A Positive Feedback Loop between YTHDC1 and EP300 Drives Tumorigenesis in Multiple Myeloma

Multiple Myeloma (MM) is an incurable malignancy of plasma cells characterized by high degree of transcriptional heterogeneity. Epigenetic reprogramming drives the dynamic transcriptional heterogeneity in MM. N⁶-methyladenosine (m⁶A) is a dynamic and reversible epigenetic process that is the most prevalent internal post-transcriptional modification in eukaryotic RNAs, mediating various biological processes. Dysregulated m⁶A modifiers, including writers, erasers, and readers, have been reported to function as oncoproteins or tumor suppressors in a cancer type-dependent manner. However, the specific roles and underlying mechanisms of m⁶A modification in MM remain elusive.

In this study, we found that YTH N⁶-Methyladenosine RNA Binding Protein C1 (YTHDC1), an m⁶A reader protein, plays a critical role in MM tumorigenesis. Analysis of public databases revealed that YTHDC1 is highly expressed among various m⁶A regulators in MM patients. Experimental results showed that YTHDC1 is significantly overexpressed in CD138⁺ cells isolated from MM patients compared to healthy donors and is associated with poor prognosis. In vitro phenotypic assays demonstrated that YTHDC1 depletion significantly inhibited MM cell proliferation, induced apoptosis, caused cell cycle arrest, and led to DNA damage. We generated xenograft models using MM cell lines with stable YTHDC1 knockdown (KD) in immune-deficient NOD CRISPR Prkdc Il2r Gamma (NCG) mice. Results demonstrated that tumor burden was significantly lower and survival time was longer in the YTHDC1 KD group compared to the control group. Furthermore, we restored YTHDC1 expression by forced expression of wild-type (WT) YTHDC1 or the enzymatically inactive YTHDC1-W377A mutant in YTHDC1 KD MM cells. Restoration of WT-YTHDC1, but not W377A YTHDC1, substantially rescued the proliferation and colony formation of YTHDC1-KD MM cells, indicating that the oncogenic function of YTHDC1 in MM is m⁶A-dependent.

Previous studies have shown that YTHDC1, as a nuclear m⁶A reader, plays distinct roles in regulating RNA metabolism. Through multiple high-throughput sequencing analyses (RNA-seq, RIP-seq, MeRIP-seq), we found that differentially expressed genes in MM were significantly enriched in pathways related to apoptosis, cell cycle, and DNA damage response, aligning with our phenotypic observations. Further analysis of MM patient gene expression profiles revealed a strong correlation between YTHDC1 and EP300. Our RNA decay assay demonstrated that YTHDC1 maintains EP300 mRNA stability by binding to its m⁶A methylation sites. Gene dependency score assessment in human cancer cell lines revealed that MM cells are the most dependent on EP300 across all cancer types. Our data indicated that the depletion of EP300 significantly inhibited MM cell survival, while the ectopic expression of EP300 substantially rescued the clonogenic and proliferative defects resulting from YTHDC1 deficiency.

EP300 encodes the adenovirus E1A-associated cellular p300 transcriptional co-activator protein, which functions as histone acetyltransferase and regulates transcription via chromatin remodeling. P300 typically mediates acetylation of histone H3 at lysine-27 and gives an epigenetic tag for transcriptional activation. Our ChIP-seq and ChIP-qPCR analyses revealed that the promoter region of YTHDC1 gene was highly enriched with histone H3 lysine 27 acetylation (H3K27ac) and p300. This suggests that YTHDC1 expression in MM cells may be transcriptionally regulated by p300-mediated H3K27ac.

In summary, this study explored the crosstalk between m⁶A epitranscriptomic and epigenetic modifications in MM, proposing a novel positive feedback loop between YTHDC1 and EP300. This loop plays a critical role in MM tumorigenesis and progression. Unlike irreversible genetic alterations, the reversibility of epigenetic modifications makes them attractive targets for drug development. Therefore, this feedback loop could serve as a novel biomarker for MM diagnosis and a promising therapeutic target.