Targeting DTX2/UFD1-mediated FTO degradation to regulate antitumor immunity

Abstract

Here, we show that vitamin E succinate (VES) acts as a degrader for the m⁶A RNA demethylase fat mass and obesity-associated protein (FTO), thus suppressing tumor growth and resistance to immunotherapy. FTO is ubiquitinated by its E3 ligase DTX2, followed by UFD1 recruitment and subsequent degradation in the proteasome. VES binds to FTO and DTX2, leading to enhanced FTO-DTX2 interaction, FTO ubiquitination, and degradation in FTO-dependent tumor cells. VES suppressed tumor growth and enhanced antitumor immunity and response to immunotherapy in vivo in mouse models. Genetic FTO knockdown or VES treatment increased m⁶A methylation in the LIF (Leukemia Inhibitory Factor) gene and decreased LIF mRNA decay, and thus sensitized melanoma cells to T cell-mediated cytotoxicity. Taken together, our findings reveal the underlying molecular mechanism for FTO protein degradation and identify a dietary degrader for FTO that inhibits tumor growth and overcomes immunotherapy resistance.

Keywords: DTX2; FTO; UFD1; m6A RNA methylation; ubiquitin-mediated proteasomal degradation.

Fig. 1.

DTX2 is an E3 ubiquitin ligase for FTO that induces FTO ubiquitination and degradation. (A) Immunoblot analysis of FTO-GFP and DTX2-Flag in FTO-GFP-overexpressed HeLa cells with or without overexpression of DTX2-Flag treated with or without MG132 (10 μM) for 6 h. GAPDH was used as a loading control. (B) Immunoblot analysis of FTO in MEL624 cells transfected with empty vector (EV) or DTX2-HA treated with cycloheximide (CHX, 100 μg/ml) over a time course. (C) Quantification of B (n =3). (D) Domain architecture of human DTX2. (E) Coimmunoprecipitation (Co-IP) assay of FTO binding with HA-tagged wild-type (WT) DTX2 or DTX2 mutants with deletions of domains in 293T cells transfected with Empty Vector (EV), △RING-HA, △WWE1-HA, △WWE2-HA, in combination with FTO-Flag and treated with MG132 (10uM) for 6 h. (F) Ubiquitination assay of 293T cells transfected with constructs expressing FTO-Flag, and WT DTX2-HA or DTX2 mutants and treated with MG132 (10 μM) for 6 h. Protein lysates were immunoprecipitated with the Flag-beads and ubiquitination was detected with the anti-Ubiquitin antibody. (G) m⁶A dot blot assay and immunoblot analysis in WM35 cells transfected with or without DTX2-HA and FTO-Flag. (H) Immunoblot analysis of FTO-Flag and FTO-Flag mutants with indicated arginine mutation in Lysine (K121R, K160R, K162R, K194R, K211R, and K216R) in 293T cells transfected WT and FTO lysine mutants. (I) m⁶A dot blot assay in 293T cells transfected with WT and FTO lysine mutants. (J) Immunoblot analysis of WT and FTO K162R in 293T cells transfected with WT and FTO K162R and treated with cycloheximide (CHX, 100 μg/ml) over a time course. (K) Quantification of J (n = 3). (L) Immunoblot analysis of Flag for WT and K162R FTO, and HA (DTX2) in 293T cells transfected with or without DTX2 in combination with FTO WT or K162R. (M) Ubiquitination assay of 293T cells transfected with or without constructs expressing DTX2-HA, FTO WT-Flag, and K162R-Flag and treated with MG132 (10 μM) for 6 h. Protein lysates were immunoprecipitated with the Flag-beads and ubiquitination was detected with the anti-Ubiquitin antibody. (N) Ubiquitination assay of HeLa cells transfected with or without HA-Ub WT or HA-Ub K48R in combination with DTX2-Flag or FTO-GFP and then treated with MG132 (10 μM) for 6 h. Protein lysates were immunoprecipitated with the anti-GFP antibody and ubiquitination was detected with the anti-HA antibody. Error bars are shown as mean ± SD (C and K). P-values are from two-tailed unpaired t test (C and K).

Fig. 2.

