Upregulation of Myt1 Promotes Acquired Resistance of Cancer Cells to Wee1 Inhibition Cody W

Published OnlineFirst October 8, 2019; DOI: 10.1158/0008-5472.CAN-19-1961

Cancer Molecular Cell Biology Research

Upregulation of Myt1 Promotes Acquired Resistance of Cancer Cells to Wee1 Inhibition Cody W. Lewis1,2,3, Amirali B. Bukhari1,2,3, Edric J. Xiao1,3, Won-Shik Choi1,2,3, Joanne D. Smith1,2,3, Ellen Homola4, John R. Mackey1,5, Shelagh D. Campbell4, Armin M. Gamper1,2,3, and Gordon K. Chan1,2,3

Abstract

Adavosertib (also known as AZD1775 or MK1775) is a tion of Cdk1 induced aberrant mitosis and cell death by small-molecule inhibitor of the protein kinase Wee1, with mitotic catastrophe. Cancer cells with intrinsic adavosertib single-agent activity in multiple solid tumors, including sar- resistance had higher levels of Myt1 compared with sensitive coma, glioblastoma, and head and neck cancer. Adavosertib cells. Furthermore, cancer cells that acquired resistance fol- also shows promising results in combination with genotoxic lowing short-term adavosertib treatment had higher levels of agents such as ionizing radiation or chemotherapy. Previous Myt1 compared with mock-treated cells. Downregulating studies have investigated molecular mechanisms of primary Myt1 enhanced ectopic Cdk1 activity and restored sensitivity resistance to Wee1 inhibition. Here, we investigated mechan- to adavosertib. These data demonstrate that upregulating Myt1 isms of acquired resistance to Wee1 inhibition, focusing on the is a mechanism by which cancer cells acquire resistance to role of the Wee1-related kinase Myt1. Myt1 and Wee1 kinases adavosertib. were both capable of phosphorylating and inhibiting Cdk1/ cyclin B, the key enzymatic complex required for mitosis, Signiﬁcance: Myt1 is a candidate predictive biomarker of demonstrating their functional redundancy. Ectopic activa- acquired resistance to the Wee1 kinase inhibitor adavosertib.

Introduction adavosertib in the clinic (1, 16); and the mechanisms underpin- ning clinical resistance are unknown. Adavosertib (also known as AZD1775 or MK1775) is a narrow In eukaryotes, Wee1 and the related Myt1 kinase (PKMYT1; spectrum inhibitor of the protein kinase Wee1 that has single- ref. 17) exhibit functionally redundant roles in the inhibition agent clinical activity in multiple solid tumors, including sarco- of the mitosis-promoting complex—Cdk1/cyclinB(18–21). Wee1 ma, glioma, head and neck cancer, and ovarian cancer (1, 2). phosphorylates Cdk1 on Y15, whereas Myt1 phosphorylates Cdk1 Wee1 activity is crucial for maintaining the S- and G –M-phase 2 on both T14 and Y15 (17, 21). When cells are ready to enter DNA damage checkpoints (3–5) and as such adavosertib sensi- mitosis, the phosphatase Cdc25C removes these inhibitory phos- tizes cancer cells to genotoxic treatments including ionizing phates from Cdk1 (22, 23). Cdk1 is rephosphorylated by Wee1 radiation, gemcitabine, cisplatin, and camptothecin (6–10). during mitotic exit—again inhibiting its activity (11, 24–26). On its own, adavosertib treatment forces S-phase HeLa (cervical Because Wee1 and Myt1 exhibit functional redundancy in cancer cells) and breast cancer cells to directly enter mitosis Cdk1 inhibition, compensatory Myt1 activation is a candidate (10–13). This causes premature condensation of underreplicated mechanism for adavosertib resistance. However, several studies chromosomes, leading to double-stranded breaks at the centro- show that knockdown or inhibition of Wee1 alone is sufﬁcient to meres (centromere fragmentation; refs. 11, 14, 15). Subsequently, abrogate the S- and G –M DNA damage checkpoints and to cause these cells arrest and die in prometaphase or following mitotic 2 cells to prematurely enter mitosis (8, 27–29). In contrast, the loss slippage (11). Nevertheless, some tumors do not respond to of Myt1 (in the presence of Wee1) neither affects the timing of mitosis nor abrogates DNA damage checkpoints (8, 27–29). 1Department of Oncology, University of Alberta, Edmonton, Alberta, Canada. These observations led some researchers to conclude that Myt1 2 Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta, Canada. is not required for Cdk1 inhibition in cancer cells. However, a 3 Cancer Research Institute of Northern Alberta, University of Alberta, Edmon- more recent study showed that Myt1 is essential for cell survival in ton, Alberta, Canada. 4Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada. 5Medical Oncology, Cross Cancer Institute, Edmon- a subset of glioblastoma cells that have downregulated Wee1 ton, Alberta, Canada. expression (30). In these glioblastoma cells, loss of Myt1 induced a mitotic arrest followed by cell death (30). In addition, Chow and Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Poon reported that the combined knockdown of Wee1 and Myt1 causes more HeLa cells to enter mitosis with damaged DNA Corresponding Author: Gordon K. Chan, University of Alberta, Cross Cancer compared with Wee1 knockdown alone (8). Furthermore, Myt1 Institute, 11560 University Avenue, Edmonton, Alberta T6G1Z2, Canada. Phone: 780-432-8433; Fax: 780-432-8428; E-mail: [email protected] knockdown enhances adavosertib-induced cell killing in cell lines derived from brain metastases (31). Cancer Res 2019;79:5971–85 Although adavosertib is in clinical development in multiple doi: 10.1158/0008-5472.CAN-19-1961 cancer types, ongoing trials include patients with advanced and 2019 American Association for Cancer Research. metastatic breast cancer. Given the high breast cancer incident and

www.aacrjournals.org 5971

Lewis et al.

mortality in North America and Europe (32), we studied adavo- Orthotopic breast cancer xenograft and drug treatments sertib resistance in breast cancer models and report that Myt1 A total of 2 106 MDA-MB-231-fluc2-tdT cells were mixed upregulation mediates intrinsic and acquired adavosertib resis- with Matrigel and 1X PBS (1:1) and injected using a 1 cc tance through the inhibition of ectopic Cdk1 activity. syringe with 26G needle in 50 mL volume orthotopically into inguinal mammary fat pad of 6- to 8-week-old female NSG (NOD/SCID gamma) mice procured from Dr. Lynne Materials and Methods Postovit's breeding colony at the University of Alberta. Tumor Cell culture volume was measured every 4 days with a Vernier caliper, HeLa cells were received directly from the ATCC, whereas and volume was calculated as [length (width)2]/2. When breast cancer cells were received from Dr. Roseline Godbout, tumors reached a volume of about 150 mm3,micewere University of Alberta, Edmonton, Alberta, Canada (also pur- randomly segregated into two groups (n ¼ 3 per group). chased from the ATCC) who amplified, aliquoted, and then Mice received daily treatment with either vehicle (0.5% froze cells at early passage (P3) in liquid nitrogen. A P3 aliquot methylcellulose dissolved in sterile water) or 60 mg/kg ada- was subsequently received and then reamplified, aliquoted, vosertib via oral gavage for 26 days. Tumors were harvested and frozen by our laboratory (P6-9). Cell lines were tested for 24 hours after last drug treatment and fixed with 10% for- Mycoplasma contamination by DAPI staining and confocal malin for 48 hours prior to embedding. All animal work was imaging. HeLa, MDA-MB-468, MDA-MB-231, SK-BR-3, and approved by the Cross Cancer Institute Animal Care Com- BT-474 cells were grown as a monolayer in high-glucose mittee in accordance with the Canadian Council on Animal DMEM supplemented with 2 mmol/L L-glutamine and 10% Care guideline. (vol/vol) FBS. T-47D and MCF7 cells were grown in high- glucose DMEM supplemented with 2 mmol/L L-glutamine, DNA microarray 10% (vol/vol) FBS, and 0.01 mg/mL insulin. MCF10A and Total RNA was isolated from frozen breast tumor biopsies, and HME-1 cells were grown in Mammary Epithelial Growth gene microarray analysis, data processing, and reverse transcrip- Medium supplemented with SingleQuots (Lonza; CC-3150). tion PCR were processed (as outlined in refs. 36–38). One Wee1 Cell lines were maintained in culture for a maximum of (A_23_P127926) and two Myt1 primers (A_24_P105102 and 2months(20–25 passages). MDA-MB-231 cells expressing A_23_P398515) were available for analysis. Myt1 primers were mCherry-H2B and EGFP-tubulin were generated as outlined by then averaged together after confirming that mRNA detection was Moudgil and colleagues (33). MDA-MB-231 cells were trans- similar by comparative analysis. DNA microarray data are depos- fected with mClover3-10aa-H2B (34). HeLa cells were trans- ited in NCBI's Gene Expression Omnibus, accession number fected with mClover3-10aa-H2B (34) and tdTomato-CENPB- GSE22820 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?). N-22 (35). All cell lines were cultured in a humidified incubator at 37 Cwith5%CO2. Immunoblot Cells were harvested and processed for Western blot as Cell synchronization described previously by Famulski and colleagues (39). Protein Cells were synchronized in G1–S phase by double thymidine extracts were separated on 12% polyacrylamide gels for 7 to block as outlined in Moudgil and colleagues (33). Cells were 15 minutes at 200 V. PageRuler Plus Prestained protein ladder treated with 2 mmol/L thymidine for 16 hours with an 8-hour (Thermo Fisher Scientific; 26619) was used as a molecular release interval between thymidine treatments. For cell synchro- weight marker. Proteins were transferred on to nitrocellulose nization described in Figs. 4 and 6A–C and Supplementary Fig. S4 for 7 to 15 minutes at 25 V by Trans-Blot Turbo Transfer (measuring premature entry into mitosis), cells were released System. Membranes were blocked with Odyssey blocking from the second thymidine block for 4 hours and then subjected buffer (LI-COR Biosciences). Membranes were probed with the to indicated treatments. following primary antibodies: anti-Wee1 antibodies (Cell Sig- naling Technology; 4936; 1:1,000 dilution), anti-Myt1 anti- Small-molecule inhibitors bodies (Cell Signaling Technology; 4282; 1:1,000 dilution), Adavosertib (Chemietek; 955365-80-7), RO-3306 (Sigma; anti-Cdc25C antibodies (Cell Signaling Technology; 4688; SML0569), and thymidine (Sigma; T1895) were prepared as 1:11,000), anti-Cdk1 antibodies (Santa Cruz Biotechnology; 10 mmol/L solutions in DMSO and then stored at 20C. sc-54; 1:500 dilution), anti-phospho-tyrosine 15 Cdk1 (Cell Signaling Technology; 9111; 1:1,000 dilution), anti-phospho- Transfections threonine 14 Cdk1 (1:1,000 dilution), anti-tubulin antibodies siRNA for Wee1 50-CAUCUCGACUUAUUGGAAAtt-30 (Sigma; T5168; 1:4,000 dilution), anti-PARP antibodies (Ambion; siRNA ID: s21), Myt1 50-GGACAGCAGCGGAUGU- (Cell Signaling Technology; 9542; 1:1,000 dilution), anti- GUUtt-30 (Ambion; siRNA ID: s194985), and 50-GCGGUAAA- pT320-PP1Ca antibody (Abcam; ab62334; 1:30,000), and GCGUUCCAUGUtt-30 (Ambion; siRNA ID: s194986) and a anti-GST antibody (Rockland; 600-401-200; 1:2,000 dilution). scrambled control siRNA 50-UGGUUUACAUGUCGACUAA-30 Membranes were then incubated with Alexa Fluor 680–conju- from Thermo Fisher Scientificwereusedataconcentrationof gated anti-rabbit (Thermo Fisher Scientific; A21109; 1:1,000 20 nmol/L (unless otherwise indicated) with 0.2% Lipofecta- dilution), anti-mouse (Thermo Fisher Scientific; A21057; mine RNAiMax (Thermo Fisher Scientific) for 24 hours prior to 1:1,000 dilution), IR800 anti-mouse (LI-COR Biosciences; treatments with adavosertib. In the siWee1 dilution assay, the 827-08364; 1:1,000 dilution), and IR800 anti-rabbit (LI-COR amount of RNAiMax Lipofectamine used was fixed at 0.2%. Biosciences; 926-32211; 1:1,000). Membranes were scanned Knockdown efficiency was analyzed by Western blot and nor- by the Odyssey Fc (LI-COR Biosciences) and then analyzed malized to tubulin or actin 48 hours after transfection. by Image Studio Lite software.

