Inactivation of Mirk/Dyrk1b Kinase Targets Quiescent ......Oct 13, 2011 · Preclinical Development...

Preclinical Development

Inactivation of Mirk/Dyrk1b Kinase Targets QuiescentPancreatic Cancer Cells

Daina Z. Ewton1, Jing Hu1, Maria Vilenchik2, Xiaobing Deng1, Kin-chun Luk2, Ann Polonskaia2,Ann F. Hoffman2, Karen Zipf2, John F. Boylan2, and Eileen A. Friedman1,3

AbstractAmajor problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive

to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or

recurrence when they reenter the cell cycle. Earlier studies showed that levels of the serine/theronine kinase

Mirk/dyrk1B are elevated up to 10-fold in quiescent G0 tumor cells. Mirk uses several mechanisms to block

cell cycling, and Mirk increases expression of antioxidant genes that decrease reactive oxygen species (ROS)

levels and increase quiescent cell viability. We now show that a novel small molecule Mirk kinase inhibitor

blocked tumor cells from undergoing reversible arrest in a quiescent G0 state and enabled some cells to exit

quiescence. The inhibitor increased cycling in Panc1, AsPc1, and SW620 cells that expressed Mirk, but not in

HCT116 cells that did not.Mirk kinase inhibition elevated ROS levels andDNAdamage detected by increased

phosphorylation of the histone protein H2AX and by S-phase checkpoints. The Mirk kinase inhibitor

increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold

by gemcitabine and cisplatin. A phenocopy of these effects occurred following Mirk depletion, showing drug

specificity. In previous studies Mirk knockout or depletion had no detectable effect on normal tissue,

suggesting that the Mirk kinase inhibitor could have a selective effect on cancer cells expressing elevated

levels of Mirk kinase. Mol Cancer Ther; 1–11. �2011 AACR.

Introduction

Quiescent cancer cells are relatively insensitive to mostchemotherapeutic drugs and radiation and can causetumor recurrence when they reenter the cell cycle. Inaddition, metastatic cancer cells in the bloodstream mayexperience a period of dormancy or quiescence whilethey adapt to their new microenvironment (1). Quiescenttumor cells degrade their polyribosomes, thus blockingtranslation and reducing total RNA and protein content.These shrunk tumor cells may be able to enter the boresof capillaries (approximately 8 mm diameter) whereascycling tumor cells are usually much larger (20–30mm). Because G0 cells have 2NDNA, but total RNA levelslower than G1 or S-phase cells, they are readily identifiedby 2-parameter flow cytometry (2). Tumor cells could

either enter an irreversible G0 state before undergoingapoptosis, terminal differentiation, or senescence, or en-ter a reversible, terminal differentiation suppressed, truequiescent G0 state or dormant state from which theycould resume cycling, like quiescent fibroblasts (3). Inour previous studies, cultures of serum-starved cancercells were enriched in G0 cells (60%–90%; refs. 2, 4) andhad up to 10-fold higher protein levels of the activatedkinase Mirk/dyrk1b and the CDK inhibitor p27kip1, aswell as 16-fold higher levels of the retinoblastoma proteinfamily member p130/Rb2 that sequesters the E2F4 tran-scription factor, thus preventing entry into cycle. Mirkinduction of a set of antioxidant genes kept G0 cells viableby reducing their reactive oxygen species (ROS) levels(2, 5). Oncogenes such as mutant ras, the rapid growth oftumor cells, and metabolic stress all raise ROS levels (6).Mirk depletion increased ROS levels in each of 4 lines ofovarian cancer cells, enabling low doses of the chemo-therapeutic drug cisplatin (which by itself increases ROSlevels) to raise cellular ROS to toxic levels (5). Pharma-cologic inhibition of Mirk kinase could prevent someantioxidant genes from being induced, raising ROSlevels, and thus increasing sensitivity to drugs such asgemcitabine, which also raise ROS levels. The difluoro-deoxycytidine gemcitabine is used for treatment of pan-creatic cancer. ROS production plays a role in the drug’scytotoxicity, as gemcitabine doubled the levels of ROS inT3M4 pancreatic cancer cells, whereas reduction of ROSby the free-radical scavenger N-acetylcysteine decreased

Authors' Affiliations: 1Pathology Department, SUNY Upstate MedicalUniversity, Syracuse, New York and 2Discovery Oncology, DiscoveryChemistry and Discovery Technologies, Hoffmann-La Roche, Nutley,New Jersey

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Eileen Friedman, SUNY Upstate Medical Univer-sity, Department of Pathology, 2305 Weiskotten Hall, 750 East AdamsStreet, Syracuse, New York 13210. Phone: 315-464-7148; Fax: 315-464-8419; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0498

�2011 American Association for Cancer Research.

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Therapeutics

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the growth inhibition caused by gemcitabine (7). More-over, when 10 pancreatic cancer cell lines were surveyedfor ROS levels, the lines with higher endogenous ROSlevels were more sensitive to the cytotoxic effects ofgemcitabine, suggesting that gemcitabine furtherincreases the already high endogenous ROS levels oftumor cells to toxic levels (7). In patients, the responserate to gemcitabine is less than 20% because of drugresistance caused by various mechanisms including re-duced gemcitabine metabolism (reviewed in ref. 8) anddecreased delivery of gemcitabine because of poor per-fusion of the tumor because of its thick desmoplasticstroma (9). Stratagems to increase delivery of gemcitabinein animal models by inhibiting stromal hedgehog signal-ing have shown some success (9). Thus, identifyingmethods to increase gemcitabine toxicity toward pancre-atic cancer cells is timely. In this study we tested whetherinactivation of Mirk kinase in quiescent cells using anovel small molecule inhibitor increased pancreaticcancer cell sensitivity to gemcitabine.

