Johnson, Amanda L.

Abstract A hallmark of GBM is its highly-immunosuppressive tumor microenvironment (TME). GBM stem-like cells (GSCs) can evade immune surveillance to allow for continued tumor growth and propagation. GSCs regulate the immune TME through recruitment and production of immunosuppressive cells and factors via tumor cell-intrinsic mechanisms. Blocking these cell-intrinsic immunosuppressive mechanisms is widely viewed as a way to enhance the effects of current immune therapies. We recently showed that TGFβ type II receptor (TGFBR2) promotes a mesenchymal transition in GBM cells characterized by high expression levels of immunosuppressive genes NT5E (CD73), ENTPD1 (CD73), LGALS1 (galectin-1), CD274 (PD-L1), PDCD1LG2 (PD-L2), and TGFB1. miRNA network analysis identified miR-16-5p and miR-124-3p as high-confidence candidates to block the expression of these immunosuppressive signaling axis in GBM cells. Transgenic miR-16-5p and miR-124-3p simultaneously reduced expression of CD274, ENTPD1, NT5E, TGFB1, and LGALS1 and increased tumor cell killing of PBMC-derived CD4+ and CD8+ T cells. We also measured an increase in drivers of the CD8+ effector state and a decrease in expression of exhaustion marker PD1 in PBMC-derived CD8+ T cells. In addition to the effects on CD4+ and CD8+ T cell activation and function, miRNA-mediated knock-down of this immunosuppressive gene signature in tumor cells also resulted in a decrease in CD206 expression and reduced release of anti-inflammatory chemokines in THP1 cells. These findings demonstrate that miRNA-based gene inhibition can block cell-intrinsic immunosuppressive pathways to enhance anti-tumor immune cell function in vitro and predicts that delivering these miRNAs using advanced nanocarriers will cooperate with current immunotherapies in vivo. Citation Format: TC Taylor, Amanda Johnson, Harmon Khela, Jack Korleski, Sophie Sall, Bachchu Lal, Karen Smith-Connor, Hernando Lopez-Bertoni. miR-16-5p and miR-124-3p block the immunosuppressive phenotype of GBM cells in vitro. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1357.

Abstract Despite aggressive therapy consisting of surgery followed by radio/chemotherapy Glioblastoma (GBM) recurs in almost all patients and, currently, there are no proven therapies to treat recurrent GBM (rGBM). Recent developments in nanomedicine provide new and promising opportunities to develop new targeted therapeutics to treat brain tumors. In this study we combine bioinformatics, forward-thinking understanding of miRNA biology and cutting-edge nucleic acid delivery vehicles to advance targeted therapeutics for rGBM. Bioinformatic analysis of RNA sequencing from GSCs and clinical rGBM specimens identified TGF-beta receptor II (TGFBR2) signaling as a targetable pathway in rGBM. Mechanistically, we show that alterations in chromatin state driven by stem-cell driving events are conducive to a therapy-resistant state induced by TGFBR2. We show that blocking TGFBR2 via molecular and pharmacological approaches decreases the stem cell capacity, cell viability and re-sensitizes clinical rGBM isolates to temozolomide (TMZ) in vitro. Network analysis of SMAD2/3 targets enriched in rGBM predicted a subset of miRNAs that can target multiple genes within this set. Notably, miR-590-3p was predicted to inhibit 37 SMAD 2/3 gene targets that are also enriched in rGBMs. Bioinformatics analysis also showed that expression of 24 of those 37 miR-590-3p targets are positively correlated with TGFBR2 expression in rGBM clinical specimens. Consistent with these predictions, miR-590-3p inhibited expression of 25 of the 37 targets across 3 distinct therapy-resistant GSC isolates. To translate these in vitro finding, we developed novel bioreducible Lipophilic poly(β-amino ester) nanoparticles (LiPBAEs) for in vivo miRNA delivery. Following direct intratumoral infusion, these nanomiRs efficiently distribute through the tumors and spatial transcriptomic analysis (STA) found a decrease in 34 out of the 37 miR- 590-3p targets in animals that received the active nanomiR compared to control miRNA. Furthermore, STA identified 3 cell populations with distinct transcriptomic profiles reflecting a gradient pharmacodynamic response within tumors that received miR-590-3p nanomiRs with decreasing target expression towards the core of the tumor closest to catheter tip placement. To test the effect of 590-3p nanomiR therapy on survival, animals bearing pre-established intracranial rGBM xenografts received three infusions of either control or miR-590-3p nanomiRs every other day beginning on post-implantation day 7. All 10 animals treated with control nanomiRs were either dead or premorbid requiring euthanasia by post-implantation day 23. In contrast, 3 of 10 animals treated with miR-590-3p nanomiRs remained alive and healthy by post-implantation day 45, at which time the experiment was terminated. These results show that miRNA-based targeted therapeutics provide new opportunities to treat rGBM and bypass the resistance that is developed to standard of care. Citation Format: Hernando Lopez-Bertoni, Jack Korleski, Sophie Sall, Kathryn Luly, Maya K Johnson, Amanda L Johnson, Harmon Khela, Bachchu Lal, TC Taylor, Jean Micheal Ashby, Hector Alonso, Alice Li, Stephany Tzeng, John Laterra, Jordan J Green. Multipronged SMAD Pathway Targeting by LiPBAE miR-590-3p NanomiRs Inhibits Recurrent Glioblastoma Growth and Prolongs Survival [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR011.