5972 Cancer Res; 79(23) December 1, 2019 Cancer Research

Myt1 Upregulation Mediates Resistance to Wee1 Inhibitor

Immunofluorescence Live cell imaging on spinning disk microscope Cells were processed for immunofluorescence as previously Cells were seeded in a 35-mm glass bottom dishes (MatTek described by Chan and colleagues (39). Cells were seeded on to Corporation). Glass bottom plates were placed on a motor- coverslips at a density of 5 104 cells/mL in a 35-mm dish. controlled stage within an incubator chamber maintained at 37C Following cell synchronization, cells were treated with either and 5% CO2. Live cell imaging was carried out with the Ultraview DMSO or adavosertib at indicated concentrations. Treatments ERS Rapid confocal imager (PerkinElmer) on an Axiovert 200M were maintained for 4 hours, and 0.1% DMSO was used as a Inverted Microscope (Carl Zeiss) and a CMOS camera (ORCA- control in all experiments. siRNA transfections were performed as Flash4.0, Hamamatsu) using the 40 objective lens. Images were outlined in the RNAi section. DNA was stained with 0.1 mg/mL captured at 5-minute interval for 24 hours using the Volocity DAPI. Coverslips were stained with the following antibodies: anti- software. The 488 nm and 561 nm lasers were set at 20% power phospho-Ser10 Histone H3 (PH3) antibodies (Abcam; ab5176; and 200 ms exposure time. Movie files were exported as OME- 1:1,000 dilution), anti-tubulin antibodies (Sigma; T5168; TIFF files and further processed in Imaris 9.0.1 (Bitplane) for 1:4,000 dilution), and anti-centromere antibody sera (gift from background subtraction and noise reduction. M. Fritzler, University of Calgary, Calgary, Canada; 1:4,000 dilution). Coverslips were mounted with 1 mg/mL Mowiol 4-88 High-content analysis (EMD Millipore) in phosphate buffer, pH 7.4. Alexa Fluor Images were taken with a high-content–automated microscopy 488–conjugated anti-mouse and anti-rabbit (1:1,000 dilution; imaging system (MetaXpress Micro XLS, software version 6, Molecular Probes) and Alexa Fluor 647–conjugated anti-human Molecular Devices, as outlined in Lewis and colleagues; ref. 11). (1:1,000 dilution; Molecular Probes) secondary antibodies were Briefly, MDA-MB-231 and HeLa cells were seeded onto a 96-well used to visualize protein localization. Images were captured at plate at a density of 4,000 cells per well. Single images were 63 magnification using a Zeiss LSM 710 Meta Confocal Micro- captured in each well with a 20 (NA 0.75) objective with the scope (Carl Zeiss). The pinhole diameter was set at 1 airy unit for equipped siCMOS camera using bandpass filters of 536/40 nm all channels, and the exposure gain for each channel was kept and 624/40 nm. On average 200 cells per well were imaged. The constant in between image acquisition of all samples. images were then manually analyzed with the MetaXpress software using the mCherry-H2B to mark changes in DNA organi- IHC zation. Mitotic timing was calculated by the interval between fi fi IHC was performed on formalin- xed, paraf n-embedded nuclear envelope breakdown (indicated by the first evidence of tissue samples using standard procedures as previously chromosome condensation) to the onset of anaphase (or chro- fl m described (40). Brie y, 4 m slices were sectioned on precleaned mosome decondensation in the case of mitotic slippage). Only Colorfrost Plus microscope slides (Thermo Fisher Scientific) using cells that entered mitosis were analyzed for mitotic timing experi- a microtome (Leica). Tissue samples were baked at 60 C for ments, and the fates of the mitotic cells (and resulting daughter fi 2 hours and deparaf nized 3 times in xylene for 10 minutes each cells) were tracked for the duration of the experiment (48 hours). and subsequently rehydrated in a gradient of ethanol washes Cell death was determined by the formation of apoptotic bodies, (100%, 80%, and 50%). Tissue sections were subjected to antigen loss of cell attachment, and/or loss of membrane integrity. retrieval in a pressure cooker using 0.05% citraconic anhydride antigen retrieval buffer (pH 7.4). Tissue samples were blocked Generation of pT14-Cdk1 antibody with 4% BSA for 30 minutes and incubated with primary antibody The phospho-specific antibody against pT14-Cdk1 was gener- against Myt1 (1:50; Cell Signaling Technology; 4282) overnight at ated by immunizing rabbits with a synthetic peptide phosphor- 4 C. Next day, endogenous peroxidase activity was blocked for 30 ylated at the T14 residue (conjugated to KLH). Sera were first minutes using 3% H2O2, followed by incubation with anti-rabbit depleted of antibodies against the unphosphorylated epitopes – horseradish peroxidase labeled secondary antibody (Dako with a nonphosphorylated peptide column. Cdk1-pT14 antibo- þ EnVision System; K4007) for 1 hour at room temperature in dies were then affinity-purified with a pT14 peptide column. þ the dark. Samples were incubated with DAB substrate chro- Specificity of the antibodies was demonstrated by no signal in mogen (Dako) for brown color development, counterstained Western blot of mutant myt1 fly extracts. with hematoxylin, and mounted with DPX mounting medium (Sigma). Images were captured using the Zeiss Axioskop2 plus Crystal violet assay upright microscope (Carl Zeiss) equipped with AxioCam color Cells were seeded into 96-well plates and transfected with fi camera. Images were then analyzed using IHC pro ler plugin (40) siRNAs against Wee1, Myt1, and scrambled control for 24 hours. fl for ImageJ as described previously (41). Brie y, images were color Cells were then treated with increasing concentrations of ada- deconvoluted to unmix pure DAB (3,3'-diaminobenzidine) and vosertib (16–4,000 nmol/L, 1:2 serial dilution). After 96-hour hematoxylin-stained areas using the nuclear-stained image treatment, media were aspirated and then cells were stained fi option in the IHC pro ler plugin. DAB-stained (brown) nuclei with 0.5% crystal violet for 20 minutes as outlined by Bukhari were marked using the threshold feature of ImageJ and assigned and colleagues (13). Crystal violet was then removed, and plates fi an automated score using the IHC pro ler macro. The automated were rinsed 3 times with water and left to air-dry for 24 hours. score is calculated based on the following formula: Crystal violet within stained cells was resuspended in 100% Score ¼½ðnumber of pixels in a zoneÞðscore of the zoneÞ/ methanol. Absorbance at 570 nm was measured using FLUOstar ðnumber of pixels in the imageÞ OPTIMA microplate reader (BMG Labtech). Percent cell survival was calculated by subtracting blank wells and then nor- Wherein the score of the zone is assigned as 4 for the high- malizing DMSO controls to 100%. The first point on each curve positive (þ3) zone, 3 for the positive (þ2) zone, 2 for the low- represents 0 nmol/L adavosertib. Graphs were plotted using positive (þ1) zone, and 1 for the negative (þ0) zone (40). GraphPad Prism V7.

www.aacrjournals.org Cancer Res; 79(23) December 1, 2019 5973

Lewis et al.

