Background Malignant gliomas (MG) are rapidly fatal despite multimodal treatments including radiation therapy, used to treat nearly all MG patients, or even the emerging cellular immunotherapies. Therapeutic resistance in glioma is related to tolerogenic STAT3 activity in both glioma cancer stem cells (GCSs) and in the tumor-associated myeloid immune cells, such as macrophages and microglia, which dominate MG microenvironment.1,2 We previously demonstrated that STAT3 activity in GSCs and tumor-associated myeloid cells can be targeted using Toll-like Receptor-9 (TLR9)-targeted oligonucleotide therapeutics such as siRNA or antisense oligonucleotides (ASO).2,3
Methods Here, we describe development of a new TLR9-targeted and double-stranded STAT3 antisense oligonucleotide (CpG-STAT3dsASO) with optimized efficacy and safety for glioma immunotherapy.
Results Compared to our benchmark ASO oligonucleotides, the LNA-modified CpG-STAT3dsASO showed enhanced target gene knockdown in human and in mouse glioma cells and also in TLR9+ immune cells, such as macrophages and microglia. When tested against orthotopic model of human U251 glioma, intracranial injections of CpG-STAT3dsASO (1 mg/kg/q2w) inhibited tumor growth and significantly extended survival of immunodeficient NSG mice compared to benchmark oligonucleotide. Next, we tested CpG-STAT3dsASO against syngeneic GL261 model in immunocompetent mice. Our results demonstrated that CpG-STAT3dsASO was more effective but also significantly better tolerated than single-stranded CpG-STAT3ASO when injected intracranially, without evidence of severe acute neural toxicities within tested dosing. All tested CpG-STAT3ASO variants induced maturation/activation of intratumoral DCs, macrophages and microglia, while reducing numbers of tumor-associated M2 macrophages and resting microglia as assessed using flow cytometry. Importantly, CpG-STAT3ASO treatments improved the ratio of intratumoral CD8 T cells to Tregs. To elucidate changes in the glioma microenvironment related to STAT3-inhibition/TLR9-activation, we performed an initial single-cell RNAseq analysis of transcriptomic profiles in immune cell subsets isolated from tumors after treatment using CpG-STAT3dsASOLNA. Our analysis indicated the reprogramming of tumor-associated myeloid cell populations within treated glioma with an increased ratio of CD8:regulatory T cells. Our results also suggested the elevation of several immune checkpoint molecules on tumor-infiltrating T cells likely as a result of IFN signaling. Importantly, our preliminary experiments demonstrated a synergy between systemic PD1 inhibition with low-dose (0.25 mg/kg) CpG-STAT3dsASO local therapy. While, neither of treatments alone was curative, the combination anti-PD1/CpG-STAT3dsASO therapy resulted in complete rejection of orthotopic GL261 tumors in the majority of treated mice (figure 1).
Conclusions We believe that further development of CpG-STAT3dsASO will pave way to clinical translation of this strategy to immunotherapy of malignant glioma.
Acknowledgements This work was supported in part by the National Cancer Institute/National Institutes of Health awards number R01CA215183 (M.K.) and P30CA033572 (COH). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Herrmann A, Cherryholmes G, Schroeder A, Phallen J, Alizadeh D, Xin H, Wang T, Lee H, Lahtz C, Swiderski P, et al. TLR9 is critical for glioma stem cell maintenance and targeting. Cancer Res 2014;74:5218–5228.
Moreira D, Adamus T, Zhao X, Su Y-L, Zhang Z, White SV, Swiderski P, Lu X, DePinho RA, Pal SK, et al. STAT3 inhibition combined with CpG immunostimulation activates antitumor immunity to eradicate genetically distinct castration-resistant prostate cancers. Clin. Cancer Res. 2018;24:5948–5962.
Adamus T, Hung C-Y, Yu C, Kang E, Hammad M, Flores L, Nechaev S, Zhang Q, Gonzaga JM, Muthaiyah K, et al. Glioma-targeted delivery of exosome-encapsulated antisense oligonucleotides using neural stem cells. Mol. Ther. Nucleic Acids. 2022;27: 611–620.
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