Approccio integrato di chemioterapia-terapia a bersaglio molecolare e immunoterapia nei sarcomi dei tessuti molli: studio preclinico e ruolo funzionale della via di segnalazione cGAS-STING.

Soft tissue sarcomas (STS) are rare and heterogeneous tumors with mesenchymal origin. Unfortunately, STS are often diagnosed in the late stages and advanced and non-resectable forms have poor prognosis. Novel effective therapeutic options are urgently needed. Our group recently showed the antitumor activity of a combined chemo-targeted approach based on the DNA-damaging agent, trabectedin (TR), and the PARP-1 enzyme inhibitor olaparib (OL) in preclinical and clinical studies. However, a different degree of drug synergism was observed in a panel of STS preclinical models and 50% of treated patients showed progressive disease at six months. With this work, we now propose to further explore a combined chemo-targeted and immunotherapy approach. In particular, considering the DNA damaging action of TR and the ability of OL to block the DNA repair machinery in tumor cells, we hypothesized that an increment in cytosolic DNA and the consequent cGAS-STING pathway activation could lead to a stimulatory environment for a subsequent natural killer (NK) cell-adoptive immunotherapy. NK are lymphocytes endowed with a peculiar anti-tumor activity based on ligands recognition on target cells and activating signals. The aim of my thesis project was to evaluate the potential anti-tumor effect of a combined chemo-targeted and immunotherapeutic approach against STS cell lines. Furthermore, a deep investigation of the molecular circuit involved in the potential priming effect on NK killing due to the chemo-targeted pre-treatment was carried out. First, we evaluated the expression of NK activating ligands on STS before and after the TR+OL treatment. We showed a variable degree of expression of MIC-A/B, ULBPs NK-activating ligands on three STS cells at basal condition and we observed that TR+OL maintained or even increased their expression. Then, we analyzed the expression and phosphorylation of key transducers in the cGAS-STING pathway verifying its activation after TR+OL treatment and we measured the increment of Interferon-β (IFN-β) production, as one of its main molecular products by ELISA assays. Moreover, by the silencing of the key cGAS-STING transcription factor IRF3, we showed that the activation of this pathway is involved in the regulation of MIC-A/B and ULBPs after TR+OL treatment. Finally, in functional in vitro assays, the three STS cell lines were challenged by drug treatment followed by NK at different NK:target ratio. A significant increase of NK killing activity was obtained after TR+OL pre-treatment if compared to non-pretreated cells. Importantly, the NK killing activity is reduced against IRF3-silenced STS cells if compared to mock-treated controls showing that cGAS-STING pathway is a key player in TR+OL priming of NK-antitumor activity. In conclusions, this in vitro study showed that the combined chemo-targeted- immunotherapy treatment is effective against STS cell lines and the molecular mechanism behind TR+OL priming effect on NK activity was mediated by the activation of the cGAS-STING DNA sensing pathway through the upregulation of NK activating ligands and IFN-β production. This novel approach deserves further investigation in vivo and in clinical settings.