Preclinical Evaluation of the WEE1 Inhibitor MK-1775 As Single-Agent Anticancer Therapy

Published OnlineFirst May 22, 2013; DOI: 10.1158/1535-7163.MCT-13-0025

Molecular Cancer Small Molecule Therapeutics Therapeutics

Preclinical Evaluation of the WEE1 Inhibitor MK-1775 as Single-Agent Anticancer Therapy

Amy D. Guertin1, Jing Li4, Yaping Liu4, Melissa S. Hurd2, Alwin G. Schuller2, Brian Long2, Heather A. Hirsch3, Igor Feldman3, Yair Benita3, Carlo Toniatti1, Leigh Zawel1, Stephen E. Fawell1, D. Gary Gilliland1, and Stuart D. Shumway1

Abstract Inhibition of the DNA damage checkpoint kinase WEE1 potentiates genotoxic chemotherapies by abrogating cell-cycle arrest and proper DNA repair. However, WEE1 is also essential for unperturbed cell division in the absence of extrinsic insult. Here, we investigate the anticancer potential of a WEE1 inhibitor, independent of chemotherapy, and explore a possible cellular context underlying sensitivity to WEE1 inhibition. We show that MK-1775, a potent and selective ATP-competitive inhibitor of WEE1, is cytotoxic across a broad panel of tumor cell lines and induces DNA double-strand breaks. MK-1775–induced DNA damage occurs without added chemotherapy or radiation in S-phase cells and relies on active DNA replication. At tolerated doses, MK-1775 treatment leads to xenograft tumor growth inhibition or regression. To begin addressing potential response markers for MK-1775 monotherapy, we focused on PKMYT1, a kinase functionally related to WEE1.

Knockdown of PKMYT1 lowers the EC50 of MK-1775 by ﬁve-fold but has no effect on the cell-based response to other cytotoxic drugs. In addition, knockdown of PKMYT1 increases markers of DNA damage, gH2AX and pCHK1S345, induced by MK-1775. In a post hoc analysis of 305 cell lines treated with MK-1775, we found that expression of PKMYT1 was below average in 73% of the 33 most sensitive cell lines. Our ﬁndings provide rationale for WEE1 inhibition as a potent anticancer therapy independent of a genotoxic partner and suggest that low PKMYT1 expression could serve as an enrichment biomarker for MK-1775 sensitivity. Mol Cancer Ther; 12(8); 1442–52. 2013 AACR.

Introduction hypothesis and show chemosensitization and radiosensi- Many commonly used anticancer drugs target DNA in tization by pharmacologic or genetic disruption of check- dividing cells and ultimately cause DNA damage. This, point kinase activity including CHK1, WEE1, ATR, ATM, in turn, triggers activation of cell-cycle checkpoints that and MK2. Inhibitors of these kinases are at various stages of preclinical and clinical development. arrest progression of the cell cycle (at the G1,S,orG2–M phases) to allow the DNA to be repaired before the cell The checkpoint kinase WEE1 catalyzes an inhibitory undergoes DNA replication and/or division. From a phosphorylation of both CDK1 (CDC2) and CDK2 on therapeutic standpoint, inhibition of checkpoint kinases tyrosine 15 (2, 3). WEE1-dependent inhibition of CDK1 that mediate cell-cycle arrest could force tumor cells to and CDK2 arrests the cell cycle in response to extrinsically continue cell division before chemically induced DNA induced DNA damage (4). WEE1 activity is also essential damage is repaired, eventually causing apoptosis or for the unperturbed cell cycle (5, 6). Cell synchronization mitotic catastrophe (1). Cell line studies support this studies in normal human fibroblasts revealed that similar amounts of WEE1 protein were detected in both S- and G2–M phases but that its greatest activity was in S-phase of Authors' Affiliations: 1Oncology Biology, 2In vivo Pharmacology, 3Infor- the cell cycle (3). Furthermore, conditional knockout of matics and Analysis at Merck Research Laboratories, Boston, Massachu- setts; and 4Discovery Profiling at Merck Research Laboratories, North WEE1 in mouse embryonic fibroblasts results in genomic Wales, Pennsylvania instability, malfunctioning checkpoints, and premature Note: Supplementary data for this article are available at Molecular Cancer mitosis (6). This phenotype was explained, in part, by Therapeutics Online (http://mct.aacrjournals.org/). recent findings that show a critical role for WEE1 in DNA synthesis. Knockdown of WEE1 led to DNA double- Current address for C. Toniatti: Institute for Applied Cancer Science, MD Anderson Cancer Center, Houston, TX; and current address for S.E. Fawell: strand breaks specifically in S-phase cells undergoing AstraZeneca, Waltham, MA. DNA replication (7, 8). Data support a model of WEE1- Corresponding Author: Stuart D. Shumway, Merck Research Labs, BMB dependent genomic stability in which WEE1 knockdown 9-126, 33 Avenue Louis Pasteur, Boston, MA 02115. Phone: 617-992- or inhibition leads to aberrantly high activity of CDK1 2471; Fax 617-992-2486; E-mail: [email protected] and/or 2, resulting in inappropriately timed firing of doi: 10.1158/1535-7163.MCT-13-0025 excessive DNA replication origins. This, in turn, quickly 2013 American Association for Cancer Research. depletes nucleotide pools and leads to stalled replication

