptoethanol (Sigma). All cell lines were passaged up to 10times, authenticated by flow cytometry (last authenticationAugust 2017), and tested for mycoplasma contamination by theMycoplasma Detection Kit-Quick Test (Biomake, last test August2017).

In vivo tumor modelsCdk8fl/fl, Cdk8fl/flMx1Cre and Cdk8fl/flNcr1Cre mice were chal-

lenged with 5 � 104 B16F10 cells by intravenous (i.v.) injectioninto the tail vein. After 23 days, the mice were sacrificedand the pulmonary tumor nodules were counted by threeindependent researchers in a blinded manner. In the AbelsonMurine Leukemia virus (A-MuLV) model newborn Cdk8fl/fl andCdk8fl/flNcr1Cre mice were injected subcutaneously (s.c.) intothe neck fold with 100 mL of replication-incompetent ecotropicretrovirus encoding for v-abl as described previously (29). Micewere checked daily for disease onset. At the first sign of thedisease, the mice were sacrificed, body and spleen weight wasdetermined, and the white blood cell count was analyzed usingthe Scil Vet ABC (Scil Animal Care). Additionally, spleen, bonemarrow, and blood were analyzed for the infiltration of B cells

(CD19þB220þ), T cells (CD3þ), and NK cells (CD3�NKp46þ)byflow cytometry. In the v-ablmodel,Cdk8fl/fl andCdk8fl/flNcr1Cremice were injected s.c. with 106v-ablþ cells into both flanks andthe tumor onset was controlled every other day. Ten days afterinjection, the mice were sacrificed and the tumor weight wasdetermined. For flow cytometric analysis of tumor infiltratingNK cells, tumors were cut into 2 to 5 mm2 pieces and digestedfor 40 minutes at 37�C in Collagenase D (1 mg/mL; Roche) andDNAse I (20 mg/mL; Roche) before filtration to obtain singe cellsuspensions.

NK-cell isolation, expansion and stimulationNK cells were isolated from spleen single-cell suspensions

using DX5-labeled MACS beads according to the manufac-turer's instructions (Miltenyi Biotec). NK cells were expandedin RPMI1640 complete medium supplemented with 5,000 U/mL rhIL2 (Proleukin, Novartis) for 7 days. The purity wasanalyzed by flow cytometry and was 85% to 95% CD3�NK1.1þ

of living cells. For Western blot analysis of lytic molecules,expanded NK cells were stimulated with 5 ng/mL rmIL12 (R&DSystems) or 50 ng/mL rmIL15 (PeproTech) for 2 hours. For thegrowth curve analysis, cells were counted daily using a cellcounting chamber (Neubauer). For antibody- or cytokine-induced IFNg , granzyme B (GZMB), and perforin production2.5 � 106 to 5 � 106 freshly isolated splenocytes were seeded inRPMI1640 complete medium on anti-NK1.1 (PK136; 10 mg/mL) precoated tubes or in RPMI1640 complete medium sup-plemented with 5,000 U/mL IL2 and 5 ng/mL IL12 or 5,000 U/mL IL2, 5 ng/mL IL12 and 50 ng/mL IL15 for 4 hours. BDGolgiStop (BD Bioscience) was added 1 hour after the startof stimulation at 37�C. After an additional 3 hours of incuba-tion, the cells were stained for CD3, DX5, NKp46, IFNg , GZMB,and perforin and analyzed by flow cytometry. For the func-tional analysis of tumor-infiltrating NK cells, tumor single-cell suspensions were incubated in RPMI complete mediumor RPMI complete medium supplemented with 5,000 U/mLIL2, 5 ng/mL IL12, and 50 ng/mL IL15 for 4 hours in thepresence of Brefeldin A (eBioscience). The IFNg production ofCD3�NKp46þ cells was analyzed by flow cytometry.

