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.

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

FRUSTERI, Cristina
2022

Abstract

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.
FLIP, immunesuppression, cytokine storm, immune dysregulation, lung cancer
File in questo prodotto:
File Dimensione Formato  
Thesis-Cristina Frusteri.pdf

embargo fino al 07/01/2024

Descrizione: PDF file of PhD thesis
Tipologia: Tesi di dottorato
Licenza: Accesso ristretto
Dimensione 3.54 MB
Formato Adobe PDF
3.54 MB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/1070306
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact