Characterization of a Novel PERK Kinase Inhibitor with Antitumor and Anti-Angiogenic Activity
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Published OnlineFirst January 18, 2013; DOI: 10.1158/0008-5472.CAN-12-3109 Cancer Tumor and Stem Cell Biology Research Characterization of a Novel PERK Kinase Inhibitor with Antitumor and Anti-Angiogenic Activity Charity Atkins1, Qi Liu1, Elisabeth Minthorn1, Shu-Yun Zhang1, David J. Figueroa1, Katherine Moss1, Thomas B. Stanley3, Brent Sanders1, Aaron Goetz2, Nathan Gaul2, Anthony E. Choudhry2, Hasan Alsaid4, Beat M. Jucker4, Jeffrey M. Axten1, and Rakesh Kumar1 Abstract The unfolded protein response (UPR) is a signal transduction pathway that coordinates cellular adaptation to microenvironmental stresses that include hypoxia, nutrient deprivation, and change in redox status. These stress stimuli are common in many tumors and thus targeting components of the UPR signaling is an attractive therapeutic approach. We have identified a first-in-class, small molecule inhibitor of the eukaryotic initiation factor 2-alpha kinase 3 (EIF2AK3) or PERK, one of the three mediators of UPR signaling. GSK2656157 is an ATP-competitive inhibitor of PERK enzyme activity with an IC50 of 0.9 nmol/L. It is highly selective for PERK with IC50 values >100 nmol/L against a panel of 300 kinases. GSK2656157 inhibits PERK activity in cells – with an IC50 in the range of 10 30 nmol/L as shown by inhibition of stress-induced PERK autophosphorylation, eIF2a substrate phosphorylation, together with corresponding decreases in ATF4 and CAAT/enhancer binding protein homologous protein (CHOP) in multiple cell lines. Oral administration of GSK2656157 to mice shows a dose- and time-dependent pharmacodynamic response in pancreas as measured by PERK autophosphoryla- tion. Twice daily dosing of GSK2656157 results in dose-dependent inhibition of multiple human tumor xenografts growth in mice. Altered amino acid metabolism, decreased blood vessel density, and vascular perfusion are potential mechanisms for the observed antitumor effect. However, despite its antitumor activity, given the on-target pharmacologic effects of PERK inhibition on pancreatic function, development of any PERK inhibitor in human subjects would need to be cautiously pursued in cancer patients. Cancer Res; 73(6); 1– 10. Ó2012 AACR. Introduction endoplasmic reticulum, triggering a coordinated cellular To cope with the relatively extreme conditions of the tumor response to alleviate the impact of the stress and enhance microenvironment, cancer cells depend on adaptive responses cell survival. Responses include an increase in the level of to cope with stress and to continue to proliferate. Multiples chaperone proteins to enhance protein refolding, degrada- lines of evidence have implicated the unfolded protein tion of the misfolded proteins, and translational arrest to response (UPR), an endoplasmic reticulum stress sensing/ decrease the burden of proteins entering the endoplasmic repair pathway, in cell survival and tumor progression (1–3). reticulumf. These pathways also regulate cell survival by – Stress stimuli that activate UPR include hypoxia, nutrient modulating apoptosis (2 4) and can trigger cell death under deprivation, increased protein synthesis, disruption of pro- conditions of prolonged endoplasmic reticulum stress. tein glycosylation (glucose deprivation), decreased luminal Three endoplasmic reticulum membrane proteins have fi endoplasmic reticulum calcium, or changes in endoplasmic been identi ed as primary effectors of the UPR: protein reticulum redox status (2, 3). These perturbations result in kinase R (PKR)-like endoplasmic reticulum kinase [PERK, the accumulation of unfolded or misfolded proteins in the also known as eukaryotic initiation factor 2-alpha kinase 3 (EIF2AK3), or pancreatic endoplasmic reticulum kinase (PEK)], inositol-requiring gene 1 a/b (IRE1), and activating Authors' Affiliations: 1GlaxoSmithKline, Oncology R&D; 2Platform Tech- transcription factor 6 (ATF6; ref. 2). These proteins are held nology & Science, Collegeville, Pennsylvania; 3Platform Technology & Science, Research Triangle Park, North Carolina; and 4Preclinical and in the inactive state by binding to the endoplasmic reticulum Translational Imaging, LAS, King of Prussia, Pennsylvania chaperone, GRP78 (BiP). Accumulation of unfolded proteins Note: Supplementary data for this article are available at Cancer Research in the endoplasmic reticulum leads to release of GRP78 from Online (http://cancerres.aacrjournals.org/). the luminal stress-sensing domains of the UPR effectors Corresponding Author: Rakesh Kumar, GlaxoSmithKline, Oncology R&D, resulting in their activation (5). 