1442 Mol Cancer Ther; 12(8) August 2013

Single-Agent Activity of the WEE1 Inhibitor MK-1775

forks that, in the absence of WEE1 activity, are sub- index was calculated as the Cell Titer Glo raw value of strates for the DNA exonuclease SLX4-MUS81 and resolve treated samples relative to vehicle-treated control wells. into DNA double-strand breaks (9). For the knockdown studies, NCI-H460 and KNS62 Deregulated WEE1 expression and activity have been were transfected with siRNA pools (SMARTpool, associated with several types of cancer. WEE1 is often Dharmacon) of either control nontargeting or PKMYT1 overexpressed in glioblastomas where elevated levels of sequences using DharmaFECT formulation 1 according WEE1 mRNA are linked to poor prognosis (10). High to manufacturer’s protocol. Cells were plated in a 6-well expression of WEE1 was found in malignant melanoma plate at 3 105 cells per well and transfected the fol- and correlated with poor disease-free survival in this lowing day with 25 nmol/L final siRNA concentration. population (11). Aberrant WEE1 expression has been Twenty-four hours after transfection, cells were rinsed implicated in additional tumor types such as hepato- with PBS, trypsinized, and seeded at 4 103 cells per cellular carcinoma (12), breast cancer (13), colon car- well in 96-well tissue culture plates. The following day cinoma (14), lung carcinoma (15), and head and neck (48 hours posttransfection) cells were treated with MK- squamous cell carcinoma (16). Advanced tumors with an 1775 or DMSO for 72 hours. To approximate cell content, increased level of genomic instability may require func- ViaLight (Lonza) was used according to the manufac- tional checkpoints to allow the repair of DNA perturba- turer’s protocol. Samples were run in triplicate, and tions that accompany genomic instability. Therefore, growth was calculated by determining the percentage WEE1 might be an attractive target in advanced tumors of the control raw value for each treatment. where its inhibition may lead to irreparable DNA damage (reviewed in ref. 17). Western blotting MK-1775 is a potent and selective ATP-competitive Cells were lysed in mammalian protein extraction small-molecule inhibitor of WEE1 (18) and is currently reagent (MPER; Thermofisher 78505) and protein concen- under clinical development as a chemosensitizer in com- tration was determined with the bicinchoninic acid (BCA) bination with chemotherapeutics (19, 20). Because of the assay. Lysates were run on SDS-PAGE and transferred DNA-damaging effects resulting from the loss of WEE1 onto nitrocellulose or polyvinylidene difluoride (PVDF) activity, we hypothesized that targeted pharmacologic membranes. Antibodies used for Western blotting at inhibition of WEE1 in the absence of chemotherapy could indicated working dilutions are from the following be a viable anticancer strategy. We show that treatment sources: total CDK1 (1:1000 dilution; CST #9112), with MK-1775 gives rise to DNA damage in S-phase cells pCDK1Y15 (1:1000; CST #9111), pCDK1T14 (1:1000; CST even in the absence of standard chemotherapeutic DNA- #2543), pCHK1S345 (1:1000; CST #2348), gH2AX (1:1000; damaging agents and that premature mitosis is not CST #2577), cyclin A (1:2000; CST #4656), and total required for its ability to inhibit cancer cell proliferation. PKMYT1 (1:1000; CST #4282) from Cell Signaling Tech- At tolerated doses, MK-1775 leads to tumor growth inhi- nologies; actin-horseradish peroxidase (HRP) from Santa bition (TGI) in multiple xenograft models. Like WEE1, Cruz Biotechnology (1:10,000; sc-1616 HRP); secondary PKMYT1 also phosphorylates and inhibits CDK1 and 2 so HRP-conjugated anti-mouse and anti-rabbit antibodies we questioned whether this kinase affected cell-based are from GE Healthcare (1:5000 each; NA9340 and responses to MK-1775 treatment. Our data suggest that NA9310). Blots were exposed with SuperSignal West low PKMYT1 expression could be a determinant of MK- Femto chemiluminescent substrate (Thermofisher Pierce). 1775 sensitivity. The results presented here support the use of the WEE1 inhibitor MK-1775 as a DNA-damaging Flow cytometry and cell synchronization anticancer therapy and suggest reduced PKMYT1 expres- Cells analyzed by flow cytometry were fixed overnight sion as a possible feature of the most responsive tumors to in ice-cold 70% ethanol and propidium iodide (PI)/RNase this agent. solution (BD Biosciences 550825) was used to determine total DNA content. To detect DNA double-strand breaks, Materials and Methods cells were stained with a fluorescein isothiocyanate Cell culture, proliferation assays, and PKMYT1 (FITC)-conjugated anti-gH2AX (S139) antibody (Millipore knockdown kit 17-344). To define the mitotic population, cells were Cancer cell lines were obtained from American Type stained with an anti-pHH3-Alexa 647 antibody directed Culture Collection (not authenticated) and grown in medi- against phospho-serine 28 (BD Biosciences 558217). um recommended by the cell line vendor. Tissue culture For synchronization studies, cells were incubated in media, serum, and supplements were purchased from serum-free medium for 36 hours, followed by replenish- Life Technologies and Sigma. For the proliferation assay ment with 20% FBS. One hour before each harvest, cells screen, cells were plated in 384-well tissue culture plates were pulsed with 10 mmol/L bromodeoxyuridine in the presence of increasing concentrations of MK-1775 (BrdUrd). Cells were fixed and stained for BrdUrd and or with dimethyl sulfoxide (DMSO) as a vehicle control. DNA content with an anti-BrdU FITC-conjugated anti- After 96 hours, Cell Titer Glo (Promega) was used accord- body and with a 7-aminoactinomycin D (7-AAD) dye, ing to manufacturer’s protocol to approximate live cell respectively, according to the instructions in the BD content. Assays were run in triplicate, and the proliferation Pharmingen FITC BrdU Flow Kit (BD Biosciences

www.aacrjournals.org Mol Cancer Ther; 12(8) August 2013 1443

Guertin et al.

559619, 557891). All cytometry data were collected on the Results BD LSR II flow cytometer using Diva software, and results Pharmacologic inhibition of WEE1 blocks were analyzed in FlowJo version 7.5. proliferation in diverse tumor cell lines A wide array of responses was observed when 522 In vivo studies cancer lines representing 16 different tumor types were CD-1 nu/nu female mice aged 5 to 6 weeks were screened with a selective inhibitor of WEE1, MK-1775 (Fig. obtained from Charles River Laboratories and housed 1A; Supplementary Fig. S1). Antiproliferative EC50 values in our animal care facility at standard laboratory condi- ranged from 0.1 mmol/L in 2% (9 of 522) to 1 mmol/L in tions and fed 2018S autoclaveable diet and water ad 19% (98 of 522) of the cell lines tested (Supplementary libitum . The protocol was approved by Merck’s Institu- Table S1). Comparing mean EC50 values of the different tional Animal Care and Use Committee. Mice were ino- tumor types revealed that, as a group, colorectal cancer 6 ¼ culated with 5 10 cells (1:1 Matrigel:PBS) for A427 and cell lines were less sensitive (mean EC50 1.16 mmol/L, LoVo models or with 1 mm3 tumor fragments for the n ¼ 66, range, 0.17–>10 mmol/L) and neuroblastoma SK-MES-1 model, subcutaneously into the right flank. tumor lines were on average more sensitive to MK-1775 3 ¼ n ¼ When tumor volume reached 200 mm ( 50), mice were treatment (mean EC50 0.28 mmol/L, 7, range, 0.12– pair-matched so each group had a similar mean and SD. 0.45 mmol/L). The sample size of the latter group is Mice received anywhere from 13 to 28 days of either limited, but the notion that neuroblastoma cells tend to vehicle (0.5% methylcellulose) or MK-1775 at 60 mg/kg, be more affected by WEE1 inhibition is consistent with both administered twice daily at a dosing volume of recent findings (21). 10 mL/kg (0.2 mL per 20 g mouse). Tumor volume and body weights were recorded biweekly. Percent TGI was WEE1 inhibition by MK-1775 causes DNA damage in calculated as 100 (100 DT/DC)ifDT > 0 where DT ¼ S-phase final mean volume initial mean volume of treated group Functional genomic screens and validation studies have and DC ¼ final mean volume initial mean volume of shown that knockdown of WEE1 leads to DNA double- vehicle control group. strand breaks and activation of the DNA damage response

A B OH ES2 A2058 A431 A427 KNS62 H460 N N N NMe MK-1775

- 50 - 50 - 50 - 50 - 50 - 50 100 200 100 200 100 200 100 200 O N (nmol/L): 100 200 100 200 N pCHK1S345

N N Y15 H pCDK1 pCDK1T14 WEE1 CDC2 CDK7 MYT1 MK-1775 Actin IC50 (nmol/L) 5.2 >1,000 >1,000 530

C DMSO MK-1775 MK-1775 4 4 10 104 10 2% 22% 1% 21% 3 3 10 103 10

2 2 10 102 10

1 1 2 h 10 101 10

0 0 10 100 10 200 400 600 800 1K 200 400 600 800 1K 100 101 102 103 104

4 10 104 4 H2AX 1% 19% H2AX 10 γ 3 3 γ 1% 18% 10 10 103

2 10 102 102

1 10 101 6 h 101

0 0 10 10 0 200 400 600 800 1K 200 400 600 800 1K 10 100 101 102 103 104

7-AAD BrdU

Figure 1. MK-1775 treatment causes DNA damage in S-phase. A, chemical structure of the WEE1 inhibitor, MK-1775. B, ES-2, A2058, A431, A427, KNS62, and NCI-H460 cells were treated with either DMSO () or increasing concentrations of MK-1775 for 2 hours. Protein lysates were analyzed by Western blotting with antibodies against the targets listed. Actin serves as a loading control. C, TOV-21G cells were treated with DMSO or 150 nmol/L MK-1775 for up to 2 or 6 hours. Cells were pulse-labeled 1 hour before harvesting with BrdUrd and analyzed by ﬂow cytometry for gH2AX versus DNA content (2 left panels) or gH2AX versus BrdUrd uptake (right). The percentage of gH2AX staining cells is indicated for each gate and separated by BrdUrd status in the right.

