Interferon regulatory factor 8 (IRF8) is a transcription factor of the IRF protein family. IRF8 was originally identified as an essentialfactor for myeloid cell lineage commitment and differentiation. Deletion of Irf8 leads to massive accumulation of CD11b⁺Gr1⁺ immature myeloid cells (IMCs), particularly the CD11b⁺Ly6C^hi/+Ly6G⁻ polymorphonuclear myeloid-derived suppressor cell-like cells (PMN-MDSCs). Under pathological conditions such as cancer, Irf8 is silenced by its promoter DNA hypermethylation, resulting in accumulation of PMN-MDSCs and CD11b⁺ Ly6G⁺Ly6C^lo monocytic MDSCs (M-MDSCs) in mice. IRF8 is often silenced in MDSCs in human cancer patients. MDSCs are heterogeneous populations of immune suppressive cells that suppress T and NK cell activity to promote tumor immune evasion and produce growth factors to exert direct tumor-promoting activity. Emerging experimental data reveals that IRF8 is also expressed in non-hematopoietic cells. Epithelial cell-expressed IRF8 regulates apoptosis and represses Osteopontin (OPN). Human tumor cells may use the IRF8 promoter DNA methylation as a mechanism to repress IRF8 expression to advance cancer through acquiring apoptosis resistance and OPN up-regulation. Elevated OPN engages CD44 to suppress T cell activation and promote tumor cell stemness to advance cancer. IRF8 thus is a transcription factor that regulates both the immune and non-immune components in human health and diseases.

Ferroptosis has emerged as a cytotoxic T lymphocyte (CTL)-induced tumor cell death pathway. The regulation of tumor cell sensitivity to ferroptosis is incompletely understood. Here, we report that interferon regulatory factor 8 (IRF8) functions as a regulator of tumor cell intrinsic ferroptosis. Genome-wide gene expression profiling identified the ferroptosis pathway as an IRF8-regulated pathway in tumor cells. IRF8.KO tumor cells acquire resistance to intrinsic ferroptosis induction and IRF8-deficient tumor cells also exhibit decreased ferroptosis in response to tumor-specific CTLs. Irf8 deletion increased p53 expression in tumor cells and knocking out p53 in IRF8.KO tumor cells restored tumor cell sensitivity to intrinsic ferroptosis induction. Furthermore, IRF8.KO tumor cells grew significantly faster than WT tumor cells in immune-competent mice. To restore IRF8 expression in tumor cells, we designed and synthesized codon usage-optimized IRF8-encoding DNA to generate IRF8-encoding plasmid NTC9385R-mIRF8. Restoring IRF8 expression via a lipid nanoparticle-encapsulated NTC9385R-mIRF8 plasmid therapy suppressed established tumor growth in vivo. In human cancer patients, nivolumab responders have a significantly higher IRF8 expression level in their tumor cells as compared to the non-responders. Our data determine that IRF8 represses p53 expression to maintain tumor cell sensitivity to intrinsic ferroptosis.

A hallmark of human colorectal cancer is lost expression of FAS, the death receptor for FASL of cytotoxic T lymphocytes (CTLs). However, it is unknown whether restoring FAS expression alone is sufficient to suppress csolorectal-cancer development. The FAS promoter is hypermethylated and inversely correlated with FAS mRNA level in human colorectal carcinomas. Analysis of single-cell RNA-Seq datasets revealed that FAS is highly expressed in epithelial cells and immune cells but down-regulated in colon-tumor cells in human colorectal-cancer patients. Codon usage-optimized mouse and human FAS cDNA was designed, synthesized, and encapsulated into cationic lipid to formulate nanoparticle DOTAP-Chol-mFAS and DOTAP-Chol-hFAS, respectively. Overexpression of codon usage-optimized FAS in metastatic mouse colon-tumor cells enabled FASL-induced elimination of FAS⁺ tumor cells in vitro, suppressed colon tumor growth, and increased the survival of tumor-bearing mice in vivo. Overexpression of codon-optimized FAS-induced FAS receptor auto-oligomerization and tumor cell auto-apoptosis in metastatic human colon-tumor cells. DOTAP-Chol-hFAS therapy is also sufficient to suppress metastatic human colon tumor xenograft growth in athymic mice. DOTAP-Chol-mFAS therapy exhibited no significant liver toxicity. Our data determined that tumor-selective delivery of FAS DNA nanoparticles is sufficient for suppression of human colon tumor growth in vivo.