Flow cytometry and cell sortingSingle-cell suspensions of splenocytes or bone marrow were

prepared. For blood analysis, the erythrocytes were lysed usingBD FACS Lysing Solution according to manufacturer's protocol(BD Bioscience). For the detection of intracellular proteins cellswere fixed and permeabilized with BD Cytofix/Cytoperm Fix-ation/Permeabilization Solution Kit (BD Bioscience) accordingto the manufacturer's instructions. The antibodies (clones)targeting following proteins were purchased from eBioscience:CD3 (17A2), CD3e (145-2C11), CD11b (M1/70), CD16/CD32 (93), CD27 (LG.7F9), CD49b (DX5), CD122 (5H4),CD226 (10E5), Gr-1 (RB6-8C5), GZMB (NGZB), IFNg(XMG1.2), KLRG1 (2F1), NKG2D (CX5), NKp46 (29A1.4),NK1.1 (PK136), perforin (eBioOMAK), and Ter119 (TER-119).Antibodies (clones) targeting following proteins were pur-chased from BD Bioscience: CD19 (1D3) and B220 (RA3-6B2). Flow cytometry experiments were performed on a BDFACSCanto II (BD Bioscience) and analyzed using BD FACS-Diva V8.0 or FlowJo V10 software. CD3�NK1.1þ cells weresorted from splenocyte single-cell suspensions using BD FAC-SAria III.

CDK8 Suppresses NK-Cell Activity

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Western blotCell lysis, SDS-PAGE, and Western blots were performed as

described previously (23). The detection of chemiluminescencewas performed using Clarity Western ECL substrate (BioRad) andthe ChemiDocT XRSþ Molecular Imager (BioRad) and analyzedand quantified by Image Lab software (BioRad). The followingCell Signaling Technology antibodies were used: anti-CDK8(#4106), anti–pSTAT1-S727 (#9177), anti-STAT1 (#9172),anti-perforin (#3693), and anti-GZMB (#4275). Anti-CDK19(#HPA007053) was purchased from Sigma-Aldrich. Anti–b-actin(Santa Cruz #47778) was used as loading control.

NK-cell cytotoxicity assayFor in vitro cytotoxicity assays, DX5-MACS–sortedNK cells were

expanded for 7 days in IL2 as described above and mixed atindicated effector:target ratios with carboxyfluorescein diacetatesuccinimidyl ester (CFSE, Molecular Probes, CellTrace CFSE CellProliferation Kit) labeled target cells. For the ex vivo cytotoxicity,assay freshly isolated splenocytes were mixed with CFSE-labeledtarget cells at ratios of 100:1, 50:1, and 10:1. After 3 to 4 hours ofincubation at 37�C, the specific target cell lysis was assessed byflow cytometry as previously described (30).

HistologyThe organs were fixedwith Roti-Histofix (Roth) and embedded

in paraffin. Sections (3 mm) were stained with hematoxylin andeosin (H&E; Microm HMS 740 Robot Stainer; Thermo Scientific)and scanned with a Zeiss AxioImager Z1.

Statistical analysisUnpaired t tests andKaplan–Meier plot analysis by the log-rank

test were performed using GraphPad Prism version 5.00 (Graph-Pad Software). Statistical significance is indicated for each exper-iment (�, P < 0.05; ��, P < 0.01; ��, P < 0.001).

ResultsCDK8 is dispensable for NK-cell development and maturation

We have previously shown that CDK8 phosphorylates STAT1–S727 in NK cells. NK cells lacking this phosphorylationsite display increased cytotoxicity and tumor surveillance (23).To thoroughly analyze the function of CDK8 in NK cells wegenerated mice lacking Cdk8 specifically in the NK-cell compart-ment (Cdk8fl/flNcr1Cre) andmice with a poly(I:C)-inducible dele-tion of Cdk8 in interferon-responsive cells (Cdk8fl/flMx1Cre). Theexpression of CDK8 in freshly isolated Cdk8fl/flNcr1Cre NK cellsand in vivo poly(I:C) pretreated Cdk8fl/flMx1Cre splenocytes wasreduced (Fig. 1A). In both mouse models, deletion of CDK8 hadno impact on the frequency and total numbers of NK cells inthe bone marrow (Fig. 1B; Supplementary Fig. S1A) and spleen(Fig. 1C; Supplementary Fig. S1B). NK cells develop in the bonemarrow and further mature in the periphery. CDK8 did not affectthe distribution of individual developmental stages of NK cellsin the bone marrow (Fig. 1D). Similarly, maturation of NK cellsin the spleen was unchanged, as the percentage of NK cells invarious maturation stages as defined by expression of CD27 andCD11b was comparable between Cdk8fl/flNcr1Cre, Cdk8fl/flMx1Creand their Cdk8fl/fl littermate controls (Fig. 1E; SupplementaryFig. S1C).