1444 Mol Cancer Ther; 12(8) August 2013 Molecular Cancer Therapeutics

Single-Agent Activity of the WEE1 Inhibitor MK-1775

(DDR). We selected a 2-hour time point to examine of MK-1775 to the culture medium, 22% of cells stained the immediate effects of pharmacologic inhibition of positive for gH2AX (Fig. 1C, middle). Chromosomal con- WEE1 in 6 cell lines of varying sensitivity to MK-1775 tent of the gH2AX-positive cells was >2N, suggesting that ¼ ¼ (Fig. 1B): ES-2 (EC50 0.26 mmol/L), A2058 (EC50 0.23 DNA damage arising from WEE1 inhibition occurs during ¼ ¼ mmol/L), A431 (EC50 0.17 mmol/L), A427 (EC50 0.12 or after the initiation of DNA synthesis in S-phase. To ¼ mmol/L), KNS62 (EC50 3.41 mmol/L), and NCI-H460 confirm this, TOV21G cells were treated with MK-1775 ¼ (EC50 3.31 mmol/L). Western blot analyses for and pulse-labeled with BrdUrd. gH2AX was detected pCHK1S345, a surrogate marker for activated DDR (22), almost exclusively in BrdUrd-positive cells (Fig. 1C, showed a dose-dependent activation of the DDR in all 6 right). This finding supports our observation that DNA cell lines, although the effect was more evident in the most double-strand breaks due to pharmacologic WEE1 inhi- sensitive cell lines, that is, ES-2, A2058, A431, and A427. As bition arise during DNA synthesis and is consistent with expected, a reduction in pCDK1Y15 was also observed in similar results using genetic disruption of WEE1 expres- all 6 cell lines, providing a link between induction of the sion (7, 8). DDR and elevated CDK activity as a result of WEE1 inhibition. Phosphorylation of CDK1 and CDK2 at T14 WEE1 inhibition by MK-1775 disrupts S-phase by PKMYT1 is also known to impair CDK1 and 2 kinase kinetics in synchronized cells activity, and MK-1775 inhibits PKMYT1 in vitro at roughly Chromosomal breaks during DNA synthesis would be 100-fold higher concentrations than those required to expected to activate the DNA replication checkpoint and inhibit WEE1 (18). However, we failed to see an MK- slow progression through S-phase. To address this, we 1775–dependent effect on pCDK1T14 at concentrations analyzed the effects of MK-1775 treatment on cell popula- that induce DNA damage. tions synchronized by serum depletion. We avoided To understand when MK-1775–induced DNA damage cell synchronization approaches targeting DNA synthesis occurs during the cell cycle, we analyzed TOV21G ovarian (e.g., double-thymidine block, aphidicolin, hydroxyurea, cancer cells by flow cytometry. In exponentially growing actinomycin D, etc.) because these methods are disruptive untreated cells, baseline staining for the DNA double- to DNA replication (causing stalled forks) and may strand break marker gH2AX was between 1% and 2% (Fig. confound analyses by inadvertently sensitizing cells to 1C, left; ref. 23). However, as little as 2 hours after addition MK-1775 treatment. Instead, we opted to induce G0

A B Time (h): 4 12 24 Vehicle MK-1775 5 5 10 5 10 100 10 4 4 10 4 80 10 10

3 3 10 3 10 60 Vehicle 10 2 10 2 2 40 10 10

1 10 1 101 % BrdUrd % 20 10 -positive cells -positive 0 50K 100K 150K 200K 250K 50K 100K 150K 200K 250K 50K 100K 150K 200K 250K

BrdU 5 5 Time (h): 04812142448121424 10 5 10 10

4 4 10 4 10 10

3 Cyclin A MK-1775 10 3 3 10 10

2 10 2 2 10 10

1 10 1 1 pChk1S345 10 10 50K 100K 150K 200K 250K 50K 100K 150K 200K 250K 50K 100K 150K 200K 250K DNA content (PI) pCDK1Y15 C + MK-1775 G S G –M γH2AX CDK1 1 2 + FBS 30.9% 48.0% 21.1% 62.8% 1345276189 011 No FBS 69.0% 17.7% 13.3% 22.7%

Figure 2. MK-1775 treatment delays DNA replication in synchronized cells. A, ES-2 cells were synchronized following 36 hours serum withdrawal. Cells were stimulated to resume cycling with 20% FBS in the added presence of either vehicle (DMSO) in lanes 1 to 6 or 500 nmol/L MK-1775 in lanes 7 to 11. Time of harvest following FBS stimulation is indicated. One hour before harvest, cells were pulse-labeled with BrdUrd and the percentage of BrdUrd-staining cells is shown in the top. Protein lysates from ES-2 cells treated in parallel were collected followed by Western blotting with the indicated antibodies. B, ﬂow cytometric analysis in select samples (4-, 12-, and 24-hour treatments) from A comparing BrdUrd staining and DNA content. C, ES-2 cells were serum-starved as above and 500 nmol/L MK-1775 was added in either the presence or absence of 20% FBS. Twenty-four hours later, DNA content and gH2AX were analyzed by ﬂow cytometry.

www.aacrjournals.org Mol Cancer Ther; 12(8) August 2013 1445

Guertin et al.

synchronization through serum withdrawal. We selected the ES-2 line for these studies because it was amenable A2058 HT-29 LoVo (625 nmol/L) (625 nmol/L) (400 nmol/L) 4 4 to synchronization by serum depletion. Complete serum 10 10 104

3 2% 3 5% 7% withdrawal for 36 hours in ES-2 cells did not reduce 10 10 103

102 102 2 viability but shifted the G0–G1 fraction to 75% to 80% 10 − MK-1775 1 1 (data not shown). Addition of 20% FBS caused vehicle- 10 10 101 0 0 10 10 100 200 400 600 800 1K 200 400 600 800 1K 200 400 600 800 1K treated ES-2 cells to almost double their S-phase popula- 104 104 104 pHH3 21% 31% 3 5% 3 46% 3 1% 4% tion by 8 hours and peak near 50% by 12 to 14 hours (Fig. 10 10 10

