Abstract 4067: Inhibition of XBP1 expression in murine macrophages by miR-214 delivered by extracellular vesicles
Authors: Searles Stephen , Alamanza Gonzalo , Zanetti Maurizio 
Published: September 2020

Recent advances in cancer therapy have focused on dysregulated local immunity, with paradigm-shifting discoveries like immune checkpoint inhibitors fundamentally altering the way we think about treating cancer. It is now well appreciated that local immune dysregulation is a viable target for cancer therapy, offering many advantages over traditional approaches. One mechanism at the origin of immune dysregulation is a sustained activation of the unfolded protein response (UPR) in the endoplasmic reticulum. The UPR is activated under conditions of stress, including hypoxia, DNA damage, and aneuploidy- stressors present within the tumor microenvironment. Unsurprisingly, the UPR is commonly dysregulated in cancer, acting to promote tumor growth. Multiple lines of evidence support this: TCGA data shows that UPR genes are upregulated in human cancer, and animal models show that tumor formation and growth rates are lower in mice lacking key UPR genes such as IRE1α and XBP1. The UPR promotes tumor growth not only cell-intrinsically, via cancer cell cytoprotection, but also cell-extrinsically, by polarizing tumor-infiltrating macrophages to a pro-tumor inhibitory/ immune stimulatory (IIS) phenotype. We recently found that the IRE1α-XBP1 branch of the UPR is responsible for the IIS signature that occurs in ER stressed macrophages. Here, we sought to develop a novel strategy to target the UPR in macrophages for the treatment of cancer. We focused on microRNAs (miRNA), since no approved small molecule inhibitors of the UPR exist at present for use in cancer patients due to toxicity. We used a proprietary technology developed by our lab that allows for the generation of “induced” extracellular vesicles (iEVs) from B cells that contain a microRNA of choice as cargo. Previously, we reported that transfection of B cells with plasmid DNA encoding for a pre-miRNA loop results in the secretion of large numbers of iEVs that are enriched in the corresponding mature miRNA. Using this technology, we engineered a plasmid DNA to code for miR-214, a miRNA known to target the 3'-UTR of XBP1 mRNA, and used this plasmid to transfected B cells, yielding iEV-214. iEV-214 were highly enriched in miR-214 relative to control iEVs, which include EVs from B cells that were not transfected (iEV-empty), transfected with a scrambled miRNA (iEV-scr), or transfected with an un-related miRNA (iEV-1291). Bone marrow-derived macrophages (BMDM) treated with iEV-214 were enriched in miR-214 relative to all of the controls, demonstrating that iEVs can transfer their miRNA payload to BMDM. When we induced ER stress in BMDM treated with iEV-214, XBP1 transcription was reduced over 50% compared to iEV-scr. Together, these data show that iEVs can transfer miR-214 into macrophages and reduce expression of XBP1 mRNA. We propose iEV-214 as a prototype of a new class of cancer therapeutics to correct UPR-mediated macrophage dysregulation.

Citation Format: Stephen Searles, Gonzalo Alamanza, Maurizio Zanetti. Inhibition of XBP1 expression in murine macrophages by miR-214 delivered by extracellular vesicles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4067.