Kinase assay (NOD/SCID gamma) mice immediately after a 26-day treatment Cdk1 kinase assays were completed as outlined in Lewis and with 60 mg/kg of adavosertib. Tumor slices were analyzed for colleagues (42, 43). Briefly, 20 ng of glutathione-S-transferase Myt1 expression and Wee1 inhibition by IHC (Fig. 1F; n ¼ 3 per (GST)fusedwitha9aminoacidPP1Ca peptide (GRPITPPRN) group). Tumors treated with adavosertib showed increased Myt1 was combined with 2,000 cells in 2x Cdk1 phospho-buffer expression (þ2; positive) compared with vehicle control–treated (100 mmol/L b-glycerophosphate, 20 mmol/L MgCl2, mice (þ1; low positive). These results strongly support Myt1 20 mmol/L NaF, and 2 mmol/L DTT) and 400 mmol/L ATP upregulation as a mechanism for acquired adavosertib resistance and then incubated at 37C for 15 minutes. Reactions were in vivo. then terminated with Laemmli sample buffer (Bio-Rad; 161- Cellular Myt1 levels determine adavosertib sensitivity 0747) and then analyzed by Western blot. The ratio of pT320- To confirm that upregulation of Myt1 can confer adavosertib PP1Ca to GST, minus "no lysate" control, was calculated for resistance, we transiently overexpressed Myt1 tagged with GFP each sample. DMSO was set to one for adavosertib serial (GFP-Myt1) or GFP alone in HeLa and MDA-MB-231 cells and dilution and HeLa for baseline Cdk1 experiment. then measured cell sensitivity to adavosertib by crystal violet Results assays (Fig. 2A). GFP-Myt1 overexpression resulted in a modest but significant increase in the adavosertib IC50s for both cell lines Upregulation of Myt1 confers resistance to Wee1 inhibition relative to GFP-transfected controls 2.2-fold and 1.7-fold increase in vitro in HeLa (P < 0.001; two-way ANOVA) and MDA-MB-231 (P ¼ To test acquired resistance to the Wee1 inhibitor adavosertib, 0.0079; two-way ANOVA), respectively. Although HeLa exhibited we derived adavosertib-resistant cell lines from HeLa (cervical a greater increase in the adavosertib IC50 compared with MDA- cancer) and MDA-MB-231 (breast cancer) cell lines. We and MB-231, HeLa also exhibited higher GFP-Myt1 expression levels others have previously shown that HeLa and MDA-MB-231 are compared with MDA-MB-231. highly sensitive to adavosertib as single-agent treatment Because increased Myt1 expression induced adavosertib resis- (10, 11, 13). We selected for resistant cells by culturing these cell tance in HeLa and MDA-MB-231, we wondered whether endog- lines in medium containing 500 nmol/L adavosertib for approx- enous Myt1 expression in other cell lines could be used as a imately 2 months (Fig. 1A). The resulting cell populations were biomarker to predict adavosertib sensitivity. Adavosertib sensi- tested for adavosertib sensitivity by crystal violet assays (13). In tivity was screened in a panel of breast cell lines (five cancer and the case of both MDA-MB-231 and HeLa, adavosertib-selected two nontumorigenic; Supplementary Table S1). HeLa and MDA- cells (denoted as "R500") showed much higher resistance to the MB-231 cells were included as positive controls for adavosertib Wee1 inhibitor compared with mock-treated (Parental, "P") cells sensitivity. Cell lines were transfected with siMyt1 or siSc and then (Fig. 1B and C). Selection increased the IC50 from 305 to 1,090 treated with adavosertib for 96 hours. Adavosertib treatment nmol/L in HeLa and from 349 to 1,217 nmol/L in MDA-MB-231. reduced cell number in a dose-dependent manner in all nine cell Because the activities of two related kinases Myt1 and Wee1 were lines tested, and as expected Myt1 knockdown (confirmed by found to be redundant in Cdk1 regulation in various organisms/ immunoblot) further reduced cell number (Fig. 2B–E; Supple- – tissues (19 21, 30), we hypothesized that upregulation of Myt1 mentary Fig. S1A–S1E). We calculated IC50 values for each cell line could underline Wee1 inhibitor resistance. We therefore com- (Supplementary Table S2). HeLa, MDA-MB-231, and HME-1 cell pared Myt1 protein levels in the derived adavosertib-resistant cell lines had IC50 values in the 300 nmol/L range, but the remaining 6 lines with the parental cell lines. Indeed, resistant cell populations cell lines (MCF10A, MDA-MB-468, SK-BR-3, MCF7, T-47D, and had increased Myt1 levels: 2.0-fold in HeLa and 3.1-fold in MDA- BT-474) had IC50 values that were approximately 2 to 4 times MB-231 relative to parental cell populations. To test whether higher. To investigate how the high IC50 values correlated with decreasing Myt1 levels could resensitize resistant cells to adavo- Myt1 levels, Myt1 protein levels (normalized to total protein sertib, we used two different siRNAs to knockdown Myt1 (siMyt1 content) were quantified in each cell line (Fig. 3A; top) and then #1 and #2) and compared adavosertib sensitivity with siRNA plotted against the corresponding IC50 values. Linear analysis – scrambled control treated cells (siSc). Myt1 knockdown in R500 revealed a strong correlation between calculated IC50 values and 2 HeLa cells decreased the IC50 from 1,261 nmol/L in siSc-treated Myt1 protein levels (Fig. 3B; top plot; R ¼ 0.6903) in agreement cells to 283 and 373 nmol/L in siMyt1 (#1 and #2)-transfected with our prediction. We also tested if the levels of Wee1 or Cdc25C cells, respectively (Fig. 1D). In R500 MDA-MB-231 cells, Myt1 (the phosphatase responsible for reversing Cdk1 phosphoryla- knockdown decreased the IC50 from 1,447 nmol/L (siSc) to 163 tion, by Wee1 and Myt1) correlated with cell sensitivity to and 371 nmol/L for siMyt1 #1 and #2, respectively (Fig. 1E). These adavosertib, but no significant correlations were observed data suggest that Myt1 upregulation is a driver of resistance in the (Fig. 3A and B). These data strongly support that Myt1 levels can R500 HeLa and MDA-MB-231 cell lines. be used as predictive biomarkers for cell sensitivity to adavosertib. Selection for Wee1 resistance leads to Myt1 upregulation in vivo siRNA knockdown of Wee1 mimics adavosertib treatment in We previously tested the efficacy of adavosertib treatment in an the presence of siMyt1 orthotopic breast cancer xenograft model (13). In that study using In addition to Wee1, adavosertib also exhibits activity against a luciferase-labeled MDA-MB-231 cell line, mice were treated with Polo-like kinse-1 (Plk1) in some cell types including small-cell 60 mg/kg of adavosertib for 26 days. Although adavosertib lung carcinoma cells (44, 45). However, adavosertib treatment treatment caused significant tumor growth delay, no tumor induces premature mitosis in cells consistent with the inhibition shrinkage was observed (13). This indicates that the tumors had of Wee1, whereas inhibition of Plk1 induces a G2 arrest (46). To at least partially acquired adavosertib resistance. To investigate confirm that the reduced cell survival observed following adavo- whether increased Myt1 expression contributed to tumor resis- sertib/siMyt1 treatment was dependent on loss of Wee1 activity tance, we harvested MDA-MB-231–derived tumors from NSG and not an off-target effect of adavosertib, we substituted the

5974 Cancer Res; 79(23) December 1, 2019 Cancer Research

Myt1 Upregulation Mediates Resistance to Wee1 Inhibitor

Figure 1. Adavosertib acquired resistance correlates with Myt1 upregulation in vitro and in vivo. A, Flow chart depicting the generation of adavosertib-resistant cell lines. B and C, Adavosertib sensitivity was tested in parental (P) and resistant (R500) HeLa (B) and MDA-MB-231 (C) cells by crystal violet assays. Error bars, SEM. Myt1 and tubulin expression was analyzed by immunoblot. Representative images and quantitation of average Myt1 levels (n ¼ 4) relative to tubulin are shown to the right. D and E, Adavosertib sensitivity was retested in HeLa (D) and MDA-MB-231 (E) cells following Myt1 knockdown by siRNA. Myt1 knockdown was confirmed by Western blot. F, MDA-MB-231 xenograft tumors of mice treated with vehicle control or 60 mg/kg adavosertib for 26 days were excised on the last day of drug administration. Paraffin-embedded tumor slices were analyzed for Myt1 expression by IHC. Scale bar, 50 mm. treatment with adavosertib for a validated siRNA-targeting Wee1 Adavosertib does not inhibit Cdk1 phosphorylation by Myt1 (siWee1; ref. 11). Select cell lines (HeLa, MDA-MB-231, MDA- Due to the role of Myt1 in cell-cycle regulation, we suspected MB-468, and T-47D) were transfected with increasing amounts of that Myt1 promotes adavosertib resistance by maintaining Cdk1 siWee1 in the background of either siSc or siMyt1 (Supplementary inhibition. Structure–function studies have reported that adavo- Fig. S2A–S2D). siWee1 transfection decreased cell survival in a sertib does not strongly interact with Myt1 and is 100 times more dose-dependent manner. However, cells transfected with siMyt1 selective toward Wee1 (9, 47). To confirm this, we treated MDA- þ siWee1 had fewer surviving attached cells compared with siSc þ MB-231 and MDA-MB-468 cells with adavosertib and then siWee1 controls in all four cell lines (P < 0.0001; two-way assayed Myt1 and Wee1 activity by examining Cdk1 phosphor- ANOVA). These data corroborate that the observed cell death ylation. In the presence of adavosertib, the cellular levels of pT14- with adavosertib and siMyt1 is due to loss of Wee1 and Myt1 Cdk1 (a surrogate biomarker for Myt1 activity) remained stable, activity. whereas the levels of pY15-Cdk1 (a surrogate biomarker of Wee1

www.aacrjournals.org Cancer Res; 79(23) December 1, 2019 5975

Lewis et al.

