Targeting METTL3 reprograms the tumor microenvironment to improve cancer immunotherapy

Abstract

The tumor microenvironment (TME) is a heterogeneous ecosystem containing cancer cells, immune cells, stromal cells, cytokines, and chemokines which together govern tumor progression and response to immunotherapies. Methyltransferase-like 3 (METTL3), a core catalytic subunit for RNA N⁶-methyladenosine (m⁶A) modification, plays a crucial role in regulating various physiological and pathological processes. Whether and how METTL3 regulates the TME and anti-tumor immunity in non-small-cell lung cancer (NSCLC) remain poorly understood. Here, we report that METTL3 elevates expression of pro-tumorigenic chemokines including CXCL1, CXCL5, and CCL20, and destabilizes PD-L1 mRNA in an m⁶A-dependent manner, thereby shaping a non-inflamed TME. Thus, inhibiting METTL3 reprograms a more inflamed TME that renders anti-PD-1 therapy more effective in several murine lung tumor models. Clinically, NSCLC patients who exhibit low-METTL3 expression have a better prognosis when receiving anti-PD-1 therapy. Collectively, our study highlights targeting METTL3 as a promising strategy to improve immunotherapy in NSCLC patients.

Keywords: METTL3; PD-L1; cancer immunotherapy; chemokines; tumor microenvironment.

Figure 1.. METTL3 inhibition downregulates pro-tumorigenic chemokines in NSCLC cells

(A) Diagram showing the process of RNA sequencing (RNA-seq) and analysis of the differentially expressed genes (DEGs) in LLC cells treated with STM2457 (5 μM, 46 h) versus DMSO. (B) m⁶A dot blot showing decreased m⁶A levels in LLC cells treated with STM2457 (5 μM, 46 h, top panel). 200 ng of each RNA was loaded as a loading control and stained with methylene blue. The relative gray level of each dot was measured by Image J (bottom panel). (C) The m⁶A content was determined using an RNA Methylation Quantification Kit. (D) Volcano plot showing DEGs from RNA-seq results of LLC cells treated as in (A). Red dots represent upregulated genes and blue dots represent downregulated genes (∣log₂(FC)∣ > 1, and p value < 0.05). FC: fold change. Statistical analysis was performed using the Wald-test with Benjamini-Hochberg correction. (E) Heatmap showing DEGs in (A), among which 275 genes were upregulated and 77 genes were downregulated. (F) Biological process analysis showing the top 10 enriched pathways of DEGs. Modified Fisher’s exact test was used to analyze the data. (G) Heatmap showing DEGs including Cxcl1, Cxcl5, and Ccl20 chemokines and genes in chemokine-related signaling pathways in LLC cells with STM2457 treatment. (H and K) Relative mRNA level of Cxcl1, Cxcl5, and Ccl20 were analyzed using RT-qPCR for LLC (H) and PC9 cells treated with STM2457 (5 μM, 46 h) (K) versus DMSO. (I and L) Immunoblotting (IB) analysis of whole cell lysates (WCL) derived from LLC (I) or H460 (L) cells treated with shMETTL3 versus shGFP. (J and M) Relative mRNA levels of CXCL1, CXCL5, and CCL20 in LLC (J) and H460 (M) cells treated with shMETTL3 vs. shGFP. (N and O) Concentrations of Cxcl1, Cxcl5, and Ccl20 (pg/ml) in the supernatants of LLC cells treated with STM2457 (5 μM, 46 h) vs. DMSO (N) or shMettl3 vs. shGFP (O) was measured by ELISA kit. (P-S) Correlation of METTL3 expression with infiltration of MDSCs (P), T_reg cells (Q), and CD8⁺ T cells in lung tumors (R) based on the immune association analysis by TIMER (http://timer.compgenomics.org/). Correlation of METTL3 expression with infiltration of NK cells in lung tumors (S) based on the immune association analysis by TISIDB (http://cis.hku.hk/TISIDB/index.php). (T and U) Quantification of METTL3 and CXCL1 staining was performed by semiquantitative scoring. n = 75, r = 0.4449, p < 0.0001; correlation coefficients were calculated by Pearson test; two-tailed p-value is shown (T). Representative image of IHC staining for METTL3 and CXCL1 expression in human NSCLC patient (U). For Figure 1. B, C, H, J, K, and M-O, data are presented as mean ± S.D. n = 3. ****p < 0.0001, ***0.0001 < p < 0.001, **0.001 < p < 0.01, *0.01 < p < 0.05. ns, not significant. (See also Figure S1)

Figure 2.. The METTL3-m⁶A-c-Myc axis increases the transcription of pro-tumorigenic chemokines

