Supplemental Figure S5 from A Bis-Indole–Derived NR4A1 Antagonist Induces PD-L1 Degradation and Enhances Antitumor Immunity

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posted on 2023-03-31, 03:44 authored by Keshav Karki, Gus A. Wright, Kumaravel Mohankumar, Un-Ho Jin, Xing-Han Zhang, Stephen Safe

Supplemental Figure S5. A-F. Gating Strategy for Teff (CD3+/CD8+) and Treg (CD3+/CD4+/CD25+/FoxP3+) cells. In panel A, cells are selected within the "cells" gate from the Forward Scatter versus Side Scatter dot plot. In panel B, cells from within the "cells" gate are plotted on a dot plot showing Side Scatter Area versus Side Scatter Height in which the single cells are selected within the "singlets" gate. In panel C, single cells from within the "cells" and "singlets" gates are plotted on a dot plot depicting Side Scatter versus Live/Dead Near IR dye. Viable, single cells are selected within the "viable" gate. In panel D, viable, single cells are shown on a dot plot depicting Side Scatter versus CD3 PE-Cy5.5 in which the CD3+ cells are selected within the "CD3+" gate. In panel E, CD3+, live, single cells are visualized on a dot plot with CD8 PE-eFluor 610 on the x-axis and CD4 Alexa Fluor 488 on the y-axis. The CD4+ cells are selected within "CD4+" gate and the CD8+ cells are selected within the "CD8+" gate. In panel E, the CD4+, CD3+, viable, single cells are plotted on a dot plot showing FoxP3 BV 421 versus CD25 Alexa Fluor 647. The regulatory T cells are selected within the "T reg cells" gate. G/H. GraphPad PRISM software was used to calculate Effective concentration (EC50) of PD-L1 mRNA (G) and PD-L1 protein (H) after treatment with different concentrations of CDIM-8 and Cl-OCH3 and the values were calculated by regression analysis using the dose-response curves generated from the experimental data.

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NIH

Texas A&M AgriLife Research

Sid Kyle Chair Endowment

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ARTICLE 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 germinal center (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 anticancer activities of NR4A1 antagonists are also accompanied by enhanced antitumor immunity in PD-L1–expressing triple-negative breast cancer and thus represent a novel class of drugs that mimic immunotherapy. These findings show that the orphan nuclear receptor NR4A1 controls PD-L1 expression and identify a chemical probe capable of disrupting this regulatory axis.

Supplemental Figure S5 from A Bis-Indole–Derived NR4A1 Antagonist Induces PD-L1 Degradation and Enhances Antitumor Immunity

Funding

NIH

Texas A&M AgriLife Research

Sid Kyle Chair Endowment

History

ARTICLE ABSTRACT

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