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Targeting Casein Kinase 1 Delta Sensitizes Pancreatic and Bladder

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Targeting Casein Kinase 1 Delta Sensitizes Pancreatic and Bladder Published OnlineFirst May 19, 2020; DOI: 10.1158/1535-7163.MCT-19-0997 MOLECULAR CANCER THERAPEUTICS | SMALL MOLECULE THERAPEUTICS Targeting Casein Kinase 1 Delta Sensitizes Pancreatic and Bladder Cancer Cells to Gemcitabine Treatment by Upregulating Deoxycytidine Kinase Francesca Vena1, Simon Bayle1, Ainhoa Nieto2, Victor Quereda1, Massimiliano Aceti1, Sylvia M. Frydman1, Samer S. Sansil3, Wayne Grant4, Andrii Monastyrskyi1, Patricia McDonald2, William R. Roush4, Mingxiang Teng5, and Derek Duckett1 ABSTRACT ◥ Although gemcitabine is the cornerstone of care for pancreatic models. Genetic studies confirmed that silencing CK1d or treat- ductal adenocarcinoma (PDA), patients lack durable responses ment with SR-3029 induced a significant upregulation of deox- and relapse is inevitable. While the underlying mechanisms ycytidine kinase (dCK), a rate-limiting enzyme in gemcitabine leading to gemcitabine resistance are likely to be multifactorial, metabolite activation. The combination of SR-3029 with gemci- there is a strong association between activating gemcitabine tabine induced synergistic antiproliferative activity and enhanced metabolism pathways and clinical outcome. This study evaluated apoptosis in both pancreatic and bladder cancer cells. Further- casein kinase 1 delta (CK1d) as a potential therapeutic target for more, in an orthotopic pancreatic tumor model, we observed PDA and bladder cancer, in which CK1d is frequently over- improved efficacy with combination treatment concomitant with expressed. We assessed the antitumor effects of genetically increased dCK expression. This study demonstrates that CK1d silencing or pharmacologically inhibiting CK1d using our in- plays a role in gemcitabine metabolism, and that the combination house CK1d small-molecule inhibitor SR-3029, either alone or in of CK1d inhibition with gemcitabine holds promise as a future combination with gemcitabine, on the proliferation and survival therapeutic option for metastatic PDA as well as other cancers of pancreatic and bladder cancer cell lines and orthotopic mouse with upregulated CK1d expression. Introduction exhibit a good initial clinical response, chemoresistance is inevitable, and patients ultimately succumb to the disease (10). Mechanistically, The development of new targeted agents that specifically inhibit 0 0 gemcitabine acts as a deoxycytidine analogue (2 ,2-difluorodeoxy- kinases involved in tumorigenesis has led to important progress in the cytidine) and belongs to the class of antimetabolite drugs. Gemcitabine treatment of various cancers (1–3). In aggressive tumor types, such as is taken up by the nucleoside transporters hENT1 and hCNT1 and pancreatic ductal adenocarcinoma (PDA), where patients are refrac- phosphorylated by deoxycytidine kinase (dCK) to the mono- and tory to all standard therapies, multiple clinical trials have been con- subsequently to the di-(dFdCDP) and tri-(dFdCTP) phosphorylated ducted in the past three decades. But results have been modest, forms (11). The dFdCTP metabolite is incorporated into DNA where it extending overall survival incrementally and with outcomes con- acts as a false nucleotide, causing replication fork collapse and cell founded by significant side-effects (4, 5), making PDA one of the death by apoptosis (12). Importantly, altered expression of enzymes most lethal human diseases, with a 5-year overall survival rate of less controlling gemcitabine metabolism provoke both gemcitabine resis- than 8% (6, 7). Therefore, an understanding of the main molecular tance and sensitivity. For example, overexpression of cytidine mechanisms that drive PDA disease and the identification of novel deaminase (CDA), the main enzyme of gemcitabine inactivation, actionable therapeutic targets are urgently needed to improve the is associated with gemcitabine resistance (13, 14), whereas upre- clinical outcomes of patients suffering with this dreadful disease (8). gulation of dCK correlates with increased response to gemcitabine Gemcitabine is the mainstay treatment for locally advanced and treatment (15–18). Furthermore, dCK overexpression has also been metastatic PDA (9). Although patients treated with gemcitabine may associated with higher overall survival and progression-free survival in patients with PDA (18). Although several strategies to improve gemcitabine efficacy have 1Department of Drug Discovery, Moffitt Cancer Center, Tampa, Florida. 2Depart- ment of Cancer Physiology, Moffitt Cancer Center, Tampa, Florida. 3Translational been evaluated in the clinic, such as combination with targeted agents, Research Core, Moffitt Cancer Center, Tampa, Florida. 