VES induces FTO protein degradation. (A) Quantification of the m⁶A/A ratios in mRNA by LC-MS/MS in MEL624 cells with or without VES treatment (n = 3). (B) Thermal shift curves of FTO from CETSA in MEL624 cells treated with or without VES (10 μM) (n = 3). (C) Molecular docking models for the interaction between human FTO (PDB: 4IE6) and VES without the aliphatic chain using Schrodinger. (D) Model for FTO binding with VES without the aliphatic chain. (E) Immunoblot analysis of WT and FTO mutants (R316Q, S318A, and Y295F) from the DARTS assay in MEL624 cells treated with or without VES. (F) Inhibitory effects of VES on FTO demethylase activity using an in vitro (cell-free) m⁶A demethylation assay. (G) IC50 values of VES (72 h) on inhibiting cell viability in CHL1 cells with or without FTO knockout (KO) (n = 3). (H) IC50 values of VES (72 h) on inhibiting cell viability in FTO-high melanoma cell lines (MEL624, CHL1) and FTO-low melanoma cell lines (SK-MEL-30. WM35, WM115) (n = 3). (I) Immunoblot analysis of FTO in MEL624 cells treated with VES at indicated doses. β-actin was used as a loading control. (J) qPCR analysis of FTO mRNA levels in cells as in I (n = 3). (K) Immunoblot analysis of FTO in MEL624 cells with or without VES (10 μM, 72 h) and MG132 (10 μM) added for 6 h prior to the end of 72 h. (L) Immunoblot analysis of FTO in MEL624 cells treated with or without VES (10 μM, 72 h) and then cycloheximide (CHX, 100 μg/ml) over a time course. (M) Quantification of L (n = 3). (N) Ubiquitination assay of 293T cells transfected with HA-Ub and FTO-GFP and treated with or without VES (25 μM, 12 h) and/or MG132 (10 μM, 6 h). Protein lysates were immunoprecipitated with the anti-GFP antibody and ubiquitination was detected with the anti-HA antibody. (O) Immunoblot analysis of WT and K162R FTO in 293T cells transfected with Flag-tagged WT FTO and FTO K162R and treated with or without VES (25 μM, 72 h). Error bars are shown as mean ± SE (F) and ± SD (A, B, G, H, J, and M). P-values are from two-tailed unpaired t tests (A, B, J, and M).

Fig. 3.

VES induces FTO protein degradation via DTX2. (A) Schematic illustration of the in situ proximity ligation assay (PLA). (B) Immunofluorescence analysis of the PLA of the interaction between DTX2 and FTO in CHL1 cells. (Scale bar, 100 μm.) (C) Quantification of the number of PLA red dots per cell in B (n = 30 cells from three biologically independent replicates). (D) Co-IP analysis of FTO binding with DTX2 in 293T cells transfected with FTO-Flag and treated with or without VES (25 μM, 12 h) with MG132 (10 μM) for 6 h. (E) Immunoblot analysis of FTO in HeLa cells with or without DTX2 knockdown and treated with or without VES (25 μM, 72 h). (F) Molecular docking showing the interaction of DTX2 with vitamin E. (G) Diagram for vitamin E binding with DTX2. (H) Immunoblot analysis of DTX2 and GAPDH from DARTS assay in 293T cells treated with VES, VE, and DS. Error bars are shown as mean ± SD (C). P-values are from two-tailed unpaired t tests (C).

Fig. 4.

UFD1 is required for VES-induced FTO protein degradation. (A) Volcano plot of FTO-interacting proteins in MEL624 cells from Co-IP followed by mass spectrometry analysis. (B) Immunoblot analysis of WT FTO, K162R FTO, and UFD1 in 293T cells transfected with or without FTO WT-Flag, FTO K162R-Flag, and UFD1-HA. (C) Immunoblot analysis of FTO in 293T cells transfected with or without UFD1 and treated with cycloheximide (CHX, 100 μg/ml) over a time course. (D) Quantification of C (n =3). (E) Immunoblot analysis of FTO in 293T cells with or without UFD1 knockdown treated with cycloheximide (CHX, 100 μg/ml) over a time course. (F) Quantification of E (n =3). (G) Co-IP assay showing the binding of FTO with DTX2 and UFD1 in HeLa cells transfected with FTO-Flag treated with or without VES (25 μM, 12 h) with MG132 (10 μM) for 6 h. (H) Co-IP assay showing the binding of FTO or FTO K162 with DTX2 or UFD1 in HeLa cells transfected with FTO WT and FTO K162R and treated with VES (25 μM, 12 h) with MG132 (10 μM) for 6 h. (I) Immunoblot analysis of FTO and UFD1 in 293T cells with or without UFD1 knockdown and treated with or without VES (10 μM, 72 h). (J) Immunoblot analysis of FTO, UFD1, and DTX2 in 293T cells with FTO-Flag overexpression and with or without overexpression of DTX2 and UFD1. (K) Immunoblot analysis of UFD1 and GAPDH from DARTS assay in MEL624 cells treated with or without VES. (L) Thermal shift curves of UFD1 from CETSA in MEL624 cells treated with or without VES (10 μM) (n = 3). (M) Schematic summary of VES-induced FTO protein degradation. Error bars are shown as mean ± SD (D, F, and L). P-values are from two-tailed unpaired t tests (D, F, and L).

Fig. 5.

