Abstract 1353: Immunomodulatory effects of valproic acid in combination with cisplatin and cetuximab in head and neck squamous cell carcinoma
Authors: Iannelli Federica , Zotti Andrea Ilaria , Roca Maria Serena , Grumetti Laura , Moccia tania , vitagliano carlo , costantini susan , capone francesca , collina francesca , Gabriele Lucia , parlato stefania , Romagnoli Giulia , Kepp Oliver , Kroemer Guido , Di Gennaro Elena , Budillon Alfredo 
Published: June 2022

Abstract
INTRODUCTION. Development of immune-check-point inhibitors (ICI) has greatly changed the treatment of recurrent or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) demonstrating durable antitumor response and overall survival (OS) improvement. However, more than 70% of patients do not respond to ICI and, recently, phase III Checkmate 651 trial did not demonstrate OS improvement of first-line ICI vs standard EXTREME regimen (cetuximab (CX) plus 5-fluorouracil/cisplatin (CDDP) in R/M HNSCC patients (ESMO2021-LBA36). We have previously showed in vitro and in vivo models of HNSCC the synergistic antitumor interaction of CDDP/CX combined with the antiepileptic valproic acid (VPA), a histone deacetylase inhibitor (HDACi) with anticancer and immunomodulatory properties (Iannelli F., 2020). Here we investigate the immunomodulatory effects of VPA/CDDP/CX combination, focusing on the induction of "immunogenic cell death" (ICD)-like features.
METHODS. ICD-related damage-associated molecular patterns (DAMPs) calreticulin (CARL), ATP, HMGB1 and Annexin-A1 (ANXA1) expression was evaluated by western blot, immunofluorescence and/or bioluminescent assays on HNSCC Cal27 and osteosarcoma U2OS cells. Maturation, migration, cytokines release, and phagocytic capacity of dendritic cells (IL4_DCs) was evaluated incubating IL4-DCs with conditioned medium from untreated/treated Cal27 cells and/or in co-culture IL4-DCs/Cal27 assays by ELISA, cytofluorimetric assays and confocal microscopy.
RESULTS. VPA/CDDP/CX combination induced in both U2OS and Cal27 cells, synergistic pre-apoptotic exposure of CARL along with HMGB1, ANXA1 and ATP release as compared with single drugs or doublet combination. Moreover, the triple combination increases IL4-DCs migration towards pretreated Cal27 cells as well as the rate of IL4-DCs phagocytosis of apoptotic bodies released by Cal27 dying cells. Triple combination also induces phenotypical maturation of DCs as shown by the increased surface expression of CD83 and CD86. Furthermore, conditioned medium from VPA/CDDP/CX treated Cal27 cells, induced increased release of immune-stimulating cytokines and chemokines IL2, IL1β, TNFα, MIP-1α and G-CSF by IL4-DCs as well as the impairment of immunosuppressive cytokines release IL-10 and VEGF, by VPA/CDDP/CX treated Cal27, co-cultured with IL4-DCs. In vivo experiments in immunocompetent syngeneic mouse model using HNSCC AT-84 cells transduced with human-EGFR are ongoing.
CONCLUSION. Overall, we demonstrated that VPA/CDDP/CX is able to enhance the immunogenicity of HNSCC model by inducing ICD. This mechanistic hypothesis will be tested by assessing ICD biomarkers on samples of patients enrolled in the V-CHANCE phase 2 clinical trial evaluating the CDDP/CX associated with VPA in R/M SCCHN patients and recently concluded (NCT02624128).
Citation Format: Federica Iannelli, Andrea Ilaria Zotti, Maria Serena Roca, Laura Grumetti, tania Moccia, carlo vitagliano, susan costantini, francesca capone, francesca collina, Lucia Gabriele, stefania parlato, Giulia Romagnoli, Oliver Kepp, Guido Kroemer, Elena Di Gennaro, Alfredo Budillon. Immunomodulatory effects of valproic acid in combination with cisplatin and cetuximab in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1353.