Figure 2. Transient overexpression of Myt1 promotes resistance to adavosertib, whereas Myt1 knockdown enhances sensitivity. A, HeLa and MDA-MB-231 cells were transiently transfected with either GFP (solid line) or GFP- Myt1 (dotted line) for 24 hours and then treated with adavosertib for 96 hours. Cell survival was analyzed by crystal violet assay. Error bars, SEM. P values are indicated (two- way ANOVA). Protein levels of Myt1, tubulin, and GFP were analyzed in both HeLa and MDA-MB-231 cell lines. Exogenous GFP-Myt1 () and endogenous Myt1 bands () are indicated. and ¥ denote untagged GFP and a GFP-Myt1 degradation product, respectively. HeLa (B), MDA-MB-231 (C), SK-BR-3 (D), and BT-474 (E) cells were transfected with siSc (solid line) or siMyt1 (dotted line) and then treated with adavosertib for 96 hours and surviving attached cells measured by crystal violet assays. Error bars, SEM. P values are indicated (two-way ANOVA). Myt1 knockdown was determined by immunoblot (right; quantitation of Myt1 relative to tubulin is shown). All experiments were repeated at least three times.

activity) declined (Supplementary Fig. S3A and S3B). These data Cells were transfected with GFP or GFP-Myt1 and then treated show that even 1,000 nmol/L adavosertib does not inhibit Myt1. with adavosertib for 4 hours following G1–S release. Lysates were then prepared and incubated with a recombinant Cdk1 substrate, Adavosertib-resistant cells have low Cdk1 activity GST-PP1Ca. Total levels of pT320 GST-PP1Ca (an indicator of To confirm that high Myt1 levels inhibit Cdk1 activity even in Cdk1 activity) were then quantified by immunoblot (Fig. 4A; the presence of adavosertib, we assayed in vitro Cdk1 activity (43). ref. 43). Adavosertib treatment increased in vitro Cdk1 activity in a First, we examined whether transient overexpression of Myt1 dose-dependent manner; however, GFP-Myt1 overexpression could suppress Cdk1 activity in HeLa and MDA-MB-231 cells. (confirm by immunoblot) significantly reduced absolute

5976 Cancer Res; 79(23) December 1, 2019 Cancer Research

Myt1 Upregulation Mediates Resistance to Wee1 Inhibitor

in vitro Cdk1 activity in both HeLa and MDA-MB-231 cells relative to cells expressing only GFP (Fig. 4B and C). Next, we tested whether adavosertib-resistant cell lines (SK-BR- 3 and BT-474) exhibited less in vitro Cdk1 activity compared with HeLa or MDA-MB-231. We ﬁrst established the baseline Cdk1 activity in cell lines in the absence of adavosertib. Cell lysates were prepared from cells 4 hours after release from G1–S arrest and normalized by total protein (Fig. 4A; no transfection). SK-BR-3 and BT-474 had 50% to 60% less in vitro Cdk1 activity compared with the more adavosertib-sensitive HeLa and MDA-MB-231 (Fig. 4D). Next, we tested the effects on Cdk1 activity of combining Myt1 knockdown with adavosertib. HeLa, MDA-MB-231, and two adavosertib-resistant cell lines (SK-BR-3 and BT-474) were transfected with siSc or siMyt1 and then treated with adavosertib (Fig. 4E). Low in vitro Cdk1 activity was observed in siSc- and siMyt1-transfected cells in the absence of adavosertib. Ada- vosertib treatment in siSc-transfected cells increased Cdk1 activity 2- to 4-fold compared with DMSO. However, the highest Cdk1 activity was observed in cells treated with adavosertib and transfected with siMyt1. Together, these data show that high Myt1 levels suppress Cdk1 activity in the presence of adavosertib. Subsequently, we treated each cell line with increasing concentrations of adavosertib (Fig. 4F). To compare the absolute change in Cdk1 activity between cell lines, the baseline Cdk1 activity (normalized to total protein; Fig. 4D) was multiplied by the change in in vitro Cdk1 activity within each cell line. We found that resistant cell lines (SK-BR-3 and BT-474) had less absolute Cdk1 activity in the presence of adavosertib compared with that of HeLa and MDA-MB-231 (Fig. 4G). To investigate whether the observed Cdk1 activity correlated with entry into mitosis, adavosertib-treated cells were stained for PH3 (a mitosis biomarker; ref. 48) and analyzed by immunoﬂuorescence microscopy. Con- sistent with in vitro Cdk1 kinase assay, even at 2,000 nmol/L adavosertib, <10% of SK-BR-3 and BT-474 cells stained positive for PH3 compared with 40% to 50% in HeLa and MDA-MB-231 (Supplementary Fig. S4A and S4B). Together, these data strongly support that high Myt1 expression drives adavosertib resistance by suppressing Cdk1 activity.

Myt1 protects cells from adavosertib-induced mitotic arrest Wee1 is required for normal mitotic exit, and the loss of Wee1 causes cells to arrest in mitosis (24–26). Similarly, Myt1 knockout in glioblastoma cells has been previously shown to prolong the duration of mitosis, leading to cell death (30). We therefore wondered whether Myt1 levels may affect how long cancer cells arrest in mitosis following adavosertib treatment. We transfected MDA-MB-231 cells expressing mCherry-H2B and GFP-tubulin with siMyt1 or siSc and then treated cells with adavosertib or DMSO. Next, we measured the duration of mitosis by timelapse microscopy (Fig. 5A and B). No signiﬁcant differences were observed between siMyt1- or siSc-transfected MDA-MB-231 cells in the absence of adavosertib (Fig. 5B; 0 nmol/L); cells exhibited normal chromosome alignment and mitotic timing. In contrast, Figure 3. adavosertib (in the background of siSc) increased the total time in High Myt1 protein levels correlate negatively with cancer cell sensitivity to mitosis compared with DMSO controls. Normal chromosome adavosertib. A, Cell extracts were prepared from cell lines and analyzed for alignment and segregation were observed in most cells at low total Myt1, Wee1, Cdc25C, and total protein by immunoblot. HeLa protein concentrations of adavosertib (250 nmol/L), but adavosertib levels were used as reference. Quantitation of Myt1, Wee1, and Cdc25C levels concentrations 500 nmol/L induced abnormal chromosome (normalized to total protein) is shown below. B, Protein levels of Myt1, condensation, which frequently was followed by in mitotic slip- Cdc25C, and Wee1 were plotted against adavosertib IC50 concentrations for various cell lines (Supplementary Table S2). Experiments were repeated at page (Supplementary Fig. S5A and S5B; two phenotypes are least three times. shown). Combined knockdown of Myt1 with adavosertib

www.aacrjournals.org Cancer Res; 79(23) December 1, 2019 5977

Lewis et al.

Figure 4.

Myt1 inhibits ectopic Cdk1 activity induced by adavosertib. A, Flow chart depicts in vitro kinase assay. Cdk1 activity in lysates from cells 4 hours after G1–S release was assessed in vitro by incubation with GST-PP1Ca (a Cdk1 substrate). Total pT320-PP1Ca peptide and GST levels were determined by immunoblot. B, In vitro Cdk1 activity (top two plots) was analyzed in HeLa and MDA-MB-231 transiently transfected with GFP or GFP-Myt1 and then treated with DMSO or adavosertib. GFP, GFP-Myt1, and tubulin levels were analyzed by immunoblot (bottom two plots). denotes GFP-Myt1, whereas and ¥ denote untagged GFP and GFP-Myt1 degradation products, respectively. C, Graph shows the quantitation of average Cdk1 activity (relative to control GFP/DMSO). Error bars, SEM. D, Baseline Cdk1

activity was analyzed in HeLa, MDA-MB-231, SK-BR-3, and BT-474 cells 4 hours after release from G1–S. Average in vitro Cdk1 activity is shown (normalized to HeLa). E, HeLa, MDA-MB-231, SK-BR-3, and BT-474 were transfected with siSc or siMyt1. Twenty-four hours after knockdown, cells were released from G1–S with DMSO or 250 nmol/L adavosertib before testing in vitro Cdk1 activity in cell lysates. Average in vitro Cdk1 activity is shown below (normalized to DMSO). F, In vitro Cdk1 activity was analyzed in HeLa, MDA-MB-231, SK-BR-3, and BT-474 treated with DMSO or adavosertib. G, Graph shows absolute in vitro Cdk1 activity (normalized to total cellular protein) in cell lines. Error bars, SEM. All experiments were repeated at least three times. , P < 0.05; , P < 0.01; , P < 0.001 (two-way ANOVA); ns, not signiﬁcant.