Activity ofNKcells is controlled by abalance between activatingand inhibitory receptors. We thus investigated the expression ofthematuration-associated inhibitory receptor KLRG1 (31) as well

as DNAM-1 and NKG2D, two activating receptors involved inantitumor responses (32), on the surface of Cdk8fl/flNcr1Cre andcontrol NK cells. We found no differences in the frequency orexpression of these receptors (Fig. 1F; Supplementary Table S1).Poly(I:C) treatment increased the frequency of KLRG1þ cells andthe expression of DNAM-1 but this effect did not rely on CDK8expression (Fig. 1F; Supplementary Table S1). We concluded thatneither NK cell–intrinsic nor –extrinsic CDK8 influences NK-celldevelopment, maturation, and expression of NK-cell receptors.

CDK8-deficient NK cells are more cytotoxic in vitroTo investigate the impact of CDK8 on NK-cell function in vitro,

we expanded NK cells in the presence of IL2 for 7 days. Thedeletion of CDK8 was confirmed (Fig. 2A). CDK8 deletion inexpanded NK cells was more efficient than in freshly isolated andunstimulated NK cells, as NK cells in vitro upregulate CDK8 (Fig.2A; Supplementary Fig. S2A). Although CDK8 has been describedas anupstreamkinaseof STAT1–S727 (19, 23),wedidnotobservean impact of CDK8 deficiency on STAT1–S727 phosphorylationin IL2-expanded NK cells (Fig. 2A). We reasoned that the CDK8paralog CDK19 may compensate for CDK8 and thus for STAT1phosphorylation. Indeed, we found enhanced expression ofCDK19 in IL2-cultured Cdk8fl/flNcr1Cre NK cells (Fig. 2B). LossofCDK8didnot alter the growthofNKcellswhen stimulatedwithIL2 (Fig. 2C). Similarly, IFNg production after short-term stim-ulation of ex vivoNK cells with IL12 or IL12þ IL15was unaffectedby the decrease in CDK8 expression. Stimulation with anti-NK1.1resulted in a 10% decrease of IFNg production byCdk8fl/flNcr1CreNK cells (Fig. 2D). This effect was not caused by differentialexpression of NK1.1 (Supplementary Fig. S2B).

The most pronounced changes upon CDK8 deletion wereobserved when we studied components of the lytic machinery.Expression of perforin was 2-fold upregulated in IL2 expandedCDK8-deficient NK cells in response to IL12 or IL15 (Fig. 2E;Supplementary Fig. S2C). This effect extended to ex vivo IL2þ IL12þ IL15 activated NK cells, confirming that our observations arenot restricted to IL2 stimulation in vitro (Fig. 2F). The expression ofGZMB was also 10% enhanced upon CDK8 deletion, but thiseffect was restricted to IL2-expanded NK cells (Fig. 2E and F;Supplementary Fig. S2C). Upon IL2 expansion, the increase inexpression of effector molecules in CDK8-deficient NK cellstranslated into enhanced cytotoxicity against YAC-1 target cellsin vitro (Fig. 2G). The enhanced killing capacity was not detectedagainst B16F10 melanoma and v-ablþ leukemic target cells (Fig.2G). This may suggest that the increased abundance of effectormolecules observed in CDK8-deficient NK cells is not sufficient toincrease their cytotoxic capacity against all target cell lines. To testwhether Cdk8fl/flNcr1CreNK cells show increased activity withoutstimulation, we performed an ex vivo cytotoxicity assay usingwhole splenocytes from na€�ve mice. Irrespective of CDK8 expres-sion, we detected no killing of RMA-S target cells after 4 hours(Supplementary Fig. S2D). In conclusion, CDK8 restrains theexpression of the lytic machinery in cytokine-activated NK cells,which leads to an enhanced cytotoxic capacity toward YAC-1 butnot B16F10 or v-ablþ target cells.