2A, top panel). However, when 500 nmol/L MK-1775 was 102 102 102 + MK-1775

1 1 1 included with the addition of 20% FBS to G0 synchronized 10 10 10 100 100 100 ES-2 cells, there was no detectable change in the S-phase 200 400 600 800 1K 200 400 600 800 1K 200 400 600 800 1K population by 12 hours and peak levels (50%) were DNA content (PI)

delayed until 24 hours post-FBS addition. Western blot 104 104 104 3 1% 3 1% 3 1% analysis presented in Fig. 2A (bottom) conﬁrmed the 10 10 10 delayed accumulation of cyclin A (indicative of S-phase), 102 102 102 − MK-1775 S345 101 101 101 a more rapid and robust induction of pCHK1 , and 100 100 100 200 400 600 800 1K 4 200 400 600 800 1K 4 200 400 600 800 1K Y15 104 10 10 inhibition of pCDK1 in MK-1775–treated relative to H2AX γ 3 3 77% 3 53% Y15 10 28% 10 10 vehicle-treated cells. Phosphorylation of pCDK1 was 2 2 102 10 10 + MK-1775

1 1 initially reduced by MK-1775 (Fig. 2A, compare lanes 1 101 10 10

0 0 100 10 10 and 7) but increased between 12 and 24 hours after 200 400 600 800 1K 200 400 600 800 1K 200 400 600 800 1K addition (Fig. 2A, compare lanes 7 and 11), although the DNA content (PI) reason for this is unknown. Although the percentage of cells in S-phase peaked near Figure 3. DNA damage underlies MK-1775–induced cytotoxicity. A2058, 50% for both vehicle- and MK-1775–treated samples by 24 HT-29, and LoVo cells were treated for 24 hours with either DMSO ( MK- hours post-FBS addition, the mean ﬂuorescent intensity of 1775) or MK-1775 at EC90 concentrations of the drug. Flow cytometry incorporated BrdUrd was far lower in MK-1775 than in was used to identify the population of cells positive for the mitotic marker S28 vehicle-treated cells, suggesting slowed DNA replication phosphorylated histone H3 (pHH3 , top) or the DNA double-strand in the MK-1775–treated population (y-axis, Fig. 2B). We break marker gH2AX (bottom). Top, the gate on the right indicates the expected mitotic population (4N DNA content) and the gate on the left also analyzed gH2AX induced by MK-1775 in serum- indicates cells positive for pHH3 with less than 4N DNA content. starved versus serum-stimulated cell populations. Fol- lowing 36-hour serum deprivation, ES-2 cells were treated with 500 nmol/L MK-1775 alone or in the presence of 20% Fig. S3). After 24 hours of treatment with MK-1775, the FBS. A 24-hour treatment with MK-1775 in the presence of percentage of pHH3-positive cells increased in 2 of the 3 20% FBS resulted in an increased S-phase population cell lines, from 2% to 26% in A2058 cells and from 5% to compared with the serum-starved control group (48% 77% in HT-29 cells (Fig. 3). Importantly, only the HT-29 compared with 18%, Fig. 2C). gH2AX was detectable in cell line contained a substantial mitotic population with approximately 3 times as many cells (63% compared with <4N DNA, which indicates premature mitosis from S- 23% gH2AX-positive) following MK-1775 treatment phase cells that have not completed DNA replication under conditions of serum stimulation (Fig. 2C and Sup- (46%, Fig. 3). Therefore, premature mitosis could be an plementary Fig. S2). These data support our own and underlying driver of cytotoxicity in some cellular con- others’ observations that disruption of WEE1 kinase activ- texts, such as HT-29, but not all, such as LoVo and possibly ity results in DNA double-strand breaks as a result of A2058. On the contrary, substantial increases in gH2AX- deregulated DNA replication. positive cell populations were observed in all 3 cell lines following MK-1775 treatment (28% in A2058, 77% in HT- DNA damage underlies MK-1775–induced 29, 53% in LoVo; Fig. 3, bottom). These data suggest that cytotoxicity induction of DNA damage rather than premature mitosis WEE1 is required for the temporal regulation of both is the primary cytotoxic consequence of WEE1 inhibition CDK2 and 1 in S- and G2 phases of the cell cycle, respec- by MK-1775 in sensitive cell lines. tively. Inhibition of WEE1, therefore, is expected to lead to both S-phase defects (DNA double-strand breaks during WEE1 inhibition by MK-1775 has anticancer activity in vivo DNA replication) and G2–M defects (premature mitosis). To question whether either or both of these events is To determine the effect that MK-1775 monotherapy necessary or sufﬁcient for MK-1775–driven cytotoxicity, treatment has on tumor growth in vivo, a maximum we examined gH2AX and phosphorylated histone H3 tolerated dose (MTD) was established at 60 mg/kg for (pHH3), a marker of mitosis, in 3 MK-1775–sensitive cell twice daily dosing. Mean body weight loss over the course lines, A2058, HT-29, and LoVo (24, 25). For each line, an of a 28-day study at this dose and schedule did not exceed approximate EC90 concentration of MK-1775 was selected 5% in the treated group (data not shown). MK-1775 on the basis of cell proliferation assays (Supplementary inhibits proliferation of the A427 non–small cell lung

1446 Mol Cancer Ther; 12(8) August 2013 Molecular Cancer Therapeutics

Single-Agent Activity of the WEE1 Inhibitor MK-1775

A B Figure 4. In vivo efﬁcacy of MK- Vehicle 1,200 – 600

1775. A, A427 xenograft bearing )

3 MK-1775 mice were dosed with either vehicle 1,000 ) Vehicle 500 3 (0.5% methylcellulose) or 60 mg/kg x28 days 800 of MK-1775. Dosing of both vehicle 400 MK-1775 and compound was twice daily for 600 28 consecutive days. Xenograft 300 tumor volumes were taken twice A427 A427 200 Final tumor 400 weekly and plotted (mean volume (mm volume SEM) against days of treatment 100 200 for vehicle (n ¼ 10) and MK-1775 (mm volume Tumor (n ¼ 10)-treated mice. B, the ﬁnal 0 0 0 7 14 21 28 tumor volume of individual Treatment groups xenografts treated for 28 days with Days of study either vehicle or MK-1775 was C D plotted. Mean tumor volume at the Vehicle Vehicle start of the study was 164 mm3 and 2,000 1,000 ) ) is indicated by a dashed line. C and 3 MK-1775 3 MK-1775 in vivo ﬁ D, additional ef cacy 1,500 x28 days 800 x13 days studies were conducted in SK- MES-1 (C) and LoVo (D) xenograft 600 models as described in A, with the 1,000 exception that MK-1775 treatment 400 SK-MES-1 stopped on day 13 in the LoVo 500 xenograft study (indicated by an 200 LoVo Tumor volume (mm volume Tumor asterisk) and tumor volumes were (mm volume Tumor * measured for an additional 2 0 0 weeks. 0 7 14 21 28 0 7 14 21 28 Days of study Days of study

