Unleashing the Power of NR4A1 Degradation as a Novel Strategy for Cancer Immunotherapy

Abstract

An effective cancer therapy requires both killing cancer cells and targeting tumor-promoting pathways or cell populations within the tumor microenvironment (TME). We purposely search for molecules that are critical for multiple tumor-promoting cell types and identified nuclear receptor subfamily 4 group A member 1 (NR4A1) as one such molecule. NR4A1 has been shown to promote the aggressiveness of cancer cells and maintain the immune suppressive TME. Using genetic and pharmacological approaches, we establish NR4A1 as a valid therapeutic target for cancer therapy. Importantly, we have developed the first-of-its kind proteolysis-targeting chimera (PROTAC, named NR-V04) against NR4A1. NR-V04 effectively degrades NR4A1 within hours of treatment in vitro and sustains for at least 4 days in vivo, exhibiting long-lasting NR4A1-degradation in tumors and an excellent safety profile. NR-V04 leads to robust tumor inhibition and sometimes eradication of established melanoma tumors. At the mechanistic level, we have identified an unexpected novel mechanism via significant induction of tumor-infiltrating (TI) B cells as well as an inhibition of monocytic myeloid derived suppressor cells (m-MDSC), two clinically relevant immune cell populations in human melanomas. Overall, NR-V04-mediated NR4A1 degradation holds promise for enhancing anti-cancer immune responses and offers a new avenue for treating various types of cancer.

Keywords: NR4A1; PROTAC degrader; cancer immunotherapy; immune modulation; melanoma.

Figure 1.

NR4A1 is elevated in several tumor-promoting immune cells within the tumor microenvironment. A. Expression of NR4A1 in T cells from blood, normal parenchyma, adjacent normal junction, or human hepatocellular carcinomas (HCC, GSE98638). B. Violin plots showing NR4A1 expression in different immune cells from human blood or melanomas. C. Violin plots showing gene expression including NR4A family and lineage markers in human melanomas (GSE120575).

Figure 2.

NR4A1-deletion leads to diminished tumor growth. A-C. Primary tumor growth curve in littermates of wild type (WT) or NR4A1^−/− mice, including (A) MC38 colon cancer, (B) Yummer1.7 melanoma and (C)B16F10 melanoma. n = 7–8 mice per group.

Figure 3.

The design and synthesis of NR4A1 PROTACs. A. Docking study revealed the carboxylic acid in celastrol is a potential tethering vector for PROTAC construction. B. The structure of synthesized PROTACs. C-D. The initial screening of NR4A1 degradation in CHL-1, a human melanoma cell line. CHL-1 cells were treated with 250 nM PROTACs for 16 hours and the degradation was determined by (C) immunoblotting and (D) densitometry. n=2.

Figure 4.

NR-V04 induces NR4A1 degradation. A. NR-V04 effectively promoted the degradation of NR4A1 in two human melanoma cell lines in 16 hours, CHL-1 (DC50 = 228.5 nM) and A375 (DC50 = 518.8 nM), while simultaneously stabilizing VHL protein. B. Celastrol treatment did not result in any significant change in the expression level of NR4A1 in the CHL-1 cell line in 16 hours. C. Time-dependent degradation of NR4A1. CHL-1 cells were treated with 500nM of NR-V04 at the indicated time points. D. NR-V04 did not induce the degradation of NR4A2 and NR4A3. A-D: n=2.

Figure 5.. NR-V04 induces a ternary complex formation and mediates NR4A1 degradation through the ubiquitin-proteasome system.

A. Proximity Ligation Assay (PLA) showing ternary complex formation induced by NR-V04. CHL-1 (left panels) and A375 (right panels) cells were treated with DMSO, 500 nM Celastrol, or 500 nM NR-V04 for 16 hrs. Representative images were shown for PLA assay (20 x magnification). B. Co-immunoprecipitation (co-IP) experiment showing complex formation between NR4A1 and VHL by NR-V04 treatment. Co-IP was performed in NR4A1-Flag overexpressed HEK293T cells that were pretreated with 0.5 μM MG132 for 10 minutes, followed by 16-hour treatment with DMSO or 500 nM NR-V04. NR4A1 was pulled down using an anti-Flag antibody conjugated to magnetic beads. C. NR-V04 induces NR4A1 degradation via VHL E3 ligase- and proteasome-dependent manner. CHL-1 cells were pretreated with 0.5 μM MG132 or 10 μM VHL 032 for 10 minutes, followed by 16-hour treatment with DMSO or 500 nM NR-V04. A-C: n=2.