In vivo tumor surveillance is enhanced by CDK8 deletion in NKcells

To evaluate the impact and relevance of our in vitro findings, weused an established in vivo model for NK cell–mediated tumorsurveillance (25) and injected the poly(I:C) pretreated Cdk8fl/fl

Witalisz-Siepracka et al.

Cancer Immunol Res; 6(4) April 2018 Cancer Immunology Research460




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Cdk8fl/ fl Cdk8fl/flNcr1Cre Cdk8fl/ fl Cdk8fl/flMx1Cre

Figure 1.

Loss of CDK8 is dispensable for NK-cell development and maturation. A, Sorted Cdk8fl/fl and Cdk8fl/flNcr1Cre splenic NK cells (left) or poly(I:C)-treated Cdk8fl/fl

andCdk8fl/flMx1Cre splenocytes (right) were isolated and the protein level of CDK8was analyzed byWestern blot. B, Frequency of Lin�(CD3�CD19�Ly-6G�Ter119�)CD122þ NK cells in bone marrow and (C) percentages of CD3�NKp46þ NK cells in the spleen were assessed by flow cytometry. D, Bone marrow Lin�CD122þ

cells were further divided into NK precursors (NKPs: NKp46�NK1.1�), immature NK cells (iNKs: NKp46�NK1.1þ), and mature NK cells (mNKs: NKp46þNK1.1þ).E, Splenic CD3�NKp46þ cells were analyzed for the expression of the maturation markers CD27 and CD11b. Shown are representative plots. The summarizeddata is presented in Supplementary Fig. S1. F, The abundance of KLRG1þ, NKG2Dþ, and DNAM-1þ cells among CD3�NKp46þ NK cells was assessed in the spleenof Cdk8fl/fl, Cdk8fl/flNcr1Cre, and poly(I:C)-treated Cdk8fl/fl and Cdk8fl/flMx1Cre mice. Shown are representative histograms. The summarized data arepresented in Supplementary Table S1. B–E, Bar graphs and values on the plots represent mean � SEM of 2 independent experiments; n ¼ 6–7.






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Figure 2.

CDK8-deficient NK cells show an increased cytotoxic capacity. A and B, IL2-expanded Cdk8fl/fl and Cdk8fl/flNcr1Cre NK cells were stimulated with IL12 or IL15 for 2hours, and the expression of (A) CDK8, pSTAT1–S727, and total STAT1 and (B) CDK19 was analyzed byWestern blot. C,MACS-purified Cdk8fl/fl and Cdk8fl/flNcr1CreNK cells were plated in IL2 and the cell numbers were determined daily by manual cell counting using a Neubauer chamber. Symbols and error bars represent meancell number � SEM of technical triplicates from 1 out of 2 independent experiments with similar outcome. D, Cdk8fl/fl and Cdk8fl/flNcr1Cre splenocytes werestimulated with anti-NK1.1, IL2þ IL12, or IL2þ IL12þ IL15 for 4 hours and the percentage of IFNgþ CD3�NKp46þNK cells was analyzed by flow cytometry. Bar graphrepresents mean percentage of IFNgþ NK cells � SEM from 2 to 3 independent experiments; n ¼ 4–6 per group. E, IL2 expanded Cdk8fl/fl and Cdk8fl/flNcr1CreNK cells were stimulated with IL12 or IL15 for 2 hours and the expression of perforin and GZMB was analyzed by Western blot. F, Cdk8fl/fl and Cdk8fl/flNcr1Cresplenocytes were stimulated with IL2 þ IL12 or IL2 þ IL12 þ IL15 for 4 hours and the expression of perforin and GZMB in CD3�NKp46þ cells was analyzed byintracellular staining. Representative histograms were overlaid. Bar graph represents mean MFI of perforin in NK cells or percentage of GZMBþ NK cells� SEM from 2 independent experiments; n¼ 4 per group. G, IL2-expanded Cdk8fl/fl and Cdk8fl/flNcr1Cre NK cells were incubated for 3 to 4 hours with CFSE-stainedYAC-1, B16F10, or v-ablþ target cells in effector-to-target ratios of 10:1, 5:1, and 1:1. The specific lysis of target cells was assessed by flow cytometry and arepresentative graph out of 2 independent experiments is shown. Symbols and error bars represent mean � SEM of technical triplicates. � , P < 0.05; �� , P < 0.01.






