JS03.6.A TUMOR ASSOCIATED MACROPHAGE DERIVED EXOSOMES MODULATE IMMUNOTHERAPEUTIC SENSITIVITY OF SHH MEDULLOBLASTOMA BY TARGETING M6A MODIFIED FOXD1

BACKGROUND Medulloblastoma (MB) is the most common malignant brain tumor in children. The infiltration of tumor-associated macrophages (TAMs) in tumor microenvironment (TME) is correlated with the poor prognosis in SHH subtype of MB patients. Epigenetic alterations on RNA m6A modification were identified in this tumor. However, it remains unclear whether the TAMs infiltration in TME was correlated with the m6A modification status during tumor progression. METHODS The expression of m6A modification-related proteins and TAMs specific marker were assessed by immunohistochemistry in 40 SHH MB primary tumors. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen target genes affected by TAM-derived exosomes in SHH MB cells. Mechanisms of m6A-modification on target gene (FOXD1) were demonstrated by WB, qPCR, RNA half-life time and RNA immunoprecipitation assays. Combination treatment with AAV2/9-shFOXD1 and PD-1 inhibitors were performed in the NeuroD2: SmoA1 mouse model. RESULTS We reported for the first time that significant infiltration of TAMs was associated with decreased expression of METTL14 in primary SHH MBs. Exosomal microRNAs (miR-320e, miR-196b-5p, and miR-628-5p) released by TAMs targeted to the 3’-UTR (3’ Untranslated Region) of METTL14 in MB cells, promoting the gene’s degradation and consequently downregulating the overall m6A level in SHH MB. MeRIP-seq and RNA-seq analyses screened out Forkhead box D1 (FOXD1) as the target gene upregulated by TAMs-derived exosomes through m6A modification in MB cells, which was correlated with poor prognosis in SHH MB patients. Further experiments in vitro verified YTHDF2 can recognize m6A-modified FOXD1 and promote its mRNA degradation. Knockdown of FOXD1 in SHH MB cells significantly promoted the release of the CD8+T cell recruiting chemokines CXCL10/11. Treatment with AAV2/9-shFOXD1 significantly enhanced the anti-tumor effect of PD-1 inhibitor in NeuroD2: SmoA1 SHH MB mice. CONCLUSION Our study revealed the m6A modification status of SHH MBs influenced by TAMs derived exosomal miRNAs. FOXD1 promoted the malignant progression of SHH MB through m6A modification. Increased immunotherapeutic sensitivity of the combination treatment with FOXD1 knockdown and anti-PD-1 blockade in vivo shed lights on a novel therapeutic strategy of SHH MB.