Background: RNA N⁶-methyladenoine (m⁶A) modification plays multifaceted roles in cancer, including tumor initiation, progression and therapy resistance. Nevertheless, the role of aberrant m⁶A in cancer stem cells (CSCs) remains unclear. Here we aim to unveil the function, mechanism and translational significance of m⁶A eraser AlkB homologue 5 (ALKBH5) in colorectal cancer (CRC) CSCs.

Methods: Expression of ALKBH5 and CSC markers in CRC patients was assessed by qPCR (n=160) and immunohistochemistry on tissue microarrays (n=156). Effect of ALKBH5 on CRC stemness and chemoresistance was studied in colon stem cell-specific ALKBH5 knockin (Alkbh5^KILgr5-Cre^ER) and knockout (Alkbh5^KOLgr5-Cre^ER) mice, colorectal CSCs and patient-derived organoids (PDOs). RNA-seq, m⁶A-seq and Ribo-seq were performed to identify ALKBH5 targets. Immunoprecipitation-Mass Spectrometry (IP-MS) was conducted to probe FAM84-binding proteins. Vesicle-like nanoparticles (VNPs)-encapsulated si-ALKBH5 were used to target ALKBH5 .

Results: ALKBH5 mRNA and protein expression positively correlated with CSC markers (LGR5, CD133) in CRC patients (all P<0.01). ALKBH5 overexpression in colorectal CSCs and PDOs enhanced self-renewal capacity and CSC marker expression, whereas ALKBH5 knockdown exerted opposite effects. In vivo limiting dilution assay validated ALKBH5 loss impaired tumorigenicity of CSCs. Importantly, colon stem cell-specific ALKBH5 knockin mice showed exacerbated azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC with higher tumor multiplicity and load (P<0.01). In contrast, AOM/DSS-induced CRC was attenuated in Alkbh5^KOLgr5-Cre^ER mice (P<0.01), inferring a role for ALKBH5 in promoting cancer-initiating capacity in colorectal CSCs. Integrative RNA-seq, m⁶A-seq and Ribo-seq identified FAM84A as a target of ALKBH5 in CSCs. ALKBH5 binds and demethylates m⁶A-modified FAM84A, causing FAM84A mRNA decay and reduced FAM84A mRNA and protein expression. FAM84A overexpression abolished ALKBH5-induced self-renewal and CSC markers, while FAM84A knockout restored stemness in ALKBH5-depleted CSCs, confirming that FAM84A is a functional target of ALKBH5 in CSCs. Mechanistically, we revealed that FAM84A interacts with β-catenin to induce ubiquitination and degradation of β-catenin by recruiting β-catenin destruction complex components (e.g. Axin2, GSK-3β). Moreover, ALKBH5 overexpression elicited fluorouracil (5-FU) and oxaliplatin resistance in CSCs and PDOs, implying ALKBH5 as a therapeutic target. Targeting ALKBH5 by knockout or VNPs-siALKBH5 exerted synergistic effects with 5-FU or oxaliplatin to trigger tumor regression in CSCs- and PDOs-derived xenografts in mice, and in ALKBH5-KO mice.

Conclusion: We identified an ALKBH5-m⁶A-FAM84A-β-catenin axis in driving CRC stemness and chemoresistance. ALKBH5 is a potential therapeutic target in overcoming chemoresistance in CRC patients.

Cong Liang

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