Urolithin A Hijacks ERK1/2‐ULK1 Cascade to Improve CD8+ T Cell Fitness for Antitumor Immunity

Abstract According to the latest evidence, the microbial metabolite Urolithin A (UA), known for its role in promoting cellular health, modulates CD8+ T cell‐mediated antitumor activity. However, the direct target protein of UA and its underlying mechanism remains unclear. Here, this research identifies ERK1/2 as the specific target crucial for UA‐mediated CD8+ T cell activation. Even at low doses, UA markedly enhances the persistence and effector functions of primary CD8+ cytotoxic T lymphocytes (CTLs) and human chimeric antigen receptor (CAR) T cells both in vitro and in vivo. Mechanistically, UA interacts directly with ERK1/2 kinases, enhancing their activation and subsequently facilitating T cell activation by engaging ULK1. The UA‐ERK1/2‐ULK1 axis promotes autophagic flux in CD8+ CTLs, enhancing cellular metabolism and maintaining reactive oxygen species (ROS) levels, as evidenced by increased oxygen consumption and extracellular acidification rates. UA‐treated CD8+ CTLs also display elevated ATP levels and enhanced spare respiratory capacity. Overall, UA activates ERK1/2, inducing autophagy and metabolic adaptation, showcasing its potential in tumor immunotherapy and interventions for diseases involving ERKs.

stained with surface antibodies, fixed with 1% formaldehyde, and followed by washing with PBS three times.Cells were then detected by flow cytometry.

The Assessment of Systemic Toxicity by Body Weight and Hematoxylin and Eosin (H&E) Staining
The systemic toxicity of the UA was also assessed.Briefly, the mice were subcutaneously inoculated with B16F10.Then, the mice were randomly divided into groups.On days 10-13 post-injection, mice were administrated with sunflower oil (Vehicle) or UA (50, 100 and 200 mg/kg) orally every day for 7 days.The body weight of treated mice was monitored.Then, the mice's major organs (heart, liver, spleen, lung, and kidney) were collected, fixed in 4% paraformaldehyde for 24 h, and embedded in paraffin.Paraffin sections (4 μm) were deparaffinized, rehydrated, stained with hematoxylin and eosin (H&E), and imaged using a panoramic scanning and Image Analysis system for High-definition pathological Section (VS120).(A) Schematic diagram of NFAT luciferase reporter assay system (left).Jurkat-NFAT-Luc cells were stimulated with 1 μg/ml anti-CD3 and 3 μg/ml anti-CD28 antibodies in the presence of various microbial metabolites at 10 μM for 6 hours, followed by luciferase detection for NFAT activity (right).Data are presented as means ± SEM (n = 3) and were analyzed by two-tailed unpaired Student's t-test.
Cytokine productions (IFN-γ, TNF-α, and IL-2) in the supernatants were detected using ELISA (D).Cytotoxicity of CAR T cells against Raji cells in vitro was assessed using flow cytometry (E).Data are presented as means ± SEM (n = 3) and were analyzed by two-tailed unpaired Student's t-test.
(F and G) 19BBz CAR-T cells were treated with DMSO or UA (10 μM) in the presence or absence of ERK inhibitor SCH772984 (SCH, 10μM) for 48 hours, followed by stimulated with Namalwa cells at E:T ratios of 1:1, 2:1 and 4:  (B) The Flag-tagged ERK1 was transfected into HEK293T cells, and the whole cell lysate was collected for a pull-down assay using control beads or UA-beads in the presence of different doses (10, 20, and 50 μM) of UA, followed by immunoblot analysis.Quantification data from three independent experiments are presented as means ± SEM (n = 3) and were analyzed by two-tailed unpaired Student's t-test.(F) OT-Ⅰ CD8 + CTLs were treated with 10 μM UA or DMSO for 48 hours in the presence or absence of chloroquine (CQ, 10 μM) for the last 4 hours and then stimulated with anti-CD3/28 for 6 hours.Production of IFN-γ, TNF-α, IL-2, and granzyme B (Gzm B) in CD8 + CTLs was assessed using flow cytometric analysis.Data are presented as means ± SEM (n = 3) and were analyzed by two-tailed unpaired Student's t-test.This experiment was repeated three times independently.
(G) OT-Ⅰ CD8 + CTLs were treated with 10 μM UA or DMSO for 48 hours in the presence or absence of 3-MA (0.5 mM) for the last 4 hours and then stimulated with anti-CD3/28 for 6 hours.Production of IFN-γ, TNF-α, IL-2, and granzyme B (Gzm B) in CD8 + CTLs was assessed using flow cytometric analysis.Data are presented as means ± SEM (n = 3) and were analyzed by two-tailed unpaired Student's t-test.This experiment was repeated three times independently.
(I) Co-immunoprecipitation analysis of Flag-tagged ULK1 (Flag-ULK1) and endogenous ERK1/2 in HEK293T cells transduced with Flag-ULK1 using anti-Flag antibodies.All immunoblots were performed three times, independently, with similar results.This experiment was repeated three times independently.

