2566 - IKBKE, the c-myc/CSF-1 Signaling Axis, and Mediating Recruitment of M2 Tumor-Associated Macrophages (TAMs) after Radiotherapy in Glioblastoma

Y. Ma¹, H. Tian², J. Lu³, D. Chen³, and W. Wen⁴; ¹Shandong Cancer Hospital and Institute, Jinan, Shandong, China, ²Qilu Hospital of Shandong University, Jinan, Shandong, China, ³Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, ⁴Department of Radiation Oncology and Shandong Provincial Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shangdong, China

Purpose/Objective(s): Glioblastoma, a formidable intracranial tumor, often necessitates radiotherapy post-surgery. Despite its therapeutic value, radiotherapy paradoxically recruits M2 tumor-associated macrophages (TAMs), contributing to a tumor-promoting immune microenvironment. Our study identifies serine/threonine kinase IKBKE as a critical player in this process, where its overexpression post-radiotherapy phosphorylates c-myc, enhancing CSF-1 expression and M2 TAM recruitment. These findings spotlight IKBKE as a potential target for sensitizing glioblastoma to radiation therapy. Materials/

Methods: A radio-resistant glioblastoma model was developed to study IKBKEs expression dynamics post-radiotherapy, employing RNA-seq, qRT-PCR, and Western blotting. The histone modification after radiotherapy, especially histone methylation, were determined by GO and GSEA enrichment analyses. The IKBKE expression was closely associated with histone demethylation using a broad-spectrum inhibitor by WB and qRT-PCR. Specific histone demethylation sites were identified using specific antibodies, inhibitors and was verified using ChIP. The recruitment of M2 TAMs was assessed using co-culture assays, transwell assay,flow cytometry, and qRT-PCR. We conducted orthotopic implantation tumor stereotactically, performed SARRP radiotherapy and MRI imaging to monitor tumor size. We validated that IKBKE mediated the recruitment of M2 TAMs after radiotherapy by multicolor immunofluorescence and flow cytometry. Endogenous and exogenous IP were used to confirm IKBKE interaction with c-myc. In vitro kinase assay and Phos-tag Acrylamide were used to demonstrate that IKBKE phosphorylates c-myc. The phosphorylation site of c-myc was confirmed by point mutations combined with in vitro kinase assays, IP and WB. The relationship of IKBKE, c-myc and FBXW7 was determined using CHX, MG132, in vivo and in vitro ubiquitination assays, immunofluorescence and IP. Chemokines were searched for using RNA-seq and identified using qRT-PCR and ELISA
Results: Radiotherapy-induced IKBKE expression is linked to enhanced histone demethylation at H3K9me3 sites, correlating with poor prognosis. IKBKEs overexpression orchestrates M2 TAM recruitment by phosphorylating c-myc at Ser96 site, altering its interaction with protein FBXW7, thereby preventing c-mycs ubiquitination and promoting CSF-1 expression. These molecular events culminate in an immunosuppressive TME conducive to tumor progression.
Conclusion: Radiotherapy promotes IKBKE by increasing histone demethylation. IKBKE inhibits the ubiquitination degradation of c-myc by directly phosphorylating it to promote CSF-1 expression to recruit M2 TAMs, resulting in a suppressive TME that promotes tumor progression.