The role of KDM5B in creating synthetic vulnerabilities in combination with radiotherapy in melanoma cells

Cutaneous melanoma is the most aggressive type of skin cancer, with survival rates declining due to tumor heterogeneity and therapy resistance. Distinct subpopulations, including slow-cycling, therapy-resistant cells with high expression of the histone demethylase KDM5B, contribute to tumor progression and poor outcomes. Intermittent cycling hypoxia, defined by repeated hypoxia followed by reoxygenation, promotes tumor plasticity and aggressiveness, yet its role in melanoma heterogeneity and resistance remains poorly understood. We established hypoxia/reoxygenation-tolerant (HRT) melanoma cell lines (Hx10) through 10 cycles of intermittent cycling hypoxia (48 h at 0.2% O₂ followed by 120 h at 20.9% O₂) under conditions of KDM5B overexpression. Radiation response was evaluated in Hx10 and nonselected control cells. To investigate adaptive mechanisms, we performed reversed-phase protein array (RPPA) screening and applied an information-theoretic approach to compute protein-specific altered signaling signatures. Pathway enrichment analyses were used to identify dysregulated subnetworks. Hx10 melanoma cells displayed increased resistance to radiation compared with nonselected control cells. Proteomic profiling identified distinct signaling signatures associated with KDM5B overexpression and adaptation to cycling hypoxia. These signatures revealed coexpressed subnetworks involving DNA repair, PI3K/AKT/mTOR, AMPK, and autophagy pathways, several of which are implicated in therapy resistance. Functional assays demonstrated that targeting either KDM5B or PI3K reduced the radioresistance of Hx10 melanoma cells. Sequential combination treatments impaired repopulation ability, particularly when KDM5B overexpression was withdrawn, indicating dependence on KDM5B for survival. Our findings provide proof-of-concept that altered signaling signatures can be used to define novel vulnerabilities in melanoma. KDM5B overexpression promotes adaptation to intermittent cycling hypoxia and confers resistance to radiation through activation of DNA repair and survival pathways. Targeting KDM5B or PI3K in combination with radiotherapy may represent a promising strategy to overcome resistance and improve treatment outcomes in melanoma.