4Department of Chem- immunotherapies, or other chemotherapies, these studies have shown istry, The Scripps Research Institute, Jupiter, Florida. 5Department of Biosta- mainly incremental improvements in outcomes for patients with tistics and Bioinformatics, Moffitt Cancer Center, Tampa, Florida. PDA (4, 19, 20). Approaches that increase gemcitabine sensitivity Note: Supplementary data for this article are available at Molecular Cancer through mechanisms that increase intracellular dFdCTP levels are Therapeutics Online (http://mct.aacrjournals.org/). highly attractive. d e F. Vena and S. Bayle contributed equally to this article. Casein kinase 1 delta and epsilon (CK1 and CK1 , respectively) are serine/threonine kinases known to play a role in multiple cellular Corresponding Author: Derek Duckett, Moffitt Cancer Center, 12902 Magnolia processes, including membrane trafficking, cytokinesis, circadian Drive, Tampa, FL 33612. Phone: 813-745-5410; E-mail: – d derek.duckett@moffitt.org rhythm, and tumorigenesis (21 24). In particular, CK1 has been shown to interact with key molecules of signaling pathways frequently Mol Cancer Ther 2020;19:1623–35 dysregulated in cancer, such as the Wnt/b-catenin, PI3K/AKT signa- doi: 10.1158/1535-7163.MCT-19-0997 lin,g and Hedgehog pathway (25–27). Importantly, alterations in the Ó2020 American Association for Cancer Research. expression levels or activity of CK1 isoforms have been found in several AACRJournals.org | 1623 Downloaded from mct.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst May 19, 2020; DOI: 10.1158/1535-7163.MCT-19-0997 Vena et al. tumor types. For instance, CK1d expression has been associated with a treatment using a Caspase 3/7 Glo Assay (Promega) as per the more aggressive disease and poorer prognosis in breast, glioblastoma, manufacturer’s instructions. All data represent the mean of three ovarian, colorectal, and pancreatic cancers (21, 28–31). Thus, CK1d independent experiments. represents an attractive therapeutic target for human cancers aber- rantly expressing CK1d. The role of CK1e isoform in cancer is less well Synergy quantification of drug combinations understood and has been correlated with both poor (22, 32, 33) and To generate CI values, different volume combinations (1:1 or 4:1) of improved overall survival (34, 35). the initial stock concentration of each drug (gemcitabine 10 mmol/L, In this study, we found CK1d expression to be correlated with SR-3029 10 mmol/L) was used to generate 10-point 1:3 dilution overall survival, tumor grade, and distal metastasis in patients with concentration response curves. Loewe additivity is a dose-effect model, PDA. Our in vitro and in vivo studies have revealed synergistic where additivity occurs in a two-drug combination if the sum of the antitumor activity between gemcitabine and our in-house CK1d ratios of the dose versus the median-effect for each individual drug is 1. inhibitor SR-3029 in both pancreatic and bladder cancer and have In this model, CI scores estimate the interaction between the two drugs. identified a distinctive mechanism responsible for SR-3029 sensitiza- ACI< 1 is considered synergistic, CI > 1 antagonistic, and CI ¼ 1is tion to gemcitabine cytotoxicity. Our findings provide a strong ratio- additive. Chou and Talalay (37) showed that Loewe equations are valid nale for combining the CK1d-specific inhibitor SR-3029 with gemci- for enzyme inhibitors with similar mechanisms of action either tabine in patients with PDA and bladder cancer. competitive or noncompetitive toward the substrate. The CI coeffi- cients were computed on the basis of the Chou–Talalay Median Effect model as outlined in CalcuSyn v2.11 (http://www.biosoft.com/w/ Materials and Methods calcusyn.htm). The degree of interaction between drugs was estimated Cell culture and reagents according to the classification presented by Chou–Talalay (37). Human PDA cell lines BxPC-3, MIAPaCa-2, and PANC-1 and human bladder cancer cell lines 5637, HT-1376, J82, T24, TCCSUP, Lentiviral transduction and UM-UC-3 were purchased from the ATCC. Cells were maintained Lentiviral particles were produced using HEK293T cells and a third- in DMEM (Gibco) or RPMI1640 (Gibco) for both BxPC-3 and generation packaging system, MISSION Lentiviral Packaging Mix MIAPaCa-2 and supplemented with 10% FBS and penicillin/strepto- (Sigma-Aldrich), as per the manufacturer's instructions. To stably fi mycin (Gibco). All cells were cultured at 37 C in a 5% CO2 incubator. express speci c luciferase or the short hairpin RNA (shRNA) against Cells have been regularly tested for Mycoplasma by qPCR and were not CK1d (shCK1d sequence: CCGGGAATTGCAGAGAATCAGACT- maintained in culture or used beyond 20 passages. CCTCGAGGAGTCTGATTCTCTGCAATTCTTTTT), PANC-1 and Gemcitabine triphosphate (dFdCTP) was purchased from Toronto T24 cells were transduced with optimized titers of lentiviruses.
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