Materials and Methods

MaterialsAffinity-purified polyclonal antibody to the Mirk

unique C terminus was described previously (10). Anti-bodies toactivatedphosphorylated gH2AX,LC3b, cleavedcaspase 3, Dyrk1A, cleaved PARP, bromodeoxyuridine(BrdU), and the senescence-associated b-galactosidaseassay kit were fromCell Signaling. Gemcitabine (Gemzar)was from Eli Lilly. All other reagents including cisplatinwere from Sigma. The human cancer cell lines wereobtained fromtheAmericanTypeCultureCollection,withnewstocks obtained in 2010 andnot further authenticated,and maintained in Dulbecco’s Modified Eagle’s Medium(DMEM) supplemented with 2 mmol/L glutamine, anti-biotics, and 10% heat-inactivated FBS. SU86.86, Panc1,AsPc1, SU86.86/shMirk, and Panc1/shMirk cells werenegative for mycoplasma (Sigma test kit).

Mirk kinase inhibitorThe Mirk kinase inhibitor was identified from a Roche

generic kinase inhibitor library by screening an Ambitkinase library (KINOMEscan) of 317 kinases. CompoundA was the most selective inhibitor among the varioushits when compared by a Caliper kinase profiling assay.A series of analogs of compound A was synthesized.These analogs were assayed for IC50 on purified Dyrk1Band Dyrk1A by using a synthetic substrate, FITC-RRRFRPASLRGPPK, and for EC50 and EC90 on SW620colon carcinoma cells (K. Luk, manuscript in prepara-tion), and the most active analog was used in the currentstudies. Structure of the Mirk kinase inhibitor is shown inFig. 2A. This analog had an EC50 of 1.9 � 0.2 mmol/L onSW620 cells and an IC50 of 68 � 48 nmol/L on Mirk/dyrk1B and 22 � 8 nmol/L on Dyrk1A. To determinespecificity, this Mirk kinase inhibitor was used at a muchhigher concentration of 10 mmol/L in a kinase assay

(Profiler Pro Kit; Caliper Life Sciences) and inhibitedthe activities of DYRK1A, ABL, FLT3, and MARK1 by88%, 64%, 56%, and 73%, respectively. The Mirk kinaseinhibitor was inactive against the other kinases in theCaliper assay panel: AKT1, AKT2, AMPK, AurA, BTK,CAMK2, CAMK4, CDK2, CHK1, CHK2, CK1d, c-Raf,c-TAK1, Erk1, Erk2, FGFR1, FYN, GSK3b, HGK, IGF1R,INSR, IRAK4, KDR, LCK, LYN, MAPKAPK2, MAP-KAPK5, MET, MSK1, MST2, p38a, p70S6K, PAK2,PIM2, PKA, PKCb, PKCz, PKD2, PKG1a, PRAK, ROCK2,RSK1, SGK1, SRC, SYK.

Induction of quiescencePanc1 or SU86.86 pancreatic cancer cells were plated at

4 � 105 cells per 60-mm dish in growth medium (DMEMþ 10% FBS) and allowed to attach overnight. Media waschanged to DMEMþ 0.2% FBS (tetracycline-negative FBSif doxycycline-inducible constructs were used) and cellswere cultured for 2 to 4 days to become quiescent, with 1mg/mL doxycycline added to induce the short hairpin(sh)Mirk constructs to deplete Mirk, as needed.

RNA interferencePancreatic cancer cell pools SU86.86/shMirk, Panc1/

shMirk, and SU86.86/sh-Luc stably expressed doxycy-cline inducible short hairpin RNAs (shRNA) to MirkmRNA sequences starting at base pair 530 in exon 4,Mirk mRNA sequences starting at base pair 1,699 in exon11, and the nonmammalian luciferase gene, respectively.Synthetic RNA interference (RNAi) duplexes to Mirkwere from Invitrogen. The RNAi 50-GTGGTGAAAGCC-TATGATCAT-30 targeted a sequence in Mirk exon 5.Transfections were done as in ref. 2.

Assay of senescenceConfluent cultures of late-passage human diploid

fibroblasts, strain BJ, were assayed for senescence-asso-ciated b-galactosidase activity at pH 6.0 by histochemis-try (11).

Quantitation of Western blotsDensitometry analysis of scanned autoradiographs

was carried out by UN-SCAN-IT gel software (Silk Sci-entific).

