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A Bis-Indole-Derived NR4A1 Antagonist Induces PD-L1 Degradation and Enhances Anti-Tumor Immunity

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A Bis-Indole-Derived NR4A1 Antagonist Induces PD-L1 Degradation and Enhances Anti-Tumor Immunity Author Manuscript Published OnlineFirst on January 7, 2020; DOI: 10.1158/0008-5472.CAN-19-2314 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. A Bis-Indole-Derived NR4A1 Antagonist Induces PD-L1 Degradation and Enhances Anti-Tumor Immunity Keshav Karki1, Gus A. Wright2, Kumaravel Mohankumar1, Un-Ho Jin1, Xing-Han Zhang1 and Stephen Safe1 Affiliations: 1 Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX, USA, 77843 2 Department of Veterinary Pathobiology, Texas A&M University, College Station, TX, USA, 77843 Running Title: NR4A1 antagonists as immunotherapy mimics Keywords: NR4A1, antagonists, mammary, tumors, immunotherapy mimics Corresponding Author: Stephen H. Safe, Ph. D., College of Veterinary Medicine & Biomedical Sciences, Department of Veterinary Physiology & Pharmacology, Texas A&M University, 4466 TAMU, College Station, TX 77843-4466 USA, Tel: 979-845-5988 / Fax: 979-862-4929, Email: [email protected] Disclosure of Conflicts of Interest: There are no conflicts of interests to declare. Word Count: 5004 Figures: 7 References: 46 1 Downloaded from cancerres.aacrjournals.org on October 6, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 7, 2020; DOI: 10.1158/0008-5472.CAN-19-2314 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT PD-L1 is expressed in tumor cells and its interaction with PD-1 plays an important role in evading immune surveillance; this can be overcome using PD-L1 or PD-1 immunotherapy antibodies. This study reports a novel approach for targeting PD- L1. In human breast cancer cell lines and 4T1 mouse mammary tumor cells PD-L1 expression was regulated by the nuclear receptor NR4A1/Sp1 complex bound to the proximal GC-rich region of the PD-L1 gene promoter. Treatment of breast cancer cells with bis-indole-derived NR4A1 antagonists including 1,1-bis(3'-indolyl)-1-(3-chloro-4- hydroxy-5-methoxyphenyl)methane (Cl-OCH3) decreased expression of PD-L1 mRNA, promoter-dependent luciferase activity and protein. In in vivo studies using a syngeneic mouse model bearing orthotopically injected 4T1 cells Cl-OCH3 decreased tumor growth and weight and inhibited lung metastasis. Cl-OCH3 also decreased expression of CD3+/CD4+/CD25+/FoxP3+ regulatory T cells and increased the Teff/Treg ratio. Therefore, the potent anti-cancer activities of NR4A1 antagonists are also accompanied by enhanced anti-tumor immunity in PD-L1-expressing triple-negative breast cancer and thus represent a novel class of drugs that mimic immunotherapy. SIGNIFICANCE Findings show that the orphan nuclear receptor NR4A1 controls PD-L1 expression and identify a chemical probe capable of disrupting this regulatory axis. 2 Downloaded from cancerres.aacrjournals.org on October 6, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 7, 2020; DOI: 10.1158/0008-5472.CAN-19-2314 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. INTRODUCTION The orphan nuclear receptors NR4A1, NR4A2 and NR4A3 are immediate early genes induced by multiple stressors, and the NR4A receptors play an important role in maintaining cellular homeostasis and in pathophysiology. There is increasing evidence that these receptors are involved in important pathways in metabolic, cardiovascular and neurological functions as well as inflammation and inflammatory diseases and in immune functions and cancer (1, 2). NR4A1 is overexpressed in colon, ovarian, pancreatic, breast (estrogen receptor positive and negative), and lung tumors, and high expression of NR4A1 in breast, colon and lung tumors predicted decreased patient survival (3-9). The functional activity of NR4A1 in cancer has been extensively investigated in cancer cell lines by either knockdown or overexpression (9-20). Results of NR4A1 knockdown studies show that this receptor regulates pathways and genes associated with solid tumor-derived cancer cell growth, survival, migration and invasion. We have also identified a series of bis-indole derived ligands including 1,1-bis(3’- indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH, CDIM-8) which binds NR4A1 (KD=100 nM) (14) and acts as an antagonist inhibiting pro-oncogenic NR4A1-regulated pathways and genes in cancer cells (14-23). Mechanistic studies show that for a number of genes including survivin, PAX3- FOXO1, and several integrins, NR4A1 acts as a nuclear co-factor that binds Sp1 or Sp4 and enhances expression of Sp1/Sp4-regulated genes (5, 17, 22, 23). Moreover, treatment with CDIM-8 and related NR4A1 antagonists inhibits expression of NR4A1/Sp-regulated genes. Although NR4A1/Sp-dependent gene regulation represents a novel pathway of gene regulation where NR4A1 does not directly bind promoter DNA, 3 Downloaded from cancerres.aacrjournals.org on