¼ cancer (NSCLC) cell line at low concentrations (EC50 116 related CDK-inhibitory kinase, PKMYT1. Phosphoryla- nmol/L) and readily induces the DDR (Fig. 1). In the A427 tion of CDK1 or CDK2 at either of 2 N-terminal sites, T14 xenograft model, MK-1775 treatment caused regression to or Y15, causes inactivation of the kinase despite the approximately 50% of the initial mean tumor volume (Fig. presence of an otherwise activating cyclin-binding part- 4A). Individual tumor analysis shows that 9 of the 10 ner. WEE1 is known to phosphorylate Y15 of CDK1 and 2, vehicle-treated A427 tumors grew between 2- and 6-fold and PKMYT1 has been shown to similarly inhibit CDK1 over their starting volume (Fig. 4B). In contrast, the final and 2 through phosphorylation at T14 and/or Y15 (26). volumes of all 10 MK-1775–treated tumors were smaller We used siRNA knockdown to determine whether than their initial volumes (Fig. 4B). Tumor growth-inhib- PKMYT1 expression can specifically alter the response itory effects of MK-1775 were observed in additional to WEE1 inhibition in 2 cell lines, NCI-H460 and KNS62. xenograft models chosen for their in vitro sensitivity: These lines were selected because they show both relative 92% TGI in the SK-MES-1 NSCLC model (Fig. 4C), 13% insensitivity to MK-1775 treatment and relatively high tumor regression at day 13 (dosing was abbreviated to 13 expression of PKMYT1 (data not shown). Cells were days) in a LoVo colorectal cancer xenograft model (Fig. transfected with a pool of 4 distinct siRNAs, all targeting 4D), 88% TGI in A431 epidermoid tumor model (data not PKMYT1, and analyzed in proliferation assays for shown), and 64% TGI in NCI-H2122 NSCLC model (data sensitivity to different cytotoxic agents (Fig. 5A). In a not shown). Collectively, these results show the anticancer representative experiment shown in Fig. 5A, MK-1775 n ¼ therapeutic potential of MK-1775 in the absence of any antiproliferative EC50s for NCI-H460 ( 3) and KNS62 additional targeted or DNA damaging agents. (n ¼ 2) shifted from 677 to 104 nmol/L and from 487 to 93 nmol/L, respectively, when PKMYT1 was knocked PKMYT1 expression can affect sensitivity to MK- down. Reduction of PKMYT1 potentiated MK-1775 an 1775 treatment average of 4.7-fold in NCI-H460 cells (n ¼ 3) and 4.9-fold The majority of cancer cell lines that we treated with in KNS62 cells (n ¼ 2). The specificity of PKMYT1-depen- MK-1775 show at least some degree of sensitivity to MK- dent potentiation of MK-1775 is confirmed by identical 1775 treatment (Supplementary Fig. S1). However, not all dose response curves for carboplatin, the MEK inhibitor cell lines are equally susceptible to WEE1 inhibition and PD-0325901, or doxorubicin in both the control and antiproliferative EC50s vary by as much as 10-fold (Sup- PKMYT1 siRNA–transfected cells (Fig. 5A). plementary Fig. S1). One potential determinant of sensi- Western blot analysis of KNS62 cells (Fig. 5B) revealed tivity to WEE1 inhibition is activity of a functionally that PKMYT1 knockdown results in slightly lower basal

www.aacrjournals.org Mol Cancer Ther; 12(8) August 2013 1447

Guertin et al.

A H460 CT siRNA

H460 PKMYT1 siRNA siRNA: CT WEE1 PKMYT1 – 100 100 100 80 80 80 80 60 60 60 60 40 40 40 40 20 20 20 20

% DMSO control 0 0 0 0 –9 –8 –7 –6 –5 –6 –5 –4 –9 –8 –7 –6 –5 –9 –8 –7 –6 –5 MK-1775 (log M) Carboplatin (log M) MEKi (log M) Doxorubicin (log M)

KNS62 CT siRNA siRNA: CT WEE1 PKMYT1 – KNS62 PKMYT1 siRNA 100 100 100 100 80 80 80 80 60 60 60 60 40 40 40 40 20 20 20 20

% DMSO control 0 0 0 0 –9 –8 –7 –6 –5 –6 –5 –4 –9 –8 –7 –6 –5 –9 –8 –7 –6 –5 MK-1775 (log M) Carboplatin (log M) MEKi (log M) Doxorubicin (log M)

B 400 nmol/L MK-1775 No tfxn CTsi PKMYT1si

Time (h): 0 2824 0 2 8 24 0 2 8 24

PKMYT1

Actin pCDK1Y15

Actin pCDK1T14

pCHK1S345

γH2AX

12345678910 11 12

Figure 5. PKMYT1 knockdown selectively increases sensitivity to MK-1775 and reduces inhibitory phosphorylation of CDKs 1 and 2. A, NCI-H460 (top) and KNS62 (bottom) cells were transfected with siRNA pools containing either 4 nontargeting control (CT) or 4 PKMYT1-targeting sequences. Knockdown was conﬁrmed by Western blotting for PKMYT1 in each cell line [see insets; siRNA pools used are nontargeting control (CT), WEE1, PKMYT1, or no transfection denoted by ]. Forty-eight hours following transfection, cells were exposed to titrations of MK-1775, carboplatin, an MEK inhibitor (PD0325901), or doxorubicin at concentrations indicated and assayed for proliferation 72 hours later. B, KNS62 cells were either untransfected (No txfn) or transfected with the nontargeting control (CT) or PKMYT1 siRNA pools used in A and 48 hours later treated with 400 nmol/L MK-1775 for the indicated times.

phosphorylation of CDK1 and 2 on Y15 and markedly both pCHK1S345 and gH2AX. This is consistent with the reduced basal phosphorylation of T14 (lane 9 vs. lanes 1 observations that MK-1775–mediated cytotoxicity arises and 5). Knockdown of PKMYT1 alone did not induce from DNA damage (Fig. 3) and that PKMYT1 knockdown gH2AX (Fig. 5B, lane 9), but in the presence of MK- further sensitizes cells to MK-1775–dependent antiproli- 1775, knockdown of PKMYT1 led to a larger increase in ferative effects (Fig. 5A).

1448 Mol Cancer Ther; 12(8) August 2013 Molecular Cancer Therapeutics

Single-Agent Activity of the WEE1 Inhibitor MK-1775

Because PKMYT1 knockdown leads to increased sen- protein levels also show good correlation (R2 ¼ 0.51), sitivity to MK-1775, we reasoned that low PKMYT1 although PKMYT1 protein levels do not necessarily pre- expression might be common among MK-1775-respon- dict PKMYT1 kinase activity in these cells. Although sive cell lines. To address this, we used the Broad-Novartis lower PKMYT1 expression did not invariably result in Cancer Cell Line Encyclopedia (CCLE), a publicly avail- greater sensitivity to MK-1775 in our cell panel of 305 able cell line database (27), to ﬁnd PKMYT1 mRNA lines, our data do support the hypothesis that low expression levels in the 522 cell lines that we had treated PKMYT1 expression is a common feature among the most with MK-1775 (Supplementary Fig. S1). PKMYT1 mRNA MK-1775–responsive cell lines. expression data were available for 305 of the 522 cancer cell lines assayed for sensitivity to MK-1775. Plotting Discussion relative PKMYT1 expression from the CCLE database MK-1775 is a potent and selective inhibitor of the WEE1 against our cell line response data at 450 nmol/L of kinase. As of this publication, it is the only WEE1 inhibitor MK-1775 failed to show a correlation between PKMYT1 that the authors are aware of currently undergoing eval- mRNA and MK-1775 sensitivity (Fig. 6A). However, 24 of uation as an anticancer agent in combination with che- the 33 cell lines (73%) that were killed by 450 nmol/L MK- motherapy in early-stage clinical trials (19, 20, 28). Previ- 1775 (response < 0.25 on an adjusted scale, indicated by a ous studies using MK-1775 have shown its potentiation dashed vertical line in Fig. 6A) had less than mean expres- of DNA damage–based therapeutics by forcing unsched- sion of PKMYT1 mRNA (i.e., 413 154). uled mitosis and ultimately resulting in apoptosis or To prospectively test the hypothesis that low PKMYT1 mitotic catastrophe (4, 18, 29–32). However, the potential expression might predict MK-1775 sensitivity, we selected therapeutic effects of WEE1 inhibition in the absence of 13 additional cell lines from the CCLE database with chemotherapies have not been widely explored. RNA varying expression levels of PKMYT1 mRNA that had interference knockdown of WEE1 is known to inhibit not previously been tested with MK-1775. The antiproli- proliferation of cancer cell lines (13, 33), and more recent- ferative EC50 values of MK-1775 across the 13 cell lines are ly, it was shown that MK-1775 alone can induce apoptosis related to PKMYT1 mRNA expression with R2 ¼ 0.496 in sarcoma cell lines treated in vitro (34). Our results (Fig. 6B, left) and PKMYT1 protein expression with R2 ¼ similarly highlight a requirement for WEE1 activity 0.310 (Fig. 6B, right). Accordingly, PKMYT1 mRNA and to maintain cellular viability and genomic stability.

