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Supplemental Figure S2 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
<p>Supplemental Figure S2. Quantitation of results in Figure 1F and 1G. and mRNA results A. Breast cancer cells were treated with Cl-OCH3 (Fig. 1F) and effects on PD-L1, NR4A1 and Sp1 were determined by western blots and quantitation of the results are summarized. B. MDA-MB-231 and 4T1 cells were treated with NR4A1 antagonists, MG132 and their combinations, whole cell lysates were analyzed by western blots (Fig. 1G) and quantitation of the results are summarized. Results of both studies (A, B) are means {plus minus} SD for at least 3 replicate determinations for each treatment groups and changes in expression were determined relative to the control group (set ratio) significantly (p<0.05) decreased effects are indicated (*) and inhibition (reversal of degradation) by MG132 is also indicated (**). C. Basal PD-L1 and NR4A1 mRNA levels in cancer cell lines were determined as described in the Methods.</p>

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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.

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