October 6, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 7, 2020; DOI: 10.1158/0008-5472.CAN-19-2314 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. there are many examples of other nuclear receptors including the steroid hormone receptors that also activate genes through interactions with Sp transcription factors bound to their cognate GC-rich promoter elements (24). The PD-L1 gene also contains a proximal GC-rich promoter sequence and recent studies in human gastric cancer cells demonstrate that PD-L1 is a Sp1-regulated gene (25). We hypothesized that PD-L1 may also be an NR4A1/Sp regulated gene that can be targeted by C-DIM/NR4A1 antagonists and thereby mimic the effects of checkpoint inhibitor antibodies. Checkpoint inhibitors represent a relatively new and effective breakthrough in cancer therapy where the blockade of cancer cell derived PD-L1 from interacting with PD-1 on T-cells reactivates tumor infiltrating lymphocytes (TIL). The effectiveness of antibody-derived checkpoint inhibitors is somewhat limited and associated with toxic side effects and development of resistance (26-32). Therefore, the major objective of this study is to determine whether PD-L1 is an NR4A1/Sp regulated gene that can be targeted by NR4A1 antagonists and thereby activate immune surveillance. Our results confirm that PD-L1 is an NR4A1/Sp1 regulated gene and treatment with NR4A1 antagonists decreased expression of PD-L1 in triple negative breast cancer cells and reactivated TILs in a syngeneic mouse model bearing 4T1 mouse mammary cancer cells. Thus, NR4A1 antagonists represent a new class of small molecules that act as immunotherapy mimics with clinical potential for treating patients expressing PD-L1 and NR4A1. 4 Downloaded from cancerres.aacrjournals.org on October 6, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 7, 2020; DOI: 10.1158/0008-5472.CAN-19-2314 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. MATERIALS AND METHODS Cell lines, antibodies, and reagents 1,1-Bis(3'-indolyl)-1-(p-hydroxyphenyl) methane (DIM-C-pPhOH; CDIM-8), and 1,1-bis(3′-indolyl)-1-(3-chloro-4-hydroxy-5-methoxyphenyl)methane (Cl-OCH3) were synthesized as described by the condensation of indole with p-hydroxybenzaldehyde or 3-chloro-4-hydroxy-5-methoxybenzaldehyde (21, 33). Indole and the substituted benzaldehydes were purchased from Sigma-Aldrich (St. Louis, MO). Human mammary breast cancer MDA-MB-231, MCF-7, MDA-MB-468, and SKBR3, cell lines and L3.6PL, Panc-1 (pancreatic cancer), SW480 (colon cancer), Rh30 (rhabdomyosarcoma), A549 (lung cancer) and 786-O (kidney cancer) cell lines were purchased from American Type Culture Collection (Manassas, VA). SW480, L3.6pL, Panc1, MCF-7, SKBR3 and MDA- MB-231 were authenticated by Biosynthesis (Lewisville, TX). Human mammary tumor Sum159PT and HS578T cell lines were kindly provided by Dr. Weston Porter, Texas A&M University and were originally purchased from ATCC. Mouse mammary tumor 4T1 and luciferase tagged 4T1-Luc cell lines were kindly provided by Dr. Mien-Chie Hung, MD Anderson Cancer Center, Houston and cells were initially purchased from ATCC. All tumor cells used in the experiments were mycoplasma negative. Mycoplasma testing was routinely carried out to ensure cell lines were mycoplasma free. MDA-MB- 231, HS578, MCF-7, MDA-MB468, SKBR3, L3.6PL, SW480, Panc-1, A549 were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. 4T1, 4T1-Luc and Sum159PT cells were maintained in (Dulbecco's Modified Eagle's Medium)/Hams F-12 50/50 mix containing 2.5 mM L-glutamine, 10% fetal bovine serum (FBS). Rh30 and 786-O were maintained in RPMI 1640-Medium supplemented with L- 5 Downloaded from cancerres.aacrjournals.org on October 6, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 7, 2020; DOI: 10.1158/0008-5472.CAN-19-2314 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. glutamine, 10% fetal bovine serum (FBS). All cells were maintained at 37°C in the presence of 5% CO2, and the solvent (dimethyl sulfoxide, DMSO) used in the experiments was ≤0.2%. Antibodies, primers and oligonucleotides are summarized in supplemental Table 1. The wild type and mutant PD-L1 promoter constructs (+66 to- 263) containing the proximal (-5 to -15) wild type GC-rich Sp1 binding (CCCGCCTCCGG), Mutant1 (CAAGCCTCCAA) and, Mutant2 (CCCGCCTCCAG) sequences were synthesized by DNA technologies (IDT, Coralville, IA). The constructs were cloned and verified by Eurofins Genomics LLC (Louisville, KY). Western blot analysis Whole cell lysates from MDA-MB-231, Sum159PT, HS578T, MCF-7, MDA-MB- 468, SKBR3, 4T1, L3.6PL, SW480, Rh30, Panc-1, A549 and 786-O were analyzed by western blots as described (21,22). Equal amounts of protein were separated by 10% and 15% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. PVDF membranes were incubated overnight at 4˚C with primary antibodies in 5% skimmed milk and incubated for 2-3 hours with secondary antibodies conjugated with HRP.
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