Effect of targeting FTO degradation in tumor growth and response to immunotherapy. (A) Cell proliferation assay in WM35 cells with or without overexpression of FTO in combination with overexpression of WT DTX2 and DTX2 mutants (n = 4). (B) Tumor volume of MEL624 cells with or without DTX2 overexpression in nude mice (n = 5). (C) Tumor volume of MEL624 cells with or without UFD1 overexpression in nude mice (n = 5). (D) Cell proliferation assay in CHL1 cells with or without FTO deletion transfected with or without DTX2 and UFD1 (n = 6). (E) Overall survival of Pan-cancer patients with high FTO protein level (n = 295) and low FTO protein level (n = 164) following anti-PD-L1 immunotherapy. (F) Overall survival of Pan-cancer patients with high DTX2 protein level (n = 107) and low DTX2 protein level (n = 317) following anti-PD-L1 immunotherapy. (G) Overall survival of Pan-cancer patients with high UFD1 protein level (n = 126) and low UFD1 protein level (n = 298) following anti-PD-L1 immunotherapy. (H) Tumor volume of B16F10 cells injected in C57BL/6 mice treated with or without VES in combination with the anti-PD-1 antibody or the isotype control antibody (n = 4). (I) Tumor weight of H. (J) Boxplot showing gene expression of DTX2 in normal and skin melanoma tissue. (K) Boxplot showing gene expression of FTO in normal and skin melanoma tissue. Error bars are shown as mean ± SD (A, D, and I), ±SE (B, C, and H). P-values are from two-tailed unpaired t tests (A, B, C, D, H, I, J, and K).

Fig. 6.

VES increased T cell–mediated cytotoxicity by targeting FTO. (A–E) Quantification of mouse B220⁺, CD3⁺, CD4⁺, CD8⁺, and CD206⁺ of CD45⁺ cells in C57BL/6 mice bearing MC38 tumors upon treatment with vehicle and VES (n = 5). (F–H) Quantification of mouse TNFα⁺, IFNγ⁺, and PD-1⁺ of CD8⁺ T cells in C57BL/6 mice bearing MC38 tumors upon treatment with vehicle and VES (n = 5). (I) Tumor volume of MC38 tumors in C57BL/6 mice treated with or without VES in combination with the anti-CD8, anti-CSF1R, or isotype control antibody. (J) Quantification of CD8⁺ of CD3⁺ T cells in activated human T cells treated with or without supernatant from MEL624 cells treated with or without VES (n = 3). (K) Quantification of CD8⁺ of CD3⁺ T cells in activated human T cells cocultured with MEL624 and pretreated with or without VES (n = 3). (L) Schematic summary of the coculture assays for T cells and GFP-labeled human cancer cells. (M) Effect of VES on the sensitivity of MEL624-GFP cells to the cytotoxicity of T cells in vitro. MEL624 cells were pretreated with or without VES for 72 h (n = 3). (N) Effect of VES pretreatment (72 h) on the sensitivity of WM35 cells with or without FTO-GFP overexpression to the cytotoxicity of T cells in vitro (n = 3). (O) Effect of DTX2 overexpression in MEL624-GFP cells on the cytotoxicity of T cells in vitro (n = 3). Error bars are shown as mean ± SD (A–H, J, K, M–O), or ±SE (I). P-values are from two-tailed unpaired t tests (A–K and M–O).

Fig. 7.

FTO inhibition enhances T cell–mediated cytotoxicity by targeting LIF. (A) Pathway analysis of differentially expressed genes from RNA seq analysis of MEL624 cells with or without FTO knockdown (GSE112902). (B) Heatmap showing shared differentially expressed genes in the JAK-STAT signaling pathway by VES or FTO knockdown in MEL624 cells. (C) Distribution of m⁶A peaks across the LIF transcript in MEL624 cells with or without FTO knockdown. (D) m⁶A IP qPCR analysis of m⁶A enrichment in the indicated gene transcripts in MEL624 cells treated with or without VES (n = 3). (E) qPCR analysis of LIF mRNA stability following treatment with actinomycin D (ActD, 2 μM) in MEL624 cells with or without VES treatment (n = 3). (F) qPCR analysis of LIF mRNA stability following treatment with actinomycin D (ActD, 2 μM) in WM35 cells with or without siRNA targeting IGF2BP1/2/3 without VES treatment (n = 3). (G) qPCR analysis of LIF mRNA level in WM35 cells with or without overexpression of FTO and DTX2 (n = 3). (H) Correlation plot of LIF mRNA level and FTO mRNA level in human melanoma tissue (n = 472). (I) qPCR analysis of LIF mRNA levels in MEL624 cells with or without LIF overexpression (n = 3). (J) Cell proliferation assay in MEL624 cells with or without LIF overexpression (n = 4). (K) Overall survival of melanoma patients with high LIF mRNA level (n = 233) and low LIF mRNA level (n = 92) following anti-PD-1 immunotherapy. (L) qPCR analysis of LIF and FTO mRNA level in MEL624 cells with or without FTO and LIF overexpression (n = 3). (M) Effect of LIF overexpression in MEL624 cells expressed with GFP or FTO-GFP on the cytotoxicity of T cells in vitro (n = 3). (N) Quantification of CD8⁺ of CD3⁺ T cells in activated human T cells cocultured with MEL624 with or without LIF overexpression (n = 3). Error bars are shown as mean ± SD (D–G, I, J, and L–N). Correlation coefficient r and P-value are from Pearson correlation analysis (H). P-values are from two-tailed unpaired t tests (D–G, I, J, and L–N).