METTL3 Inhibits Antitumor Immunity by Targeting m6A-BHLHE41-CXCL1/CXCR2 Axis to Promote Colorectal Cancer

Abstract

Background & aims: N⁶-Methyladenosine (m⁶A) is the most prevalent RNA modification and recognized as an important epitranscriptomic mechanism in colorectal cancer (CRC). We aimed to exploit whether and how tumor-intrinsic m⁶A modification driven by methyltransferase like 3 (METTL3) can dictate the immune landscape of CRC.

Methods: Mettl3 knockout mice, CD34⁺ humanized mice, and different syngeneic mice models were used. Immune cell composition and cytokine level were analyzed by flow cytometry and Cytokine 23-Plex immunoassay, respectively. M⁶A sequencing and RNA sequencing were performed to identify downstream targets and pathways of METTL3. Human CRC specimens (n = 176) were used to evaluate correlation between METTL3 expression and myeloid-derived suppressor cell (MDSC) infiltration.

Results: We demonstrated that silencing of METTL3 in CRC cells reduced MDSC accumulation to sustain activation and proliferation of CD4⁺ and CD8⁺ T cells, and eventually suppressed CRC in Apc^Min/+Mettl3^+/- mice, CD34⁺ humanized mice, and syngeneic mice models. Mechanistically, METTL3 activated the m⁶A-BHLHE41-CXCL1 axis by analysis of m⁶A sequencing, RNA sequencing, and cytokine arrays. METTL3 promoted BHLHE41 expression in an m⁶A-dependent manner, which subsequently induced CXCL1 transcription to enhance MDSC migration in vitro. However, the effect was negligible on BHLHE41 depletion, CXCL1 protein or CXCR2 inhibitor SB265610 administration, inferring that METTL3 promotes MDSC migration via BHLHE41-CXCL1/CXCR2. Consistently, depletion of MDSCs by anti-Gr1 antibody or SB265610 blocked the tumor-promoting effect of METTL3 in vivo. Importantly, targeting METTL3 by METTL3-single guide RNA or specific inhibitor potentiated the effect of anti-programmed cell death protein 1 (anti-PD1) treatment.

Conclusions: Our study identifies METTL3 as a potential therapeutic target for CRC immunotherapy whose inhibition reverses immune suppression through the m⁶A-BHLHE41-CXCL1 axis. METTL3 inhibition plus anti-PD1 treatment shows promising antitumor efficacy against CRC.

Keywords: Colorectal Cancer; Immunotherapy; MDSC; METTL3; N(6)-Methyladenosine.