5978 Cancer Res; 79(23) December 1, 2019 Cancer Research

Myt1 Upregulation Mediates Resistance to Wee1 Inhibitor

Figure 5. Myt1 knockdown and adavosertib cooperatively lead to mitotic arrest and cell death. A, MDA-MB-231 cells expressing GFP-tubulin (green) and mCherry-H2B (red) were transfected with siSc or siMyt1 for 24 hours and then treated with adavosertib or vehicle control (DMSO). Cells were then analyzed by timelapse imaging. Representative images are shown. B and C, Whisker–box plots show the duration of mitosis for MDA-MB-231 (B) and HeLa (C) treated as indicated (0 nmol/L ¼ DMSO). D and E, Donut plots showing the proportion of cell survival (white) and cell death in mitosis (red) or interphase (black) following mitotic arrest. The number of cells analyzed is indicated in the inner circle of the donut plots. , P < 0.001; , P < 0.0001 (two-way ANOVA); ns, not signiﬁcant.

www.aacrjournals.org Cancer Res; 79(23) December 1, 2019 5979

Lewis et al.

(125–250 nmol/L) prolonged mitosis more than adavosertib transfected with siSc or siMyt1 showed no signs of abnormalities. alone (Fig. 5A; P < 0.0001). siMyt1 combined with adavosertib Likewise, most adavosertib/siSc-treated cells had intact chromo- also increased the percentage of cells that exhibited abnormal somes with only a small number exhibiting signs of chromosome chromosome condensation and/or mitotic slippage. No increases shattering. In contrast, treatment with adavosertib resulted in in the duration of mitosis were observed after siMyt1 transfection chromosome shattering in nearly all mitotic spreads from Myt1 and adavosertib concentrations 500 nmol/L. To compare the knockdown cells. effects on mitosis in MDA-MB-231 with another cell line, we Finally, to further investigate the phenomenon of centromere repeated the experiment in HeLa cells expressing GFP-H2B fragmentation, a HeLa cell line expressing GFP-H2B and tdTo- (Fig. 5C). Like MDA-MD-231, adavosertib/siSc increased the mato-CENP-B was established to monitor chromosomes and duration of mitosis in HeLa compared with DMSO/siSc. Unlike centromeres, respectively. Asynchronous cells were transfected in MDA-MB-231, siMyt1 significantly enhanced the duration of with siMyt1 or siSc and then treated with either DMSO or mitosis at all concentrations of adavosertib tested. 250 nmol/L adavosertib for 24 hours. No centromere fragmen- We measured the percentage of cell death in MDA-MB-231 and tation was observed in cells treated with siSc/DMSO, siMyt1/ HeLa cells by timelapse microscopy for up to 48 hours (Fig. 5D DMSO, or siSc/250 nmol/L adavosertib (Fig. 6D and E; Supple- and E). Few cell deaths were observed in siSc/DMSO controls for mentary Table S3). However, combining 250 nmol/L adavosertib either cell line; however, 18% of MDA-MD-231 and 25% of HeLa with siMyt1 resulted in centromeres clustering away from the bulk cells died during mitosis or after slippage in siMyt1/DMSO- of the chromosomes in 26.7% of cells (Fig. 6D; Supplementary treated cells. Consistent with cell survival assay data (Fig. 2B Table S3). Cell death was observed in 100% of cells exhibiting and C), we observed that adavosertib alone induced cell death centromere fragmentation (Fig. 6E). To confirm that centromere in a dose-dependent manner in both MDA-MB-231 and HeLa, fragmentation was dependent on upregulated Cdk1 activity which was further enhanced by siMyt1. Mitotic cell death com- following loss of Wee1 and Myt1 activity, we treated cells with monly occurred at high concentrations of adavosertib (500 the small-molecule Cdk1 inhibitor RO3306. We found that nmol/L) in siSc-transfected cells and at lower doses of adavosertib RO3306 completely suppressed centromere fragmentation in (125–250 nmol/L) in siMyt1-transfected cells. These data suggest siMyt1/adavosertib-treated cells (Supplementary Table S3). that loss of both Wee1 and Myt1 prolongs mitosis and induces Collectively, these findings may indicate that the ability of Myt1 mitotic cell death compared with loss of either kinase alone. to inhibit Cdk1 during DNA replication protects cells from undergoing centromere fragmentation when Wee1 is inhibited Myt1 knockdown induces centromere fragmentation in cancer by adavosertib. cells treated with adavosertib Centromere fragmentation occurs when cells enter mitosis High Myt1 expression is associated with a worse clinical without completing DNA synthesis (11, 14). We previously outcome in breast cancer reported that 20% of HeLa cells treated with 1,000 nmol/L Because most of our data were derived from breast cancer cell adavosertib underwent mitosis accompanied with centromere lines, we wanted to know if Myt1 was overexpressed in tumors fragmentation (11); however, at concentrations close to the IC50 from patients with breast cancer. We compared Myt1 mRNA levels of HeLa (250 nmol/L), centromere fragmentation was rarely in breast cancer tissues (176 samples; refs. 36–38) against normal observed. Here, we show that after Myt1 knockdown, abnormal breast tissue (10 samples) by cDNA microarray (36–38) and chromosome condensation and alignment (characteristic signs of found that median mRNA levels of Myt1 were approximately centromere fragmentation; refs. 11, 14) are observed in both 14-fold higher in cancer tissue compared with normal tissue (P ¼ MDA-MB-231 and HeLa cells treated with only 250 nmol/L 0.0004; Student t test; Fig. 7A; Supplementary Fig. S6A and S6B). adavosertib. HeLa and MDA-MB-231 cells were transfected with Nevertheless, we noted that Myt1 mRNA levels varied greatly siSc or siMyt1, synchronized in G1–S, and then released into among samples. We then correlated Myt1 expression with overall media containing adavosertib or DMSO for 4 hours (11). Cells disease grade, mitotic grade, and hormone receptor status (basal were then fixed and stained for centromeres, microtubules, and like vs. ER positive; Fig. 7B–D; Supplementary Fig. S6C–S6E). We DNA and analyzed by immunofluorescence microscopy (Fig. 6A). found that higher Myt1 expression was associated with a higher In the absence of adavosertib, <2% of HeLa and MDA-MB-231 overall disease grade (P < 0.0001; Student t test), a higher mitotic cells transfected with siSc or siMyt1 entered mitosis; observed grade (P < 0.0001; Student t test), and basal-like (triple negative) mitotic cells exhibited normal centromere localization, chromo- hormone receptor status (P ¼ 0.009; Student t test). We next some morphology, and mitotic spindles. Treatment with evaluated whether Myt1 expression was associated with either 250 nmol/L adavosertib in the presence of siSc increased the disease recurrence (disease-free survival) or overall patient sur- percentage of mitotic cells to 12% and 17% in HeLa and MDA- vival (Fig. 7E and F). We therefore dichotomized the samples into MB-231, respectively (Fig. 6A and B). Of these mitotic cells high and low Myt1 expression groups with the low group repre- observed in the presence of adavosertib alone, most exhibited senting the bottom quarter percentile of the samples, which is abnormal chromosome condensation and had centromeres and comparable with Myt1 expression in normal tissue. We found that microtubules that clustered away from the bulk of the chromo- higher Myt1 expression was associated with both a worse disease- somes, consistent with centromere fragmentation (Fig. 6A and B). free survival (P ¼ 0.0118; Mantel–Cox test) and overall survival However, 250 nmol/L Adavosertib in the presence of siMyt1 (P ¼ 0.0121; Mantel–Cox test). Because our sample size (n ¼ 176) increased centromere fragmentation 11-fold in HeLa and 4-fold was relatively small, we accessed the cBioPortal database to in MDA-MD-231 (Fig. 6B). compare high and low Myt1-expressing breast cancers to confirm To acquire higher-resolution images of the chromosomes from our findings. An analysis of 1,423 additional samples confirmed mitotic cells, HeLa cells were prepared for karyotype analysis high Myt1 levels were strongly associated with a lower overall (Fig. 6C). The chromosomes in DMSO-treated cells that were survival (Supplementary Fig. S6F; P < 0.0001; Mantel–Cox test;

5980 Cancer Res; 79(23) December 1, 2019 Cancer Research

Myt1 Upregulation Mediates Resistance to Wee1 Inhibitor

Figure 6.

Myt1 knockdown enhances adavosertib-induced centromere fragmentation. A, siSc- or siMyt1-transfected HeLa and MDA-MB-231 cells were released from G1–S phase into media containing either DMSO or 250 nmol/L adavosertib for 4 hours. Cells were then ﬁxed and stained for tubulin (green), centromeres (red), and DNA (blue) and analyzed by confocal microscopy. Representative mitotic cells for each treatment are shown. Scale bar, 5 mm. B, Donut plots show the proportions of interphase (white), mitotic (red), centromere-fragmented (blue), and dead cells (black). The number of cells analyzed is indicated in the inner circle of the donut plots. C, Metaphase spreads of HeLa cell chromosomes following treatment conditions described in A. Scale bar, 10 mm. D, HeLa cells expressing GFP-H2B (green) and tdTomato-CENP-B (red) were analyzed by live cell imaging. Scale bar, 10 mm. E, Dendrograms show the cell fates of HeLa following transfection with siMyt1 and 250 nmol/L adavosertib treatment (n ¼ 39). Each line represents a single cell, and forked lines indicate cell division. Capped black lines indicate cell death, whereas uncapped lines represent cells that either survived treatment or exited the imaging ﬁeld prior to the end of the experiment.

www.aacrjournals.org Cancer Res; 79(23) December 1, 2019 5981

Lewis et al.