1250 South Collegeville Road, Collegeville, PA 19426. Phone: 610-917- PERK is a type I endoplasmic reticulum transmembrane 4855; Fax: 610-917-4181; E-mail: [email protected] protein containing a stress-sensing domain facing the endo- doi: 10.1158/0008-5472.CAN-12-3109 plasmic reticulum lumen and a cytosolic kinase domain (6, 7). Ó2012 American Association for Cancer Research. Release of GRP78 from the stress-sensing domain of PERK www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst January 18, 2013; DOI: 10.1158/0008-5472.CAN-12-3109 Atkins et al. results in oligomerization and autophosphorylation (8, 9). components offers a novel therapeutic approach for cancers, PERK phosphorylates the eukaryotic initiation factor 2a especially those of secretory nature (adenocarcinoma, neuro- (eIF2a) at serine-51 (10). This site is also phosphorylated by endocrine tumors, and multiple myeloma) as well as those with other PERK family members [general control nonderepressed greater hypoxia (pancreatic adenocarcinoma and glioblasto- 2 (GCN2), protein kinase RNA-activated (PKR), and heme- ma). This manuscript describes the biologic characterization regulated kinase (HRI)] in response to different stimuli. Phos- of a novel, highly selective inhibitor of PERK and its potential phorylation of eIF2a converts it to an inhibitor of eIF2B, which utility as an anticancer agent. hinders the assembly of the 40S ribosome translation initiation complex and consequently reduces the rate of translation initiation. However, translation of certain messages encoding Materials and Methods downstream effectors of eIF2a, ATF4, and CAAT/enhancer Reagents binding protein (C/EBP) homologous protein (CHOP), which GSK2656157 was synthesized at GlaxoSmithKline (GSK). It modulate cellular survival pathways, is actually increased upon was dissolved in dimethyl sulfoxide (DMSO) to a stock con- endoplasmic reticulum stress. A second proposed PERK sub- centration of 10 mmol/L, and stored in the dark at À20 C. strate, Nrf2 (NF-E2-related factor 2), regulates cellular redox Compound stock solution was thawed at room temperature potential and contributes to cell adaptation (11). It is thought before dilution into aqueous media at selected concentrations that the normal function of PERK is to protect secretory cells for use in biologic assays. For in vivo studies, GSK2656157 was from endoplasmic reticulum stress. PERK knockout mice formulated in 0.5% hydroxypropyl methyl cellulose, 0.1% exhibit diabetes due to pancreatic islet cell death, skeletal tween-80 in water (pH 6.75). Tunicamycin (Molecular BioPro- abnormalities, and growth retardation (12–14). These features ducts), thapsigargin (Sigma-Aldrich), and cyclohexamide are similar to those seen in patients with Wolcott–Rallison (Tocris Bioscience) were commercially obtained. EasyTag syndrome, who carry inactivating germ line mutations in the EXPRESS S35 Protein Labeling Mix, [35S]-, 2 mCi (74 MBq) was PERK gene (15, 16). purchased from Perkin Elmer. PERK has also been shown to have endoplasmic reticulum NuPAGE SDS-PAGE, transfer solutions and consumables stress independent effects. PERK regulates proliferation of beta were purchased from Invitrogen. Odyssey blocking buffer was cells during embryonic and neonatal development and is purchased from LI-COR Biosciences. RNeasy RNA isolation kits essential for viability of acinar cells in mouse exocrine pan- were purchased from Qiagen. Quantitative Pathway PCR creas, neither of which is associated with endoplasmic retic- Arrays were purchased from SABiosciences. ulum stress response (13, 14, 17). PERK is also required for endoplasmic reticulum functions including proinsulin traffick- Animals ing and quality control in beta cells (18, 19). Similarly, PERK Eight- to twelve-week-old naive female CD-1, female nu/nu modulates proliferation and differentiation of osteoblasts as CD-1 mice (Charles River Laboratories) and severe combined well as secretion of type I collagen (20). immunodeficient (SCID) mice (Taconic Farms) were used. All Disruption of PERK kinase activity in human tumor cell animal studies were conducted after review by the Institutional lines has been shown to increase apoptosis under hypoxia Animal Care and Use Committee at GSK and in accordance in vitro and impair tumor growth in vivo (21). Tumor cells with the GSK Policy on the Care, Welfare and Treatment of experience episodes of hypoxia and nutrient deprivation dur- Laboratory Animals. ing their growth due to inadequate blood supply and aber- rant blood vessel function (22, 23). Thus, they are likely to Cell lines and culture be dependent on active UPR signaling to facilitate their Cell lines were purchased from the American Type Culture growth. Consistent with this, mouse fibroblasts derived from Collection and were routinely cultured in the recommended À À À À À À PERK / , XBP1 / , and ATF4 / mice and fibroblasts