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Full Text (PDF) Published OnlineFirst October 23, 2018; DOI: 10.1158/1535-7163.MCT-18-0863 Small Molecule Therapeutics Molecular Cancer Therapeutics Targeting of Hematologic Malignancies with PTC299, A Novel Potent Inhibitor of Dihydroorotate Dehydrogenase with Favorable Pharmaceutical Properties Liangxian Cao1, Marla Weetall1, Christopher Trotta1, Katherine Cintron1, Jiyuan Ma1, Min Jung Kim1, Bansri Furia1, Charles Romfo1, Jason D. Graci1, Wencheng Li1, Joshua Du1, Josephine Sheedy1, Jean Hedrick1, Nicole Risher1, Shirley Yeh1, Hongyan Qi1, Tamil Arasu1, Seongwoo Hwang1,William Lennox1, Ronald Kong1, Janet Petruska1,Young-Choon Moon1, John Babiak1, Thomas W. Davis1, Allan Jacobson2, Neil G. Almstead1, Art Branstrom1, Joseph M. Colacino1, and Stuart W. Peltz1 Abstract PTC299 was identified as an inhibitor of VEGFA mRNA engagement in the DHODH conformation in situ is translation in a phenotypic screen and evaluated in the required for its optimal activity. PTC299 has broad and clinic for treatment of solid tumors. To guide precision potent activity against hematologic cancer cells in preclin- cancer treatment, we performed extensive biological char- ical models, reflecting a reduced pyrimidine nucleotide acterization of the activity of PTC299 and demonstrated salvage pathway in leukemia cells. Archived serum samples that inhibition of VEGF production and cell proliferation from patients treated with PTC299 demonstrated increased by PTC299 is linked to a decrease in uridine nucleotides levels of dihydroorotate, the substrate of DHODH, indi- by targeting dihydroorotate dehydrogenase (DHODH), a cating target engagement in patients. PTC299 has advan- rate-limiting enzyme for de novo pyrimidine nucleotide tages over previously reported DHODH inhibitors, includ- synthesis. Unlike previously reported DHODH inhibitors ing greater potency, good oral bioavailability, and lack of that were identified using in vitro enzyme assays, PTC299 off-target kinase inhibition and myelosuppression, and is a more potent inhibitor of DHODH in isolated mito- thus may be useful for the targeted treatment of hemato- chondria suggesting that mitochondrial membrane lipid logic malignancies. Introduction fourth enzymatic step, the ubiquinone-mediated oxidation of dihydroorotate to orotate in the de novo pyrimidine synthesis De novo pyrimidine nucleotide biosynthesis is activated in pathway (6). DHODH is a validated therapeutic target for the proliferating cancer cells in response to an increased demand for treatment of autoimmune diseases such as rheumatoid arthritis synthesis of DNA, RNA, and phospholipids by activation of (7, 8). Inhibition of DHODH was shown in preclinical studies to oncogenic pathways such as Myc, PI3K, PTEN, and mTOR (1– prevent growth of many types of cancers (9–13). Moreover, it was 5). Dihydroorotate dehydrogenase (DHODH), located on the demonstrated that inhibition of DHODH induces differentiation surface of the inner mitochondrial membrane, catalyzes the of acute myeloid leukemia (AML) cells (14–16). Pharmacologic inhibition of de novo pyrimidine synthesis sensitizes triple-nega- tive breast cancer cells to genotoxic chemotherapy agents and 1PTC Therapeutics, Inc., South Plainfield, New Jersey. 2Department of Microbi- reduces chemotherapy resistance (2). DHODH inhibitors such as ology and Physiological Systems, University of Massachusetts Medical School, teriflunomide, brequinar, and vidofludimus have been consid- Worcester, Massachusetts. ered for use in oncology (17–20), but their use may be limited due Note: Supplementary data for this article are available at Molecular Cancer to a narrow therapeutic window as a result of lesser potency and/ Therapeutics Online (http://mct.aacrjournals.org/). or off-target activities such as their inhibition of kinases (21, 22). Current address for T. Arasu: US Food and Drug Administration, 629 Cranbury There is great interest in the discovery and development of new Road, 2nd Floor, East Brunswick, NJ 08816; and current address for T.W. Davis: DHODH inhibitors for therapeutic uses (23, 24). These efforts PMV Pharmaceuticals Inc., Cedar Brook Corporate Center, 8 Clarke Drive, have mainly relied on in vitro enzyme assays to define and Cranbury Township, NJ 08512. optimize activity, in which the purified enzyme conformation Corresponding Author: Liangxian Cao, PTC Therapeutics, Inc., 100 Corporate may differ from that found in the cells due to lack of membrane fi CT, South Plain eld, NJ 07080. Phone: 908-912-9132; Fax: 908-222-0567; lipids in the assay (25). Recently, it was demonstrated that E-mail: [email protected] DHODH attaches to the inner mitochondrial membrane by doi: 10.1158/1535-7163.MCT-18-0863 binding charged phospholipids which stabilize the flexible sub- Ó2018 American Association for Cancer Research. strate- and drug-binding site, suggesting that the design and www.aacrjournals.org 3 Downloaded from mct.