ALK regulates macrophage polarization via the USP7/SOX9/MFAP2-mediated glycolytic pathway to promote neuroblastoma progression.

Abstract

Neuroblastoma (NB) is a common pediatric malignancy in which activating mutations of anaplastic lymphoma kinase (ALK) drive tumor progression, yet the underlying mechanisms remain incompletely understood. Here, we demonstrate that ALK signaling promotes glycolysis and M2 macrophage polarization through the USP7-SOX9-MFAP2 axis. Using qRT-PCR, western blot, and co-culture systems, we found that ALK inhibition with lorlatinib reduces lactate production, downregulates M2 markers, and upregulates M1 markers in NB cells and NB mouse models. Mechanistically, Co-IP and ubiquitination assays revealed that ALK recruits and activates the deubiquitinase USP7, which deubiquitinates and stabilizes SOX9. Dual-luciferase reporter and ChIP-qPCR analyses further demonstrated that SOX9 directly binds to the MFAP2 promoter to activate its transcription. Functional assays showed that elevated MFAP2 enhances glycolysis, leading to increased lactate and immunosuppressive factor secretion, which in turn polarizes tumor-associated macrophages toward the pro-tumor M2 phenotype. Importantly, in vivo experiments confirmed that MFAP2 overexpression partially reverses the anti-tumor effects of lorlatinib. These findings identify the ALK/USP7/SOX9/MFAP2 cascade as a critical regulator of metabolic reprogramming and immune evasion in NB, and suggest that targeting this axis may represent a promising therapeutic strategy for high-risk NB.