andCdk8fl/flMx1Cremice i.v. with B16F10melanoma cells. Tumorburden in the lungs was analyzed 23 days thereafter. The numberof pulmonary tumor nodules inmice lacking CDK8 in interferon-responsive cells was diminished (Fig. 3A). To confirm that the

observed effect on tumor surveillance is mediated by NK cells, weinjected the B16F10 cells i.v. intoCdk8fl/flNcr1Cremice. Mice withdiminished CDK8 in NKp46þ cells were better able to suppressformation of B16F10 lung tumor nodules than were littermate

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Figure 3.

Mice lacking CDK8 in NK cells showreduced B16F10 lung metastasis. A,Cdk8fl/fland Cdk8fl/flMx1Cremice weretreated with poly(I:C) as described inthe Materials and Methods section. Onday 16, 5� 104 B16F10 melanoma cellswere injected i.v. into the mice. After23 days, the number of pulmonarytumor nodules was assessed.B, 5 � 104 B16F10 melanoma cellswere injected i.v. into Cdk8fl/fl andCdk8fl/flNcr1Cre mice. After 23 days,the number of tumor nodules in thelung was assessed. A, B, Shown arerepresentative lung pictures (left) andbar graphs representing mean � SEMfrom 3 independent experiments;n ¼ 11–12. � , P < 0.05.

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Figure 4.

Loss of CDK8 in NK cells results inimproved antitumor response againstv-ablþ leukemic cells. Cdk8fl/fl andCdk8fl/flNcr1Cremicewere injected s.c.with 106 v-ablþ cells and after 10 daysthe tumor weight was assessed andtumor infiltrating NK cells wereanalyzed by flow cytometry. Shownare (A) representative tumor picturesand (B) bar graph representingmean tumor weight relative to bodyweight � SEM from 2 independentexperiments; n ¼ 12–15. C,Representative plots of tumorinfiltrating CD3�NKp46þ cellspregated on CD19� cells from Cdk8fl/fl

(left panel) and Cdk8fl/flNcr1Cre mice(right panel) are shown and (D) theirpercentage among CD19� cells ispresented as bar graph showing mean� SEM from 2 independentexperiments; n ¼ 11. E and F, Tumorsingle-cell suspensions were leftuntreated or stimulatedwith IL2þ IL12þ IL15 for 4 hours and the percentageof IFNgþCD3�NKp46þ NK cells wasanalyzed by flow cytometry. E,Representative histograms ofunstimulated and stimulated tumorsamples were overlaid. F, The bargraph represents mean percentageof IFNgþ NK cells � SEM from 1experiment; n ¼ 4 per group;� , P < 0.05; �� , P < 0.01.






controls (Fig. 3B). This led us to conclude that the radical decreasein CDK8 expression in the NK-cell compartment results inenhanced in vivo tumor surveillance despite the absence of observ-able changes in vitro. In vitro changes were probably masked byexperimental conditions including prolonged cytokine stimula-tion. Similar effects were seen when we injected v-ablþ trans-formed cells s.c. into Cdk8fl/fl and Cdk8fl/flNcr1Cre mice. Again,depletionofCDK8 inNK cells restrained tumor growth, asCdk8fl/fl

Ncr1Cre mice showed smaller tumors than their littermate con-trols (Fig. 4A and B). To investigate themechanismunderlying theimproved tumor surveillance, we analyzed NK-cell infiltrationinto the solid tumors by flow cytometry. Indeed, Cdk8fl/flNcr1CreNK cells infiltrated the tumors more efficiently (Fig. 4C and D).However, we could not detect differences in IFNg productionby tumor infiltrating NK cells between the Cdk8fl/fl and Cdk8fl/fl

Ncr1Cre mice (Fig. 4E and F). We thus concluded that enhancedNK-cell functionality as well as increased frequency of NK cells inthe tumor environment both contribute to improved control oftumor growth in vivo.