Figure S1 .
Figure S1.Urolithin A promotes activation of the TCR signaling, related to Figure 1

Figure S2 .
Figure S2.Urolithin A treatment intrinsically promotes the survival and effector function of CD8 + CTLs in vivo, related to Figure 1

Figure S3 .
Figure S3.Urolithin A treatment regulates CD8 + T cell function through the ERK pathway, related to Figure 2

Figure
Figure S4.Urolithin A treatment enhances CAR T cell function through ERK, related to Figure 3 1 for 18 hours.Cytokine production (IFN-γ, TNF-α, and IL-2) of 19BBz CAR T cells in the supernatant was detected using ELISA (F).Cytotoxicity of 19BBz CAR T cells against Namalwa cells was determined in vitro using flow cytometry (G).Data are presented as means ± SEM (n = 3) and were analyzed by two-tailed unpaired Student's t-test (F) and two-way ANOVA (G).All results are representative of at least three independent experiments.* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; ns, no statistically significant.

( C )
Interactions of UA and ERK1 by docking analysis.Schematic representation of UA binding to ERK1 was shown.(D to F) The molecular dynamics (MD) simulations of UA-ERK1 interaction.Representative conformations of the apo ERK (D) and ERK bound with UA (E).The protein structure is shown on the surface, with red representing O atoms, blue representing N atoms, white representing H atoms, and C atoms shown in green in D and cyan in E. The side chains of residue Thr 202 and PTR 204 are demonstrated in sticks.Comparing D and E, the side chain of Thr 202 in C is buried deeper by the surrounding residues, while its counterpart in E is more exposed to the solvent.Solvent accessible surface area (SASA) plot of ERK1 and complex with UA (F).(G) Viability of OT-Ⅰ CTLs treated with different doses of Tat-ERK (1, 5, 10, 20, and 50 μM) peptide for 48 hours.Data are presented as means ± SEM (n = 3) and were analyzed by two-tailed unpaired Student's t-test.(H) OT-Ⅰ CTLs were treated with UA (10 μM) in the presence of different doses (5, 10, and 20 μM) of Tat-ERK peptide in vitro for 48 hours and then stimulated with anti-CD3/CD28 antibodies for 6 hours.Flow cytometric analysis assessed the production of IFN-γ, TNF-α, IL-2, and granzyme B in CD8+ CTLs.Data are presented as means ± SEM (n = 3) and were analyzed by two-tailed unpaired Student's t-test.(I) OT-Ⅰ CTLs were treated with UA (10 μM) in the presence of different doses (5, 10, and 20 μM) of Tat-ERK peptide in vitro for 48 hours.Cytotoxicity of the treated CTLs against 10 nM OVA257-264 peptide-pulsed EL4 targets was determined in vitro.Data are presented as means ± SEM (n = 3) and were analyzed by two-tailed unpaired Student's t-test.were performed three times, independently, with similar results.(D) OT-Ⅰ CD8 + CTLs were treated with 10 μM UA or DMSO for 48 hours, followed by immunoblot analysis.Quantification data of indicated protein (normalized to GAPDH) from three independent experiments are presented as means ± SEM (n = 3) and were analyzed by two-tailed unpaired Student's t-test.(E) OT-Ⅰ CD8 + CTLs were treated with 10 μM UA or DMSO in the presence or absence of PD0325901 (ERKi, 10 μM) or Tat-ERK (10 μM) for 48 hours and transferred to B16-MO5-Fluc lung metastases-bearing C57BL/6 mice.Autophagosomes in transferred CD8 + CTLs were assessed using flow cytometry.Data are presented as means ± SEM (n = 4) and were analyzed by two-tailed unpaired Student's t-test.