Flow cytometryFor analysis of DNA content only, cells were fixed with

70% ethanol and then treated with RNase A, before aminimum of 10,000 propidium iodide (PI) stained cellswere analyzed by the LSR II. For determination of DNAand RNA content to distinguish G0 from G1 cells, 2-parameter cell-cycle analyses were carried out on cellsfixed in ice cold 70% ethanol, then washed and Hoechst33258 added to bind to DNA and block DNA staining byPyronin Y, followed by Pyronin Y exactly as detailed (2).Separate and simultaneous analyses of intracellular DNAcontent and BrdU uptake were carried out on a FACScanflow cytometer (Becton Dickinson). After a 1-hour BrdU

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pulse (10 mmol/L), harvested cells were fixed with 70%ethanol, treated with an anti–BrdU-FITC conjugated an-tibody (BD Biosciences), and costained with 0.01 mg/mLof PI (BD Biosciences). PI and BrdU were excited at488 nm. A total of 3 � 104 cells/sample were analyzedat a rate of 100 to 200 cells/s. Data were analyzed byFlowJo software.

Results

Pancreatic cancer cells can enter a reversible G0

quiescent phase where they exhibit low levels ofapoptosis and senescenceQuiescent cells degrade their polyribosomes allowing

G0 cells to be distinguished from G1 cells by their 2NDNA content, but lower RNA content, by 2-parameterflow cytometry. Tumor cell lines that contained an activeMirk kinase—SU86.86, Panc1, AsPc1, SW620—werestudied (2), as Mirk is activated by oncogenic K-rassignaling (12). Quiescence was induced in SU86.86 pan-creatic cancer cells by 3 days of serum starvation, accu-mulating 50% to 60% of cells in G0, whereas in culturesmaintained in log phase growth only approximately 5%of cells were in G0 (Fig. 1A). The arrest was reversible,because only 1% of cells remained in G0 after quiescentcultures were placed in fresh growth media containing10% FBS for 24 hours. Most cells had traversed the cellcycle and were either arrested in G2 þ M by the mitoticinhibitor nocodazole or had slipped through mitoticarrest to a 4Nþ state (Fig. 1A). Only after PI staining,analysis of parallel cultures by 1-parameter flow cyto-metry for DNA content showed similar findings thatunder serum deprivation most cells have a 2N DNAcontent (G0 or G1); most quiescent cells released intogrowth medium moved through the cell cycle to bearrested by nocodazole in G2 þ M, and few apoptoticsub-G0–G1 cells were seen. Similarly, 50% to 60% of Panc1pancreatic cancer cells were accumulated in G0 by 3 daysof serum starvation and the arrest was reversible whenthe quiescent cells were released into fresh growth me-dium (Fig. 2B, insets). To further define the quiescentstate, biochemical markers for apoptosis, senescence, andautophagy were examined. Few sub-G0–G1 SU86.86 cellsor Panc1 cells were seen by either cytometric analysis inquiescent cultures, consistent with only a 2-fold increasein the low level of the apoptotic protein cleaved caspase 3,(Fig. 1A, right). Next, measurement of senescence-asso-ciated b-galactosidase activity at pH 6.0 (13) by histo-chemistry showed only 0.5% (1 of 194) and 1.8% (3 of 163)senescent cells in cultures of quiescent SU86.86 cells andquiescent Panc1 cells, respectively. The positive controlwas confluent, late-passage BJ human diploid fibroblastcultures containing 66% (125 of 189) senescent BJ fibro-blasts. Autophagy has been convincingly associated withovarian cancer cell dormancy as cells reduce their rRNAand protein levels (14). Panc1 cells and SU86.86 cells inlog phase growth exhibited low levels of the autophagicmarker LC3b-II, which were strongly increased when

cells became quiescent (Fig. 1A, right; controls for autop-hagy induction in Supplementary Fig. S1). Thus, fewquiescent cells were lost by apoptosis or senescence,but cells underwent autophagy to reduce their size andmetabolic activity.

G0 cells are maintained after many cycling cells arekilled by gemcitabine

Quiescent cancer cells in vivo are a clinical problembecause of lack of sensitivity to chemotherapeutic drugs.To test whether the quiescent G0 cancer cells found intissue culture had this property and so modeled the invivo state, SU86.86 cells were either treated with gemci-tabine at the same time as they were made quiescent byculture in low serummedium (Fig. 1B, left), or first madequiescent and then treated with gemcitabine (Fig. 1B,right). After 24 hours, the fraction of G0 cells increasedcompared with cultures in normal growth medium.However, most gemcitabine-treated cells under quiescentculture conditions initially accumulated at the G1–S-phase boundary, probably at early S-phase checkpoints,as described by others (15, 16). Loss of cycling cells andappearance of sub-G0–G1 apoptotic cells were seen with 3to 5 days gemcitabine treatment (Fig. 1B, left). When theculture was already quiescent, then treated with gemci-tabine for 1 to 5 days, the G0 fraction increased and deadsub-G0 cells accumulated as the number of viable cellsdecreased (Fig. 1B, right). Thus, SU86.86 cell culturesthat remained after gemcitabine treatment containedG0 quiescent cells. Entry into quiescence may be a generalstress response as ovarian cancer cells starved of FBS ornutrients accumulated in G0 (17).

