Inhibition of Wee1 Sensitizes Cancer Cells to Antimetabolite Chemotherapeutics in Vitro and in Vivo, Independent of P53 Functionality
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Published OnlineFirst October 11, 2013; DOI: 10.1158/1535-7163.MCT-13-0424 Molecular Cancer Small Molecule Therapeutics Therapeutics Inhibition of Wee1 Sensitizes Cancer Cells to Antimetabolite Chemotherapeutics In Vitro and In Vivo, Independent of p53 Functionality Annemie A. Van Linden1, Dmitry Baturin1, James B. Ford1, Susan P. Fosmire1, Lori Gardner1, Christopher Korch2,3, Philip Reigan3,4, and Christopher C. Porter1,3 Abstract Inhibition of Wee1 is emerging as a novel therapeutic strategy for cancer, and some data suggest that cells with dysfunctional p53 are more sensitive to Wee1 inhibition combined with conventional chemotherapy than those with functional p53. We and others found that Wee1 inhibition sensitizes leukemia cells to cytarabine. Thus, we sought to determine whether chemosensitization by Wee1 inhibition is dependent on p53 dysfunction and whether combining Wee1 inhibition is tolerable and effective in vivo. Synergistic inhibition of proliferation with a Wee1 inhibitor in clinical development, MK1775, and cytarabine was observed in all acute myelogenous leukemia (AML) cell lines tested, regardless of p53 functionality. Mechanistic studies indicate that inhibition of Wee1 abrogates the S-phase checkpoint and augments apoptosis induced by cytarabine. In AML and lung cancer cell lines, genetic disruption of p53 did not alter the cells’ enhanced sensitivity to antimetabolites with Wee1 inhibition. Finally, mice with AML were treated with cytarabine and/or MK1775. The combination of MK1775 and cytarabine was well tolerated in mice and enhanced the antileukemia effects of cytarabine, including survival. Thus, inhibition of Wee1 sensitizes hematologic and solid tumor cell lines to antimetabolite chemotherapeutics, whether p53 is functional or not, suggesting that the use of p53 mutation as a predictive biomarker for response to Wee1 inhibition may be restricted to certain cancers and/or chemotherapeutics. These data provide preclinical justification for testing MK1775 and cytarabine in patients with leukemia. Mol Cancer Ther; 12(12); 2675–84. Ó2013 AACR. Introduction Wee1 is a cell-cycle checkpoint protein downstream of Cell-cycle checkpoint and DNA damage response pro- Chk1 that is activated during the normal cell cycle, as well teins are critical mediators of successful DNA replication as in the context of DNA damage. The primary function of in the presence and absence of genotoxic stress. Cancer WEE1 is inhibitory phosphorylation of cyclin—depen- cells are particularly dependent on these processes, a dent kinases (CDK) at tyrosine 15 (Y15), thereby inhibiting phenomenon that could be exploited therapeutically (1, cell-cycle progression (6, 7). Most studies of Wee1 have 2). For example, Chk1 has been studied extensively as an focused on the phosphorylation of CDK1 in the context of adjuvant therapeutic target in combination with antican- DNA damage, which prevents progression through mito- cer therapy, including radiation and chemotherapy (3, 4). sis with levels of DNA damage that would result in mitotic This strategy is expected to be particularly effective in catastrophe. Like Chk1, inhibition of Wee1 in combination tumors with disrupted p53 function, as they are highly with DNA-damaging agents has been explored as a ther- apeutic strategy for tumors with dysregulated p53. dependent upon the G2–M checkpoint mediated by Chk1 (5). Indeed, in published reports, inhibition of Wee1 with small-molecule inhibitors in combination with DNA- damaging agents, including doxorubicin, has shown Authors' Affiliations: Departments of 1Pediatrics and 2Medicine, Univer- some specificity for TP53-mutated tumor models (8–11). sity of Colorado, School of Medicine; 3University of Colorado Cancer Using RNA interference screens, we and others have Center; and 4Department of Pharmaceutical Sciences, University of Color- ado, School of Pharmacy, Aurora, Colorado recently identified Wee1 as a critical mediator of acute myelogenous leukemia (AML) cell survival after treat- Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). ment with cytarabine, an antimetabolite that induces S- phase arrest, and a key component of successful AML Corresponding Author: Christopher C. Porter, University of Colorado School of Medicine,12800 East 19th Avenue, RC1N 4101, Aurora, CO therapy (12, 13). The addition of the Wee1 inhibitor, 80045. Phone: 303-724-4665; Fax: 303-724-4013; E-mail: MK1775 (8), to cytarabine impairs the cell-cycle check- [email protected] point and induces more apoptosis than cytarabine alone doi: 10.1158/1535-7163.MCT-13-0424 (13). Notably, our data were generated in cell lines that