Fig 1: 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.
Fig 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.
Fig 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.
Fig 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.
Fig 5: 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.
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