Although tumor transplantation models are well defined,fast, and versatile, they fail to mimic the slow growth of a tumorin vivo. To study the influence of CDK8 in such a setting, weinjected newborn Cdk8fl/fl and Cdk8fl/flNcr1Cre mice with theAbelson Murine Leukemia virus (A-MuLV). The virus induces aslowly progressing pro-B cell leukemia that is controlled in anNK cell–dependent manner (33). As depicted in Fig. 5A, mice

expressing little CDK8 in NK cells showed prolonged survival.Nonetheless, at the time of sacrifice, the symptoms of leukemiawere comparable in both groups, as assessed by spleen weight,white blood cell count (Fig. 5B), and infiltration of malignant Bcells in the bone marrow (Fig. 5C and D). In this model, theenhanced tumor surveillance was not paralleled by an increasednumber of immune cells; the frequency of T and NK cells inbone marrow, spleen, and blood of the leukemic mice wascomparable (Fig. 5E). This led us to conclude that in the slowlyevolving leukemia model, the increased capability of Cdk8fl/fl

Ncr1Cre NK cells to kill emerging tumor cells resulted inprolonged survival of the affected mice (Fig. 5A). Our resultssuggest that targeting CDK8 could improve NK-cell function inthe context of tumor immunotherapy.

DiscussionImmunotherapy can improve life expectancy and survival of

patients suffering from cancer. A greater variety of anticancertreatment strategies, including those that target the immunesystem, is needed (34). We here show that deletion of CDK8 inNK cells enhanced antitumor responses against B16F10 melano-ma cells and v-abl–transformed lymphoma cells. In addition,CDK8 deletion prolonged disease latency in a slowly progressingmodel system for leukemia. These results define CDK8 as worthtargeting in our battle against cancer.

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Figure 5.

Loss of CDK8 in NK cells provides a survival benefit in a chronic leukemia model. Newborn Cdk8fl/fl (n ¼ 10) and Cdk8fl/flNcr1Cre (n ¼ 8) mice were injected s.c.with a replication-incompetent ecotropic retrovirus encoding for v-abl (A-MuLV). A, The Kaplan–Meier plot summarizes 2 independent experiments. B, Thespleen/body weight ratio and the white blood cell count (WBC) at the survival endpoint are represented by bar graphs showing mean � SEM of 2 independentexperiments. C, Representative plots of bone marrow–infiltrating B cells (CD19þB220þ) are shown, and the values represent mean � SEM of one representativeout of 2 independent experiments (n ¼ 4–5). D, H&E stains of bone marrow from leukemia-bearing Cdk8fl/fl and Cdk8fl/flNcr1Cre mice were performed at thedisease endpoint. E, The frequency of CD3�NKp46þ NK and CD3þ T cells amongst splenic, bone marrow or blood lymphocytes of leukemia-bearing mice at thedisease endpoint were determined by flow cytometry. Bar graph represents mean � SEM of one representative out of 2 independent experiments (n ¼ 4–5).� , P < 0.05.