are Ó2013 American Association for Cancer Research. reported to have normal p53 function. www.aacrjournals.org 2675 Downloaded from mct.aacrjournals.org on September 26, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst October 11, 2013; DOI: 10.1158/1535-7163.MCT-13-0424 Van Linden et al. Therefore, we sought to determine whether the function Vectors of p53 influences the sensitivity to Wee1 inhibition with MSCV-ires-GFP (MiG), MSCV-DDp53-GFP (DDp53), chemotherapy in a broad panel of AML cell lines with and MSCV-DNp53-GFP (DNp53) plasmids (provided by various molecular abnormalities. In contrast with data Dr. DeGregori) were packaged into viral particles and from solid tumor models sensitized to DNA-damaging transduced into OCI-AML3 cells as previously described agents (8–11), we found that the functionality of p53 has (15). Transduced cells were sorted for GFP using a no bearing on the chemosensitization of AML cells to MoFlow fluorescence-activated cell sorter (Dako Cytoma- cytarabine, as all of the cell lines tested were sensitized tion). Nonsilencing short hairpin RNA (shRNA) and to cytarabine with Wee1 inhibition. Mechanistic studies shRNA-targeting p53 from the TRC collection (16) were indicate that inhibition of Wee1 abrogates the S-phase purchased from the Functional Genomics Facility of the checkpoint and augments apoptosis induced by cytar- University of Colorado Cancer Center (Boulder, CO) and abine. Furthermore, in isogenic models, in which wild- packaged as previously described (17). Transduced cells type p53 activity was impaired by RNA interference or were selected in puromycin (Sigma-Aldrich). dominant negative p53 constructs, we did not find enhanced chemosensitization with impaired p53. Also, Antibodies, chemicals, and reagents in contrast with data from solid tumor models, we did Antibody directed against p21CIP1 was purchased from not observe chemosensitization to doxorubicin with BD Biosciences; antibodies against phosphorylated CDK1 Wee1 inhibition in AML cells, even in cells with non- (Y15), CDK2 (Y15), and histone H3 (S10) were purchased functional p53. In addition, we found that the chemo- from Cell Signaling Technology. Cytarabine and doxoru- sensitization to antimetabolite chemotherapeutics is not bicin were purchased from Sigma-Aldrich and diluted in limited to leukemia, as lung cancer cells were equally water. Nutlin-3 was purchased from Cayman Chemical sensitized to cytarabine and pemetrexed, whether p53 and diluted in dimethyl sulfoxide (DMSO). Pemetrexed function was impaired or not. Finally, in mice with was purchased from Selleck. MK1775 was provided by the AML, we found that the combination of Wee1 inhibition National Cancer Institute (Bethesda, MD) and Merck with cytarabine slowed disease progression and pro- Sharp & Dohme Corporation. longed survival better than cytarabine alone. These data support the development of clinical trials of antimetab- Animal studies olite chemotherapeutics and Wee1 inhibition for Female C57BL/6J mice, 6 to 8 weeks old, were pur- patients with cancers; however, distinct from DNA- chased from The Jackson Laboratory. All mice were damaging agents that induce the G2–M checkpoint, our housed in sterile micro-isolators in the Center for Com- data do not support the use of TP53 mutation as a parative Medicine at the University of Colorado Denver biomarker to predict beneficial effects of Wee1 inhibi- (Denver, CO). Mice were treated with cytarabine 50 mg/ tion when combined with antimetabolites that induce kg/d by intraperitoneal injection and/or MK1775 40 mg/ the S-phase checkpoint. kg/d by oral gavage. One million luciferase expressing AML cells (18) were injected into unirradiated recipients to induce leukemia. Luciferase activity was measured 5 Materials and Methods minutes after injection of luciferin using a Xenogen Cell lines and tissue culture IVIS2000 imaging system. Animal studies were approved Cell lines were generous gifts from the laboratories of by the Institutional Animal Care and Use Committee of Drs. Douglas Graham and James DeGregori (both at the University of Colorado Denver. University of Colorado School of Medicine, Aurora, CO). Cell lines were DNA fingerprinted by multiplex PCR Data analyses using the Profiler Plus or Identifier Kits (ABI) and con- Excel and GraphPad Prism 5 (GraphPad Software) were firmed to match published or internal databases as pre- used for data sorting, analysis, and graphical depiction of viously described (14), before storage of stock vials in data. Student t test was used to compare two samples; liquid nitrogen. All cells were cultured at 37C in humid- ANOVA and Bonferroni post-test were used to compare ified air supplemented with 5% CO2, in RPMI supple- more than two samples. The Mantel–Cox (log-rank) test mented with 10% FBS and antibiotics, except