Characterization of Mirk kinase inhibitor in cellsMirk kinase phosphorylates several different cell-cycle

regulators to maintain cells in quiescence. Mirk destabi-lizes cyclin D isoforms by phosphorylation at a knownubiquitination site, preventing passage throughG1 (4, 18).Mirk phosphorylates and stabilizes the CDK inhibitorp27, which is elevated in quiescent cells (2, 19, 20). BothMirk/dyrk1B and Dyrk1A phosphorylate LIN52-ser28,which is required for the assembly of the DREAM com-plex consisting of p130/Rb2 and bound E2F4 and DP1, aswell as the core proteins LIN9, LIN37, LIN52, LIN54, andRBBP4 (21). Assembly of the DREAM complex is neces-sary for cells to enter quiescence. As a result, depletion ofMirk allowed SU86.86 pancreatic cancer cells to escapequiescence by increasing phosphorylation of p130/Rb2,causing loss of sequestered E2F4 and thus disassembly ofthe DREAM complex (2). The Mirk kinase inhibitorshould have a similar effect if it targets Mirk within cells.The effect of the Mirk kinase inhibitor on cell cycling wascompared in 2 colon cancer cell lines, SW620 cells whichexpress Mirk/dyrk1B kinase and Dyrk1A and, as a con-trol for off-target effects, HCT116 cells which only expressDyrk1A (Fig. 2A, Western blot, bottom). Cycling cellswere treated with the inhibitor for 24 hours followed by a1-hour BrdU pulse. Flow cytometry analysis showed that

Mirk/dyrk1B in Pancreatic Cancer Cell Quiescence

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the fraction of S-phase cells was increased 29% by theMirk kinase inhibitor in SW620 cells, whereas no changewas seen in parallel cultures of HCT116 cells, which donot express Mirk/dyrk1B (Fig. 2A). Although the inhib-itor was effective against each kinase in vitro, Mirk/dyrk1B, not Dyrk1A, was responsible for the change inthe cycling of these tumor cells.

The Mirk kinase inhibitor blocked accumulation ofquiescent cancer cellsThe Mirk role in allowing cycling tumor cells to enter a

reversible quiescent state was compared in Panc1 andAsPc1 cells, which express active Mirk kinase (19). Panc1cells represent 10% of the pancreatic cancers with anamplified Mirk gene (22), whereas the Mirk gene is notamplified in AsPc1 cells (23). The Mirk kinase inhibitorwas added to log phase cultures at the time when theywere switched to fresh DMEM þ 0.2% FBS (Fig. 2B andC). In parallel cultures without inhibitor, approximatelyhalf of the Panc1 or AsPc1 cells accumulated in G0,whereas in cultures treatedwith theMirk kinase inhibitoronly 14% of Panc1 cells and 29% of AsPc1 cells werefound in G0. In 4 replicate experiments the Mirk kinaseinhibitor blocked these cancer cells from remaining in G0

during serum starvation. Treatment with the Mirk kinaseinhibitor, like Mirk depletion (2), increased amounts ofcyclin D1 by 3-fold (data not shown), consistent with cellsescaping into cycle. Thus, Mirk kinase activity was nec-essary for Panc1 and AsPc1 pancreatic cancer cells toaccumulate in a quiescent state. In contrast, treatment ofeither of the 2 strains of human diploid fibroblasts withthe Mirk kinase inhibitor at a range of concentrations didnot increase their cycling (Hu and Friedman, manuscriptin preparation). Diploid fibroblasts express very lowlevels of Mirk like most other normal tissues (10), incontrast to the elevated levels of Mirk found in mostpancreatic cancers (19).

Mirk depletion in Panc1 cells also reduces thefraction of quiescent cellsIf the increase in cycling caused by the Mirk kinase

inhibitor in cancer cells was due to off-target effects, Mirkdepletion would not alter the fraction of G0 cells. Panc1/shMirk cells were serum-starved; at the same timethey were depleted of Mirk by induction of the shRNA

construct to exon 11. Even though Mirk depletionrequired the turnover of the rather stable Mirk protein(t1/2 > 5 hours), Mirk depletion, like Mirk kinase inhibitortreatment, increased the fraction of cells in S and G2 þ Mphases, showing that the increase in cell cycling was notdue to off-target effects (Fig. 2B, bottom, insets).

Inhibition of Mirk kinase allows some quiescenttumor cells to enter cycle

Panc1, AsPc1, and SU86.86 cultures were serum-starved to accumulate G0 cells. In each line, addition offresh nutrients plus very limited FBS (0.2%) was enoughto move a mean of 15% � 4% of the cells out of G0.However, approximately twice as many cells left G0, 29%� 5%, if the quiescent cultures were switched to freshDMEM þ 0.2% FBS also containing the Mirk kinaseinhibitor (Fig. 2D). Because Mirk phosphorylates at least3 cell-cycle regulators, each of which mediates arrest inG0, inhibition of one or more of these activities may besufficient to move cells into cycle.

DNA damage in quiescent pancreatic cancer cells isincreased either by treatment with the Mirk kinaseinhibitor or by Mirk depletion through targetingeither exon 11 or exon 5

Mirk increases expression of the antioxidants superox-ide dismutases (SOD2 and SOD3), which reduce super-oxide levels, and the ferroxidase ceruloplasmin thatprevents the formation of hydroxyl ions, probablythrough its transcriptional coactivator functions (24).As a result, Mirk depletion increased total ROS (2).Similarly, treatment of Panc1 cells by the Mirk kinaseinhibitor led to a dose-dependent increase in total ROS,which activate the DCFA fluorochrome (Fig. 3A, right),and an increase in superoxide ions, which activate thedihydroethidium fluorochrome (Fig. 3A, left). The in-crease in superoxides by the Mirk kinase inhibitor showsits specificity in intact cells. Mirk increases expression ofSOD2 and SOD3, but none of the other 4 kinases that areblocked by the Mirk kinase inhibitor in vitro have anyreported antioxidant activity (Materials and Methods).Increased ROS levels can lead to cell death, and time-lapse studies of Mirk kinase inhibitor treated Panc1 cellsshowed flattening and intracellular vesicle formationwithin 40 hours, with marked cell loss by 86 hours

