An optimized retinoic acid-inducible gene I agonist M8 induces immunogenic cell death markers in human cancer cells and dendritic cell activation
RIG-I is a cytosolic RNA sensor that recognizes short 5′ triphosphate RNA, commonly generated during virus infection. Upon activation, RIG-I initiates antiviral immunity, and in some circumstances, induces cell death. Because of this dual capacity, RIG-I has emerged as a promising target for cancer immunotherapy. Previously, a sequence-optimized RIG-I agonist (termed M8) was generated and shown to stimulate a robust immune response capable of blocking viral infection and to function as an adjuvant in vaccination strategies. Here, we investigated the potential of M8 as an anti-cancer agent by analyzing its ability to induce cell death and activate the immune response. In multiple cancer cell lines, M8 treatment strongly activated caspase 3-dependent apoptosis, that relied on an intrinsic NOXA and PUMA-driven pathway that was dependent on IFN-I signaling. Additionally, cell death induced by M8 was characterized by the expression of markers of immunogenic cell death-related damage-associated molecular patterns (ICD-DAMP)—calreticulin, HMGB1 and ATP—and high levels of ICD-related cytokines CXCL10, IFNβ, CCL2 and CXCL1. Moreover, M8 increased the levels of HLA-ABC expression on the tumor cell surface, as well as up-regulation of genes involved in antigen processing and presentation. M8 induction of the RIG-I pathway in cancer cells favored dendritic cell phagocytosis and induction of co-stimulatory molecules CD80 and CD86, together with increased expression of IL12 and CXCL10. Altogether, these results highlight the potential of M8 in cancer immunotherapy, with the capacity to induce ICD-DAMP on tumor cells and activate immunostimulatory signals that synergize with current therapies.
KeywordsRIG-I Interferons Cancer immunotherapy Immunogenic cell death Dendritic cells
Antigen processing machinery
American Type Culture Collection
Bcl-2-associated X protein
Bcl-2 homology 3
Caspase recruitment domain
Carbonyl cyanide 3-chlorophenylhydrazone
C-C motif chemokine ligand 2
Cell trace far red
C-X-C motif ligand 1
C-X-C motif chemokine 10
Damage-associated molecular patterns
Kaighn’s modification of Ham’s F-12 medium
Glyceraldehyde 3-phosphate dehydrogenase
Human leucocyte antigens-A, B, and C loci
High-mobility group box 1
Immunogenic cell death
Indoleamine-pyrrole 2,3-dioxygenase 1
Interferon α and β receptor subunit 1
Interferon regulatory factor 3
Mitochondrial antiviral signaling protein
Myeloid-derived suppressor cells
Monocyte-derived dendritic cells
Pathogen-associated molecular patterns
Poly (ADP-ribose) polymerase
Pattern recognition receptors
Proteasome subunit beta type-8, -9, -10
p53 upregulated modulator of apoptosis
Retinoic acid-inducible gene-I
Receptor-interacting protein 1
Relative light units
Short interfering RNA
Antigen peptide transporter 1, 2
TANK-binding kinase 1
Vesicular stomatitis virus
Conception and design: LC, AZ, JH, DO. Collection and assembly of data: LC, AZ, EV, MM, MF, EP, GP, CK. Data analysis and interpretation: LC, MJ, DO, AZ, AS, and JH. Manuscript writing: LC, AZ, JH. Final approval of manuscript: all authors.
This research was supported by grants from Fondazione Cenci Bolognetti, the Italian Association for Cancer Research (AIRC) (IG16901), and NIH Grants 1R561AI108861-01A1 and 7R21CA192185. Elisabetta Vulpis is supported by an AIRC fellowship for Italy.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
PBMC were freshly isolated from peripheral blood samples of anonymous volunteer healthy donors at the Transfusion Center of Sapienza University of Rome. Written informed consent was obtained from all blood donors to the use of their blood for scientific purposes.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee of the Sapienza University of Rome (Rif.3373/250914) and with the 1964 Helsinki declaration. Institutional Review Board approval was not required for this kind of study.
Cell line authentication
Primary Mel1007 and metastatic melanoma Mel120 cells were a kind gift of Dr. G. Parmiani (Milan, Italy), A549, HCT116 and PC3 were all from ATCC. All the experiments were performed with cells at low passage numbers (≤ 10). Cell identity was monitored based on morphology and growth rate. Mycoplasma was routinely tested.
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