Inhibiting PIK3C3/VPS34 enhances anti-GD2 immunotherapy in neuroblastoma

Anti-GD2 immunotherapy has improved survival in children with high-risk neuroblastoma, yet relapse and refractory disease remain major challenges, underscoring the need for strategies that extend therapeutic benefit. We investigated whether inhibition of PIK3C3/VPS34, a class III PtdIns3K that regulates autophagy and endosomal trafficking, could potentiate anti-GD2 therapy. Inducible PIK3C3/VPS34 knockdown induced apoptosis, impaired spheroid growth, and suppressed tumor progression. Notably, PIK3C3/VPS34 depletion enhanced anti-GD2 antibody-driven NK cell cytotoxicity and increased tumor cell-surface GD2 expression. In vivo, combined PIK3C3/VPS34 inhibition and anti-GD2 therapy achieved durable tumor suppression, which was associated with increased infiltration of NK cells and T cells, enhanced T-cell activation, reduced immunosuppressive myeloid populations, enhanced pro-inflammatory macrophage polarization, and elevated production of immune-recruiting chemokines. Pharmacological PIK3C3/VPS34 inhibition recapitulated the effects of genetic depletion, inducing neuroblastoma cell death and enhancing anti-GD2 antibody-dependent NK cell cytotoxicity in vitro. Enhanced NK cell-mediated killing was associated with marked enrichment of GD2 at the tumor cell surface. PIK3C3/VPS34 inhibition caused a disproportionate increase in surface relative to total cellular GD2 levels, while combined inhibition of PIK3C3/VPS34 and lysosomal function resulted in additional GD2 accumulation, supporting a role for endolysosomal processing in controlling GD2 abundance. Together, these findings demonstrate that PIK3C3/VPS34 inhibition enhances the efficacy of anti-GD2 immunotherapy through complementary tumor-intrinsic and immune-mediated mechanisms, including direct tumor cell killing, increased cell-surface GD2 expression, and enhanced antitumor immune responses within the tumor microenvironment, supporting PIK3C3/VPS34 inhibition as a promising therapeutic strategy for high-risk neuroblastoma.Abbreviations: ADCC: antibody-dependent cellular cytotoxicity; ANOVA: analysis of variance; BafA1: bafilomycin A1; dox: doxycycline; ELISA: enzyme-linked immunosorbent assay; FVS780: fixable viability stain 780; FACS: fluorescence-activated cell sorting; GD2: disialoganglioside 2; G-MDSCs: granulocytic myeloid-derived suppressor cells; HCQ: hydroxychloroquine; IP: intraperitoneal; MHC-I: major histocompatibility complex class I; PtdIns3K: class III phosphatidylinositol 3-kinase; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; shRNA: short hairpin RNA; TME: tumor microenvironment; Tregs: regulatory T cells