Figure 7. High Myt1 expression is associated with a worse clinical outcome in breast cancer. Patient breast cancer tissue was analyzed for Myt1 mRNA expression by cDNA microarray (average of two primers; Supplementary Fig. S6). A, Normalized Myt1 expression in breast cancer tissue (n ¼ 176) was compared with normal breast tissue (n ¼ 10). B–D, Overall disease grade (B), mitotic grade (C), and hormone status (D) were compared with Myt1 expression. Tumor samples were grouped into high (above the 25th percentile; n ¼ 132) and low (below the 25th percentile; n ¼ 44) Myt1-expressing samples. E and F, Kaplan–Meier curves show overall survival (E) and relapse-free survival (F) of patients with breast cancer with tumors that express high and low levels of Myt1. G, Adavosertib induces ectopic Cdk1 activity in cells with low Myt1 expression (i) but not cells with high Myt1 expression (ii). iii, Cdk1 regulation is complex, and other cell-cycle regulators may also contribute to cell sensitivity to adavosertib.

refs. 49, 50). Previous studies have reported that Wee1 is overexpression, these data suggest that Myt1 overexpression may be an expressed in various cancers including breast cancer (3, 4, 51–54). important mechanism promoting cancer development. In this study, no differences in Wee1 expression were observed between breast cancer and normal tissue samples (Supplementary Fig. S6G). Breast cancer cells with Wee1 levels above the median Discussion expression were associated with a higher overall and mitotic grade Clinical trials show that adavosertib treatment responses are (Supplementary Fig. S6H and S6I), but there were no differences variable, and some cancers do not respond to adavosertib (1, 16); in hormone status, overall survival, and disease-free survival however, the mechanisms of drug resistance are unknown. We (Supplementary Fig. S6J–S6L). Although it is worth pointing out ﬁnd that cancer cells can acquire resistance to adavosertib through that we examined only Myt1 mRNA levels, not Myt1 protein the upregulation of Myt1. HeLa and MDA-MB-231 cell lines,

5982 Cancer Res; 79(23) December 1, 2019 Cancer Research

Myt1 Upregulation Mediates Resistance to Wee1 Inhibitor

which initially exhibited high sensitivity to Wee1 inhibition, after induced by Wee1 inhibition (10, 11, 13). Mitotic catastrophe is selection acquired resistance to adavosertib that was marked by a not defined by a molecular pathway, but it can be identified by 2- to 3-fold increase in Myt1 expression (Fig. 1B and C). In mitotic abnormalities including premature mitosis, centromere addition, in vivo experiments using an orthotopic breast cancer fragmentation (14, 15), and mitotic exit delays (57–59). We xenograft model demonstrated that tumors following 26 days of observed that the type and incidence of these mitotic abnormal- adavosertib treatment had increased Myt1 expression compared ities prior to cell death varied depending on the concentration of with control-treated tumor tissue (Fig. 1F). Together, these data adavosertib used and cellular Myt1 levels. Despite HeLa and strongly suggest that Myt1 upregulation is a mechanism for tumor MDA-MB-231 cells having similar Myt1 protein levels and ada- cells to acquire resistance to Wee1 inhibition. However, our data vosertib IC50s, we did note that siMyt1 transfection in the pres- do not exclude the possibility that other proteins or pathways may ence of adavosertib had a more significant effect on the duration also promote resistance to adavosertib. Recently, proteomics of mitosis in HeLa compared with MDA-MB-231 (Fig. 5). This study showed that primary resistance to adavosertib is also difference may be explained by other biological differences that associated with the upregulation of the mTOR pathway in may exist between the two cell lines. Apart from Myt1, differences small-cell lung carcinoma cells (55). in the expression (or activity) of other cell-cycle regulators may To validate that Myt1 upregulation had a direct role in adavo- cause longer mitotic delays following adavosertib treatment sertib resistance, we tested the effects of Myt1 knockdown and (Fig. 7G). The cellular levels of mitotic cyclins and their degra- overexpression. Myt1 knockdown resensitized resistant HeLa and dation rate in anaphase are known to affect the duration of MDA-MB-231 cell lines to adavosertib (Fig. 1D and E), which sug- mitosis in cells treated with other antimitotic drugs (58, 60). The gested that Myt1 upregulation was required to sustain resistance in upregulation of mitotic cyclins (cyclin A/B) and the downregula- these cells. Furthermore, Myt1 knockdown also enhanced adavo- tion of Cdk-interacting protein p21 have been suggested to sertib sensitivity in breast cancer cell lines initially exhibiting correlate with increased cancer cell sensitivity adavosertib (10). intrinsic resistance to the Wee1 inhibitor (Fig. 2B–E; Supplementary Alternatively, differences in the activity of the upstream kinase/ Fig. S1). Moreover, we were able to induce adavosertib resistance phosphatase signaling pathway that regulates Wee1, Myt1, or directly in parental HeLa and MDA-MB-231 cells by transiently Cdc25C could also affect the duration of mitosis in these cells. It is overexpressing GFP-Myt1 (Fig. 2A). Collectively, these data argue also possible that the loss of Myt1 and Wee1 activity may induce that Myt1 upregulation directly drives adavosertib resistance. mitotic catastrophe by an unknown mechanism that is indepen- Our results suggest that Myt1 expression level is a candidate dent of Cdk1 activity. predictive biomarker for tumor sensitivity to adavosertib. We In the case of SK-BR-3 and BT-474, the amount of Myt1 compared adavosertib sensitivity in cancer cell lines and identi- expressed was enough to prevent premature mitosis in most cells fied a strong correlation (R2 ¼ 0.69) between adavosertib resis- treated with adavosertib. At the highest concentration tested tance and Myt1 protein expression (Fig. 3; Supplementary (2,000 nmol/L), less than 10% of SK-BR-3 or BT-474 prematurely Table S2), in agreement with a study suggesting a negative entered mitosis. In contrast, 10% to 20% of HeLa and MDA-MB- correlation of Myt1 mRNA levels with Wee1 inhibitor sensitivi- 231 underwent premature mitosis at 250 nmol/L, and 40% to ty (31). Although Wee1 and Cdc25C are important regulators of 50% of cells entered mitosis at 2,000 nmol/L adavosertib. Cdk1 activity (22, 23, 56), we did not observe any significant Although Myt1 did not protect most HeLa and MDA-MB-231 correlation between the expression of these proteins and adavo- cells from mitotic catastrophe at high concentrations of adavosertib sensitivity. sertib (500–2,000 nmol/L), Myt1 was essential for cell survival at Developmental studies in model organisms have shown that lower, more clinically relevant concentrations. Cells treated with Wee1 and Myt1 are at least partially redundant in the regulation of 125 to 250 nmol/L adavosertib progressed through mitosis sig- Cdk1 (18–20). This redundancy is important because ectopic nificantly slower than those treated with DMSO, but cell death Cdk1 activity is lethal due to the promotion of replication stress, was relatively low (10%–20% cell death at 250 nmol/L; Fig. 5). premature entry into mitosis, and the dysregulation of mitotic Myt1 knockdown in combination with 125 to 250 nmol/L processes (10, 12, 21). We confirmed that Myt1 retained its adavosertib caused cells to arrest in mitosis 2 to 3 times longer activity even in the presence of adavosertib, indicating that Myt1 than in the case of adavosertib alone and caused cell death to could compensate in Cdk1 regulation when Wee1 is inhibited increase to 75%. Similarly, 10% of HeLa and 17% of MDA-MB- (Supplementary Fig. S3; refs. 9, 47). However, we found that in 231 cells underwent premature mitosis associated with centro- cells with low levels of Myt1, such as HeLa and MDA-MB-231, mere fragmentation when treated with 250 nmol/L adavosertib Myt1 activity is insufficient to suppress ectopic Cdk1 activity when (Fig. 6). However, Myt1 knockdown in combination with ada- Wee1 is inhibited (Fig. 4E–G). Transient overexpression of GFP- vosertib induced mitosis associated with centromere fragmenta- Myt1 in HeLa and MDA-MB-231 reduced in vitro Cdk1 activity tion in 75% of HeLa and 50% of MDA-MB-231 cells. Together, relative to GFP controls (Fig. 4B and C), which argues that higher these data show that Myt1 and Wee1 cooperatively suppress cell Myt1 levels can induce resistance in these cells by inhibiting Cdk1. death by mitotic catastrophe. Likewise, SK-BR-3 and BT-474 have high Myt1 baseline levels and Our findings establish Myt1 level as a candidate predictive exhibit low Cdk1 activity in the presence of adavosertib (Fig. 4D– biomarker for tumor response after adavosertib treatment. This G). Myt1 knockdown enhanced in vitro Cdk1 activity induced by could have wide-ranging clinical implications as adavosertib adavosertib in all cell lines tested (Fig. 4E), which further argues enters the clinic. Currently, adavosertib is undergoing phase I/II that Myt1 at least partially inhibits Cdk1 activity if Wee1 is clinical trials against different cancer types alone and in combi- inhibited. Together, these data corroborate that Myt1 promotes nation with different anticancer agents. Although tumor response resistance to adavosertib through the inhibition of Cdk1. is observed in some patients, cancer progression continues in Our data suggest Myt1 inhibition of ectopic Cdk1 activity many other patients treated with adavosertib (1, 61). To our protects cells from mitotic catastrophe, the mode of cell death knowledge, clinical studies on adavosertib do not take Myt1

www.aacrjournals.org Cancer Res; 79(23) December 1, 2019 5983

Lewis et al.