(A) Schematic workflow for analysis of potential transcription factors regulating DEGs of chemokines and their-related pathways. (B) RT-qPCR validation for the relative mRNA level of 3 transcription factors predicted in (A) in LLC cells treated with STM2457 (5 μM, 46 h) vs. DMSO. (C) IB analysis of WCL derived from LLC cells treated with shMettl3 vs. shGFP. (D) Relative mRNA levels of c-Myc and Klf4 in LLC cells as in (C). (E) IB analysis of WCL derived from H460 cells treated with shc-Myc vs. shGFP. (F) Relative mRNA levels of CXCL1, CXCL5, and CCL20 in H460 cells as in (E). (G and H) MeRIP-qPCR analysis of the m⁶A level of and c-Myc in H460 cells treated with shMETTL3 vs. shGFP (G) or STM2457 (5 μM, 46 h) vs. DMSO (H). The house-keeping gene HPRT as a negative control. (I) Table showing the results of JASPAR analysis of potential c-Myc binding sites on the promoter regions of CXCL1, CXCL5, and CCL20. (J-L) ChIP-qPCR assays confirmed that c-Myc could bind to the promoter regions of CXCL1 (J), CXCL5 (K), and CCL20 (L) in H460 cells. (M-O) ChIP-qPCR assays showing less c-Myc enrichment on the promoter regions of CXCL1 (M), CXCL5 (N), and CCL20 (O) in H460 cells treated with shMETTL3 vs. shGFP. (P) IB analysis of WCL derived from H460 cells treated with shMETTL3, shc-Myc and shc-Myc & shMETTL3 vs. shGFP. (Q) Relative mRNA levels of CXCL1, CXCL5, and CCL20 were detected using RT-qPCR in H460 cells as in (P). For Figure 2. B, D, F, G, H, J-O, and Q, data are presented as mean ± S.D. n = 3. ****p < 0.0001, ***0.0001 < p < 0.001, **0.001 < p < 0.01, *0.01 < p < 0.05. ns, not significant. (See also Figure S2)

Figure 3.. METTL3 destabilizes PD-L1 in NSCLC cells

(A) Quantification of METTL3 and PD-L1 staining was performed by semiquantitative scoring. n = 142, Pearson r = −0.3884, two-tailed p < 0.0001. (B) Representative IHC staining images for high METTL3 expression and low PD-L1 expression in human NSCLC patient #1 and patient #2. (C and D) IB analysis (C) and relative Pd-l1 mRNA levels (D) in LLC cells treated with shMettl3 vs. shGFP. (E and F) IB analysis (E) and relative Pd-l1 mRNA levels (F) in LLC cells treated with STM2457 vs. DMSO. (G and H) IB analysis (G) and relative PD-L1 mRNA levels (H) in H460 cells treated with shMETTL3 vs. shGFP. (I and J) IB analysis (I) and relative PD-L1 mRNA levels (J) in H460 cells treated with STM2457 vs. DMSO. (K and L) IB analysis (K) and relative PD-L1 mRNA levels (L) in H460 cells infected with control, HA-METTL3-WT, or HA-METTL3-CD lentiviruses. For Figure 3. D, F, H, J, and L, data are presented as mean ± S.D. n = 3. ****p < 0.0001, ***0.0001 < p < 0.001, **0.001 < p < 0.01, *0.01 < p < 0.05. (See also Figure S3)

Figure 4.. METTL3 promotes m⁶A modification of PD-L1 mRNA

(A) m⁶A-RIP-qPCR analysis showed m⁶A enrichment was mainly located on CDS #2 and 3’UTR m⁶A motifs, but not on CDS #1 m⁶A motif on PD-L1 mRNA in H460 cells. (B and C) Inhibition of METTL3 by shRNAs (B) or STM2457 (C) significantly decreased the relative m⁶A enrichment on PD-L1 CDS #2 and 3’UTR m⁶A motifs. (D) The graph showing the constructs containing PD-L1 CDS #2 & 3’UTR wild-type, CDS #2 mutant, 3’UTR mutant, or CDS #2 & 3’UTR mutant (Mut). (E) Luciferase activity of PD-L1 CDS #2 & 3’UTR WT-fused dual-luciferase reporter in HEK293T transiently transfected with the indicated constructs. (F) The relative luciferase activity in HEK293T cells transiently transfected with Flag-METTL3 together with PD-L1 CDS #2 & 3’UTR WT, CDS #2 Mut, 3’UTR Mut, or CDS #2 & 3’UTR Mut fused dual-luciferase reporters. For Figure 4. A-C, E, and F, data are presented as mean ± S.D. n = 3. ****p < 0.0001, ***0.0001 < p < 0.001, **0.001 < p < 0.01, *0.01 < p < 0.05. ns, not significant. (See also Figure S4)

Figure 5.. METTL3 knockdown by shRNA reshapes the TME to potentiate PD-1 blockade in subcutaneous LLC lung tumor model

