ALKBH5 Functions As an Oncogene in Multiple Myeloma

Background: RNA N ⁶-methyladenosine (m ⁶A) plays a critical role in regulating gene expression and determining cell fate. The dysregulation of m ⁶A modulators, including α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5), has been reported to promote tumor development through their enzymatic function. However, the functions of mRNA m ⁶A and its modulators in multiple myeloma (MM) are largely unknown.

Methods: We queried publicly available MM datasets to study the expression profile of m ⁶A modulators (METTL3, METTL14, WTAP, FTO, and ALKBH5) in MM and their relationships with clinical outcomes in patients with MM. Both gain- and loss-of-function studies were performed to investigate the role of ALKBH5 in MM. The cell proliferation assay, colony formation assay, Annexin V apoptosis analysis, and 5-ethynyl-2′-deoxyuridine (EdU) assay were performed to evaluate the functions of ALKBH5 in MM in vitro. Human MM cell line xenograft models were constructed to examine the effects of ALKBH5 knockdown or overexpression on MM growth in vivo. The rescue assay using catalytically inactive mutant ALKBH5-H204A was conducted to determine whether demethylation activity was required for the function of ALKBH5 in MM. Then, we performed RNA sequencing and m ⁶A sequencing to explore the key targets that mediated ALKBH5 function in MM. We investigated the gene regulatory mechanism of ALKBH5 in MM by m ⁶A immunoprecipitation assay, RNA immunoprecipitation assay, RNA decay assay, dual-luciferase reporter assay, and so forth. Gene set enrichment analysis and Western blotting were employed to determine the downstream signaling pathways regulated by ALKBH5 and the recognized target.

Results: ALKBH5 was overexpressed in MM and associated with a poor prognosis in patients with MM. The increased ALKBH5 expression was required for the survival and growth of MM cells in vitro and in vivo. Mechanistically, m ⁶A demethylation activity was required for ALKBH5 to exert tumorigenic effects in MM. Tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1) was identified as a functionally important target of ALKBH5. ALKBH5 regulated TRAF1 expression via affecting mRNA stability of TRAF1 in an m ⁶A- and YTHDF2-dependent manner. ALKBH5 promoted MM cell growth and survival partly through TRAF1-mediated activation of NF-κB and MAPK signaling pathways.

Conclusion: Our findings showed that ALKBH5 played an oncogenic role in MM and highlighted that ALKBH5 could potentially be a novel therapeutic target in MM.