Figure 1. Pancreatic cancer cells can enter a reversible quiescent G0 state, and G0 cells are enriched in cultures after gemcitabine treatment. A, top, SU86.86pancreatic cancer cells in log phase growth, cultured in DMEM þ 0.2% FBS for 3 days to accumulate G0 quiescent cells, and quiescent cells (Quies)released for 24 hours into fresh growth medium containing 100 ng/mL nocodazole (Noco) to arrest cycling cells in G2 þM, with analysis only for DNA contentfollowing PI staining. The quiescent-> released culture contains no nocodazole. Bottom, similar experiment with cells designated as G0 (arrow), G1, S,G2þM, and >4N DNA content (G0, gold; G1, dark blue; S, purple; G2þM, red) asmeasured by 2 component analysis by flow cytometry after staining DNAwithHoechst dye and RNA with Pyronin Y. RNAase treatment eliminated the Pyronin Y binding. Right, cultures of log and quiescent (qui) SU86.86 cellswere analyzed by Western blotting for cleaved caspase 3 forms of 19 and 17 kDa, and the autophagy markers formed I and II of LC3b. B, left, cyclingSU86.86 cells (8% G0, arrow) were placed in DMEM þ 0.2% FBS to induce quiescence and concurrently treated with 1 mmol/L gemcitabine for 1 to 5 dayswith analysis by Hoechst/Pyronin staining so that G0 cells were detected (20% G0, 60% in G1 and at G1–S boundary; the original data were in color:G0 gold and G1 dark blue, so the G1 cells are rendered a darker gray than the G0 cells when the figure was presented as shades of gray) or by PI staining thatallows apoptotic sub-G0–G1 cells to be quantitated (*, with 3% 9%, 18%, and 25% apoptotic sub-G0–G1 cells after days 0, 3, 4, or 5 days treatment with 1mmol/L gemcitabine, respectively). Right, SU86.86 cells were placed in suspension culture to make cells quiescent. Cultures were trypsinized to single cells,washed, pooled, and 105 cells resuspended in 20 mL DMEM þ 0.4% bovine serum albumin per non–tissue culture 100-mm plates. One set of cultures wastreated with 0.3 mmol/L gemcitabine for 5 days with viable cell numbers determined daily by exclusion of trypan blue dye (n ¼ 3). S, S phase.






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Figure 2. The Mirk kinase inhibitor exhibits specificity by increasing cycling only of cancer cells expressing Mirk/Dyrk1B and blocks many Panc1 andAsPc1 cells from entering quiescence, while allowing some cancer cells to escape quiescence. A, SW620 (Dyrk1A and Mirk/dyrk1B expressing) and HCT116(only Dyrk1A expressing) cells treated with 1 mmol/L Mirk kinase inhibitor for 24 hours with a 1-hour pulse of BrdU, before analysis by flow cytometryafter PI staining of DNA. Bottom, lysates of SW620 cells and HCT116 cells were immunoblotted for Mirk/Dyrk1B and the related kinase Dyrk1A. Dyrk1Bmigrates as a 69/65 kDa dimer and was not detected in HCT116 cells. Structure of the Mirk kinase inhibitor is shown. B, analysis by 2-parameter flowcytometry. Insets on right show the DNA distribution by Hoechst staining. Top, Panc1 cells in log phase growth. Middle, quiescent Panc1 cells. Inset showsDNA profile of cultures released for 48 hours into growth medium plus 100 ng/mL nocodazole. Bottom, log phase Panc1 cells were switched to DMEMþ 0.2%FBS and cultured for 3 days with 0.25 mmol/L Mirk kinase inhibitor; right, Panc1/sh-Mirk pancreatic cancer cells depleted of Mirk while being made quiescentby culture in DMEM þ 0.2% FBS for 3 days. C, top, log phase AsPc1 cells cultured in DMEM þ 0.2% FBS for 2 days. Bottom, parallel cultures treated with0.25 mmol/L of Mirk kinase inhibitor for 2 days. Mirk kinase inhibitor treatment for 3 days left 10% of cells in G0. D, quiescent Panc1, AsPc1, and SU86.86cells were switched to fresh DMEM þ 0.2% FBS with a parallel set also treated with 0.25 mmol/L Mirk kinase inhibitor before analysis by 2-parameterflow cytometry as in Fig. 1A. The fractions of cells in G0 are listed. One of the duplicate experiments with similar results is shown.

