Abstract

Background: Glioblastoma (GBM) exhibits marked cellular heterogeneity and resistance to therapy. Calcium (Ca2+) signaling at endoplasmic reticulum (ER)–mitochondria contact sites has emerged as a key regulator of mitochondrial function and cell fate; however, its lineage-specific role and therapeutic relevance in GBM remain unclear.Methods:ITPR1expression was analyzed using single-cell and bulk RNA sequencing (RNA-seq) datasets and validated by immunohistochemistry and survival analyses. Functional studies were conducted using genetic silencing or CRISPR-mediated activation ofITPR1, combined with DRP1 knockdown, Ca2+imaging, transmission electron microscopy, co-immunoprecipitation, mitochondrial fractionation, and mitochondrial functional assays. Therapeutic efficacy was evaluated in orthotopic GBM xenograft models treated with 2-aminoethoxydiphenyl borate (2-APB), temozolomide (TMZ), or their combination.Results: ITPR1 was enriched in mesenchymal-like malignant cell states and associated with higher tumor grade, recurrence, and poor prognosis.ITPR1knockdown suppressed GBM cell proliferation and tumor growth while promoting intrinsic apoptosis. Mechanistically, loss of ITPR1 impaired ER-to-mitochondria Ca2+transfer, disrupted ER–mitochondria contacts, and altered mitochondrial ultrastructure. This was accompanied by reduced DRP1 Ser616 phosphorylation and mitochondrial recruitment, as well as decreased autophagy and mitophagy activity. Consequently,ITPR1knockdown led to mitochondrial depolarization, increased mitochondrial reactive oxygen species (ROS) accumulation, and activation of mitochondria-dependent apoptosis. Conversely, DRP1 knockdown attenuated the mitochondrial and pro-survival effects induced byITPR1overexpression. In vivo, combined treatment with 2-APB and TMZ resulted in greater tumor suppression and prolonged survival compared with either treatment alon