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst October 23, 2018; DOI: 10.1158/1535-7163.MCT-18-0863 Cao et al. development of novel inhibitors that additionally engage in lipid (Crown Bioscience); otherwise we did not perform any additional interactions may represent a promising approach to targeting cell line authentication in house. The information on the source of DHODH in vivo (26). cells and when they were obtained can be found in Supplemen- PTC299 was identified as an inhibitor of the translation of tary Table S1. VEGFA mRNA using PTC Therapeutics' proprietary GEMS tech- Stable cell lines were generated as reported previously (27). nology phenotypic screening platform (27). PTC299 was in Briefly, plasmid DNA was transfected into HT1080 cells using clinical trials for the treatment of solid tumors (28, 29) and is Fugene-6 transfection reagent (Roche Diagnostics GmbH), fol- currently being reevaluated for use in hematologic cancers. To lowed by selection with 200 mg/mL hygromycin for luciferase- guide safe and effective precision cancer treatment, we performed stable cell lines or 200 mg/mL Zeocin (Invitrogen) for V5 tag stable extensive biochemical characterization of PTC299 and demon- cells in the culture medium. strated that it interacts with and inhibits DHODH, a rate-limiting enzyme in the de novo biosynthesis of pyrimidine nucleotides (5). Western blot analysis Inhibition of VEGFA production by PTC299 is a downstream Western blot was performed as reported previously (27). Anti- effect of inhibiting de novo pyrimidine synthesis as it can be body (C1) specific for VEGFA was purchased from Santa Cruz completely rescued by exogenously added uridine but not by Biotechnologies (1:200 dilution); antibody specific for human other nucleosides. Collectively, PTC299 represents a novel class of b-actin was purchased from Abcam (1:10,000 dilution); antibody potent DHODH inhibitors, and our data demonstrate the poten- specific for V5 tag was purchased from Invitrogen (1:5,000 tial of PTC299 as a treatment option to address unmet needs in dilution). Antibody specific for DHODH was purchased from cancers, such as leukemia and lymphoma, that have lower levels Proteintech (Cat#: 14877-1-AP, 1:500 dilution); and antibody of uridine salvage activity and thus rely on the de novo biosynthesis specific for human prohibitin was purchased from Thermo of pyrimidine nucleotides for survival and rapid proliferation. Scientific (Cat#: PA5-12274 and PA5-14133, 1:1,000 dilution). Immunodetection was done using the corresponding secondary Materials and Methods antibodies conjugated with infrared dyes or horseradish peroxide. The expression levels of proteins were detected with Odyssey General methods (LI-COR) or using enhanced chemiluminescence (Pierce). DNA constructs were generated using standard procedures as reported previously (27). VEGFA 50- and 30-untranslated regions Determination of ELISA EC and cytotoxicity CC values (UTR) were amplified from human genomic DNA (Clontech), 50 50 All ELISAs were performed using commercially available ELISA while the VEGFA165 open reading frame from RNA derived from kits (R&D Systems) according to the manufacturer's instructions. HeLa cells incubated under hypoxia (1% oxygen) was amplified The screen of 240 cell lines was performed at Crown Bioscience. by RT-PCR. DNAs generated with PCR were confirmed by DNA Cells were treated with PTC299 at a series of doses for 72 hours, and sequencing. the inhibition of cell proliferation was determined using a standard PTC299, PTC-371, GSK983, and vidofludimus (4SC-101) were assay, CellTiter-Glo Luminescent Cell Viability Assay (Promega), synthesized at PTC Therapeutics; for details, see the Supplemen- that measures total cellular adenosine triphosphate (ATP) con- tary Materials and Methods in the Supplementary Data File. centrations as an indicator of cell viability. Data generated from Brequinar (SML0113) and teriflunomide (SML0936) were pur- ELISA or cytotoxicity studies were plotted with Prism software. A chased from Sigma, whereas pyrazofurin-MP (P61-B01A) was sigmoidal dose response with a variable slope regression curve was purchased from TriLink Biotechnologies. generated for each compound. Maximal and minimal inhibitions were set at 100% and 0%, respectively, and CC50 values were Cell culture calculated after curve fitting using the Prism software. Tumor cell lines, except for MOLM-13 which were obtained from Dr. Kensuke Kojima in July 2015 (Saga University, Japan) 35S-labeling and immunoprecipitation 0 and Huh-7 which were obtained in February 2001 from the Stuart HT1080 cells harboring
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