Figure 6. Low PKMYT1 expression A 1,000 may underlie sensitivity to MK- 900 1775. A, relative PKMYT1 mRNA expression (CCLE database, 800 Broad-Novartis) was plotted on the 700 y-axis against response to 450 600 nmol/L MK-1775 treatment in 305 500 cell lines, each represented by a single dot. The response to MK- 400 * 1775 on the x-axis is an adjusted (CCLE) 300 growth value based on a 96-hour PKMYT1 mRNA 200 proliferation assay. A value of 1 indicates no change in growth rate 100 relative to DMSO treated cells and 0 a value of 0.25 (vertical dashed line) 0 0.2 0.4 0.6 0.8 1 1.2 or less indicates a negative growth rate or cell death. Mean relative Increasing MK-1775 sensitivity PKMYT1 expression among the 305 cell lines is 413 (arbitrary units, B indicated by dotted horizontal line 25 R 2 R 2 marked by an asterisk). B, thirteen = 0.496 100 = 0.310 cell lines not included in the post

T 80 hoc analysis above were selected 24 for analysis of PKMYT1 expression 60 and sensitivity to MK-1775. The protein 40 PKMYT1 PKMYT1 23 EC50 values (mmol/L) from a C mRNA proliferation assay for each cell line 20 were plotted against relative 22 PKMYT1 mRNA Ct values (left) or 0.1 0.2 0.3 0.4 0.1 0.2 0.3 0.4 PKMYT1 protein signal intensity by Western blot analysis (right, not a MK-1775 MK-1775 linear scale). EC50 (µmol/L) EC50 (µmol/L)

www.aacrjournals.org Mol Cancer Ther; 12(8) August 2013 1449

Guertin et al.

Furthermore, we provide the first demonstration of TGI (internal response data and CCLE expression data), this with MK-1775 monotherapy and suggest that low levels suggests that PKMYT1 expression could be one of mul- of PKMYT1 mRNA or protein could be a possible indi- tiple prognostic factors when trying to predict the out- cator of sensitivity to WEE1 inhibition by MK-1775. come of WEE1 inhibition. In the only other instance where MK-1775 effects were MK-1775 has been widely studied in preclinical xeno- studied independently of a DNA damaging partner, graft models as a chemotherapy or radiation sensitizer. Kreahling and colleagues attribute the cell-based cytotox- These studies generally show that MK-1775 monotherapy icity of MK-1775 to induction of premature mitosis (34). is not an effective anticancer treatment. It should be This alone, however, is unlikely to account for the noted, however, that MK-1775 is dosed below its mono- antiproliferative activity of MK-1775 in their study. For therapy MTD of 60 mg/kg twice daily in these studies, example, Kreahling and colleagues reported a prolifer- arguably accounting for the differing single-agent anti- ative EC50 of 169 nmol/L for MK-1775 in the more sen- cancer activity observed between previous studies and sitive HT1080 cell line, yet only 5% of the population was our work presented here. Importantly, in a study of pHH3-positive after 500 nmol/L MK-1775 treatment (34). patient-derived pancreatic carcinoma xenograft models, Forced mitosis, as evidenced by increased pHH3 staining, control groups receiving MK-1775 single-agent treat- is not consistent with proliferation-based sensitivities to ment at doses considerably below MTD showed surpris- MK-1775. Recent findings underscore a critical role for ing TGI (39). Unlike responses in the gemcitabine and WEE1 in regulating appropriate initiation and progres- MK-1775 combination arm, the anticancer activity of sion of DNA replication forks and thereby maintaining MK-1775 was not dependent on p53 mutational status genomic integrity by preventing DNA double-strand (39), consistent with our own work (both A427 and LoVo breaks during DNA replication (7–9, 35). Kreahling and xenograft models are wild-type for TP53) and that of colleagues did not examine markers of DNA damage Kreahling and colleagues (34). following MK-1775 treatment so the relative contribution Recent studies have described synergy between inhi- of premature mitosis versus DNA damage cannot be bitors of WEE1 and CHK1 kinases (40, 41). Genomic appreciated. We also found that some cell lines display damage resulting from deregulated DNA replication, a large increase in pHH3 staining in S-phase cells, indic- determined by gH2AX staining in S-phase cells, is not ative of premature mitosis (e.g., HT-29, Fig. 3), but we also only a hallmark of the response to WEE1 monotherapy found that premature mitosis was not a requirement of (described here) but also both the combination of the sensitivity to WEE1 inhibition (e.g., LoVo, Fig. 3). Because WEE1 and CHK1 inhibitors and CHK1 inhibitor mono- a strong induction of DNA damage accompanied MK- therapy (42). Our own work supports the in vivo com- 1775–driven cytotoxicity, regardless of the effect on mitot- bination benefit from combined WEE1 and CHK1 inhi- ic indices, our results suggest that DNA damage rather bition (data not shown; ref. 43). Notably, however, than premature mitotic entry is the dominant, although when MK-1775 and MK-8776 (formerly SCH-900776) not exclusive, mechanism underlying effectiveness of are co-administered, the combination MTD requires WEE1 inhibition. both a dose reduction (60 mg/kg each drug alone to PKMYT1 and WEE1 both catalyze inhibitory phosphor- 40 mg/kg in combination) and a schedule reduction ylations on CDK1 and 2. Our observations that low (twice daily dosing each drug alone to twice weekly PKMYT1 mRNA expression is common among the most dosing in combination), reflecting increased toxicity of sensitive cell lines to MK-1775 and that knockdown of the combination (data not shown and (43)). A compar- PKMYT1 can sensitize less responsive cell lines to MK- ison of the TGI of the MK-1775 and MK-8776 regimen at 1775 together suggest functional redundancy between combination MTD versus MK-1775 alone at monother- PKMYT1 and WEE1. In support of this, siRNA studies apy MTD suggests that despite the strong in vitro syn- have shown that knockdown of PKMYT1 leads to similar, ergy of the WEE1 and CHK1 inhibitor combination, the although less pronounced, abrogation of G2 cell-cycle 2 treatments have similar therapeutic indices (91% TGI arrest and sensitization to DNA-damaging agents for combination vs. 13% regression for MK-1775 alone in (31, 36, 37). Furthermore, and similar to WEE1, overex- LoVo colorectal xenograft model; 70% TGI for combi- pression of PKMYT1 is sufficient to induce a G2 cell-cycle nation vs. 89% TGI for MK-1775 alone in SK-MES-1 delay in HeLa cells (38). Interestingly, this study found NSCLC xenograft model; 59% TGI for combination that the interaction of PKMYT1 with the CDK1–cyclin B1 vs. 90% TGI for MK-1775 alone in A-431 epidermoid complex, rather than PKMYT1 phosphorylation of CDK1– xenograft model). Future studies will be required to cyclin B1, was responsible for the cell-cycle delay. This determine whether a specific cellular context or altered argues in favor of PKMYT1 expression rather than dosing approach for combined WEE1 and CHK1 inhi- PKMYT1 activity as a potential indicator of MK-1775 bitors provides an advantage over either single-agent sensitivity. In our evaluation of sensitivity and PKMYT1 treatment. Regardless, our work corroborates find- mRNA expression among 305 cell lines, we found that ings that WEE1 activity is essential to genomic stability many lines with relatively low levels of PKMYT1 did not and that WEE1 inhibition constitutes a viable thera- respond to MK-1775 treatment (Fig. 6A). Despite the peutic consideration based on anticancer efficacy of caveats inherent in comparing two independent data sets MK-1775 monotherapy.

