Abstract

Background/Objectives: Pituitary neuroendocrine tumors (PitNETs) frequently exhibit invasive behaviors that complicate clinical treatment. While cyclin-dependent kinase 5 (CDK5) and lymphokine-activated killer T-cell-originated protein kinase (PBK, also known as PDZ-binding kinase) are implicated in tumor progression, their reciprocal regulatory mechanism remains unclear. This study aims to elucidate the CDK5-PBK interaction in PitNETs and identify potential therapeutic agents targeting this pathway. Methods: We utilized proximity labeling and phospho-specific assays to characterize the CDK5 and PBK interaction in PitNET cell lines. Immunohistochemical analysis was performed on patient tumor tissues to evaluate clinical relevance. Artificial intelligence (AI)-based virtual screening was employed to discover dual-target inhibitors. The therapeutic efficacy of the identified compound, proguanil hydrochloride, was subsequently evaluated using in vitro functional assays, alongside in vivo xenograft animal models. Results: We identified a mutual phosphorylation loop between CDK5 (at S159) and PBK (at T9) that activates insulin signaling, thereby promoting cellular proliferation and invasion in PitNETs. Patient tumor analysis revealed that the co-expression of phosphorylated CDK5 (S159) and PBK (T9) significantly correlates with tumor invasiveness (p < 0.001). Through AI screening, proguanil hydrochloride was identified as a candidate dual-target inhibitor. In vitro assays confirmed that it effectively reduces tumor cell growth, while in vivo xenograft studies validated its capacity to inhibit tumor progression. Conclusions: The CDK5-PBK mutual phosphorylation axis serves as a key driver of invasiveness in PitNETs. Proguanil hydrochloride represents a promising candidate dual-target therapeutic agent capable of disrupting this pathway to suppress tumor growth.

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