(A and B) Kaplan-Meier plots showing the survival of NSCLC patients with anti-PD-1 therapy from GSE190265, p = 0.029 (A) and GSE190266, p = 0.033 (B) datasets based on METTL3 expression levels. Two-sided log-rank test. (C) Treatment plan for C57BL/6J mice bearing subcutaneous shMettl3- or shGFP-treated LLC tumors with indicated treatments. (D and E) Tumor growth curves (D) and tumor weight (E) of shGFP- or shMettl3-treated tumors as in (C). n = 5 mice per group. Two-way ANOVA test for (D). (F) Mean fluorescence intensity (MFI) of PD-L1 in CD45⁻ cells as in (C). (G-K) Quantification of tumor-infiltrating CD8⁺CD3⁺ T cells (G), IFN-γ⁺CD8⁺ T cells (H), NK cells (I), MDSCs (J), and FoxP3⁺CD25⁺CD4⁺ T_reg cells (K) from tumors as in (C). For Figure 5. E-K, data are presented as mean ± S.D. n = 5. ****p < 0.0001, ***0.0001 < p < 0.001, **0.001 < p < 0.01, *0.01 < p < 0.05. ns, not significant. (See also Figure S5)

Figure 6.. Mettl3 inhibition reduces tumorigenesis and improves anti-PD-1 immunotherapy in Kras^LSL-G12D/+;Trp53^fl/fl (KP) mouse model

(A) Schematic treatments for Kras^LSL-G12D/+;Trp53^fl/fl (KP) lung tumor model. KP mice were intranasally injected with Ad-Cre (2 × 10⁶ pfu/mouse) for 5 weeks, followed by treatment with either control, PD-1 mAb, STM2457, or combination. (B) Representative H&E-stained images of lungs in KP mice as in (A). (C and D) Quantification of all tumor sizes in cross-sectional area of three nonconsecutive sections from each mouse (C) and percentage of total lung cross-sectional area occupied by tumor of three nonconsecutive sections from each mouse (D) of KP mice as in (A). (E-G) Relative mRNA levels of Cxcl1 (E), Cxcl5 (F), Ccl20 (G) in tumor-burdened lungs as in (A). (H) PD-L1 Mean Fluorescence Intensity (MFI) of CD45⁻ cells in tumor-burdened lungs as in (A). (I) Schematic treatments for lung tumors in KP and KPM mice. KP or KPM mice were intranasally injected with Ad-Cre (2 × 10⁶ pfu/mouse) for 5 weeks, followed by treatment with control or anti-PD-1 antibodies (8 treatments in 4 weeks). (J) Representative H&E-stained images of lungs in KP and KPM mice as in (I). (K and L) Quantification of all tumor sizes in cross-sectional area of three nonconsecutive sections from each mouse (K) and percentage of total lung cross-sectional area occupied by tumor of three nonconsecutive sections from each mouse (L) of KP and KPM mice as in (I). (M-P) Relative mRNA levels of Cxcl1 (M), Cxcl5 (N), Ccl20 (O), and Pd-ll (P) in tumor-burdened lungs as in (I). (Q-U) Quantification of tumor-infiltrating CD8⁺CD3⁺ T cells (Q), IFN-γ⁺CD8⁺ T cells (R), NK cells (S), MDSCs (T), and FoxP3⁺CD25⁺CD4⁺T_reg cells (U) from tumor-burdened lungs as in (I). For Figure 6. C-H and K-U data were presented as mean ± S.D. n = 5. ****p < 0.0001, ***0.0001 < p < 0.001, **0.001 < p < 0.01, *0.01 < p < 0.05. ns, not significant. (See also Figure S6)

Figure 7.. Inhibition of METTL3 with STM2457 sensitizes tumor to anti-PD-1 immunotherapy in subcutaneous and orthotopic lung cancer models

(A) Schematic treatments for C57BL/6J mice bearing subcutaneous LLC-Luc tumors. (B-D) Tumor volume (B), Kaplan-Meier survival curve (C), and final tumor weight (D) for mice as in (A). Tumor volumes were measured every two days and plotted individually. n = 8 per group for (B and C); n = 5 per group for (D). Two-sided log-rank test (C). (E-G) Relative mRNA levels of Cxcl1 (E), Cxcl5 (F), and Ccl20 (G) were analyzed using RT-qPCR in LLC-Luc tumors as in (A). (H-K) Quantification of tumor-infiltrating CD8⁺CD3⁺ T cells (H), IFN-γ⁺CD8⁺ T cells (I), NK cells (J), and FoxP3⁺CD25⁺ CD4⁺T_reg cells (K) from tumors as in (A). (L) Representative images of LLC-Luc tumor-burdened lungs treated with control, PD-1 mAb, STM2457 and combination. (M) The tumor volume was measured using digital caliper of mice with indicated treatment as in (L). (N-Q) Relative mRNA levels of Cxcl1 (N), Cxcl5 (O), and Ccl20 (P) in tumor-burdened lungs as in (L). (Q) PD-L1 Mean Fluorescence Intensity (MFI) of CD45⁻ cells in tumor-burdened lungs as in (L) (R-V) Quantification of tumor-infiltrating CD8⁺CD3⁺ T cells (R), IFN-γ⁺CD8⁺ T cells (S), NK cells (T), MDSCs (U) and FoxP3⁺CD25⁺ CD4⁺T_reg cells (V) from tumors as in (L). For Figure 7. D-K and M-V data are presented as mean ± S.D., n = 5. ****p < 0.0001, ***0.0001 < p < 0.001, **0.001 < p < 0.01, *0.01 < p < 0.05. ns, not significant. (See also Figure S7)