YTHDF2 promotes ATP synthesis and immune evasion in B cell malignancies

RNA epigenetics plays crucial roles in physiological and pathological processes. As the most abundant methylation of mRNA, 0.2-0.7% of the total adenosine, m6A is dynamically deposited co-transcriptionally by the methyltransferase complex, reversely removed by demethylases recognized and directly regulated by “reader” proteins. As the first identified m6A 'reader' protein, YTHDF2 recognizes and destabilizes m6A modified mRNAs by inducing mRNA decay or degradation in P-body and plays indispensable oncogenic or tumor suppressive roles in multiple malignant contexts by mediating distinct m6A modified mRNAs. Here, we report that YTHDF2 promotes the development of B cell malignancies by facilitating energy supplements and immune evasion. RNA-sequencing (RNA-seq) data reveal that a set of ATP production- and immune response-related genes are consistently and significantly dysregulated upon YTHDF2 knockdown (KD) in both B cell acute lymphoblastic leukemia (B-ALL) patient-derived xenotransplant (PDX) cells (IAH8R) and diffuse large B cell lymphoma (DLBCL) cells (SU-DHL-4). RNA immunoprecipitation sequencing (RIP-seq) of either wildtype or mutant YTHDF2 proteins identify target genes directly bind to YTHDF2 dependent on the RNA binding pockets. Methylation RIP-seq (MeRIP-seq) assays identify either m6A-moidfied genes or m5C-modified mRNAs, which is further confirmed by bisulfite-converted mRNA sequencing (BS-RNA-seq) in malignant B cells. Mechanically, we find that YTHDF2 enhances ATP synthesis by sustaining mRNA stabilities of target genes as an m5C reader protein by interacting with PABPC1, a well-know m5C-modified mRNA stabilizer. Further RNA-seq upon PABPC1 KD and RIP-seq assays for PABPC1 reveal the overlapped genes which are also regulated and bound with YTHDF2 via m5C modification. Strikingly, RNA-seq and m6A-MeRIP-seq data show that YTHDF2 promotes immune evasion by destabilizing CD19 and MHC-II molecules (e.g., HLA-DMA and HLA-DMB) in an m6A-dependent manner. Single cell RNA-seq (scRNA-seq) data further reveal the immune microenvironment and cell proportions in bone marrow of Ythdf2 conditional KO mouse models (Ythdf2fl/fl). Our proof-of-concept study discovers that a YTHDF2 selective inhibitor (e.g. CCI-38) significantly inhibits cell metabolism and immune evasion in malignant B cells (KOPN-8) by RNA-seq. Collectively, our data show that YTHDF2 promotes ATP synthesis and immune evasion and serves as a promising therapeutic target in B cell malignancies. Overall design: [Dataset 1] 22 samples. Duplicates for RNA-seq samples in B-ALL PDX cells (IAH8R) and DLBCL cells (SU-DHL-4) upon YTHDF2 KD or PABPC1 KD by two different shRNAs compared to controls (shNS) respectively. Duplicates for RNA-seq samples when treated with CCI-38 or DMSO in malignant B cells (KOPN-8). [Dataset 2] 10 samples. Duplicates for BS-seq or MeRIP-seq against m6A or m5C. [Dataset 3] 13 samples. Duplicates for RIP-seq samples in malignant B cells carrying CRISPR-Cas9 and sgRNA target endogenous YTHDF2, which were further overexpressed with flag tagged wildtype or mutant YTHDF2 proteins (synonymous substitution for sgRNA sequence). 2-3 replicates for RIP-seq samples in IAH8R cells with forced expression of flag tagged PABPC1. [Dataset 4] 1 single cell RNA-seq sample. We employ a conditional Ythdf2 knockout mouse (Ythdf2fl/fl) carrying Mx1-Cre or not, and intravenously treat the mice with poly I:C each other day for a total of 5 times. Freshly collected bone marrow cells are stained with a specific Total seq type C harshtag for each mouse (2 wildtype bone marrow, 2 Ythdf2 knockout bone marrow). After staining with featured barcodes, equal numbers of cells are mixed together and proceed to 10X Genomics single cell platform and collect 10000 cells for the next library preparation and sequencing.