1450 Mol Cancer Ther; 12(8) August 2013 Molecular Cancer Therapeutics

Single-Agent Activity of the WEE1 Inhibitor MK-1775

Disclosure of Potential Conflicts of Interest H.A. Hirsch, I. Feldman, Y. Benita, C. Toniatti, L. Zawel, S.E. Fawell, S.D. A.D. Guertin is employed (other than primary affiliation; e.g., consult- Shumway ing) as a scientist, J. Li is employed as a director of Biology Discovery, and Writing, review, and/or revision of the manuscript: A.D. Guertin, A.G. G. Gilliland as senior vice president with Merck & Co., Inc. No potential Schuller, H.A. Hirsch, Y. Benita, C. Toniatti, L. Zawel, S.E. Fawell, conflicts of interest were disclosed by the other authors. G. Gilliland, S.D. Shumway Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): A.D. Guertin, L. Zawel Authors' Contributions Study supervision: J. Li, B. Long, C. Toniatti, L. Zawel, S.E. Fawell, S.D. Conception and design: A.D. Guertin, B. Long, C. Toniatti, L. Zawel, S.E. Shumway Fawell, S.D. Shumway The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Development of methodology: J. Li, Y. Liu, Y. Benita, L. Zawel advertisement Acquisition of data (provided animals, acquired and managed patients, in accordance with 18 U.S.C. Section 1734 solely to indicate provided facilities, etc.): A.D. Guertin, J. Li, Y. Liu, M.S. Hurd, A.G. this fact. Schuller, B. Long, L. Zawel, S.D. Shumway Analysis and interpretation of data (e.g., statistical analysis, biostatis- Received January 11, 2013; revised April 30, 2013; accepted May 16, 2013; tics, computational analysis): A.D. Guertin, J. Li, Y. Liu, A.G. Schuller, published OnlineFirst May 22, 2013.

References 1. Medema RH, Macurek L. Checkpoint control and cancer. Oncogene targets in head and neck cancer. Mol Cell Proteomics 2011;10: 2012;31:2601–13. M111.011635. 2. Parker LL, Piwnicaworms H. Inactivation of the P34(Cdc2)-cyclin-B 17. Sorensen CS, Syljuasen RG. Safeguarding genome integrity: the complex by the human WEE1 tyrosine kinase. Science 1992;257: checkpoint kinases ATR, CHK1 and WEE1 restrain CDK activity during 1955–7. normal DNA replication. Nucleic Acids Res 2012;40:477–86. 3. Watanabe N, Broome M, Hunter T. Regulation of the human WEE1Hu 18. Hirai H, Iwasawa Y, Okada M, Arai T, Nishibata T, Kobayashi M, et al. Cdk tyrosine 15-kinase during the cell-cycle. EMBO J 1995;14: Small-molecule inhibition of WEE1 kinase by MK-1775 selectively 1878–91. sensitizes p53-deficient tumor cells to DNA-damaging agents. Mol 4. Hamer PCD, Mir SE, Noske D, Van Noorden CJF, Wurdinger T. WEE1 Cancer Ther 2009;8:2992–3000. kinase targeting combined with DNA-damaging cancer therapy cat- 19. Stathis A, Oza A. Targeting WEE1-like protein kinase to treat cancer. alyzes mitotic catastrophe. Clin Cancer Res 2011;17:4200–7. Drug News Perspect 2010;23:425–9. 5. Mcgowan CH, Russell P. Human WEE1 kinase inhibits cell-division by 20. Schellens JHM, Shapiro G, Pavlick AC, Tibes R, Leijen S, Tolaney SM, phosphorylating P34(Cdc2) exclusively on Tyr15. EMBO J 1993;12: et al. Update on a phase I pharmacologic and pharmacodynamic study 75–85. of MK-1775, a WEE1 tyrosine kinase inhibitor, in monotherapy and 6. Tominaga Y, Li C, Wang R-H, Deng C-X. Murine WEE1 plays a critical combination with gemcitabine, cisplatin, or carboplatin in patients with role in cell cycle regulation and pre-implantation stages of embryonic advanced solid tumors (2011 ASCO Annual Meeting). J Clin Oncol development. Int J Biol Sci 2006;2:161–70. 29:2011, 2011 (suppl; abstr 3068). 7. Beck H, Nahse V, Larsen MSY, Groth P, Clancy T, Lees M, et al. 21. Russell MR, Levin K, Rader J, Belcastro L, Li Y, Martinez D, et al. Regulators of cyclin-dependent kinases are crucial for maintaining Combination therapy targeting the Chk1 and WEE1 kinases shows genome integrity in S phase. J Cell Biol 2010;188:629–38. therapeutic efficacy in neuroblastoma. Cancer Res 2013;73:776–84. 8. Dominguez-Kelly R, Martin Y, Koundrioukoff S, Tanenbaum ME, Smits 22. Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, et al. Chk1 is VAJ, Medema RH, et al. WEE1 controls genomic stability during an essential kinase that is regulated by Atr and required for the G(2)/M replication by regulating the Mus81-Eme1 endonuclease. J Cell Biol DNA damage checkpoint. Genes Dev 2000;14:1448–59. 2011;194:567–79. 23. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double- 9. Beck H, Nahse-Kumpf V, Larsen MS, O'Hanlon KA, Patzke S, Holm- stranded breaks induce histone H2AX phosphorylation on serine 139. berg C, et al. Cyclin-dependent kinase suppression by WEE1 kinase J Biol Chem 1998;273:5858–68. protects the genome through control of replication initiation and 24. Gurley LR, D'Anna JA, Barham SS, Deaven LL, Tobey RA. Histone nucleotide consumption. Mol Cell Biol 2012;32:4226–36. phosphorylation and chromatin structure during mitosis in Chinese 10. Mir SE, Hamer PCD, Krawczyk PM, Balaj L, Claes A, Niers JM, et al. In hamster cells. Eur J Biochem 1978;84:1–15. silico analysis of kinase expression identifies WEE1 as a gatekeeper 25. Paulson JR, Taylor SS. Phosphorylation of histones 1 and 3 and against mitotic catastrophe in glioblastoma. Cancer Cell 2010;18: nonhistone high mobility group 14 by an endogenous kinase in HeLa 244–57. metaphase chromosomes. J Biol Chem 1982;257:6064–72. 11. Magnussen GI, Holm R, Emilsen E, Rosnes AKR, Slipicevic A, Florenes 26. Mueller PR, Coleman TR, Kumagai A, Dunphy WG. Myt1 - a mem- VA. High expression of WEE1 is associated with poor disease-free brane-associated inhibitory kinase that phosphorylates Cdc2 on both survival in malignant melanoma: potential for targeted therapy. PLoS threonine-14 and tyrosine-15. Science 1995;270:86–90. One 2012;7:e38254. 27. Broad Institute. Available from: http://www.broadinstitute.org/ccle/ 12. Masaki T, Shiratori Y, Rengifo W, Igarashi K, Yamagata M, Kurokohchi home. Accessed May 2012. K, et al. Cyclins and cyclin-dependent kinases: comparative study of 28. Mizuarai S, Yamanaka K, Itadani H, Arai T, Nishibata T, Hirai H, et al. hepatocellular carcinoma versus cirrhosis. Hepatology 2003;37: Discovery of gene expression-based pharmacodynamic biomarker for 534–43. a p53 context-specific anti-tumor drug WEE1 inhibitor. Mol Cancer 13. Iorns E, Lord CJ, Grigoriadis A, McDonald S, Fenwick K, Mackay A, 2009;8:34. et al. Integrated functional, gene expression and genomic analysis for 29. Hirai H, Arai T, Okada M, Nishibata T, Kobayashi M, Sakai N, et al. MK- the identification of cancer targets. PLoS One 2009;4:e5120. 1775, a small molecule WEE1 inhibitor, enhances antitumor efficacy of 14. Backert S, Gelos M, Kobalz U, Hanski ML, Bohm C, Mann B, et al. various DNA-damaging agents, including 5-fluorouracil. Cancer Biol Differential gene expression in colon carcinoma cells and tissues Ther 2010;9:514–22. detected with a cDNA array. Int J Cancer 1999;82:868–74. 30. Aarts M, Sharpe R, Garcia-Murillas I, Gevensleben H, Hurd MS, Shum- 15. Yoshida T, Tanaka S, Mogi A, Shitara Y, Kuwano H. The clinical way SD, et al. Forced mitotic entry of S-phase cells as a therapeutic significance of Cyclin B1 and WEE1 expression in non-small-cell lung strategy induced by inhibition of WEE1. Cancer Discov 2012;2:524–39. cancer. Ann Oncol 2004;15:252–6. 31. Indovina P, Giordano A. Targeting the checkpoint kinase WEE1 Selec- 16. Wu ZX, Doondeea JB, Gholami AM, Janning MC, Lemeer S, Kramer K, tive sensitization of cancer cells to DNA-damaging drugs. Cancer Biol et al. Quantitative chemical proteomics reveals new potential drug Ther 2010;9:523–5.

