Surgery is the first-line and only curative treatment for localized paragangliomas (PPGLs). However, in metastatic cases, surgical intervention is rarely feasible, and effective therapeutic options remain limited, resulting in a poor prognosis for patients with aggressive or metastatic (m)PPGL. This underscores the urgent need to identify novel treatment strategies. In this study, we investigate the antitumor potential of targeting two key dysregulated pathways in PPGL: PI3K signaling and cell cycle regulation.

We assessed the efficacy of buparlisib, a PI3Kα inhibitor (PI3Ki), and ribociclib, a CDK4/6 inhibitor (CDK4/6i), as monotherapies and in combination, using functional assays in representative in vitro models of PPGL. These models included two rodent cell lines (MPC and PC12) as well as rat- and patient-derived primary PPGL cells. The combination therapy was further evaluated in vivo in PC12-derived mouse xenografts. Transcriptomic analysis of treated PC12 cells identified a mitotic gene signature uniquely dysregulated by combination treatment, which was validated in PPGL cells through qRT-PCR and functional assays assessing mitotic spindle defects. Additionally, RNA-Seq analysis of human PPGLs revealed an upregulation of this gene signature in mPPGLs compared to non-metastatic cases, a finding further confirmed by qRT-PCR in independent patient samples.

Dual inhibition of PI3K and CDK4/6 resulted in superior suppression of cell proliferation and invasion compared to monotherapies, while also inducing apoptosis in both 2D and 3D organotypic cultures of PPGL cell lines. Synergistic effects were observed in primary organotypic cultures derived from both rat and patient PPGLs, as well as in PC12-derived xenografts, where tumor growth was significantly reduced, even at lower drug doses. RNA-Seq analysis identified key mediators of the combination treatment response, including mitotic spindle pathways and FOXM1 signaling, both of which were experimentally validated. Bioinformatics analyses further confirmed the activation of the mitotic gene network in human mPPGLs.

Combined inhibition of PI3K and CDK4/6 targets mitotic spindle regulation and exerts potent antitumor effects in PPGL models. Given the upregulation of mitosis-associated genes in mPPGL, this combination therapy holds promise as a novel approach for treating aggressive forms of these tumors.