expression levels into account prior to treating patients with this Authors' Contributions inhibitor. However, Myt1 expression should be assessed in pre- Conception and design: C.W. Lewis, W.-S. Choi, J.D. Smith, A.M. Gamper, and posttreatment tissue samples from participants in these G.K. Chan studies to evaluate if there is a relationship between Myt1 expres- Development of methodology: C.W. Lewis, E. Homola Acquisition of data (provided animals, acquired and managed patients, sion and the clinical response to adavosertib. If a correlation fi provided facilities, etc.): C.W. Lewis, A.B. Bukhari, E.J. Xiao, W.-S. Choi, between high Myt1 levels and adavosertib resistance is identi ed, J.D. Smith, A.M. Gamper, G.K. Chan Myt1 expression levels could be used to stratify patients with Analysis and interpretation of data (e.g., statistical analysis, biostatistics, cancer in a future clinical trial on adavosertib. Our data show that computational analysis): C.W. Lewis, A.B. Bukhari, E.J. Xiao, W.-S. Choi, Myt1 is overexpressed in tumor tissue (relative to normal J.D. Smith, A.M. Gamper, G.K. Chan tissue; Fig. 7A). Likewise, Myt1 is also reported to be upregulated Writing, review, and/or revision of the manuscript: C.W. Lewis, A.B. Bukhari, J.D. Smith, J.R. Mackey, S.D. Campbell, A.M. Gamper, G.K. Chan in other cancer types such as colorectal cancer and glioblasto- Administrative, technical, or material support (i.e., reporting or organizing mas (30, 62). If high Myt1 levels are indeed a predictive biomarker data, constructing databases): G.K. Chan for tumor response to adavosertib, it is unlikely that adavosertib Study supervision: A.M. Gamper, G.K. Chan on its own will be effective in targeting these tumor types. Other (antibodies for detecting Cdk1 phosphorylation on T14): Furthermore, the finding that Myt1 overexpression is associated S.D. Campbell with poor breast cancer prognosis suggests that those patients with breast cancer most in need of new therapies are least likely to Acknowledgments benefit from adavosertib. We thank Michael Weinfeld (and laboratory members), Michael Hendzel, and Chan laboratory members for their helpful discussion. We also Combining adavosertib with a small-molecule Myt1 inhibitor thank Xuejun Sun and Geraldine Barron for assistance in the cell imaging will likely prove beneficial in overcoming resistance. However, facility. C.W. Lewis is supported by the NSERC Alexander Graham Bell there are additional reasons why a Myt1 inhibitor may be ben- Canada Graduate Scholarship and Izaak Walton Killam Memorial Scholar- eficial in the clinic. Myt1 level is a candidate prognostic biomark- ship. A.B. Bukhari is supported by Alberta Cancer Foundation's Dr. Cyril er, because high level in breast cancer is associated with a higher M. Kay Graduate Scholarship. J.D. Smith was supported by the Queen tumor grade, higher mitotic grade, triple-negative status, reduced Elizabeth II Graduate Scholarship. E.J. Xiao was supported by a NSERC – USRA award. The Chan laboratory was funded by NSERC (RGPIN-2016- overall survival, and increased disease relapse (Fig. 7B F). In 06466), CIHR (PJT-159585), and the Cancer Research Society (19060). The addition, Myt1 upregulation is associated with cancer cell metas- Gamper laboratory was funded by a startup grant from the University of tasis and lower overall survival in colorectal cancers (62). Myt1 is Alberta and a CIHR grant (PJT-159585). also a crucial survival factor in a subset of glioblastomas (30). Together, these data suggest that Myt1 may be a driver of tumor The costs of publication of this article were defrayed in part by the payment aggressiveness, which further provides a rationale for developing of page charges. This article must therefore be hereby marked advertisement Myt1 inhibitors. in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Disclosure of Potential Conflicts of Interest Received June 27, 2019; revised September 4, 2019; accepted October 4, 2019; No potential conflicts of interest were disclosed. published first October 8, 2019.

References 1. Do K, Wilsker D, Ji J, Zlott J, Freshwater T, Kinders RJ, et al. Phase I study of 10. Aarts M, Sharpe R, Garcia-Murillas I, Gevensleben H, Hurd MS, Shumway single-agent AZD1775 (MK-1775), a Wee1 kinase inhibitor, in patients SD, et al. Forced mitotic entry of S-phase cells as a therapeutic strategy with refractory solid tumors. J Clin Oncol 2015;33:3409–15. induced by inhibition of WEE1. Cancer Discov 2012;2:524–39. 2. Sanai N, Li J, Boerner J, Stark K, Wu J, Kim S, et al. Phase 0 trial of AZD1775 11. Lewis CW, Jin Z, Macdonald D, Wei W, Qian XJ, Choi WS, et al. Prolonged in first-recurrence glioblastoma patients. Clin Cancer Res 2018;24:3820–8. mitotic arrest induced by Wee1 inhibition sensitizes breast cancer cells to 3. PosthumaDeBoer J, Wurdinger T, Graat HC, van Beusechem VW, Helder paclitaxel. Oncotarget 2017;8:73705–22. MN, van Royen BJ, et al. WEE1 inhibition sensitizes osteosarcoma to 12. Duda H, Arter M, Gloggnitzer J, Teloni F, Wild P, Blanco MG, et al. radiotherapy. BMC Cancer 2011;11:156. A mechanism for controlled breakage of under-replicated chromo- 4. Mir SE, De Witt Hamer PC, Krawczyk PM, Balaj L, Claes A, Niers JM, et al. In somes during mitosis. Dev Cell 2016;39:740–55. silico analysis of kinase expression identifies WEE1 as a gatekeeper against 13. Bukhari AB, Lewis CW, Pearce JJ, Luong D, Chan GK, Gamper AM. mitotic catastrophe in glioblastoma. Cancer Cell 2010;18:244–57. Inhibiting Wee1 and ATR kinases produces tumor-selective synthetic 5. Zheng H, Shao F, Martin S, Xu X, Deng CX. WEE1 inhibition targets cell lethality and suppresses metastasis. J Clin Invest 2019;29:1329–44. cycle checkpoints for triple negative breast cancers to overcome cisplatin 14. Beeharry N, Rattner JB, Caviston JP, Yen T. Centromere fragmentation is a resistance. Sci Rep 2017;7:43517. common mitotic defect of S and G2 checkpoint override. Cell Cycle 2013; 6. Osman AA, Monroe MM, Ortega Alves MV, Patel AA, Katsonis P, Fitzgerald 12:1588–97. AL, et al. Wee-1 kinase inhibition overcomes cisplatin resistance associated 15. Brinkley BR, Zinkowski RP, Mollon WL, Davis FM, Pisegna MA, Pershouse with high-risk TP53 mutations in head and neck cancer through mitotic M, et al. Movement and segregation of kinetochores experimentally arrest followed by senescence. Mol Cancer Ther 2015;14:608–19. detached from mammalian chromosomes. Nature 1988;336:251–4. 7. Hirai H, Arai T, Okada M, Nishibata T, Kobayashi M, Sakai N, et al. 16. Van Linden AA, Baturin D, Ford JB, Fosmire SP, Gardner L, Korch C, et al. MK-1775, a small molecule Wee1 inhibitor, enhances anti-tumor Inhibition of Wee1 sensitizes cancer cells to antimetabolite chemother- efficacy of various DNA-damaging agents, including 5-fluorouracil. apeutics in vitro and in vivo, independent of p53 functionality. Mol Cancer Cancer Biol Ther 2010;9:514–22. Ther 2013;12:2675–84. 8. Chow JP, Poon RY. The CDK1 inhibitory kinase MYT1 in DNA damage 17. Mueller PR, Coleman TR, Kumagai A, Dunphy WG. Myt1: a membrane- checkpoint recovery. Oncogene 2013;32:4778–88. associated inhibitory kinase that phosphorylates Cdc2 on both threonine- 9. Hirai H, Iwasawa Y, Okada M, Arai T, Nishibata T, Kobayashi M, et al. 14 and tyrosine-15. Science 1995;270:86–90. Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensi- 18. Ayeni JO, Varadarajan R, Mukherjee O, Stuart DT, Sprenger F, Srayko M, tizes p53-deficient tumor cells to DNA-damaging agents. Mol Cancer Ther et al. Dual phosphorylation of cdk1 coordinates cell proliferation with key 2009;8:2992–3000. developmental processes in Drosophila. Genetics 2014;196:197–210.