www.aacrjournals.org Mol Cancer Ther; 12(8) August 2013 1451

Guertin et al.

32. Wang YL, Decker SJ, Sebolt-Leopold J. Knockdown of Chk1, WEE1 with the intracellular trafficking of Cdc2-cyclin B1 complexes. Mol Cell and Myt1 by RNA interference abrogates G(2) checkpoint and induces Biol 1999;19:5113–23. apoptosis. Cancer Biol Ther 2004;3:305–13. 39. Rajeshkumar NV, De Oliveira E, Ottenhof N, Watters J, Brooks D, 33. Murrow LM, Garimella SV, Jones TL, Caplen NJ, Lipkowitz S. Iden- Demuth T, et al. MK-1775, a potent WEE1 inhibitor, synergizes with tification of WEE1 as a potential molecular target in cancer cells by gemcitabine to achieve tumor regressions, selectively in p53-defi- RNAi screening of the human tyrosine kinome. Breast Cancer Res cient pancreatic cancer xenografts. Clin Cancer Res 2011;17: Treat 2010;122:347–57. 2799–806. 34. Kreahling JM, Gemmer JY, Reed D, Letson D, Bui M, Altiok S. MK1775, 40. Davies KD, Cable PL, Garrus JE, Sullivan FX, von Carlowitz I, Huerou a selective WEE1 inhibitor, shows single-agent antitumor activity YL, et al. Chk1 inhibition and WEE1 inhibition combine synergistically against sarcoma cells. Mol Cancer Ther 2012;11:174–82. to impede cellular proliferation. Cancer Biol Ther 2011;12:1–9. 35. Martin Y, Dominguez-Kelly R, Freire R. Novel insights into maintaining 41. Carrassa L, Chila R, Lupi M, Ricci F, Celenza C, Mazzoletti M, et al. genomic integrity: WEE1 regulating Mus81/Eme1. Cell Div 2011;6:21. Combined inhibition of Chk1 and WEE1 in vitro synergistic effect 36. Tibes R, Bogenberger JM, Chaudhuri L, Hagelstrom RT, Chow D, translates to tumor growth inhibition in vivo. Cell Cycle 2012;11: Buechel ME, et al. RNAi screening of the kinome with cytarabine in 2507–17. leukemias. Blood 2012;119:2863–72. 42. Davies KD, Humphries MJ, Sullivan FX, von Carlowitz I, Le Huerou Y, 37. El Touny LH, Banerjee PP. Identification of both Myt-1 and Wee-1 as Mohr PJ, et al. Single-agent inhibition of Chk1 is antiproliferative in necessary mediators of the p21-independent inactivation of the Cdc- human cancer cell lines in vitro and inhibits tumor xenograft growth in 2/cyclin B1 complex and growth inhibition of TRAMP cancer cells by vivo. Oncol Res 2011;19:349–63. genistein. Prostate 2006;66:1542–55. 43. Guertin AD, Martin MM, Roberts B, Hurd M, Qu X, Miselis NR, et al. 38. Liu F, Rothblum-Oviatt C, Ryan CE, Piwnica-Worms H. Overproduc- Unique functions of CHK1 and WEE1 underlie synergistic anti-tumor tion of human Myt1 kinase induces a G(2) cell cycle delay by interfering activity upon pharmacologic inhibition. Cancer Cell Int 2012;12:45.

1452 Mol Cancer Ther; 12(8) August 2013 Molecular Cancer Therapeutics

Preclinical Evaluation of the WEE1 Inhibitor MK-1775 as Single-Agent Anticancer Therapy

Amy D. Guertin, Jing Li, Yaping Liu, et al.

Mol Cancer Ther 2013;12:1442-1452. Published OnlineFirst May 22, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-13-0025

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2013/05/22/1535-7163.MCT-13-0025.DC1

Cited articles This article cites 40 articles, 16 of which you can access for free at: http://mct.aacrjournals.org/content/12/8/1442.full#ref-list-1

Citing articles This article has been cited by 29 HighWire-hosted articles. Access the articles at: http://mct.aacrjournals.org/content/12/8/1442.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/12/8/1442. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.