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(Supplementary Fig. S2). Death occurred by apoptosis, atleast in part, because after treatment with theMirk kinaseinhibitor, activation of caspase 3 was seen in 20 cell linesderived from pancreatic, colon, or breast cancers (datanot shown).ROS are known to damage cellular components such as

DNA. Histone protein H2AX molecules within the chro-matin at a double-stranded DNA break site becomephosphorylated on a serine near the carboxyl terminus.Many phosphorylated H2AX (gH2AX) molecules arefound at such a break, creating a focal site for accumu-lation of proteins involved in DNA repair and chromatinremodeling within a short time of DNA damage (25).Panc1 cells and AsPc1 cells were treated with a range ofconcentrations of the Mirk kinase inhibitor for 2 dayswhile beingmade quiescent by serum starvation (Fig. 3B).In Panc1 cells, a sharp 8-fold increase in double-strandedDNA breaks identified by gH2AX and increased cleavageof apoptotic proteins PARP and caspase 3 occurred whenthe inhibitor concentration reached 0.25 mmol/L, with aconcomitant loss in activation of the survival protein Akt.Increased levels of gH2AX were also seen at nuclearfoci in Mirk kinase inhibitor treated cells by fluorescencemicroscopy (not shown). The addition of gemcitabinetogether with the Mirk inhibitor resulted in more intenseH2AX phosphorylation indicating that the inhibitor

sensitizes Panc1 cells to gemcitabine (Fig. 3B, right lanes).At more elevated levels of theMirk inhibitor (0.5 mmol/L)increased autophagy was seen, with more cleavage ofLC3b-II. No alteration in the amount of Mirk protein wasobserved with the inhibitor, as expected, because theinhibitor functions by binding to Mirk kinase. In AsPc1cells, Mirk kinase inhibitor treatment also led to DNAbreaks identified by phosphorylated gH2AX and to ap-optosis as measured by increased levels of the cleavedPARP and caspase 3, with the maximum effect at 0.25 to0.5 mmol/L (Fig. 3B).

To test the possibility that the double-stranded DNAbreaks and apoptosis seen after treatment of quiescentcancer cells with the Mirk kinase inhibitor were due tooff-target effects, their analysis was repeated after deple-tion ofMirk. Panc1 cells weremade quiescent while beingdepleted of Mirk by doxycycline induction of a stablytransfected shRNA construct to exon 11 (Fig. 4A) or bytransient transfection of synthetic RNAi duplexes to Mirkexon 5 (Fig. 4B). When Mirk was depleted in G0 cells thelow levels of gH2AX increased (Fig. 4A and B, quiescentlanes). The gH2AX levels were 2- to 5-fold higher whentheMirk-depleted cells were released from quiescence byculture in fresh growth medium for 24 hours, and someunrepaired gH2AX marked lesions remained after 48hours (Fig. 4A, 0 drug). Gemcitabine treatment is known

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Figure 3.Mirk kinase inhibitor increases ROS levels and leads to damaged DNA. A, Panc1 cells were made quiescent in DMEMþ 0.2% FBS containing 0.1 to2.5 mmol/L Mirk kinase inhibitor for 3 days and then assayed for release of superoxides by incubation with dihydroethidium, which was oxidized by superoxideto 2-hydroxyethidium (left) or by incubation with CM-H2DCFA (right), which reacted with hydroxyl radicals and other ROS species. Data from duplicateexperiments are averaged (mean� SE). B, quiescent Panc1 cells were treated 2 days with 0.06 to 1.0 mmol/L Mirk kinase inhibitor with 0.1 mmol/L gemcitabineadded to parallel cultures. Lysates were examined by Western blotting for histone H2AX phosphorylation, Akt phosphorylation, levels of Mirk and actin,the apoptotic markers cleaved PARP and cleaved caspase 3 of 17 and 19 kDa, and the autophagy markers forms I and II of LC3b. Right, similar studywith AsPc1 cells.






to create gH2AX by the indirect mechanism of replicationfork collapse (26). As the positive control, gemcitabine-treated cycling Mirk-depleted cells had higher gH2AXlevels than undepleted cells. Doxycycline itself had noeffect on gH2AX formationwhen a doxycycline-inducibleshRNA to the unexpressed luciferase gene was examined(Supplementary Fig. S4). Thus, inhibiting Mirk kinaseor depleting it in quiescent tumor cells led to increasedDNA damage, which could be increased by gemcitabinetreatment.

DNA damage activates a signaling pathway throughATR that arrests cells at S-phase checkpoints (16, 27).When quiescent Panc1 cells, either undepleted or Mirk-depleted, were allowed to reenter cycle by a switch togrowth medium containing the mitotic inhibitor nocoda-zole, the undepleted cells traversed one cell cycle to arrestin G2 þ M, whereas the Mirk-depleted cells were unableto completely traverse S-phase indicating that Mirk de-pletion alone in quiescent cells caused DNA damage.When quiescent Panc1 cells, undepleted or Mirk-deplet-ed, were allowed to reenter cycle by a switch to growthmedium containing gemcitabine, many undepleted cellsarrested throughout S-phase at checkpoints as expected(Fig. 4C, bottom). In contrast, the Mirk-depleted Panc1cells arrested at very early S-phase checkpoints, suggest-ing more DNA damage. Similarly, gemcitabine inducedmore S-phase checkpoints in a second pancreatic cancer

cell line, Mirk-depleted SU86.86 cells compared withundepleted cells, when both were released from quies-cence (Supplementary Fig. S3). Thus, Mirk depletion inquiescent tumor cells led to DNA damage that wasunrepaired when cells reentered the cell cycle, sensitizingthem to additional DNA damage by gemcitabine.

