Targeting immune dysregulation mediated by FLIP and putative FLIP-related pathways to develop new therapeutic approaches

Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP) is an anti-apoptotic protein that has been reported to be highly expressed in several cancer settings and induced in different infectious diseases to block caspase-mediated cell death. Myeloid derived suppressor cells (MDSCs) represent a heterogeneous myeloid cell population, comprising both myeloid-cell progenitors and fully differentiated cells, whose role in altering the immune responses was observed in various pathological contexts. In a variety of tumor settings, a high number of circulating MDSCs is associated with poorer prognosis and weaker response to treatment. Signal transducer and activator of transcription 3 (STAT3) pathway represents one those upregulated in immunosuppressive, c-FLIP-over-expressing monocytes and, therefore, it is an ideal target to control MDSC-associated functions. Moreover, STAT3 pathway is relevant to differentiate myeloid cells into a cytokine-producing source during the cytokine release syndrome (CRS). By availing of a transgenic (Tg) mouse model that over-expresses viral form of FLIP (v-FLIP) in the myeloid cell lineage, in this study we demonstrated that treatments to target STAT3 were able to mitigate the FLIP-mediated immune dysregulation. These data suggest that STAT3-targeting approaches might be promising in a variety of diseases in which MDSCs can play a pathogenic role. To investigate further the molecular role of c-FLIP behind the mechanisms of immunomodulation in myeloid cell subset we demonstrated the nuclear co-localization c-FLIP-p50 NF-kB subunit. When c-FLIP is over-expressed, it can translocate to the nucleus in a complex with p50 protein suggesting the acquisition of transcriptional functions. We demonstrated a physical interaction c-FLIP-p50 in both cytoplasmic and nuclear fractions, and revealed, through ChIP-seq analysis, several DNA sequences involved in immune system processes that were identified as c-FLIP-dependent. Since FLIP expression was linked to viral replication, we hypothesized its involvement in the progression of COVID-19 infection caused by SARS-CoV-2 virus. Indeed, we found a direct correlation between c-FLIP expression and immunosuppressive activity of monocytes circulating in the blood of COVID-19 patients, similarly to what previously reported by our laboratory in oncology patients. Immunosuppression by COVID-19 patient monocytes could be indeed reverted by STAT3 inhibitors. Finally, we investigated c-FLIP as predictive biomarker of outcome following immunotherapy. In a non-small-cell lung cancer (NSCLC) cohort undergoing to immune-checkpoint inhibitors (ICIs) therapy, we demonstrated that c-FLIP assessment was able to predict the clinical response, indicating c-FLIP as promising predictive biomarker to optimize the current oncology treatments. Collectively, our results contribute to deepen the knowledge about c-FLIP regulation in immune cells, which can be used to develop selective inhibitors to restrain MDSC suppressive activity under different pathological conditions.