We have previously shown that mice harboring a point muta-tion in a STAT1 serine phosphorylation site (Stat1S727A) showincreased protection against tumor spread by enhanced NK cell–mediated cytotoxicity (23). We and others have identified CDK8as an upstream kinase for STAT1–S727 phosphorylation (19, 23).CDK8 knockdown in an immortalized NK cell line also increasescytotoxicity in vitro (23). Our findings described in this studylargely recapitulated and extended our previous observationswith one exception: we were intrigued to see that the STAT1–S727 phosphorylation is preserved upon CDK8 deletion inIL2-expanded NK cells. In the long-term absence of CDK8 in NKcells, as in a knockout mouse model, CDK19, which is a paralogkinase of CDK8, becomes upregulated and might phosphorylateSTAT1–S727. It is unknown if and howCDK19 influences NK-cellbiology. The compensatory upregulation seems to be cell typespecific as no such effects have been observed in human coloncancer cell lines upon CDK8 knockout (35). Even though CDK19may compensate for the lack of CDK8, we do not know howCDK8 deletion in the presence of an intact STAT1–S727 phos-phorylation enhances cytotoxicity. An intact STAT1 transactiva-tion domain is critical for efficient recruitment of CDK8 (19).Therefore, we speculate that the enhanced cytotoxicity ofStat1S727ANK cells recapitulates the reduced recruitment of CDK8to specific STAT1-dependent genes. Alternatively, the effects ofCDK8 deletion may be unrelated to STAT1-dependent transcrip-tion and the enhanced NK-cell surveillance may be caused byother mechanisms. CDK8, which interacts with a variety oftranscription factors (1), also phosphorylates SMAD proteinsdownstream of the cytokine TGFb (8), a suppressor of NK-cellfunction (36). CDK8 may regulate NK-cell activity in a SMAD-dependent manner and the deletion of CDK8 could impair TGFbsignaling.Moreover, CDK8deficiencydrives epigenetic changes inchromatin: loss of CDK8 in intestinal cells causes a reduction inhistone trimethylation on H3K27 due to the repression of EZH2activity (37). As EZH2-deficient NK cells show enhanced cyto-toxicity against tumor cells (38),we speculate thatCDK8-deficientNK cells also harbor changes in H3K27 trimethylation that coulddrive increased NK-cell activity. Besides acting on specific tran-scription factors, CDK8 regulates transcription in a globalmannerby taking part in the mediator complex together with MED12,MED13, and cyclin C (1). It remains unclear whether the effects ofCDK8 deletion in NK cells involve the mediator complex. Noinformation is available on other mediator subunits for NK-cellbiology. MED12 mutations have been described in post-trans-plant T lymphoproliferative disorders (39) and cyclin C has beenidentified as tumor suppressor in T-cell acute lymphoblastic

leukemia (40), suggesting a complex role of themediator complexin the regulation of T lymphocytes.

CDK8 is deregulated in several types of cancer, including breastand colon cancer, and is thus a promising therapeutic target (14).In summary, we have shown CDK8 does not influence NK celldevelopment and proliferation, but instead suppresses NK-cellantitumor activity in vivo. Although the molecular mechanismsbehind these effects remain unclear, our results implicate thattargeting CDK8 in cancer patients holds high therapeutic poten-tial. Blocking or degrading CDK8 will not only act on the tumorcells but also enhance NK-cell lytic responses.

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

Authors' ContributionsConception and design: A. Witalisz-Siepracka, D. Gotthardt, V. SexlDevelopment of methodology: D. Gotthardt, V. SexlAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):A.Witalisz-Siepracka,D.Gotthardt,M. Prchal-Murphy,Z. Didara, I. Menzl, L. Edlinger, E.M. PutzAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Witalisz-Siepracka, D. Gotthardt, Z. Didara,L. Edlinger, V. SexlWriting, review, and/or revision of the manuscript: A. Witalisz-Siepracka,D. Gotthardt, I. Menzl, E.M. Putz, V. SexlAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Z. Didara, D. PrinzStudy supervision: V. Sexl

AcknowledgmentsThe work was supported by the Austrian Science Fund FWF (grant P28571 to

V. Sexl, the Schroedinger Fellowship J-3635 to E.M. Putz, and the PhD program"Inflammation and Immunity" FWF W1212 to V. Sexl).

We would like to thank the European Conditional Mouse MutagenesisConsortium for providing the Cdk8tm1a(EUCOMM)Hmgu mouse strain andMohammed Selloum (Yann Herault Group) for providing the Cdk8tm1c

(EUCOMM)Hmgu mouse strain. We are grateful to Thomas R€ulicke and ThomasKolbe for their help in acquiring the mice and to all members of the mousefacility. We thank all members of the SFB F61, in particular Thomas Decker andMathias M€uller, for discussions and scientific input. We are indebted to SabineFajmann and Petra Kudweis for excellent technical assistance.

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 April 10, 2017; revised September 15, 2017; accepted January 26,2018; published OnlineFirst January 31, 2018.

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2018;6:458-466. Published OnlineFirst January 31, 2018.Cancer Immunol Res Agnieszka Witalisz-Siepracka, Dagmar Gotthardt, Michaela Prchal-Murphy, et al.

Specific CDK8 Deletion Enhances Antitumor Responses−NK Cell

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