The Mirk kinase inhibitor sensitizes cells togemcitabine and cisplatin

Although the lowest concentration of Mirk kinaseinhibitor that optimally induced DNA damage and ap-optosis in quiescent Panc1 cells was 0.25 mmol/L, thelower concentration of 0.12 mmol/L was sufficient whengemcitabine was added at the same time, suggesting anadditive effect (Fig. 3B). Thus, treatment with the Mirkkinase inhibitor should increase the toxicity of gemcita-bine. Two assays were used: metabolism of MTT tomeasure growth arrest after 2 days of exposure and viablecell number by dye exclusion analysis after 4 days oftreatment. Gemcitabine at 0.3 mmol/L caused a smallgrowth arrest, decreasing cell numbers by only 25%,whereas addition of Mirk kinase inhibitor up to 0.12mmol/L decreased cell number by MTT metabolism to65% of control values. After 4 days, 0.3 mmol/L gemci-tabine reduced viable cell numbers by 3-fold, whereasaddition of Mirk kinase inhibitor further reduced cellnumber by approximately 2-fold (Fig. 5A). Similarly,

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Figure 4. Mirk depletion, either by induction of a stably transfected shRNA to Mirk exon 11 or by transient transfection of RNAi duplexes to exon 5,induced double-stranded DNA breaks in quiescent (quies) tumor cells. Mirk depletion allows Panc1 cells to escape quiescence and increases the S-phasecheckpoints induced by gemcitabine. A, Panc1-shMirk cells were depleted of Mirk by doxycycline induction of shRNA to Mirk exon 11. Cells were releasedfrom quiescence by culture in growth medium containing 0 to 3 mmol/L gemcitabine. After 24 and 48 hours, lysates were examined as in Fig. 3B.B, quiescent Panc1 cells were depleted of Mirk by synthetic RNAi duplexes to a sequence in exon 5. Cells were released into growth medium containing0 to 1 mmol/L gemcitabine for 24 hours, then lysates were examined as in A. C, Panc1/sh-Mirk pancreatic cancer cells depleted of Mirk (þDOX) inDMEM þ 0.2% FBS for 3 days and then switched to growth medium containing 0.3 mmol/L gemcitabine, with 100 ng/mL nocodazole or nocodazolealone. After 48 hours, DNA content was determined by flow cytometry after PI staining.

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addition of Mirk kinase inhibitor to 0.1 mmol/L gemci-tabine for 2 days decreased cell number by MTT metab-olism by 4-fold, from 16% to 65% of control values, andafter 4 days increased cell kill measured by direct cellcounting by approximately 2-fold. Thus, pharmacologicinhibition of Mirk kinase increased the tumor cell kill bygemcitabine. Similar studies tested whether the Mirkkinase inhibitor would potentiate the toxicity of cisplatintoward Panc1 cells and SW620 colon carcinoma cells. TheEC50 of cisplatin toward Panc1 cells was decreased fromapproximately 4 to 0.7 mmol/L, whereas the EC50 ofcisplatin toward SW620 cells was decreased from approx-imately 2.5 to 0.4 mmol/L (Fig. 5B). Thus, the small-molecule Mirk kinase inhibitor increased gemcitabineor cisplatin toxicity by an average of approximately 5-fold in 2 different tumor cell lines. These results are

consistent with our earlier studies in which Mirk deple-tion enhanced the sensitivity of ovarian cancer cells tocisplatin (5).

Mirk depletion sensitizes quiescent pancreaticcancer cells to killing by gemcitabine

To confirm the specificity of the Mirk kinase inhibitortargeting in intact cells, the capacity of Mirk depletion tosensitize 2 pancreatic cancer cell lines to gemcitabine wastested by using cell viability assays to ensure that celldeath, not growth arrest, was measured. QuiescentPanc1/shMirk cells were depleted of Mirk while beingtreated over 4 days with gemcitabine. Depletion of Mirkled to approximately a 2-fold loss of Panc1 cell viability, astatistically significant difference with P ¼ 0.0051 by theStudent paired t test (Fig. 6A). Trypan blue exclusion

Figure 5. The Mirk kinase inhibitorenhances the toxicity ofgemcitabine in quiescentpancreatic cancer cells andenhances the toxicity of cisplatinfor cycling cancer cells. A, top,quiescentPanc1cellswere treated2 days with 0.06 to 2 mmol/L Mirkkinase inhibitor with 0.3 mmol/L(left) or 0.1 mmol/L (right)gemcitabine added to one set ofparallel plates. Cell growthmeasured by metabolism of MTT,n ¼ 3 per point, mean � SE.Bottom, quiescent Panc1 cellswere treated4dayswith0.03 to 0.5mmol/L Mirk kinase inhibitor with0.3 mmol/L (left) or 0.1 mmol/L(right) gemcitabine added to oneset of parallel plates. Cell growthmeasured by counting viable cellnumbers after trypan blueexclusion; mean � SD is shown ifSD > 5%. One of the duplicateexperiments with similar results isshown. B, cycling SW620 coloncarcinoma cells and Panc1 cellswere treated with a range ofcisplatin concentrations and 0.6mmol/L Mirk kinase inhibitor for4 days in triplicate in 96-wellplates. Cell growth was analyzedby metabolism of MTT andnormalized to 0% kill for theinhibitor alone, and to 100% for themaximum effect on growth.