5984 Cancer Res; 79(23) December 1, 2019 Cancer Research

Myt1 Upregulation Mediates Resistance to Wee1 Inhibitor

19. Okumura E, Fukuhara T, Yoshida H, Hanada Si S, Kozutsumi R, Mori M, immunohistochemistry images of human tissue samples. PLoS One 2014; et al. Akt inhibits Myt1 in the signalling pathway that leads to meiotic 9:e96801. G2/M-phase transition. Nat Cell Biol 2002;4:111–6. 41. Kumar G, Tajpara P, Bukhari AB, Ramchandani AG, De A, Maru GB. Dietary 20. Palmer A, Gavin AC, Nebreda AR. A link between MAP kinase and p34 curcumin post-treatment enhances the disappearance of B(a)P-derived (cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and DNA adducts in mouse liver and lungs. Toxicol Rep 2014;1:1181–94. inactivates the p34(cdc2) inhibitory kinase Myt1. EMBO J 1998;17: 42. Lewis CW, Taylor RG, Golsteyn RM. Measurement of Cdk1/cyclin B kinase 5037–47. activity by specific antibodies and western blotting. Methods Mol Biol 21. Jin Z, Homola E, Tiong S, Campbell SD. Drosophila myt1 is the major cdk1 2016;1342:337–48. inhibitory kinase for wing imaginal disc development. Genetics 2008;180: 43. Lewis CW, Taylor RG, Kubara PM, Marshall K, Meijer L, Golsteyn RM. A 2123–33. western blot assay to measure cyclin dependent kinase activity in cells or 22. Russell P, Nurse P. cdc25þ functions as an inducer in the mitotic control of in vitro without the use of radioisotopes. FEBS Lett 2013;587:3089–95. fission yeast. Cell 1986;45:145–53. 44. Wright G, Golubeva V, Remsing Rix LL, Berndt N, Luo Y, Ward GA, et al. 23. Timofeev O, Cizmecioglu O, Settele F, Kempf T, Hoffmann I. Cdc25 Dual targeting of WEE1 and PLK1 by AZD1775 elicits single agent cellular phosphatases are required for timely assembly of CDK1-cyclin B at the anticancer activity. ACS Chem Biol 2017;12:1883–92. G2/M transition. J Biol Chem 2010;285:16978–90. 45. Serpico AF, D'Alterio G, Vetrei C, Della Monica R, Nardella L, Visconti R, 24. Chow JP, Poon RY, Ma HT. Inhibitory phosphorylation of cyclin- et al. Wee1 rather than Plk1 is inhibited by AZD1775 at therapeutically dependent kinase 1 as a compensatory mechanism for mitosis exit. relevant concentrations. Cancers 2019;11:pii: E819. Mol Cell Biol 2011;31:1478–91. 46. Gheghiani L, Loew D, Lombard B, Mansfeld J, Gavet O. PLK1 activation in 25. Visconti R, Della Monica R, Palazzo L, D'Alessio F, Raia M, Improta S, et al. late G2 sets up commitment to mitosis. Cell Rep 2017;19:2060–73. The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness 47. Zhu JY, Cuellar RAD, Berndt N, Lee HE, Olesen SH, Martin MP, et al. and sensitivity to antimicrotubule cancer drugs. Cell Death Differ 2015;22: Structural basis of Wee kinases functionality and inactivation by diverse 1551–60. small molecule inhibitors. J Med Chem 2017;60:7863–75. 26. Visconti R, Palazzo L, Della Monica R, Grieco D. Fcp1-dependent dephos- 48. Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, phorylation is required for M-phase-promoting factor inactivation at et al. Mitosis-specific phosphorylation of histone H3 initiates primarily mitosis exit. Nat Commun 2012;3:894. within pericentromeric heterochromatin during G2 and spreads in an 27. Coulonval K, Kooken H, Roger PP. Coupling of T161 and T14 phosphor- ordered fashion coincident with mitotic chromosome condensation. ylations protects cyclin B-CDK1 from premature activation. Mol Biol Cell Chromosoma 1997;106:348–60. 2011;22:3971–85. 49. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al. 28. Nakajima H, Yonemura S, Murata M, Nakamura N, Piwnica-Worms H, Integrative analysis of complex cancer genomics and clinical profiles using Nishida E. Myt1 protein kinase is essential for Golgi and ER assembly the cBioPortal. Sci Signal 2013;6:pl1. during mitotic exit. J Cell Biol 2008;181:89–103. 50. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio 29. Wells NJ, Watanabe N, Tokusumi T, Jiang W, Verdecia MA, Hunter T. The C- cancer genomics portal: an open platform for exploring multidimensional terminal domain of the Cdc2 inhibitory kinase Myt1 interacts with Cdc2 cancer genomics data. Cancer Discov 2012;2:401–4. complexes and is required for inhibition of G(2)/M progression. J Cell Sci 51. Iorns E, Lord CJ, Grigoriadis A, McDonald S, Fenwick K, Mackay A, et al. 1999;112:3361–71. Integrated functional, gene expression and genomic analysis for the 30. Toledo CM, Ding Y, Hoellerbauer P, Davis RJ, Basom R, Girard EJ, et al. identification of cancer targets. PLoS One 2009;4:e5120. Genome-wide CRISPR-Cas9 screens reveal loss of redundancy between 52. Murrow LM, Garimella SV, Jones TL, Caplen NJ, Lipkowitz S. Identi- PKMYT1 and WEE1 in glioblastoma stem-like cells. Cell Rep 2015;13: fication of WEE1 as a potential molecular target in cancer cells by RNAi 2425–39. screening of the human tyrosine kinome. Breast Cancer Res Treat 2010; 31. Guertin AD, Li J, Liu Y, Hurd MS, Schuller AG, Long B, et al. Preclinical 122:347–57. evaluation of the WEE1 inhibitor MK-1775 as single-agent anticancer 53. De Witt Hamer PC, Mir SE, Noske D, Van Noorden CJ, Wurdinger T. WEE1 therapy. Mol Cancer Ther 2013;12:1442–52. kinase targeting combined with DNA-damaging cancer therapy catalyzes 32. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. mitotic catastrophe. Clin Cancer Res 2011;17:4200–7. Cancer incidence and mortality worldwide: sources, methods and major 54. Magnussen GI, Holm R, Emilsen E, Rosnes AK, Slipicevic A, Florenes VA. patterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359–86. High expression of Wee1 is associated with poor disease-free survival in 33. Moudgil DK, Westcott N, Famulski JK, Patel K, Macdonald D, Hang H, et al. malignant melanoma: potential for targeted therapy. PLoS One 2012;7: A novel role of farnesylation in targeting a mitotic checkpoint protein, e38254. human Spindly, to kinetochores. J Cell Biol 2015;208:881–96. 55. Sen T, Tong P, Diao L, Li L, Fan Y, Hoff J, et al. Targeting AXL and mTOR 34. Bajar BT, Wang ES, Lam AJ, Kim BB, Jacobs CL, Howe ES, et al. Improving pathway overcomes primary and acquired resistance to WEE1 inhibition in brightness and photostability of green and red fluorescent proteins for live small-cell lung cancer. Clin Cancer Res 2017;23:6239–53. cell imaging and FRET reporting. Sci Rep 2016;6:20889. 56. Russell P, Nurse P. Negative regulation of mitosis by wee1þ, a gene 35. Gao L, Shao L, Higgins CD, Poulton JS, Peifer M, Davidson MW, et al. encoding a protein kinase homolog. Cell 1987;49:559–67. Noninvasive imaging beyond the diffraction limit of 3D dynamics in 57. Uetake Y, Sluder G. Prolonged prometaphase blocks daughter cell prolif- thickly fluorescent specimens. Cell 2012;151:1370–85. eration despite normal completion of mitosis. Curr Biol 2010;20:1666–71. 36. Liu RZ, Graham K, Glubrecht DD, Germain DR, Mackey JR, Godbout R. 58. Gascoigne KE, Taylor SS. Cancer cells display profound intra- and Association of FABP5 expression with poor survival in triple-negative interline variation following prolonged exposure to antimitotic drugs. breast cancer: implication for retinoic acid therapy. Am J Pathol 2011; Cancer Cell 2008;14:111–22. 178:997–1008. 59. Orth JD, Loewer A, Lahav G, Mitchison TJ. Prolonged mitotic arrest triggers 37. Liu RZ, Graham K, Glubrecht DD, Lai R, Mackey JR, Godbout R. A fatty partial activation of apoptosis, resulting in DNA damage and p53 induc- acid-binding protein 7/RXRbeta pathway enhances survival and prolifer- tion. Mol Biol Cell 2012;23:567–76. ation in triple-negative breast cancer. J Pathol 2012;228:310–21. 60. Gascoigne KE, Taylor SS. How do anti-mitotic drugs kill cancer cells? J Cell 38. Germain DR, Graham K, Glubrecht DD, Hugh JC, Mackey JR, Godbout R. Sci 2009;122:2579–85. DEAD box 1: a novel and independent prognostic marker for early 61. Leijen S, van Geel RM, Pavlick AC, Tibes R, Rosen L, Razak AR, et al. Phase I recurrence in breast cancer. Breast Cancer Res Treat 2011;127:53–63. study evaluating WEE1 inhibitor AZD1775 as monotherapy and in com- 39. Famulski JK, Vos LJ, Rattner JB, Chan GK. Dynein/Dynactin-mediated bination with gemcitabine, cisplatin, or carboplatin in patients with transport of kinetochore components off kinetochores and onto spindle advanced solid tumors. J Clin Oncol 2016;34:4371–80. poles induced by nordihydroguaiaretic acid. PLoS One 2011;6:e16494. 62. Jeong D, Kim H, Kim D, Ban S, Oh S, Ji S, et al. Protein kinase, membrane- 40. Varghese F, Bukhari AB, Malhotra R, De A. IHC profiler: an open associated tyrosine/threonine 1 is associated with the progression of source plugin for the quantitative evaluation and automated scoring of colorectal cancer. Oncol Rep 2018;39:2829–36.

www.aacrjournals.org Cancer Res; 79(23) December 1, 2019 5985

Upregulation of Myt1 Promotes Acquired Resistance of Cancer Cells to Wee1 Inhibition

Cody W. Lewis, Amirali B. Bukhari, Edric J. Xiao, et al.

Cancer Res 2019;79:5971-5985. Published OnlineFirst October 8, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-1961

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2019/10/08/0008-5472.CAN-19-1961.DC1

Cited articles This article cites 62 articles, 23 of which you can access for free at: http://cancerres.aacrjournals.org/content/79/23/5971.full#ref-list-1

Citing articles This article has been cited by 2 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/79/23/5971.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://cancerres.aacrjournals.org/content/79/23/5971. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.