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

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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 Ó2019 American Association for Cancer Research. metastatic breast cancer. Given the high breast cancer incident and www.aacrjournals.org 5971 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst October 8, 2019; DOI: 10.1158/0008-5472.CAN-19-1961 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 incu- bator 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-
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