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assays showed that induced depletion of Mirk in quies-cent SU86.86 cells led to more than a 3-fold increase inkilling by 0.3 to 10 mmol/L gemcitabine, a statisticallysignificant difference with P ¼ 0.0064 by the Studentpaired t test (Fig. 6B). Similar results occurred by deplet-ing Mirk by targeting a different sequence (Supplemen-tary Fig. S4). Placing anchorage-dependent fibroblasts insuspension culture can induce quiescence (3), and loss ofadhesion along with serum starvation increased the frac-tion of G0 pancreatic cancer cells to approximately 70%.Quiescent SU86.86 cells and Panc1 cells in suspensionreformed small clumps within a day and were killed bylower concentrations of gemcitabine than adherent cells(Supplementary Fig. S5). Thus, Mirk depletion, like Mirkkinase inhibition, increased the killing of Panc1 andSU86.86 pancreatic cancer cells by gemcitabine.

Discussion

In this study pharmacologic inhibition of Mirk kinasesensitized pancreatic cancer cells to gemcitabine and

cisplatin, increasing cell kill up to 5-fold. This drugsensitization was also seen following depletion of Mirkusing different targets of RNA interference and thusconfirmed the correct targeting by the kinase inhibitorwithin cells. Significantly, because Mirk is most highlyexpressed in quiescent pancreatic cancer cells, targetingMirk may enable gemcitabine to kill these out of cyclecells in 2 ways: by damaging them in G0 by increasingtheir ROS levels and then by forcing them back intocycle before repair. In support of this hypothesis,pharmacologic inhibition of Mirk kinase blocked mostcycling Panc1 and AsPc1 cells from becoming quiescentin response to suboptimal growth conditions, andmoved some pancreatic cancer cells out of the quiescentstate. Mirk transcriptional coactivator activity increasesexpression of antioxidant genes, thus reducing ROSlevels and increasing quiescent tumor cell viability(2). In this study, inhibition of Mirk kinase in quiescentcells increased their ROS levels. Mirk induces SOD2and SOD3, which reduce superoxides, and the Mirkkinase inhibitor increased superoxide levels, againshowing inhibitor specificity. ROS damage DNA, anda marker of double-stranded DNA breaks, phosphory-lated histone H2AX, was elevated in quiescent Panc1,SU86.86, and AsPc1 pancreatic cancer cells in whichMirk kinase was inhibited or Mirk was depleted. Loss ofthe Mirk capacity to increase antioxidant gene expres-sion would lead to more ROS and thus to more DNAdamage. Targeting Mirk may appear controversialbecause a recent survey of the kinome by a syntheticlethal RNAi screening strategy revealed several kinases,but not but Mirk/dyrk1B, capable of sensitizing Mia-PaCa cells to gemcitabine (28). Because Mirk is notexpressed in MiaPaCa cells, the omission is understand-able. MiaPaCa cells model the 10% of resected humanpancreatic adenocarcinomas that do not express Mirkprotein (19). In addition, most screening studies usecycling cells, and Mirk protein levels and kinase activ-ities are highest in cells in G0 and quite low in cyclingcells (29).

Mirk is an unusual kinase in that it is widelyexpressed at very low levels in most normal adulttissues suggesting a noncritical function (10). In supportof this interpretation, depletion of the already low levelsof Mirk in fibroblasts caused no detectable decrease inviability as measured by clonogenic assays (12), or bydye exclusion tests (17). Mirk levels are elevated inskeletal muscle and testes, but embryonic knockout ofthe Mirk/dyrk1B gene resulted in viable, fertile micewith normal skeletal muscle development, so othergenes must compensate for loss of Mirk (30, 31). Incontrast, the Mirk gene is amplified in a subset ofpancreatic cancers, Mirk expression is upregulated inother pancreatic cancers (19), and Mirk/dyrk1B med-iates survival in pancreatic cancer cells (19). This dif-ference in sensitivity to Mirk depletion suggests thattargeting Mirk in pancreatic cancers might limit mosteffects to the cancer cells themselves.

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Figure 6. Depletion of Mirk by targeting 2 different exons enhances thetoxicity of gemcitabine in 2 different lines of pancreatic cancer cellsmade quiescent. A, Mirk was depleted by shRNA directed to exon 11 for3 days in quiescent Panc1 cells, which were then treated for 4 days withgemcitabine in fresh medium. Cell number quantitated by crystal violetstaining, with all values plus doxycycline statistically different fromuninduced values by the Student paired t test with P ¼ 0.0051. B,quiescent SU86.86/sh-Mirk cells depleted of Mirk by shRNA to exon 4for 4 days with gemcitabine added. Trypan blue exclusion assays weredone (n ¼ 3), with P ¼ 0.0064 by the Student paired t test. Mean � SD isshown if SD > 5%.

Ewton et al.





Disclosure of Potential Conflicts of Interest

E.A. Friedman received a commercial research grant and honorariafrom Speakers Bureau.

Acknowledgments

The authors thank Dr. Dave Heimbrook for helpful discussions.

Grant Support

This work was supported by NIH grant 5R21CA135164 (E.A. Friedman), theJones/Rohner Foundation (E.A. Friedman), and Hoffmann La Roche.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 6, 2011; revised August 22, 2011; accepted August 24,2011; published OnlineFirst August 30, 2011.

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Published OnlineFirst August 30, 2011.Mol Cancer Ther Daina Z. Ewton, Jing Hu, Maria Vilenchik, et al. Pancreatic Cancer CellsInactivation of Mirk/Dyrk1b Kinase Targets Quiescent

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