Lovalvo, I. | bioRxiv Science

bioRxiv2025

Druggable genome CRISPRi screen in 3D hydrogels reveals regulators of cortactin-driven actin remodeling in invading glioblastoma cells

To identify new therapeutic targets that limit glioblastoma (GBM) invasion, we applied druggable-genome CRISPR screens to patient-derived GBM cells in micro-dissectible biomimetic 3D hydrogel platforms that permit separation and independent analysis of core vs. invasive fractions. We identified 12 targets whose suppression limited invasion, of which ACP1 (LMW-PTP) and Aurora Kinase B (AURKB) were validated in neurosphere assays. Proximity labeling analysis identified cortactin as an ACP1- AURKB link, as cortactin undergoes serine phosphorylation by AURKB and tyrosine dephosphorylation by ACP1. Suppression of ACP1 or AURKB in culture and in vivo shifted the balance of cortactin phosphorylation in GBM and reduced actin polymerization and actin-cortactin co-localization. Additional biophysical analysis implicated AURKB in GBM cell adhesion and cortical stiffness, and ACP1 in resistance to mechanical stress and shape plasticity needed for 3D migration. These findings reveal a novel targetable axis that balances kinase and phosphatase activities to regulate actin polymerization during GBM invasion.

cancer biology↗

bioRxiv2024

Replicating retroviral delivery of an IL-15 superagonist improves antitumor immunity and long-term survival in poorly immunogenic glioblastoma models

Glioblastoma (GBM) is the most lethal primary brain neoplasm due to its highly immunosuppressive microenvironment and resistance to conventional therapies. To overcome this challenge, we engineered a replicating retrovirus (RRV) to deliver a superagonist interleukin-15 receptor-linked fusion protein (RLI) directly to tumor cells, engineering them into local immunotherapy biofactories. This strategy leverages the tumor-selective replication of RRV to achieve localized and sustained RLI expression within the tumor microenvironment. In two orthotopic poorly immunogenic GBM mouse models, intratumoral administration of RRV RLI significantly reduced tumor growth and prolonged survival compared to controls, with some mice achieving long-term remission and demonstrating immunologic memory upon rechallenge. Transcriptomic and flow cytometric analyses revealed that RRV RLI treatment enhanced infiltration and activation of CD8 T cells, NK cells, and upregulated antigen presentation pathways within the tumor microenvironment. Depletion studies indicated that the therapeutic efficacy of RRV RLI is dependent on both CD4 and CD8 T cells. Notably, combining RRV RLI with the GBM standard of care chemotherapeutic agent temozolomide (TMZ) synergistically improved survival outcomes. Subsequent single-cell RNA and T cell receptor sequencing identified enhanced effector cell activation, antigen presentation, and clonal T cell expansion in the combination therapy group. Further T cell receptor analysis and clustering implied a tumor-specific immune response rather than one targeting the viral delivery vehicle, suggesting that this therapeutic approach could be reapplied without eliciting anti-vector immunity. Our findings suggest that RRV-mediated delivery of RLI effectively transforms GBM tumors into immunostimulatory hubs, eliciting a potent anti-tumor immune response. This novel viral immunotherapy holds significant promise for clinical translation in the treatment of GBM and other difficult-to-treat solid tumors.

cancer biology↗