Figure S7 .
Figure S7.UA-ERK1/2-ULK1 cascade-mediated autophagy regulates cellular metabolism, related to Figure6(A to C) OT-Ⅰ CD8 + CTLs were treated with DMSO and UA (10μM) for 48 hours, followed by RNA-seq analysis.The data are from one experiment with three technical replicates per sample.GSVA analysis of RNA-seq data of DMSO-and UA-treated OT-Ⅰ CD8 + CTLs (A).Gene Ontology (GO) Biological Process enrichment results in the differentially expressed genes (DEGs) with RNA-seq data of DMSO-and UA-treated OT-Ⅰ CD8 + CTLs.Heat map illustrating the average transcript expression of the indicated genes in different metabolic pathways(C).Rows represent averaged z-scores.

Figure S8 .
Figure S8.UA-ERK1/2-ULK1 cascade-mediated autophagy regulates cellular metabolism and ROS levels, related to Figure 6 (A) OT-Ⅰ CD8 + CTLs cells were treated with 10 μM UA or DMSO for 48 hours in the presence or absence of ULK1 inhibitor MRT68921 (ULK1i, 5 μM) for the last 4 hours.Oxygen consumption rate (OCR) was measured in treated CD8+ CTLs under basal and stimulated conditions with oligomycin, FCCP, and rotenone.Basal respiration, maximal respiration, spare respiratory capacity statistics, and ATP production were shown.Data are presented as means ± SEM (n = 3) and were analyzed by two-tailed unpaired Student's t-test.(B) OT-Ⅰ CD8 + CTLs cells were treated with 10 μM UA or DMSO for 48 hours in the presence or absence of ULK1 inhibitor MRT68921 (ULK1i, 5 μM) for the last 4 hours.Extracellular acidification rate (ECAR) of the treated OT-Ⅰ CTLs was measured under basal and stimulated conditions with glucose, rotenone, and 2-deoxy-glucose (2-DG).Basal and maximal ECAR were shown.Data are presented as means ± SEM (n = 3) and were analyzed by two-tailed unpaired Student's t-test.

Figure S9 .
Figure S9.No significant tissue damage was observed in mice after the oral administration of Urolithin A, related to Figure 7A C57BL/6 mice were inoculated subcutaneously with 2 × 10 5 B16F10 melanoma cells on day 0 and treated with daily intragastric administration of UA at different doses (50, 100, and 200 mg/kg, sunflower oil as vehicle control) from day 12 to 19 (n = 4 mice per group).(A) The body weight of the indicated groups was monitored.Data are presented as means ± SEM (n = 4 mice per group) and analyzed by the Two-way ANOVA.(B) The heart, liver, spleen, lung, and kidney weights of the indicated groups were measured after mice sacrifice.Data are presented as means ± SEM (n = 4 mice per group) were analyzed by two-tailed unpaired Student's t-test (C) Representative H&E staining images of indicated organs were shown (Black bar = 50 μm).

Figure
Figure S12.Urolithin A exhibits antitumor activity in MC38 tumor mice, related to Figure7(A to E) C57BL/6 mice were subcutaneously inoculated with 2 × 10 5 MC38 cells on day 0 and treated with daily intragastric administration of UA at 50 mg/kg from day 13 to 19 (A).Tumor volumes were monitored (n = 5 mice per group) (B).Tumor images and tumor-to-body weight ratios were shown (n = 5 mice per group)(C).Numbers of CD8 + T cells in the tumor and spleen were analyzed using flow cytometry (n = 5 mice per group)(D).Expression of IFN-γ, TNF-α, IL-2, and Granzyme B in tumorinfiltrating CD8 + T cells was assessed using flow cytometry (n = 4 mice per group)(E).Data are presented as means ± SEM and were analyzed by two-way ANOVA (B) and two-tailed unpaired Student's t-test(C to E).(F to H) C57BL/6 mice bearing B16-MO5-Fluc lung metastases were treated with sunflower oil or UA (F).Tumor growth was assessed by detecting luciferase activity in the lung (G).and survival curve (H) was monitored (n = 5 mice per group).Data are presented as means ± SEM and were analyzed by two-tailed unpaired Student's t-test (G) and Log-rank test (H).