m⁶A-dependent upregulation of DDX21 by super-enhancer-driven IGF2BP2 and IGF2BP3 facilitates progression of acute myeloid leukaemia

Background: Acute myeloid leukaemia (AML) is a haematological malignancy with unfavourable prognosis. Despite the effectiveness of chemotherapy and targeted therapy, relapse or drug resistance remains a major threat to AML patients. N6-methyladenosine (m⁶A) RNA methylation and super-enhancers (SEs) are extensively involved in the leukaemogenesis of AML. However, the potential relationship between m⁶A and SEs in AML has not been elaborated.

Methods: Chromatin immunoprecipitation (ChIP) sequencing data from Gene Expression Omnibus (GEO) cohort were analysed to search SE-related genes. The mechanisms of m⁶ A-binding proteins IGF2BP2 and IGF2BP3 on DDX21 were explored via methylated RNA immunoprecipitation (MeRIP) assays, RNA immunoprecipitation (RIP) assays and luciferase reporter assays. Then we elucidated the roles of DDX21 in AML through functional assays in vitro and in vivo. Finally, co-immunoprecipitation (Co-IP) assays, RNA sequencing and ChIP assays were performed to investigate the downstream mechanisms of DDX21.

Results: We identified two SE-associated transcripts IGF2BP2 and IGF2BP3 in AML. High enrichment of H3K27ac, H3K4me1 and BRD4 was observed in IGF2BP2 and IGF2BP3, whose expression were driven by SE machinery. Then IGF2BP2 and IGF2BP3 enhanced the stability of DDX21 mRNA in an m⁶A-dependent manner. DDX21 was highly expressed in AML patients, which indicated a poor survival. Functionally, knockdown of DDX21 inhibited cell proliferation, promoted cell apoptosis and led to cell cycle arrest. Mechanistically, DDX21 recruited transcription factor YBX1 to cooperatively trigger ULK1 expression. Moreover, silencing of ULK1 could reverse the promoting effects of DDX21 overexpression in AML cells.

Conclusions: Dysregulation of SE-IGF2BP2/IGF2BP3-DDX21 axis facilitated the progression of AML. Our findings provide new insights into the link between SEs and m⁶A modification, elucidate the regulatory mechanisms of IGF2BP2 and IGF2BP3 on DDX21, and reveal the underlying roles of DDX21 in AML.