Figure 6.. The pharmacokinetic (PK) and pharmacodynamic (PD) study of NR-V04.

A. PK parameters of NR-V04 in WT mice. n = 3. B. NR-V04 induces long-lasting degradation of NR4A1 in MC38 tumors. Mice bearing MC38 tumors were treated with two-dose administration of NR-V04 via i.p. injection at 1.8 mg/kg, and tumors were collected at indicated timepoints. Tumor lysates were analyzed by immunoblotting with each lane representing an individual tumor lysate. n=2 C. Immunoblotting showing NR4A1 degradation in MC38 tumors upon termination. MC38 tumor-bearing mice were treated with vehicle, 1.8 mg/kg NR-V04 or 0.75 mg/kg celastrol treatment (equivalent to 1.67 μmol/kg), every 4 days until experimental end points. Tumor tissues were collected for lysate collection and immunoblotting (n = 4).

Figure 7.. NR-V04 exhibits outstanding anti-tumor effects in several tumor models.

A-F. Tumor-bearing mice were treated with 1.8 mg/kg NR-V04 or vehicle via i.p. injection on day 7 when tumors were palpable. The treatment was repeated every four days until the tumors reached the endpoint size of 2 cm in diameter. (A) NR-V04 inhibited MC38 colon adenocarcinoma growth, n=4 per group; (B) NR-V04 inhibited Yummer 1.7 melanoma growth, n=4 per group. (C) NR-V04 inhibited B16F10 melanoma growth, n=5 per group. (D) NR-V04 failed to inhibit B16F10 melanoma growth in NR4A1^−/− mice, n=5 per group. (E) NR-V04 failed to inhibit MC38 colon adenocarcinoma growth in NSG mice, n=4. (F) NR-V04 failed to inhibit B16F10 melanoma in NSG mice, n=5. Two-way ANOVA was performed for all tumor growth curves with P values indicated.

Figure 8.. Effect of NR-V04 on immune cells in the TME. A-B and E-K.

Tumor-bearing mice were treated with 1.8 mg/kg NR-V04 or vehicle via i.p. injection when tumor size reached 1 cm in diameter, with two treatments started as day 1 and repeated on day 4. Tumors were collected and single cells were isolated from tissues for flow cytometry analysis. A-B. NR-V04 treatment increases B cell percentage in the TME, but not in spleen and blood in mice inoculated with B16F10 melanoma, n=7. C-D. NR4A1 depletion induces B cell proliferation. B cells isolated from spleen were labeled with cell trace violet, untreated or treated with B16F10 lysis, following with the cotreatment of DMSO or 500 nM NR-V04 for 24 hours (n=3). B cell proliferation was determined by flow cytometry. E. NR-V04 fails to inhibit B16F10 melanoma growth in mice deficient of mature B cells (n=5). F-G. NR-V04 treatment increased effector memory CD8 T cell percentage in spleen, but not in tumors and blood in B16F10 melanoma (n=7). H-I. NR-V04 treatment decreased mMDSC percentage in tumor and blood, but not in spleen in B16F10 melanoma, n=7. J-K. NR-V04 treatment increased B cell percentage in the TME, but not in spleen and blood in Yumm 1.7 melanoma (n=7). E. Two-way ANOVA was performed for the tumor growth curve with P values indicated. Others are shown as the mean ± SD. A two-sided unpaired t test was performed, with P values indicated.

Figure 9.. NR-V04 has minimal toxicity.

A. Schematic of the toxicity testing. Male and female mice were treated with two doses of 2 mg/kg NR-V04 and two doses of 5 mg/kg NR-V04 over two weeks. Blood samples were collected on day 0, 7, 14, and 42 for hematology analysis, and body weight was measured twice per week. After day 42, all mice were euthanized, and tissues (kidney, liver, and small intestine) were harvested for H&E staining (n=3). B. Mice did not experience significant weight loss with NR-V04 treatment during the 42-day period. C-G. Hematology analysis of different blood cell components after NR-V04 or vehicle treatment, including (C) whole blood cells, (D) lymphocytes, (E) neutrophils, (F) red blood cells, and (G) platelets. H. NR-V04 impacts on tissue histology, including representative kidney, liver, and small intestine.